Rothia species glutamine endopeptidases and use thereof

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Apr. 5, 2012, is named 20120406_SequenceListing_TextFile.sub.--701586.sub.--066292_US.txt and is 180 kilobytes in size.

BACKGROUND OF INVENTION

Celiac disease, also called celiac sprue or gluten-sensitive enteropathy, is a disease which develops in susceptible individuals in response to the intake of dietary gluten. The disease is caused by an immune reaction to gluten, most noticeably, to gliadin-derived peptides. These peptides elicit an immune response damaging microvilli which are tiny protrusions that line the small intestine. Their destruction causes malabsorption of nutrients leading to a variety of generalized gastro-intestinal disease symptoms such as diarrhea and abdominal pain. Additional and secondary symptoms include weight loss, fatigue, anemia, osteopenia and skin and tooth enamel defects.

A related disease is dermatitis herpetiformis, which is a chronic eruption characterized by clusters of intensely pruritic vesicles, papules, and urticaria-like lesions. IgA deposits occur in almost all normal-appearing and perilesional skin. Asymptomatic gluten-sensitive enteropathy is found in 75 to 90% of these patients and in some of their relatives. Onset is usually gradual. Itching and burning are severe, and scratching often obscures the primary lesions with eczematization of nearby skin, leading to an erroneous diagnosis of eczema. Strict adherence to a gluten-free diet for prolonged periods may control the disease in some patients, obviating or reducing the requirement for drug therapy. Dapsone, sulfapyridine and colchicines are sometimes prescribed for relief of itching.

Gluten allergy and gluten intolerance are related ailments which result from an overreaction of a subject's immune system to gluten and gliadin that are normally considered harmless. The symptoms are very similar to celiac sprue or gluten-sensitive enteropathy but without the enteropathy. Afflicted subjects have an abundance of IgG and IgA antibodies against .alpha./.beta.-gliadin. Typical symptoms are abdominal pain, gas, bloating and diarrhea; there is a general feeling of sickness and fatigue after grain-based products are consumed. Severe allergy can led to Gluten-sensitive idiopathic neuropathy where the typical symptoms are ataxia and peripheral neuropathies because the primary tissue targeted are the central nervous system and peripheral nerves.

There is currently no good marketed treatment for celiac disease or the various gluten and gliadin allergy related diseases. In most cases, the symptoms are reversible and can be avoided if the patients refrain from the intake of gluten. Complete elimination of gluten from the diet is not easy to achieve and maintain. Glutens are abundantly contained in dietary products made of wheat, barley and rye. Moreover, gluten is also widely used, for example in commercial soups, sauces, ice creams, hot dogs, and other foods, that patients need detailed lists of foodstuffs to avoid and expert advice from a dietitian familiar with celiac disease. Ingesting even small amounts of gluten may prevent remission or induce relapse. Supplementary vitamins, minerals, and hematinics may also be required, depending on deficiency. A few patients respond poorly or not at all to gluten withdrawal, either because the diagnosis is incorrect or because the disease is refractory. In the latter case, oral corticosteroids (e.g., prednisone 10 to 20 mg bid) may induce response.

The gluten-free diet advice is to be followed for a lifetime, and intake of gluten, even in small amounts, can cause an immediate immunological response. In view of the serious and widespread nature of Celiac Sprue, the development of a non-dietary therapy would allow patients to lead a more normal life and find a broad application in the gluten-sensitive patient population. The present invention addresses such needs.

Current approaches geared towards the development of treatment options for celiac disease and allergic gluten sensitivity focus on enzyme preparations that are able to digest the immunogenic gluten/gliadin oligopeptides into smaller fragments that do not elicit an immune response. Gluten proteins are remarkably resistant to digestive enzymes operating in the gastro-intestinal tract due to the low content of lysine/arginine and the high proline content. Enzymes capable of gluten digestion are considered an attractive therapeutic option.

SUMMARY OF THE INVENTION

Embodiments of the invention are based on the discovery of specific enzymatic activities in human whole saliva and dental plaques. The specific enzymatic activities are from glutamine endopeptidase enzyme(s) found in Rothia species bacteria living in the human mouth and dental plaque therein. The glutamine endopeptidase enzyme can cleave the peptide bond after the Gln within the Xaa-Pro-Gln (-XPQ- motif), where Xaa is any amino acid, Pro is proline and Gln is glutamine. This tripeptide motif is also particularly abundant in known celiac T-cell gluten epitopes. The inventors showed that the saliva-associated glutamine endopeptidase enzymes can degrade gluten/gliadins in vitro. Glutens and gliadins are proline and glutamine rich proteins that are the cause of the immune response in Celiac Sprue, gluten allergy and dermatitis herpetiformis. The discovery of this enzyme provides the use of the enzyme for non-dietary therapies of Celiac Sprue, gluten allergy and dermatitis herpetiformis.

Embodiments of the invention provide an isolated glutamine endopeptidase enzyme having enzymatic activity to break down glutens into small peptide fragments. In some embodiments, the enzyme has an apparent molecular weight of about 70-75 lcDa as determined by gliadin zymograms or sodium dodecyl sulfate polyacrylamide gel electrophoresis, has a functional pH range of 3-10 as determined by detectable Z-YPQ-pNA cleaving activity within a 24 hour digestion period, complete digestion is achieved at 72 hours under the described assay conditions, has a functional pH range of 7-10 as determined by substantially complete Z-YPQ-pNA cleavage within a 1 hour digestion period, cleaves the peptide bond after XPY and XPQ motifs in glutens, is 100% inhibited by 1 mM of EDTA or PMSF, is a metal-ion dependent protease, is precipitated by 25-45% ammonium sulphate, and is negatively charged at pH>5.0. In one embodiment, the enzyme is derived from a Rothia species bacterium.

In one embodiment, provided herein is a formulation for use in treatment of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis, the formulation comprises an effective dose of an extract from a Rothia species bacteria or an isolated glutamine endopeptidase enzyme and a pharmaceutically acceptable excipient, wherein the extract from the Rothia species bacteria contains a glutamine endopeptidase enzyme.

Embodiments of the invention also provide a method of treating Celiac Sprue, gluten allergy and/or dermatitis herpetiformis in a subject in need thereof, the method comprises administering to a subject when consuming a gluten-containing foodstuff an effective dose of an extract from a Rothia species bacteria, an isolated glutamine endopeptidase enzyme, or a formulation comprising an isolated glutamine endopeptidase enzyme; wherein the extract from the Rothia species bacteria contains a glutamine endopeptidase enzyme that attenuates gluten toxicity in the subject.

In one embodiment, provided herein is a method of detoxifying gluten, the method comprises contacting gluten-containing foodstuff with an effective dose of an extract from a Rothia species bacterium, an isolated glutamine endopeptidase enzyme, or a formulation comprising an isolated glutamine endopeptidase enzyme, wherein the extract from the Rothia species bacteria contains a glutamine endopeptidase enzyme. In one embodiment, the extract is a purified sample of the enzyme.

In one embodiment, the subject has been diagnosed with Celiac Sprue, gluten allergy and/or dermatitis herpetiformis.

In one embodiment, provided herein is a method of predicting/diagnosing Celiac Sprue, gluten allergy and/or dermatitis herpetiformis in a subject in need thereof, the method comprises (a) contacting a biological sample from the subject with a fixed amount of gliadin or synthetic gliadin-derived enzyme substrate for a 24 hour period; (b) measuring the amount of gliadin degradation; and (c) comparing the amount of gliadin degradation for the biological sample with that obtained for a control assay, wherein the control assay is a mixture of a same fixed amount of gliadin with an isolated glutamine endopeptidase enzyme or a formulation that contains a glutamine endopeptidase enzyme for a 24 hour period, wherein the extent of gliadin degradation of less than 50% of that of the control assay indicates the subject likely have Celiac Sprue, gluten allergy and/or dermatitis herpetiformis. The biological sample can be whole saliva or dental plaque derived from the subject. The fixed amount of gliadin used in the assay is such that when an equivalent corresponding sample from a healthy subject, e.g. saliva or dental plaque, is mixed with the fixed amount of gliadin, 100% of the gliadin is digested within 24 hours under the same assay conditions. A healthy subject is one who does not have, diagnosed with or have symptoms associated with Celiac Sprue, gluten allergy, gluten intolerance and/or dermatitis herpetiformis as determined by the various methods known in the art and also described herein. For the control assay, the amount of an isolated glutamine endopeptidase enzyme or a formulation that contains a glutamine endopeptidase enzyme used is that which will digest 100% of the fixed amount of gliadin within a 24 hour period. In one embodiment, the diagnostic assay and the control assay are conducted in parallel under the same conditions.

In one embodiment, the glutamine endopeptidase enzyme is derived from the Rothia species described herein appears in the 70-75 kDa region in a gliadin zymogram, is active in a saliva sample, is a metal-ion dependent protease, and attenuates gluten toxicity by cleaving the peptide bond after glutamine at -XPQ- and XPY motifs in gluten-containing foodstuff, wherein X=any amino acids, P=proline, Q=glutamine and Y=tyrosine.

In another embodiment, the glutamine endopeptidase enzyme from the Rothia species described herein appears in the 70-75 kDa region in a gliadin zymogram and is active in a sample of dental plaque.

In another embodiment, the glutamine endopeptidase enzyme from the Rothia species described herein appears in the 70-75 kDa region in a gliadin zymogram and is heat labile. Boiling at 100.degree. C. for 5 minutes abolishes the endopeptidase activity.

In one embodiment, the glutamine endopeptidase enzyme from the Rothia species has a pH optimum range between 7-10 for its enzymatic activity, i.e., digestion of proteins with -XPQ- and -XPY- motifs results in smaller protein fragments.

In one embodiment, the glutamine endopeptidase enzyme comprises at least 45% amino acid sequence identity to SEQ. ID. NO: 1. In another embodiment, the glutamine endopeptidase enzyme comprises SEQ. ID. NO: 1. The enzyme can be conjugated with other molecules to increase stability, e.g., to PEG. In another embodiment, the glutamine endopeptidase enzyme consists essentially of SEQ. ID. NO: 1. In yet another embodiment, the glutamine endopeptidase enzyme consists of SEQ. ID. NO: 1.

In one embodiment, the glutamine endopeptidase enzyme is a recombinantly synthesized glutamine endopeptidase enzyme. In some embodiments, the recombinantly synthesized glutamine endopeptidase enzyme comprising at least 45% amino acid sequence identity or similarity to SEQ. ID. NO: 1 is used. In some embodiments of the methods described, the recombinant enzyme has modifications that increase the enzyme stability, enzyme activity and potency such that a smaller amount of enzyme is necessary to achieve the desired gluten digestion. Modifications can include but are not limited to changes in amino acid changes, amino acid modifications (e.g., acetylation, PEGylation), and fusion protein.

In some embodiments, the Rothia species bacteria is Rothia mucilaginosa ot 681 (strain WSA-2B), Rothia species ot 188 (strain WSA-8) Rothia mucilaginosa ATCC 25296 and Rothia dentocariosa ATCC 17931. These Rothia species bacteria can grow on gluten-limited media. Extracts from these bacteria exhibit glutamine endopeptidase activity.

In some embodiments, the extract from Rothia species bacteria is selected from a group consisting a clarified lysate of a Rothia species bacteria, a 25-45% ammonium sulphate precipitate of the lysate of a Rothia species bacteria where the precipitate has been resuspended in buffer and desalted, the supernatant fluid of a suspension of a Rothia species bacteria, and a suspension of a Rothia species bacteria.

In one embodiment, the extract from Rothia species bacteria, the glutamine endopeptidase enzyme or formulation containing the glutamine endopeptidase enzyme is administered just before, during, or just after consumption of gluten-containing foodstuff.

In one embodiment, the extract from the Rothia species bacteria, the glutamine endopeptidase enzyme enzyme or formulation containing the glutamine endopeptidase enzyme enzyme is administered orally.

In one embodiment, the extract from the Rothia species bacteria, the glutamine endopeptidase enzyme enzyme or formulation containing the glutamine endopeptidase enzyme enzyme is admixed to the gluten-containing foodstuff.

In one embodiment, the extract from the Rothia species bacteria, the glutamine endopeptidase enzyme enzyme or formulation containing the glutamine endopeptidase enzyme enzyme comprises an enteric coating.

In one embodiment, the extract from the Rothia species bacteria, the glutamine endopeptidase enzyme enzyme or formulation containing the glutamine endopeptidase enzyme enzyme is a lyophilized preparation.

In one embodiment, the extract from the Rothia species bacteria, the glutamine endopeptidase enzyme enzyme or formulation containing the glutamine endopeptidase enzyme enzyme is formulated for oral administration.

In one embodiment, the effective dose of the extract from the Rothia species bacteria, the glutamine endopeptidase enzyme enzyme or formulation containing the glutamine endopeptidase enzyme enzyme ranges from 0.01 mg to 500 mg/kg body weight.

BRIEF DESCRIPTION OF TILE DRAWINGS

FIG. 1 demonstrates the identification of seven aerobic and ten anaerobic strains that grew well on gluten-limited agar where gluten is the only nitrogen source. None of the strains grew on agar containing the same ingredients without gluten (not shown). Note: predominant aerobic species: Rothia; predominant anaerobic species: Bifidobacterium.

FIG. 2 shows the degradation of gliadins by the mixture of bacteria in dental plaque (FIG. 2A) or strain WSA-8 (FIG. 2B) suspended in saliva ion buffer. Both cell suspensions had an OD.sub.620 of 1.0. Gliadin was added to a final concentration of 250 .mu.g/ml (SIGMA-ALDRICH.RTM. Cat. No. G3375). After various incubation time points, 100 .mu.l aliquots were removed, boiled and subjected to SDS PAGE. Lane 1: molecular weight standard; lanes 2-7, cell/gliadin mixtures incubated for 0, 2, 4, 6, 24 and 48 h, respectively. Arrow points to the major constituent in the gliadin mixture. Note that gliadins are faster degraded by strain WSA-8 (FIG. 2B) than by the mixture of micoorganisms present in dental plaque (FIG. 2A).

FIG. 3 shows the degradation of gliadins by strains WSA-2B (FIG. 3A) and WSA-8 (FIG. 3B). Cells were grown on gluten limited agar and suspended in saliva ion buffer to an OD.sub.620=1.0. Lane 1: molecular weight standard, lanes 2-7: Cell/gliadin mixture incubated for 0, 5, 15, 30, 60 and 120 min, respectively; Lanes 8 and 9: gliadins incubated for 0 and 120 min in boiled cell suspensions; lanes 10 and 11: cell suspensions without added gliadins; Lanes 12 and 13: gliadins incubated for 0 and 2 hr in saliva ion buffer. Arrow points to the major constituent in the gliadin mixture. Note: both strains rapidly degraded gliadins, WSA-8 a little faster than WSA-2B.

FIG. 4A-4D shows the relationship between cell density and proteolytic activity. WSA-2B or WSA-8 cells were suspended in saliva ion buffer to a final concentration of OD.sub.620=0.15, 0.3, 0.6, and 1.2. Z-KPQ-pNA or Z-YPQ-pNA was added as enzymatic substrates to final concentrations of 200 .mu.M. Note that the rate of substrate hydrolysis increased with increasing cell density. The fact that both KPQ and YPQ were cleaved signifies that the amino acid at position p3 has little influence on enzyme recognition. As expected, boiled cell suspensions (OD.sub.620=1.2) were devoid of enzymatic activities.

FIG. 5 shows the gliadin zymography (8%) of WSA-2B and WSA-8. Strains were cultured on Brucella agar (BA) or gluten-limited agar (GA), and suspended in saliva ion buffer. In each lane cells from 150 .mu.l suspension (OD.sub.620=5.0) were loaded. Lane 1: molecular weight standard; lane 2: WSA-2B grown on BA; lane 3: WSA-2B grown on GA; lane 4: WSA-8 grown on BA; lane 5: WSA-8 grown on GA. Clear bands indicate the presence of an enzyme with gliadin-degrading activity. Note that the molecular weight of the enzymes is approximately 70 kDa.

FIG. 6 shows the total ion chromatogram of an in-gel tryptic digest of the WSA-8 glutamine endopeptidase enzyme. The chromatogram shows multiple peptide fragments.

FIG. 7 shows the hydrolysis of Z-YPQ-pNA (200 .mu.M) by a cell suspension of WSA-2B (FIG. 7A) or WSA-8 (FIG. 7B) in saliva ion buffer in the absence and presence of various protease inhibitors. Saliva ion buffer contains 50 mM KCl, 1 mM K.sub.2HPO4, 1 mM CaCl.sub.2 and 0.1 mM MgCl.sub.2 (pH 6.5). Cells were preincubated with the inhibitors for 15 min at room temperature prior to the addition of substrate. Z-YPQ-pNA hydrolysis was followed at 405 nm. Initial velocities of the enzymatic reaction are plotted. Note that PMSF and EDTA completely inhibited YPQ-cleavage activities.

FIG. 8 shows the gliadin zymography of WSA-8 cells with and without PMSF. A cell suspension of WSA-8 (OD.sub.620nm=5.0) was incubated for 15 min at room temperature with and without added PMSF, a serine protease inhibitor (final concentration 1 mM). Following incubation, cells (150 .mu.l) were harvested and subjected to gliadin zymography. Lane 1, Molecular weight standard; lane 2: WSA-8 without PMSF; lane 3, WSA-8 with PMSF. Note: pre-incubation with PMSF abolishes all activity.

FIG. 9 shows the gliadin substrate hydrolysis by three commercially obtained strains: Kocuria varians ATCC 15306; R. mucilaginosa ATCC 25296; and R. dentocariosa ATCC 17931. Strain WSA-8 was included for comparison. Note that R. dentocariosa but not the phylogenetically closely related species Kocuria varians cleaves Z-YPQ-pN. None of the strains exhibited noticeable activity towards the QQP, PFP or PPF substrates.

FIG. 10 shows the gliadin zymography (6%) of Rothia strains developed at low and neutral pH (pH 7.0). Per lane 150 .mu.l cells (OD.sub.620=5.0) were loaded after cell disruption. Lane 1, MW standard, lanes 2 and 6, strain WSA-2B (R. mucilaginosa); lanes 3 and 7: strain WSA-8 (Rothia spp. of 188), lanes 4 and 8: strain WSA-26 (R. mucilaginosa); lanes 5 and 9: R. mucilaginosa (ATCC 25296). After electrophoresis, the gel was cut in the middle and one half was renatured and developed at pH=3 (left panel) and the other half at pH=7 (right panel). Note that at pH=3, the protease produced by WSA-8 had retained some of its activity.

FIG. 11 shows the effects of pH on WSA-8 glutamine endopeptidase enzyme activity. WSA-8 cells grown on Brucella agar were suspended to a final OD.sub.620 of 1.2 in 20 mM Tris buffers ranging in pH from 2.0 to 10.0. The synthetic substrate Z-YPQ-pNA was added to a final concentration of 200 .mu.M. Substrate hydrolysis was assessed at 405 nm hourly for the first 6 hours followed by a reading at 24 h and 72 h.

FIGS. 12A and 12B show the gliadin 33-mer fragmentation by WSA-8. Cells grown on agar were harvested and suspended in saliva ion buffer to a final OD.sub.620 of 1.2 and incubated with gliadin 33-mer (final concentration 250 .mu.g/ml). After 0 h, 2 h and 5 h, aliquots were removed, boiled, filtered and analyzed by RP-HPLC. Peaks 1 to 11, as indicated by arrows, were collected for structural analysis by LC-ESI-MS/MS (FIG. 12A). Sequences of the peptides of peak 1 to 11 are shown in FIG. 12B; larger arrows indicate QPQ cleavage sites, and narrow arrows indicate LPY, QPF and PFP cleavage sites on the 33-mer.

FIGS. 13A and 13B show the gliadin 26-mer fragmentation by WSA-8. Cells grown on agar were harvested and suspended in saliva ion buffer to a final OD.sub.620 of 1.2 and incubated with gliadin 26-mer (final concentration 250 .mu.g/ml). After 0 h, 2 h and 5 h, aliquots were removed, boiled, filtered and analyzed by RP-HPLC. Peaks 1 to 10, as indicated by arrows, were collected for structural analysis by LC-ESI-MS/MS (FIG. 13A). Sequences of the peptides of peak 1 to 10 are shown in FIG. 13B; larger arrows indicate QPQ cleavage sites, and narrow arrows indicate LPY, QPF and PFP cleavage sites on the 33-mer.

FIGS. 14A and 14B show the gliadin 33-mer fragmentation by R. mucilaginosa ATCC 25296. Cells grown on agar were harvested and suspended in saliva ion buffer to a final OD.sub.620 of 1.2 and incubated with gliadin 33-mer (final concentration 250 .mu.g/ml). After 0 h, 2 h and 5 h, aliquots were removed, boiled, filtered and analyzed by RP-HPLC. Peaks 1 to 11, as indicated by arrows, were collected for structural analysis by LC-ESI-MS/MS (FIG. 14A). Sequences of the peptides of peak 1 to 11 are shown in FIG. 14B; larger arrows indicate QPQ cleavage sites, and narrow arrows indicate LPY, QPF and PFP cleavage sites on the 33-mer.

FIGS. 15A and 15B show the gliadin 26-mer fragmentation by R. mucilaginosa ATCC 25296. Cells grown on agar were harvested and suspended in saliva ion buffer to a final OD.sub.620 of 1.2 and incubated with gliadin 26-mer (final concentration 250 .mu.g/ml). After 0 h, 2 h and 5 h, aliquots were removed boiled, filtered and analyzed by RP-HPLC. Peaks 1 to 7, as indicated by arrows, were collected for structural analysis by LC-ESI-MS/MS (FIG. 15A). Sequences of the peptides of peak 1 to 7 are shown in FIG. 15B; larger arrows indicate QPQ cleavage sites, and narrow arrows indicate LPY, QPF and PFP cleavage sites on the 33-mer.

FIG. 16 shows the DEAE chromatogram of R. mucilaginosa ATCC 25296 sonicated cell supernatant fractions enriched for enzyme activity using ammonium sulfate fractionation. A total amount of 670 mg protein was loaded onto the column. Dotted trace: total protein (A214 nm); solid trace: Z-YPQ-pNA hydrolysis activity.

FIG. 17A shows the SDS PAGE of partially purified R. mucilaginosa enzyme(s). R. mucilaginosa cell extract (20 .mu.g protein/lane): Lane 1: protein standard (5 ul, Bio-Rad all blue); lane 2: empty; lane 3: P-0a; lane 4: P-0b; lane 5: P-0c, lane 6: P-1; lane 7: P-2: lane 8: R. mucilaginosa extract before DEAE fractionation; lane 10 (zymogram): R. mucilaginosa cells (OD.sub.620=5.0, 300 .mu.l).

FIG. 17B shows the gliadin zymography of DEAE fractions of partially purified R. mucilaginosa enzyme(s). R. mucilaginosa cell extract (20 .mu.g protein/lane): Lane 1: protein standard (5 ul, Bio-Rad all blue); lane 2: empty; lane 3: P-0a; lane 4: P-0b; lane 5: P-0c, lane 6: P-1; lane 7: P-2: lane lane 9: R. mucilaginosa extract before DEAE fractionation; lane 10 (zymogram). R. mucilaginosa cells (OD.sub.620=5.0, 300 .mu.l).

FIG. 17C shows the SDS PAGE of P-0b and P-1 fractions following further purification by gelfiltration and anion exchange (AE) chromatography (see Materials and Methods for experimental details). Arrows (a and b) point to two active protease bands exhibiting apparent molecular weights of 75 and 70 kD, respectively.

FIG. 17D shows the zymography of the same samples loaded in FIG. 17C, confirming gliadin-degrading activity in the enriched fractions.

FIG. 17E shows the evaluation of cleavage specificity of the semi-pure enzyme preparation (DEAF P1 purified by gel filtration and HiTrap AE QXL, FIGS. 17 C and D right lanes). Final enzyme concentration: 122 ug/ml. Note that both Z-YPQ-pNA and Z-LPY-pNA are hydrolysed. No hydrolysis occurred in the absence of enzyme (not shown). Enzymatic kinetic rates were higher towards Z-YPQ-pNA than towards Z-LPY-pNA. Both substrates were completely hydrolyzed after 16 h (last measurement).

FIG. 18 shows the amino acid sequence of R. mucilaginosa neprilysin (SED. ID. NO: 1) and the conserved regions that are important for the glutamine endopeptidase enzyme activity (in bold).

FIG. 19 shows the amino acid sequence alignment of closely related sequences in Table 6. The sequences are ZP.sub.--05367591 (SEQ. ID. NO: 1), YP.sub.--003363565 (SEQ. ID. NO: 23), ZP.sub.--07073157 (SEQ. ID. NO: 24), ZP.sub.--06905919 (SEQ. ID. NO: 25), YP.sub.--003315199 (SEQ. ID. NO: 26), YP.sub.--003325693 (SEQ. ID. NO: 27), YP.sub.--003636471 (SEQ. ID. NO: 28), ZP.sub.--06830706 (SEQ. ID. NO: 29) and ZP.sub.--07359309 (SEQ. ID. NO: 30) in the order of appearance.

FIG. 20 shows the amino acid sequence alignment of closely related sequences of bacteria neprilysins ZP.sub.--05367591 (SEQ. ID. NO: 1), YP.sub.--003363565 (SEQ. ID. NO: 48), YP.sub.--003325693 (SEQ. ID. NO: 49), YP.sub.--003636471 (SEQ. ID. NO: 50), ZP.sub.--07359309. (SEQ. ID. NO: 51), YP.sub.--003275516 (SEQ. ID. NO: 52), YP.sub.--001131754 (SEQ. ID. NO: 53), YP.sub.--003379113 (SEQ. ID. NO: 54), YP.sub.--951040.1 (SEQ. ID. NO: 55), YP.sub.--002882628 (SEQ. ID. NO: 56), ZP.sub.--04382847 (SEQ. ID. NO: 57), YP.sub.--705057.1 (SEQ. ID. NO: 58), YP.sub.--003199539 (SEQ. ID. NO: 59), ZP.sub.--06184089 (SEQ. ID. NO: 60), YP.sub.--003645328 (SEQ. ID. NO: 61), ZP.sub.--03393889 (SEQ. ID. NO: 62), and YP.sub.--002322079 (SEQ. ID. NO: 63) in the order of appearance respectively.

FIG. 21A-21C show the comparison of 33-mer degradation by R. mucilaginosa ATCC 25296 (Rm) and R. dentocariosa ATCC 17931 (Rd). Cells were suspended in saliva ion buffer to an OD620 of 1.2 and 33-mer was added to a final concentration of 250 .mu.g/ml. At 0 h (solid line), 2 h (dashed line) and 5 h (dotted line) 100 .mu.l sample aliquots were analyzed by RP-HPLC. Note that Rd (middle panel) is unable to cleave the 33-mer.

FIG. 22A-22C show the comparison of 26-mer degradation by R. mucilaginosa ATCC 25296 (Rm) and R. dentocariosa ATCC 17931 (Rd). Cells were suspended in saliva ion buffer to an OD of 1.2 and 26-mer was added to a final concentration of 250 ug/ml. At 0 h (solid line), 2 h (dashed line) and 5 h (dotted line) 100 ul sample aliquots were analyzed by RP-HPLC. Note that Rd (middle panel) is unable to cleave the 26-mer.

FIG. 23 shows the gliadin zymogram of R. dentocariosa (Rd) and R. mucolaginosa (Rm). Lane 1, MW standard; lane 2, empty; lane 3: Rd cells harvested from 150 .mu.l OD=5.0; lane 3, Rd cells from 300 .mu.l OD=5.0, lane 4, Rm cells from 150 .mu.l 013=5.0; lane 5, Rm cells from 300 .mu.l OD=5.0. Note: no gliadin-degrading enzymes noticeable in Rd cell suspensions.

FIG. 24 shows Z-YPQ-pNA and Z-LPY-pNA hydrolysis by R. mucilaginosa (Rm) and R. dentocariosa (Rd). Note: R. dentocariosa is unable to cleave Z-LPY-pNA (picture taken after 72 h).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that human whole saliva (WS) and dental plaque contain enzymatic activities that cleaves the Xaa-Pro-Gln (-XPQ-) bond after Gln, where Xaa is any amino acid, Pro is proline and Gln is glutamine. This tripeptide is also particularly abundant in known celiac T-cell gluten epitopes. The inventors showed that the saliva-associated glutamine endopeptidase enzyme(s) can degrade gluten/gliadins in vitro. Gluten/gliadins are proline and glutamine rich proteins that are the cause of the adverse immune response in Celiac Sprue, gluten allergy and dermatitis herpetiformis. The discovery of this enzyme provides the use of the enzyme(s) for non-dietary therapies of Celiac Sprue, gluten allergy and dermatitis herpetiformis.

In order to isolate and identify the glutamine endopeptidase enzyme(s), the inventors started with isolating and identifying the gliadin-degrading bacteria from human WS and also dental plague. These gliadin-degrading bacteria are Rothia species bacteria (FIG. 1). The inventors further isolated, purified and functionally characterized the glutamine endopeptidase enzyme from Rothia mucilaginosa ATCC 25296 that was naturally associated with the oral cavity (FIGS. 16 and 17A-E). The functional enzyme characterizations included the approximate molecular weight of the enzyme as determined by gliadin zymograms and by SDS-PAGE, cleavage specificity of this protease, capacity to degrade toxic gliadin epitopes, pH activity and inhibitor sensitivity profiles. Enzymes were obtained by chromatography and zymography and structurally characterized by LC-ESI-MS/MS. The inventors have identified the enzyme neprilysin as a gluten/gliadin-degrading glutamine endopeptidase enzyme protease from R. mucilaginosa.

Accordingly, embodiments of the invention provide an isolated glutamine endopeptidase enzyme. In one embodiment, the isolated glutamine endopeptidase enzyme is purified from a Rothia species bacterium. In one embodiment, the isolated glutamine endopeptidase enzyme is a recombinantly synthesized glutamine endopeptidase enzyme. In one embodiment, the enzyme is a protein.

In some embodiments, the enzyme has an apparent molecular weight of about 70-75 kDa as determined by gliadin zymograms. It also has an apparent molecular weight of about 70-75 kDa as determined by SDS-PAGE. In some embodiments, the apparent molecular weight of the enzyme is determined by gel filtration chromatography, which is a technique known to one skilled in the art.

In some embodiments, the enzyme has a functional pH range of 3-10. Within this range of pHs, there is detectable Z-YPQ-pNA and gliadin peptides cleaving activities within a 24 hour digestion period. Complete digestion is achieved at 72 hours under the described assay conditions. At a pH range of 7-10, there is substantially complete Z-YPQ-pNA cleavage within a 1 hour digestion period. Detectable Z-YPQ-pNA and gliadin peptides cleaving activities refers to at least 10% of the substrate used in the assay, i.e., Z-YPQ-pNA, 33-mer or 26-mer, is digested to smaller peptide fragments. Z-YPQ-pNA and the 26 mer and 33 mer gliadin peptides can be used as the substrates for assaying glutamine endopeptidase enzyme activity. Enzyme activity assay is assessed by measuring proteolytic activities towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic, highly immunogenic, gliadin peptides (33-mer of .alpha.2-gliadin and 26-mer of .gamma.-gliadin) as described. Detectable Z-YPQ-pNA and gliadin peptides cleaving activities refers to at least 10% of the substrate used in the assay, i.e., Z-YPQ-pNA, 33-mer or 26-mer, is digested to smaller peptide fragments. Methods of detecting smaller peptide fragments are well known to one skilled in the art, e.g., by RP-HPLC and mass spectrometery as described herein. In some embodiments, the enzyme is active at pH 7.0, 7.2, 7.5, 7.7, 8.0, 8.2, 8.5, 8.7, 9.0, 9.2, 9.5, 9.7 and 10, including all the intermediate pHs between 7.0 to 10.0, wherein 100% of the substrate is cleaved within a 1 hour digestion period.

In one embodiment, the enzyme cleaves the peptide bond after a -XPY- or -XPQ- motif in glutens. In another embodiment, the enzyme cleaves the peptide bond immediately after a -XPQ- motif and proline is the amino acid at the P1' position after the motif.

In some embodiments, the enzyme does not cleave the peptide bonds after the QPF, PFP, QQP, and PPF motifs in glutens.

In one embodiment, the enzyme is inhibited by an agent selected from the group consisting of EDTA, PMSF, AEBSF, omapatrilat, opiorphin, RB-101, and UK-414,495. The concentration of the inhibiting agent ranges from about 0.01 .mu.M to about 1.0 mM and the percent inhibition ranges from about 10% to 100% depending on the concentration of inhibition agent used. In some embodiments, the inhibiting agent ranges from about 0.01 .mu.M to about 0.1 mM, from about 0.01 .mu.M to about 0.05 mM, from about 0.01 .mu.M to about 0.5 mM, from about 0.1 .mu.M to about 0.5 mM, from about 0.1 .mu.M to about 1 mM, from about 1 .mu.M to about 0.1 mM, from about 1 .mu.M to about 0.05 mM, from about 1 .mu.M to about 0.5 mM, from about 1 .mu.M to about 1 mM, from about 5 .mu.M to about 0.1 mM, from about 5 .mu.M to about 0.05 mM, from about 5 .mu.M to about 0.5 mM, from about 5 .mu.M to about 1 mM, from about 10 .mu.M to about 0.1 mM, from about 10 .mu.M to about 0.05 mM, from about 10 .mu.M to about 0.5 mM, from about 10 .mu.M to about 1 mM, from about 0.1 mM to about 0.5 mM, from about 0.1 mM to about 1 mM, and from about 0.5 .mu.M to about 1.0 mM. In some embodiments, the percent inhibition is about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 99%. In one embodiment, the enzyme is 100% inhibited by 1 mM EDTA or PMSF.

In some embodiments, the enzyme is a metal-ion dependent protease. In one embodiment, the enzyme is a zinc-dependent protease. In one embodiment, there is one zinc molecule per molecule of enzyme.

In one embodiment, the enzyme is a metal-ion dependent serine protease.

In some embodiments, the enzyme is precipitated by 25-45% ammonium sulphate in a lysate containing the enzyme. The enzyme is negatively charged at pH P>5.0. Examples of a lysate containing the enzyme include but are not limited to a R. mucilaginosa bacterial lysate and the yeast lysate where yeast is the protein expression host for the recombinantly synthesized enzyme

In some embodiments, the isolated glutamine endopeptidase enzyme is isolated and purified from a Rothia species bacterium, such as R. mucilaginosa ATCC 25296, WSA-8, R. mucilaginosa of 681, R. mucilaginosa DY-18, R. dentocariosa M567 and R. dentocariosa ATCC 17931.

In another embodiment, the isolated glutamine endopeptidase enzyme is isolated from a bacterium selected from the group consisting of R. mucilaginosa ATCC 25296, WSA-8, R. mucilaginosa of 681, R. mucilaginosa DY-18, R. dentocariosa M567, R. dentocariosa ATCC 17931, R. mucilaginosa DY-18, Xylanimonas cellulosilytica DSM 15894, Cellulomonas flavigena DSM 20109, Actinomyces viscosus C505, Gordonia bronchialis DSM 43247, Mycobacterium gilvum PYR-GCK, Kribbella flavida DSM 17836, Mycobacterium vanbaalenii PYR-1, Beutenbergia cavernae DSM 12333, Rhodococcus jostii RHA 1, Nakamurella multipartita DSM 44233, Mobiluncus mulieris 28-1, Tsukamurella paurometabola DSM 20162, Corynebacterium amycolatum SK46, Bifidobacterium longum subsp. infantis ATCC 15697, Sanguibacter keddieii ATCC 51767 and Cellulomonas flavigena ATCC 482.

The inventors showed that cell suspensions from the commercial strain of R. dentocariosa ATCC 17931 exhibited glutamine endopeptidase enzyme activity in FIG. 9 using synthetic peptides such as Z-YPQ-pNA as the enzyme substrate. However, in separate experiments, the inventers also showed that cell suspensions of R. dentocariosa ATCC 17931 did not exhibit any detectable glutamine endopeptidase enzyme activity when using the oligopeptides 33-mer or the 26-mer as enzyme substrates (see FIG. 21-24). The observed differences could be due to the different assay methods used and the fact that cell suspensions were used. The smaller synthetic peptides may have fitted better into the enzyme active site than the oligopeptides. These smaller synthetic peptides and the 33-mer asd 26-mer oligopeptides do not accurately reflex the natural substrate of the enzyme. The glutamine endopeptidase enzyme in R. dentocariosa ATCC 17931 had only 76% identity to SEQ. ID. NO: 1 and is classified as a metalloendopeptidase PepO rather than a neprilysin. A metalloendopeptidase PepO may have different substrate specificity compared to a neprilysin and this can also account for the negative results obtained for the cell suspension of R. dentocariosa ATCC 17931 with the 33-mer and the 26-mer as shown in FIGS. 21-24.

Many proteins exist in an active (mature state) and in an inactive (pro-protein state). It is also a known fact that an enzyme's activity is regulated by its environment. The presence of inhibitory factors of the glutamine endopeptidase enzyme in the cell suspension in FIG. 22-24 could produce the negative results obtained.

In some embodiments, the enzyme comprises at least 60% amino acid sequence identity or similarity to NEIVFPAAILQPP (SEQ. ID. NO: 31), FDDQGSRYDGDG (SEQ. ID. NO: 32), DPHSPDEF (SEQ. ID. NO: 33), NGVVRNIDEFY (SEQ. ID. NO: 34), and RVRIW (SEQ. ID. NO: 35), and has a "bHEbbHbc" motif, wherein H=histidine, E=glutamate, `b` is an uncharged amino acid residue, and `c` a hydrophobic amino acid residue, wherein "bHEbbHbc" motif forms part of the metal-binding site, wherein the enzyme cleaves Z-YPQ-pNA or gliadin peptides substrates within a 24 hour digestion period at a pH range from 3-10.

In some embodiments, the enzyme consists essentially of at least 60% amino acid sequence identity or similarity to NEIVFPAAILQPP (SEQ. ID. NO: 31), FDDQGSRYDGDG (SEQ. ID. NO: 32), DPHSPDEF (SEQ. ID. NO: 33), NGVVRNIDEFY (SEQ. ID. NO: 34), and RVRIW (SEQ. ID. NO: 35), and has a "bHEbbHbc" motif, wherein H=histidine, E=glutamate, `b` is an uncharged residue, and `c` a hydrophobic residue, the "bHEbbHbc" motif forms part of the metal-binding site.

In other embodiments, the enzyme has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to NEIVFPAAILQPP (SEQ. ID. NO: 31), FDDQGSRYDGDG (SEQ. ID. NO: 32), DPHSPDEF (SEQ. ID. NO: 33), NGVVRNIDEFY (SEQ. ID. NO: 34), and RVRIW (SEQ. ID. NO: 35). More preferably, the enzyme has at least 70% similarity, at least 80% similarity, at least 90% similarity, at least 95% similarity or at least 99% similarity to NEIVFPAAILQPP (SEQ. ID. NO: 31), FDDQGSRYDGDG (SEQ. ID. NO: 32), DPHSPDEF (SEQ. ID. NO: 33), NGVVRNIDEFY (SEQ. ID. NO: 34), and RVRIW (SEQ. ID. NO: 35).

In some embodiments, the "bHEbbHbc" motif is IGHEIGHGF (SEQ. ID. NO: 47).

In some embodiments, the sequences NEIVFPAAILQPP (SEQ. ID. NO: 31), FDDQGSRYDGDG (SEQ. ID. NO: 32), DPHSPDEF (SEQ. ID. NO: 33), NGVVRNIDEFY (SEQ. ID. NO: 34), and RVRIW (SEQ. ID. NO: 35) form part of the enzyme active site, for example, in binding the oligopeptide substrate, co-ordinating the metal ion and the nucleophile exchange.

In another embodiment, the enzyme further comprises at least 60% amino acid sequence identity or similarity to VNGKWL (SEQ. ID. NO: 36), EIPADRP (SEQ. ID. NO: 37), RIGALY (SEQ. ID. NO: 38), EIAPIL (SEQ. ID. NO: 39), and QSGLGLPDESYYREE (SEQ. ID. NO: 40).

In another embodiment, the enzyme further consists essentially of at least 60% amino acid sequence identity or similarity to VNGKWL (SEQ. ID. NO: 36), EIPADRP (SEQ. ID. NO: 37), RIGALY (SEQ. ID. NO: 38), EIAPIL (SEQ. ID. NO: 39), and QSGLGLPDESYYREE (SEQ. ID. NO: 40).

In other embodiments, the enzyme has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to VNGKWL (SEQ. ID. NO: 36), EIPADRP (SEQ. ID. NO: 37), RIGALY (SEQ. ID. NO: 38), EIAPIL (SEQ. ID. NO: 39), and QSGLGLPDESYYREE (SEQ. ID. NO: 40). More preferably, the enzyme has at least 70% similarity, at least 80% similarity, at least 90% similarity, at least 95% similarity or at least 99% similarity to VNGKWL (SEQ. ID. NO: 36), EIPADRP (SEQ. ID. NO: 37), RIGALY (SEQ. ID. NO: 38), EIAPIL (SEQ. ID. NO: 39), and QSGLGLPDESYYREE (SEQ. ID. NO: 40).

In some embodiments, the sequences VNGKWL (SEQ. ID. NO: 36), EIPADRP (SEQ. ID. NO: 37), RIGALY (SEQ. ID. NO: 38), EIAPIL (SEQ. ID. NO: 39) and QSGLGLPDESYYREE (SEQ. ID. NO: 40) are important for co-ordinating the protein to fold its three dimentional shape for the endopeptidase enzyme activity.

In some embodiments, the enzyme further comprises at least 60% amino acid sequence identity or similarity to FYGKTLSGTQQIRE (SEQ. ID. NO: 41), RWKRGV (SEQ. ID. NO: 42), LDWMT (SEQ. ID. NO: 43), WRDFSAL (SEQ. ID. NO: 44), MTPQTVNAYY (SEQ. ID. NO: 45) and NEIVFPAAILQPP (SEQ. ID. NO: 31).

In some embodiments, the enzyme further consists essentially of at least 60% amino acid sequence identity or similarity to FYGKTLSGTQQIRE (SEQ. ID. NO: 41), RWKRGV (SEQ. ID. NO: 42), LDWMT (SEQ. ID. NO: 43), WRDFSAL (SEQ. ID. NO: 44), MTPQTVNAYY (SEQ. ID. NO: 45) and NEIVFPAAILQPP (SEQ. ID. NO: 31).

In other embodiments, the enzyme has at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to FYGKTLSGTQQIRE (SEQ. ID. NO: 41), RWKRGV (SEQ. ID. NO: 42), LDWMT (SEQ. ID. NO: 43), WRDFSAL (SEQ. ID. NO: 44), MTPQTVNAYY (SEQ. ID. NO: 45) and NEIVFPAAILQPP (SEQ. ID. NO: 31). More preferably, the enzyme has at least 70% similarity, at least 80% similarity, at least 90% similarity, at least 95% similarity or at least 99% similarity to FYGKTLSGTQQIRE (SEQ. ID. NO: 41), RWKRGV (SEQ. ID. NO: 42), LDWMT (SEQ. ID. NO: 43), WRDFSAL (SEQ. ID. NO: 44), MTPQTVNAYY (SEQ. ID. NO: 45) and NEIVFPAAILQPP (SEQ. ID. NO: 31).

In some embodiments, the sequences FYGKTLSGTQQIRE (SEQ. ID. NO: 41), RWKRGV (SEQ. ID. NO: 42), LDWMT (SEQ. ID. NO: 43), WRDFSAL (SEQ. ID. NO: 44), MTPQTVNAYY (SEQ. ID. NO: 45) and NEIVFPAAILQPP (SEQ. ID. NO: 31) are important for co-ordinating the protein to fold its three dimentional shape for the endopeptidase enzyme activity.

In some embodiments, the enzyme is a protein comprising at least 45% amino acid sequence identity or similarity to SEQ. ID. NO: 1. In other embodiments, the enzyme is a protein consisting of at least 45% amino acid sequence identity or similarity to SEQ. ID. NO: 1. In other embodiments, the enzyme is a protein consisting essentially of at least 45% amino acid sequence identity or similarity to SEQ. ID. NO: 1.

In other embodiments, the enzyme has at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to SEQ. ID. NO: 1. More preferably, the enzyme has at least 50% similarity, at least 55% similarity, at least 60% similarity, at least 65% similarity, at least 70% similarity, at least 75% similarity, at least 80% similarity, at least 85% similarity, at least 90% similarity, at least 95% similarity or at least 99% similarity to SEQ. ID. NO: 1.

In some embodiments, the enzyme comprises a functional fragment of a whole intact protein, wherein the functional fragment cleaves the peptide bond after a -XPY- or -XPQ- motif in glutens. In one embodiment, the whole intact protein is SEQ. ID. NO: 1. In one embodiment, the functional fragment cleaves the peptide bond immediately after a -XPQ- motif and proline is the amino acid at the P1' position after the motif.

In another embodiment, the functional fragment comprises at least 20 contiguous amino acid residues. In other embodiments, the functional fragment comprises at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, at least 360, at least 380, at least 400, at least 420, at least 440, at least 460, at least 480, at least 500, at least 520, at least 540, at least 560, at least 580, at least 600, at least 620, at least 640, or at least 660 contiguous amino acid residues.

The functional fragment can be assayed for the peptide bond cleavage using any methods known in the art, including but not limited to those described herein where Z-YPQ-pNA, Z-LPY-pNA, 26 mer and 33 mer gliadin peptides are used as substrates. In one embodiment, the functional fragment has at least 20% activity compared to the whole intact protein using the same assay method and substrate. In other embodiments, the functional fragment has at least 30% activity, at least 40% activity, at least 50% activity, at least 60% activity, at least 70% activity, at least 80% activity, at least 90% activity, at least 95% activity, at least 99% activity compared to the whole intact protein using the same assay method and substrate.

In one embodiment, the enzyme is neprilysin.

In another embodiment, the enzyme is neprilysin isolated from a bacterium selected from the group consisting of R. mucilaginosa ATCC 25296, WSA-8, R. mucilaginosa of 681, R. mucilaginosa DY-18, R. dentocariosa M567, R. dentocariosa ATCC 17931, R. mucilaginosa DY-18, Xylanimonas cellulosilytica DSM 15894, Cellulomonas flavigena DSM 20109, Actinomyces viscosus C505, Gordonia bronchialis DSM 43247, Mycobacterium gilvum PYR-GCK, Kribbella flavida DSM 17836, Mycobacterium vanbaalenii PYR-1, Beutenbergia cavernae DSM 12333, Rhodococcus jostii RHA1, Nakamurella multipartita DSM 44233, Mobiluncus mulieris 28-1, Tsukamurella paurometabola DSM 20162, Corynebacterium amycolatum SK46, Bifidobacterium longum subsp. infantis ATCC 15697, Sanguibacter keddieii ATCC 51767 and Cellulomonas flavigena ATCC 482.

Neprilysin belongs to the peptidase M13 superprotein family (pfam01431: Peptidase_M13). Members of this family are typically type-II membrane anchored enzymes which are known, or believed to activate or inactivate oligopeptide (pro)-hormones such as opioid peptides, or in the bacteria, the protein member is believed to be involved with milk protein cleavage. Other members of this superfamily include endothelin-converting enzyme, metalloendopeptidase, metalloendopeptidase PepO, and zinc metalloprotease.

The neprilysin (NEP) family of zinc metallopeptidases includes neprilysin, endothelin-converting enzyme-2 (ECE-2), PEX, damage induced neuronal endopeptidase (DINE), Kell and several neprilysin-like proteins. The best characterised of this family is neprilysin. Neprilysin (EC=3.4.24.11) is also known as membrane metallo-endopeptidase, neutral endopeptidase (NEP), CD10, and common acute lymphoblastic leukemia antigen (CALLA). Neprilysin is expressed at the cell surface of a variety of cell types.

Enzymatically, neprilysin functions both as an endopeptidase with a thermolysin-like specificity and as a dipeptidylcarboxypeptidase. Neprilysin are oligopeptidases, the enzyme digests oligo- and polypeptides, but not proteins. It is a zinc-dependent metalloprotease enzyme and binds one zinc ion per protein molecule.

Structurally, neprilysin consists of a short cytoplasmic domain, a membrane-spanning region and a large extracellular domain. The cytoplasmic domain contains a conformationally-restrained octapeptide, which is thought to act as a stop transfer sequence that prevents proteolysis and secretion. The protein fold of the peptidase domain for neprilysin resembles that of thermolysin, also an enzyme member of the Peptidase_M13 super family. The active site residues for members of the NEP family and thermolysin typically occurs in the motif HEXXH. In crystallographic studies, the HEXXH motif forms parts of the metal-binding site. The HEXXH motif is relatively common, but can be more stringently defined for metalloproteases as `abXHEbbHbc`, where `a` is most often valine or threonine and forms part of the S1' subsite in thermolysin and neprilysin, `b` is an uncharged residue, and `c` a hydrophobic residue. Proline is never found in this site, possibly because it would break the helical structure adopted by this motif in metalloproteases (Rawlings N D and Barrett A J., 1995, Meth. Enzymol. 248 183-228). Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain occur by a mechanism in which water acts as a nucleophile, one or two metal ions hold the water molecule in place, and charged amino acid side chains are ligands for the metal ions.

In one embodiment, the isolated glutamine endopeptidase enzyme is not thermolysin.

Proteins having at least 50% sequence identity or similarity to SEQ. ID. NO: 1 are shown in Table 6, in FIG. 19 and FIG. 20. Sequence alignment of SEQ. ID. NO: 1, a neprilysin isolated from R. mucilaginosa ATCC 25296 using the BLASTP algorithm produced over 100 similar sequences in the public bacteria databases alone; the similar sequences had at least 45% similarity to SEQ. ID. NO: 1.

SEQ. ID. NO: 1 is the amino acid sequence of neprilysin predicted in R. mucilaginosa ATCC 25296 contig00029, whole genome shotgun sequence (RefSeq: ZP.sub.--05367591).

TABLE-US-00001 1 MTTNSGITKE WVDETVKPGD DFFRHVNGKW LATHEIPADR PKDGGLYTLR DNAEKHVREL 61 VEKIAKEQPE SRIGALYNSF MDVEKIEADG LEPLLKEIAP ILNSATPSHL AVTLALLSRA 121 GLPQLFAWYT SNDPKDPKNY TFFLYQSGLG LPDESYYREE KHEAACAAYV EHIARMFQLT 181 GLAEGFGLTP EQAAQLVFTH ESELARLHWN VVENRDAEAT YNPYQATELD EKFPGFPFSQ 241 WLLALGADPE TLGQVIVAQP SFFEGAAKLF TSIPLMSWKL WAVWTVLRSR APFMYDELVQ 301 ESFNFYGKTL SGTQQIRERW KRGVGAVEKA LGEEIGQEYV AVHFPPSHKE KMLVLVGNLL 361 EAYRESIESL DWMTEATRQK ALEKLSKFVT KIGYPDKWRD FSALELVPGD LFENLRRTGA 421 FDADWLIARK GQPVDKAEWL MTPQTVNAYY MPPANEIVFP AAILQPPYFN PDADDAANYG 481 NIGMIIGHEI GHGFDDQGSR YDGDGKLESW WTEEDYAKFK ERTAALVEQY NAYVPVGLDP 541 KFHVNGELTL GENIGDLAGM SIALKAYRLA LKKQGIESLA DAPVIDGMTG IQRFFFSNAR 601 GWCTKSRPQH AEVMISVDPH SPDEFRVNGV VRNIDEFYEA FGVSEGDALY LAPEERVRIW

The coding sequence of neprilysin within the R. mucilaginosa ATCC 25296 contig00029 is found at region 5574 to 7556 of the contig00029 RefSeq: NZ_ACV001000010.1. (SEQ ID NO 22)

TABLE-US-00002 1 atgactacta actctggaat cactaaagaa tgggtggatg aaaccgtcaa gccgggcgac 61 gatttcttcc gccacgtcaa cggcaagtgg cttgctaccc acgaaatccc ggcggaccgc 121 cccaaggacg gcggcctgta caccctccgc gataacgcag agaagcacgt gcgtgagctg 181 gtggagaaga tcgcgaagga gcagccggag tcccgcatcg gcgcgctgta caactccttc 241 atggatgttg agaagattga ggcggacggc ctggaacctc tgctgaagga aatcgccccg 301 attctgaact cggcaacccc ctcccacctg gctgtgacct tggcgctgct gtctcgtgcg 361 ggtctgccgc agctgttcgc ctggtacacc agcaacgacc cgaaggaccc gaagaattac 421 acgttcttcc tgtaccagtc gggcctgggt ctgccggatg aatcctacta ccgtgaagag 481 aagcacgagg ctgcatgcgc ggcgtatgtt gagcatattg cccgcatgtt ccagctgacc 541 ggtctggctg agggcttcgg tctcaccccg gagcaggcgg ctcagctggt gttcacccac 601 gagtctgagc tggctcgtct gcactggaac gtcgtggaga accgcgacgc tgaggcgacc 661 tacaacccgt accaggcgac cgagctggac gagaagttcc ccggcttccc gttctcgcag 721 tggctgctgg ctctgggtgc tgacccggag accctgggtc aggttattgt ggctcagccg 781 tccttctttg agggtgcggc gaagctgttc acctccatcc cgctgatgag ctggaagctg 841 tgggctgtgt ggactgttct gcgttcgcgt gcgccgttca tgtacgacga gctggttcag 901 gagagcttca acttctacgg caagaccctt tccggtactc agcagattcg tgagcgttgg 961 aagcgcggcg tgggcgctgt cgagaaggct ctgggtgagg agattggcca ggagtacgta 1021 gctgtgcact tcccgccctc gcacaaggag aagatgctgg ttctggtcgg caacctcctt 1081 gaggcgtacc gcgagtctat tgagtcgctg gactggatga ctgaggcaac ccgtcagaag 1141 gcgctggaga agctgtcgaa gttcgtcacc aagatcggtt accccgataa gtggcgtgac 1201 ttctccgcgc tggagctcgt tcccggtgac ctgttcgaga acctgcgccg caccggtgcg 1261 ttcgatgctg actggctgat tgcccgtaag ggtcagccgg tggataaggc ggagtggctg 1321 atgactccgc agaccgtgaa cgcgtactac atgccgccgg cgaatgagat tgtgttcccg 1381 gcagcgattc tgcagccgcc gtacttcaac ccggatgctg acgatgcggc gaactacggc 1441 aatatcggca tgattattgg ccacgagatt ggtcacggtt ttgacgatca gggttcccgc 1501 tatgacggtg acggcaagct ggagagctgg tggactgagg aggattacgc gaagttcaag 1561 gagcgtaccg cagccctggt ggagcagtac aacgcgtacg ttccggtggg tctggacccg 1621 aagttccacg tgaacggtga gctgactctg ggcgagaaca ttggcgacct ggctggcatg 1681 tcgattgcgt tgaaggcgta ccgtctggct ttgaagaagc agggcattga gtcgctggct 1741 gacgcgccgg tgattgacgg catgaccggt attcagcgtt tcttcttctc gaatgctcgc 1801 ggctggtgca cgaagtcccg cccgcagcat gctgaggtga tgatttcggt ggatccgcat 1861 tcgccggatg agttccgtgt gaacggtgtg gtgcgcaata ttgatgagtt ctatgaggcg 1921 tttggcgtct ctgagggcga tgcactgtac ctggctccgg aggagcgcgt gcgcatctgg 1981 tag

In one embodiment, provided herein is a recombinantly synthesized glutamine endopeptidase enzyme that comprises at least 45% amino acid sequence identity or similarity to SEQ. ID. NO: 1 is used. In another embodiment, provided herein is a recombinantly synthesized glutamine endopeptidase enzyme that consist of at least 45% amino acid sequence identity or similarity to SEQ. ID. NO: 1. In yet another embodiment, provided herein is a recombinantly synthesized glutamine endopeptidase enzyme is SEQ. ID. NO: 1. In yet another embodiment, provided herein is a recombinantly synthesized glutamine endopeptidase enzyme that consists essentially of at least 45% amino acid sequence identity or similarity to SEQ. ID. NO: 1.

In other embodiments, the recombinantly synthesized glutamine endopeptidase enzyme has at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity to SEQ. ID. NO: 1. More preferably, the enzyme has at least 50% similarity, at least 55% similarity, at least 60% similarity, at least 65% similarity, at least 70% similarity, at least 75% similarity, at least 80% similarity, at least 85% similarity, at least 90% similarity, at least 95% similarity or at least 99% similarity to SEQ. ID. NO: 1.

One skilled in the art would be able to use standard recombination molecular techniques to synthesize recombinantly neprilysin. For example, design PCR primers based on SEQ. ID. NO: 1 for PCR cloning the coding sequence from the genomic DNA sequence of a Rothia species bacterium. One skilled in the art would also know to include modifications to the coding sequence for efficient protein synthesis and purification in non-bacteria systems such as yeast and mammalian cell lines. Modifications to the coding sequence can include but are not limited to removal of signal peptide, addition or change of signal peptide, change to the preferred codon usage of protein synthesis host and fusion protein formation.

In some embodiments, one skilled in the art would also know to include modifications to the coding sequence for increasing the enzyme stability, enzyme activity and enzyme potency such that a smaller amount of enzyme is necessary to achieve the desired gluten digestion. Modifications can include but are not limited to changes in amino acid changes, amino acid modifications (e, g., acetylation, PEGylation), and fusion protein formation.

The isolated glutamine endopeptidase enzyme that is purified from a Rothia species bacteria or is a recombinantly synthesized enzyme is useful in non-dietary based methods related to the treatment, prevention of additional immune reaction and diagnosis of Celiac sprue, gluten allergy and/or dermatitis herpetiformis as well as the detoxifying gluten-containing foodstuff.

The present invention provides methods for treating the symptoms of Celiac sprue, gluten allergy and/or dermatitis herpetiformis by decreasing the levels of toxic gluten oligopeptides in foodstuffs, either prior to or after ingestion by a subject. The gluten oligopeptides are "toxic" to these subjects, causing an autoimmune response by the body's immune system to synthesize antibodies to against itself, resulting in loss of nutrient-absorbing villi in the small intestines. A well studied gluten oligopeptide is the 33-mer gliadin oligopeptide, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ. ID. NO: 2) (Lu Shan, et al., 2002, Science, 297:2275-2279; Frits Koning, et al., 2003, Science 299:513). Proline/glutamine-rich proteins are digested by trypsin, chymotrypsin, elastase and carboxypeptidase in the gut and smaller oligopeptides are produced which are resistant to further digestion by endogenous trypsin, chymotrypsin, elastase and carboxypeptidase in the gut. These digestion-resistant gluten oligopeptides are presumably toxic because they bind to HLA-DQ2 and stimulate T cell infiltration in the small intestines. By digestion with an extract from a Rothia species bacteria, these toxic oligopeptides are cleaved into fragments, thereby preventing or relieving their toxic effects in Celiac Sprue, gluten allergy or dermatitis herpetiformis subjects. Digestion of the toxic gluten oligopeptides to small not-toxic fragments can also be achieved with contacting the gluten oligopeptides with a Rothia species bacterium or with an isolated glutamine endopeptidase that is derived a Rothia species bacterium. The glutamine endopeptidase derived from a Rothia species bacterium has been shown to cleave an internal peptide bond after glutamine at a Xaa-Pro-Gln (XPQ) type motif in a peptide, e.g. gluten oligopeptides: Z-KPQ-pNA (benzyloxycarbonyl-lysine-proline-glutamine-paranitroanilide) and Z-YPQ-Pna (benzyloxycarbonyl-tyrosine-proline-glutamine-paranitroanilide)- . Gluten oligopeptides tend to be rich in proline and glutamine. In the 33-mer gliadin oligopeptide, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ. ID. NO: 2), there are eight glutamine endopeptidase cleavage sites of the Xaa-Pro-Gln (XPQ) type, namely 1 FPQ, 4 QPQ and 3 YPQ sites. X or Xaa=any amino acids, P or Pro=proline, Q or Gln=glutamine, Y=lysine, F=phenylalanine, Z=benzyloxycarbonyl group and pNA is para-nitroanilide. Furthermore, there are three sequences of the Xaa-Pro-Tyr (XPY) type where Y=Tyr (tyrosine).

In the human body microorganisms outnumber human eukaryotic cells by an order of magnitude. The preponderance of these microorganisms can be found in the gastrointestinal (GI) tract, where they live for the most part in symbiosis with the host. Due to its rich colonization, the GI tract has been considered a "super organ" with functions provided not only by host--but by bacteria-derived proteins as well. The mutually beneficial relationship between the host and its colonizers is most evident in the biology of digestion. Many complex carbohydrates cannot be degraded by the arsenal of human digestive enzymes but can be hydrolyzed by bacterial glycosidases yielding catabolic compounds that can subsequently be utilized by the host. This represents a symbiotic relationship where the moist and nutrient-rich environment of the GI tract offers an ideal habitat for microbial colonization and at the same time the host benefits from optimal energy recovery from ingested food stuff (Camp et al., 2009, Gastroenterology 136(6):1989-2002).

Dietary gluten comprises a family of proteins that is subdivided into gliadins and glutenins, nutrients that are abundantly present in the Western diet. Gluten is fairly difficult to digest because of its unusual amino acid content and sequence. The predominant amino acids in the gluten sequences are proline and glutamine, which are not recognized by the typical proteolytic enzymes secreted by the stomach and the pancreas. In .about.0.5 and 2% of the human population the undigestable gluten fragments cause an immunologic response leading to celiac disease. Most of those diagnosed with celiac disease require a strict life long adherence to a gluten free diet. This is extremely difficult to maintain since even minor gluten contamination are present in many foods not overtly known for gluten content. One of the therapeutic strategies to counteract or prevent the deleterious effect of these minor amounts of dietary gluten focuses on proteolytic enzymes to aid in their degradation, thus preventing their antigenic presentation to and activation of intestinal T cells. We discovered that potent gluten digestive enzymes are naturally associated with the upper GI tract, i.e., with microorganisms colonizing the oral cavity (Helmerhorst et al., 2010, PLoS One, in press). Such microorganisms may actually play a role in gluten digestion which and this role has so far not been recognized. In addition, such enzymes should be explored to investigate their potential clinical usefulness in the protection against celiac disease in subjects at risk. Host resident gluten-degrading microorganisms are apparently a viable source of novel enzyme(s) of tremendous interest. These enzymes offer the additional advantage to be potentially exploited as probiotic agents to generate more long lasting changes in the GI gluten digestive capacity of celiac patients.

Accordingly, the present invention provides a method of treating Celiac Sprue, gluten allergy and/or dermatitis herpetiformis to a subject in need thereof, the method comprises administering to the subject an effective dose of an extract from a Rothia species bacterium; wherein the extract from the Rothia species bacteria contains a glutamine endopeptidase enzyme that attenuates gluten toxicity in the subject.

In some embodiments, the extract from Rothia species bacteria is selected from a group consisting of an isolated glutamine endopeptidase enzyme, a clarified lysate of a Rothia species bacteria, a 25-45% ammonium sulphate precipitate of the lysate of a Rothia species bacteria where the precipitate has been resuspended in buffer and desalted, the supernatant fluid of a suspension of a Rothia species bacteria, and a suspension of a Rothia species bacteria. In one embodiment, the isolated glutamine endopeptidase enzyme is purified from a Rothia species bacterium. In another embodiment, the isolated glutamine endopeptidase enzyme is a recombinantly synthesized glutamine endopeptidase enzyme. In another embodiment, the isolated glutamine endopeptidase enzyme is neprilysin. In another embodiment, the isolated glutamine endopeptidase enzyme is selected from the proteins in Table 6. In yet another embodiment, the isolated glutamine endopeptidase enzyme is SEQ. ID. NO: 1.

In one embodiment, the extract comprises the purified enzyme and a pharmaceutically acceptable carrier. For example, the enzyme can be at least 20% pure, at least 35% pure, at least 45% pure, at least 55% pure, at least 65% pure, at least 75% pure, at least 85% pure, at least 95% pure, at least 95% pure, at least 99% pure, wherein all the percentages between 20 and 99 are explicitly included. The extraction can be further purified, for example, from the 70-75 kDa extraction using standard purification schemes known in the art, e.g. size exclusion chromatography to isolate the 70-75 kDa fractions from a clarified crude extract of a Rothia species bacteria cell lysate. The bacteria can be lysed by standard methods known in the art, e.g. with lysozymes and treatment in a par bomb. The lysate can then be clarified by ultracentrifugation at 100,000.times.G force for 1 hour at 4.degree. C. The clarified lysate can then be concentrated and then fractioned with commercially available gel filtration matrix such as SEPHACRYL.RTM. (S-100/200/300/400/500) from GE Healthcare Life Sciences. Fractions with glutamine endopeptidase activity can be determined by methods known in the art and those described herein. One skilled in the art will be able to make minor modification for the enzyme being studied.

In one embodiment, the glutamine endopeptidase enzyme is purified in the following method: R. mucilaginosa ATCC cells were cultured from Brucella agar plates (Hardy Diagnostics, Santa Maria, Calif.) in 4 liter BHI for 24 h at 37.degree. C. while shaking. Cells were harvested and suspended in 50 mM TrisHCl and 50 mM NaCl (pH 8.0) and concentrated to a final O.D. of 67 at 620 nm. Cells were sonicated for 20 times at a power setting of 7 using the Branson cell lysis sonifier the degree of lysis was monitored spectrophotometrically and sonication was terminated when the turbidity was reduced by 90%. Thesonicate was centrifuged at 31,000.times.g for 20 min. The supernatant was collected and precipitated with 25-45% saturated ammonium sulfate. The precipitate was collected by centrifugation at 10,000.times.g for 20 min, and the pellet was dissolved, concentrated and desalted using centrifuge tubes with a 50 kD MW cut-off (MILLIPORE.RTM.). An aliquot of 670 mg protein was obtained. This protein was applied to a DEAE SEPHAROSE.RTM. Fast Flow column (GE Healthcare) of 2.6 cm.times.82.5 cm connected to an FPLC system (Pharmcia Biotech). Chromatographic separation of proteins was achieved at a flow rate of 0.7 ml/min and applying a gradient of 0-10% buffer B (containing 50 mM Tris HCl and 1M NaCl (pH 8.0) from 0 to 70 min; 10-35% buffer B from 70-2070 min, and 35-100% buffer B from 2070 to 2427 min. Fractions containing 24 ml were collected and protease activities were measured by mixing 200 .mu.l of each fraction with 3 .mu.l Z-YPQ-pNA (final concentration 150 mM). Active fractions were desalted, concentrated and 6.5 mg of protein was loaded onto a G-100 gel filtration column (SEPHADEX.RTM. G-100, Pharmacia fine Chemical Piscataway, N.J.) of 2.6 cm.times.82.5 cm. Samples were eluted at a flow rate of 0.5 ml/min. Collected fractions with activity were concentrated as described above and subjected either to a 1-ml column of HITRAP QFF anion-exchange chromatography (GE Healthcare, City, State) or to a 1-ml column of HITRAP QXL anion-exchange chromatography (GE Healthcare) Samples were eluted with a linear gradient of buffer B (formulation as described above). Fractions were again evaluated for activity, concentrated and analyzed for protein composition by SDS PAGE.

In one embodiment, the method is practiced when the subject is consuming any gluten-containing foodstuff. In another embodiment, the method is practiced prior to the consumption of gluten-containing foodstuff, wherein the subject is about to have some gluten-containing food or the subject suspects that there might be gluten or wheat-derived ingredients in the food that the subject is about to be consumed. In another embodiment, the method is practiced whenever food is consumed or three times a day with the three major meals of a day: breakfast, lunch and dinner.

Accordingly, in some embodiments, the extract from Rothia species bacteria is administered just before, during, or just after consumption of gluten-containing foodstuff.

In one embodiment, the extract from Rothia species bacteria is administered prior to consumption of gluten-containing foodstuff.

In one embodiment, the extract from Rothia species bacteria is administered in a gluten-containing foodstuff, e.g., incorporated into the gluten-containing foodstuff.

In one embodiment, the extract from Rothia species bacteria is administered from 1 hour prior to 1 hour after the subject has consumed a gluten-containing foodstuff.

In one embodiment, the extract from Rothia species bacteria is administered just before, during, or just after consumption of gluten-containing foodstuff.

Accordingly, the present invention also provides a method of detoxifying gluten-containing foodstuff, the method comprising contacting gluten-containing foodstuff with an effective dose of an extract from a Rothia species bacterium, wherein the extract from the Rothia species bacteria contains a glutamine endopeptidase enzyme. Detoxifying gluten-containing foodstuff has the same meaning as attenuating gluten toxicity. The goal is to reduce the amount of proline and glutamine rich oligopeptides that elicit immune responses characteristics of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis.

In some aspects, the methods described herein comprise administering to a subject an effective dose of a Rothia species bacterium, an extract from a Rothia species bacteria or an isolated glutamine endopeptidase.

In other aspects, the methods described herein comprise contacting the gluten-containing foodstuff with an effective dose of a Rothia species bacterium, an extract from Rothia species bacteria or an isolated glutamine endopeptidase. In one embodiment, the contacting is performed in vitro prior to consumption of the gluten-containing food stuff. In another embodiment, the contacting is performed in vivo prior to, concurrent with or after consumption of the gluten-containing foodstuff. For example, the effective dose of a Rothia species bacterium, an extract from Rothia species bacteria or an isolated glutamine endopeptidase can be in the form of a lyophilized powder that is sprinkled upon the gluten-containing foodstuff, similar to putting grated cheese on pasta.

In another embodiment, the Rothia species bacteria is R. mucilaginosa ot 681 (strain WSA-2B, aka WSB 26), Rothia species ot 188 (strain WSA-8), R. mucilaginosa ATCC 25296 and R. dentocariosa ATCC 17931. These Rothia species bacteria can grow on gluten-limited media (FIG. 1). Extracts from these bacteria exhibit glutamine endopeptidase activities. Gluten-limited agar formulation contains per liter: Gluten: 23 g, Sodium chloride: 5.0 g, soluble starch: 1.0 g, Agar No. 2: 12.0 g, Sodium bicarbonate: 0.4 g, Glucose: 1.0 g, Sodium pyruvate: 1.0 g, Cysteine HCl monohydrate: 0.5 g, L-Arginine: 1.0 g, Soluble pyrophosphate: 0.25 g, Sodium succinate: 0.5 g, Haemin: 0.01 g, Vit K: 0.001 g.

In one embodiment, the glutamine endopeptidase enzyme appears in the region of 70-75 kDa in a 6% gliadin zymogram (see FIG. 5). Therefore, in one embodiment, the apparent molecular size of the glutamine endopeptidase enzyme present in the extract, derived or isolated from a Rothia species bacterium is 70-75 kDa. Zymography is an electrophoretic technique, based on SDS-PAGE that includes a substrate copolymerized with the polyacrylamide gel, for the detection of enzyme activity. Samples are prepared in the standard SDS-PAGE treatment buffer but without boiling, and without a reducing agent. Following electrophoresis, the SDS is removed from the gel (or zymogram) by incubation in unbuffered Triton X-100, followed by incubation in an appropriate digestion buffer, e.g., 20 mM Tris, pH=8.0, for an optimized length of time at 37.degree. C. or other optimum temperature for the enzyme. The zymogram is subsequently stained (commonly with Amido Black or Coomassie Brilliant Blue), and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme. Zymography is an established method in the filed of Enzymology, e.g., in Lantz M S, Ciborowski P (1994) Methods Enzymol. 235: 563-594; and Snoek-van Beurden P A, Von den Hoff J W (2005) Biotechniques 38: 73-83. These references are hereby incorporated by reference in their entirety. One skilled in the art will be able to make minor modification for the enzyme being studied.

In one embodiment, the glutamine endopeptidase enzyme is active in a saliva sample. The normal pH range of human saliva is between 5 and 8. Accordingly, the glutamine endopeptidase enzyme present in the extract, derived or isolated from a Rothia species bacterium is active in a pH range of between about 5 and about 8. In one embodiment, the glutamine endopeptidase enzyme has a functional pH range of 3-10 within which there is detectable Z-YPQ-pNA cleaving activity within a 24 hour digestion period according to the assay method described herein. In another embodiment, the glutamine endopeptidase enzyme has a functional pH range of 7-10 within which there is substantially complete Z-YPQ-pNA cleavage within a 1 hour digestion period according to the assay method described herein.

In another embodiment, the glutamine endopeptidase enzyme is a metal-ion dependent protease (see FIG. 7). Addition of a divalent cation metal chelator, ethylenediaminetetraacetic acid (EDTA) completely inhibited YPQ cleavage activity. This indicates that the enzyme is a metal-ion dependent enzyme. In one embodiment, the glutamine endopeptidase enzyme present in the extract, derived or isolated from a Rothia species bacteria is inhibited by 1 mM 1-10 Phenanthroline. In one embodiment, the glutamine endopeptidase enzyme present in the extract, derived or isolated from a Rothia species bacterium is inhibited by 1 mM EDTA. Addition of the serine protease inhibitor, phenylmethanesulphonylfluoride or phenylmethylsulphonyl fluoride (PMSF) completely inhibited YPQ cleavage activity. In one embodiment, the glutamine endopeptidase enzyme present in the extract, derived or isolated from a Rothia species bacterium is inhibited by 0.1-1 mM PMSF. In one embodiment, the glutamine endopeptidase enzyme present in the extract, derived or isolated from a Rothia species bacterium is inhibited by 0.1-1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF).

In one embodiment, the glutamine endopeptidase enzyme attenuates gluten toxicity by cleaving the peptide bond after glutamine at -XPQ- motifs in gluten-containing foodstuff, wherein X=any amino acids, P=proline, and Q=glutamine, or at XPY where Y=tyrosine.

Accordingly, in some embodiments, the glutamine endopeptidase of a Rothia species described herein is capable of cleaving of any of the following peptides, including known T cell epitopes in gluten, under optimal conditions: QLQPFPQPQLPY (SEQ. ID. NO: 3) or PFPQPQLPY (SEQ. ID. NO: 4), PQPQLPYPQPQLPY (SEQ. M. NO: 5) or PQPQLPYPQ (SEQ. ID. NO: 6), QPQQSFPQQQ (SEQ. ID. NO: 7) or PQQSFPQQQ (SEQ. ID. NO: 8), QLQPFPQPELPY (SEQ. ID. NO: 9), PQPELPYPQPELPY (SEQ. ID. NO: 10), QPQQSFPEQQ (SEQ. ID. NO: 11); IQPQQPAQL (SEQ. ID. NO: 12); QQPQQPYPQ (SEQ. ID. NO: 13); SQPQQQFPQ (SEQ. ID. NO: 14); QQPFPQQPQ (SEQ. ID. NO: 15); or PFSQQQQPV (SEQ. ID. NO: 16), including 33-mer from alpha-gliadin, LQLQPF(PQPQLPY).sub.3PQPQPF (SEQ. ID. NO: 2), and the 26-mer from gamma-gliadin, FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ. ID. NO: 17). In some embodiments, the glutamine endopeptidase of a Rothia species described herein have a kcat/Km of at least about 2.5 s.sup.-1 M.sup.-1, usually at least about 250 s.sup.-1 M.sup.-1 and preferably at least about 25000 s.sup.-1 M.sup.-1 for cleaving of any of the peptides described herein. A glutamine endopeptidase of a Rothia species described herein have a specificity kcat/Km>2 mM.sup.-1 s.sup.-1 for the quenched fluorogenic substrate Abz-QPQQP-Tyr(NO.sub.2)-D. Methods of assaying such enzymatic activities are known to those skilled in the art, e.g., by HPLC or fluorescence spectroscopy and as described in U.S. Pat. No. 7,534,426, the reference is hereby incorporated by reference in its entirety. For the fluorescence spectroscopy-based assays, suitable fluorophores can be attached to the amino- and carboxy-termini of the peptides.

In one embodiment, the effective dose of the extract from the Rothia species bacterium is administered orally. In other embodiments, the effective dose of the Rothia species bacteria or the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium is administered orally.

In one embodiment, the extract from the Rothia species bacteria is admixed to the gluten-containing foodstuff. In other embodiments, the Rothia species bacteria or the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium is admixed to the gluten-containing foodstuff. For example, the extract, the bacteria or enzyme is mixed with the gluten-containing foodstuff prior to ingesting.

In one embodiment, the extract from the Rothia species bacteria is formulated with a pharmaceutically acceptable excipient or carrier. In other embodiments, the Rothia species bacteria or the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium is formulated with a pharmaceutically acceptable excipient or carrier.

In one embodiment, the extract from the Rothia species bacteria is contained in a formulation that comprises an enteric coating. In other embodiments, the Rothia species bacteria or the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium is contained in a formulation that comprises an enteric coating.

In one embodiment, the extract from the Rothia species bacteria is a lyophilized preparation. In other embodiments, the Rothia species bacteria, the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium, recombinant enzyme, or various supernatants of the Rothia spp. lysate is lyophilized. Lyophilization or freeze-drying is a means of drying, achieved by freezing the wet substance and causing the ice to sublime directly to vapor by exposing it to a low partial pressure of water vapor. In practice, the substance may not be completely frozen, especially if non-aqueous solutions are present, and most lyophilization processes are completed by a period of desorption drying. The purpose of freeze-drying is to increase the shelf life, or preserve a specimen, be it food, microbial organisms, or, in some circumstances to decrease the size of the product. For various purposes, such as stable storage, the extract, bacteria or isolated enzyme can be lyophilized. Lyophilization is preferably performed on an initially concentrated preparation, e.g. of at least about 1 mg/ml for extract or isolated enzyme preparation and 1000 bacteria/ml. PEG can be added to improve the enzyme stability. In some embodiments, lyophilized extract, bacteria or isolated enzyme is without loss of specific activity. The lyophilized extract, bacteria or isolated enzyme and excipients is useful in the production of enteric-coated capsules or tablets, e.g., a single capsule or tablet can contain at least about 1 mg usually at least about 10 mg of Rothia species bacterial extract or isolated glutamine endopeptidase enzyme, and may contain at least 100 mg glutamine endopeptidase, at least about 200 mg, at least about 300 mg, at least about 400 mg, at least about 500 mg, up to about 1000 mg protein, including all the numbers between 1-1000 mg. Wherein lyophilized bacteria comprises the enteric-coated capsules or tablets, a single capsule or tablet can contain at least about 1000, at least about 10,000, at least about 100,000, at least about 1 billion Rothia species bacteria, including all the numbers between 1-1 billion. As described in detail here, enteric coatings can be applied, where a substantial fraction of the activity is retained, and is stable for at least about 1 month at 4.degree. C. The method of lyophilizing bacteria is known to one skilled in the art, e.g. U.S. Pat. Nos. 4,205,132, 4,444,760, 5,192,743, 5,529,915, 6,750,330, and 7,572,893, all of which are incorporated by reference inn their entirety.

In one embodiment, the extract from the Rothia species bacteria is formulated for oral administration. In other embodiments, the Rothia species bacteria or the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium is formulated for oral administration. For example, as capsules of a lyophilized preparation described. One or two capsule is taken with gluten-containing foodstuff.

In one embodiment, the effective dose of the extract from the Rothia species bacteria from ranges 0.01 mg to 500 mg/kg body weight. In another embodiment, wherein the Rothia species bacteria are used, the effective dose of the Rothia species bacteria ranges from 1000 to 1 billion Rothia species bacteria. In another embodiment, wherein the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium is used, the effective dose of the enzyme is from 0.01 mg to 500 mg/kg body weight.

In one embodiment, the subject has been diagnosed with Celiac Sprue, gluten allergy/gluten intolerance and/or dermatitis herpetiformis. In another embodiment, the subject is a mammal, preferably a human. Current diagnosis methods for Celiac sprue include but are not limited to one or more of serological tests, e.g. anti-gliadin antibodies, anti-transglutaminase antibodies, anti-endomysial antibodies; endoscopic evaluation, e.g. to identify celiac lesions; histological assessment of small intestinal mucosa, e.g. to detect villous atrophy, crypt hyperplasia, infiltration of intra-epithelial lymphocytes; and any GI symptoms dependent on inclusion of gluten in the diet.

In one embodiment of the methods described herein further comprises administering an effective dose of prolyl endopeptidase ranging from 0.01 mg to 500 mg/kg body weight. Prolyl endopeptidase (PREP or PEP) or prolyl oligopeptidase (EC 3.4.21.26), (sometimes also known as post-proline cleaving enzyme) is a large cytosolic enzyme that belongs to a distinct class of serine peptidases. The enzyme cleaves peptide bonds at the C-terminal side of proline residues. Its activity is confined to action on oligopeptides of less than 10 kDa and it has an absolute requirement for the trans-configuration of the peptide bond preceding proline. Some types of prolyl endopeptidase have been used in studies to decrease the propensity of gluten-containing wheat products to aggravate coeliac disease (Stepniak D, et al., 2006, Am J Physiol Gastrointest Liver Physiol 291 (4): G621-9), e.g. PEP derived or isolated from Flavobacterium meningosepticum, Sphingomonas capsulate, Penicillium citrinum, Lactobacillus helveticus and Myxococcus Xanthus in U.S. Patent Application No: 20060002917 and 20080193436, and in U.S. Pat. Nos. 7,563,864, 7,303,871, and 7320788. These references are hereby incorporated by reference in their entirety.

In one embodiment, the glutamine endopeptidase enzyme is isolated from a Rothia species bacterium by conventional protein purification methods known to those skilled in the art, e.g. as described in the Current Protocols in Molecular Biology and the Current Protocols in Protein Sciences. The protein fraction of an extract from a Rothia species bacterium can be concentrated by ammonium sulphate precipitation, and then purified by ion exchange chromatography on DEAE SEPHAROSE.RTM. CL-6B and gel filtration on SEPHADEX.RTM. G-100. Sample fractions are taken at each step and assayed for -XPQ- cleavage activity in order to follow the location of the enzyme. Such -XPQ- cleavage activity assays are well known in the art and are also described here.

In one embodiment, the present invention provides a pharmaceutical formulation for use in treatment of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis comprising an effective dose of an extract from a Rothia species bacteria and a pharmaceutically acceptable excipient, wherein the extract from the Rothia species bacteria contains a glutamine endopeptidase enzyme that attenuates gluten toxicity in the subject.

In another embodiment, the pharmaceutical formulation comprising an effective dose of a Rothia species bacteria and a pharmaceutically acceptable excipient, wherein the Rothia species bacteria contains a glutamine endopeptidase enzyme that attenuates gluten toxicity in the subject.

In another embodiment, the pharmaceutical formulation comprising an effective dose of an isolated glutamine endopeptidase enzyme and a pharmaceutically acceptable excipient, wherein the glutamine endopeptidase enzyme that attenuates gluten toxicity in the subject. In one embodiment, the isolated glutamine endopeptidase enzyme is derived or isolated from a Rothia species bacterium. In one embodiment, the isolated glutamine endopeptidase enzyme is a recombinant protein. In one embodiment, the isolated glutamine endopeptidase enzyme is neprilysin.

In some embodiments of the pharmaceutical formulations described herein, the glutamine endopeptidase enzyme appears in the region of 70-75 kDa in a gliadin zymogram, is active in a saliva sample, is a metal-ion dependent protease, is stable to acid conditions, and detoxifies gluten by cleaving the peptide bond after glutamine at -XPQ- motifs in gluten-containing foodstuff, wherein X=any amino acids, P=proline, and Q=glutamine. In one embodiment, glutamine endopeptidase enzyme is active in a buffer that mimics the ion composition of saliva, e.g. saliva ion buffer described herein.

In some embodiments of the pharmaceutical formulations described herein, the Rothia species bacteria is R. mucilaginosa ot 681 (strain WSA-2B) Rothia species ot 188 (strain WSA-8), R. mucilaginosa ATCC 25296 and/or R. dentocariosa ATCC 17931. In some embodiments, the pharmaceutical formulations comprises more than one Rothia species bacteria or glutamine endopeptidase isolated from more than one type of bacteria described herein.

In some embodiments of the pharmaceutical formulations described herein, the extract, Rothia species bacteria or the isolated glutamine endopeptidase enzyme is lyophilized.

In some embodiments of the pharmaceutical formulations described herein, the effective dose of the extract ranges from 0.01 mg to 500 mg/kg body weight when the formulation comprises the extract and/or isolated glutamine endopeptidase enzyme, and 1000 to 1 billion bacteria when the formulation comprises the Rothia species bacteria.

In some embodiments of the pharmaceutical formulations described herein, the formulation is suitable for oral administration, e.g., an emulsion, a suspension, a tablet or a capsule.

In some embodiments of the pharmaceutical formulations described herein, the formulation comprises an enteric coating.

In some embodiments of the pharmaceutical formulations described herein, the formulation further comprises an effective dose of prolyl endopeptidase ranging from 0.01 mg to 500 mg/kg body weight. The prolyl endopeptidase can be isolated from F. meningosepticum, S. capsulate, P. citrinum, L.s helveticus and M. Xanthus. In other embodiments, the pharmaceutical formulations can comprise more that one prolyl endopeptidases, wherein the prolyl endopeptidases are from several origins or sources, e.g. from a formulation comprising prolyl endopeptidases isolated from F. meningosepticum and S. capsulate.

In one embodiment, the present invention provides a method of predicting/diagnosing Celiac Sprue, gluten allergy and/or dermatitis herpetiformis in a subject in need thereof, the method comprises determining the extent of digestion of a fixed amount of gliadin within 24 hour period by a biological sample obtained from a subject, for example, unstimulated whole saliva, stimulated whole saliva or dental plaque wherein when less than 50% of the fixed amount of gliadin digested indicates the subject likely have Celiac Sprue, gluten allergy/intolerance and/or dermatitis herpetiformis.

In one embodiment, the subject expresses the HLA-DQ2, DQ2.5, DQ2.2/DQ7.5 or DQ8 allele and/or has the HLA-DQ2, DQ2.5, DQ2.2/DQ7 or DQ8 antigen. Such subjects would be considered at risk of developing Celiac Sprue, gluten allergy/intolerance and/or dermatitis herpetiformis.

In another embodiment, the subject exhibits at least one symptom that is known to be associated Celiac Sprue, gluten allergy and/or dermatitis herpetiformis, e.g. itchy skin with no obvious rash or insect bites or those described herein. Such subjects would be considered at risk of developing Celiac Sprue, gluten allergy/intolerance and/or dermatitis herpetiformis.

In one embodiment, the subject is related to another subject who is diagnosed with Celiac Sprue and/or dermatitis herpetiformis. The relationship can be immediate and direct, e.g. as in father, mother, siblings; or indirect, e.g. as in cousins, aunt, uncle, grandparents. Such subjects would be considered at risk of developing Celiac Sprue, gluten allergy/intolerance and/or dermatitis herpetiformis.

For the diagnostic method, the fixed amount of gliadin is 250 .mu.g/ml. The gliadin used can be commercially available gliadin extract from wheat (SIGMA-ALDRICH.RTM. Cat. No. G3375). The gliadin extract is a mixture of gliadins with the most prominent constituent being about 37 kDa in size (as evidenced from the gel electrophoresis). Methods of gliadin extract use are will known in the art and are also described in experiments in FIGS. 2, 3, 5, and 8 of the Example 1 section.

In one embodiment, a biological sample obtained from a subject is a sample of whole saliva or dental plaques. In one embodiment, the sample of whole saliva is unstimulated whole saliva. Unstimulated whole saliva is saliva that had naturally accumulated in the oral cavity between swallowings and it is collected by expectoration in graduated cylindrical tubes places on ice.

In another embodiment, the sample of whole saliva is stimulated whole saliva. Stimulated whole saliva is collected when donors are chewing on a 1 g bolus of tasteless paraffin wax (parafilm). The masticatory stimulated saliva is collected in graduated cylindrical tubes places on ice.

In one embodiment, the dental plaques are supragingival plaque samples. These are collected from interproximal dental spaces with an explorer 24 hr after refraining from oral hygiene and are suspended in saliva ion buffer to an OD.sub.620nm of .about.1.0 prior to mixing with the gliadin.

In one embodiment, the saliva or dental plaque is suspended in saliva ion buffer and mixed with a gliadin-derived enzymatic substrate, such as A-Xaa-Pro-Gln-B where A is an N-terminal protective group, e.g. benzyloxycarbonyl and B is the reporter group, e.g. paranitroanilide, and Xaa is an amino acid present in zero, 1 or more copies. Saliva or plaque suspended in saliva ion buffer is then incubated with this substrate to allow cleavage of the peptide bond after glutamine. This cleavage is indicative of glutamine endopeptidase activity, and is monitored spectrophotometrically, luminometrically or fluorimetrically by asy methods known to one skilled in the art. Subjects showing statistically significant differences (P<0.05) from values obtained from a healthy pool of subjects will be considered at risk for displaying or developing Celiac Sprue and/or dermatitis herpetiformis and/or gluten allergy.

In another embodiment, gliadin-degrading protease(s) in the saliva and/or plaque samples from patients suffering from Celiac Sprue and/or dermatitis herpetiformis and/or gluten allergy or of those at risk of developing the disease is visualized and quantitated by gliadin zymography. Gliadin zymography is a technique similar to gelatin zymography, except that gliadin is incorporated in the gel as the enzymatic substrate instead of gelatin. Aliquots of 100 ul of saliva or suspended plaque sample will be dried and suspended in sample buffer containing 0.125 M Tris-HCl, 20% (v/v) glycerol, 4% sodium dodecyl sulfate, and 0.005% (w/w) bromophenol blue. Gel electrophoresis will be carried out at 4.degree. C. at a constant voltage of 100 V, followed by renaturing of the gel in 2.5% (v/v) triton X-100 and developing of enzyme activity for 24 h at 37 C. in 20 mM Tris buffer (pH=7.5). Protease band intensities are quantitated by densitometric analysis. The absence of one or more protease bands in the overall gliadin zymogram protease pattern will be considered a diagnostic marker for displaying or developing Celiac Sprue and/or dermatitis herpetiformis and/or gluten allergy.

In another embodiment, the amount of Rothia species will be quantitated in saliva or dental plaque samples using strain-specific complimentary .sup.32P-labeled DNA or RNA probes against unique 16S DNA domains or by generating oligolabeled DNA fragments (Feinberg et al., Anal. Biochem 132: 6-13 (1983). A 200 .mu.l aliquot of whole saliva or suspended dental plaque samples will be mixed with 150 .mu.l 10 mM Tris and 1 mM EDTA. From this mixture, 200 .mu.l will be mixed with 100 .mu.l 0.5M NaOH. Rothia-specific DNA and RNA levels will be quantitated following Nothern and Southern blot analysis known to those skilled in the art. Measures of quantitation will be based on the pixel intensities of the read-out system. DNA/RNA will also be quantitated using TAQMAN.RTM.-derivatized probes instead of radiolabeled probes. In this case, DNA/RNA is isolated from Rothia species, followed by quantative PCR and read-out of a fluorescent signal the intensity of which is related to the numbers of Rothia DNA/RNA present in the sample. Rothia levels will be expressed relative to total bacterial DNA in the saliva or plaque sample, which will be quantitated using a probe complimentary to a highly conserved DNA domain. Subjects showing statistically significant differences in Rothia levels (P<0.05) from values obtained from a healthy pool of subjects will be considered at risk for displaying or developing Celiac Sprue and/or dermatitis herpetiformis and/or gluten allergy.

The method of assessing the degree and mode of digestion can be determined by protein gel electrophoresis or mass spectrometry respectively that are known in the art and are described herein.

In one embodiment, the invention provides a kit for predicting/diagnosing Celiac Sprue, gluten allergy/intolerance and/or dermatitis herpetiformis in a subject in need thereof, comprising: an amount of gliadin substrate and reagents to assay for the endopeptidase activity by determining the amount of undigested gliadin.

In a further embodiment, the kit provides materials, reagents and instructions such as containers and buffers for performing the assay, and a chart for comparing the results and making a decision based on the results. For example, the kit can have a measured quantity of saliva ion buffer, e.g. 5 ml in a screw-cap container, measured amount of a gliadin solution, a measured amount of reagents to assay for undigested gliadin and instruction and a chart of possible color results. In using this exemplary kit, this entire volume of 5 ml is emptied into the buccal cavity of a subject and the subject swishes the buffer vigorously for 1 minute and spits buffer back into the original container. Next, the measured amount of a gliadin solution is added. The container is capped tightly, the contents mixed by repeated inverting the container for 1 minute and left at room temperature for 24 hours. At the end of that period, the measured amount of reagents to assay for undigested gliadin is added and mixed. In one embodiment, the reagents to assay for undigested gliadin produces a color read out. The color read out of the container is observed and compared to a chart that is provided with the kit. As another example, the kit can have a measured quantity of saliva ion buffer, a graduated container for collecting saliva, measured amount of a gliadin solution, a measured amount of reagents to assay for undigested gliadin and instruction and a chart of possible color results. In using this exemplary kit, the subject collects the required amount of saliva in the graduated container for collecting saliva, and then the saliva is diluted with the measured quantity of saliva ion buffer and mixed with the measured amount of a gliadin solution and left at room temperature for 24 hours before assaying for the amount of undigested gliadin.

DEFINITIONS OF TERMS

As used herein, the term "treat", "treating" or "treatment" means to stabilize or improve the clinical symptoms of the subject. "Treat", "treating" or "treatment" also means to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition, at bringing about ameliorations of the symptoms of the pathology. Evidence of therapeutic effect may be any diminution in the severity of disease, particularly as measured by the severity of symptoms such as fatigue, chronic diarrhea, malabsorption of nutrients, weight loss, abdominal distension, anemia, and other symptoms of Celiac Sprue. Other disease indicia include the presence of antibodies specific for glutens, the presence of antibodies specific for tissue transglutaminase, the presence of pro-inflammatory T cells and cytokines, damage to the villus structure of the small intestine as evidenced by histological or other examination, enhanced intestinal permeability, and the likes. In one embodiment, the symptom of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis is alleviated by at least 20%, at least 30%, at least 40%, or at least 50%. In one embodiment, the symptom of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis is alleviated by more that 50%. In one embodiment, the symptom of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis is alleviated by 80%, 90%, or greater.

As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

The term "consisting of" in reference to the isolated enzyme, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment

As used herein, the term "effective dose" refers to an amount of a biologically active molecule or conjugate thereof sufficient to exhibit a detectable therapeutic effect, e.g. reduction in the symptoms associated with Celiac sprue, gluten allergy and/or dermatitis herpetiformis, e.g. fatigue, chronic diarrhea, malabsorption of nutrients, weight loss, abdominal distension, anemia, the presence of antibodies specific for tissue transglutaminase, the presence of pro-inflammatory T cells and cytokines, and damage to the villus structure of the small intestines. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and can vary depending on factors known in the art, such as, for example, the subject's history and age, the stage of pathological processes, and the administration of other agents or therapeutics that inhibit pathological processes in Celiac sprue, gluten allergy and/or dermatitis herpetiformis.

As used herein, in one embodiment, the term "an extract from a Rothia species" refers to a clarified aqueous solution that formerly comprised a Rothia species for example, a suspension of Rothia species in phosphate buffered saline (PBS) that was agitated for 1 hour at room temperature and then centrifuged at 1000.times.G for 10 minutes to sediment the bacteria. The supernatant PBS fluid is "an extract from a Rothia species". Similarly, a clarified saliva sample is "an extract from a Rothia species".

In another embodiment, "an extract from a Rothia species" can also mean a clarified periplasmic extraction of a Rothia species, for example, a suspension of Rothia species in phosphate buffered saline (PBS) with 20% or 500 mM sucrose and is then agitated for 1 hour at 4.degree. C. and then centrifuged at 1000.times.G for 10 minutes to sediment the bacteria. In the presence of high sucrose concentration, the bacteria undergo osmotic shock. Such methods of making periplasm extracts are well known to those skilled in the art, e.g. as described in U.S. Pat. No. 5,856,142, this reference is hereby incorporated by reference in its entirety.

In another embodiment, "an extract from a Rothia species" can also mean a clarified cell lysate of a Rothia species, wherein the bacteria are lysed in a suitable buffer and the lysate is centrifuged at 20,000.times.G for 30 minutes to sediment the cell debris. In other embodiments, ultracentrifugation clarified cell lysate of a Rothia species and a chromatography fraction containing a 70-75 kDa protein with a glutamine endopeptidase activity as assayed by gliadin zymography and other methods described herein are also considered "extracts from a Rothia species".

As used herein, the term "a glutamine endopeptidase" refers to a proteolytic peptidase that breaks peptide bonds of non-terminal amino acids (i.e. within the molecule) at the -XPQ- or -Xaa-Pro-Gln- triplet sequence and the breakage occurs immediately after the glutamine residue. X or Xaa=any amino acids, P or Pro=proline, and Q or Gln=glutamine.

As used herein, the term "attenuates gluten toxicity" in the context of a glutamine endopeptidase refers to the endopeptidase enzyme reduces, weakens or lessen in the amount, degree, and/or the density of toxic gluten oligopeptides production from gluten-containing foodstuff before or after the normal digestion of gluten-containing foodstuff by endogenous trypsin, chymotrypsin, elastase and carboxypeptidase in the gut. This is achieved by digesting the toxic gluten oligopeptides to smaller peptide fragments that are lacking the T cell epitopes in glutens. The activity of a glutamine endopeptidase from Rothia species described herein, before and/or after the digestion of gluten oligopeptides produced by endogenous trypsin, chymotrypsin, elastase and carboxypeptidase would result in less than 10% of the post-digestion products being longer than PQPQLPYPQ (SEQ. ID. NO: 6) which has nine amino acid residues. This can be assessed by the longer retention times on a C18 reverse phase HPLC column monitored at A.sub.215 and such methods of well known to one skilled in the art.

The assessment of "attenuation of gluten toxicity" can be determined by measuring the ability of the extract of Rothia species or isolated glutamine endopeptidase from Rothia species described herein to increase the concentration of free NH.sub.2-termini in a reaction mixture containing 1 mg/ml of undigested or trypsin/chymotrypsin/elastase/carboxypeptidase pre-digested gluten substrate and 10 .mu.g/ml of the extract or glutamine endopeptidase from a Rothia species, incubated at 37.degree. C. for 1 hour. An attenuation of gluten toxicity activity useful in the practice of the present invention will increase the concentration of the free amino termini under such conditions, usually by at least about 25%, more usually by at least about 50%, and preferably by at least about 100%. Additionally, there would be a reduction in the residual molar concentration of oligopeptides greater than about 1000 Da in a 1 mg/ml trypsin/chymotrypsin/elastase/carboxypeptidase pre-digested gluten substrate after a 1 hour incubation with 10 .mu.g/ml of the extract or enzyme by at least about 2-fold, usually by at least about 5-fold, and preferably by at least about 10-fold. The concentration of such oligopeptides can be estimated by methods known in the art, for example size exclusion chromatography and the like.

In another embodiment, "attenuates gluten toxicity" also refers to reducing the ability of a gluten oligopeptide to bind to HLA-DQ. The ability of a substrate to bind to HLA-DQ is indicative of its toxicity; fragments smaller than about 8 amino acids are generally not stably bound to Class II MHC. The detoxification of whole gluten can be monitored by polyclonal T cell lines derived from intestinal biopsies of celiac or gluten allergic patients, by LC-MS-MS and by ELISA assays using monoclonal antibodies capable of recognizing sequences specific to gliadin.

For example, an extract of a Rothia species or the isolated glutamine endopeptidase from Rothia species described herein can reduce the potency by which a trypsin/chymotrypsin/elastase/carboxypeptidase pre-digested gluten substrate can antagonize binding of PQPELPYPQPQLP (SEQ. ID. NO: 18) to HLA-DQ2. Treatment with an extract of Rothia species bacteria or the isolated glutamine endopeptidase from Rothia bacteria described herein that digests toxic oligopeptides, by reducing the concentration of the toxic oligopeptides, prevents a mixture containing them from competing with a test peptide for MHC binding. Such a competition assay can be performed by incubating 1 mg/ml trypsin/chymotrypsin/elastase/carboxypeptidase pre-digested gluten substrate with 10 .mu.g/ml of the extract or enzyme, and the ability of the resulting solution to displace radioactive PQPELPYPQPQPLP (SEQ. ID. NO: 19) pre-bound to HLA-DQ2 molecules can then be quantified, with a reduction of displacement, relative to a non-treated control, indicative of utility in the methods of the present invention.

In yet another embodiment, "attenuates gluten toxicity" also refers to reducing the anti-tTG antibody and/or anti-gliadin antibodies response to a "gluten challenge diet" in a Celiac sprue or gluten allergic/gluten intolerance patient by at least about 2-fold, more usually by at least about 5-fold, and preferably by at least about 10-fold. A "gluten challenge diet" is defined as the intake of 100 g bread per day for 3 days by an adult Celiac Sprue or gluten allergic patient previously on a gluten-free diet. The anti-tTG antibody (ATA) and anti-gliadin antibodies (AGA) response can be measured in peripheral blood using standard clinical diagnostic procedures, as known in the art.

As used herein, the term "admix" in the context of gluten-containing foodstuff refers to mixing or blending with gluten-containing foodstuff.

As used herein, the term "glutens" refers to a mixture of proteins, including gliadins and glutelins, found in wheat grains and other grain, which are not soluble in water and which give wheat dough its elastic texture. "Glutens" also refer to the prolamins that are found in rye, barley, and oats.

As used herein, the term "glutelin" refers to prolamin-like proteins that are found in grass seeds, e.g. wheat, and they are soluble in dilute acids or bases, detergents, chaotropic or reducing agents. "Glutelin" tend to be rich in prolines and glutamine.

As used herein, the term "prolamins" refers to a group of plant storage proteins having high proline content and is found in the seeds of cereal grains such as wheat (gliadin), barley (hordein), rye (secalin), corn (zein) and as a minor protein, avenin in oats. They are characterized by a high glutamine and proline content and are generally soluble only in strong alcohol solutions. Some prolamins, notably gliadin from wheat, and similar proteins found in the grass seed of the Triticeae species can induce coeliac disease in genetically predisposed individuals.

As used herein, the term "gliadin" refers to the alcohol-soluble, glutamine and proline-rich prolamin glycoprotein found in wheat. This is one of the proteins that induce coeliac disease in genetically predisposed individuals. In other embodiments, gliadins also encompass proline-rich prolamin glycoproteins from other sources. Examples of gliadin sequences include but are not limited to wheat alpha gliadin sequences, for example as provided in GENBANK accession numbers AJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607; AJ133606; AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307; U51306; U51304; U51303; U50984; and U08287. A sequence of wheat omega gliadin is set forth in Genbank accession number AF280605.

As used herein, the term "gluten-containing foodstuff" refers to food and/or ingredients of food that has gluten and other proteins found in wheat, barley, rye, and oats. "Gluten-containing foodstuff" also to refer to food and/or ingredients of foods that are made of wheat, barley, rye, and oats.

As used herein, the term "consuming gluten-containing foodstuff" refers to ingesting food made of wheat, rye, barley, and oats, e.g. pizza, cake, etc. as well as ingesting food made with ingredients that are made with wheat, rye, barley, and oats, e.g. soy sauce and chocolate cookie dough ice cream.

As used herein, the term "diagnosed of Celiac sprue, gluten allergy/gluten intolerance and/or dermatitis herpetiformis" refers to having the symptoms associated with Celiac sprue, gluten allergy and/or dermatitis herpetiformis, e.g. fatigue, chronic diarrhea, malabsorption of nutrients, weight loss, abdominal distension, anemia, the presence of antibodies specific for tissue transglutaminase (ATA), antibodies specific for .alpha./.beta.,.gamma.-gliadin (AGA), the presence of pro-inflammatory T cells and cytokines, and damage to the villus structure of the small intestines.

As used herein, the term "toxic gluten oligopeptides" refers are peptides derived during normal human digestion of gliadins and related storage proteins from dietary cereals, e.g. wheat, rye, barley, and the like. Such oligopeptides are believed to act as antigens for T cells in Celiac Sprue. For binding to Class H MHC proteins, immunogenic peptides are usually from about 8 to 20 amino acids in length, more usually from about 10 to 18 amino acids. Such peptides may include XPQ and XPY motifs, such as the motif PQPQLPYPQ (SEQ. ID. NO: 6). Determination of whether an oligopeptide is immunogenic for a particular patient is readily determined by standard T cell activation and other assays known to those of skill in the art. Other examples of immunogenic gliadin oligopeptides are described in Wieser (1995) Baillieres Clin Gastroenterol 9(2):191-207, incorporated herein by reference. "Toxic gluten oligopeptides" also refers are peptides that comprise known T cell epitopes in gluten, e.g. QLQPFPQPQLPY (SEQ. ID. NO: 3) or PFPQPQLPY (SEQ. ID. NO: 4), PQPQLPYPQPQLPY (SEQ. ID. NO: 5) or PQPQLPYPQ (SEQ. ID. NO: 6), QPQQSFPQQQ (SEQ. ID. NO: 7) or PQQSFPQQQ (SEQ. ID. NO: 8), QLQPFPQPELPY (SEQ. ID. NO: 9), PQPELPYPQPELPY (SEQ. ID. NO: 10), QPQQSFPEQQ (SEQ. ID. NO: 11); IQPQQPAQL (SEQ. ID. NO: 12); QQPQQPYPQ (SEQ. ID. NO: 13); SQPQQQFPQ (SEQ. ID. NO: 14); QQPFPQQPQ (SEQ. ID. NO: 15); or PFSQQQQPV (SEQ. ID. NO: 16), including 33-mer from alpha-gliadin, LQLQPF(PQPQLPY).sub.3PQPQPF (SEQ. ID. NO: 2), and the 26-mer from gamma-gliadin, FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ. ID. NO: 17).

The term "isolated" refers to the enzyme protein which is substantially or essentially free from bacterial components which normally accompany or interact with the enzyme as found in the bacteria.

As used herein, the term "inhibited" or "inhibition" when used in the context with the glutamine endopeptidase activity means the reduction of the cleavage of -XPQ-containing peptides by at least about 50%, about 60%, about 70%, about 80%, about 90%, about 100% by any assay described herein or known in the art, wherein X is any amino acid, P is proline and Q is glutamine.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, for example the carrier does not decrease the impact of the agent on the treatment. In other words, a carrier is pharmaceutically inert.

As used herein, "identity" means the percentage of identical amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ea., Oxford University Press, New York, 1988; Biocomputing: Informatics and--14 Genome Projects, Smith, D. W., ea., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988)). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs such as BLASTP.

The terms "identical" or percent "identity", in the context of two or more polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., amino acid sequence of the enzyme described herein), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This term also refers to, or can be applied to, the compliment of a test sequence. The term also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement).

Programs for searching for alignments are well known in the art, e.g., BLAST and the like. For example, if the target species is human, a source of such amino acid sequences or gene sequences (germline or rearranged antibody sequences) can be found in any suitable reference database such as GENBANK, the NCBI protein databank, VBASE, a database of human antibody genes, and the Kabat database of immunoglobulins or translated products thereof. If the alignments are done based on the nucleotide sequences, then the selected genes should be analyzed to determine which genes of that subset have the closest amino acid homology to the originating species antibody. It is contemplated that amino acid sequences or gene sequences which approach a higher degree homology as compared to other sequences in the database can be utilized and manipulated in accordance with the procedures described herein. Moreover, amino acid sequences or genes which have lesser homology can be utilized when they encode products which, when manipulated and selected in accordance with the procedures described herein, exhibit specificity for the predetermined target antigen. In certain embodiments, an acceptable range of homology is greater than about 50%. It should be understood that target species can be other than human.

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25: 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215: 403-410, 1990, respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The terms "similar" or percent "similar", in the context of two or more polypeptide sequences, refers to two or more sequences or subsequences that are similar or have a specified percentage of amino acid residues are similar (i.e., about 60% similarity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher similarity over a specified region (e.g., amino acid sequence of the enzyme described herein), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Similarity occurs when the amino acids are not the same but are "conservative amino acid substitution of those in the reference protein, e.g., the neprilysin sequence from R. mucilaginosa. The term also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, similarity exists over a region that is at least about 25 amino acids or nucleotides in length.

As used herein, the term "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge and size. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

As used herein, the term "substantially complete" in reference to Z-YPQ-pNA cleavage means "the same as complete, total or very close to complete", such as 99%, 99.3%, 99.5%, 99.99%, and 100% Z-YPQ-pNA cleavage.

As used herein, the term "amino acid" is intended to include not only the L-, D- and nonchiral forms of naturally occurring amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine, valine), but also modified amino acids, amino acid analogs, and other chemical compounds which can be incorporated in conventional oligopeptide synthesis, e.g., 4-nitrophenylalanine, isoglutamic acid, isoglutamine, .epsilon.-nicotinoyl-lysine, isonipecotic acid, tetrahydroisoquinoleic acid, .alpha.-aminoisobutyric acid, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine, 4-aminobutyric acid, and the like.

Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

The term "functional fragment" in reference to the enzyme refers to functional portion of the enzyme and not the whole intact enzyme. The functional fragment has an amino acid residue sequence that is shorter than that of a whole intact enzyme described herein. For example, if the enzyme is neprilysin, the whole intact enzyme is 660-amino acid long, while a functional fragment is only a portion of the 660 amino acid polypeptide, such as, only 100-amino acid long. "Functional fragments" have peptide bond cleavage activities; they cleave the peptide bond after a -XPY- or -XPQ- motif in glutens.

As used herein, the term "active" when used with the enzyme refers to the enzyme cleaving activity. Therefore when an enzyme is "active", it means that the enzyme exhibit detectable cleaving activity, preferably cleaving the peptide bond after a -XPY- or -XPQ- motif in glutens.

Celiac Sprue, Gluten Allergy and/or Dermatitis Herpetiformis

Celiac sprue, also known as celiac disease, gluten-sensitive enteropathy, and gluten-induced enteropathy, is a chronic disease of the digestive tract that interferes with the digestion and absorption of nutrients from food. People with celiac sprue cannot tolerate gluten. Celiac disease is an inherited, autoimmune disease in which the lining of the small intestine is damaged from eating gluten and other proteins found in wheat, barley, rye, and possibly oats. There is a propensity of Celiac disease in individuals who possess the HLA-DQ8 class II antigen receptor gene. The exact cause of celiac disease is unknown although it is believed that intestinal damage is caused by interactions between specific gliadin oligopeptides and the HLA-DQ2, DQ2.5, DQ2.2/DQ7 or DQ8 antigen, which in turn induce proliferation of T lymphocytes in the sub-epithelial layers. T helper 1 cells and cytokines apparently play a major role in a local inflammatory process leading to villus atrophy of the small intestine. The intestines contain projections, called villi that absorb nutrients. The lining villi become damaged due to the body's immune reaction. In undiagnosed or untreated celiac disease, these villi become flattened. Because the lining of the intestine contains essential enzymes for digestion and absorption, its destruction leads to malabsorption, a difficulty in absorption of food and essential nutrients. As result, Celiac sprue is often considered a malabsorption disorder. This affects the ability to absorb nutrients properly. The disease can develop at any point in life, from infancy to late adulthood. Those with a family member with celiac disease are at greater risk for developing the disease. The disorder is most common in Caucasians and those of European ancestry. Women are affected more commonly than men.

The symptoms of celiac disease can vary significantly from person to person. This is part of the reason the diagnosis is frequently delayed. For example, one person may have constipation, a second may have diarrhea, and a third may have no irregularity in stools.

A non-limiting list of gastrointestinal symptoms include abdominal pain, abdominal distention, bloating, gas, indigestion, constipation, decreased appetite that may also be increased or unchanged, diarrhea, chronic or occasional lactose intolerance which is common upon diagnosis, but usually goes away following treatment, nausea and vomiting, stools that float, are foul smelling, bloody, or "fatty", and unexplained weight loss although people can be overweight or of normal weight upon diagnosis.

A non-limiting list of nonintestinal symptoms include anemia (low blood count), bone and joint pain, bone disease such as osteoporosis, kyphoscoliosis, and fracture, breathlessness due to anemia, bruising easily, dental enamel defects and discoloration, depression, fatigue, growth delay in children, hair loss, hypoglycemia due to low blood sugar, irritability and behavioral changes, malnutrition, mouth ulcers, muscle cramps, nosebleeds, seizures, short stature, unexplained skin disorders (dermatitis herpetiformis), swelling which can be general or abdominal, and vitamin or mineral deficiency which can include single or multiple nutrient (for example, iron, folate, vitamin K).

There is currently no treatment for celiac disease except the advice to follow a lifelong gluten-free diet. This allows the intestinal villi to heal. Patients are advised to eliminate foods, beverages, and medications that contain wheat, barley, rye, and possibly oats. The health care provider may prescribe vitamin and mineral supplements to correct nutritional deficiencies. Occasionally, corticosteroids (such as prednisone) may also be prescribed for short-term use or in patients suffering from refractory sprue. Following a well-balanced, gluten-free diet is generally the only treatment needed to stay well.

The current diagnosis method includes a complete blood count (CBC) to detect signs of anemia, testing for an increase in alkaline phosphatase level which may indicate bone loss, testing for low cholesterol and albumin levels which may be signs of malabsorption and malnutrition, testing for an increase in liver enzymes and abnormal blood clotting, and detection of specific antibodies to tissue transglutaminase and gliadin. The health care provider will order these antibody test if Celiac sprue is suspected. If the tests are positive, upper endoscopy is usually performed to sample a piece of tissue (biopsy) from the first part of the small intestine (duodenum). An endoscopy with enteroscopy, particularly of the lower sections of the intestine most commonly affected, will show a flattening of the villi. A follow-up biopsy or blood work may be ordered several months after the diagnosis and treatment. These confirm the disease. Normal results mean that the patient has responded have responded to treatment, thereby confirming the diagnosis.

Formulation of Pharmaceutical Compositions and Applications Thereof

The extract from the Rothia species bacteria, Rothia species bacteria or the glutamine endopeptidase enzyme derived or isolated from a Rothia species bacterium can be incorporated into a variety of formulations for therapeutic administration in accordance with the present invention. For example, a simple formulation can incorporate an extract, Rothia species bacteria, or enzyme described herein with an excipient combined in solution, then frozen and lyophilized. The resulting powder can be formulated in a capsule, sachet, pill, and the like, and may further be formulated to comprise an enteric coating.

In one embodiment, the extract, Rothia species bacteria, or enzyme described herein are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, or liquid forms, such as tablets, capsules, powders, granules, solutions, gels, and microspheres. As such, administration of the extract, Rothia species bacteria, or the enzyme described herein can be achieved by oral administration.

In pharmaceutical dosage forms, the extract, Rothia species bacteria, or enzyme described herein can be administered alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds to provide a cocktail of activities. The following methods and excipients are exemplary and are not to be construed as limiting the invention. The currently pharmaceutically active compounds used in the treatment and alleviation of symptoms of Celiac Sprue includes the following: an inhibitor of tissue transglutaminase (see U.S. Pat. No. 7,579,313), an anti-inflammatory agent, an anti-ulcer agent, a mast cell-stabilizing agent, and/or and anti-allergy agent. Examples of such agents include HMG-CoA reductase inhibitors with anti-inflammatory properties such as compactin, lovastatin, simvastatin, pravastatin and atorvastatin; anti-allergic histamine H1 receptor antagonists such as acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine and mizolastine; leukotriene receptor antagonists such as montelukast and zafirlukast; COX2 inhibitors such as celecoxib and rofecoxib; p38 MAP kinase inhibitors such as BIRB-796; and mast cell stabilizing agents such as sodium chromoglycate (chromolyn), pemirolast, proxicromil, repirinast, doxantrazole, amlexanox nedocromil and probicromil.

In one embodiment, the formulation or administration protocol combines an extract, Rothia species bacteria, and/or glutamine endopeptidase enzyme described herein with an inhibitor of transglutaminase 2 (TG2) (see U.S. Pat. No. 7,579,313). Such formulations can provide additional protection from gluten mediated enteropathy, as TG2 has been shown to have a significant pro-inflammatory effect on gluten peptides in the celiac gut. In particular, TG2 inhibitors containing halo-dihydroisoxazole, diazomethylketone or dioxoindole moieties are useful for this purpose.

In one embodiment, the formulation or administration protocol combines an extract, Rothia species bacteria, and/or glutamine endopeptidase enzyme described herein with an anti-inflammatory agent, e.g. a statin; p38 MAP kinase inhibitor; anti-TNFalpha agent; etc.

In another embodiment, the formulation comprises a cocktail of an extract, Rothia species bacteria, and/or glutamine endopeptidase enzyme described herein and a selection of several proteases such as the prolyl endopeptidases from Flavobacterium meningosepticum, Sphingomonas capsulate, Penicillium citrinum, Lactobacillus helveticus and Myxococcus Xanthus described in U.S. Patent Application Nos: 20060002917 and 20080193436, and in U.S. Pat. Nos. 7,563,864, 7,303,871, and 7320788. These references are hereby incorporated by reference in its entirety.

In one embodiment, the formulation comprises an extract from a Rothia species bacterium, the glutamine endopeptidase enzyme described herein or a Pegylated form thereof. PEGylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity) increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

Methods of PEGylating proteins are known to one of ordinary skill in the art, e.g. U.S. Pat. No. 7,585,837 and also described herein. The reference is hereby incorporated by reference in its entirety. The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals. PEGs that are activated at each terminus with the same reactive moiety are known as "homobifunctional", whereas if the functional groups present are different, then the PEG derivative is referred as "heterobifunctional" or "heterofunctional." The chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates and carbonates. In the second generation PEGylation chemistry more efficient functional groups such as aldehyde, esters, amides etc made available for conjugation. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and NHS esters.

Pharmaceutical formulations can be administered by any known route. By way of example, the composition can be administered by a mucosal, pulmonary, topical, or other localized or systemic route (e.g., enteral and parenteral). The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection, infusion and other injection or infusion techniques, without limitation.

For oral preparations, the extract, Rothia species bacteria, or enzyme described herein can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as microcrystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrants, such as corn starch, potato starch or croscarmellose sodium; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives, colorants, and flavoring agents.

For enteral administration, a composition can be incorporated into an inert carrier in discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active compound; as a powder or granules; or a suspension or solution in an aqueous liquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a draught. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.

A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.

A syrup or suspension can be made by adding the extract, Rothia species bacteria, or enzyme described herein to a concentrated, aqueous solution of a sugar, e.g., sucrose, to which can also be added any accessory ingredients. Such accessory ingredients may include flavoring, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredient, e.g., as a polyhydric alcohol, for example, glycerol or sorbitol.

Formulations for oral administration can be presented with an enhancer. Orally-acceptable absorption enhancers include surfactants such as sodium lauryl sulfate, palmitoyl carnitine, Laureth-9, phosphatidylcholine, cyclodextrin and derivatives thereof; bile salts such as sodium deoxycholate, sodium taurocholate, sodium glycochlate, and sodium fusidate; chelating agents including citric acid and salicylates; and fatty acids (e.g., oleic acid, lauric acid, acylcarnitines, mono- and diglycerides). Other oral absorption enhancers include benzalkonium chloride, benzethonium chloride, CHAPS (3-(3-cholamidopropylt-dimethylammonio-1-propanesulfonate), Big-CHAPS(N,N-bis(3-D-gluconamidopropylt-cholamide), chlorobutanol, octoxynol-9, benzyl alcohol, phenols, cresols, and alkyl alcohols. An especially preferred oral absorption enhancer for the present invention is sodium lauryl sulfate.

In one embodiment of the invention, the formulations comprising an extract, a Rothia species bacteria, or an enzyme described herein and the oral formulations comprise enteric coatings, so that the extract, Rothia species bacteria, or enzyme described herein is delivered to the intestinal tract. Enteric formulations are often used to protect an active ingredient from the strongly acid contents of the stomach. Such formulations are created by coating a solid dosage form with a film of a polymer that is insoluble in acid environments, and soluble in basic environments. Exemplary films are cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate, methacrylate copolymers, and cellulose acetate phthalate.

As regards formulations for administering the extract, a Rothia species bacterium, or an enzyme described herein, one particularly useful embodiment is a tablet formulation comprising the extract, the Rothia species bacteria, or the enzyme described herein with an enteric polymer casing. An example of such a preparation can be found in WO2005/021002. The active material in the core can be present in a micronized or solubilized form. In addition to active materials the core can contain additives conventional to the art of compressed tablets. Appropriate additives in such a tablet can comprise diluents such as anhydrous lactose, lactose monohydrate, calcium carbonate, magnesium carbonate, dicalcium phosphate or mixtures thereof; binders such as microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxypropyl-cellulose, polyvinylpyrrolidone, pre-gelatinised starch or gum acacia or mixtures thereof; disintegrants such as microcrystalline cellulose (fulfilling both binder and disintegrant functions) cross-linked polyvinylpyrrolidone, sodium starch glycollate, croscarmellose sodium or mixtures thereof; lubricants, such as magnesium stearate or stearic acid, glidants or flow aids, such as colloidal silica, talc or starch, and stabilisers such as desiccating amorphous silica, colouring agents, flavours etc. Preferably the tablet comprises lactose as diluent. When a binder is present, it is preferably hydroxypropylmethyl cellulose. Preferably, the tablet comprises magnesium stearate as lubricant. Preferably the tablet comprises croscarmellose sodium as disintegrant. Preferably, the tablet comprises microcrystalline cellulose.

The diluent can be present in a range of 10-80% by weight of the core. The lubricant can be present in a range of 0.25-2% by weight of the core. The disintegrant can be present in a range of 1-10% by weight of the core. Microcrystalline cellulose, if present, can be present in a range of 10-80% by weight of the core.

The extract, the Rothia species bacteria, or the enzyme described herein preferably comprises between 10 and 50% of the weight of the core, more preferably between 15 and 35% of the weight of the core (calculated as free base equivalent). The core can contain any therapeutically suitable dosage level of the active ingredient, but preferably contains up to 150 mg as free base of the active ingredient. Particularly preferably, the core contains 20, 30, 40, 50, 60, 80 or 100 mg as free base of the active ingredient. The active ingredient can be present as the free base, or as any pharmaceutically acceptable salt. If the active ingredient is present as a salt, the weight is adjusted such that the tablet contains the desired amount of active ingredient, calculated as free base of the salt. Preferably, the active ingredient is present as a hydrochloride salt.

The core can be made from a compacted mixture of its components. The components can be directly compressed, or can be granulated before compression. Such granules can be formed by a conventional granulating process as known in the art. In an alternative embodiment, the granules can be individually coated with an enteric casing, and then enclosed in a standard capsule casing.

The core is surrounded by a casing which comprises an enteric polymer. Examples of enteric polymers are cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, ethylhydroxycellulose phthalate, polyvinylacetate pthalate, polyvinylbutyrate acetate, vinyl acetate-maleic anhydride copolymer, styrene-maleic mono-ester copolymer, methyl acrylate-methacrylic acid copolymer or methacrylate-methacrylic acid-octyl acrylate copolymer. These can be used either alone or in combination, or together with other polymers than those mentioned above. The casing can also include insoluble substances which are neither decomposed nor solubilised in living bodies, such as alkyl cellulose derivatives such as ethyl cellulose, crosslinked polymers such as styrene-divinylbenzene copolymer, polysaccharides having hydroxyl groups such as dextran, cellulose derivatives which are treated with bifunctional crosslinking agents such as epichlorohydrin, dichlorohydrin or 1,2-,3,4-diepoxybutane. The casing can also include starch and/or dextrin.

Preferred enteric coating materials are the commercially available EUDRAGIT.RTM. enteric polymers such as EUDRAGIT.RTM. L, EUDRAGIT.RTM. S and EUDRAGIT.RTM. NE used alone or with a plasticiser. Such coatings are normally applied using a liquid medium, and the nature of the plasticiser depends upon whether the medium is aqueous or non-aqueous. Plasticisers for use with aqueous medium include propylene glycol, triethyl citrate, acetyl triethyl citrate or CITROFLEX.RTM. or CITROFLEX.RTM. A2. Non-aqueous plasticisers include these, and also diethyl and dibutyl phthalate and dibutyl sebacate. A preferred plasticiser is triethyl citrate. The quantity of plasticiser included will be apparent to those skilled in the art.

The casing can also include an anti-tack agent such as talc, silica or glyceryl monostearate. Preferably the anti-tack agent is glyceryl monostearate. Typically, the casing can include around 5-25 wt % Plasticiser and up to around 50 wt % of anti tack agent, preferably 1-10 wt % of anti-tack agent.

If desired, a surfactant can be included to aid with forming an aqueous suspension of the polymer. Many examples of possible surfactants are known to the person skilled in the art. Preferred examples of surfactants are polysorbate 80, polysorbate 20, or sodium lauryl sulphate. If present, a surfactant can form 0.1-10% of the casing, preferably 0.2-5% and particularly preferably 0.5-2%

In one embodiment, there is a seal coat included between the core and the enteric coating. A seal coat is a coating material which can be used to protect the enteric casing from possible chemical attack by any alkaline ingredients in the core. The seal coat can also provide a smoother surface, thereby allowing easier attachment of the enteric casing. A person skilled in the art would be aware of suitable coatings. Preferably the seal coat is made of an Opadry coating, and particularly preferably it is Opadry White OY-S-28876.

In an example, lactose monohydrate, microcrystalline cellulose, the active ingredient--e.g. the extract form Rothia species, the hydroxypropyl methyl cellulose and half of the croscarmellose sodium are screened into a 10 Liter Fielder high-shear blender (any suitable high shear blender could be used) and blended for 5 minutes at 300 rpm with the chopper off. The mixture is then granulated by the addition of about 750 ml water whilst continuing to blend. The granules are dried in a Glatt 3/5 fluid bed drier, screened by Comil into a Pharmatec 5 Liter bin blender and then blended with any lactose anhydrous given in the formula plus the remainder of the croscarmellose sodium over 5 minutes at 20 rpm. Magnesium stearate is screened into the blender and the mixing process continued for a further 1 minute at 10 rpm. The lubricated mix is compressed using a Riva Piccolla rotary tablet press fitted with 9.5 mm round normal convex punches (any suitable tablet press could be used). The sealcoat, and subsequently the enteric coat, are applied by spraying of an aqueous suspension of the coat ingredients in a Manesty 10 coater using parameters for the coating process as recommended by the manufacturers of the coating polymers (again, any suitable coater could be used).

Other enteric formulations comprise engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings and can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches. The polymers maintain contact with intestinal epithelium for extended periods of time and actually penetrate it, through and between cells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623): 410-414. Drug delivery systems can also utilize a core of superporous hydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh et al. (2001) J Control Release 71(3):307-18. Other enteric-coated preparations of this sort can be prepared by one skilled in the art, using these materials or their equivalents.

The compositions can be formulated as a sustained release composition. For example, sustained-release means or delivery devices are known in the art and include, but are not limited to, sustained-release matrices such as biodegradable matrices or semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules that comprise the extract, Rothia species bacteria, or enzyme described herein

A sustained-release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of one of polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).

Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomally entrapped an extract, Rothia species bacteria, or enzyme described herein. Such liposomes can be prepared by methods known per se: DE 3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) anilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy. Other biodegradable polymers and their use are described, for example, in detail in Brem et al. (1991, J. Neurosurg. 74:441-446). For examples of sustained release compositions, see U.S. Pat. No. 3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22:547 (1983) and R. Langer et al., Chem. Tech. 12:98 (1982).

Methods for preparing liposomes and microspheres for administration to a patient are known to those of skill in the art. U.S. Pat. No. 4,789,734, the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).

Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS19:155-157 (1998), the contents of which are hereby incorporated by reference.

Preferred micro particles are those prepared from biodegradable polymers, such as polyglycolide, polylactide and copolymers thereof. Those of skill in the art can readily determine an appropriate carrier system depending on various factors, including the desired rate of drug release and the desired dosage.

Formulations are typically provided in a unit dosage form, where the term "unit dosage form," refers to physically discrete units suitable as unitary dosages for the subjects, each unit containing a predetermined quantity of the extract, Rothia species bacteria, or enzyme described herein in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents that are inherently nontoxic and nontherapeutic, are commercially available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available. Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2 dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.

In one embodiment, other ingredients may be added to pharmaceutical formulations, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA, and sugar alcohols such as mannitol or sorbitol.

In one embodiment, the pharmaceutical formulation to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).

Depending on the subject and condition being treated and on the administration route, an extract, Rothia species bacteria, or enzyme described herein can be administered in dosages of 0.01 mg to 500 mg/kg body weight per day, e.g. about 20, 100, 250, 500 or more mg/day or about 0.5, 1, 1.5, or more g/day for an average person for the extract or the enzyme and 1000 to 1 million bacteria per dose per day for the Rothia species bacteria. A typical dose of the extract or enzyme described herein in subjects will be in at least about 1 mg/adult subject, more usually at least about 10 mg/adult subject; and usually at least about 50, 150, 250, 500 or more mg/adult subject; usually not more than about 5 g, not more than about 1 g, or not more than about 500 mg/adult subject. Efficient proteolysis of gluten in vivo for an adult can, depending on diet and other factors, require at least about 500 units of a therapeutically efficacious glutamine endopeptidase from Rothia species bacteria described herein. In some embodiments, low dose of glutamine endopeptidase, such as 1000 units, can be used. In other embodiments, such as for the rapid detoxification of 5-10 g ingested gluten, as much as 20,000-50,000 units, or as much as 1,000,000 Units can be provided in unit dose form. One unit is defined as the amount of enzyme required to hydrolyze 1 .mu.mol of Z-KPQ-pNA or Z-YPQ-pNA per min under specified conditions. Most glutamine endopeptidases have specific activities in the range of 5-50 units/mg protein. For barley EP-B2 (whose specific activity of a PEP is in the 1000 Units/mg range, as measured with Cbz-Phe-Arg-pNA), low dose glutenase may consist of 10,000-100,000 Units, whereas high-dose PEPs contains as much as 1,000,000 Units. It will be understood by those of skill in the art that the dose can be raised, but that additional benefits may not be obtained by exceeding the useful dosage. Dosages will be appropriately adjusted for pediatric formulation. In children the effective dose may be lower, for example at least about 0.1, 0.5, 1, 10, 20, 100, 150, 250 or more mg.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. The dose levels can also depend on whether the extract, Rothia species bacteria, or enzyme is use, the severity of the symptoms and the susceptibility of the subject to side effects. The isolated enzyme can be more potent than the extract or the bacteria. Moreover, treatment of a subject with a therapeutically effective dose can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the extract, Rothia species bacteria, or enzyme encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art, or as described herein. Preferred dosages for a given enzyme are readily determinable by those of skill in the art by a variety of means.

The therapeutic effect can be measured in terms of clinical outcome or can be determined by immunological or biochemical tests. For example, in the treatment of Celiac sprue, suppression of the deleterious T-cell activity can be measured by enumeration of reactive Th1 cells, by quantitating the release of cytokines at the sites of lesions, or using other assays for the presence of autoimmune T cells known in the art. Alternatively, one can look for a reduction in symptoms of a disease, e.g. as set forth in Pyle et al, Clin. Gastroenterol. Hepatol. 3:679-686, 2005.

Various methods for administration may be employed, it being appreciated that the formulations of the extract, Rothia species bacteria, or enzyme described herein provided by the present invention provide improved formulations for oral administration. For example, in the treatment of Celiac Sprue with an extract, Rothia species bacteria, or enzyme described herein, the present invention provides unit dose forms of the extract, Rothia species bacteria, or enzyme described herein suitable for administration with meals. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose can be larger, followed by smaller maintenance doses. The dose can be administered as infrequently as weekly or biweekly, or more often fractionated into smaller doses and administered daily, with meals, semi-weekly, or otherwise as needed to maintain an effective dosage level.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of the invention or to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, and the like), but some experimental errors and deviations may be present. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

The methods of the invention are used to treat foods to be consumed or that are consumed by individuals having from Celiac Sprue and/or dermatitis herpetiformis by delivering an effective dose of an extract, Rothia species bacteria, or enzyme described herein. If the extract, Rothia species bacteria, or enzyme described herein is administered directly to a human subject, then the active agent(s) are contained in a pharmaceutical formulation. Alternatively, the desired effects can be obtained by incorporating the extract, Rothia species bacteria, or enzyme described herein into food products. Diagnosis of suitable subjects can utilize a variety of criteria known to those of skill in the art. A quantitative increase in antibodies specific for gliadin, and/or tissue transglutaminase is indicative of the disease. Family histories and the presence of the HLA alleles HLA-DQ2 and/or HLA-DQ8 are indicative of a susceptibility to the disease (Fernando Fernandez-Banares, 2006, Eur. J. Gastoent. Hepatology, 17:1333-8).

The therapeutic effect can be measured in terms of clinical outcome or can be determined by immunological or biochemical tests. Suppression of the deleterious T-cell activity can be measured by enumeration of reactive Th1 cells, by quantitating the release of cytokines at the sites of lesions, or using other assays for the presence of autoimmune T cells known in the art, e.g. is US Patent Application 20080299108. Alternatively, one can look for a reduction in symptoms of a disease.

Various methods for administration may be employed, preferably using oral administration, for example with meals. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose can be larger, followed by smaller maintenance doses. The dose can be administered as infrequently as weekly or biweekly, or more often fractionated into smaller doses and administered daily, with meals, semi-weekly, or otherwise as needed to maintain an effective dosage level.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in celiac sprue, gluten allergy, celiac disease, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 18th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); The ELISA guidebook (Methods in molecular biology 149) by Crowther J. R. (2000); Fundamentals of RIA and Other Ligand Assays by Jeffrey Travis, 1979, Scientific Newsletters; Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987)); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.); Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.); Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005); Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean.+-.1%.

The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

The present invention can be defined in any of the following alphabetized paragraphs: [A] An isolated glutamine endopeptidase enzyme that cleaves a peptide bond after XPQ and XPY motifs in glutens. [B] The isolated enzyme of paragraph [A], wherein the enzyme has an apparent molecular weight of about 70-75 kDa as determined by gliadin zymograms or by SDS-PAGE. [C] The isolated enzyme of paragraph [A] or [B], wherein the enzyme has a functional pH range of 3-10 as determined by detectable Z-YPQ-pNA cleaving activity within a 24 hour digestion period and a functional pH range of 7-10 as determined by substantially complete Z-YPQ-pNA cleavage within a 1 hour digestion period. [D] The isolated enzyme of any one of paragraphs [A]-[C], wherein the enzyme is 100% inhibited by 1 mM of EDTA or PMSF. [E] The isolated enzyme of any one of paragraphs [A]-[D], wherein the enzyme is derived from a Rothia species bacteria., wherein the Rothia species bacteria is selected from the group consisting of R. mucilaginosa ot 681 (strain WSA-2B), R. mucilaginosa ATCC 25296 and Rothia species ot 188 (strain WSA-8). [F] The isolated enzyme of any one of paragraphs [A]-[E], wherein the enzyme is a recombinantly synthesized enzyme. [G] The isolated enzyme of any one of paragraphs [A]-[F], wherein the enzyme has an amino acid sequence that show at least 45% identity or at least 60% similarity to SEQ. ID. NO: 1. [H] The isolated enzyme of any one of paragraphs [A]-[F], wherein the enzyme comprises SEQ. ID. NO: 1. [I] The isolated enzyme of any one of paragraphs [A]-[F], wherein the enzyme consists essentially of SEQ. ID. NO: 1. [J] The isolated enzyme of any one of paragraphs [A]-[F], wherein the enzyme is SEQ. ID. NO: 1. [K] A formulation for use in the treatment of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis, the formulation comprising an effective dose of an isolated enzyme of any one of paragraphs [A]-[J] and a pharmaceutically acceptable carrier. [L] A formulation for use in treatment of Celiac Sprue, gluten allergy and/or dermatitis herpetiformis, comprising: an effective dose of an extract from a Rothia species bacteria and a pharmaceutically acceptable excipient, wherein the extract from the Rothia species bacteria contains a glutamine endopeptidase enzyme. [M] The formulation of paragraph [K] or [L], wherein the enzyme is stable in acid conditions. [N] The formulation of paragraph [L] or [M], wherein the formulation is suitable for oral administration. [O] The formulation of any one of paragraphs [L]-[N], wherein the formulation comprises an enteric coating. [P] The formulation of any one of paragraphs [L]-[O] further comprises an effective dose of prolyl endopeptidase ranging from 0.01 mg to 500 mg/kg body weight. [Q] The use of the formulation of any one of paragraphs [L]-[P] for digesting gluten-containing food stuff. [R] The use of the formulation of paragraph [Q], wherein the formulation is administered within one hour of eating. [S] A method of detoxifying gluten, the method comprising contacting gluten-containing foodstuff with an effective dose of an isolated enzyme of any one of claims [A]-[J] or a formulation of any one of paragraphs [L]-[P]. [T] The method paragraph [S], wherein the contacting is performed in vitro prior to consumption of the gluten-containing food stuff. [U] The method of paragraph [S], wherein the contacting is performed in vivo by administration of the effective dose prior to, concurrent with or after consumption of the gluten-containing food stuff. [V] A method of treating Celiac Sprue, gluten allergy and/or dermatitis herpetiformis in a subject in need thereof, the method comprising administering to the subject an effective dose of an isolated enzyme of any one of paragraphs [A]-[J] or a formulation of any one of paragraphs [L]-[P], wherein gluten toxicity is attenuated in the subject. [W] The method of paragraph [V], wherein the effective dose is administered prior to consumption of gluten-containing foodstuff. [X] The method of paragraph [V], wherein the effective dose is administered in a gluten-containing foodstuff. [Y] The method of paragraph [V], wherein the effective dose is administered from 1 hour prior to 1 hour after the subject has consumed a gluten-containing foodstuff. [Z] The method of paragraph [V], wherein the effective dose is administered just before, during, or just after consumption of gluten-containing foodstuff. [AA] The method of any one of paragraphs [V], [W], [Y]-[Z], wherein the effective dose is administered orally. [BB] The method of paragraph [X], wherein the effective dose is admixed to the gluten-containing foodstuff. [CC] The method of any one of paragraphs [V]-[BB], wherein one determines that the subject has been diagnosed with Celiac Sprue, gluten allergy and/or dermatitis herpetiformis. [DD] An assay for diagnosing Celiac Sprue, gluten allergy and/or dermatitis herpetiformis in a subject comprising a) contacting a biological sample from the subject with a fixed amount of gliadin for a 24 hour period; b) measuring the amount of gliadin degradation; and c) comparing the amount of gliadin degradation for the biological sample with that obtained for a control assay, wherein the control assay is a mixture of a same fixed amount of gliadin with an isolated enzyme of paragraphs [A]-[J] or a formulation of paragraphs [L]-[P] for a 24 hour period, wherein the extent of gliadin degradation of less than 50% of that of the control assay indicates the subject likely have Celiac Sprue, gluten allergy and/or dermatitis herpetiformis. [EE] The method of paragraph [DD], wherein the subject is at risk of developing Celiac Sprue, gluten allergy and/or dermatitis herpetiformis. [FF] The method of paragraph [DD] or [EE], wherein the fixed amount of gliadin is 250 .mu.g/ml. [GG] The method of any one of paragraphs [DD]-[FF], wherein the determining is performed by protein gel electrolysis. [HH] The method of any one of paragraphs [DD]-[FF], wherein the determining is performed by mass spectrometry. [II] The method of any one of paragraphs [DD]-[HH], wherein the biological sample is whole saliva. [JJ] The method of paragraph [II], wherein the saliva is unstimulated saliva. [KK] The method of paragraph [II], wherein the saliva is stimulated saliva. [LL] The method of any one of paragraphs [DD]-[HH], wherein the biological sample is dental plaque, wherein the dental plaque is suspended in saliva ion buffer to an OD.sub.620 of .about.1.0 prior to mixing with the gliadin. [MM] A kit for predicting/diagnosing Celiac Sprue, gluten allergy and/or dermatitis herpetiformis in a subject in need thereof, comprising a gliadin and a reagent for assaying undigested gliadin containing a vial containing an isolated enzyme of paragraphs [A]-[J] or a formulation of paragraphs [L]-[P].

This invention is further illustrated by the following example which should not be construed as limiting. The contents of all references cited throughout this application, as well as the figures and table are incorporated herein by reference.

EXAMPLES

Materials and Methods

Collection of dental plaque and whole saliva samples--Prior to sample collection informed consent was obtained from the participating subject according to protocols approved by the Institutional Review Board at Boston University. The subject presented with good oral health without overt signs of gingival inflammation or other oral or systemic conditions. Supragingival plaque was collected from interproximal dental spaces with an explorer 24 h after refraining from oral hygiene. The plaque material was suspended in 500 .mu.l saliva ion buffer, the composition of which is 50 mM KCl, 1.5 mM potassium phosphate, 1 mM CaCl.sub.2 and 0.1 mM MgCl.sub.2, pH 7.0. Masticatory stimulated whole saliva (WS) (5 ml) was obtained by expectoration as described previously (Campese et al., 2009, Arch. Oral. Biol. 54:345-53).

Plating of oral microorganisms on Brucella-limited agar and gluten-limited agar--An aliquot of 50 .mu.l of 1:1000 diluted dental plaque or WS suspensions were plated on gluten-limited agar (GA), the formula for each liter is: 23 g wheat gluten (Sigma), 5 g sodium chloride, 1 g soluble starch, 12 g Agar No. 2, 0.4 g sodium bicarbonate, 1 g glucose, 1 g sodium pyruvate, 0.5 g cysteine hydrochloride monohydrate, 0.01 g haemin, 0.001 g vitamin K, 1 g L-arginine, 0.25 g soluble pyrophosphate and 0.5 g socium succinate. Incubations were carried out at 37.degree. C. under aerobic conditions or in a sealed pot that was rendered anaerobic using GASPAK.RTM. pouches (Beckton-Dickinson, Franklin Lakes, Md.). Individual colonies were transferred to GA plates, and after 48 h incubation were subcultured on Brucella agar (Hardy Diagnostics, Santa Maria, Calif.). Subculturing on Brucella agar plates was continued until cultures that were macroscopically and microscopically pure were obtained. The strains were then plated once more on GA to confirm growth on this selective agar formulation. For long term storage, bacteria were kept at -80.degree. C. in a glycerol/BHI broth mixture (20/80% v/v). To the stocks of anaerobic microorganisms DMSO was added to a final concentration of 5%.

Microbial speciation and identification by 16S rRNA--Microbial colonies with gliadin-degrading activity were identified by 16S rRNA analysis. DNA extraction was performed using the ULTRACLEANT.TM. Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) following the manufacturer's instructions for the isolation of genomic DNA from Gram-positive bacteria. Purified DNA was sequenced using an ABI prism cycle-sequencing kit (BIGDYE.RTM. Terminator Cycle Sequencing kit) on an ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, Calif.). Reactions used a quarter-dye chemistry as previously described (Paster et al. 2001, J. Bacteriol. 183:3770-83; Aas et al., 2005, J Clin. Microbiol. 43:5721-32). Partial sequences were identified by BLASTN analysis against the Human Oral Microbiome Database containing sequencing of over 35,000 clones and isolates. Sequences were assembled from the ABI electropherogram files using Sequencher 4.9 (Gene Codes Corporation, Ann Arbor, Mich.).

Degradation of paranitroanilide-derived substrates--Four gliadin-derived substrates, Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, were chemically synthesized (Anaspec, Fremont, Calif.) and dissolved in 50-75% dimethyl sulfoxide to 20 mM. The dental plaque suspension was diluted in saliva ion buffer to an OD.sub.620 of 1.2. Bacterial strains were grown on Brucella agar for 24 or 48 h, harvested with a cotton swab and suspended in saliva ion buffer to an OD.sub.620 of 1.2. An aliquot of 200 .mu.l of dental plaque or bacterial suspensions was added to 2 .mu.l of the paranitroanilide-derivatized substrates in a 96-well microliter plate (final concentration of substrates is 200 nM). Z-YPQ-pNA, Z-PPF-pNA and Z-PFP-pNA showed mild precipitation upon mixing with the plaque suspension in saliva ion buffer which did not interfere with efficient substrate hydrolysis. Enzyme activity was monitored spectrophotometrically at 405 nm. For some experiments, measurements were carried out in the kinetic mode. All values were corrected for the lowest absorbance values measured after addition of the enzyme source to the substrate.

Degradation of gliadins in-solution--A mixture of gliadins was purchased from SIGMA (Cat. No. G3375, St. Louis, Mo.) and dissolved to 5 mg/ml in 60% (v/v) ethanol Gliadins were added to suspensions of dental plaque and bacterial strains WSA-2B and WSA-8 (OD.sub.620=.about.1.0). Experimental incubation time points were 0, 2, 4, 6, 24 and 72 hr, or 0, 5, 15, 30, 60 and 120 min. After the indicated incubation time intervals, 100 .mu.l aliquots were removed and boiled to inactivate enzyme activity. EDTA was added to a final concentration of 2.5 mM, samples were dried using a speed-vac (Savant, Thermo Electron, Waltham, Mass.) and analyzed on pre-cast 12% gels (NOVEX, INVITROGEN, Carlsbad, Calif.). Electrophoresis, gel straining and destaining were carried out as described (Helmerhorst et al., 2010, PLoS One, in press).

Degradation of gliadins in-gel (gliadin zymography)--Bacteria were suspended in saliva ion buffer to a final OD.sub.620 of 5.0. A 150 .mu.l aliquot was centrifuged. The bacterial pellet was suspended in 20 .mu.l zymogram buffer and analyzed by gliadin zymography as described (Helmerhorst et al., 2010, PLoS One, in press). In some experiments the zymogram gel contained 6% instead of 8% acrylamide. With the lower percentage of acrylamide, a better separation of the enzymes in the 70 kD region was achieved. After electrophoresis was completed, the zymogram gel was further processed by incubation in renaturing and developing buffers (INVITROGEN, Carlsbad, Calif.). Enzymatic activities were revealed by staining with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 10% (v/v) acetic acid and 40% (v/v) methanol and destaining in the same solution not containing the dye.

Degradation of 33-mer and 26-mer gliadin domains and RP-HPLC analysis--Synthetic highly immunogenic peptides derived from .alpha.-gliadin (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; a 33-mer, SEQ. ID. NO: 2; Shan et al., 2002, Science 297(5590):2275-9) or .gamma.-gliadin (FLQPQQPFPQQPQQPYPQQPQQPFPQ; 26-mer, SEQ. ID. NO: 17; Shan et al., 2005, J Proteome Res. 4:1732-41) were synthesized at a purity of 95% (21st Century Biochemicals, Marlboro, Mass.). Both peptides were dissolved in milliQ water at 10 mg/ml, the concentration was verified by measurement of the OD at 215 nm (.epsilon.=20). The 33-mer or the 26-mer was added to a suspension of strains WSA-8 or Rothia mucilaginosa ATCC 25296 in saliva ion buffer (OD.sub.620 1.2). After t=0 h, 2 h and 5 h incubation, 100 .mu.l aliquots were removed and boiled to inactivate enzyme activity. The 100 .mu.l aliquots were mixed with 900 .mu.l buffer A containing 0.1% (v/v) trifluoroacetic acid (TFA), filtered over a 0.22 .mu.m filter (Pall Cooperation, Ann Arbor, Mich.). RP-HPLC was carried out using a HPLC Model 715 (Gilson, Middleton, Wis.) and a C-18 column (TSK-GEL 5 .mu.m, ODS-120T, 4.6.times.250 mm, TOSOHaas, Montgomeryville, Pa.). Peptides were eluted sing a linear gradient from 0% to 55% buffer B containing 80% (v/v) acetonitrile and 0.1% (v/v) TFA over a 75 min time interval at a flow rate of 1.0 ml/min (Helmerhorst et al., 2010, PLoS One, in press). The eluate was monitored at 219 and 230 nm and eluting fractions were collected using peak width and peak sensitivity settings of 1.2 and 5, respectively (Unipoint version 3.3, Gilson).

Mass Spectrometric Characterization of 33-mer/26-mer fragments using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS)--Mass spectrometry was conducted using a capillary nano-flow liquid chromatography and electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS) as previously described (Sun et al., 2009, Faseb J 23:2691-701). In brief, HPLC fractions containing individual gliadin degradation peptides were concentrated under vacuum and suspended in 5% acetonitrile in 0.1% formic acid. 1-3 .mu.l samples were injected using an autosampler (Micro AS, Thermo Finnigan, San Jose, Calif.). Separation/elution of peptides was achieved using an in-line capillary C-18 column (Magic C-18, Micron Bioresource) applying a gradient from 5 to 95% acetonitrile in 0.1% formic acid over a 35 min time interval at a flow rate of 250 n1/min.

The raw MS/MS data of the mixture of gliadins were searched against an in-house generated database containing the sequences of just these two peptides using SEQUEST software (Bioworks Browser 3.3.1, Thermo-Finnigan). X-corr values applied were 1.5, 2.2 and 3.5 for Z=1, 2, and 3, resp. DCn and peptide probabilities were set at >0.1 and <0.05, respectively. The suitability of the selected settings to avoid false positive identifications has been reported (Helmerhorst et al., 2010, PLoS One, in press).

Chromatographic separation of R. mucilaginosa enzymes--R. mucilaginosa ATCC 25296 cells were cultured from Brucella agar plates (Hardy Diagnostics, Santa Maria, Calif.) in 4 liter BHI for 24 h at 37.degree. C. while shaking. Cells were harvested and suspended in 50 mM Tris-HCl and 50 mM NaCl (pH 8.0) and concentrated to a final O.D. of 67 at 620 nm. Cells were sonicated for 20 times at a power setting of 7 using the Branson cell lysis sonifier the degree of lysis was monitored spectrophotometrically and sonication was terminated when the turbidity was reduced by 90%. The sonicate was centrifuged at 31,000.times.g for 20 min. The supernatant was removed and subjected to ammonium sulfate precipitation. The active fraction was found to be enriched in the precipitate obtained using 25-45% saturated ammonium sulfate. This precipitate was collected by centrifugation at 10,000.times.g for 20 min. The precipitate was dissolved, concentrated and desalted using centrifuge tubes with a 50 kD MW cut-off (MILLIPORE.RTM.). An aliquot of 670 mg protein was applied to a DEAE SEPHAROSE.RTM. Fast Flow column (GE Healthcare) of 2.6 cm.times.82.5 cm connected to an FPLC system (Pharmacia Biotech). Chromatographic separation of proteins was achieved at a flow rate of 0.7 ml/min and applying a gradient of 0-10% buffer B (50 mM Tris-HCl and 1M NaCl (pH 8.0) from 0 to 70 min; 10-35% buffer B from 70-2070 min, and 35-100% buffer B from 2070 to 2427 min. Fractions of 24 ml were collected and protease activities were measured by mixing 200 .mu.l of each fraction with 3 .mu.l Z-YPQ-pNA (final concentration 150 mM). Active fractions were desalted, concentrated and 6.5 mg of protein was loaded onto a G-100 gel filtration column (SEPHADEX.RTM. G-100, Pharmacia fine Chemical Piscataway, N.J.) of 2.6 cm.times.82.5 cm. Samples were eluted at a flow rate of 0.5 ml/min. Collected fractions with activity were concentrated as described above and subjected either to a 1-ml column of HITRAP QFF anion-exchange chromatography (GE Healthcare, City, State) or to a 1-ml column of HITRAP QXL anion-exchange chromatography (GE Healthcare). Fractions were again evaluated for activity, concentrated and analyzed for protein composition by SDS PAGE.

Gliadin zymography, in-gel digestion and LC-ESI-MS/MS characterization of enzymes--Active chromatographic fractions that showed a reduction in protein complexity compared to the starting material were subjected to gliadin zymography for activity analysis and to achieve further separation of proteins by electrophoresis. Active bands were excised with a scalpel on a clean glass plates and transferred to individual EPPENDORF tubes labeled (a) for the upper band and (b) for the lower band. From several repeat experiments, six upper and six lower band gel slices were separately processed. Proteins in the gel slices were digested in-gel with trypsin and analyzed by LC-ESI-MS/MS (Taplin Mass Spectrometry facility, Harvard Medical School, Boston, Mass.). Data were searched against a R. mucilaginosa database which was downloaded from NCBI using BIOWORKS software version 3.1. Peptide filter criteria applied were delta CN>0.1, peptide probability <0.5 and Xcorr values 2.2 and 3.5 for Z=2+ and Z=3+ for fully tryptic peptides and 2.4 and 3.75 for Z=2+ and Z=3+ for partially tryptic peptides.

Example 1

Some types of prolyl glutamine endopeptidase isolated from wheat, designate EP-B2 have been used in studies to decrease the propensity of gluten-containing wheat products to aggravate coeliac disease (Vora et al., Biotechnol Bioeng. 2007 Sep. 1; 98(1):177-85 and Gass et al., Gastroenterology. 2007 August; 133(2):472-80). The inventors have discovered that human whole saliva and dental plaque contain enzymatic activities that can cleave the Xaa-Pro-Gln (-XPQ-) bond after Gln, where Xaa is any amino acid, Pro is proline and Gln is glutamine (Helmerhorst et al., J. Biol. Chem. 29:19957-66, 2008). This tripeptide is also particularly abundant in known celiac T-cell gluten epitopes. Based on this, the inventors tested to determine whether the saliva-associated enzymes can degrade gluten/gliadins. This was confirmed experimentally by showing that plaque bacterial suspensions cleave gliadin. To isolate the microorganisms producing the gliadin-degrading enzymes, dental plaque was cultured on selective agar media containing only gluten as the protein source. Strains that can grow in this type of agar were further tested for their capacity to cleave gliadin-derived enzymatic substrates and gliadin in solution and in gel. Two microorganisms showing by far the highest gliadin-degrading activities were R. mucilaginosa ot 681 (WSA-2B=WSA-26) and Rothia species ot 188 (strain WSA-8) (see FIGS. 1, 2 and 3). Plaque consists of >600 different species (at the World Wide Web site of "homd" organization). The identification of Rothia species is significant, considering that these species are fairly uncommon in oral specimens as they rank approximately at #200 in order of abundance. Rothia showed preferential cleavage after the -XPQ- sequence (FIG. 4). The highly immunogenic 33-mer gliadin oligopeptide, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ. ID. NO: 2), contains eight potential cleavage sites of the Xaa-Pro-Gln (XPQ) type, namely one FPQ, four QPQ and three YPQ sites. Gliadin zymography was conducted to gain insight into the approximate molecular weight of the glutamine endopeptidase enzymes from WSA-2B and WSA-8. The enzymes produced by WSA-2B and WSA-8 differ slightly in molecular weight, but both appeared in the 70-75 kDa region (8% zymogram results presented in FIG. 5; 6% zymogram results presented in FIG. 10). A series of protease inhibitors were used to determine the class of the gliadin degrading enzymes. Complete inhibition of activity of the glutamine endopeptidase activity was achieved with PMSF and EDTA (FIG. 7) indicating that the enzymes are metal-ion dependent proteases.

The enzyme responsible for cleaving gliadin-derived enzymatic substrate can be extracted from whole unlysed bacteria. Clarified supernatant fluid of a suspension of whole bacteria exhibited the cleaving property. Increase cleaving activity can be obtained by lysing the bacteria, e.g., through sonication. These indicate that the enzyme is secreted into the periplasmic space and exterior as well as located in the interior of the bacterium cell or be membrane associated and released upon membrane disruption.

In addition, two commercially available Rothia species were tested for their capacity to cleave gliadin-derived enzymatic substrates. Both R. mucilaginosa ATCC 25296, Rothia dentocariosa ATCC 17931 were exhibited the cleaving property towards Z-YPQ-pNA (FIG. 9).

Given the efficient growth of Rothia species on gluten-limited agar, and their capacity to efficiently degrade gliadin, the exploitation of Rothia species and the enzymes produced by these microorganisms for therapeutic and diagnostic applications in celiac disease as well as gluten allergy in envisioned.

Example 2

Selection and 16S RNA speciation of gluten-degrading oral bacteria--Gluten is a collection of glutenins and gliadins of varying lengths and compositions. All gluten proteins are rich in glutamine and proline residues (Wieser, 2007, Food Microbial. 24:115-9). Experiments were conducted to explore if gluten-limited agar (GA) is suitable to select for oral microorganismsms capable of metabolizing gluten. Bacteria from dental plaque and whole saliva were plated on GA and subcultured on Brucella agar to purity. A total of 7 aerobic strains and 10 anaerobic strains were harvested applying the selective plating strategy (FIG. 1). The strains did not grow on control agar formulations that contained all the ingredients of the GA agar except wheat gluten (data not shown). The strains harvested from the oral specimens were unique in terms of their capacity to utilize gluten as a substrate. The 17 strains were identified by 16S rRNA analysis. The RNA typing results revealed that some of the strains were actually the same species. For instance, strains WSA-2B and WSA-26 were both typed as R. mucilaginosa of 681; strains WSAN-14, -16, and -24 were identified as Bifidobacterium longum; and strains PAN-5, -8, -18, and -19 were typed as Bifidobacterium dentium. Strain WSA-27 contained two species (contaminated) and strain PA-10 was a non-oral microorganism (contaminant). Both strains were excluded from further analysis.

Hydrolysis of paranitroanilide-derivatized substrates--In earlier work the inventors have demonstrated that human dental plaque bacteria cleave the synthetic substrates Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA (Helmerhorst et al., 2010, PLoS One, in press). The time span needed for the complete hydrolysis of all four substrates was 24 h. Cleavage of these four substrates by the 15 oral species as well as by mixed dental plaque were assessed (Table 4). Interestingly, none of the anaerobic strains cleaved any of the four substrates. On the other hand, in the aerobic category, R. mucilaginosa and Rothia ot188, but not the Streptococcus or Staphylococcus were particularly efficient in cleaving Z-YPQ-pNA. In contrast to Z-YPQ-pNA, the substrates Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA were not cleaved, not even upon prolonged incubation times. For two of the strains, WSA-2B (R. mucilaginosa) and WSA-8 (Rothia ot188) the precise time course of Z-YPQ-pNA cleavage was investigated and compared to the cleavage of another substrate of the XPQ type, namely Z-KPQ-pNA. Both substrates were hydrolyzed in a cell-density and time-dependent fashion (FIG. 4). At the highest cell densities evaluated (OD.sub.620 1.2) WSA-2B completely hydrolysed Z-YPQ-pNA and Z-KPQ-pNA after 6 h, whereas strain WSA-8 cleavage of these substrates was completed after 3 h and 1 h, respectively.

Gliadin degradation in solution--The inventors compared gliadin degradation by plaque bacteria and by strain WSA-8 (FIGS. 2 and 3). For a proper comparison, whole plaque and WSA-8 bacteria were suspended in saliva ion buffer to the same optical density (OD.sub.620 1.2). A mixture of gliadins (SIGMA) was added to the suspension to a final concentration of 250 .mu.g/ml. After incubation for various time intervals at 37.degree. C., 100 .mu.l aliquots were removed from the incubation mixture and boiled to inactivate the enzyme. SDS-PAGE analysis shows that the major protein in the gliadin preparation, exhibiting a molecular weight of approximately 37 kD, was susceptible to degradation, albeit at a fairly low rate, in mixed dental plaque (FIGS. 2 and 3). Gliadins were however highly susceptible to the proteases produced by strain WSA-8, as evidenced from the fact that within 2 h of incubation the added amount of gliadin (250 .mu.g/ml) was completely degraded. The precise time course for gliadin degradation by WSA-8 was established by sampling at shorter time intervals within the 2 h incubation time period (FIG. 4B). Data indicated that 50% of the added gliadin amount was degraded by WSA-8 in about 30 minutes. Similar results were obtained with gliadins incubated with strain WSA-2B (data not shown). These results demonstrate that Rothia species do not only cleave synthetic gliadin tripeptide substrates but are also highly effective in degrading gluten. Furthermore, their activities far exceed the activities present in mixed dental plaque.

Determination of enzyme molecular weight by gliadin zymography--The next series of experiments were designed to gain more insight into enzyme characteristics of the Rothia organisms. First, R. mucilaginosa strain 25296 was obtained from the American Type Culture Collection (ATCC) for comparison to the strains that were isolated from the oral cavity. Strains WSA-2B, -8-26 and the ATCC strain were subjected to gliadin zymography. In this study, a zymogram with 6% instead of 8% acrylamide was employed in the separating gel. With the lower percent acrylamide a better resolution of proteins in the 70 kD region was achieved. The zymogram that was developed at neutral pH showed that all strains express gliadin-degrading enzymes appearing as clear bands in the zymogram. As expected, the protease patterns of strains WSA-2B, WSA-26 and the R. mucilaginosa ATCC strain were quite similar, showing a major double band in the 70-75 kD region in addition to some activity in the higher molecular weight regions (FIG. 5 and FIG. 10). Strain WSA-8 displayed a single prominent protease band with an electrophoretic mobility around 70 kD (FIG. 5 and FIG. 10).

Inhibitor sensitivity of the gliadin-degrading enzymes--Strains WSA-2B and WSA-8 were chosen to investigate the sensitivity of the enzymes to a series of protease inhibitors. The inhibitors tested were EDTA and phophoamidon inhibiting metallo proteases, PMSF and AEBSF inhibiting serine proteases, aprotinin, an inhibitor of trypsin-like enzymes, 2-PDS being an inhibitor of cysteine proteases and pepstatin A inhibiting aspartyl proteases. Inhibitory effects were monitored toward the capacity to hydrolyze Z-YPQ-pNA. The initial rate of substrate proteolysis (Vi) was determined during the first 30 minute incubation time interval. From the slopes obtained in the absence and presence of inhibitors, it could be established that EDTA and PMSF were the most effective inhibitors, abolishing enzyme activity in both strains completely. AEBSF, a PMSF analog, was also effective yielding 97% inhibition in WSA-2B and 78% inhibition in WSA-8. The other inhibitors yielded <30% inhibition (FIG. 7). The inhibitory effect of PMSF toward gliadin degradation was confirmed in a gliadin zymogram, showing that the protease band is not detectable in cells that were pre-incubated with PMSF (FIG. 8). The inhibitory effect of EDTA signifies a strong metal ion requirement for the enzymes in question and the inhibition by PMSF and AEBSF classifies the gliadin-degrading enzymes as proteases.

pH activity analysis. To further investigate the pH range over which strain WSA-8 was active, we studied Z-YPQ-pNA substrate hydrolysis by WSA-8 cells suspended in 20 mM Tris ranging in pH from 2.0 to 10.0. The WSA-8 enzymes showed optimal activities at pH values >7.0, similar to the observations made with mixed dental plaques suspensions (Helmerhorst et al., 2010, PLoS One, in press). Substrate hydrolysis rates showed reductions at pH 6, 5 and 4 parallel with decreasing pH values. At pH 3.0 reactions proceeded at a very slow pace, but after 72 h, complete substrate hydrolysis was observed (FIG. 11). At pH 2.0, no activity was observed over the 72 h time span examined (FIG. 11).

Degradation of the 33-mer and 26-mer--Within the gliadin sequences, certain peptide regions are particularly immunogenic and resistant to degradation by human-derived digestive enzymes. These are a 33-mer peptide (also denoted as "superantigen"; Schuppan et al., 2009, Gastroenterology 137:1912-33; (Hausch et al., 2002, Am J Physiol Gastrointest Liver Physiol 283(4):G996-G1003) present in alpha-gliadins, and a 26-mer peptide from gamma gliadins (Shan et al., 2002, Science 297(5590):2275-9). Both peptides are rich in the XPQ sequences which are the prospective primary enzymatic targets. The 33-mer and the 26-mer were incubated (final concentrations 250 .mu.g/ml) with suspensions of WSA-8 and R. mucilaginosa (ATCC 25296). After 0, 2 h, and 5 h incubation, 100 .mu.l aliquots were removed, boiled and analyzed by RP-HPLC. The intact 33-mer peptide eluted after 66 min (FIG. 12A, peak off scale). In the presence of WSA-8 cells the 33-mer was proteolytically cleaved, as evidenced by disappearance of the peak at 66 min and appearance of degradation fragments. Complete degradation was observed between 2 h-5 h incubation. The degradation fragments were collected and structurally characterized by LC-ESI-MS/MS analysis. From the N- and C-termini of these superantigen peptides, the enzymatic cleavage site specificities could be derived. Consistent with our initial hypothesis, prominent cleavage was observed after QPQ.dwnarw. (three sites, indicated with the larger arrows, FIG. 12B). Interestingly, cleavage at XPQ.dwnarw.P did not occur indicating that a proline residue in the p1' position prevents enzyme recognition and/or effective cleavage activity by WSA-8 (FIG. 12B). A novel, recurring cleavage site specificity was noted after LPY.dwnarw. and this specific activity was confirmed with the enzymatic substrate Z-LPY-pNA (data not shown). Cleavage was furthermore observed after QPF.dwnarw. and after PFP.dwnarw. which was somewhat unexpected based on the inactivity of WSA-8 towards the synthetic enzyme substrates Z-PPF-pNA and Z-PFP-pNA (Table 4). It was noted that extension of the incubation time from 2 to 5 h led to the cleavage of fragments originally eluting in peaks 9 and 10 to smaller peptides eluting now in peaks 1 to 4 demonstrating extensive degradation of the 33-mer peptide by WSA-8.

Proteolytic fragmentation analysis of the 26-mer (FIG. 13) revealed cleavage activities after XPQ (the larger arrows, FIG. 13B) with only one of such tripeptide not cleaved in the N-terminal domain after Q5. QPY was cleaved possibly by the same enzyme(s) which recognizes LPY in the 33-mer. Seven additional cleavage sites were observed with varying cleavage site specificities (FIG. 13B).

Results of the proteolysis of the 33-mer and 26-mer by R. mucilaginosa ATCC 25296 are shown in FIGS. 14 and 15, respectively. As for WSA-8, XPQ and LPY were again identified as prominent protease target sites (FIGS. 14B and 15B). Interestingly, while WSA-8 was unable to cleave XPQ when proline occupied the P1' position, R. mucilaginosa was able to target XPQ.dwnarw.P as evidenced from three peptides resulting from this cleavage eluting in peak 3. Cleavage at these sites is significant since it is well known that proline in the p1' position frequently interferes with efficient degradation by proteolytic enzymes. This indicated that the enzymes produced by R. mucilaginosa exhibit unusually efficient cleavage capacities towards these gluten domains.

Isolation of the enzyme from R. mucilaginosa ATCC 25296--R. mucilaginous cells were sonicated and proteins of interest were partially isolated by step-wise salt precipitation with ammonium sulfate. FIG. 16 shows the DEAE chromatogram of the enzyme fraction partially enriched by ammonium sulfate precipitation and the protease activity in the DEAE eluate. The total protein pattern (dotted trace, measured at 214 nm) of the eluate is complex as expected and most of the peaks were off-scale. The proteolytic cleavage of z-YPQ-pNA was assessed in all fractions collected and indicated that enzymatic activity was present in peaks P-0, P-1 and P-2. The P-0 peak eluting in the void volume before the gradient with buffer B contained proteins not binding to the DEAE resin. Activity analysis indicated that three different sub-peaks were present in P-0 which were separately collected and designated P-0a, P-0b and P-0c. The peaks were turbid in solution, suggesting the presence of hydrophobic components or lipoproteins. This is not unexpected since the proteins originated from a cell sonicate. Peaks P1 and P2 eluted from the DEAE column within the gradient and should be considered anionic proteins. FIG. 17A shows the SDS PAGE and FIG. 17B shows the gliadin zymography of the collected DEAE peaks. Strong gliadin-degrading activity was associated with P-0b and P1 in the 70-75 kD region of the zymogram and bands with similar electrophoretic mobility were also present in the SDS gel (boxed). Interestingly, P2 did not show activity in the zymogram (repeatedly observed) and was not considered for further purification.

Fractions P-0b and P1 were concentrated and subjected to G-100 gel filtration chromatography. Unfortunately this step yielded only minimal further purification of individual proteins (data not shown). To improve purification the P-0b and P-1 fractions were then further subjected to HiTrap QQF and HiTrap QXL chromatographic resins which both represent strong anion-exchange resins. The NaCl concentration in buffer A was reduced to 25 mM to facilitate protease binding to the anion exchange column since no binding was observed to cation-exchange resins. Fractions collected from both columns were evaluated for Z-YPQ-pNA hydrolysis activity. Selected peaks were subsequently analyzed by SDS-PAGE (FIG. 17C) and gliadin zymography (FIG. 17D). The results show that this anion exchange separation yielded partially pure and enzyme enriched preparations. The specific activity in these fractions was >50 fold increased compared to the starting material applied to the DEAE column. The clear bands observed in the zymogram gel confirmed proteolytic activity towards gliadin. Various repeats of these experiments resulted in 6 experimental samples all showing a double band in the zymograms at 70 and 75 kD with proteolytic activity. The upper 75 kD band was designated (a) and the lower 70 kD band (b). Six upper and six lower bands were excised, digested in-gel with trypsin and analyzed by LC-ESI-MS/MS.

Identification of Neprilysin--Table 5 summarizes proteins that were identified by >2 peptides and exhibited molecular weight values between 50 and 80 kD. Indicated are the protein names, whether or not the protein is an enzyme (based on functional assignments listed at NCBI), the NCBI accession number, the calculated molecular weight based on the cumulative mass of the amino acid residues in the protein sequence, and the number of times the proteins was found in the six gel slices. When the sequences were carefully analyzed it was noted that oligopeptide-binding protein was highly homologous with extracellular solute-binding proteins. While repeatedly identified, these proteins belong to a superfamily of proteins that are functionally involved in oligopeptide transport. Most other proteins identified were likewise involved in cell metabolic processes unrelated to proteolytic activity. In fact, only one of the proteins identified, neprilysin, is a peptidase and hence a proteolytic enzyme. It exhibits a calculated molecular weight of 74 kD exactly matching the 70-75 kD weight range noted in the gels. The primary amino acid sequence of R. mucilaginosa neprilysin is shown in FIG. 18. It was identified both in band (a) and in band (b). Its appearances in both bands indicate different isoforms of the enzyme. In band (a) it was found only in 1 of the 6 gel samples, but by a very high number of peptides (9). In band (b) it was present in 2 of the 6 gel slices and identified by the presence of multiple peptides (7 peptides).

Neprilysin belongs to the peptidase M13 super family. M13 peptidases are well-studied metallo-proteases found in a wide range of organisms including mammals and bacteria. The metal ion dependency of this enzyme is consistent with the current observation that proteolytic activity towards gliadins is completely inhibited in the presence of EDTA (FIG. 7). In mammals M13 proteases participate in processes such as cardiovascular development, blood-pressure regulation, nervous control of respiration, and regulation of the function of neuropeptides in the central nervous system. In bacteria they may be used for digestion of milk. The present report provides evidence that neprilysin can play an additional role in the digestion of dietary gluten. This finding opens new avenues for the clinical exploitation of this enzyme in the treatment of celiac disease.

CONCLUSION

Gluten proteins are primarily found in barley and wheat and they cause celiac disease in genetically predisposed subjects. Gluten, by virtue of being rich in glutamine and proline residues, is notoriously difficult to digest by human digestive enzymes. Glutamine endoproteases and gliadin degrading activity were reported in human dental plaque. The present study was initiated to isolate the responsible microorganisms and to functionally and structurally characterize the gliadin degrading enzymes. The ultimate goal is to explore the therapeutic usefulness of such enzymes in the treatment of celiac disease.

Oral specimens containing a mixture of microorganisms were cultured and sub-cultured on Brucella agar and gluten-limited agar to identify and isolate gluten-degrading strains. Enzyme activities in pure bacterial suspensions was assessed by measuring proteolytic activities towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic, highly immunogenic, gliadin peptides (33-mer of .alpha.2-gliadin and 26-mer of .gamma.-gliadin). Gliadin zymography was utilized to obtain the approximate molecular weights of the enzyme(s), their pH activity range and inhibitor profiles. Bacteria of interest were speciated by 16S RNA analysis. Enzymes were purified from sonicated cell culture supernatants by DEAE anion-exchange chromatography, G-100 gel filtration chromatography and HiTrap anion-exchange chromatography. Enzyme activity in collected fractions was monitored using the synthetic peptide Z-YPQ-pNA and the overall protein profile was assessed by SDS-PAGE. Samples enriched in microbial enzymes were subjected to gliadin zymography, active bands were excised, trypsinized and analyzed by LC-ESI-MS/MS. Principal findings: Bacteria with strong gliadin degrading activities were identified as R. mucilaginosa and Rothia spp of 188. Cell suspensions degraded Z-YPQ-pNA but not Z-QQP-pNA, Z-PPF-pNA or Z-PFP-pNA. Importantly, Rothia cells cleaved the 33-mer and the 26-mer gliadin immunogenic domains which are otherwise indigestible by gastro-intestinal enzymes of mammalian origin. Analysis of the sites cleaved using peptide isolation by RP-HPLC and structural characterization byLC-ESI-MS/MS confirmed the recognition of the XPQ.dwnarw. sequence by both Rothia species. The sequence of XPX.dwnarw.P was recognized by R. mucilaginosa only. Another identified prominent cleavage site was after LPY.dwnarw.. Gliadin zymography yielded evidence for the presence of two major enzyme bands of .about.70 and .about.75 kD in R. mucilaginosa and one such band of .about.70 kD in Rothia ot188. The enzymes were active over a broad pH range (pH 3-10) as assessed by Z-YPQ-pNA hydrolysis, and optimal activities were observed at pH>7.0. The most efficient inhibitors of enzyme activity were EDTA and PMSF (100% inhibition). DEAE chromatographic separation of sonicated Rothia cell supernatant yielded peaks with enzyme activity in the void (P0) as well as in early-eluting peaks (P1 and P2). LC-ESI-MS/MS analysis of protease enriched fractions following excision from the zymogram yielded the identification of the enzyme neprilysin in both zymogram bands. The theoretical mass of neprilysin (74 kD) closely matches the experimental MW of the enzyme in the SDS and zymogram gels.

TABLE-US-00003 TABLE 1 Comparison of amino acid sequences of salivary basic proline-rich protein 2 (PRB2) from human saliva and wheat omega-5 gliadin protein from Triticum aestivum* (SEQ. ID. NO: 20 and 21 respectively in the order of appearance) >sp|P02812|PRB2_HUMAN Basic salivary proline-rich protein 2 OS = Homo sapiens GN = PRB2 PE = 1 SV = 3 MLLILLSVALLALSSAQNLNEDVSQEESPSLIAGNPQGAPPQGGNKPQGPPSPPGKPQGP PPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDKSRSPRSPPGKPQG PPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDNKSRSSRSPPGKP QGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDNKSQSARSPPG KPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKSQGPPPPGKPQGPPPQGGSKSRSSRSP PGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGGSKSRSAR SPPGKPQGPPQQEGNNPQGPPPPAGGNPQQPQAPPAGQPQGPPRPPQGGRPSRPPQ >tr|Q402I5|Q402I5_WHEAT Omega-5 gliadin OS = Triticum aestivum PE = 4 SV = 1 MKTFIIFVLLAMAMNIASASRLLSPRGKELHTPQEQFPQQQQFPQPQQFPQQQIPQQHQI PQQPQQFPQQQQFLQQQQIPQQQIPQQHQIPQQPQQFPQQQQFPQQHQSPQQQFPQQQFP QQKLPQQEFPQQQISQQPQQLPQQQQIPQQPQQFLQQQQFPQQQPPQQHQFPQQQLPQQQ QIPQQQQIPQQPQQIPQQQQIPQQPQQFPQQQFPQQQFPQQQFPQQEFPQQQQFPQQQIA RQPQQLPQQQQIPQQPQQFPQQQQFPQQQSPQQQQFPQQQFPQQQQLPQKQFPQPQQIPQ QQQIPQQPQQFPQQQFPQQQQFPQQQEFPQQQFPQQQFHQQQLPQQQFPQQQFPQQQFPQ QQQFPQQQQLTQQQFPRPQQSPEQQQFPQQQFPQQPPQQFPQQQFPIPYPPQQSEEPSPY QQYPQQQPSGSDVISISGL *XPQ sequences are highlighted in red and underlined.

TABLE-US-00004 TABLE 2 Characteristics of gliadins from Triticum aestivum and human salivary basic proline-rich proteins.sup.1 Protein # a.a. % Q % P %(Q + P) #XPQ .alpha./.beta.-gliadins 293 34 15 49 16-23 .gamma.-gliadins 290 31 16 47 2-39 .omega.5-gliadins 439 51 19 70 72 .omega.-gliadins 306 24 19 43 8-38 PRB1 393 16 37 53 47-48 PRB2 416 15 37 52 50 PRB3 309 14 35 49 20 PRB4 310 14 34 48 21 .sup.1Excluding fragments and clones, there were total of 11 sequences for alpha/beta-gliadin, 154 sequences for gamma-gliadins, 1 sequence for omega-5-gliadin and 3 sequences for omega-gliadins.

TABLE-US-00005 TABLE 3 Cleavage specificities of trypsin and of enzymes associated with dental plaque bacteria towards gliadins. Total # gliadin peptides Cleavage sites (% of total).sup.b Enzyme source observed.sup.a XXR/K XPQ XQP XFP XPF Other Pure Trypsin 71 40 2 0 1 3 25 Plaque 94 0 32 26 12 7 17 bacteria enzyme mixture .sup.aBy LC-ESI-MS/MS, based on Xcorr values of 2.2 and 3.5 for doubly and triply charged peptides, resp. .sup.bOnly C-terminal cleavages were considered. Note: prominent cleavage activity against XPQ and XQP in the gliadin sample incubated with plaque supernatant enzymes.

TABLE-US-00006 TABLE 4 Enzymatic characteristics of the selected aerobic and anaerobic strains growing on gluten agar. Growth on Oral Hydrolysis of gluten-based substrates.sup.b Aer/Anaer Strain ID/type.sup.a gluten agar Species YPQ QQP PPF PFP Aerobic WSA-2B = Rothia mucilaginosa ot 681 Y Y +++ -- -- -- WSA-7A = Streptococcus mitis ot 677 Y Y -- -- -- -- WSA-8 = Rothia sp. ot 188 Y Y +++ -- -- -- WSA-10 = Staphylococcus epidermis ot 601 Y Y -- -- -- -- WSA-26 = Rothia mucilaginosa ot 681 Y Y +++ -- -- -- Anaerobic WSAN-14 = Bifidobacterium longum ATCC 15697 Y Y -- -- -- -- WSAN-16 = Bifidobacterium longum ATCC 15697 Y Y -- -- -- -- WSAN-24 = Bifidobacterium longum ATCC 15697 Y Y -- -- -- -- WSAN-25 = Veilonella atypica ot 524 Y Y -- -- -- -- PAN-0 = Streptococcus pneumoniae ot 734 Y Y -- -- -- -- PAN-5 = Bifidobacterium dentium ot 588 Y Y -- -- -- -- PAN-8 = Bifidobacterium dentium ot 588 Y Y -- -- -- -- PAN-18 = Bifidobacterium dentium ot 588 Y Y -- -- -- -- PAN-19 = Bifidobacterium dentium ot 588 Y Y -- -- -- -- PAN-23 = Bifidobacterium dentium ot 588 Y Y -- -- -- -- Whole Plaque mixture Y Y +++ +++ +++ +++ Note: WSA-2B = WSA-26 = Rothia mucilaginosa. For subsequent experiments strains WSA-2B (Rothia mucilaginosa ot 681) and WSA-8 (Rothia sp. ot 188) were selected. .sup.aSpeciation carried out by 16S RNA analysis .sup.bFinal concentration of substrate: 200 .mu.M. Bacteria added in saliva ion buffer, OD = 1.2. Hydrolysis measured at 405 nm after 24 h incubation.

TABLE-US-00007 TABLE 5 Proteins with MW between 50 and 80 kD identified by .gtoreq.2 peptides in band (a) and band (b) by LC-ESI-MS/MS Max # Times peptides identified in Band Protein Enzyme ID/Acc# (kD) MW found 6 samples (a)~75 kD oligopeptide-binding protein [Rothia mucilaginosa ATCC 25296] * N ZP_05368706 62 7 3 *** L-lactate permease [Rothia mucilaginosa ATCC 25296] N ZP_05367274 56 2 1 PTS system, glucose subfamily, IIBCA component N ZP_05368708 72 2 1 [Rothia mucilaginosa ATCC 25296] cytochrome D ubiquinol oxidase subunit 1 N ZP_05367386 58 2 2 *** [Rothia mucilaginosa ATCC 25296] serine/threonine-protein kinase PknB [Rothia mucilaginosa N ZP_05367629 75 2 1 ATCC 25296] 2-oxoglutarate dehydrogenase, E2 component, dihydrolipoamide N ZP_05368146 55 5 4 *** succinyltransferase [Rothia mucilaginosa ATCC 25296] neprilysin [Rothia mucilaginosa ATCC 25296] ** Y ZP_05367591 74 9 1 extracellular solute-binding protein, family 5 [Rothia mucilaginosa N ZP_05368703 62.0538 2 1 ATCC 25296] * extracellular solute-binding protein, family 5 [Rothia mucilaginosa N ZP_05368704 61.911 2 1 ATCC 25296] * penicillin-binding protein [Rothia mucilaginosa ATCC 25296] N ZP_05367785 76 5 1 putative ABC transporter substrate-binding protein N ZP_05368162 75 2 1 [Rothia mucilaginosa ATCC 25296] sodium/proline symporter [Rothia mucilaginosa ATCC 25296] N ZP_05367828 52 2 1 (b)~70 kD ABC transporter, ATP-binding protein [Rothia mucilaginosa N ZP_05368390 67 3 1 ATCC 25296] penicillin-binding protein [Rothia mucilaginosa ATCC 25296] N ZP_05367785 77 11 3 *** PTS system, glucose subfamily, IIBCA component N ZP_05368708 72 3 1 [Rothia mucilaginosa ATCC 25296] iiabc fructose/xylitol-pts [Rothia mucilaginosa ATCC 25296] N ZP_05367712 68 10 2 *** putative ABC transporter transmembrane protein N ZP_05368162 69 5 1 [Rothia mucilaginosa ATCC 25296] cytochrome D ubiquinol oxidase subunit 1 [Rothia mucilaginosa N ZP_05367386 58 3 3 *** ATCC 25296] oligopeptide-binding protein [Rothia mucilaginosa ATCC 25296] * N ZP_05368706 62 8 3 *** 2-oxoglutarate dehydrogenase, E2 component, dihydrolipoamide N ZP_05368146 55 3 5 *** succinyltransferase [Rothia mucilaginosa ATCC 25296] ABC transporter, ATP-binding protein [Rothia mucilaginosa N ZP_05368611 62 2 1 ATCC 25296] oxidoreductase, molybdopterin binding [Rothia mucilaginosa N ZP_05367779 59 2 1 ATCC 25296] neprilysin [Rothia mucilaginosa ATCC 25296] ** Y ZP_05367591 74 7 2 *** sodium/proline symporter [Rothia mucilaginosa ATCC 25296] N ZP_05367828 52 2 1 extracellular solute-binding protein, family 5 [Rothia mucilaginosa N ZP_05368703 62.053 9 1 ATCC 25296] * extracellular solute-binding protein, family 5 [Rothia mucilaginosa N ZP_05368704 61.911 10 3 *** ATCC 25296] * putative ABC transporter substrate-binding protein N ZP_05368612 75 4 1 [Rothia mucilaginosa ATCC 25296] extracellular solute-binding protein, family 5 N ZP_05368701 61.766 9 1 [Rothia mucilaginosa ATCC 25296] * extracellular solute-binding protein, family 5 N ZP_05368702 62.938 3 2 *** [Rothia mucilaginosa ATCC 25296] * extracellular solute-binding protein, family 5 N ZP_05368705 62.0708 5 1 [Rothia mucilaginosa ATCC 25296] * * structurally related proteins ** peptidases *** proteins identified in more than 1 sample Note: Multiple proteins were identified in bands (a) and (b), likely because of the close proximity of both bands in the zymogram. The only enzyme identified was neprilysin.

TABLE-US-00008 TABLE 6 Protein searched: Neprilysin from Rothia mucilaginosa, ZP_05367591 (exact same aa) (same and alike Accession Percent aa) number Species Protein name identical Percent positives ZP_05367591 Rothia mucilaginosa neprilysin predicted 100 100 YP_003363565 Rothia mucilaginosa metalloendopeptidase 99 99 ZP_07073157 Rothia dentocariosa metalloendopeptidase PepO 76 87 ZP_06905919 Rothia dentocariosa metalloendopeptidase PepO 76 87 YP_003315199 Sanguibacter keddieii endothelin-converting enzyme 53 69 YP_003325693 Xylanimonas neprilysin 53 70 cellulosilytica YP_003636471 Cellulomonas flavigena neprilysin 52 67 ZP_06830706 Rhodococcus equi metalloendopeptidase PepO 52 66 ZP07359309 Actinomyces viscosus neprilysin 50 64

TABLE-US-00009 TABLE 7 Updated characteristics of gliadins from Triticum aestivum and human salivary basic proline-rich proteins.sup.1 Protein # a.a.sup.a % Q % P %(Q + P) #XPQ .alpha./.beta.-gliadins .sup. 288.sup.b 34 15 49 7-22 .gamma.-gliadins 276.sup.c 31 16 47 2-38 .omega.-gliadins .sup. 356.sup.d 24 19 43 8-72 PRB1 392 16 37 53 47 PRB2 416 15 37 52 50 PRB3 309 14 35 49 20 PRB4 310 14 34 48 21 .sup.aIncluding signal peptides .sup.b,c,dAverage number of amino acids in .alpha./.beta.-gliadins (58 entries), .gamma.-gliadins (110 entries), .omega.-gliadins (8 entries)

SEQUENCE LISTINGS

1

1361660PRTRothia mucilaginosa 1Met Thr Thr Asn Ser Gly Ile Thr Lys Glu Trp Val Asp Glu Thr Val1 5 10 15Lys Pro Gly Asp Asp Phe Phe Arg His Val Asn Gly Lys Trp Leu Ala 20 25 30Thr His Glu Ile Pro Ala Asp Arg Pro Lys Asp Gly Gly Leu Tyr Thr 35 40 45Leu Arg Asp Asn Ala Glu Lys His Val Arg Glu Leu Val Glu Lys Ile 50 55 60Ala Lys Glu Gln Pro Glu Ser Arg Ile Gly Ala Leu Tyr Asn Ser Phe65 70 75 80Met Asp Val Glu Lys Ile Glu Ala Asp Gly Leu Glu Pro Leu Leu Lys 85 90 95Glu Ile Ala Pro Ile Leu Asn Ser Ala Thr Pro Ser His Leu Ala Val 100 105 110Thr Leu Ala Leu Leu Ser Arg Ala Gly Leu Pro Gln Leu Phe Ala Trp 115 120 125Tyr Thr Ser Asn Asp Pro Lys Asp Pro Lys Asn Tyr Thr Phe Phe Leu 130 135 140Tyr Gln Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr Arg Glu Glu145 150 155 160Lys His Glu Ala Ala Cys Ala Ala Tyr Val Glu His Ile Ala Arg Met 165 170 175Phe Gln Leu Thr Gly Leu Ala Glu Gly Phe Gly Leu Thr Pro Glu Gln 180 185 190Ala Ala Gln Leu Val Phe Thr His Glu Ser Glu Leu Ala Arg Leu His 195 200 205Trp Asn Val Val Glu Asn Arg Asp Ala Glu Ala Thr Tyr Asn Pro Tyr 210 215 220Gln Ala Thr Glu Leu Asp Glu Lys Phe Pro Gly Phe Pro Phe Ser Gln225 230 235 240Trp Leu Leu Ala Leu Gly Ala Asp Pro Glu Thr Leu Gly Gln Val Ile 245 250 255Val Ala Gln Pro Ser Phe Phe Glu Gly Ala Ala Lys Leu Phe Thr Ser 260 265 270Ile Pro Leu Met Ser Trp Lys Leu Trp Ala Val Trp Thr Val Leu Arg 275 280 285Ser Arg Ala Pro Phe Met Tyr Asp Glu Leu Val Gln Glu Ser Phe Asn 290 295 300Phe Tyr Gly Lys Thr Leu Ser Gly Thr Gln Gln Ile Arg Glu Arg Trp305 310 315 320Lys Arg Gly Val Gly Ala Val Glu Lys Ala Leu Gly Glu Glu Ile Gly 325 330 335Gln Glu Tyr Val Ala Val His Phe Pro Pro Ser His Lys Glu Lys Met 340 345 350Leu Val Leu Val Gly Asn Leu Leu Glu Ala Tyr Arg Glu Ser Ile Glu 355 360 365Ser Leu Asp Trp Met Thr Glu Ala Thr Arg Gln Lys Ala Leu Glu Lys 370 375 380Leu Ser Lys Phe Val Thr Lys Ile Gly Tyr Pro Asp Lys Trp Arg Asp385 390 395 400Phe Ser Ala Leu Glu Leu Val Pro Gly Asp Leu Phe Glu Asn Leu Arg 405 410 415Arg Thr Gly Ala Phe Asp Ala Asp Trp Leu Ile Ala Arg Lys Gly Gln 420 425 430Pro Val Asp Lys Ala Glu Trp Leu Met Thr Pro Gln Thr Val Asn Ala 435 440 445Tyr Tyr Met Pro Pro Ala Asn Glu Ile Val Phe Pro Ala Ala Ile Leu 450 455 460Gln Pro Pro Tyr Phe Asn Pro Asp Ala Asp Asp Ala Ala Asn Tyr Gly465 470 475 480Asn Ile Gly Met Ile Ile Gly His Glu Ile Gly His Gly Phe Asp Asp 485 490 495Gln Gly Ser Arg Tyr Asp Gly Asp Gly Lys Leu Glu Ser Trp Trp Thr 500 505 510Glu Glu Asp Tyr Ala Lys Phe Lys Glu Arg Thr Ala Ala Leu Val Glu 515 520 525Gln Tyr Asn Ala Tyr Val Pro Val Gly Leu Asp Pro Lys Phe His Val 530 535 540Asn Gly Glu Leu Thr Leu Gly Glu Asn Ile Gly Asp Leu Ala Gly Met545 550 555 560Ser Ile Ala Leu Lys Ala Tyr Arg Leu Ala Leu Lys Lys Gln Gly Ile 565 570 575Glu Ser Leu Ala Asp Ala Pro Val Ile Asp Gly Met Thr Gly Ile Gln 580 585 590Arg Phe Phe Phe Ser Asn Ala Arg Gly Trp Cys Thr Lys Ser Arg Pro 595 600 605Gln His Ala Glu Val Met Ile Ser Val Asp Pro His Ser Pro Asp Glu 610 615 620Phe Arg Val Asn Gly Val Val Arg Asn Ile Asp Glu Phe Tyr Glu Ala625 630 635 640Phe Gly Val Ser Glu Gly Asp Ala Leu Tyr Leu Ala Pro Glu Glu Arg 645 650 655Val Arg Ile Trp 660233PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 2Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro1 5 10 15Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro 20 25 30Phe312PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 3Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr1 5 1049PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 4Pro Phe Pro Gln Pro Gln Leu Pro Tyr1 5514PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 5Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr1 5 1069PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 6Pro Gln Pro Gln Leu Pro Tyr Pro Gln1 5710PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 7Gln Pro Gln Gln Ser Phe Pro Gln Gln Gln1 5 1089PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 8Pro Gln Gln Ser Phe Pro Gln Gln Gln1 5912PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 9Gln Leu Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr1 5 101014PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 10Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Glu Leu Pro Tyr1 5 101110PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 11Gln Pro Gln Gln Ser Phe Pro Glu Gln Gln1 5 10129PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 12Ile Gln Pro Gln Gln Pro Ala Gln Leu1 5139PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 13Gln Gln Pro Gln Gln Pro Tyr Pro Gln1 5149PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 14Ser Gln Pro Gln Gln Gln Phe Pro Gln1 5159PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 15Gln Gln Pro Phe Pro Gln Gln Pro Gln1 5169PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 16Pro Phe Ser Gln Gln Gln Gln Pro Val1 51726PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 17Phe Leu Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Tyr1 5 10 15Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln 20 251813PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 18Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Gln Leu Pro1 5 101914PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 19Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Gln Pro Leu Pro1 5 1020416PRTHomo sapiens 20Met Leu Leu Ile Leu Leu Ser Val Ala Leu Leu Ala Leu Ser Ser Ala1 5 10 15Gln Asn Leu Asn Glu Asp Val Ser Gln Glu Glu Ser Pro Ser Leu Ile 20 25 30Ala Gly Asn Pro Gln Gly Ala Pro Pro Gln Gly Gly Asn Lys Pro Gln 35 40 45Gly Pro Pro Ser Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro Gln Gly 50 55 60Gly Asn Gln Pro Gln Gly Pro Pro Pro Pro Pro Gly Lys Pro Gln Gly65 70 75 80Pro Pro Pro Gln Gly Gly Asn Lys Pro Gln Gly Pro Pro Pro Pro Gly 85 90 95Lys Pro Gln Gly Pro Pro Pro Gln Gly Asp Lys Ser Arg Ser Pro Arg 100 105 110Ser Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro Gln Gly Gly Asn Gln 115 120 125Pro Gln Gly Pro Pro Pro Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro 130 135 140Gln Gly Gly Asn Lys Pro Gln Gly Pro Pro Pro Pro Gly Lys Pro Gln145 150 155 160Gly Pro Pro Pro Gln Gly Asp Asn Lys Ser Arg Ser Ser Arg Ser Pro 165 170 175Pro Gly Lys Pro Gln Gly Pro Pro Pro Gln Gly Gly Asn Gln Pro Gln 180 185 190Gly Pro Pro Pro Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro Gln Gly 195 200 205Gly Asn Lys Pro Gln Gly Pro Pro Pro Pro Gly Lys Pro Gln Gly Pro 210 215 220Pro Pro Gln Gly Asp Asn Lys Ser Gln Ser Ala Arg Ser Pro Pro Gly225 230 235 240Lys Pro Gln Gly Pro Pro Pro Gln Gly Gly Asn Gln Pro Gln Gly Pro 245 250 255Pro Pro Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro Gln Gly Gly Asn 260 265 270Lys Ser Gln Gly Pro Pro Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro 275 280 285Gln Gly Gly Ser Lys Ser Arg Ser Ser Arg Ser Pro Pro Gly Lys Pro 290 295 300Gln Gly Pro Pro Pro Gln Gly Gly Asn Gln Pro Gln Gly Pro Pro Pro305 310 315 320Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro Gln Gly Gly Asn Lys Pro 325 330 335Gln Gly Pro Pro Pro Pro Gly Lys Pro Gln Gly Pro Pro Pro Gln Gly 340 345 350Gly Ser Lys Ser Arg Ser Ala Arg Ser Pro Pro Gly Lys Pro Gln Gly 355 360 365Pro Pro Gln Gln Glu Gly Asn Asn Pro Gln Gly Pro Pro Pro Pro Ala 370 375 380Gly Gly Asn Pro Gln Gln Pro Gln Ala Pro Pro Ala Gly Gln Pro Gln385 390 395 400Gly Pro Pro Arg Pro Pro Gln Gly Gly Arg Pro Ser Arg Pro Pro Gln 405 410 41521439PRTTriticum aestivum 21Met Lys Thr Phe Ile Ile Phe Val Leu Leu Ala Met Ala Met Asn Ile1 5 10 15Ala Ser Ala Ser Arg Leu Leu Ser Pro Arg Gly Lys Glu Leu His Thr 20 25 30Pro Gln Glu Gln Phe Pro Gln Gln Gln Gln Phe Pro Gln Pro Gln Gln 35 40 45Phe Pro Gln Gln Gln Ile Pro Gln Gln His Gln Ile Pro Gln Gln Pro 50 55 60Gln Gln Phe Pro Gln Gln Gln Gln Phe Leu Gln Gln Gln Gln Ile Pro65 70 75 80Gln Gln Gln Ile Pro Gln Gln His Gln Ile Pro Gln Gln Pro Gln Gln 85 90 95Phe Pro Gln Gln Gln Gln Phe Pro Gln Gln His Gln Ser Pro Gln Gln 100 105 110Gln Phe Pro Gln Gln Gln Phe Pro Gln Gln Lys Leu Pro Gln Gln Glu 115 120 125Phe Pro Gln Gln Gln Ile Ser Gln Gln Pro Gln Gln Leu Pro Gln Gln 130 135 140Gln Gln Ile Pro Gln Gln Pro Gln Gln Phe Leu Gln Gln Gln Gln Phe145 150 155 160Pro Gln Gln Gln Pro Pro Gln Gln His Gln Phe Pro Gln Gln Gln Leu 165 170 175Pro Gln Gln Gln Gln Ile Pro Gln Gln Gln Gln Ile Pro Gln Gln Pro 180 185 190Gln Gln Ile Pro Gln Gln Gln Gln Ile Pro Gln Gln Pro Gln Gln Phe 195 200 205Pro Gln Gln Gln Phe Pro Gln Gln Gln Phe Pro Gln Gln Gln Phe Pro 210 215 220Gln Gln Glu Phe Pro Gln Gln Gln Gln Phe Pro Gln Gln Gln Ile Ala225 230 235 240Arg Gln Pro Gln Gln Leu Pro Gln Gln Gln Gln Ile Pro Gln Gln Pro 245 250 255Gln Gln Phe Pro Gln Gln Gln Gln Phe Pro Gln Gln Gln Ser Pro Gln 260 265 270Gln Gln Gln Phe Pro Gln Gln Gln Phe Pro Gln Gln Gln Gln Leu Pro 275 280 285Gln Lys Gln Phe Pro Gln Pro Gln Gln Ile Pro Gln Gln Gln Gln Ile 290 295 300Pro Gln Gln Pro Gln Gln Phe Pro Gln Gln Gln Phe Pro Gln Gln Gln305 310 315 320Gln Phe Pro Gln Gln Gln Glu Phe Pro Gln Gln Gln Phe Pro Gln Gln 325 330 335Gln Phe His Gln Gln Gln Leu Pro Gln Gln Gln Phe Pro Gln Gln Gln 340 345 350Phe Pro Gln Gln Gln Phe Pro Gln Gln Gln Gln Phe Pro Gln Gln Gln 355 360 365Gln Leu Thr Gln Gln Gln Phe Pro Arg Pro Gln Gln Ser Pro Glu Gln 370 375 380Gln Gln Phe Pro Gln Gln Gln Phe Pro Gln Gln Pro Pro Gln Gln Phe385 390 395 400Pro Gln Gln Gln Phe Pro Ile Pro Tyr Pro Pro Gln Gln Ser Glu Glu 405 410 415Pro Ser Pro Tyr Gln Gln Tyr Pro Gln Gln Gln Pro Ser Gly Ser Asp 420 425 430Val Ile Ser Ile Ser Gly Leu 435221983DNARothia mucilaginosa 22atgactacta actctggaat cactaaagaa tgggtggatg aaaccgtcaa gccgggcgac 60gatttcttcc gccacgtcaa cggcaagtgg cttgctaccc acgaaatccc ggcggaccgc 120cccaaggacg gcggcctgta caccctccgc gataacgcag agaagcacgt gcgtgagctg 180gtggagaaga tcgcgaagga gcagccggag tcccgcatcg gcgcgctgta caactccttc 240atggatgttg agaagattga ggcggacggc ctggaacctc tgctgaagga aatcgccccg 300attctgaact cggcaacccc ctcccacctg gctgtgacct tggcgctgct gtctcgtgcg 360ggtctgccgc agctgttcgc ctggtacacc agcaacgacc cgaaggaccc gaagaattac 420acgttcttcc tgtaccagtc gggcctgggt ctgccggatg aatcctacta ccgtgaagag 480aagcacgagg ctgcatgcgc ggcgtatgtt gagcatattg cccgcatgtt ccagctgacc 540ggtctggctg agggcttcgg tctcaccccg gagcaggcgg ctcagctggt gttcacccac 600gagtctgagc tggctcgtct gcactggaac gtcgtggaga accgcgacgc tgaggcgacc 660tacaacccgt accaggcgac cgagctggac gagaagttcc ccggcttccc gttctcgcag 720tggctgctgg ctctgggtgc tgacccggag accctgggtc aggttattgt ggctcagccg 780tccttctttg agggtgcggc gaagctgttc acctccatcc cgctgatgag ctggaagctg 840tgggctgtgt ggactgttct gcgttcgcgt gcgccgttca tgtacgacga gctggttcag 900gagagcttca acttctacgg caagaccctt tccggtactc agcagattcg tgagcgttgg 960aagcgcggcg tgggcgctgt cgagaaggct ctgggtgagg agattggcca ggagtacgta 1020gctgtgcact tcccgccctc gcacaaggag aagatgctgg ttctggtcgg caacctcctt 1080gaggcgtacc gcgagtctat tgagtcgctg gactggatga ctgaggcaac ccgtcagaag 1140gcgctggaga agctgtcgaa gttcgtcacc aagatcggtt accccgataa gtggcgtgac 1200ttctccgcgc tggagctcgt tcccggtgac ctgttcgaga acctgcgccg caccggtgcg 1260ttcgatgctg actggctgat tgcccgtaag ggtcagccgg tggataaggc ggagtggctg 1320atgactccgc agaccgtgaa cgcgtactac atgccgccgg cgaatgagat tgtgttcccg 1380gcagcgattc tgcagccgcc gtacttcaac ccggatgctg acgatgcggc gaactacggc 1440aatatcggca tgattattgg ccacgagatt ggtcacggtt ttgacgatca gggttcccgc 1500tatgacggtg acggcaagct ggagagctgg tggactgagg aggattacgc gaagttcaag 1560gagcgtaccg cagccctggt ggagcagtac aacgcgtacg ttccggtggg tctggacccg 1620aagttccacg tgaacggtga gctgactctg ggcgagaaca ttggcgacct ggctggcatg 1680tcgattgcgt tgaaggcgta ccgtctggct ttgaagaagc agggcattga gtcgctggct 1740gacgcgccgg tgattgacgg catgaccggt attcagcgtt tcttcttctc gaatgctcgc 1800ggctggtgca cgaagtcccg cccgcagcat gctgaggtga tgatttcggt ggatccgcat 1860tcgccggatg agttccgtgt gaacggtgtg gtgcgcaata ttgatgagtt ctatgaggcg 1920tttggcgtct ctgagggcga tgcactgtac ctggctccgg aggagcgcgt gcgcatctgg 1980tag 198323660PRTRothia mucilaginosa 23Met Thr Thr Asn Ser Gly Ile Thr Lys Glu Trp Val Asp Glu Thr Val1 5 10 15Lys Pro Gly Asp Asp Phe Phe Arg His Val Asn Gly Lys Trp Leu Ala 20 25 30Thr His Glu Ile Pro Ala Asp Arg Pro Lys Asp Gly Gly Leu Tyr Thr 35 40 45Leu Arg Asp Asn Ala Glu Lys His Val Arg Glu Leu Val Glu Lys Ile 50 55 60Ala Lys Glu Gln Pro Glu Ser Arg Ile Gly Ala Leu Tyr Asn Ser Phe65 70 75 80Met Asp Val Glu Lys Ile Glu Ala Asp Gly Leu Glu Pro Leu Leu Lys 85 90 95Glu Ile Ala Pro Ile Leu Asn Ser Ala Thr Pro Ser His Leu Ala Val 100 105 110Thr Leu Ala Leu Leu Ser Arg Ala Gly Leu Pro Gln Leu Phe Ala Trp 115 120 125Tyr Thr Ser Asn Asp Pro Lys Asp Pro Lys Asn Tyr Thr Phe Phe Leu 130 135 140Tyr Gln Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr Arg Glu Glu145 150 155 160Lys His Glu Ala Ala Cys Ala Ala Tyr Val Glu His Ile Ala Arg Met 165 170

175Phe Glu Leu Thr Gly Leu Ala Glu Gly Phe Gly Leu Thr Pro Glu Gln 180 185 190Ala Ala Gln Leu Val Phe Thr His Glu Ser Glu Leu Ala Arg Leu His 195 200 205Trp Asn Val Val Glu Asn Arg Asp Ala Glu Ala Thr Tyr Asn Pro Tyr 210 215 220Gln Ala Thr Glu Leu Asp Glu Lys Phe Pro Gly Phe Pro Phe Ser Gln225 230 235 240Trp Leu Leu Ala Leu Gly Ala Asp Pro Glu Thr Leu Gly Gln Val Ile 245 250 255Val Ala Gln Pro Ser Phe Phe Glu Gly Ala Ala Lys Leu Phe Thr Ser 260 265 270Ile Pro Leu Met Ser Trp Lys Leu Trp Ala Val Trp Thr Val Leu Arg 275 280 285Ser Arg Ala Pro Phe Met Tyr Asp Glu Leu Val Gln Glu Ser Phe Asn 290 295 300Phe Tyr Gly Lys Thr Leu Ser Gly Thr Gln Gln Ile Arg Glu Arg Trp305 310 315 320Lys Arg Gly Val Gly Ala Val Glu Lys Ala Leu Gly Glu Glu Ile Gly 325 330 335Gln Glu Tyr Val Ala Val His Phe Pro Pro Ser His Lys Glu Lys Met 340 345 350Leu Val Leu Val Gly Asn Leu Leu Glu Ala Tyr Arg Glu Ser Ile Glu 355 360 365Ser Leu Asp Trp Met Thr Glu Ala Thr Arg Gln Lys Ala Leu Glu Lys 370 375 380Leu Ser Lys Phe Val Thr Lys Ile Gly Tyr Pro Asp Lys Trp Arg Asp385 390 395 400Phe Ser Ala Leu Glu Leu Ala Pro Gly Asp Leu Phe Glu Asn Leu Arg 405 410 415Arg Thr Gly Ala Phe Asp Ala Asp Trp Leu Ile Ala Arg Lys Gly Gln 420 425 430Pro Val Asp Lys Ser Glu Trp Leu Met Thr Pro Gln Thr Val Asn Ala 435 440 445Tyr Tyr Met Pro Pro Ala Asn Glu Ile Val Phe Pro Ala Ala Ile Leu 450 455 460Gln Pro Pro Tyr Phe Asn Pro Asp Ala Asp Asp Ala Ala Asn Tyr Gly465 470 475 480Asn Ile Gly Met Ile Ile Gly His Glu Ile Gly His Gly Phe Asp Asp 485 490 495Gln Gly Ser Arg Tyr Asp Gly Asp Gly Lys Leu Glu Ser Trp Trp Thr 500 505 510Glu Glu Asp Tyr Ala Lys Phe Lys Glu Arg Thr Ser Ala Leu Val Glu 515 520 525Gln Tyr Asn Ala Tyr Val Pro Val Gly Leu Asp Pro Lys Phe His Val 530 535 540Asn Gly Glu Leu Thr Leu Gly Glu Asn Ile Gly Asp Leu Ala Gly Met545 550 555 560Ser Ile Ala Leu Lys Ala Tyr Cys Leu Ala Leu Lys Lys Gln Gly Ile 565 570 575Glu Ser Leu Ala Asp Ala Pro Val Ile Asp Gly Met Thr Gly Ile Gln 580 585 590Arg Phe Phe Phe Ser Asn Ala Arg Gly Trp Cys Thr Lys Ser Arg Pro 595 600 605Gln His Ala Glu Val Met Ile Ser Val Asp Pro His Ser Pro Asp Glu 610 615 620Phe Arg Val Asn Gly Val Val Arg Asn Ile Asp Glu Phe Tyr Glu Ala625 630 635 640Phe Gly Val Ser Glu Gly Asp Ala Leu Tyr Leu Ala Pro Glu Glu Arg 645 650 655Met Arg Ile Trp 66024663PRTRothia dentocariosa 24Met Asp Asn Ala Lys Asn Ser Gly Ile Thr Thr Glu Trp Val Asp Asn1 5 10 15Ala Val Lys Pro Gly Asp Asp Phe Phe Arg His Val Asn Gly Arg Trp 20 25 30Leu Asp Thr Tyr Glu Ile Pro Ala Asp Arg Ser Lys Asp Gly Gly Leu 35 40 45Tyr Thr Leu Arg Asp Asp Ala Glu Lys His Val Arg Glu Ile Val Glu 50 55 60Arg Ile Ala Lys Glu Gln Pro Glu Ser Arg Ile Gly Ala Leu Tyr Asn65 70 75 80Ser Phe Met Asp Thr Asp Lys Ile Glu Ser Asp Gly Leu Glu Pro Leu 85 90 95Met Arg Glu Val Ala Pro Ile Leu Asn Ala Ala Thr Ser Asp Gln Leu 100 105 110Ala Val Thr Leu Ala Leu Leu Ser Arg Ala Gly Leu Ala Gln Leu Leu 115 120 125Gly Trp Tyr Thr Ser Ile Asp Gln Lys Asp Pro Glu His Tyr Val Phe 130 135 140Tyr Leu Thr Gln Ala Gly Leu Gly Leu Pro Asp Glu Ala Tyr Tyr Arg145 150 155 160Glu Glu Lys Tyr Glu Gln Val Cys Ala Ala Tyr Ile Glu His Ile Ala 165 170 175Thr Met Phe Glu Leu Thr Gly Phe Ala Glu Ser Phe Asn Leu Thr Pro 180 185 190Met Glu Ala Ala Gln Leu Ile Phe Ser His Glu Thr Glu Ile Ala Ala 195 200 205His His Trp Asp Val Val Lys Asn Arg Asp Ala Glu Ala Lys Tyr Asn 210 215 220Pro Val Lys Ala Thr Glu Leu Asp Glu Lys Phe Pro Gly Phe Pro Leu225 230 235 240Gln Gln Trp Leu Leu Ala Leu Gly Ala Asp Pro Glu Glu Leu Gly Thr 245 250 255Val Ile Val Ser Gln Pro Ser Phe Leu Glu Gly Val Ala Asn Met Trp 260 265 270Gln Ser Thr Pro Leu Met Thr Trp Lys Leu Trp Ala Leu Trp Cys Ala 275 280 285Ile Arg Ser Arg Ala Pro Tyr Leu Pro Asp Ala Phe Val Gln Glu Asn 290 295 300Phe Asn Phe Tyr Gly Arg Thr Leu Ser Gly Thr Glu Glu Leu Arg Glu305 310 315 320Arg Trp Lys Arg Gly Val Ala Ala Val Glu Asn Ala Leu Asp Gln Glu 325 330 335Leu Gly Lys Glu Tyr Val Ala Val His Phe Pro Pro Glu His Lys Glu 340 345 350Lys Met Leu Lys Leu Val Asn Asn Leu Leu Glu Ala Tyr Arg Arg Ser 355 360 365Ile Thr Asn Leu Asp Trp Met Thr Glu Ala Thr Arg Glu Lys Ala Leu 370 375 380Glu Lys Leu Ser Lys Phe Val Thr Lys Ile Gly Tyr Pro Asp Glu Trp385 390 395 400Arg Asp Tyr Ser Lys Leu Thr Leu Val Pro Gly Asp Leu Phe Glu Asn 405 410 415Leu Arg Arg Thr Ala Ala Phe Asn Ser Asp Phe Met Ile Asp Arg Ala 420 425 430Gly Asp Pro Val Asp Lys Asn Glu Trp Leu Met Ser Pro Gln Thr Val 435 440 445Asn Ala Tyr Tyr Met Pro Pro Ala Asn Glu Ile Val Phe Pro Ala Ala 450 455 460Ile Leu Arg Pro Pro Phe Phe Asp Pro Glu Ala Asp Asp Ala Ala Asn465 470 475 480Tyr Gly Gly Ile Gly Met Val Ile Gly His Glu Ile Gly His Gly Phe 485 490 495Asp Asp Lys Gly Ala Leu Tyr Asp Gly Asp Gly Ala Leu Asn Asn Trp 500 505 510Trp Thr Glu Glu Asp Phe Ala Glu Phe Thr Lys Arg Thr Ser Ala Leu 515 520 525Val Gln Gln Tyr Asn Ala Tyr Thr Pro Ala Asn Leu Asp Pro Gln Lys 530 535 540Phe Gln Val Asn Gly Glu Leu Thr Leu Gly Glu Asn Ile Gly Asp Leu545 550 555 560Ser Gly Leu Ser Ile Ala Leu Arg Ala Tyr Glu Ile Ala Leu Ala Glu 565 570 575Gln Gly Ile Thr Ser Leu Glu Asp Ala Pro Val Ile Asp Asp Met Thr 580 585 590Ala Ala Gln Arg Leu Phe Trp Ser Thr Ala Gln Gly Trp Arg Thr Lys 595 600 605Ser Arg Pro Gln His Ala Glu Met Met Ile Ser Val Asp Pro His Ser 610 615 620Pro Asp Glu Phe Arg Val Asn Gly Val Val Arg Asn Ile Asp Glu Phe625 630 635 640Tyr Asp Ala Phe Asp Val Pro Glu Gly Ser Lys Leu Tyr Leu Ala Pro 645 650 655Glu Asp Arg Val Arg Ile Trp 66025663PRTRothia dentocariosa 25Met Asp Asn Ala Lys Asn Ser Gly Ile Thr Thr Glu Trp Val Asp Asn1 5 10 15Ala Val Lys Pro Gly Asp Asp Phe Phe Arg His Val Asn Gly Arg Trp 20 25 30Leu Asp Thr Tyr Glu Ile Pro Ala Asp Arg Ser Lys Asp Gly Gly Leu 35 40 45Tyr Thr Leu Arg Asp Asp Ala Glu Lys His Val Arg Glu Ile Val Glu 50 55 60Arg Ile Ala Lys Glu Gln Pro Glu Ser Arg Ile Gly Ala Leu Tyr Asn65 70 75 80Ser Phe Met Asp Thr Asp Lys Ile Glu Ser Asp Gly Leu Glu Pro Leu 85 90 95Met Arg Glu Val Ala Pro Ile Leu Asn Ala Ala Thr Ser Glu Gln Leu 100 105 110Ala Val Thr Leu Ala Leu Leu Ser Arg Ala Gly Leu Ala Gln Leu Leu 115 120 125Gly Trp Tyr Thr Ser Ile Asp Gln Lys Asp Pro Glu His Tyr Val Phe 130 135 140Tyr Leu Thr Gln Ala Gly Leu Gly Leu Pro Asp Glu Ala Tyr Tyr Arg145 150 155 160Glu Glu Lys Tyr Glu Gln Val Cys Ala Ala Tyr Ile Glu His Ile Ala 165 170 175Thr Met Phe Glu Leu Thr Gly Leu Ala Glu Ser Phe Asn Leu Thr Pro 180 185 190Met Glu Ala Ala Gln Leu Ile Phe Ser His Glu Thr Glu Ile Ala Ala 195 200 205His His Trp Asp Val Val Lys Asn Arg Asp Ala Glu Ala Lys Tyr Asn 210 215 220Pro Val Lys Ala Pro Glu Leu Asp Glu Lys Phe Pro Gly Phe Pro Leu225 230 235 240Gln Gln Trp Leu Leu Ala Leu Gly Ala Asp Pro Lys Glu Leu Gly Thr 245 250 255Val Ile Val Ser Gln Pro Ser Phe Leu Glu Gly Val Ala Ser Met Trp 260 265 270Gln Ser Thr Pro Leu Met Thr Trp Lys Leu Trp Ala Leu Trp Cys Ala 275 280 285Ile Arg Ser Arg Ala Pro Tyr Leu Pro Asp Ala Phe Val Gln Glu Asn 290 295 300Phe Asn Phe Tyr Gly Arg Thr Leu Ser Gly Thr Glu Glu Leu Arg Glu305 310 315 320Arg Trp Lys Arg Gly Val Ala Ala Val Glu Asn Ala Leu Asp Gln Glu 325 330 335Leu Gly Lys Glu Tyr Val Ala Val His Phe Pro Pro Glu His Lys Glu 340 345 350Lys Met Leu Lys Leu Val Asn Asn Leu Leu Glu Ala Tyr Arg Arg Ser 355 360 365Ile Thr Asn Leu Asp Trp Met Thr Glu Ala Thr Arg Glu Lys Ala Leu 370 375 380Glu Lys Leu Ser Lys Phe Val Thr Lys Ile Gly Tyr Pro Asp Glu Trp385 390 395 400Arg Asp Tyr Ser Lys Leu Thr Leu Val Pro Gly Asp Leu Phe Glu Asn 405 410 415Leu Arg Arg Ala Ala Ala Phe Asn Ser Asp Phe Met Ile Asp Arg Ala 420 425 430Gly Asp Pro Val Asp Lys Asn Glu Trp Leu Met Ser Pro Gln Thr Val 435 440 445Asn Ala Tyr Tyr Met Pro Pro Ala Asn Glu Ile Val Phe Pro Ala Ala 450 455 460Ile Leu Arg Pro Pro Phe Phe Asp Pro Glu Ala Asp Asp Ala Ala Asn465 470 475 480Tyr Gly Gly Ile Gly Met Val Ile Gly His Glu Ile Gly His Gly Phe 485 490 495Asp Asp Lys Gly Ala Leu Tyr Asp Gly Asp Gly Ala Leu Asn Asn Trp 500 505 510Trp Thr Glu Glu Asp Phe Ala Glu Phe Thr Lys Arg Thr Ser Ala Leu 515 520 525Val Gln Gln Tyr Asn Ala Tyr Thr Pro Ala Asn Leu Asp Pro Gln Lys 530 535 540Phe Arg Val Asn Gly Glu Leu Thr Leu Gly Glu Asn Ile Gly Asp Leu545 550 555 560Ser Gly Leu Ser Ile Ala Leu Arg Ala Tyr Glu Ile Ala Leu Ala Glu 565 570 575Gln Gly Ile Thr Ser Leu Glu Asp Ala Pro Val Ile Asp Gly Met Thr 580 585 590Ala Ala Gln Arg Leu Phe Trp Ser Thr Ala Gln Gly Trp Arg Thr Lys 595 600 605Ser Arg Pro Gln His Ala Glu Met Met Ile Ser Val Asp Pro His Ser 610 615 620Pro Asp Glu Phe Arg Val Asn Gly Val Val Arg Asn Ile Asp Glu Phe625 630 635 640Tyr Asp Ala Phe Asp Val Pro Glu Gly Ser Lys Leu Tyr Leu Ala Pro 645 650 655Glu Asp Arg Val Arg Ile Trp 66026673PRTSanguibacter keddieii 26Met Thr Thr Asn Glu Lys Asn Ala Ala Gln Glu His Asp Leu Arg Pro1 5 10 15Gly Thr Thr Ser Gly Ile Asp Val Ser Ala Leu Asp His Asp Val Arg 20 25 30Pro Gln Asp Asp Leu Tyr Arg His Val Asn Gly Arg Trp Ile Ala Ser 35 40 45His Val Ile Pro Ala Asp Arg Ala Ser Asp Gly Gly Phe Arg Arg Leu 50 55 60His Asp Glu Ala Glu Ala His Val Arg Gln Ile Ile Glu Glu Leu Gly65 70 75 80Gly Arg Asp Asp Leu Ser Gly Glu Glu Ala Gln Ile Gly Ala Leu Tyr 85 90 95Ala Ser Phe Met Asp Thr Ala Thr Val Glu Ala Ala Gly Val Asp Pro 100 105 110Leu Ala Gln Asp Leu Ala Leu Val Ser Ser Ala Ser Thr Gln Ala Glu 115 120 125Leu Thr Gly Ser Leu Gly Ala Leu Gln Arg Thr Gly Gly Ala Gly Ala 130 135 140Phe Gly Phe Trp Val Asp Asn Asp Ala Lys Asp Pro Glu Lys Tyr Val145 150 155 160Val His Leu Tyr Gln Ala Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr 165 170 175Arg Glu Asp Lys Tyr Ala Glu Thr Arg Glu Ala Tyr Val Ala His Val 180 185 190Ala Arg Met Leu Glu Leu Ser Gly Thr Val Ala Ala Glu Ala Ser Ala 195 200 205Asp Ala Ala Ala Arg Val Leu Ala Leu Glu Thr Arg Leu Ala Thr His 210 215 220His Trp Asp Val Val Arg Asp Arg Asp Ser Glu Leu Thr Tyr Asn Pro225 230 235 240Thr Thr Leu Ala Ala Leu Ala Glu Thr Ala Pro Gly Phe Asp Trp Arg 245 250 255Ala Trp Ala Val Ala Leu Gly Ala Pro Glu Gly Ala Leu Asp Asp Val 260 265 270Val Val Arg Glu Pro Asp Phe Phe Thr Gly Phe Ala Ser Leu Trp Thr 275 280 285Ser Glu Pro Leu Glu Asp Trp Lys Thr Trp Met Val Tyr His Leu Val 290 295 300Ser Ala Arg Ala Pro Tyr Leu Thr Asp Glu Leu Val Glu Ala Asn Phe305 310 315 320Asp Phe Tyr Gly Arg Thr Leu Ser Gly Thr Gln Glu Val Arg Glu Arg 325 330 335Trp Lys Arg Gly Val Ser Leu Val Glu Gly Ala Leu Gly Glu Ala Val 340 345 350Gly Lys Val Tyr Val Asp Arg His Phe Pro Pro Thr His Lys Ser Arg 355 360 365Met Asp Val Leu Val Ala Asn Leu Ile Glu Ala Tyr Arg Gln Ser Ile 370 375 380Thr Gln Leu Asp Trp Met Gly Gln Glu Thr Lys Thr Lys Ala Leu Ala385 390 395 400Lys Leu Ser Ala Phe Thr Pro Lys Val Gly Tyr Pro Val Arg Trp Arg 405 410 415Asp Tyr Ser Ala Leu Val Val Asp Ala Gly Asp Leu Val Gly Asn Val 420 425 430Arg Arg Ser Asn Ser Val Asp Leu Asp Arg Glu Leu Ala Lys Ile Gly 435 440 445Gln Pro Ile Asp Arg Asp Glu Trp His Met Thr Pro Gln Thr Val Asn 450 455 460Ala Tyr Tyr Asn Pro Ser Met Asn Glu Ile Val Phe Pro Ala Ala Ile465 470 475 480Leu Arg Pro Pro Phe Phe Asp Pro Glu Ala Glu Asp Ala Ala Asn Tyr 485 490 495Gly Gly Ile Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp 500 505 510Asp Gln Gly Ser Lys Tyr Asp Gly Ala Gly Arg Leu Glu Asp Trp Trp 515 520 525Thr Gly Ser Asp Arg Ala Glu Phe Glu Lys Arg Thr Ala Ala Leu Ile 530 535 540Glu Gln Tyr Asp Ala Phe His Pro Ala Gln Leu Asp Ala Asp Arg Thr545 550 555 560Val Asn Gly Ala Leu Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly Gly 565 570 575Leu Ser Ile Ala Leu Lys Ala Tyr Glu Ile Ser Leu Gly Thr Asp Leu 580 585 590Ser Ala Ala Pro Ser Val Asp Gly Leu Ser Gly Thr Glu Arg Val Phe 595 600 605Leu Ser Trp Ala Gln Val Trp Gln Thr Thr Met Arg Asp Glu Ala Met 610 615 620Val Gln Arg Leu Ala Thr Asp Pro His Ser Pro Ala Glu Phe Arg Cys625 630 635 640Asn Gly Val Val Ser Asn Leu Asp Glu Phe His Thr Thr Tyr Asn Val 645 650

655Gln Pro Gly Asp Ala Leu Tyr Leu Ala Pro Glu Asp Arg Val Arg Ile 660 665 670Trp27671PRTXylanimonas cellulosilytica 27Met Thr Ile Glu Ala Leu Arg Ser Gly Ile Asp Leu Thr Ala Leu Asp1 5 10 15Pro Ala Thr Arg Pro Gln Asp Asp Leu Tyr Arg His Val Asn Gly Thr 20 25 30Trp Ile Ala Thr His Glu Ile Pro Ala Asp Arg Ser Met Asp Gly Ala 35 40 45Phe Met Lys Leu Arg Asp Leu Ser Glu Glu Arg Val Arg Ala Ile Ile 50 55 60Thr Asp Leu Ala Gly Ser Ser Asp Thr Glu Pro Gly Thr Thr Ala Gly65 70 75 80Lys Ile Gly Asp Leu Tyr Ser Ser Phe Met Asp Thr Ala Arg Ile Ala 85 90 95Ala Leu Gly Ala Thr Pro Leu Ala Ala Asp Leu Ala Leu Ile Ala Gly 100 105 110Ala Thr Ser Gln Ala Glu Leu Thr Gly Val Leu Gly Ala Leu Gln Arg 115 120 125Thr Gly Gly Gly Gly Ala Val Gly Phe Tyr Val Asp Asn Asp Ala Ala 130 135 140Asp Pro Glu Gln Tyr Arg Val Tyr Leu Cys Gln Ser Gly Leu Gly Leu145 150 155 160Pro Asp Glu Ala Tyr Tyr Arg Glu Asp Gln Tyr Ala Pro Ile Arg Glu 165 170 175Lys Tyr Val Pro His Val Ala Arg Met Leu Arg Leu Gly Gly Ala Ala 180 185 190Glu Leu Leu Gly Val Asp Ala Asp Asp Ala Ala Ala Arg Val Val Ala 195 200 205Leu Glu Ser Lys Leu Ala Gly His His Trp Asp Val Val Lys Asp Arg 210 215 220Asp Ala Thr Leu Thr Tyr Asn Pro Thr Thr Ile Ala Glu Leu Ala Val225 230 235 240Thr Ala Pro Gly Phe Asp Trp Gln Ala Trp Ala Ile Ala Leu Gly Ala 245 250 255Pro Thr Gly Ala Leu Asp Ala Leu Val Val Arg Glu Pro Ser Tyr Ala 260 265 270Glu Gly Leu Ala Ala Leu Trp Gln Ser Glu Pro Leu Glu Asp Trp Gln 275 280 285Val Trp Ala Ala Tyr Arg Leu Val Thr Ala Arg Ala Pro Tyr Leu Thr 290 295 300Asp Glu Ile Val Glu Ala Asn Phe Asp Phe Tyr Gly Arg Thr Leu Ser305 310 315 320Gly Ala Gln Glu Val Arg Glu Arg Trp Lys Arg Gly Val Ala Leu Val 325 330 335Glu Gly Ala Leu Gly Glu Ala Val Gly Glu Gln Tyr Val Ala Arg His 340 345 350Phe Pro Pro Ala His Lys Ala Arg Met Asp Glu Leu Val Ala Asn Leu 355 360 365Val Ala Ala Tyr Arg Glu Ser Ile Glu Gly Leu Asp Trp Met Thr Asp 370 375 380Glu Thr Lys Ala Lys Ala Leu Ala Lys Leu Glu Lys Phe Thr Pro Lys385 390 395 400Ile Gly Tyr Pro Val Lys Trp Arg Asp Tyr Ser Ala Leu Val Ile Asp 405 410 415Ala Asp Asp Leu Val Gly Asn Val Arg Arg Ser Asn Ala Phe Asp Leu 420 425 430Asp His Glu Leu Gly Lys Val Gly Lys Pro Leu Asp Arg Asp Glu Trp 435 440 445Phe Met Thr Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Gly Met Asn 450 455 460Glu Ile Val Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp Pro465 470 475 480Glu Ala Asp Asp Ala Val Asn Tyr Gly Gly Ile Gly Gly Val Ile Gly 485 490 495His Glu Ile Gly His Gly Phe Asp Asp Gln Gly Ser Lys Tyr Asp Gly 500 505 510Asp Gly Arg Leu Gln Asp Trp Trp Thr Ala Glu Asp Arg Ala Glu Phe 515 520 525Glu Lys Arg Thr Gly Ala Leu Ile Ala Gln Tyr Asp Ala Phe Val Pro 530 535 540Glu Gln Leu Gly Ala Asp Gly Ala His Val Asn Gly Ser Leu Thr Ile545 550 555 560Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile Ala Ile Arg Ala 565 570 575Tyr Arg Ile Ala Leu Ala Ala Ala Arg Glu Gly Ala Thr Leu Asp Asp 580 585 590Ala Pro Val Ile Asp Gly Leu Thr Gly Leu Glu Arg Val Phe Leu Gly 595 600 605Trp Ala Gln Val Trp Gln Ser Lys Gly Arg Asp Glu Glu Val Leu Arg 610 615 620Arg Leu Ala Thr Asp Pro His Ser Pro Asn Glu Phe Arg Cys Asn Gly625 630 635 640Ile Val Arg Asn Val Asp Glu Phe His Asp Ala Tyr Gly Val Arg Glu 645 650 655Gly Asp Ala Leu Trp Leu Ala Pro Glu Asp Arg Val Arg Ile Trp 660 665 67028662PRTCellulomonas flavigena 28Met Thr Arg Ser Gly Val Pro Leu Asp Asp Leu Asp Pro Ser Val Arg1 5 10 15Pro Gln Asp Asp Leu Asp Leu Phe Val Asn Gly Arg Trp Ala Ala Ser 20 25 30Tyr Val Ile Pro Pro Asp Arg Ser Met Asp Gly Pro Phe Arg Ala Leu 35 40 45Tyr Asp Glu Ala Glu Arg Gln Val Leu Asp Ile Ile Thr Asp Ala Ala 50 55 60Gln Ala Ala Gly Glu Gly Asp Gly Val Glu Ala Lys Ile Gly Ala Leu65 70 75 80Tyr Ala Ser Phe Met Asp Thr Asp Ala Val Arg Ala Ala Gly Val Glu 85 90 95Pro Leu Arg Glu Asp Leu Ala Leu Val Asp Ala Ala Thr Thr Pro Ala 100 105 110Glu Leu Thr Val Ala Val Gly Arg Leu Gln Arg Thr Gly Ala Leu Ser 115 120 125Ala Val Asp Leu Tyr Val Asp Asn Asp Ala Lys Asp Pro Asp Ser Tyr 130 135 140Val Val His Leu Val Gln Gly Gly Leu Gly Leu Pro Asp Glu Ala Tyr145 150 155 160Tyr Arg Glu Glu Gln His Ala Ala Val Arg Glu Lys Tyr Leu Pro His 165 170 175Val Ala Arg Met Leu Arg Leu Ala Ala Pro Val Ser Gly Val Val Ala 180 185 190Ala Gly Asp Ala Asp Asp Leu Ala Ala Arg Val Val Ala Leu Glu Ser 195 200 205Arg Ile Ala Ala His His Trp Asp Val Val Lys Asp Arg Asp Ala Glu 210 215 220Leu Thr Tyr Asn Ala Leu Thr Leu Ala Glu Leu Ala Ala Arg Ala Pro225 230 235 240Gly Phe Asp Trp Arg Ala Trp Ala Glu Ala Leu Gly Ala Pro Ala Gly 245 250 255Ala Leu Asp Arg Leu Val Val Arg Glu Pro Ser Phe Ala Glu Gly Leu 260 265 270Ala Ala Leu Trp Thr Glu Val Pro Val Ala Asp Trp Gln Ala Trp Ala 275 280 285Thr Tyr His Val Val Ser Ser Arg Ala Pro Tyr Leu Thr Asp Glu Leu 290 295 300Val Glu Ala Asn Phe Asp Phe Tyr Gly Arg Thr Leu Ser Gly Ala Pro305 310 315 320Glu Leu Arg Asp Arg Trp Lys Arg Gly Val Ser Leu Val Gln Gly Ala 325 330 335Leu Gly Glu Ala Val Gly Lys Val Tyr Val Glu Arg His Phe Pro Pro 340 345 350Ser His Lys Glu Arg Met Asp Glu Leu Val Ala Asn Leu Val Glu Ala 355 360 365Tyr Arg Arg Ser Ile Thr Glu Leu Glu Trp Met Gly Glu Glu Thr Arg 370 375 380Gln Arg Ala Leu Glu Lys Leu Ala Arg Phe Thr Pro Lys Ile Gly Tyr385 390 395 400Pro Ala Arg Trp Arg Asp Tyr Ser Ala Leu Glu Val Arg Ala Asp Asp 405 410 415Leu Val Gly Asn Val Arg Arg Ser Asn Ala Phe Asp Leu Asp Arg Glu 420 425 430Leu Gly Lys Ile Gly Arg Pro Ile Asp Arg Asp Glu Trp Phe Met Thr 435 440 445Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Gly Met Asn Glu Ile Val 450 455 460Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp Ala Glu Ala Asp465 470 475 480Asp Ala Ala Asn Tyr Gly Gly Ile Gly Ala Val Ile Gly His Glu Ile 485 490 495Gly His Gly Phe Asp Asp Gln Gly Ser Lys Tyr Asp Gly Asp Gly Arg 500 505 510Leu Val Asp Trp Trp Thr Ala Glu Asp Arg Ala Glu Phe Glu Arg Arg 515 520 525Thr Lys Ser Leu Val Asp Gln Tyr Ala Gln Tyr Ser Pro Arg Gln Leu 530 535 540Gly Gly Ser His Arg Val Asn Gly Glu Leu Thr Ile Gly Glu Asn Ile545 550 555 560Gly Asp Leu Gly Gly Leu Ser Ile Ala Val Arg Ala Tyr Glu Ile Ala 565 570 575Leu Gly His Pro Leu Asp Glu Ala Pro Val Leu Asp Gly Tyr Thr Gly 580 585 590Leu Gln Arg Leu Phe Met Gly Trp Ala His Ser Trp Arg Thr Lys Gly 595 600 605Arg Asp Glu Glu Val Ile Arg Arg Leu Ala Thr Asp Pro His Ser Pro 610 615 620Asp Glu Phe Arg Cys Asn Gly Val Val Arg Asn Ile Asp Glu Phe Tyr625 630 635 640Thr Ala Phe Asp Val Gln Pro Asp Asp Ala Leu Trp Leu Asp Pro Glu 645 650 655Gln Arg Val Arg Ile Trp 66029652PRTRhodococcus equi 29Met Glu Cys Met Thr Arg Ser Gly Ile Asp Leu Thr His Ile Asp Ala1 5 10 15Gly Thr Arg Pro Gln Asp Asp Leu Phe Val His Val Asn Gly Lys Trp 20 25 30Leu Asp Glu Tyr Glu Ile Pro Ala Asp Arg Ala Val Asp Gly Ala Phe 35 40 45Arg Thr Leu Tyr Asp Lys Ala Glu Glu Asp Val Lys Thr Leu Ile Gln 50 55 60Glu Ala Ser Glu Ser Gly Ala Ala His Gly Thr Asp Ala Gln Lys Ile65 70 75 80Gly Asp Leu Tyr Ser Ser Phe Met Asp Ala Asp Ala Val Glu Ala Ala 85 90 95Gly Leu Ser Pro Ile Ala Glu Glu Leu Ala Ala Val Ala Asn Ala Ala 100 105 110Asp Leu Ser Glu Leu Ala Gly Ile Val Gly Arg Leu Gln Arg Thr Gly 115 120 125Val Gly Gly Gly Ile Gly Gln Tyr Val Asp Thr Asp Ala Lys Asp Ser 130 135 140Ser Arg Tyr Leu Val His Phe Thr Gln Ser Gly Ile Gly Leu Pro Asp145 150 155 160Glu Ser Tyr Tyr Arg Glu Asp Asn Tyr Ala Pro Ile Arg Asp Ala Tyr 165 170 175Val Glu His Ile Arg Lys Met Phe Glu Leu Ala Gly Ile Glu Tyr Asp 180 185 190Ala Gln Arg Val Phe Asp Leu Glu Thr Ala Ile Ala Gly Gly His Trp 195 200 205Asp Val Val Lys Arg Arg Asp Ala Glu Leu Gly Tyr Asn Leu Val Thr 210 215 220Leu Asp Asp Leu Arg Ser Gln Tyr Gly Gly Phe Asp Trp Asp Ala Trp225 230 235 240Ile Ser Gly Leu Gln Ala Thr Pro Glu Gln Leu Ala Glu Ile Val Val 245 250 255Arg Gln Pro Gly Phe Val Glu Ser Phe Thr Glu Leu Trp Thr Ser Arg 260 265 270Pro Leu Glu Asp Trp Lys Ala Trp Ala Thr Trp Arg Val Leu His Ser 275 280 285Arg Ala Ala Phe Leu Thr Glu Ala Ile Val Ala Glu Asp Phe Ala Phe 290 295 300Phe Gly Thr Thr Leu Ser Gly Thr Gln Glu Asn Arg Glu Arg Trp Lys305 310 315 320Arg Gly Val Ser Leu Val Gln Asp Leu Leu Gly Glu Ala Val Gly Lys 325 330 335Leu Tyr Val Glu Arg His Phe Pro Pro Glu Ser Lys Ala Arg Met Gln 340 345 350Glu Leu Val Ala Asn Leu Gln Glu Ala Tyr Arg Arg Asn Ile Ser Asp 355 360 365Leu Glu Trp Met Ser Pro Ala Thr Arg Gln Ala Ala Leu Ala Lys Leu 370 375 380Asp Lys Phe Thr Pro Lys Ile Gly Tyr Pro Asp Lys Trp Arg Asp Tyr385 390 395 400Ser Ala Val Glu Ile Asp Pro Ala Asp Leu Val Gly Asn Tyr Arg Ser 405 410 415Gly Phe Ala Ala Glu His Asp Arg Asp Ile Ala Lys Leu Gly Gly Pro 420 425 430Val Asp Arg Asp Glu Trp Phe Met Thr Pro Gln Thr Val Asn Ala Tyr 435 440 445Tyr Asn Pro Gly Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln 450 455 460Pro Pro Phe Phe Asp Pro Glu Ala Asp Asp Ala Ala Asn Tyr Gly Gly465 470 475 480Ile Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln 485 490 495Gly Ala Lys Tyr Asp Gly Asp Gly Asn Met Val Asp Trp Trp Thr Asp 500 505 510Asp Asp Arg Thr Glu Phe Gly Lys Arg Thr Lys Ala Leu Ile Glu Gln 515 520 525Tyr Asn Asp Phe Glu Pro Lys Asp Leu Pro Gly His His Val Asn Gly 530 535 540Glu Phe Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile545 550 555 560Ala Leu Glu Ala Tyr Arg Ile Ala Thr Glu Gly Thr Glu Ala Pro Val 565 570 575Leu Asp Glu Leu Asp Gly Leu Gln Arg Val Phe Phe Gly Trp Ala Gln 580 585 590Val Trp Arg Thr Lys Ala Arg Lys Glu Glu Ala Leu Arg Arg Leu Ala 595 600 605Val Asp Pro His Ser Pro Pro Glu Phe Arg Cys Asn Gly Val Ile Arg 610 615 620Asn Leu Asp Ser Phe His Asp Ala Phe Asp Val Gln Pro Gly Asp Ala625 630 635 640Leu Tyr Leu Glu Pro Glu Lys Arg Val Lys Ile Trp 645 65030726PRTActinomyces viscosus 30Met Thr Asp Ala Cys Ala Thr Ser Pro Thr Pro Thr Gly Pro Ala Ser1 5 10 15Asp Ser Pro Ala Gln His Ser Gln Pro Ala Ser Ser Ser Pro Ala Ala 20 25 30Asp Ala Ile Leu Ala Gly Val Leu Glu Thr Ala His Thr Asp Thr Ser 35 40 45Val Arg Pro Gln Asp Asp Leu Phe Arg Phe Val Asn Gly Gln Trp Leu 50 55 60Thr Thr Ala Glu Ile Pro Ala Asp Arg Pro Ser Ser Gly Ala Phe Thr65 70 75 80Thr Leu Arg Asp Glu Ser Glu Ala Ala Cys Arg Gln Ile Val Glu Glu 85 90 95Leu Ala Glu Gln Phe Ser Ser Val Ala Pro Glu Gly Ala Ala Glu Val 100 105 110Leu Ser Thr Asn Arg Gly Arg Val Gly Ala Leu Tyr Gln Ala Phe Met 115 120 125Asp Glu Ala His Leu Glu Glu Leu Gly Ala Glu Pro Leu Ala Glu Glu 130 135 140Leu Ala Pro Val Leu Gly Ala Ser Ser Lys Glu Glu Leu Ala Arg Ala145 150 155 160Leu Gly Glu Met Thr Pro Val Gly Phe Met Gly Val Val Gly Ala Asp 165 170 175Val Glu Val Asp Ile Asn Asp Pro Glu Arg Tyr Thr Ser Trp Val Gly 180 185 190Gln Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr Arg Glu Glu Ala 195 200 205Gln Ala Pro Leu Arg Gln Ala Tyr Val Glu His Val Ala Arg Met Met 210 215 220Ala Leu Ala Gly Leu Thr Asp Ser Phe Gly Ala Ser Gly Glu Asp Leu225 230 235 240Ala Glu Arg Val Met Ala Val Glu Thr Ala Leu Ala Lys Gly His Trp 245 250 255Asp Arg Val Thr Cys Arg Asp Val Glu Lys Met Asn Asn Pro Met Ser 260 265 270Trp Gln Gln Ile Val Asp Ser Ala Pro Asp Leu Pro Trp Asp Glu Trp 275 280 285Arg Glu Gly Ile Arg Ala Ala Ala Arg Ser Ala Gly Ile Glu Gln Thr 290 295 300Ala Phe Leu Glu Glu Ala Ile Val Thr Gln Pro Asp Tyr Leu Pro His305 310 315 320Ala Ala Gly Val Trp Gln Glu Thr Asp Leu Glu Asp Leu Lys Val Trp 325 330 335Val Ala Trp His Val Val His Gly Arg Ala Ser Leu Leu Ser Gly Ala 340 345 350Phe Val Glu Glu Ser Phe Asp Phe Tyr Gly Arg Thr Leu Gln Gly Thr 355 360 365Asp Glu Leu Arg Pro Arg Trp Lys Arg Gly Val Gly Leu Val Glu Ser 370 375 380Cys Leu Gly Glu Ala Leu Gly Glu Ile Tyr Val Glu Arg His Phe Pro385 390 395 400Pro Ser His Lys Ser Ala Met Glu Ala Leu Val Gly Arg Leu Ile Glu 405 410 415Ala Tyr His Gln Ser Ile Ser Ser Leu Glu Trp Met Ser Pro Ala Thr 420 425 430Arg Glu Arg Ala Leu Glu Lys Leu Ala Leu Phe Thr Pro Lys Ile Gly 435 440 445Tyr Pro

Val Arg Trp Arg Asp Tyr Ser Ala Val Glu Val Val Pro Gly 450 455 460Asp Val Leu Ala Ser Val Arg Ser Val Glu Arg Ala Asp Met Ala Tyr465 470 475 480Ser Leu Asn Lys Leu Thr Lys Pro Val Asp Arg Asp Glu Trp His Met 485 490 495Thr Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Thr Met Asn Glu Ile 500 505 510Val Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp Pro Gln Ala 515 520 525Asp Asp Ala Val Asn Tyr Ala Gly Ile Gly Ala Val Ile Gly His Glu 530 535 540Ile Gly His Gly Phe Asp Asp Gln Gly Ser Thr Phe Asp Gly Thr Gly545 550 555 560Lys Val Ser Asp Trp Trp Thr Gln Glu Asp Arg Glu Ala Phe Thr Glu 565 570 575Arg Thr Ser Ala Leu Ile Ser Gln Tyr Asp Ala Tyr Thr Pro Lys Val 580 585 590Val Val Ala Lys His Arg Glu Ala Gly Thr Ala Gln Glu Glu Ile Pro 595 600 605His Val Asn Gly Ala Leu Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly 610 615 620Gly Leu Gly Ile Ala Leu Lys Ala Tyr Ser Leu Ala Leu Ala Asp Ala625 630 635 640Gly Ile Pro Ser Val Asp Glu Ala Pro Val Ile Asp Gly Leu Thr Gly 645 650 655Leu Gln Arg Phe Phe Tyr Ser Trp Ala Arg Ile Trp Arg Ser Lys Ser 660 665 670Arg Pro Asp Tyr Ala Glu Leu Leu Leu Thr Val Asp Pro His Ser Pro 675 680 685Ala Glu Phe Arg Cys Asn Gly Ile Val Arg Asn Val Asp Ala Phe Tyr 690 695 700Lys Ala Phe Ala Val Asp Ser Asp Asp Ala Leu Trp Leu Pro Pro Asn705 710 715 720Glu Arg Val Ser Ile Trp 7253113PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 31Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln Pro Pro1 5 103212PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 32Phe Asp Asp Gln Gly Ser Arg Tyr Asp Gly Asp Gly1 5 10338PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 33Asp Pro His Ser Pro Asp Glu Phe1 53411PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 34Asn Gly Val Val Arg Asn Ile Asp Glu Phe Tyr1 5 10355PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 35Arg Val Arg Ile Trp1 5366PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 36Val Asn Gly Lys Trp Leu1 5377PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 37Glu Ile Pro Ala Asp Arg Pro1 5386PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 38Arg Ile Gly Ala Leu Tyr1 5396PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 39Glu Ile Ala Pro Ile Leu1 54015PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 40Gln Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr Arg Glu Glu1 5 10 154114PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 41Phe Tyr Gly Lys Thr Leu Ser Gly Thr Gln Gln Ile Arg Glu1 5 10426PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 42Arg Trp Lys Arg Gly Val1 5435PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 43Leu Asp Trp Met Thr1 5447PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 44Trp Arg Asp Phe Ser Ala Leu1 54510PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 45Met Thr Pro Gln Thr Val Asn Ala Tyr Tyr1 5 10468PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 46Xaa Gln Pro Gln Gln Pro Tyr Asp1 5479PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 47Ile Gly His Glu Ile Gly His Gly Phe1 548660PRTRothia mucilaginosa 48Met Thr Thr Asn Ser Gly Ile Thr Lys Glu Trp Val Asp Glu Thr Val1 5 10 15Lys Pro Gly Asp Asp Phe Phe Arg His Val Asn Gly Lys Trp Leu Ala 20 25 30Thr His Glu Ile Pro Ala Asp Arg Pro Lys Asp Gly Gly Leu Tyr Thr 35 40 45Leu Arg Asp Asn Ala Glu Lys His Val Arg Glu Leu Val Glu Lys Ile 50 55 60Ala Lys Glu Gln Pro Glu Ser Arg Ile Gly Ala Leu Tyr Asn Ser Phe65 70 75 80Met Asp Val Glu Lys Ile Glu Ala Asp Gly Leu Glu Pro Leu Leu Lys 85 90 95Glu Ile Ala Pro Ile Leu Asn Ser Ala Thr Pro Ser His Leu Ala Val 100 105 110Thr Leu Ala Leu Leu Ser Arg Ala Gly Leu Pro Gln Leu Phe Ala Trp 115 120 125Tyr Thr Ser Asn Asp Pro Lys Asp Pro Lys Asn Tyr Thr Phe Phe Leu 130 135 140Tyr Gln Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr Arg Glu Glu145 150 155 160Lys His Glu Ala Ala Cys Ala Ala Tyr Val Glu His Ile Ala Arg Met 165 170 175Phe Glu Leu Thr Gly Leu Ala Glu Gly Phe Gly Leu Thr Pro Glu Gln 180 185 190Ala Ala Gln Leu Val Phe Thr His Glu Ser Glu Leu Ala Arg Leu His 195 200 205Trp Asn Val Val Glu Asn Arg Asp Ala Glu Ala Thr Tyr Asn Pro Tyr 210 215 220Gln Ala Thr Glu Leu Asp Glu Lys Phe Pro Gly Phe Pro Phe Ser Gln225 230 235 240Trp Leu Leu Ala Leu Gly Ala Asp Pro Glu Thr Leu Gly Gln Val Ile 245 250 255Val Ala Gln Pro Ser Phe Phe Glu Gly Ala Ala Lys Leu Phe Thr Ser 260 265 270Ile Pro Leu Met Ser Trp Lys Leu Trp Ala Val Trp Thr Val Leu Arg 275 280 285Ser Arg Ala Pro Phe Met Tyr Asp Glu Leu Val Gln Glu Ser Phe Asn 290 295 300Phe Tyr Gly Lys Thr Leu Ser Gly Thr Gln Gln Ile Arg Glu Arg Trp305 310 315 320Lys Arg Gly Val Gly Ala Val Glu Lys Ala Leu Gly Glu Glu Ile Gly 325 330 335Gln Glu Tyr Val Ala Val His Phe Pro Pro Ser His Lys Glu Lys Met 340 345 350Leu Val Leu Val Gly Asn Leu Leu Glu Ala Tyr Arg Glu Ser Ile Glu 355 360 365Ser Leu Asp Trp Met Thr Glu Ala Thr Arg Gln Lys Ala Leu Glu Lys 370 375 380Leu Ser Lys Phe Val Thr Lys Ile Gly Tyr Pro Asp Lys Trp Arg Asp385 390 395 400Phe Ser Ala Leu Glu Leu Ala Pro Gly Asp Leu Phe Glu Asn Leu Arg 405 410 415Arg Thr Gly Ala Phe Asp Ala Asp Trp Leu Ile Ala Arg Lys Gly Gln 420 425 430Pro Val Asp Lys Ser Glu Trp Leu Met Thr Pro Gln Thr Val Asn Ala 435 440 445Tyr Tyr Met Pro Pro Ala Asn Glu Ile Val Phe Pro Ala Ala Ile Leu 450 455 460Gln Pro Pro Tyr Phe Asn Pro Asp Ala Asp Asp Ala Ala Asn Tyr Gly465 470 475 480Asn Ile Gly Met Ile Ile Gly His Glu Ile Gly His Gly Phe Asp Asp 485 490 495Gln Gly Ser Arg Tyr Asp Gly Asp Gly Lys Leu Glu Ser Trp Trp Thr 500 505 510Glu Glu Asp Tyr Ala Lys Phe Lys Glu Arg Thr Ser Ala Leu Val Glu 515 520 525Gln Tyr Asn Ala Tyr Val Pro Val Gly Leu Asp Pro Lys Phe His Val 530 535 540Asn Gly Glu Leu Thr Leu Gly Glu Asn Ile Gly Asp Leu Ala Gly Met545 550 555 560Ser Ile Ala Leu Lys Ala Tyr Cys Leu Ala Leu Lys Lys Gln Gly Ile 565 570 575Glu Ser Leu Ala Asp Ala Pro Val Ile Asp Gly Met Thr Gly Ile Gln 580 585 590Arg Phe Phe Phe Ser Asn Ala Arg Gly Trp Cys Thr Lys Ser Arg Pro 595 600 605Gln His Ala Glu Val Met Ile Ser Val Asp Pro His Ser Pro Asp Glu 610 615 620Phe Arg Val Asn Gly Val Val Arg Asn Ile Asp Glu Phe Tyr Glu Ala625 630 635 640Phe Gly Val Ser Glu Gly Asp Ala Leu Tyr Leu Ala Pro Glu Glu Arg 645 650 655Met Arg Ile Trp 66049671PRTXylanimonas cellulosilytica 49Met Thr Ile Glu Ala Leu Arg Ser Gly Ile Asp Leu Thr Ala Leu Asp1 5 10 15Pro Ala Thr Arg Pro Gln Asp Asp Leu Tyr Arg His Val Asn Gly Thr 20 25 30Trp Ile Ala Thr His Glu Ile Pro Ala Asp Arg Ser Met Asp Gly Ala 35 40 45Phe Met Lys Leu Arg Asp Leu Ser Glu Glu Arg Val Arg Ala Ile Ile 50 55 60Thr Asp Leu Ala Gly Ser Ser Asp Thr Glu Pro Gly Thr Thr Ala Gly65 70 75 80Lys Ile Gly Asp Leu Tyr Ser Ser Phe Met Asp Thr Ala Arg Ile Ala 85 90 95Ala Leu Gly Ala Thr Pro Leu Ala Ala Asp Leu Ala Leu Ile Ala Gly 100 105 110Ala Thr Ser Gln Ala Glu Leu Thr Gly Val Leu Gly Ala Leu Gln Arg 115 120 125Thr Gly Gly Gly Gly Ala Val Gly Phe Tyr Val Asp Asn Asp Ala Ala 130 135 140Asp Pro Glu Gln Tyr Arg Val Tyr Leu Cys Gln Ser Gly Leu Gly Leu145 150 155 160Pro Asp Glu Ala Tyr Tyr Arg Glu Asp Gln Tyr Ala Pro Ile Arg Glu 165 170 175Lys Tyr Val Pro His Val Ala Arg Met Leu Arg Leu Gly Gly Ala Ala 180 185 190Glu Leu Leu Gly Val Asp Ala Asp Asp Ala Ala Ala Arg Val Val Ala 195 200 205Leu Glu Ser Lys Leu Ala Gly His His Trp Asp Val Val Lys Asp Arg 210 215 220Asp Ala Thr Leu Thr Tyr Asn Pro Thr Thr Ile Ala Glu Leu Ala Val225 230 235 240Thr Ala Pro Gly Phe Asp Trp Gln Ala Trp Ala Ile Ala Leu Gly Ala 245 250 255Pro Thr Gly Ala Leu Asp Ala Leu Val Val Arg Glu Pro Ser Tyr Ala 260 265 270Glu Gly Leu Ala Ala Leu Trp Gln Ser Glu Pro Leu Glu Asp Trp Gln 275 280 285Val Trp Ala Ala Tyr Arg Leu Val Thr Ala Arg Ala Pro Tyr Leu Thr 290 295 300Asp Glu Ile Val Glu Ala Asn Phe Asp Phe Tyr Gly Arg Thr Leu Ser305 310 315 320Gly Ala Gln Glu Val Arg Glu Arg Trp Lys Arg Gly Val Ala Leu Val 325 330 335Glu Gly Ala Leu Gly Glu Ala Val Gly Glu Gln Tyr Val Ala Arg His 340 345 350Phe Pro Pro Ala His Lys Ala Arg Met Asp Glu Leu Val Ala Asn Leu 355 360 365Val Ala Ala Tyr Arg Glu Ser Ile Glu Gly Leu Asp Trp Met Thr Asp 370 375 380Glu Thr Lys Ala Lys Ala Leu Ala Lys Leu Glu Lys Phe Thr Pro Lys385 390 395 400Ile Gly Tyr Pro Val Lys Trp Arg Asp Tyr Ser Ala Leu Val Ile Asp 405 410 415Ala Asp Asp Leu Val Gly Asn Val Arg Arg Ser Asn Ala Phe Asp Leu 420 425 430Asp His Glu Leu Gly Lys Val Gly Lys Pro Leu Asp Arg Asp Glu Trp 435 440 445Phe Met Thr Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Gly Met Asn 450 455 460Glu Ile Val Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp Pro465 470 475 480Glu Ala Asp Asp Ala Val Asn Tyr Gly Gly Ile Gly Gly Val Ile Gly 485 490 495His Glu Ile Gly His Gly Phe Asp Asp Gln Gly Ser Lys Tyr Asp Gly 500 505 510Asp Gly Arg Leu Gln Asp Trp Trp Thr Ala Glu Asp Arg Ala Glu Phe 515 520 525Glu Lys Arg Thr Gly Ala Leu Ile Ala Gln Tyr Asp Ala Phe Val Pro 530 535 540Glu Gln Leu Gly Ala Asp Gly Ala His Val Asn Gly Ser Leu Thr Ile545 550 555 560Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile Ala Ile Arg Ala 565 570 575Tyr Arg Ile Ala Leu Ala Ala Ala Arg Glu Gly Ala Thr Leu Asp Asp 580 585 590Ala Pro Val Ile Asp Gly Leu Thr Gly Leu Glu Arg Val Phe Leu Gly 595 600 605Trp Ala Gln Val Trp Gln Ser Lys Gly Arg Asp Glu Glu Val Leu Arg 610 615 620Arg Leu Ala Thr Asp Pro His Ser Pro Asn Glu Phe Arg Cys Asn Gly625 630 635 640Ile Val Arg Asn Val Asp Glu Phe His Asp Ala Tyr Gly Val Arg Glu 645 650 655Gly Asp Ala Leu Trp Leu Ala Pro Glu Asp Arg Val Arg Ile Trp 660 665 67050662PRTCellulomonas flavigena 50Met Thr Arg Ser Gly Val Pro Leu Asp Asp Leu Asp Pro Ser Val Arg1 5 10 15Pro Gln Asp Asp Leu Asp Leu Phe Val Asn Gly Arg Trp Ala Ala Ser 20 25 30Tyr Val Ile Pro Pro Asp Arg Ser Met Asp Gly Pro Phe Arg Ala Leu 35 40 45Tyr Asp Glu Ala Glu Arg Gln Val Leu Asp Ile Ile Thr Asp Ala Ala 50 55 60Gln Ala Ala Gly Glu Gly Asp Gly Val Glu Ala Lys Ile Gly Ala Leu65 70 75 80Tyr Ala Ser Phe Met Asp Thr Asp Ala Val Arg Ala Ala Gly Val Glu 85 90 95Pro Leu Arg Glu Asp Leu Ala Leu Val Asp Ala Ala Thr Thr Pro Ala 100 105 110Glu Leu Thr Val Ala Val Gly Arg Leu Gln Arg Thr Gly Ala Leu Ser 115 120 125Ala Val Asp Leu Tyr Val Asp Asn Asp Ala Lys Asp Pro Asp Ser Tyr 130 135 140Val Val His Leu Val Gln Gly Gly Leu Gly Leu Pro Asp Glu Ala Tyr145 150 155 160Tyr Arg Glu Glu Gln His Ala Ala Val Arg Glu Lys Tyr Leu Pro His 165 170 175Val Ala Arg Met Leu Arg Leu Ala Ala Pro Val Ser Gly Val Val Ala 180 185 190Ala Gly Asp Ala Asp Asp Leu Ala Ala Arg Val Val Ala Leu Glu Ser 195 200 205Arg Ile Ala Ala His His Trp Asp Val Val Lys Asp Arg Asp Ala Glu 210 215 220Leu Thr Tyr Asn Ala Leu Thr Leu Ala Glu Leu Ala Ala Arg Ala Pro225 230 235 240Gly Phe Asp Trp Arg Ala Trp Ala Glu Ala Leu Gly Ala Pro Ala Gly 245 250 255Ala Leu Asp Arg Leu Val Val Arg Glu Pro Ser Phe Ala Glu Gly Leu 260 265 270Ala Ala Leu Trp Thr Glu Val Pro Val Ala Asp Trp Gln Ala Trp Ala 275 280 285Thr Tyr His Val Val Ser Ser Arg Ala Pro Tyr Leu Thr Asp Glu Leu 290 295 300Val Glu Ala Asn Phe Asp Phe Tyr Gly Arg Thr Leu Ser Gly Ala Pro305 310 315 320Glu Leu Arg Asp Arg Trp Lys Arg Gly Val Ser Leu Val Gln Gly Ala 325 330 335Leu Gly Glu Ala Val Gly Lys Val Tyr Val Glu Arg His Phe Pro Pro 340 345 350Ser His Lys Glu Arg Met Asp Glu Leu Val Ala Asn Leu Val Glu Ala 355 360 365Tyr Arg Arg Ser Ile Thr Glu Leu Glu Trp Met Gly Glu Glu Thr Arg 370 375 380Gln Arg Ala Leu Glu Lys Leu Ala Arg Phe Thr Pro Lys Ile Gly Tyr385 390 395 400Pro Ala Arg Trp Arg Asp Tyr Ser Ala Leu Glu Val Arg Ala Asp Asp 405 410 415Leu Val Gly Asn Val Arg Arg Ser Asn Ala Phe Asp Leu Asp Arg Glu 420 425 430Leu Gly Lys Ile Gly Arg Pro Ile Asp Arg Asp Glu Trp Phe Met Thr 435 440 445Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Gly Met Asn Glu Ile Val 450 455 460Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp Ala Glu Ala Asp465 470 475 480Asp Ala Ala Asn Tyr Gly Gly Ile Gly Ala Val Ile Gly His Glu Ile 485 490 495Gly His Gly Phe Asp Asp Gln Gly Ser Lys Tyr Asp Gly Asp Gly Arg 500

505 510Leu Val Asp Trp Trp Thr Ala Glu Asp Arg Ala Glu Phe Glu Arg Arg 515 520 525Thr Lys Ser Leu Val Asp Gln Tyr Ala Gln Tyr Ser Pro Arg Gln Leu 530 535 540Gly Gly Ser His Arg Val Asn Gly Glu Leu Thr Ile Gly Glu Asn Ile545 550 555 560Gly Asp Leu Gly Gly Leu Ser Ile Ala Val Arg Ala Tyr Glu Ile Ala 565 570 575Leu Gly His Pro Leu Asp Glu Ala Pro Val Leu Asp Gly Tyr Thr Gly 580 585 590Leu Gln Arg Leu Phe Met Gly Trp Ala His Ser Trp Arg Thr Lys Gly 595 600 605Arg Asp Glu Glu Val Ile Arg Arg Leu Ala Thr Asp Pro His Ser Pro 610 615 620Asp Glu Phe Arg Cys Asn Gly Val Val Arg Asn Ile Asp Glu Phe Tyr625 630 635 640Thr Ala Phe Asp Val Gln Pro Asp Asp Ala Leu Trp Leu Asp Pro Glu 645 650 655Gln Arg Val Arg Ile Trp 66051726PRTActinomyces viscosus 51Met Thr Asp Ala Cys Ala Thr Ser Pro Thr Pro Thr Gly Pro Ala Ser1 5 10 15Asp Ser Pro Ala Gln His Ser Gln Pro Ala Ser Ser Ser Pro Ala Ala 20 25 30Asp Ala Ile Leu Ala Gly Val Leu Glu Thr Ala His Thr Asp Thr Ser 35 40 45Val Arg Pro Gln Asp Asp Leu Phe Arg Phe Val Asn Gly Gln Trp Leu 50 55 60Thr Thr Ala Glu Ile Pro Ala Asp Arg Pro Ser Ser Gly Ala Phe Thr65 70 75 80Thr Leu Arg Asp Glu Ser Glu Ala Ala Cys Arg Gln Ile Val Glu Glu 85 90 95Leu Ala Glu Gln Phe Ser Ser Val Ala Pro Glu Gly Ala Ala Glu Val 100 105 110Leu Ser Thr Asn Arg Gly Arg Val Gly Ala Leu Tyr Gln Ala Phe Met 115 120 125Asp Glu Ala His Leu Glu Glu Leu Gly Ala Glu Pro Leu Ala Glu Glu 130 135 140Leu Ala Pro Val Leu Gly Ala Ser Ser Lys Glu Glu Leu Ala Arg Ala145 150 155 160Leu Gly Glu Met Thr Pro Val Gly Phe Met Gly Val Val Gly Ala Asp 165 170 175Val Glu Val Asp Ile Asn Asp Pro Glu Arg Tyr Thr Ser Trp Val Gly 180 185 190Gln Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr Arg Glu Glu Ala 195 200 205Gln Ala Pro Leu Arg Gln Ala Tyr Val Glu His Val Ala Arg Met Met 210 215 220Ala Leu Ala Gly Leu Thr Asp Ser Phe Gly Ala Ser Gly Glu Asp Leu225 230 235 240Ala Glu Arg Val Met Ala Val Glu Thr Ala Leu Ala Lys Gly His Trp 245 250 255Asp Arg Val Thr Cys Arg Asp Val Glu Lys Met Asn Asn Pro Met Ser 260 265 270Trp Gln Gln Ile Val Asp Ser Ala Pro Asp Leu Pro Trp Asp Glu Trp 275 280 285Arg Glu Gly Ile Arg Ala Ala Ala Arg Ser Ala Gly Ile Glu Gln Thr 290 295 300Ala Phe Leu Glu Glu Ala Ile Val Thr Gln Pro Asp Tyr Leu Pro His305 310 315 320Ala Ala Gly Val Trp Gln Glu Thr Asp Leu Glu Asp Leu Lys Val Trp 325 330 335Val Ala Trp His Val Val His Gly Arg Ala Ser Leu Leu Ser Gly Ala 340 345 350Phe Val Glu Glu Ser Phe Asp Phe Tyr Gly Arg Thr Leu Gln Gly Thr 355 360 365Asp Glu Leu Arg Pro Arg Trp Lys Arg Gly Val Gly Leu Val Glu Ser 370 375 380Cys Leu Gly Glu Ala Leu Gly Glu Ile Tyr Val Glu Arg His Phe Pro385 390 395 400Pro Ser His Lys Ser Ala Met Glu Ala Leu Val Gly Arg Leu Ile Glu 405 410 415Ala Tyr His Gln Ser Ile Ser Ser Leu Glu Trp Met Ser Pro Ala Thr 420 425 430Arg Glu Arg Ala Leu Glu Lys Leu Ala Leu Phe Thr Pro Lys Ile Gly 435 440 445Tyr Pro Val Arg Trp Arg Asp Tyr Ser Ala Val Glu Val Val Pro Gly 450 455 460Asp Val Leu Ala Ser Val Arg Ser Val Glu Arg Ala Asp Met Ala Tyr465 470 475 480Ser Leu Asn Lys Leu Thr Lys Pro Val Asp Arg Asp Glu Trp His Met 485 490 495Thr Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Thr Met Asn Glu Ile 500 505 510Val Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp Pro Gln Ala 515 520 525Asp Asp Ala Val Asn Tyr Ala Gly Ile Gly Ala Val Ile Gly His Glu 530 535 540Ile Gly His Gly Phe Asp Asp Gln Gly Ser Thr Phe Asp Gly Thr Gly545 550 555 560Lys Val Ser Asp Trp Trp Thr Gln Glu Asp Arg Glu Ala Phe Thr Glu 565 570 575Arg Thr Ser Ala Leu Ile Ser Gln Tyr Asp Ala Tyr Thr Pro Lys Val 580 585 590Val Val Ala Lys His Arg Glu Ala Gly Thr Ala Gln Glu Glu Ile Pro 595 600 605His Val Asn Gly Ala Leu Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly 610 615 620Gly Leu Gly Ile Ala Leu Lys Ala Tyr Ser Leu Ala Leu Ala Asp Ala625 630 635 640Gly Ile Pro Ser Val Asp Glu Ala Pro Val Ile Asp Gly Leu Thr Gly 645 650 655Leu Gln Arg Phe Phe Tyr Ser Trp Ala Arg Ile Trp Arg Ser Lys Ser 660 665 670Arg Pro Asp Tyr Ala Glu Leu Leu Leu Thr Val Asp Pro His Ser Pro 675 680 685Ala Glu Phe Arg Cys Asn Gly Ile Val Arg Asn Val Asp Ala Phe Tyr 690 695 700Lys Ala Phe Ala Val Asp Ser Asp Asp Ala Leu Trp Leu Pro Pro Asn705 710 715 720Glu Arg Val Ser Ile Trp 72552670PRTGordonia bronchialis 52Met Thr Ala Ser Asp Thr Thr Leu Asn Ser Ser Ser Arg Asn Ser Gly1 5 10 15Leu Asp Leu Glu Trp Val Asp Asp Ser Val Arg Val Gln Asp Asp Leu 20 25 30Phe Ala His Val Asn Gly Lys Trp Leu Glu Arg His Val Ile Pro Glu 35 40 45Asp Arg Ala Ile Asp Gly Ala Phe His Val Leu Arg Asp Arg Ala Glu 50 55 60Glu Asn Val Arg Asp Ile Ile Thr Glu Cys Ala Glu Ser Ser Pro Pro65 70 75 80Ser Gly Ser Asp Ala Leu Lys Ile Gly Asp Leu Phe Ala Ser Phe Met 85 90 95Asp Thr Asp His Ile Asp Ala Leu Gly Ile Gly Pro Ile Arg Gly Glu 100 105 110Leu Asp Lys Ile Ala Gly Ile Asp Ser Thr Thr Ala Leu Ala Arg Arg 115 120 125Met Gly Arg Leu Thr Arg His Gly Val Gly Gly Leu Phe Gly Phe Tyr 130 135 140Val Asp Thr Asp Ala Arg Gln Ser Asp Arg Tyr Leu Val His Ile Val145 150 155 160Gln Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr His Pro Asp Ala 165 170 175Asp Gly Asn Asp Ile His Ala Glu Thr Arg Ala Ala Tyr Ala Ala His 180 185 190Val Glu Arg Met Phe Ala Leu Ala Gly Phe Asp Ala Pro Ala Asp Arg 195 200 205Ala Ala Thr Val Leu Asp Leu Glu Thr Ala Ile Ala Glu His His Trp 210 215 220Asp Val Val Ala Arg Arg Asp Ala Glu Arg Thr Tyr Asn Leu Met Ser225 230 235 240Leu Ala Glu Phe Glu Ser Ala Ala Ala Gly Phe Asp Leu Gly Glu Trp 245 250 255Phe Gly Gly Leu Gly Thr Val Gly Asp Ala Thr Phe Ala Glu Val Val 260 265 270Val Gly Gln Pro Ser Phe Val Ser Gly Ala Ala Gly Leu Ile Ala Ser 275 280 285Arg Pro Leu Asp Asp Trp Lys Thr Trp Leu Ala Trp Arg Leu Leu Arg 290 295 300Ser Ala Ala Pro Tyr Leu Ser Ser Glu Phe Val Glu Glu Asn Phe Asp305 310 315 320Phe Tyr Gly Arg Thr Leu Ser Gly Ala Gln Thr Asn Arg Asp Arg Trp 325 330 335Lys Arg Gly Val Ser Phe Val Glu Gly Ala Met Gly Phe Ala Val Gly 340 345 350Lys Leu Tyr Val Asp Lys His Phe Pro Pro Glu Ala Lys Ala Arg Met 355 360 365Asp Glu Leu Ile Ala Asn Leu Val Ala Ala Tyr Arg Arg Asn Ile Thr 370 375 380Asp Leu Glu Trp Met Thr Pro Glu Thr Arg Thr Lys Ala Leu Ala Lys385 390 395 400Leu Asp Lys Phe Thr Pro Lys Ile Gly Tyr Pro Ala Thr Trp Arg Asp 405 410 415Tyr Gly Ala Leu Ile Val Asp Arg Gly Asp Leu Ile Gly Asn Val Ala 420 425 430Arg Ala Ser Ser Phe Glu Gln Glu Arg Glu Phe Ala Lys Ile Gly Ala 435 440 445Pro Val Asp Arg Asp Glu Trp Phe Met Thr Pro Gln Thr Val Asn Ala 450 455 460Tyr Tyr Asn Pro Gly Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu465 470 475 480Gln Pro Pro Phe Phe Asp Pro Asp Ala Asp Asp Ala Ala Asn Tyr Gly 485 490 495Gly Ile Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp 500 505 510Gln Gly Ala Lys Tyr Asp Gly Asp Gly Asn Leu Val Asn Trp Trp Ser 515 520 525Asp Ala Asp Arg Glu Glu Phe Ser Ser Arg Thr Ala Lys Leu Ile Glu 530 535 540Gln Tyr Gly Glu Phe Thr Pro Glu Gly Leu Asp Pro Lys Tyr Lys Val545 550 555 560Asn Gly Gly Phe Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu 565 570 575Ser Ile Ala Leu Val Ala Tyr Gln Leu Ala Thr Glu Gly Thr Thr Pro 580 585 590Pro Val Ile Asp Gly Leu Thr Gly Val Gln Arg Val Phe Tyr Ser Trp 595 600 605Ala Glu Ile Trp Arg Thr Lys Thr Arg Glu Ala Glu Ala Ile Lys Arg 610 615 620Leu Ser Ile Asp Pro His Ser Pro Pro Glu Phe Arg Cys Asn Gly Val625 630 635 640Val Arg Asn Ile Asp Val Phe Tyr Asp Ala Phe Asp Val Lys Pro Gly 645 650 655Asp Thr Leu Tyr Leu Asp Glu Ala Asp Arg Val Arg Ile Trp 660 665 67053666PRTMycobacterium gilvum 53Met Thr Val Glu Ala Thr Ser Asp Thr Ser Ala Lys Ser Gly Ile Asp1 5 10 15Leu Arg Tyr Val Asp Ala Asp Ala Arg Pro Gln Asp Asp Leu Phe Gly 20 25 30His Val Asn Gly Arg Trp Leu Ala Glu Tyr Gln Ile Pro Gly Asp Arg 35 40 45Ala Thr Asp Gly Ala Phe Arg Thr Leu Tyr Asp Arg Ala Glu Glu Gln 50 55 60Ile Arg Asp Leu Ile Thr Glu Ala Ala Ser Ala His Ala Ala Glu Gly65 70 75 80Thr Asp Gln Gln Arg Ile Gly Asp Leu Tyr Ala Ser Phe Met Asp Glu 85 90 95Gln Thr Val Arg Asp Arg Gly Leu Ala Pro Leu Leu Asp Glu Leu Gly 100 105 110Ala Ile Asp Ala Ala Gly Ser Pro Asp Ala Leu Ala Glu Val Leu Gly 115 120 125Ser Leu Gln Arg Thr Gly Ile Gly Gly Ala Thr Gly Leu Tyr Val Asp 130 135 140Thr Asp Ser Lys Asn Ser Thr Arg Tyr Leu Leu His Leu Thr Gln Ser145 150 155 160Gly Ile Gly Leu Pro Asp Glu Ser Tyr Phe Arg Glu Glu Gln His Ala 165 170 175Glu Ile Leu Ala Ala Tyr Pro Gly His Ile Ala Ala Met Phe Gly Leu 180 185 190Val Leu Gly Gly Asp Pro Gly Glu His Ala Ala Thr Ala Gln Arg Ile 195 200 205Val Ala Leu Glu Thr Lys Leu Ala Ala Ala His Trp Asp Val Val Lys 210 215 220Arg Arg Asp Ala Asp Leu Thr Tyr Asn Leu Arg Thr Phe Ala Glu Leu225 230 235 240Thr Asp Glu Ser Pro Gly Phe Asp Trp Thr Arg Trp Leu Gly Gly Leu 245 250 255Gly Ala Asp Arg Asp Lys Ala Ala Asp Val Val Val Arg Gln Pro Asp 260 265 270Tyr Leu Thr Ala Phe Ala Ser Leu Trp Ser Gly Ser Ser Leu Glu Asp 275 280 285Trp Lys Asp Trp Leu Arg Trp Arg Val Ile His Gly Arg Ala Phe Leu 290 295 300Leu Thr Asp Glu Leu Ile Ala Glu Asp Phe Ser Phe Tyr Gly Lys Arg305 310 315 320Leu Ser Gly Thr Glu Glu Ile Arg Asp Arg Trp Lys Arg Gly Val Ser 325 330 335Val Val Glu Ala Leu Met Gly Glu Ala Leu Gly Lys Leu Tyr Val Glu 340 345 350Arg His Phe Pro Pro Gln Ala Lys Ala Arg Met Asp Glu Leu Val Ala 355 360 365Asn Leu Arg Glu Ala Tyr Arg Val Ser Ile Asn Thr Leu Asp Trp Met 370 375 380Thr Pro Gln Thr Arg Glu Lys Ala Leu Val Lys Leu Asp Lys Phe Thr385 390 395 400Pro Lys Ile Gly Tyr Pro Asn Thr Trp Arg Asp Tyr Ser Ala Leu Val 405 410 415Ile Glu Arg Asp Asp Leu Tyr Gly Asn Tyr Arg Arg Gly Tyr Ala Leu 420 425 430Glu Tyr Asp Arg Asp Leu Ala Lys Leu Gly Gly Pro Val Asp Arg Asp 435 440 445Glu Trp Phe Met Thr Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Gly 450 455 460Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe465 470 475 480Asp Ala Asp Ala Asp Asp Ala Ala Asn Tyr Gly Gly Ile Gly Ala Val 485 490 495Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln Gly Ala Lys Tyr 500 505 510Asp Gly Asp Gly Asn Leu Val Asp Trp Trp Thr Asp Glu Asp Arg Ala 515 520 525Glu Phe Gly Lys Arg Thr Thr Ala Leu Ile Glu Gln Tyr Glu Gln Phe 530 535 540Thr Pro Arg Gly Leu Glu Pro Ser His His Val Asn Gly Ala Phe Thr545 550 555 560Val Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile Ala Leu Leu 565 570 575Ala Tyr Arg Leu Ser Leu Lys Gly Glu Pro Ala Pro Val Ile Asp Gly 580 585 590Leu Thr Gly Val Gln Arg Val Phe Tyr Gly Trp Ala Gln Val Trp Arg 595 600 605Thr Lys Ser Arg Glu Ala Glu Ala Ile Arg Arg Leu Ala Val Asp Pro 610 615 620His Ser Pro Pro Glu Phe Arg Cys Asn Gly Val Ile Arg Asn Met Asp625 630 635 640Ala Phe Tyr Asp Ala Phe Asp Val Asp Pro Glu Asp Ala Leu Tyr Leu 645 650 655Glu Pro Gln Arg Arg Val His Ile Trp Asn 660 66554651PRTKribbella flavida 54Met Thr Ala Gly Ile Asp Ile Thr Gly Leu Ser Thr Thr Val Arg Pro1 5 10 15Gln Asp Asp Leu Tyr Arg His Ala Asn Gly Arg Trp Leu Asp Glu His 20 25 30Glu Ile Pro Ala Asp Lys Ala Ile Tyr Gly Ala Phe His Ala Leu Gly 35 40 45Asp Thr Ala Glu Leu Asn Val Arg Ser Ile Val Glu Arg Thr Leu Asp 50 55 60Ala Gly His Pro Glu Gly Ser Glu Ala Arg Lys Ile Ala Asp Leu Tyr65 70 75 80Arg Ser Phe Leu Asp Glu Asp Thr Val Glu Arg Leu Gly Ala Asp Pro 85 90 95Ile Ala Asp Gln Leu Ala Leu Ala Gly Ser Ile Glu Asp Arg Asp Ala 100 105 110Leu Val Ala Ala Leu Gly Thr Leu Glu Leu Gln Gly Val Gly Gly Ile 115 120 125Phe His Tyr Trp Val Asp Val Asp Glu Lys Lys Ser Asp Gln Tyr Val 130 135 140Val Tyr Leu Thr Gln Gly Gly Leu Ser Leu Pro Asp Glu Ser Tyr Tyr145 150 155 160Arg Asp Asp Ala Phe Gln Glu Gln Arg Thr Ala Tyr Val Ala His Val 165 170 175Ala Arg Met Leu Gly Leu Ala Gly Leu Ala Asp Ala Glu Gly Ala Ala 180 185 190Glu Arg Ile Leu Ala Leu Glu Thr Arg Leu Ala Ala Gly His Trp Asp 195 200 205Val Val Lys Asn Arg Asp Val Thr Ala Thr Tyr Asn Lys Phe Asp Arg 210 215 220Ala Arg Leu Asp Ala Leu Met Pro Gly Phe Asp Trp Ser Arg Trp Leu225 230 235

240Pro Asn Ala Gly Val Pro Glu Ser Ala Phe Glu Gln Val Val Val Arg 245 250 255Gln Pro Asp Tyr Phe Thr Ser Ala Ala Glu Ala Leu Gln Glu Leu Glu 260 265 270Leu Asp His Trp Lys Glu Trp Leu Ser Trp Arg Ile Val His Ser Ala 275 280 285Ala Pro Leu Leu Ser Ser Ala Phe Val Ala Glu Asn Phe Glu Phe Tyr 290 295 300Gly Arg Thr Leu Thr Gly Ala Pro Glu Leu Arg Glu Arg Trp Lys Arg305 310 315 320Gly Leu Gly Val Val Gly Ser Ala Leu Gly Glu Ala Val Gly Gln Leu 325 330 335Tyr Val Ala Glu Phe Phe Pro Pro Val Ala Lys Ala Arg Met Val Glu 340 345 350Leu Val Gly Asn Leu Val Glu Ala Tyr Arg Gln Arg Ile Glu Ala Leu 355 360 365Asp Trp Met Ser Pro Glu Thr Arg Gln Arg Ala Leu Asp Lys Leu Gly 370 375 380Arg Phe Thr Pro Lys Ile Gly Tyr Pro Asp Lys Trp Arg Asp Tyr Ser385 390 395 400Ala Leu Glu Val Ala Pro Asp Asp Leu Val Gly Asn Val Arg Arg Ser 405 410 415Val Ala Val Glu Thr Ala Arg Glu Leu Ala Lys Leu Gly Gly Pro Val 420 425 430Asp Arg Thr Glu Trp Gln Met Thr Pro Gln Thr Val Asn Ala Tyr Tyr 435 440 445Asn Pro Ala Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln Pro 450 455 460Pro Phe Phe Ala Leu Asp Ala Asp Asp Ala Leu Asn Tyr Gly Ala Ile465 470 475 480Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln Gly 485 490 495Ser Arg Tyr Asp Gly Asp Gly Asn Ile Ser Asp Trp Trp Thr Asp Glu 500 505 510Asp Arg Ala Ala Phe Glu Val Arg Ala Asn Arg Leu Val Glu Gln Tyr 515 520 525Asp Ala Leu Glu Pro Ala Glu Ala Pro Gly Gln His Val Asn Gly Ala 530 535 540Leu Thr Leu Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile Ala545 550 555 560Tyr Thr Ala Tyr Glu Ile Ser Leu Ala Gly Ala Glu Ala Pro Val Ile 565 570 575Asp Gly Leu Thr Gly Ala Glu Arg Phe Phe Leu Ala Trp Ala Asn Ala 580 585 590Trp Ser Thr Lys Thr Arg Pro Ala Glu Val Val Arg Arg Leu Ala Ile 595 600 605Asp Pro His Ser Pro Pro Glu Phe Arg Cys Asn Ala Val Val Arg Asn 610 615 620Ile Asp Ala Phe His Glu Ala Phe Gly Val Gly Pro Asp Asp Ala Met625 630 635 640Trp Leu Ala Pro Glu Gln Arg Val Arg Ile Trp 645 65055673PRTMycobacterium vanbaalenii 55Met Thr Val Glu Ala Thr Arg Asn Leu Lys Ser Gly Ile Asp Leu Arg1 5 10 15Tyr Val Asp Thr Asp Ala Arg Pro Gln Asp Asp Leu Phe Gly His Val 20 25 30Asn Gly Arg Trp Leu Ala Glu Tyr Gln Ile Pro Ala Asp Arg Ala Thr 35 40 45Asp Gly Ala Phe Arg Thr Leu His Asp Arg Ala Glu Glu Gln Ile Arg 50 55 60Asp Leu Ile Thr Glu Val Ala Gly Ser Gly Ala Ala Arg Gly Thr Asp65 70 75 80Glu Gln Arg Ser Ser Gly Ser Ile Asp Ala Ser Arg Ile Gly Asp Leu 85 90 95Tyr Ala Ser Phe Met Asp Glu Asp Thr Val Arg Ala Arg Gly Leu Thr 100 105 110Pro Leu Arg Asp Glu Leu Ala Ala Val Asp Ala Ala Asp Thr Pro Glu 115 120 125Ala Leu Ala Arg Val Leu Gly Ala Leu Gln Arg Val Gly Val Gly Gly 130 135 140Gly Thr Gly Leu Tyr Val Asp Thr Asp Ser Lys Asn Ser Ser Arg Tyr145 150 155 160Leu Leu His Leu Ser Gln Ser Gly Ile Gly Leu Pro Asp Glu Ser Tyr 165 170 175Phe Arg Glu Glu Arg His Ala Glu Ile Leu Ala Ala Tyr Pro Gly His 180 185 190Ile Ala Ala Met Phe Gly Leu Val Tyr Gly Gly Asp Pro Asp Asp His 195 200 205Ala Ala Thr Ala Asp Arg Ile Val Ala Leu Glu Thr Lys Ile Ala Ala 210 215 220Ala His Trp Asp Val Val Lys Arg Arg Asp Ala Asp Leu Thr Tyr Asn225 230 235 240Leu Arg Thr Phe Ala Glu Val Thr Asp Gln Ala Pro Gly Phe Asp Trp 245 250 255Pro Gly Trp Leu Ala Gly Leu Gly Gly Thr Ala Glu Gln Ala Ala Glu 260 265 270Val Val Val Arg Gln Pro Asp Tyr Leu Thr Ala Phe Ala Gly Leu Trp 275 280 285Ala Asp Glu Ser Leu Glu Asp Trp Lys Asn Trp Leu Arg Trp Arg Val 290 295 300Ile His Ala Arg Ala Phe Leu Leu Thr Asp Glu Leu Ile Ala Glu Asp305 310 315 320Phe Ser Phe Tyr Gly Arg Arg Leu Ser Gly Thr Glu Glu Ile Arg Glu 325 330 335Arg Trp Lys Arg Gly Val Ser Val Val Glu Ser Leu Met Gly Glu Ala 340 345 350Leu Gly Arg Leu Tyr Val Glu Arg His Phe Pro Pro His Ala Lys Ala 355 360 365Arg Met Asp Glu Leu Val Asp Asn Leu Arg Glu Ala Tyr Arg Val Ser 370 375 380Ile Asn Ser Leu Asp Trp Met Thr Pro Gln Thr Arg Glu Lys Ala Leu385 390 395 400Val Lys Leu Asp Lys Phe Thr Pro Lys Ile Gly Tyr Pro Asn Lys Trp 405 410 415Arg Asp Tyr Ser Ala Leu Val Ile Ala Arg Asp Asp Leu Tyr Gly Asn 420 425 430Tyr Gln Arg Gly Tyr Ala Leu Glu Tyr Asp Arg Asp Leu Ala Lys Leu 435 440 445Gly Gly Pro Val Asp Arg Asp Glu Trp Phe Met Thr Pro Gln Thr Val 450 455 460Asn Ala Tyr Tyr Asn Pro Gly Met Asn Glu Ile Val Phe Pro Ala Ala465 470 475 480Ile Leu Gln Pro Pro Phe Phe Asp Ala Asp Ala Asp Asp Ala Ala Asn 485 490 495Tyr Gly Gly Ile Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe 500 505 510Asp Asp Gln Gly Ala Lys Tyr Asp Gly Asp Gly Asn Leu Val Asn Trp 515 520 525Trp Thr Asp Gln Asp Arg Asp Glu Phe Gly Leu Arg Thr Lys Ala Leu 530 535 540Ile Glu Gln Tyr Glu Glu Leu Val Pro Arg Gly Leu Glu Pro Ser His545 550 555 560His Val Asn Gly Ala Phe Thr Val Gly Glu Asn Ile Gly Asp Leu Gly 565 570 575Gly Leu Ser Ile Ala Leu Leu Ala Tyr Arg Leu Ser Leu Lys Gly Glu 580 585 590Pro Ala Pro Val Ile Asp Gly Leu Thr Gly Glu Gln Arg Val Phe Phe 595 600 605Gly Trp Ala Gln Val Trp Arg Thr Lys Ser Arg Glu Ala Glu Ala Ile 610 615 620Arg Arg Leu Ala Val Asp Pro His Ser Pro Pro Glu Phe Arg Cys Asn625 630 635 640Ala Val Val Arg Asn Met Asp Ala Phe Tyr Asp Ala Phe Glu Val Asp 645 650 655Glu Asp Asp Glu Leu Tyr Leu Glu Pro Gln Arg Arg Val His Ile Trp 660 665 670Asn56661PRTBeutenbergia cavernae 56Met Thr Thr Thr Glu Ala Ala Pro Ser Pro Ala Ala Asp Asp Pro Thr1 5 10 15Ala Ala Ala Asp Pro Ala Val Arg Pro Gln Asp Asp Leu Tyr Arg His 20 25 30Val Asn Gly Thr Trp Leu Arg Thr His Glu Ile Pro Ala Asp Arg Ala 35 40 45Arg Asp Gly Val Val Phe Asp Leu Arg Asp Gln Ala Glu Leu Asp Val 50 55 60Arg Ala Ile Val Glu Glu Ser Gly Ala Gly Thr Leu Asp Ala Pro Asp65 70 75 80Ala Asp Glu Ala Arg Lys Ile Ala Ala Leu Tyr Arg Ser Phe Met Asp 85 90 95Thr Glu Ala Ile Glu Ala Ala Gly Val Ala Pro Leu Ala Pro Asp Leu 100 105 110Ala Leu Ile Glu Asn Ala Thr Asp Arg Ser Ala Leu Ala Arg Ala Met 115 120 125Gly Thr Leu Gln Arg Thr Gly Val Gly Gly Ala Leu Gly Met Trp Val 130 135 140Asp Thr Asp Ala Asp Asp Pro Thr Ala Tyr Arg Ala Tyr Leu Tyr Gln145 150 155 160Ser Gly Leu Gly Leu Pro Asp Glu Ser Tyr Tyr Arg Ser Glu Glu Gln 165 170 175Ala Thr Thr Arg Glu Ala Tyr Val Ala His Val Ala Arg Met Leu Arg 180 185 190Leu Ala Gly Val Ala Ala Asp Ala Glu Ala Asp Ala Val Ala Gly Arg 195 200 205Ile Met Asp Leu Glu Thr Arg Leu Ala Ala Ala His Trp Asp Arg Val 210 215 220Arg Asp Arg Asp Ala Val Ala Thr His Asn Pro Thr Ala Trp Ala Glu225 230 235 240Leu Ala Asp Val Ala Arg Gly Phe Asp Ala Asp Ala Trp Ala Glu Gly 245 250 255Leu Arg Val Pro Ala Asp Ala Phe Asp Val Val Val Leu Arg Glu Pro 260 265 270Ser Phe Phe Ala Ala Leu Gly Glu Ala Trp Thr Asp Val Pro Leu Glu 275 280 285Gln Trp Arg Glu Trp Leu Thr Trp Arg Val Val Arg Ser Arg Ala Pro 290 295 300Tyr Leu Thr Asp Glu Ile Val Thr Ala Asn Phe Asp Phe Tyr Gly Arg305 310 315 320Thr Leu Thr Gly Ala Gln Glu Leu Arg Glu Arg Trp Lys Arg Gly Val 325 330 335Ala Leu Val Glu Gly Ala Leu Gly Glu Ala Val Gly Arg Ile Tyr Val 340 345 350Gln Arg His Phe Pro Pro Thr His Lys Ala Arg Met Asp Thr Leu Val 355 360 365Ala Asn Leu Val Glu Ala Tyr Arg Arg Ser Ile Thr Ser Leu Asp Trp 370 375 380Met Gly Pro Asp Thr Arg Glu Arg Ala Leu Ala Lys Leu Ala Ala Phe385 390 395 400Thr Pro Lys Ile Gly Tyr Pro Val Arg Trp Arg Asp Tyr Ser Ser Leu 405 410 415Glu Leu Val Pro Gly Asp Val Val Ala Asn Val Arg Ala Ala Ser Thr 420 425 430Tyr Glu Leu Asp Arg Asp Leu Ala Lys Ile Gly Arg Pro Val Asp Arg 435 440 445Asp Glu Trp Phe Met Pro Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro 450 455 460Gly Met Asn Glu Val Val Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe465 470 475 480Phe Asp Pro Asp Ala Ala Asp Ala Ala Asn Tyr Gly Ser Ile Gly Ala 485 490 495Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln Gly Ser Arg 500 505 510Tyr Asp Gly Asp Gly Arg Leu Ala Asp Trp Trp Thr Pro Glu Asp Arg 515 520 525Ala Glu Phe Glu Thr Arg Thr Ala Ala Leu Val Ala Gln Tyr Asp Ala 530 535 540Phe Ser Pro Ala Gln Leu Asp Gly Ser Arg His Val Ser Gly Gly Leu545 550 555 560Thr Val Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ala Ile Ala Val 565 570 575Asp Ala Tyr Glu Ile Ala Leu Gly Arg Arg Pro Asp Arg Ala Glu Leu 580 585 590Arg Glu Leu Phe Ala Ser Trp Ala Val Ser Trp Arg Glu Lys Gly His 595 600 605Asp Ala Glu Val Ile Arg Leu Leu Thr Ile Asp Pro His Ser Pro Pro 610 615 620Glu Phe Arg Cys Asn Gly Val Val Ala Asn Leu Asp Ala Phe Ala Glu625 630 635 640Ala Phe Asp Val Gln Pro Gly Asp Gly Leu Trp Ile Asp Pro Gln Asp 645 650 655Arg Val Arg Ile Trp 66057652PRTRhodococcus erythropolis 57Met Thr Thr Ala His Arg Ser Gly Ile Asp Leu Thr His Leu Asp Gly1 5 10 15Asp Thr Arg Ala Gln Asp Asp Leu Phe Val His Val Asn Gly Lys Trp 20 25 30Leu Ala Glu Tyr Glu Ile Pro Ala Asp Arg Ala Val Asp Gly Ala Phe 35 40 45Arg Thr Leu Tyr Asp Lys Ala Glu Glu Asp Val Gln Thr Ile Ile Thr 50 55 60Glu Ala Ala Glu Ser Asn Ser Ala Ala Gly Thr Asp Ala Gln Arg Ile65 70 75 80Gly Asp Leu Tyr Ser Ser Phe Met Asp Ala Asp Ala Val Glu Ala Ala 85 90 95Gly Leu Gly Pro Ile Asp Asp Glu Leu Asp Ala Val Ala Asp Ala Pro 100 105 110Asp Arg Ser Ser Leu Ala Thr Val Ile Gly Arg His Gln Arg Val Gly 115 120 125Val Gly Gly Ala Ile Ser His Tyr Val Asp Thr Asp Ala Lys Asn Ser 130 135 140Glu Arg Tyr Leu Ile His Phe Ser Gln Ser Gly Ile Ser Leu Pro Asp145 150 155 160Glu Ser Tyr Tyr Arg Glu Asp Asn Tyr Ala Glu Ile Arg Glu Lys Tyr 165 170 175Val Ala His Ile Asp Lys Met Phe Thr Leu Ala Gly Val Gly Tyr Asp 180 185 190Ala Gln Arg Val Phe Glu Leu Glu Lys Lys Ile Ala Ala Gly His Trp 195 200 205Asp Val Val Lys Arg Arg Asp Ala Asp Leu Ser Tyr Asn Leu Val Gly 210 215 220Phe Glu Glu Leu Thr Glu Gln Asn Pro Gly Leu Asp Trp Ala Ala Trp225 230 235 240Leu Ser Gly Leu Gly Ala Asp Ser Glu Lys Phe Ala Glu Ile Val Val 245 250 255Arg Gln Pro Asp Phe Leu Arg Thr Phe Thr Ala Leu Trp Ala Ser Glu 260 265 270Ser Leu Asp Asp Trp Lys Ala Trp Ala Ala Trp Lys Val Leu His Ala 275 280 285Arg Ser Pro Tyr Leu His Ser Ala Leu Val Asp Glu Thr Phe Ala Phe 290 295 300Tyr Gly Thr Thr Leu Ser Gly Thr Glu Glu Asn Arg Glu Arg Trp Lys305 310 315 320Arg Gly Val Ser Leu Val Gln Asp Leu Leu Gly Glu Ala Val Gly Lys 325 330 335Leu Tyr Val Asp Arg His Phe Pro Ala Glu Ala Lys Thr Arg Met Leu 340 345 350Glu Leu Val Ala Asn Leu Gln Glu Ala Tyr Arg Arg Asn Ile Ser Asp 355 360 365Leu Glu Trp Met Ser Pro Gln Thr Arg Glu Ala Ala Leu Ala Lys Leu 370 375 380Glu Lys Phe Thr Pro Lys Ile Gly Tyr Pro Asp Lys Trp Arg Asp Tyr385 390 395 400Ala Gly Leu Glu Ile Ser Ala Thr Asp Leu Val Gly Asn Tyr Arg Arg 405 410 415Gly Tyr Ala Ala Glu Tyr Asp Arg Asp Leu Ala Lys Leu Gly Gly Pro 420 425 430Val Asp Arg Asp Glu Trp Phe Met Thr Pro Gln Thr Val Asn Ala Tyr 435 440 445Tyr Asn Pro Gly Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln 450 455 460Pro Pro Phe Phe Asp Ala Ala Ala Asp Asp Ala Ala Asn Tyr Gly Gly465 470 475 480Ile Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln 485 490 495Gly Ala Lys Tyr Asp Gly Asp Gly Asn Met Val Asp Trp Trp Thr Asp 500 505 510Glu Asp Arg Ser Glu Phe Gly Lys Arg Thr Lys Ala Leu Ile Glu Gln 515 520 525Tyr Asn Glu Phe Glu Pro Lys Glu Leu Pro Gly His His Val Asn Gly 530 535 540Glu Phe Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile545 550 555 560Ala Ile Ala Ala Tyr Lys Ile Ala Thr Glu Gly Thr Glu Pro Pro Val 565 570 575Ile Asp Gly Leu Thr Gly Leu Gln Arg Val Phe Phe Gly Trp Ala Gln 580 585 590Val Trp Arg Thr Lys Ala Arg Glu Ala Glu Ala Leu Arg Arg Leu Ala 595 600 605Val Asp Pro His Ser Pro Pro Glu Phe Arg Cys Asn Gly Val Val Arg 610 615 620Asn Leu Asp Thr Phe His Glu Glu Phe Asp Val Lys Pro Gly Asp Ala625 630 635 640Leu Tyr Leu Glu Pro Glu Lys Arg Val Lys Ile Trp 645 65058652PRTRhodococcus jostii 58Met Glu Arg Met Thr Thr Ala Gln Arg Ser Gly Ile Asp Leu Thr His1 5 10 15Leu Asp Asn Asp Thr Arg Ala Gln Asp Asp Leu Phe Val His Val Asn 20 25 30Gly Lys Trp Ile Asp Asp Tyr Glu Ile Pro Ala Asp Arg Ala Ile Asp 35 40 45Gly Ala Phe Arg Thr Leu Tyr Asp Lys Ala Glu Val Asp Val

Gln Thr 50 55 60Ile Ile Glu Glu Ala Ala Asp Ser Gly Ala Ala Pro Gly Thr Asp Ala65 70 75 80Gln Arg Ile Gly Asp Leu Tyr Gly Ser Phe Met Asp Ala Asp Val Val 85 90 95Glu Ala Ala Gly Leu Ala Pro Ile Ala Gly Glu Leu Ala Asp Val Ala 100 105 110Ser Ala Ala Asp Leu Ser Ala Leu Ala Ala Val Ile Gly Arg Gln Gln 115 120 125Arg Thr Gly Val Gly Gly Ala Val Gly His Tyr Val Asp Thr Asp Ala 130 135 140Lys Asn Ser Glu Arg Tyr Leu Val His Phe Ser Gln Ser Gly Ile Gly145 150 155 160Leu Pro Asp Glu Ser Tyr Tyr Arg Gln Asp Asp His Ala Glu Ile Arg 165 170 175Ala Ala Tyr Val Lys His Ile Ala Lys Met Phe Ala Leu Ala Gly Val 180 185 190Asp Tyr Asp Ala Gln Arg Val Phe Asp Leu Glu Thr Lys Ile Ala Ala 195 200 205Gly His Trp Asp Val Val Lys Arg Arg Asp Ala Glu Leu Ser Tyr Asn 210 215 220Leu Leu Thr Leu Asp Gln Leu Pro Ala Gly Leu Asp Trp Ser Ser Trp225 230 235 240Ile Gly Ala Leu Gly Gly Thr Ser Glu Gln Phe Ala Glu Ile Val Val 245 250 255Arg Gln Pro Asp Phe Leu Thr Thr Leu Thr Glu Leu Trp Thr Ser Glu 260 265 270Asp Ile Asp Asp Trp Lys Ala Trp Ala Thr Trp Asn Val Ile Arg Ser 275 280 285Arg Ala Pro Tyr Leu Thr Gln Ala Leu Val Asp Glu Asn Phe Ala Phe 290 295 300Tyr Gly Lys Thr Leu Thr Gly Ala Glu Glu Ile Arg Glu Arg Trp Lys305 310 315 320Arg Gly Val Ser Leu Val Gln Asp Leu Leu Gly Glu Ala Val Gly Lys 325 330 335Leu Tyr Val Glu Arg His Phe Pro Ala Asp Ala Lys Ala Arg Met Gln 340 345 350Glu Leu Val Ala Asn Leu Gln Glu Ala Tyr Arg Arg Asn Ile Ser Asp 355 360 365Leu Asp Trp Met Ser Pro Glu Thr Arg Gln Ala Ala Leu Arg Lys Leu 370 375 380Glu Lys Phe Thr Pro Lys Ile Gly Tyr Pro Asp Lys Trp Arg Asp Tyr385 390 395 400Ser Ala Val Thr Ile Ser Arg Asp Asp Leu Val Gly Asn Tyr Arg Ser 405 410 415Gly Tyr Ala Ala Glu Tyr Asp Arg Asp Leu Ala Lys Leu Gly Gly Pro 420 425 430Val Asp Arg Asp Glu Trp Phe Met Thr Pro Gln Thr Val Asn Ala Tyr 435 440 445Tyr Asn Pro Gly Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln 450 455 460Pro Pro Phe Phe Asp Ala Ala Ala Asp Asp Ala Ala Asn Tyr Gly Gly465 470 475 480Ile Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln 485 490 495Gly Ala Lys Tyr Asp Gly Asp Gly Asn Met Val Asp Trp Trp Thr Asp 500 505 510Asp Asp Arg Thr Glu Phe Gly Lys Arg Thr Lys Ala Leu Ile Glu Gln 515 520 525Tyr Asn Glu Phe Glu Pro Lys Ala Leu Pro Gly His Asn Val Asn Gly 530 535 540Glu Phe Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile545 550 555 560Ala Ile Ala Ala Tyr Arg Ile Ala Thr Glu Gly Ser Glu Pro Glu Val 565 570 575Leu Asp Gly Leu Thr Gly Ile Gln Arg Val Phe Phe Gly Trp Ala Gln 580 585 590Val Trp Arg Thr Lys Ala Arg Asp Ala Glu Ala Leu Arg Arg Leu Ala 595 600 605Val Asp Pro His Ser Pro Pro Glu Phe Arg Cys Asn Gly Val Val Arg 610 615 620Asn Leu Asp Thr Phe His Asp Ala Phe Asp Val Lys Pro Gly Asp Ala625 630 635 640Leu Tyr Leu Asp Gln Glu Glu Arg Val Lys Ile Trp 645 65059651PRTNakamurella multipartita 59Met Thr Ala Ser Pro Ala Ala Asp Val Ile Val Pro Arg Pro Gln Asp1 5 10 15Asp Leu Phe Arg His Val Asn Gly Pro Trp Leu Ala Thr Ala Glu Ile 20 25 30Pro Ala Asp Arg Ser Ala Asp Gly Ala Phe Tyr Gln Leu Arg Asp Glu 35 40 45Ala Glu Lys Asp Ser Arg Ala Ile Ile Glu Asp Ala Ala Ala Ala Ala 50 55 60Asp Gly Ala Glu Pro Gly Ser Pro Val Gln Leu Ile Gly Asp Leu Tyr65 70 75 80Arg Ser Phe Met Asp Val Glu Ala Val Glu Arg Gln Gly Leu Ala Pro 85 90 95Ile Ala Ala Arg Leu Thr Glu Val Glu Gly Val Asp Ser Pro Ala Ala 100 105 110Leu Met Arg Thr Leu Gly Arg Leu Arg Arg Ser Gly Val Gly Gly Ala 115 120 125Phe Ala Ile Asp Val Asp Thr Asp Pro Gly Asp Pro Asp Arg Tyr Val 130 135 140Leu Asn Leu Tyr Gln Gly Gly Ile Gly Leu Pro Asp Glu Ser Tyr Tyr145 150 155 160Ser Asp Ala Ala His Ala Asp Val Leu Ser Ala Tyr Ala Ala Phe Leu 165 170 175Pro Ser Ile Leu Glu Leu Ala Gly Ile Pro Glu Ser Ala Gly Ala Gly 180 185 190Ala Ala Val Val Glu Leu Glu Thr Ala Val Ala Ala Gly His Trp Asp 195 200 205Arg Val Arg Ser Arg Asp Ser Ser Gln Thr Tyr Asn Pro Lys Asp Arg 210 215 220Ala Gly Leu Asp Ala Leu Leu Pro Gly Pro Leu Trp Asp Ala Trp Leu225 230 235 240Asp Gly Leu Gly Ala Asp Pro Ser Val Leu Asp Gln Val Val Val Arg 245 250 255Gln Pro Asp Tyr Phe Thr Ala Leu Ala Ala Leu Leu Thr Pro Asp His 260 265 270Leu Pro Ala Trp Arg Ala Trp Leu Ser Trp Gln Ile Val Arg Ser Leu 275 280 285Ala Pro Leu Gly Pro Ala Glu Leu Val Glu Lys Asn Phe Asp Phe Tyr 290 295 300Gly Arg Thr Leu Ser Gly Thr Pro Glu Leu Arg Glu Arg Trp Lys Arg305 310 315 320Gly Val Gly Phe Val Glu Met Ala Ala Asn Glu Ala Val Gly Arg Leu 325 330 335Tyr Val Glu Arg His Phe Pro Pro Glu Ser Lys Arg Arg Met Asp Glu 340 345 350Leu Val Ala Asn Leu Leu Ala Ala Tyr Arg Thr Glu Ile Gly Lys Leu 355 360 365Pro Trp Met Gly Glu Gln Thr Arg Ala Arg Ala Leu Glu Lys Leu Asp 370 375 380Ala Phe Thr Pro Lys Ile Gly Tyr Pro Ala Arg Trp Arg Asp Tyr Thr385 390 395 400Ala Leu Thr Val Ala Ala Asp Asp Leu Ile Gly Asn Ala Ala Arg Ala 405 410 415Ala Ala Phe Glu Leu Asp Arg Glu Leu Gly Lys Leu Gly Gly Pro Val 420 425 430Asp Arg Asp Glu Trp Phe Met Ser Pro Gln Thr Val Asn Ala Tyr Tyr 435 440 445Asn Pro Gly Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln Pro 450 455 460Pro Phe Phe Asp Pro Glu Ala Asp Asp Ala Val Asn Tyr Gly Gly Ile465 470 475 480Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln Gly 485 490 495Ser Lys Tyr Asp Gly Arg Gly Ala Leu Gln Asp Trp Trp Thr Pro Ala 500 505 510Asp Arg Ala Ala Phe Glu Gln Leu Thr Gly Arg Leu Ile Asp Gln Tyr 515 520 525Ser Ala Leu Glu Pro Lys Asn Thr Pro Gly His His Val Asn Gly Ala 530 535 540Leu Thr Ile Gly Glu Asn Ile Gly Asp Val Gly Gly Leu Gly Ile Ala545 550 555 560Tyr Gln Ala Trp Arg Ile Ser Leu Gly Asp Gln Ser Ala Pro Val Ile 565 570 575Asp Gly Leu Thr Gly Ala Gln Arg Phe Phe Arg Ser Trp Ala Thr Val 580 585 590Trp Arg Leu Lys Met Arg Glu Ala Glu Gln Val Arg Met Leu Ser Ile 595 600 605Asp Pro His Ser Pro Ala Glu Phe Arg Cys Asn Gln Val Val Arg Asn 610 615 620Ile Ala Glu Phe His Glu Ala Phe Asp Thr Arg Pro Thr Asp Gly Leu625 630 635 640Trp Leu Asp Glu Gln Asp Arg Val Arg Ile Trp 645 65060667PRTMobiluncus mulieris 60Met Thr Lys Asn Pro Leu Leu Gly Ala Leu Asp Pro Asn Glu Phe Asp1 5 10 15Asn Asn Ile Arg Pro Gln Asp Asp Leu Tyr Arg Phe Val Asn Gly Lys 20 25 30Trp Ile Glu Ala Cys Gln Ile Pro Ala Asp Lys Ser Ala Asp Gly Ser 35 40 45Phe Tyr Val Leu Thr Glu Asn Ser Glu Lys Gln Val Arg Ala Ile Ile 50 55 60Glu Asp Cys Val Glu Gly Lys Ile Ala Gly Glu Leu Ala Gly Lys Val65 70 75 80Ala Ala Leu Tyr Gly Ser Phe Met Asp Glu Ala Gln Val Glu Ala Asp 85 90 95Ser Ala Pro Ser Leu Glu Arg Leu Phe Gly Pro Leu Arg Thr Ala Lys 100 105 110Thr Lys Gly Asp Leu Arg Asp Leu Trp Ala Lys Thr Trp Lys Met Gly 115 120 125Thr Tyr Asp Gly Phe Phe Ser Met Gly Ile Asp Ile Asp Leu Asn Asn 130 135 140Pro Asp Cys Tyr Ile Asn Tyr Phe Ser Gln Asp Gly Ile Gly Leu Pro145 150 155 160Glu Arg Ala Tyr Phe Leu Glu Glu Lys His Arg Glu Val Leu Gln Ala 165 170 175Tyr Arg Glu His Val Gly Arg Met Phe Ser Leu Ser Gly Phe Met Ser 180 185 190Ser Glu Ser Gln Ser Gln Ala Ala Gly Asn Thr Val Val Asp Phe Glu 195 200 205Thr Glu Ile Ala Arg Leu His Trp Asp Asn Val Lys Thr Arg Asp Thr 210 215 220Asp Ala Thr Asn Asn Pro Met Thr Trp Ala Glu Leu Ala Ala Lys Leu225 230 235 240Pro Val Phe Asp Leu Asp Ala Trp Arg Ala Ala Ser Arg Leu Pro Glu 245 250 255Lys Met Phe Thr Gln Val Asn Val Gly Met Pro Asp Phe Phe Ala Gly 260 265 270Met Asp Gln Leu Trp Ala Glu Thr Asp Phe Glu Thr Leu Arg Leu Trp 275 280 285Met Thr Trp Gln Ala Leu Asn Gly Gln Val Ser Leu Leu Ser Asn Ala 290 295 300Leu Val Gln Ala Asn Phe Glu Phe Tyr Gly Lys Arg Leu Ala Gly Arg305 310 315 320Glu Val Ile Arg Asp Arg Trp Lys Arg Gly Val Ser Leu Ala Ser Ser 325 330 335Val Met Gly Glu Ala Leu Gly Gln Leu Tyr Val Ala Arg His Phe Pro 340 345 350Pro Asp Ser Lys Glu Lys Met Ser Arg Leu Val Asp Asn Leu Ile Ala 355 360 365Ala Tyr Arg Glu Ser Ile Ser Ser Leu Glu Trp Met Gly Ser Glu Thr 370 375 380Arg Ala Lys Ala Leu Glu Lys Leu Ser Leu Phe Thr Pro Lys Ile Gly385 390 395 400Tyr Pro Asp Lys Trp Arg Asn Tyr Glu Lys Leu Asp Val Ser Ala Pro 405 410 415Thr Leu Val Glu Lys Val Ala Ser Ala Ser Gln Phe Gly Thr Asp Phe 420 425 430Trp Ile Asp Lys Leu Gly Gly Pro Val Asp His Thr Ile Trp His Met 435 440 445Thr Pro Gln Thr Val Asn Ala Tyr Tyr Asn Pro Thr Asp Asn Glu Ile 450 455 460Val Phe Pro Ala Gly Ile Leu Gln Pro Pro Phe Phe Asp Pro Gln Ala465 470 475 480Asp Asp Ala Val Asn Tyr Gly Ser Ile Gly Ala Val Ile Gly His Glu 485 490 495Ile Gly His Gly Phe Asp Asp Gln Gly Ala Lys Phe Asp Gly His Gly 500 505 510Lys Val Glu Asn Trp Trp Thr Glu Thr Asp Leu Lys Glu Phe Glu Lys 515 520 525Arg Thr Arg Ala Leu Ile Ala Gln Tyr Asp Gln Tyr Val Pro Arg Gly 530 535 540Leu Pro Glu Glu Phe His Val Asn Gly Ala Leu Thr Ile Gly Glu Asn545 550 555 560Ile Gly Asp Leu Gly Gly Leu Asp Ile Ala Trp Lys Ala Tyr Leu Leu 565 570 575Ala Leu Lys Asp Gln Gly Ile Asn Asp Pro Ala Asp Ala Pro Val Ile 580 585 590Glu Gly Tyr Thr Gly Ala Gln Arg Phe Phe Tyr Ser Trp Ala Leu Ser 595 600 605Trp Gln Asn Lys Thr Arg Val Glu Ala Ala Lys Gln Leu Ile Ala Ile 610 615 620Asp Pro His Ser Pro Ala Glu Phe Arg Cys Asn Gly Val Val Ala Asn625 630 635 640Leu Asp Leu Phe Ala Lys Thr Phe Gly Leu Lys Pro Gly Asp Asp Leu 645 650 655Trp Ile Glu Pro Glu Asn Arg Val Arg Ile Trp 660 66561676PRTTsukamurella paurometabola 61Met Thr Ser Glu Pro Ala Leu Asp Leu Ser Phe Val Asp Pro Asp Ile1 5 10 15Arg Val Gln Asp Asp Leu Phe Gly His Val Asn Gly Lys Trp Leu Asp 20 25 30Gly His Glu Ile Pro Ala Asp Arg Ser Ser Asp Gly Ala Phe Tyr Ala 35 40 45Leu Arg Asp Ala Ala Glu Leu His Val Lys Glu Ile Val Glu Glu Ala 50 55 60Ala Ala Asp Ala Ala Ala Thr Gly Asp Ala Ala Lys Ile Gly Asp Leu65 70 75 80Tyr Ser Ser Phe Met Asp Thr Ala Ala Ile Glu Ala Ala Gly Val Ala 85 90 95Pro Val Ala Asp Glu Ile Ala Glu Ile Arg Trp Ala Asp Ser Ala Thr 100 105 110Asp Leu Ala Thr Val Leu Gly Arg Leu Gln Arg Thr Gly Val Gly Gly 115 120 125Leu Leu Gly Tyr Tyr Val Asp Thr Asp Ala Lys Arg Ser Asp Arg Tyr 130 135 140Leu Val Gln Val Thr Gln Ser Gly Ile Ser Leu Pro Asp Glu Ala Tyr145 150 155 160Tyr Arg Asp Glu Gln Tyr Ala Pro Leu Arg Ala Lys Phe Thr Ala His 165 170 175Val Ala Asp Thr Phe Arg Leu Ala Thr Arg Leu Ala Pro Ala Leu Leu 180 185 190Glu Gly Ile Val Thr Ala Gly Glu Glu Asp Ala Ala Ala Ala Ser Val 195 200 205Leu Asp Leu Glu Thr Ala Ile Ala Ala Gly His Trp Asn Val Val Asp 210 215 220Arg Arg Asp Ala Asp Lys Ser Tyr Asn Leu Arg Thr Phe Ala Asp Leu225 230 235 240Gly Thr Glu Ala Pro Ala Leu Gln Leu Arg Ala Trp Thr Ala Ala Leu 245 250 255Thr Ala Ala Glu Ala Phe Ala Glu Val Phe Ala Glu Val Asn Val Arg 260 265 270Gln Pro Ser Phe Val Thr His Ala Ala Ala Leu Leu Ser Asp Arg Pro 275 280 285Leu Ala Gln Trp Arg Thr Trp Leu Ala Trp Arg Val Leu Arg Ala Arg 290 295 300Ser Pro Tyr Leu Ser Asp Glu Leu Val Ala Ala Asn Phe Ala Phe Tyr305 310 315 320Gly Thr Ala Leu Thr Gly Ala Thr Glu Asn Arg Glu Arg Trp Lys Arg 325 330 335Gly Val Gly Leu Val Glu Glu His Leu Gly Phe Ala Val Gly Glu Leu 340 345 350Tyr Thr Ala Arg His Phe Pro Ala Asp Ser Lys Ala Arg Met Gln Ala 355 360 365Leu Val Ala Asp Leu Val Glu Ala Tyr Arg Arg Arg Ile Val Glu Leu 370 375 380Pro Trp Met Thr Pro Ala Thr Arg Glu Arg Ala Leu Glu Lys Leu Gly385 390 395 400Lys Phe Thr Pro Lys Ile Gly Tyr Pro Asp Ala Ala Arg Asp Tyr Ser 405 410 415Ser Leu Gln Ile Gln Arg Asp Asp Leu Leu Gly Asn Val Arg Arg Gly 420 425 430Asn Ala Ala Glu His Asp Arg Glu Phe Ala Lys Ile Gly Ala Pro Val 435 440 445Asp Arg Asp Glu Trp Phe Met Thr Pro Gln Thr Val Asn Ala Tyr Tyr 450 455 460Asn Pro Gly Met Asn Glu Ile Val Phe Pro Ala Ala Ile Leu Gln Pro465 470 475 480Pro Phe Phe Ala Pro Thr Ala Asp Asp Ala Val Asn Phe Gly Gly Ile 485 490 495Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln Gly 500 505 510Ala Lys Tyr Asp Gly Asp Gly Asn Leu Glu Asp Trp Trp Thr Asp Thr 515 520 525Asp Arg Glu Glu Phe Gly Lys Arg Thr Arg Ala Leu Ile Glu Gln Tyr 530 535 540Asp Glu Leu Thr Pro Arg Glu Leu

Gly Ala Asp Ser Glu His His Val545 550 555 560Asn Gly Gly Phe Thr Val Gly Glu Asn Ile Gly Asp Leu Gly Gly Leu 565 570 575Gly Ile Ala Leu Val Ala Tyr Gly Ile Ala Arg Glu Arg Ala Gly Gly 580 585 590Thr Val Asp Asp Pro Ser Thr Glu Ala Asp Gly Leu Thr Gly Leu Gln 595 600 605Arg Val Phe Tyr Ser Trp Gly Gln Ile Trp Arg Gly Lys Ser Arg Pro 610 615 620Glu Glu Ala Ile Arg Arg Leu Ala Ile Asp Pro His Ser Pro Pro Glu625 630 635 640Phe Arg Cys Asn Ala Ile Ala Ser Asn Leu Asp Glu Phe Tyr Ser Ala 645 650 655Phe Gly Val Thr Glu Gly Asp Thr Leu Phe Leu Ala Pro Glu Arg Arg 660 665 670Val Ser Ile Trp 67562677PRTCorynebacterium amycolatum 62Met Thr Asn Ser Thr Asp Thr Glu Thr Gln Ser Ala Thr Ser Gln Phe1 5 10 15Ser Thr Thr Pro Arg Pro Gln Asp Asp Leu Tyr Leu Ala Ile Asn Gly 20 25 30Glu Trp Ile Ser Asn His Val Ile Pro Ala Asp Arg Pro Ile Asp Gly 35 40 45Ala Phe Tyr Lys Leu Arg Asp Glu Ala Glu Glu Asn Val Arg Glu Leu 50 55 60Ile Thr Thr Ala Ala Asp Ala Asp Pro Asn Ser Arg Val Ala Arg Leu65 70 75 80Tyr Asn Ser Phe Met Asp Glu Ala Ala Ile Asn Asp Ala Gly Ala Ala 85 90 95Pro Leu Ala Gln Asp Leu Glu Met Ile Ala Ser Ala Glu Asn Ala His 100 105 110Glu Leu Ala Leu Ala Leu Gly Arg Leu Asp Arg Leu Gly Val Gly Gly 115 120 125Ala Leu Gly Tyr Trp Val Glu Lys Asp Ser Gly Ser Asp Leu Asp Ala 130 135 140Leu Tyr Leu Leu Gln Ser Gly Leu Gly Leu Pro Asp Glu Ala Tyr Tyr145 150 155 160Arg Glu Ser Gly His Ala Asp Thr Leu Ala Ala Tyr Glu Gln His Val 165 170 175Ala Ala Met Leu Lys Leu Leu Ser Ser His Asp Asp Gly Ala Gly Asp 180 185 190Leu Ser Thr Ala Leu Ala Ala Phe Glu Leu Val Asp Pro Ala Ala Ala 195 200 205Ala Ala Arg Ile Val Ala Phe Glu Lys Arg Ile Ala Ala Gly His Trp 210 215 220Asn Val Val Asp Thr Arg Asp Ala Leu Lys Thr Tyr Asn Lys Thr Ala225 230 235 240Ile Ala Asp Leu Pro Thr Gly Phe Pro Val Ala Glu Trp Leu Ala Ala 245 250 255Thr Gly Val Asn Glu Thr Asn Gln Thr Ala Ile Asp Thr Ile Val Val 260 265 270Met Met Pro Ser Tyr Phe Glu His Leu Ser Lys Leu Trp Gln Asp Thr 275 280 285Asp Leu Ala Asp Leu Arg Leu Trp Ala Leu Trp Arg Val Leu His Gln 290 295 300Arg Ala Ala Tyr Leu Ser Asp Asp Phe Ser Ala Glu Ser Phe Asn Phe305 310 315 320Tyr Gly Arg Thr Leu Gln Gly Ser Thr Glu Gln Arg Ala Arg Trp Lys 325 330 335Arg Gly Val Ala Phe Ala Asp Gly Ala Val Gly His Asp Val Gly Lys 340 345 350Leu Tyr Val Glu Lys His Phe Pro Pro Glu Tyr Lys Glu Gln Val Leu 355 360 365Glu Leu Val Asp Tyr Leu Leu Ala Ala Tyr Arg Glu Arg Ile Ser Gln 370 375 380Leu Pro Trp Met Thr Lys Ala Thr Gln Glu Arg Ala Leu Glu Lys Leu385 390 395 400Ser Leu Phe Lys Ala Lys Ile Gly Tyr Pro Glu Arg Trp Arg Asp Tyr 405 410 415Ser Ala Met Glu Leu Gly Gly Ser Leu Met Asp Asn Ala Arg Ala Ala 420 425 430Ser Ala Phe Ala His Asp Tyr Glu Val Ala Lys Leu Gly Thr Pro Ala 435 440 445Asn Arg Asp Glu Trp His Gly Thr Pro Gln Thr Val Asn Ala Phe Tyr 450 455 460Asn Pro Val Val Asn Asp Ile Thr Phe Pro Ala Ala Ile Leu Gln Pro465 470 475 480Pro Phe Phe Ser Pro Asp Ala Ser Pro Ala Glu Asn Phe Gly Gly Ile 485 490 495Gly Ala Val Ile Gly His Glu Ile Gly His Gly Phe Asp Asp Gln Gly 500 505 510Ser Gln Tyr Asp Gly His Gly Asn Leu His Gln Trp Trp Thr Asp Glu 515 520 525Asp Arg Ala Ala Phe Glu Lys Leu Thr Ser Ala Leu Val Asp Gln Tyr 530 535 540Glu Gly Leu Val Pro Gln Ala Leu Arg Glu Gln Ala Glu Glu Gly Ala545 550 555 560Asp Leu Pro Gly Val Asn Gly Arg Phe Thr Leu Gly Glu Asn Ile Gly 565 570 575Asp Leu Gly Gly Leu Gly Ile Ala Val Val Ala Phe Arg Arg Phe Leu 580 585 590Ala Ala Arg Gly Lys Glu Leu Gly Leu Glu Asp Ala Pro Glu Thr Tyr 595 600 605Arg Asp Leu Phe Lys Gln Trp Ala Leu Val Trp Arg Ser Lys Ile Arg 610 615 620Pro Glu Phe Ala Arg Gln Leu Leu Ala Ile Asp Pro His Ser Pro Ala625 630 635 640Glu Phe Arg Cys Asn Val Ile Ala Ser Asn Ile Asp Glu Phe His Glu 645 650 655Ala Phe Gly Thr Ser Ala Gly Asp Gly Met Trp Arg Glu Pro Asn Glu 660 665 670Arg Val Asn Ile Trp 67563724PRTBifidobacterium longum 63Met Thr Asn Glu Thr Glu Thr Leu Thr Ser Gly Ile Asp Pro Ala Ser1 5 10 15Phe Ser Ser Val Ile Lys Pro Thr Asn Asp Leu Phe Arg Tyr Val Asn 20 25 30Gly Pro Trp Ile Asp Thr Tyr Arg Leu Pro Asp Asp Arg Ser Arg Tyr 35 40 45Gly Ser Phe Asp Lys Leu Ala Glu Asp Ala Glu Asn Gln Val Arg Asp 50 55 60Ile Leu Asp Ala Asp Asp Cys Pro Ala Thr Lys Ser Gln Ala Leu Tyr65 70 75 80His Ser Phe Leu Asn Thr Asp Ala Ile Glu Ala Ala Gly Leu Asp Pro 85 90 95Ile Arg Asp Glu Leu Asp Leu Ile Asp Ser Ala Ile Asp Lys Ala Ala 100 105 110Leu Thr Arg Val Leu Gly Thr Ile Asn Pro Ala Gly Gly Pro Asp Leu 115 120 125Phe Gly Leu Ala Val Tyr Gly Asp Pro Gly Asp Pro Asp Arg Asn Ile 130 135 140Ala His Leu Glu Gln Ala Gly Leu Cys Leu Pro Asp Glu Ala Tyr Tyr145 150 155 160Arg Glu Asp His Tyr Val Pro Val Arg Glu Ala Tyr Val Ala Met Val 165 170 175Ala Thr Gln Leu Val Asn Ala Gly Tyr Ala Pro Ala Ala Glu Ser Asn 180 185 190Gly Gly Asp Glu Leu Pro Ala Asn Thr Gly Asp Asp Glu Ser Asn Ala 195 200 205Pro Thr Thr Val Pro Ser Glu Ala Ala Leu Asp Met Ala Arg His Phe 210 215 220Leu Ala Val Glu Thr Lys Ile Ala Ala Asn His Trp Asp Asn Val Ala225 230 235 240Thr Arg Asp Ser Val Lys Thr Tyr Asn Pro Thr Asp Tyr Ala Asp Leu 245 250 255Ala Ala Thr Leu Lys Asn Phe Asp Leu Ala Ser Trp Ile Asp Ala Trp 260 265 270Gln Thr Ala Tyr Asp Ala Thr Glu Ala Ala Lys Ala Gln Ser Ile Asp 275 280 285Phe Lys Ser Val Phe Ala Arg Ile Ile Val His Glu Pro Ser Phe Leu 290 295 300Thr Gly Leu Asp Ala Phe Trp Ala Glu Ala Asp Leu Ala Asp Leu Lys305 310 315 320Leu Trp Ala Arg Val His Met Ile Leu Gly Phe Ala Ser Ser Leu Ser 325 330 335Arg Asp Phe Asp Thr Thr Asn Phe Asp Phe Tyr Gly Lys Val Leu Ser 340 345 350Gly Ala Lys Lys Gln Arg Asp Arg Trp Lys Arg Ala Val Ser Leu Val 355 360 365Asn Gly Val Cys Gly Glu Asp Val Gly Arg Glu Tyr Ala Arg Leu His 370 375 380Phe Pro Glu Ser Ser Lys Arg Arg Met Glu Glu Leu Val Ala Asn Leu385 390 395 400Ile Asp Ala Tyr Arg Val Ser Ile Ala Asn Ser Asp Trp Leu Gly Glu 405 410 415Asp Thr Lys Ala Lys Ala Leu Glu Lys Ile Ser Lys Phe Thr Pro Lys 420 425 430Ile Gly Tyr Thr Asn His Trp Arg Asp Tyr Ser Ala Leu Ser Val Ser 435 440 445Ala Asp Ala Leu Pro Ala Glu Asn Ala Lys Ala Ala Asn Leu Tyr Glu 450 455 460Thr Gly Tyr Gln Leu Ala Lys Val Gly Lys Ala Val Asp Lys Asp Glu465 470 475 480Trp Leu Met Asn Pro Gln Thr Val Asn Ala Tyr Tyr Glu Pro Ser Met 485 490 495Asn Val Ile Val Phe Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp 500 505 510Pro Lys Ala Glu Asp Ala Ala Asn Tyr Gly Gly Ile Gly Ala Val Ile 515 520 525Gly His Glu Ile Gly His Gly Phe Asp Asp Gln Gly Ser Gln Tyr Asp 530 535 540Gly Asp Gly Lys Leu Asn Asn Trp Trp Thr Asp Glu Asp Arg Lys Asn545 550 555 560Phe Glu Ala Arg Thr Gly Ala Leu Ile Ala Gln Tyr Asn Ser Phe Val 565 570 575Pro Leu Gln Leu Ala Glu Lys Tyr Ala Asp Glu Ser Asp Lys Ala Pro 580 585 590His Val Asn Gly Ala Leu Thr Ile Gly Glu Asn Ile Gly Asp Leu Gly 595 600 605Gly Val Asn Ile Ala Leu Lys Ala Tyr Ala Phe Ala Leu Gly Lys Ala 610 615 620Ala Gly Lys Pro Asp Ala Glu Glu Asp Gly Ser Pro Ala Ala Ile Lys625 630 635 640Ala Leu Leu Asp Thr Ala Pro Glu Met Asp Gly Phe Thr Gly Leu Gln 645 650 655Arg Phe Phe Leu Ser Tyr Ala Ser Ile Trp Arg Thr Lys Asn Arg Asp 660 665 670Glu Leu Ala Glu Gln Tyr Leu Gln Ile Asp Pro His Ser Pro Ala Glu 675 680 685Phe Arg Thr Asn Gly Ile Ala Ser Asn Val Asp Leu Phe Tyr Asp Ala 690 695 700Phe Gly Val Thr Glu Gly Asp Ala Met Trp Leu Ala Pro Lys Asp Arg705 710 715 720Val Ser Ile Trp647PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 64Leu Pro Tyr Pro Gln Pro Gln1 5657PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 65Leu Pro Tyr Pro Gln Pro Gln1 5667PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 66Leu Pro Tyr Pro Gln Pro Gln1 5676PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 67Pro Gln Pro Gln Pro Phe1 56811PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 68Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 106911PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 69Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 107011PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 70Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 10717PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 71Pro Gln Pro Gln Leu Pro Tyr1 5727PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 72Pro Gln Pro Gln Leu Pro Tyr1 5737PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 73Pro Gln Pro Gln Leu Pro Tyr1 57414PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 74Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 107514PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 75Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 107610PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 76Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln1 5 107713PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 77Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe1 5 107810PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 78Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr1 5 107917PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 79Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro1 5 10 15Gln8017PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 80Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro1 5 10 15Gln8116PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 81Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe1 5 10 158213PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 82Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr1 5 108310PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 83Pro Gln Gln Pro Gln Gln Pro Tyr Pro Gln1 5 108413PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 84Pro Gln Gln Pro Gln Gln Pro Tyr Pro Gln Gln Pro Gln1 5 10858PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 85Pro Gln Gln Pro Gln Gln Pro Tyr1 5868PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 86Gln Pro Gln Gln Pro Tyr Pro Gln1 58710PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 87Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln1 5 108810PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 88Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln1 5 108910PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 89Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro1 5 109012PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 90Pro Tyr Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln1 5 109110PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 91Pro Phe Pro Gln Gln Pro Gln Gln Pro Tyr1 5 109213PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 92Gln Pro Tyr Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln1 5 109313PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 93Phe Leu Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln1 5 109410PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 94Phe Leu Gln Pro Gln Gln Pro Phe Pro Gln1 5 109516PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 95Phe Leu Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro Gln Gln Pro Tyr1 5 10 159610PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 96Tyr Pro Gln Gln Pro Gln Gln Pro Phe Pro1 5 10978PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 97Phe Leu Gln Pro Gln Gln Pro Phe1 5987PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 98Leu Pro Tyr Pro Gln Pro Gln1 5997PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 99Leu Pro Tyr Pro Gln Pro Gln1 51007PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 100Leu Pro Tyr Pro Gln Pro Gln1 510111PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 101Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 1010211PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 102Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 1010311PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 103Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 101047PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 104Pro Gln Pro Gln Leu Pro Tyr1 51057PRTArtificial SequenceDescription of Artificial Sequence Synthetic

peptide 105Pro Gln Pro Gln Leu Pro Tyr1 51067PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 106Pro Gln Pro Gln Leu Pro Tyr1 510714PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 107Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 1010814PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 108Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln1 5 1010912PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 109Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln1 5 1011012PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 110Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln1 5 1011110PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 111Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln1 5 101129PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 112Leu Pro Tyr Pro Gln Pro Gln Pro Phe1 511313PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 113Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe1 5 1011410PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 114Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr1 5 1011510PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 115Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr1 5 1011617PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 116Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro1 5 10 15Gln11717PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 117Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro1 5 10 15Gln11816PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 118Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe1 5 10 1511913PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 119Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr1 5 1012013PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 120Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr1 5 1012127PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 121Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro1 5 10 15Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr 20 2512220PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 122Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro1 5 10 15Gln Leu Pro Tyr 2012322PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 123Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu1 5 10 15Pro Tyr Pro Gln Pro Gln 2012410PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 124Pro Gln Gln Pro Gln Gln Pro Tyr Pro Gln1 5 101258PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 125Gln Pro Gln Gln Pro Tyr Pro Gln1 51268PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 126Gln Pro Phe Pro Gln Gln Pro Gln1 512710PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 127Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro1 5 1012810PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 128Pro Gln Gln Pro Gln Gln Pro Phe Pro Gln1 5 101297PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 129Pro Gln Gln Pro Phe Pro Gln1 51308PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 130Gln Pro Gln Gln Pro Phe Pro Gln1 51317PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 131Pro Gln Gln Pro Phe Pro Gln1 51328PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 132Gln Pro Gln Gln Pro Phe Pro Gln1 513310PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 133Gln Pro Gln Gln Pro Phe Pro Gln Gln Pro1 5 101348PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 134Tyr Gln Pro Gln Gln Pro Phe Pro1 51359PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 135Leu Gln Pro Gln Gln Pro Phe Pro Gln1 513610PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 136Phe Leu Gln Pro Gln Gln Pro Phe Pro Gln1 5 10