Enzymatic compositions for carbohydrate antigen cleavage, methods, uses, devices and systems related thereto

Abstract

Provided herein are enzymatic compositions for carbohydrate antigen cleavage, methods, uses, devices and systems related thereto. Specifically, the compositions comprise two enzymes, GalNAc deacetylase and galactosaminidase, and the compositions may also comprise a crowding agent. In addition, it was found that the compositions described herein are active at temperatures and pH levels suitable for cell survival.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 719,272, filed on August 17, 2018, entitled “ENZYMATIC COMPOSITIONS FOR CARBOHYDRATEANTIGEN CLEAVAGE, METHODS, USES, APPARATUSES AND SYSTEMS ASSOCIATED THEREWITH.” Technical Field

[0003] The present invention relates to the field of enzyme compositions, in particular to an enzyme composition for cleaving an antigen, and provides a method, use, device and system for cleaving an antigen using the composition.

[0004] background

[0005] Correct blood type matching is a primary requirement in transfusion medicine, as the plasma of individuals with type A blood contains antibodies against B antigens, and vice versa, so incompatible transfusions can lead to complement activation and red blood cell (RBC) lysis (Daniels 2010). These cell-surface antigens are carbohydrate structures ending in α-1,3-linked N-acetylgalactosamine (GalNAc) or galactose (Gal) in type A and B blood, respectively. Type O RBCs, on the other hand, do not contain these terminal sugars and can be universally transfused (Garratty 2008). Therefore, a good supply of type O RBCs is required in blood banks for emergency situations where the patient's blood type is unknown or unclear. However, supplies are often limited.

[0006] Goldstein first proposed and demonstrated the concept of enzymatic removal of GalNAc or Gal structures from A RBCs or B RBCs as a means of converting A RBCs or B RBCs into O RBCs (Goldstein 1982; US4609627 and CA2272925). Using α-galactosidase from green coffee beans, type B RBCs were converted into type O RBCs, followed by successful blood transfusion (Kruskall2000). However, the amount of enzyme required makes this method impractical. The conversion of type A is more challenging, mainly because type A blood has many subtypes with different internal connections (Clausen 1989). Similarly, α-galactosidase has been used to remove type B antigens (e.g., see EP2243793). By using a tetrasaccharide substrate to screen a bacterial library with A and B conversion activity, an important step towards practical conversion including type A has been taken. Two new glycosidase families have been discovered that exhibit high antigen-cleavage activity at neutral pH: CAZy GH109 α-N-acetylgalactosaminidases and GH110 α-galactosidases (Liu 2007). Both enzymes transform their corresponding RBCs, completely removing their respective antigens. However, transformation still requires large amounts of enzyme, particularly type A (60 mg enzyme / blood unit), which has limited further development. Enzymes with higher efficiency in cleaving carbohydrate antigens from cells would be useful.

[0007] Overview

[0008] The present invention is based in part on the surprising discovery that the combination of galactosaminidase and GalNAc deacetylase as described herein is several orders of magnitude more effective than previously identified A antigen cleavage enzymes. For example, under some conditions, some GalNAc deacetylases and galactosaminidase are able to cleave A antigen at or below 1 μ / ml. In addition, the cleavage efficiency of the enzyme combination is maintained at a pH suitable for maintaining red blood cell viability (i.e., a pH of about 6.5 to about 7.5). In addition, it was found that the enzyme is active at temperatures between 4°C and 37°C, which also applies to blood collection, washing, and storage protocols. In addition, the efficiency of the enzyme is further improved by adding a crowding agent (e.g., dextran). It has also been recognized that the same two-step cleavage process can be applied to donor organs. The enzymes as described herein were primarily tested on samples with a hematocrit of 10%, because these enzymes are better used with packaged red blood cell (rbc) bags (approximately 220 ml) and the required amount is calculated, which contains a level of about 80% hematocrit.

[0009] In some embodiments, in the absence of a crowding agent: 3 μg / ml, 10% hematocrit, 1 hour, 37° C., >5.3 mg of each enzyme / packaged rbc bag can be used to cleave A antigen from red blood cells, and in other embodiments, with a crowding agent: 0.5 μg / ml, 10% hematocrit, 1 hour, 37° C., >0.9 mg of each enzyme / packaged rbc bag can be used to cleave A antigen from red blood cells. However, one skilled in the art will appreciate that more enzyme can be used to reduce the possible time to process the blood, or less enzyme can be used as long as the cells are incubated for a longer time.

[0010] According to one embodiment, a composition is provided, comprising: (a) a purified GalNAc deacetylase protein; and (b) a purified galactosamidase protein.

[0011] According to one embodiment, a composition is provided, comprising: (a) a purified GalNAc deacetylase protein selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 17; SEQ ID NO: 23; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34 and SEQ ID NO: 35; and (b) a purified galactosaminidase protein selected from one or more of the following: SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 36 and SEQ ID NO: 37.

[0012] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0013] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 85% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 85% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0014] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 80% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 80% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0015] According to another embodiment, a composition is provided, comprising: a purified enzyme having GalNAc deacetylase activity consisting essentially of an amino acid sequence at least 75% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31, and 32-35; and a purified enzyme having galactosaminidase activity consisting essentially of an amino acid sequence at least 75% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36, and 37.

[0016] According to another embodiment, a composition is provided, comprising an enzyme selected from one or more of (a), and one or more of (b): (a) the purified GalNAc deacetylase protein is the purified GalNAc deacetylase protein of Flavonifractor plautii of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5; and (b) the purified galactosamidase protein is the purified galactosamidase protein of Flavonifractor plautii of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 10.

[0017] According to another embodiment, a composition is provided, comprising one or more enzymes selected from the group consisting of: (a) a purified GalNAc deacetylase protein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 17, and SEQ ID NO: 32; and (b) a purified galactosamidase protein of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 36, and SEQ ID NO: 37 from Fusobacterium prausnitzii.

[0018] According to another embodiment, a composition is provided, comprising one or more enzymes selected from the group consisting of: (a) purified GalNAc deacetylase proteins of SEQ ID NO: 17 and SEQ ID NO: 32, purified Clostridium tertium GalNAc deacetylase proteins; and (b) purified galactosamidase proteins of SEQ ID NO: 19 and SEQ ID NO: 36, purified Clostridium tertium galactosamidase proteins.

[0019] The GalNAc deacetylase and galactosamidase composition may be capable of cleaving A antigen at 1 μg / ml or less. The GalNAc deacetylase and galactosamidase composition may have A antigen cleavage activity at a pH of about 6.5 to about 7.5. The GalNAc deacetylase and galactosamidase composition may have A antigen cleavage activity at a temperature of 4°C to 37°C.

[0020] The composition may include: (a) the purified GalNAc deacetylase and the purified galactosamidase may be immobilized; (b) the purified GalNAc deacetylase may be immobilized; or (c) the purified galactosamidase may be immobilized.

[0021] The immobilized enzyme can be attached to a surface, which can be selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) tubes; (d) columns; and (e) matrices. The composition can also include a crowding agent. The crowding agent can be selected from one or more of the following: dextran, dextran sulfate, dextrin, pullulan, poly (ethylene glycol), polysucrose TM and inert proteins.

[0022] According to another embodiment, a purified enzyme is provided that includes the GalNAc deacetylase of Fusobacterium prausnitzii of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.

[0023] According to another embodiment, a purified enzyme is provided that includes the galactosamidase of Fusobacterium prausnitzii of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10.

[0024] According to another embodiment, a purified enzyme is provided, which includes the GalNAc deacetylase of Clostridium tertiotrophoside of SEQ ID NO: 17 or SEQ ID NO: 32.

[0025] According to another embodiment, a purified enzyme is provided, which includes the galactosamidase of Clostridium tertiotrophoside of SEQ ID NO: 19 or SEQ ID NO: 36.

[0026] According to another embodiment, an isolated nucleic acid sequence encoding a GalNAc deacetylase is provided, which is selected from one or more of the following: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 16; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 28 and SEQ ID NO: 30.

[0027] According to another embodiment, an isolated nucleic acid sequence encoding a galactosamidase is provided, which is selected from one or more of the following: SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 18 and SEQ ID NO: 20.

[0028] According to another embodiment, a vector is provided, comprising the nucleic acid described herein. The vector may further comprise one or more heterologous nucleic acid sequences selected from the group consisting of a protein tag and a cleavage site.

[0029] The protein tag may be selected from one or more of the following: albumin binding protein (ABP); alkaline phosphatase (AP); AU1 epitope; AU5 epitope; Avi tag; bacteriophage T7 epitope (T7 tag); bacteriophage V5 epitope (V5 tag); biotin-carboxyl carrier protein (BCCP); blue tongue virus tag (B tag); single domain camelid antibody (C tag); calmodulin binding peptide (CBP or calmodulin tag); chloramphenicol acetyltransferase (CAT); cellulose binding domain (CBP); chitin binding domain (CBD); choline binding domain (CBD); dihydrofolate reductase (DHFR); DogTag; E2 epitope; E tag; FLAG epitope (FLAG tag); galactose binding protein (GBP); green fluorescent protein (GFP); Glu-Glu (EE tag); glutathione S transferase (GST); human influenza hemagglutinin (HA); HaloTag TM ; Alternating histidine and glutamine tag (HQ tag); Alternating histidine and asparagine tag (HN tag); Histidine affinity tag (HAT); Horseradish peroxidase (HRP); HSV epitope; Isopeptag (Isopep tag); Ketosterol isomerase (KSI); KT3 epitope; LacZ; Luciferase; Maltose binding protein (MBP); Myc epitope (Myc tag); NE tag; NusA; PDZ domain; PDZ ligand; Polyarginine (Arg tag); Polyaspartic acid (Asp tag); Polycysteine ​​(Cys tag); Polyglutamic acid (Glu tag); Polyhistidine (His tag); Polyphenylalanine (Phe tag); Profinity eXact; Protein C; Rho1D4 tag; S1 tag; S tag; Softag 1; Softag 3; SnoopTagJr; SnoopTag; Spot tag; SpyTag (Spy tag); Streptavadin binding peptide (SBP); Staphylococcal protein A (Protein A); Staphylococcal protein G (Protein G); Strep tag; Streptavadin (SBP tag); Strep tag II; Sdy tag; small ubiquitin-like modifier (SUMO); tandem affinity purification (TAP); T7 epitope; tetracysteine ​​tag (TC tag); thioredoxin (Trx); TrpE; Ty tag; ubiquitin; universal; V5 tag; VSV-G or VSV tag and Xpress tag.

[0030] According to another embodiment, a method for enzymatically cleaving A antigen from blood, red blood cells, or a donor organ is provided, the method comprising: (a) combining a GalNAc deacetylase protein and a galactosamidase protein with: (i) blood comprising type A antigen; (ii) type A or type AB red blood cells; or (iii) a donor organ displaying type A antigen; and (b) incubating the enzyme with: (i) the blood; (ii) the type A or type AB red blood cells; or (iii) the donor organ for a period of time sufficient to allow the enzyme to cleave the A antigen from the blood, the red blood cells, or the donor organ.

[0031] The GalNAc deacetylase may be a purified protein selected from one or more of the following: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:17; SEQ ID NO:23; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34 and SEQ ID NO:35; and the galactosaminidase may be a purified protein selected from one or more of the following: SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:36 and SEQ ID NO:37.

[0032] The composition may include: a purified enzyme having GalNAc deacetylase activity, which consists essentially of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 2, 4, 5, 17, 23, 29, 31 and 32-35; and a purified enzyme having galactosaminidase activity, which consists essentially of an amino acid sequence at least 90% identical to the sequence shown in one of SEQ ID NOs: 7, 9, 10, 19, 21, 36 and 37.

[0033] The GalNAc deacetylase may be the purified GalNAc deacetylase protein of Fusobacterium prausnitzii of SEQ ID NO: 4 or SEQ ID NO: 5, and the galactosamidase may be the purified galactosamidase protein of Fusobacterium prausnitzii of SEQ ID NO: 9 or SEQ ID NO: 10.

[0034] The method may further comprise adding a crowding agent. The crowding agent may be selected from one or more of the following: dextran; dextran sulfate; dextrin; pullulan; poly (ethylene glycol); polysucrose TM ; Hyperbranched glycerol and inert protein.

[0035] The method may further comprise washing the blood, the red blood cells or the donor organ to remove GalNAc deacetylase, galactosamidase and the crowding agent.

[0036] The GalNAc deacetylase and the galactosamidase may be able to cleave A antigen at 1 μg / ml or less. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.5 to about 7.5. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 37°C.

[0037] According to another embodiment, a blood collection and storage system is provided, comprising: (a) a purified GalNAc deacetylase protein; and (b) a purified galactosamidase protein.

[0038] The system may further comprise a surface on which the enzyme is immobilized, the surface being selected from one or more of: (a) a bead or microsphere; (b) a container; (c) a tube; (d) a column; or (e) a matrix.

[0039] According to another embodiment, a blood collection and storage device is provided, comprising: (a) a surface; (b) a purified GalNAc deacetylase protein immobilized on the surface; and (c) a purified galactosamidase protein immobilized on the surface.

[0040] The surface of the device on which the enzyme is immobilized can be selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) tubes; (d) columns; or (e) matrices. The container can be a bag.

[0041] The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 100 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 90 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 80 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 70 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 60 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 50 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 40 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 30 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 20 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 15 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 14 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 13 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 12 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 11 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 10 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 9 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 8 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 7 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 6 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be capable of cleaving the A antigen at 5 μg / ml or below.The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 4 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 3 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 2 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 1 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.9 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.8 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.7 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.6 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.5 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.4 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.3 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.2 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.1 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.09 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.08 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.07 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.06 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.05 μg / ml or less. The GalNAc deacetylase and the galactosaminidase may be capable of cleaving the A antigen at 0.04 μg / ml or below.The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.03 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.02 μg / ml or less. The GalNAc deacetylase and the galactosamidase may be able to cleave the A antigen at 0.01 μg / ml or less.

[0042] The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.5 to about 7.5. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.0 to about 8.0. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.8 to about 7.8. The GalNAc deacetylase and the galactosamidase may have A-antigen cleavage activity at a pH of about 6.9 to about 7.9. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a pH of about 6.4 to about 7.8.

[0043] The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 37°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 3°C to 38°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 40°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 4°C to 37°C. The GalNAc deacetylase and the galactosamidase may have A antigen cleavage activity at a temperature of 5°C to 37°C.

[0044] The purified GalNAc deacetylase and the purified galactosamidase may be immobilized. The purified GalNAc deacetylase may be immobilized. The purified galactosamidase may be immobilized. The immobilized enzyme may be attached to a surface. The surface may be selected from one or more of the following: beads or microspheres; a container; a tube; a column; or a matrix. The surface may be selected from one or more of the following: a container; a tube; a column; or a matrix. The container may be a bag.

[0045] According to another embodiment, a purified enzyme of the GalNAc deacetylase of Fusobacterium prausnitzii comprising SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 is provided.

[0046] According to another embodiment, a purified enzyme comprising the galactosamidase of Fasciobacterium prausnitzii of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 10 is provided.

[0047] According to another embodiment, a purified enzyme is provided, comprising the purified Clostridium tertiotrophoside GalNAc deacetylase and galactosamidase fusion protein of SEQ ID NO: 14.

[0048] According to another embodiment, a vector comprising a nucleic acid described herein and a heterologous nucleic acid sequence is provided.

[0049] According to another embodiment, the method can be performed in vitro or ex vivo. As used herein, ex vivo means that the method is performed outside the organism. For example, ex vivo will include ex vivo lung perfusion (EVLP) and the processing of donated blood. As used herein, ex vivo refers to an experiment or measurement or treatment performed in or on a tissue or cell (e.g., red blood cells or donor organs) of an organism from an external environment, wherein the tissue or cell is under minimal or some condition change in vivo. Sequence Listing <110> The University of British Columbia <120> Enzymatic compositions for carbohydrate antigen cleavage, methods, uses, devices and systems related thereto <130> P1600PC00 <140> NOT YET ASSIGNED <141> 2019-08-16 <150> US 62 / 719,272 <151> 2018-08-17 <160> 108 <170> Notepad <210> 1 <211> 2319 <212> DNA <213> Flavonifractor plautii <400> 1 atgagaaatc gaaggaaagc tgtttcgctt ctaacgggcc tactcgtgac ggcccagtta 60 tttccaaccg cggcgcttgc ggcagactcc agcgagtccg cattgaacaa ggcccccgga 120 tatcaggatt ttcccgccta ttacagcgac agtgcgcatg ccgatgacca ggtgactcac 180 ccggacgtag ttgtcctgga agaaccgtgg aacggctatc gctattgggc cgtttatacg 240 cccaacgtga tgcggatctc catctacgaa aacccgtcca tcgttgcctc cagcgacgga 300 gtgcattggg tagaaccgga ggggctttcc aatcccattg agccgcagcc gcccagcacc 360 cgctaccaca actgcgacgc tgatatggtc tataacgcgg aatacgatgc catgatggcc 420 tattggaact gggcggatga ccagggcgga ggcgttgggg ccgaagtccg gctgcggatt 480 tcctatgacg gcgtacattg gggcgtcccc gtgacttatg atgagatgac ccgcgtatgg 540 tcgaagccca cctccgacgc ggagcgtcag gttgcggatg gagaggatga cttcattacc 600 gccattgctt ctccagaccg ctacgatatg ctctctccca ctattgtcta cgatgacttc 660 cgggatgtgt tcatcctgtg ggccaacaat accggcgacg tggggtatca gaatggtcag 720 gcgaacttcg tggaaatgcg ttattcggac gacgggatca cctggggtga gccagtccgc 780 gtcaacggct tcctggggct tgacgagaat gggcagcagt tggccccctg gcatcaggat 840 gtccagtatg ttccagattt gaaggagttt gtttgtattt cccagtgctt tgccggccga 900 aatccggatg gctctgtcct gcacctgacc acatcaaagg atggagtcaa ctgggagcag 960 gtgggcacca agcccctgct gtcccccggg ccagacggca gttgggatga tttccagatc 1020 tatcgctcca gtttttacta tgagccaggc agttccgccg gagatggtac catgcgcgtc 1080 tggtacagtg ccctgcagaa ggacaccaat aacaagatgg tcgcggattc ctccgggaat 1140 ctgaccattc aggccaaaag tgaggatgac cgcatctgga ggatcggcta tgcggaaaac 1200 agttttgttg agatgatgcg cgtgctgctg gatgaccccg gctacacgac gcccgccctg 1260 gtttccggca attcccttat gctgagtgct gagaccactt cccttcccac aggggatgtc 1320 atgaagctgg aaaccagttt cgcgcctgtg gacacctctg atcaggtcgt gaaatatacc 1380 tccagtgatc cggatgtggc gacggtggat gagtttggaa ccattacagg cgtttctgtc 1440 ggttcagcgc gcatcatggc ggagacccgg gagggcctgt ccgacgacct tgaaattgca 1500 gtggtggaga atccgtacac gctgattccc cagtccaata tgacggcaac cgccaccagc 1560 gtctacggcg ggacgacgga gggccccgcc tccaatgtcc tcgatggaaa cgtccgcaca 1620 atatggcata ccaactatgc tcccaaagat gaactgccgc agagtatcac cgtttccttt 1680 gaccagccct ataccgtcgg ccgcttcgtc tataccccac gtcaaaacgg gacaaatggc 1740 ataatttcgg agtatgagct atacgccatc caccaggacg gcagcaagga cctagtcgcc 1800 tccggctcag actgggcgct cgatgccaag gataaaaccg tgagctttgc accggtagaa 1860 gccgtcggcc tggagctcaa ggcgattgcc ggcgcaggtg ggttcggtac tgccgccgaa 1920 ctcaatgtgt atgcgtatgg tccaatcgag cctgcgcccg tatatgtccc ggtggatgac 1980 cgggatgctt ccctggtgtt tacgggtgca tggaatagcg acagcaacgg aagcttttat 2040 gaagggacgg cccgttatac caacgagatc ggcgcgtccg tggagttcac atttgtgggg 2100 acggccattc ggtggtatgg tcaaaatgat gtaaatttcg gcgctgcgga ggtatacgtg 2160 gacggcgttc tggcagggga ggtaaatgtg tatgggccgg cggcggctca gcagcttcta 2220 tttgaggcgg acggtctggc ctatgggaag cataccatcc gcatcgtctg tgtgtctccg 2280 gtggttgact tcgactattt ttcgtatgtg ggagaataa 2319 <210> 2 <211> 772 <212> PRT <213> Flavonifractor plautii <400> 2 Met Arg Asn Arg Arg Lys Ala Val Ser Leu Leu Thr Gly Leu Leu Val 1 5 10 15 Thr Ala Gln Leu Phe Pro Thr Ala Ala Leu Ala Ala Asp Ser Ser Glu 20 25 30 Ser Ala Leu Asn Lys Ala Pro Gly Tyr Gln Asp Phe Pro Ala Tyr Tyr 35 40 45 Ser Asp Ser Ala His Ala Asp Asp Gln Val Thr His Pro Asp Val Val 50 55 60 Val Leu Glu Glu Pro Trp Asn Gly Tyr Arg Tyr Trp Ala Val Tyr Thr 65 70 75 80 Pro Asn Val Met Arg Ile Ser Ile Tyr Glu Asn Pro Ser Ile Val Ala 85 90 95 Ser Ser Asp Gly Val His Trp Val Glu Pro Glu Gly Leu Ser Asn Pro 100 105 110 Ile Glu Pro Gln Pro Pro Ser Thr Arg Tyr His Asn Cys Asp Ala Asp 115 120 125 Met Val Tyr Asn Ala Glu Tyr Asp Ala Met Met Ala Tyr Trp Asn Trp 130 135 140 Ala Asp Asp Gln Gly Gly Gly Val Gly Ala Glu Val Arg Leu Arg Ile 145 150 155 160 Ser Tyr Asp Gly Val His Trp Gly Val Pro Val Thr Tyr Asp Glu Met 165 170 175 Thr Arg Val Trp Ser Lys Pro Thr Ser Asp Ala Glu Arg Gln Val Ala 180 185 190 Asp Gly Glu Asp Asp Phe Ile Thr Ala Ile Ala Ser Pro Asp Arg Tyr 195 200 205 Asp Met Leu Ser Pro Thr Ile Val Tyr Asp Asp Phe Arg Asp Val Phe 210 215 220 Ile Leu Trp Ala Asn Asn Thr Gly Asp Val Gly Tyr Gln Asn Gly Gln 225 230 235 240 Ala Asn Phe Val Glu Met Arg Tyr Ser Asp Asp Gly Ile Thr Trp Gly 245 250 255 Glu Pro Val Arg Val Asn Gly Phe Leu Gly Leu Asp Glu Asn Gly Gln 260 265 270 Gln Leu Ala Pro Trp His Gln Asp Val Gln Tyr Val Pro Asp Leu Lys 275 280 285 Glu Phe Val Cys Ile Ser Gln Cys Phe Ala Gly Arg Asn Pro Asp Gly 290 295 300 Ser Val Leu His Leu Thr Thr Ser Lys Asp Gly Val Asn Trp Glu Gln 305 310 315 320 Val Gly Thr Lys Pro Leu Leu Ser Pro Gly Pro Asp Gly Ser Trp Asp 325 330 335 Asp Phe Gln Ile Tyr Arg Ser Ser Phe Tyr Tyr Glu Pro Gly Ser Ser 340 345 350 Ala Gly Asp Gly Thr Met Arg Val Trp Tyr Ser Ala Leu Gln Lys Asp 355 360 365 Thr Asn Asn Lys Met Val Ala Asp Ser Ser Gly Asn Leu Thr Ile Gln 370 375 380 Ala Lys Ser Glu Asp Asp Arg Ile Trp Arg Ile Gly Tyr Ala Glu Asn 385 390 395 400 Ser Phe Val Glu Met Met Arg Val Leu Leu Asp Asp Pro Gly Tyr Thr 405 410 415 Thr Pro Ala Leu Val Ser Gly Asn Ser Leu Met Leu Ser Ala Glu Thr 420 425 430 Thr Ser Leu Pro Thr Gly Asp Val Met Lys Leu Glu Thr Ser Phe Ala 435 440 445 Pro Val Asp Thr Ser Asp Gln Val Val Lys Tyr Thr Ser Ser Asp Pro 450 455 460 Asp Val Ala Thr Val Asp Glu Phe Gly Thr Ile Thr Gly Val Ser Val 465 470 475 480 Gly Ser Ala Arg Ile Met Ala Glu Thr Arg Glu Gly Leu Ser Asp Asp 485 490 495 Leu Glu Ile Ala Val Val Glu Asn Pro Tyr Thr Leu Ile Pro Gln Ser 500 505 510 Asn Met Thr Ala Thr Ala Thr Ser Val Tyr Gly Gly Thr Thr Glu Gly 515 520 525 Pro Ala Ser Asn Val Leu Asp Gly Asn Val Arg Thr Ile Trp His Thr 530 535 540 Asn Tyr Ala Pro Lys Asp Glu Leu Pro Gln Ser Ile Thr Val Ser Phe 545 550 555 560 Asp Gln Pro Tyr Thr Val Gly Arg Phe Val Tyr Thr Pro Arg Gln Asn 565 570 575 Gly Thr Asn Gly Ile Ile Ser Glu Tyr Glu Leu Tyr Ala Ile His Gln 580 585 590 Asp Gly Ser Lys Asp Leu Val Ala Ser Gly Ser Asp Trp Ala Leu Asp 595 600 605 Ala Lys Asp Lys Thr Val Ser Phe Ala Pro Val Glu Ala Val Gly Leu 610 615 620 Glu Leu Lys Ala Ile Ala Gly Ala Gly Gly Phe Gly Thr Ala Ala Glu 625 630 635 640 Leu Asn Val Tyr Ala Tyr Gly Pro Ile Glu Pro Ala Pro Val Tyr Val 645 650 655 Pro Val Asp Asp Arg Asp Ala Ser Leu Val Phe Thr Gly Ala Trp Asn 660 665 670 Ser Asp Ser Asn Gly Ser Phe Tyr Glu Gly Thr Ala Arg Tyr Thr Asn 675 680 685 Glu Ile Gly Ala Ser Val Glu Phe Thr Phe Val Gly Thr Ala Ile Arg 690 695 700 Trp Tyr Gly Gln Asn Asp Val Asn Phe Gly Ala Ala Glu Val Tyr Val 705 710 715 720 Asp Gly Val Leu Ala Gly Glu Val Asn Val Tyr Gly Pro Ala Ala Ala 725 730 735 Gln Gln Leu Leu Phe Glu Ala Asp Gly Leu Ala Tyr Gly Lys His Thr 740 745 750 Ile Arg Ile Val Cys Val Ser Pro Val Val Asp Phe Asp Tyr Phe Ser 755 760 765 Tyr Val Gly Glu 770 <210> 3 <211> 2238 <212> DNA <213> Flavonifractor plautii <400> 3 gcagactcca gcgagtccgc attgaacaag gcccccggat atcaggattt tcccgcctat 60 tacagcgaca gtgcgcatgc cgatgaccag gtgactcacc cggacgtagt tgtcctggaa 120 gaaccgtgga acggctatcg ctattgggcc gtttatacgc ccaacgtgat gcggatctcc 180 atctacgaaa acccgtccat cgttgcctcc agcgacggag tgcattgggt agaaccggag 240 gggctttcca atcccattga gccgcagccg cccagcaccc gctaccacaa ctgcgacgct 300 gatatggtct ataacgcgga atacgatgcc atgatggcct attggaactg ggcggatgac 360 cagggcggag gcgttggggc cgaagtccgg ctgcggattt cctatgacgg cgtacattgg 420 ggcgtccccg tgacttatga tgagatgacc cgcgtatggt cgaagcccac ctccgacgcg 480 gagcgtcagg ttgcggatgg agaggatgac ttcattaccg ccattgcttc tccagaccgc 540 tacgatatgc tctctcccac tattgtctac gatgacttcc gggatgtgtt catcctgtgg 600 gccaacaata ccggcgacgt ggggtatcag aatggtcagg cgaacttcgt ggaaatgcgt 660 tattcggacg acgggatcac ctggggtgag ccagtccgcg tcaacggctt cctggggctt 720 gacgagaatg ggcagcagtt ggccccctgg catcaggatg tccagtatgt tccagatttg 780 aagggagtttg tttgtatttc ccagtgcttt gccggccgaa atccggatgg ctctgtcctg 840 cacctgacca catcaaagga tggagtcaac tgggagcagg tgggcaccaa gcccctgctg 900 tcccccgggc cagacggcag ttgggatgat ttccagatct atcgctccag tttttactat 960 gagccaggca gttccgccgg agatggtacc atgcgcgtct ggtacagtgc cctgcagaag 1020 gacaccaata acaagatggt cgcggattcc tccgggaatc tgaccattca ggccaaaagt 1080 gaggatgacc gcatctggag gatcggctat gcggaaaaca gttttgttga gatgatgcgc 1140 gtgctgctgg atgaccccgg ctacacgacg cccgccctgg tttccggcaa ttcccttatg 1200 ctgagtgctg agaccacttc ccttcccaca ggggatgtca tgaagctgga aaccagtttc 1260 gcgcctgtgg acacctctga tcaggtcgtg aaatatacct ccagtgatcc ggatgtggcg 1320 acggtggatg agtttggaac cattacaggc gtttctgtcg gttcagcgcg catcatggcg 1380 gagacccggg agggcctgtc cgacgacctt gaaattgcag tggtggagaa tccgtacacg 1440 ctgattcccc agtccaatat gacggcaacc gccaccagcg tctacggcgg gacgacggag 1500 ggccccgcct ccaatgtcct cgatggaaac gtccgcacaa tatggcatac caactatgct 1560 cccaaagatg aactgccgca gagtatcacc gtttcctttg accagcccta taccgtcggc 1620 cgcttcgtct ataccccacg tcaaaacggg acaaatggca taatttcgga gtatgagcta 1680 tacgccatcc accaggacgg cagcaaggac ctagtcgcct ccggctcaga ctgggcgctc 1740 gatgccaagg ataaaaccgt gagctttgca ccggtagaag ccgtcggcct ggagctcaag 1800 gcgattgccg gcgcaggtgg gttcggtact gccgccgaac tcaatgtgta tgcgtatggt 1860 ccaatcgagc ctgcgcccgt atatgtcccg gtggatgacc gggatgcttc cctggtgttt 1920 acgggtgcat ggaatagcga cagcaacgga agcttttatg aagggacggc ccgttatacc 1980 aacgagatcg gcgcgtccgt ggagttcaca tttgtgggga cggccattcg gtggtatggt 2040 caaaatgatg taaatttcgg cgctgcggag gtatacgtgg acggcgttct ggcaggggag 2100 gtaaatgtgt atgggccggc ggcggctcag cagcttctat ttgaggcgga cggtctggcc 2160 tatgggaagc ataccatccg catcgtctgt gtgtctccgg tggttgactt cgactatttt 2220 tcgtatgtgg gagaataa 2238 <210> 4 <211> 745 <212> PRT <213> Flavonifractor plautii <400> 4 Ala Asp Ser Ser Glu Ser Ala Leu Asn Lys Ala Pro Gly Tyr Gln Asp 1 5 10 15 Phe Pro Ala Tyr Tyr Ser Asp Ser Ala His Ala Asp Asp Gln Val Thr 20 25 30 His Pro Asp Val Val Val Leu Glu Glu Pro Trp Asn Gly Tyr Arg Tyr 35 40 45 Trp Ala Val Tyr Thr Pro Asn Val Met Arg Ile Ser Ile Tyr Glu Asn 50 55 60 Pro Ser Ile Val Ala Ser Ser Asp Gly Val His Trp Val Glu Pro Glu 65 70 75 80 Gly Leu Ser Asn Pro Ile Glu Pro Gln Pro Pro Ser Thr Arg Tyr His 85 90 95 Asn Cys Asp Ala Asp Met Val Tyr Asn Ala Glu Tyr Asp Ala Met Met 100 105 110 Ala Tyr Trp Asn Trp Ala Asp Asp Gln Gly Gly Gly Val Gly Ala Glu 115 120 125 Val Arg Leu Arg Ile Ser Tyr Asp Gly Val His Trp Gly Val Pro Val 130 135 140 Thr Tyr Asp Glu Met Thr Arg Val Trp Ser Lys Pro Thr Ser Asp Ala 145 150 155 160 Glu Arg Gln Val Ala Asp Gly Glu Asp Asp Phe Ile Thr Ala Ile Ala 165 170 175 Ser Pro Asp Arg Tyr Asp Met Leu Ser Pro Thr Ile Val Tyr Asp Asp 180 185 190 Phe Arg Asp Val Phe Ile Leu Trp Ala Asn Asn Thr Gly Asp Val Gly 195 200 205 Tyr Gln Asn Gly Gln Ala Asn Phe Val Glu Met Arg Tyr Ser Asp Asp 210 215 220 Gly Ile Thr Trp Gly Glu Pro Val Arg Val Asn Gly Phe Leu Gly Leu 225 230 235 240 Asp Glu Asn Gly Gln Gln Leu Ala Pro Trp His Gln Asp Val Gln Tyr 245 250 255 Val Pro Asp Leu Lys Glu Phe Val Cys Ile Ser Gln Cys Phe Ala Gly 260 265 270 Arg Asn Pro Asp Gly Ser Val Leu His Leu Thr Thr Ser Lys Asp Gly 275 280 285 Val Asn Trp Glu Gln Val Gly Thr Lys Pro Leu Leu Ser Pro Gly Pro 290 295 300 Asp Gly Ser Trp Asp Asp Phe Gln Ile Tyr Arg Ser Ser Phe Tyr Tyr 305 310 315 320 Glu Pro Gly Ser Ser Ala Gly Asp Gly Thr Met Arg Val Trp Tyr Ser 325 330 335 Ala Leu Gln Lys Asp Thr Asn Asn Lys Met Val Ala Asp Ser Ser Gly 340 345 350 Asn Leu Thr Ile Gln Ala Lys Ser Glu Asp Asp Arg Ile Trp Arg Ile 355 360 365 Gly Tyr Ala Glu Asn Ser Phe Val Glu Met Met Arg Val Leu Leu Asp 370 375 380 Asp Pro Gly Tyr Thr Thr Pro Ala Leu Val Ser Gly Asn Ser Leu Met 385 390 395 400 Leu Ser Ala Glu Thr Thr Ser Leu Pro Thr Gly Asp Val Met Lys Leu 405 410 415 Glu Thr Ser Phe Ala Pro Val Asp Thr Ser Asp Gln Val Val Lys Tyr 420 425 430 Thr Ser Ser Asp Pro Asp Val Ala Thr Val Asp Glu Phe Gly Thr Ile 435 440 445 Thr Gly Val Ser Val Gly Ser Ala Arg Ile Met Ala Glu Thr Arg Glu 450 455 460 Gly Leu Ser Asp Asp Leu Glu Ile Ala Val Val Glu Asn Pro Tyr Thr 465 470 475 480 Leu Ile Pro Gln Ser Asn Met Thr Ala Thr Ala Thr Ser Val Tyr Gly 485 490 495 Gly Thr Thr Glu Gly Pro Ala Ser Asn Val Leu Asp Gly Asn Val Arg 500 505 510 Thr Ile Trp His Thr Asn Tyr Ala Pro Lys Asp Glu Leu Pro Gln Ser 515 520 525 Ile Thr Val Ser Phe Asp Gln Pro Tyr Thr Val Gly Arg Phe Val Tyr 530 535 540 Thr Pro Arg Gln Asn Gly Thr Asn Gly Ile Ile Ser Glu Tyr Glu Leu 545 550 555 560 Tyr Ala Ile His Gln Asp Gly Ser Lys Asp Leu Val Ala Ser Gly Ser 565 570 575 Asp Trp Ala Leu Asp Ala Lys Asp Lys Thr Val Ser Phe Ala Pro Val 580 585 590 Glu Ala Val Gly Leu Glu Leu Lys Ala Ile Ala Gly Ala Gly Gly Phe 595 600 605 Gly Thr Ala Ala Glu Leu Asn Val Tyr Ala Tyr Gly Pro Ile Glu Pro 610 615 620 Ala Pro Val Tyr Val Pro Val Asp Asp Arg Asp Ala Ser Leu Val Phe 625 630 635 640 Thr Gly Ala Trp Asn Ser Asp Ser Asn Gly Ser Phe Tyr Glu Gly Thr 645 650 655 Ala Arg Tyr Thr Asn Glu Ile Gly Ala Ser Val Glu Phe Thr Phe Val 660 665 670 Gly Thr Ala Ile Arg Trp Tyr Gly Gln Asn Asp Val Asn Phe Gly Ala 675 680 685 Ala Glu Val Tyr Val Asp Gly Val Leu Ala Gly Glu Val Asn Val Tyr 690 695 700 Gly Pro Ala Ala Ala Gln Gln Leu Leu Phe Glu Ala Asp Gly Leu Ala 705 710 715 720 Tyr Gly Lys His Thr Ile Arg Ile Val Cys Val Ser Pro Val Val Asp 725 730 735 Phe Asp Tyr Phe Ser Tyr Val Gly Glu 740 745 <210> 5 <211> 760 <212> PRT <213> Flavonifractor plautii <400> 5 Met Gly His His His His His His His His His Ser Ser Gly Ala 1 5 10 15 Asp Ser Ser Glu Ser Ala Leu Asn Lys Ala Pro Gly Tyr Gln Asp Phe 20 25 30 Pro Ala Tyr Tyr Ser Asp Ser Ala His Ala Asp Asp Gln Val Thr His 35 40 45 Pro Asp Val Val Val Leu Glu Glu Pro Trp Asn Gly Tyr Arg Tyr Trp 50 55 60 Ala Val Tyr Thr Pro Asn Val Met Arg Ile Ser Ile Tyr Glu Asn Pro 65 70 75 80 Ser Ile Val Ala Ser Ser Asp Gly Val His Trp Val Glu Pro Glu Gly 85 90 95 Leu Ser Asn Pro Ile Glu Pro Gln Pro Pro Ser Thr Arg Tyr His Asn 100 105 110 Cys Asp Ala Asp Met Val Tyr Asn Ala Glu Tyr Asp Ala Met Met Ala 115 120 125 Tyr Trp Asn Trp Ala Asp Asp Gln Gly Gly Gly Val Gly Ala Glu Val 130 135 140 Arg Leu Arg Ile Ser Tyr Asp Gly Val His Trp Gly Val Pro Val Thr 145 150 155 160 Tyr Asp Glu Met Thr Arg Val Trp Ser Lys Pro Thr Ser Asp Ala Glu 165 170 175 Arg Gln Val Ala Asp Gly Glu Asp Asp Phe Ile Thr Ala Ile Ala Ser 180 185 190 Pro Asp Arg Tyr Asp Met Leu Ser Pro Thr Ile Val Tyr Asp Asp Phe 195 200 205 Arg Asp Val Phe Ile Leu Trp Ala Asn Asn Thr Gly Asp Val Gly Tyr 210 215 220 Gln Asn Gly Gln Ala Asn Phe Val Glu Met Arg Tyr Ser Asp Asp Gly 225 230 235 240 Ile Thr Trp Gly Glu Pro Val Arg Val Asn Gly Phe Leu Gly Leu Asp 245 250 255 Glu Asn Gly Gln Gln Leu Ala Pro Trp His Gln Asp Val Gln Tyr Val 260 265 270 Pro Asp Leu Lys Glu Phe Val Cys Ile Ser Gln Cys Phe Ala Gly Arg 275 280 285 Asn Pro Asp Gly Ser Val Leu His Leu Thr Thr Ser Lys Asp Gly Val 290 295 300 Asn Trp Glu Gln Val Gly Thr Lys Pro Leu Leu Ser Pro Gly Pro Asp 305 310 315 320 Gly Ser Trp Asp Asp Phe Gln Ile Tyr Arg Ser Ser Phe Tyr Tyr Glu 325 330 335 Pro Gly Ser Ser Ala Gly Asp Gly Thr Met Arg Val Trp Tyr Ser Ala 340 345 350 Leu Gln Lys Asp Thr Asn Asn Lys Met Val Ala Asp Ser Ser Gly Asn 355 360 365 Leu Thr Ile Gln Ala Lys Ser Glu Asp Asp Arg Ile Trp Arg Ile Gly 370 375 380 Tyr Ala Glu Asn Ser Phe Val Glu Met Met Arg Val Leu Leu Asp Asp 385 390 395 400 Pro Gly Tyr Thr Thr Pro Ala Leu Val Ser Gly Asn Ser Leu Met Leu 405 410 415 Ser Ala Glu Thr Thr Ser Leu Pro Thr Gly Asp Val Met Lys Leu Glu 420 425 430 Thr Ser Phe Ala Pro Val Asp Thr Ser Asp Gln Val Val Lys Tyr Thr 435 440 445 Ser Ser Asp Pro Asp Val Ala Thr Val Asp Glu Phe Gly Thr Ile Thr 450 455 460 Gly Val Ser Val Gly Ser Ala Arg Ile Met Ala Glu Thr Arg Glu Gly 465 470 475 480 Leu Ser Asp Asp Leu Glu Ile Ala Val Val Glu Asn Pro Tyr Thr Leu 485 490 495 Ile Pro Gln Ser Asn Met Thr Ala Thr Ala Thr Ser Val Tyr Gly Gly 500 505 510 Thr Thr Glu Gly Pro Ala Ser Asn Val Leu Asp Gly Asn Val Arg Thr 515 520 525 Ile Trp His Thr Asn Tyr Ala Pro Lys Asp Glu Leu Pro Gln Ser Ile 530 535 540 Thr Val Ser Phe Asp Gln Pro Tyr Thr Val Gly Arg Phe Val Tyr Thr 545 550 555 560 Pro Arg Gln Asn Gly Thr Asn Gly Ile Ile Ser Glu Tyr Glu Leu Tyr 565 570 575 Ala Ile His Gln Asp Gly Ser Lys Asp Leu Val Ala Ser Gly Ser Asp 580 585 590 Trp Ala Leu Asp Ala Lys Asp Lys Thr Val Ser Phe Ala Pro Val Glu 595 600 605 Ala Val Gly Leu Glu Leu Lys Ala Ile Ala Gly Ala Gly Gly Phe Gly 610 615 620 Thr Ala Ala Glu Leu Asn Val Tyr Ala Tyr Gly Pro Ile Glu Pro Ala 625 630 635 640 Pro Val Tyr Val Pro Val Asp Asp Arg Asp Ala Ser Leu Val Phe Thr 645 650 655 Gly Ala Trp Asn Ser Asp Ser Asn Gly Ser Phe Tyr Glu Gly Thr Ala 660 665 670 Arg Tyr Thr Asn Glu Ile Gly Ala Ser Val Glu Phe Thr Phe Val Gly 675 680 685 Thr Ala Ile Arg Trp Tyr Gly Gln Asn Asp Val Asn Phe Gly Ala Ala 690 695 700 Glu Val Tyr Val Asp Gly Val Leu Ala Gly Glu Val Asn Val Tyr Gly 705 710 715 720 Pro Ala Ala Ala Gln Gln Leu Leu Phe Glu Ala Asp Gly Leu Ala Tyr 725 730 735 Gly Lys His Thr Ile Arg Ile Val Cys Val Ser Pro Val Val Asp Phe 740 745 750 Asp Tyr Phe Ser Tyr Val Gly Glu 755 760 <210> 6 <211> 3159 <212> DNA <213> Flavonifractor plautii <400> 6 gcggcgcctg caacggacac cggcaacgca ggactgattg cagaaggtga ttatgccatt 60 gccggcaatg gcgtccgcgt cacttatgac gcggacgggc agacaatcac tctgtaccgc 120 acagagggat ctgggcttat ccagatgagc aagccttctc cattgggagg gccagtgatt 180 ggagggcagg aggttcagga cttcagccat atttcatgtg atgtggagca gagcaccagc 240 ggagtgatgg gcagcggtca gagaatgacc attacctctc agagcatgag cacgggccta 300 attcgtacct atgtgctgga gacctctgat atcgaggagg gtgtggtata tactgcaaca 360 tcctatgagg caggagcttc tgatgtggaa gtgtcttggt tcattggcag tgtgtatgag 420 ctttatggtg cggaagatcg tatctggagt tataacggcg gcggtgaggg gccgatgcac 480 tactatgata cgcttcaaaa gattgacctg accgactctg gcaagttcag tagggagaat 540 aaacaggatg acacggctgc aagtattcct gtgtcagata tttacattgc tgatggaggg 600 attaccgttg gcgatgcttc tgcaaccaga agggaggtac atactccggt tcaggaaacc 660 agtgattcag ctcaagtttc tatcgggtgg ccaggcaaag tcattgccgc cggaagcgtg 720 atcgaaattg gtgagagctt tgctgtagtc catccgggtg actattataa cggcttgaga 780 ggttacaaaa atgcaatgga tcacttgggc gtgattatgc ctgcacctgg ggatattcct 840 gatagcagct atgatctccg atgggaaagc tggggctggg ggtttaactg gacgatcgat 900 ttaataatcg gcaaattgga tgaacttcag gcagccggag tcaagcagat cactttggat 960 gatggttggt ataccaatgc aggagactgg gccttaaatc cagaaaagtt tccaaatgga 1020 gcctccgatg cgtgcggct gacagatgca attcatgagc atggtatgac tgcactcctt 1080 tggtggagac cttgtgacgg cgggatcgat agtatactct atcagcaaca ccctgaatat 1140 ttcgttatgg atgcagatgg aagacctgca aggcttccta ctcctggtgg tgggaccaat 1200 cccagcttgg gatatgcact ttgccctatg gcggatggtg cgattgcaag ccaagttgac 1260 tttgtaaacc gtgcaatgaa tgattggggg ttcgatggct tcaagggaga tttgtgtgg 1320 agtatgcctg aatgctacaa tcctgcacat aaccacgcct cgccagaaga atccactgaa 1380 aagcaatccg agatataccg cgtctcttat gaggctatg tggccaacga ccccaatgtg 1440 ttcaatttgt tgtgcaactg cggtacgccc caggactact atagtttacc atatatgaca 1500 cagattgcta cggctgaccc cacttctgtg gatcaaacaa ggagacgcgt gaaagcctac 1560 aaggcactga tgggagatta tttccctgtt acagccgacc acaataacat ctggtatcca 1620 agtgccgtcg gtacgggctc tgttctcatt gaaaaacgtg accttagcgg tactgccaag 1680 gaagaattg aaaaatggct tgggattgcg gatacagttc agttgcagaa aggccggttt 1740 attggcgatc tttacagtta tggttttgac ccttacgaaa cctatgtggt ggagaaagac 1800 ggggttatgt actatgcctt ctacaaagat gggagcaaat atagccccac tggctatcca 1860 gatattgagt tgaaggggct agatccaaat aaaatgtata ggattgttga ctatgtcaat 1920 gatcgtgtcg tggcaacaaa cctgatgggt gataacgctg tattcaatac acgtttttcc 1980 gactatctac tggttaaagc ggtggaaatt tcggaaccgg atccagaacc tgttgaccct 2040 gattatggtt tcacctctgt tgatgacaga gacgaggctc ttatttacac agggacatgg 2100 catgatgaca ataacgcatc tttcagcgaa gggactgcac gttataccaa cagtacggat 2160 gcttcggttg tattctcctt tactggaact tccattcgct ggtatggcca gagggatacc 2220 aattttggca cggcagaagt ttatttggac gatgaactga aaacaacagt tgatgcgaat 2280 ggggccgcag aagcaggcgt atgtcttttt gaggcgcttg atcttccggc tgccgagcat 2340 accattaaaa ttgtgtgcaa gagcggagtg attgatattg accgctttgc atatgaagct 2400 gctacccttg aacccatcta tgaaaaggtc gatgcgctct cggatcggat cacttatgtt 2460 gggaattggg aagagtatca caacagcgag ttctacatgg gaaacgcaat gcgcacagac 2520 gaagccggcg cttatgctga actgactttc cgtggtacag ccgtacgcct gtatgcagag 2580 atgagcttca attttggcac tgcagatgtc tatttagacg gagagttagt ggaaaacata 2640 atcctatacg gccaggaagc aactgggcag ctaatgtttg agcgtacggg actggaggaa 2700 ggagaacata ccattcgcct tgtacaaaac gcctggaaca tcaatttgga ctatatttct 2760 tatctaccag agcaagatca accaacgccg ccggagacga cggttactgt tgatgcaatg 2820 gacgcccaac tggtgtatac aggcgtatgg aatgatgact atcatgacgt ctttcaggaa 2880 ggaaccgccc gttatgccag tagtgccggc gcctcggtcg agttcgaatt tactggaagc 2940 gaaatccgtt ggtatggaca aaatgattcc aacttcggtg ttgccagcgt ttatatcgat 3000 aatgagtttg tgcagcaggt aaatgttaac ggagctgcgg ctgtgggaaa gcttttgttt 3060 caaaaggctg atctaccagc cggttcgcac acgatccgca ttgtgtgcga tactccggtt 3120 attgatttgg actatttgac ttataccact aacgcataa 3159 <210> 7 <211> 1078 <212> PRT <213> Flavonifractor plautii <400> 7 Met Arg Gly Lys Lys Phe Ile Ser Leu Thr Leu Ser Thr Met Leu Cys 1 5 10 15 Leu Gln Leu Leu Pro Thr Ala Ser Phe Ala Ala Ala Pro Ala Thr Asp 20 25 30 Thr Gly Asn Ala Gly Leu Ile Ala Glu Gly Asp Tyr Ala Ile Ala Gly 35 40 45 Asn Gly Val Arg Val Thr Tyr Asp Ala Asp Gly Gln Thr Ile Thr Leu 50 55 60 Tyr Arg Thr Glu Gly Ser Gly Leu Ile Gln Met Ser Lys Pro Ser Pro 65 70 75 80 Leu Gly Gly Pro Val Ile Gly Gly Gln Glu Val Gln Asp Phe Ser His 85 90 95 Ile Ser Cys Asp Val Glu Gln Ser Thr Ser Gly Val Met Gly Ser Gly 100 105 110 Gln Arg Met Thr Ile Thr Ser Gln Ser Met Ser Thr Gly Leu Ile Arg 115 120 125 Thr Tyr Val Leu Glu Thr Ser Asp Ile Glu Glu Gly Val Val Tyr Thr 130 135 140 Ala Thr Ser Tyr Glu Ala Gly Ala Ser Asp Val Glu Val Ser Trp Phe 145 150 155 160 Ile Gly Ser Val Tyr Glu Leu Tyr Gly Ala Glu Asp Arg Ile Trp Ser 165 170 175 Tyr Asn Gly Gly Gly Glu Gly Pro Met His Tyr Tyr Asp Thr Leu Gln 180 185 190 Lys Ile Asp Leu Thr Asp Ser Gly Lys Phe Ser Arg Glu Asn Lys Gln 195 200 205 Asp Asp Thr Ala Ala Ser Ile Pro Val Ser Asp Ile Tyr Ile Ala Asp 210 215 220 Gly Gly Ile Thr Val Gly Asp Ala Ser Ala Thr Arg Arg Glu Val His 225 230 235 240 Thr Pro Val Gln Glu Thr Ser Asp Ser Ala Gln Val Ser Ile Gly Trp 245 250 255 Pro Gly Lys Val Ile Ala Ala Gly Ser Val Ile Glu Ile Gly Glu Ser 260 265 270 Phe Ala Val Val His Pro Gly Asp Tyr Tyr Asn Gly Leu Arg Gly Tyr 275 280 285 Lys Asn Ala Met Asp His Leu Gly Val Ile Met Pro Ala Pro Gly Asp 290 295 300 Ile Pro Asp Ser Ser Tyr Asp Leu Arg Trp Glu Ser Trp Gly Trp Gly 305 310 315 320 Phe Asn Trp Thr Ile Asp Leu Ile Ile Gly Lys Leu Asp Glu Leu Gln 325 330 335 Ala Ala Gly Val Lys Gln Ile Thr Leu Asp Asp Gly Trp Tyr Thr Asn 340 345 350 Ala Gly Asp Trp Ala Leu Asn Pro Glu Lys Phe Pro Asn Gly Ala Ser 355 360 365 Asp Ala Leu Arg Leu Thr Asp Ala Ile His Glu His Gly Met Thr Ala 370 375 380 Leu Leu Trp Trp Arg Pro Cys Asp Gly Gly Ile Asp Ser Ile Leu Tyr 385 390 395 400 Gln Gln His Pro Glu Tyr Phe Val Met Asp Ala Asp Gly Arg Pro Ala 405 410 415 Arg Leu Pro Thr Pro Gly Gly Gly Thr Asn Pro Ser Leu Gly Tyr Ala 420 425 430 Leu Cys Pro Met Ala Asp Gly Ala Ile Ala Ser Gln Val Asp Phe Val 435 440 445 Asn Arg Ala Met Asn Asp Trp Gly Phe Asp Gly Phe Lys Gly Asp Tyr 450 455 460 Val Trp Ser Met Pro Glu Cys Tyr Asn Pro Ala His Asn His Ala Ser 465 470 475 480 Pro Glu Glu Ser Thr Glu Lys Gln Ser Glu Ile Tyr Arg Val Ser Tyr 485 490 495 Glu Ala Met Val Ala Asn Asp Pro Asn Val Phe Asn Leu Leu Cys Asn 500 505 510 Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu Pro Tyr Met Thr Gln Ile 515 520 525 Ala Thr Ala Asp Pro Thr Ser Val Asp Gln Thr Arg Arg Arg Val Lys 530 535 540 Ala Tyr Lys Ala Leu Met Gly Asp Tyr Phe Pro Val Thr Ala Asp His 545 550 555 560 Asn Asn Ile Trp Tyr Pro Ser Ala Val Gly Thr Gly Ser Val Leu Ile 565 570 575 Glu Lys Arg Asp Leu Ser Gly Thr Ala Lys Glu Glu Tyr Glu Lys Trp 580 585 590 Leu Gly Ile Ala Asp Thr Val Gln Leu Gln Lys Gly Arg Phe Ile Gly 595 600 605 Asp Leu Tyr Ser Tyr Gly Phe Asp Pro Tyr Glu Thr Tyr Val Val Glu 610 615 620 Lys Asp Gly Val Met Tyr Tyr Ala Phe Tyr Lys Asp Gly Ser Lys Tyr 625 630 635 640 Ser Pro Thr Gly Tyr Pro Asp Ile Glu Leu Lys Gly Leu Asp Pro Asn 645 650 655 Lys Met Tyr Arg Ile Val Asp Tyr Val Asn Asp Arg Val Val Ala Thr 660 665 670 Asn Leu Met Gly Asp Asn Ala Val Phe Asn Thr Arg Phe Ser Asp Tyr 675 680 685 Leu Leu Val Lys Ala Val Glu Ile Ser Glu Pro Asp Pro Glu Pro Val 690 695 700 Asp Pro Asp Tyr Gly Phe Thr Ser Val Asp Asp Arg Asp Glu Ala Leu 705 710 715 720 Ile Tyr Thr Gly Thr Trp His Asp Asp Asn Asn Ala Ser Phe Ser Glu 725 730 735 Gly Thr Ala Arg Tyr Thr Asn Ser Thr Asp Ala Ser Val Val Phe Ser 740 745 750 Phe Thr Gly Thr Ser Ile Arg Trp Tyr Gly Gln Arg Asp Thr Asn Phe 755 760 765 Gly Thr Ala Glu Val Tyr Leu Asp Asp Glu Leu Lys Thr Thr Val Asp 770 775 780 Ala Asn Gly Ala Ala Glu Ala Gly Val Cys Leu Phe Glu Ala Leu Asp 785 790 795 800 Leu Pro Ala Ala Glu His Thr Ile Lys Ile Val Cys Lys Ser Gly Val 805 810 815 Ile Asp Ile Asp Arg Phe Ala Tyr Glu Ala Ala Thr Leu Glu Pro Ile 820 825 830 Tyr Glu Lys Val Asp Ala Leu Ser Asp Arg Ile Thr Tyr Val Gly Asn 835 840 845 Trp Glu Glu Tyr His Asn Ser Glu Phe Tyr Met Gly Asn Ala Met Arg 850 855 860 Thr Asp Glu Ala Gly Ala Tyr Ala Glu Leu Thr Phe Arg Gly Thr Ala 865 870 875 880 Val Arg Leu Tyr Ala Glu Met Ser Phe Asn Phe Gly Thr Ala Asp Val 885 890 895 Tyr Leu Asp Gly Glu Leu Val Glu Asn Ile Ile Leu Tyr Gly Gln Glu 900 905 910 Ala Thr Gly Gln Leu Met Phe Glu Arg Thr Gly Leu Glu Glu Gly Glu 915 920 925 His Thr Ile Arg Leu Val Gln Asn Ala Trp Asn Ile Asn Leu Asp Tyr 930 935 940 Ile Ser Tyr Leu Pro Glu Gln Asp Gln Pro Thr Pro Pro Glu Thr Thr 945 950 955 960 Val Thr Val Asp Ala Met Asp Ala Gln Leu Val Tyr Thr Gly Val Trp 965 970 975 Asn Asp Asp Tyr His Asp Val Phe Gln Glu Gly Thr Ala Arg Tyr Ala 980 985 990 Ser Ser Ala Gly Ala Ser Val Glu Phe Glu Phe Thr Gly Ser Glu Ile 995 1000 1005 Arg Trp Tyr Gly Gln Asn Asp Ser Asn Phe Gly Val Ala Ser Val 1010 1015 1020 Tyr Ile Asp Asn Glu Phe Val Gln Gln Val Asn Val Asn Gly Ala 1025 1030 1035 Ala Ala Val Gly Lys Leu Leu Phe Gln Lys Ala Asp Leu Pro Ala 1040 1045 1050 Gly Ser His Thr Ile Arg Ile Val Cys Asp Thr Pro Val Ile Asp 1055 1060 1065 Leu Asp Tyr Leu Thr Tyr Thr Thr Asn Ala 1070 1075 <210> 8 <211> 3159 <212> DNA <213> Flavonifractor plautii <400> 8 gcggcgcctg caacggacac cggcaacgca ggactgattg cagaaggtga ttatgccatt 60 gccggcaatg gcgtccgcgt cacttatgac gcggacgggc agacaatcac tctgtaccgc 120 acagagggat ctgggcttat ccagatgagc aagccttctc cattgggagg gccagtgatt 180 ggagggcagg aggttcagga cttcagccat atttcatgtg atgtggagca gagcaccagc 240 ggagtgatgg gcagcggtca gagaatgacc attacctctc agagcatgag cacgggccta 300 attcgtacct atgtgctgga gacctctgat atcgaggagg gtgtggtata tactgcaaca 360 tcctatgagg caggagcttc tgatgtggaa gtgtcttggt tcattggcag tgtgtatgag 420 ctttatggtg cggaagatcg tatctggagt tataacggcg gcggtgaggg gccgatgcac 480 tactatgata cgcttcaaaa gattgacctg accgactctg gcaagttcag tagggagaat 540 aaacaggatg acacggctgc aagtattcct gtgtcagata tttacattgc tgatggaggg 600 attaccgttg gcgatgcttc tgcaaccaga agggaggtac atactccggt tcaggaaacc 660 agtgattcag ctcaagtttc tatcgggtgg ccaggcaaag tcattgccgc cggaagcgtg 720 atcgaaattg gtgagagctt tgctgtagtc catccgggtg actattataa cggcttgaga 780 ggttacaaaa atgcaatgga tcacttgggc gtgattatgc ctgcacctgg ggatattcct 840 gatagcagct atgatctccg atgggaaagc tggggctggg ggtttaactg gacgatcgat 900 ttaataatcg gcaaattgga tgaacttcag gcagccggag tcaagcagat cactttggat 960 gatggttggt ataccaatgc aggagactgg gccttaaatc cagaaaagtt tccaaatgga 1020 gcctccgatg cgttgcggct gacagatgca attcatgagc atggtatgac tgcactcctt 1080 tggtggagac cttgtgacgg cgggatcgat agtatactct atcagcaaca ccctgaatat 1140 ttcgttatgg atgcagatgg aagacctgca aggcttccta ctcctggtgg tgggaccaat 1200 cccagcttgg gatatgcact ttgccctatg gcggatggtg cgattgcaag ccaagttgac 1260 tttgtaaacc gtgcaatgaa tgattggggg ttcgatggct tcaagggaga ttatgtgtgg 1320 agtatgcctg aatgctacaa tcctgcacat aaccacgcct cgccagaaga atccactgaa 1380 aagcaatccg agatataccg cgtctcttat gaggctatg tggccaacga ccccaatgtg 1440 ttcaatttgt tgtgcaactg cggtacgccc caggactact atagtttacc atatatgaca 1500 cagattgcta cggctgaccc cacttctgtg gatcaaacaa ggagacgcgt gaaagcctac 1560 aaggcactga tgggagatta tttccctgtt acagccgacc acaataacat ctggtatcca 1620 agtgccgtcg gtacgggctc tgttctcatt gaaaaacgtg accttagcgg tactgccaag 1680 gaagaattg aaaaatggct tgggattgcg gatacagttc agttgcagaa aggccggttt 1740 attggcgatc tttacagtta tggttttgac ccttacgaaa cctatgtggt ggagaaagac 1800 ggggttatgt actatgcctt ctacaaagat gggagcaaat atagccccac tggctatcca 1860 gatattgagt tgaaggggct agatccaaat aaaatgtata ggattgttga ctatgtcaat 1920 gatcgtgtcg tggcaacaaa cctgatgggt gataacgctg tattcaatac acgtttttcc 1980 gactatctac tggttaaagc ggtggaaatt tcggaaccgg atccagaacc tgttgaccct 2040 gattatggtt tcacctctgt tgatgacaga gacgaggctc ttatttacac agggacatgg 2100 catgatgaca ataacgcatc tttcagcgaa gggactgcac gttataccaa cagtacggat 2160 gcttcggttg tattctcctt tactggaact tccattcgct ggtatggcca gagggatacc 2220 aattttggca cggcagaagt ttatttggac gatgaactga aaacaacagt tgatgcgaat 2280 ggggccgcag aagcaggcgt atgtcttttt gaggcgcttg atcttccggc tgccgagcat 2340 accattaaaa ttgtgtgcaa gagcggagtg attgatattg accgctttgc atatgaagct 2400 gctacccttg aacccatcta tgaaaaggtc gatgcgctct cggatcggat cacttatgtt 2460 gggaattggg aagagtatca caacagcgag ttctacatgg gaaacgcaat gcgcacagac 2520 gaagccggcg cttatgctga actgactttc cgtggtacag ccgtacgcct gtatgcagag 2580 atgagcttca attttggcac tgcagatgtc tatttagacg gagagttagt ggaaaacata 2640 atcctatacg gccaggaagc aactgggcag ctaatgtttg agcgtacggg actggaggaa 2700 ggagaacata ccattcgcct tgtacaaaac gcctggaaca tcaatttgga ctatatttct 2760 tatctaccag agcaagatca accaacgccg ccggagacga cggttactgt tgatgcaatg 2820 gacgcccaac tggtgtatac aggcgtatgg aatgatgact atcatgacgt ctttcaggaa 2880 ggaaccgccc gttatgccag tagtgccggc gcctcggtcg agttcgaatt tactggaagc 2940 gaaatccgtt ggtatggaca aaatgattcc aacttcggtg ttgccagcgt ttatatcgat 3000 aatgagtttg tgcagcaggt aaatgttaac ggagctgcgg ctgtgggaaa gcttttgttt 3060 caaaaggctg atctaccagc cggttcgcac acgatccgca ttgtgtgcga tactccggtt 3120 attgatttgg actatttgac ttataccact aacgcataa 3159 <210> 9 <211> 1052 <212> PRT <213> Flavonifractor plautii <400> 9 Ala Ala Pro Ala Thr Asp Thr Gly Asn Ala Gly Leu Ile Ala Glu Gly 1 5 10 15 Asp Tyr Ala Ile Ala Gly Asn Gly Val Arg Val Thr Tyr Asp Ala Asp 20 25 30 Gly Gln Thr Ile Thr Leu Tyr Arg Thr Glu Gly Ser Gly Leu Ile Gln 35 40 45 Met Ser Lys Pro Ser Pro Leu Gly Gly Pro Val Ile Gly Gly Gln Glu 50 55 60 Val Gln Asp Phe Ser His Ile Ser Cys Asp Val Glu Gln Ser Thr Ser 65 70 75 80 Gly Val Met Gly Ser Gly Gln Arg Met Thr Ile Thr Ser Gln Ser Met 85 90 95 Ser Thr Gly Leu Ile Arg Thr Tyr Val Leu Glu Thr Ser Asp Ile Glu 100 105 110 Glu Gly Val Val Tyr Thr Ala Thr Ser Tyr Glu Ala Gly Ala Ser Asp 115 120 125 Val Glu Val Ser Trp Phe Ile Gly Ser Val Tyr Glu Leu Tyr Gly Ala 130 135 140 Glu Asp Arg Ile Trp Ser Tyr Asn Gly Gly Gly Glu Gly Pro Met His 145 150 155 160 Tyr Tyr Asp Thr Leu Gln Lys Ile Asp Leu Thr Asp Ser Gly Lys Phe 165 170 175 Ser Arg Glu Asn Lys Gln Asp Asp Thr Ala Ala Ser Ile Pro Val Ser 180 185 190 Asp Ile Tyr Ile Ala Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Ala 195 200 205 Thr Arg Arg Glu Val His Thr Pro Val Gln Glu Thr Ser Asp Ser Ala 210 215 220 Gln Val Ser Ile Gly Trp Pro Gly Lys Val Ile Ala Ala Gly Ser Val 225 230 235 240 Ile Glu Ile Gly Glu Ser Phe Ala Val Val His Pro Gly Asp Tyr Tyr 245 250 255 Asn Gly Leu Arg Gly Tyr Lys Asn Ala Met Asp His Leu Gly Val Ile 260 265 270 Met Pro Ala Pro Gly Asp Ile Pro Asp Ser Ser Tyr Asp Leu Arg Trp 275 280 285 Glu Ser Trp Gly Trp Gly Phe Asn Trp Thr Ile Asp Leu Ile Ile Gly 290 295 300 Lys Leu Asp Glu Leu Gln Ala Ala Gly Val Lys Gln Ile Thr Leu Asp 305 310 315 320 Asp Gly Trp Tyr Thr Asn Ala Gly Asp Trp Ala Leu Asn Pro Glu Lys 325 330 335 Phe Pro Asn Gly Ala Ser Asp Ala Leu Arg Leu Thr Asp Ala Ile His 340 345 350 Glu His Gly Met Thr Ala Leu Leu Trp Trp Arg Pro Cys Asp Gly Gly 355 360 365 Ile Asp Ser Ile Leu Tyr Gln Gln His Pro Glu Tyr Phe Val Met Asp 370 375 380 Ala Asp Gly Arg Pro Ala Arg Leu Pro Thr Pro Gly Gly Gly Thr Asn 385 390 395 400 Pro Ser Leu Gly Tyr Ala Leu Cys Pro Met Ala Asp Gly Ala Ile Ala 405 410 415 Ser Gln Val Asp Phe Val Asn Arg Ala Met Asn Asp Trp Gly Phe Asp 420 425 430 Gly Phe Lys Gly Asp Tyr Val Trp Ser Met Pro Glu Cys Tyr Asn Pro 435 440 445 Ala His Asn His Ala Ser Pro Glu Glu Ser Thr Glu Lys Gln Ser Glu 450 455 460 Ile Tyr Arg Val Ser Tyr Glu Ala Met Val Ala Asn Asp Pro Asn Val 465 470 475 480 Phe Asn Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu 485 490 495 Pro Tyr Met Thr Gln Ile Ala Thr Ala Asp Pro Thr Ser Val Asp Gln 500 505 510 Thr Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Met Gly Asp Tyr Phe 515 520 525 Pro Val Thr Ala Asp His Asn Asn Ile Trp Tyr Pro Ser Ala Val Gly 530 535 540 Thr Gly Ser Val Leu Ile Glu Lys Arg Asp Leu Ser Gly Thr Ala Lys 545 550 555 560 Glu Glu Tyr Glu Lys Trp Leu Gly Ile Ala Asp Thr Val Gln Leu Gln 565 570 575 Lys Gly Arg Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Phe Asp Pro Tyr 580 585 590 Glu Thr Tyr Val Val Glu Lys Asp Gly Val Met Tyr Tyr Ala Phe Tyr 595 600 605 Lys Asp Gly Ser Lys Tyr Ser Pro Thr Gly Tyr Pro Asp Ile Glu Leu 610 615 620 Lys Gly Leu Asp Pro Asn Lys Met Tyr Arg Ile Val Asp Tyr Val Asn 625 630 635 640 Asp Arg Val Val Ala Thr Asn Leu Met Gly Asp Asn Ala Val Phe Asn 645 650 655 Thr Arg Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile Ser Glu 660 665 670 Pro Asp Pro Glu Pro Val Asp Pro Asp Tyr Gly Phe Thr Ser Val Asp 675 680 685 Asp Arg Asp Glu Ala Leu Ile Tyr Thr Gly Thr Trp His Asp Asp Asn 690 695 700 Asn Ala Ser Phe Ser Glu Gly Thr Ala Arg Tyr Thr Asn Ser Thr Asp 705 710 715 720 Ala Ser Val Val Phe Ser Phe Thr Gly Thr Ser Ile Arg Trp Tyr Gly 725 730 735 Gln Arg Asp Thr Asn Phe Gly Thr Ala Glu Val Tyr Leu Asp Asp Glu 740 745 750 Leu Lys Thr Thr Val Asp Ala Asn Gly Ala Ala Glu Ala Gly Val Cys 755 760 765 Leu Phe Glu Ala Leu Asp Leu Pro Ala Ala Glu His Thr Ile Lys Ile 770 775 780 Val Cys Lys Ser Gly Val Ile Asp Ile Asp Arg Phe Ala Tyr Glu Ala 785 790 795 800 Ala Thr Leu Glu Pro Ile Tyr Glu Lys Val Asp Ala Leu Ser Asp Arg 805 810 815 Ile Thr Tyr Val Gly Asn Trp Glu Glu Tyr His Asn Ser Glu Phe Tyr 820 825 830 Met Gly Asn Ala Met Arg Thr Asp Glu Ala Gly Ala Tyr Ala Glu Leu 835 840 845 Thr Phe Arg Gly Thr Ala Val Arg Leu Tyr Ala Glu Met Ser Phe Asn 850 855 860 Phe Gly Thr Ala Asp Val Tyr Leu Asp Gly Glu Leu Val Glu Asn Ile 865 870 875 880 Ile Leu Tyr Gly Gln Glu Ala Thr Gly Gln Leu Met Phe Glu Arg Thr 885 890 895 Gly Leu Glu Glu Gly Glu His Thr Ile Arg Leu Val Gln Asn Ala Trp 900 905 910 Asn Ile Asn Leu Asp Tyr Ile Ser Tyr Leu Pro Glu Gln Asp Gln Pro 915 920 925 Thr Pro Pro Glu Thr Thr Val Thr Val Asp Ala Met Asp Ala Gln Leu 930 935 940 Val Tyr Thr Gly Val Trp Asn Asp Asp Tyr His Asp Val Phe Gln Glu 945 950 955 960 Gly Thr Ala Arg Tyr Ala Ser Ser Ala Gly Ala Ser Val Glu Phe Glu 965 970 975 Phe Thr Gly Ser Glu Ile Arg Trp Tyr Gly Gln Asn Asp Ser Asn Phe 980 985 990 甘氨酸-缬氨酸-丙氨酸-丝氨酸-缬氨酸-酪氨酸-异亮氨酸-天冬氨酸-天冬酰胺-谷氨酸-苯丙氨酸-缬氨酸-谷氨酰胺-谷氨酰胺-缬氨酸-天冬酰胺 995 1000 1005 缬氨酸-天冬酰胺-甘氨酸-丙氨酸-丙氨酸-丙氨酸-缬氨酸-甘氨酸-赖氨酸-亮氨酸-亮氨酸-苯丙氨酸-谷氨酰胺-赖氨酸-丙氨酸 1010 1015 1020 天冬氨酸-亮氨酸-脯氨酸-丙氨酸-甘氨酸-丝氨酸-组氨酸-苏氨酸-异亮氨酸-精氨酸-异亮氨酸-缬氨酸-半胱氨酸-天冬氨酸-苏氨酸 1025 1030 1035 脯氨酸-缬氨酸-异亮氨酸-天冬氨酸-亮氨酸-天冬氨酸-酪氨酸-亮氨酸-苏氨酸-酪氨酸-苏氨酸-苏氨酸-天冬酰胺-丙氨酸 104-0 1045 1050 <210> 10 <211> 1067 <212> PRT <213> Flavonifractor plautii <400> 10 甲硫氨酸-甘氨酸-组氨酸-组氨酸-组氨酸-组氨酸-组氨酸-组氨酸-组氨酸-组氨酸-组氨酸-丝氨酸-丝氨酸-甘氨酸-丙氨酸 1 5 10 15 丙氨酸-脯氨酸-丙氨酸-苏氨酸-天冬氨酸-苏氨酸-甘氨酸-天冬酰胺-丙氨酸-甘氨酸-亮氨酸-异亮氨酸-丙氨酸-谷氨酸-甘氨酸-天冬氨酸 20 25 30 酪氨酸-丙氨酸-异亮氨酸-丙氨酸-甘氨酸-天冬酰胺-甘氨酸-缬氨酸-精氨酸-缬氨酸-苏氨酸-酪氨酸-天冬氨酸-丙氨酸-天冬氨酸-甘氨酸 35 40 45 谷氨酰胺-苏氨酸-异亮氨酸-苏氨酸-亮氨酸-酪氨酸-精氨酸-苏氨酸-谷氨酸-甘氨酸-丝氨酸-甘氨酸-亮氨酸-异亮氨酸-谷氨酰胺-甲硫氨酸 50 55 60 丝氨酸-赖氨酸-脯氨酸-丝氨酸-脯氨酸-亮氨酸-甘氨酸-甘氨酸-脯氨酸-缬氨酸-异亮氨酸-甘氨酸-甘氨酸-谷氨酰胺-谷氨酸-缬氨酸 65 70 75 80 Gln Asp Phe Ser His Ile Ser Cys Asp Val Glu Gln Ser Thr Ser Gly 85 90 95 Val Met Gly Ser Gly Gln Arg Met Thr Ile Thr Ser Gln Ser Met Ser 100 105 110 Thr Gly Leu Ile Arg Thr Tyr Val Leu Glu Thr Ser Asp Ile Glu Glu 115 120 125 Gly Val Val Tyr Thr Ala Thr Ser Tyr Glu Ala Gly Ala Ser Asp Val 130 135 140 Glu Val Ser Trp Phe Ile Gly Ser Val Tyr Glu Leu Tyr Gly Ala Glu 145 150 155 160 Asp Arg Ile Trp Ser Tyr Asn Gly Gly Gly Glu Gly Pro Met His Tyr 165 170 175 Tyr Asp Thr Leu Gln Lys Ile Asp Leu Thr Asp Ser Gly Lys Phe Ser 180 185 190 Arg Glu Asn Lys Gln Asp Asp Thr Ala Ala Ser Ile Pro Val Ser Asp 195 200 205 Ile Tyr Ile Ala Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Ala Thr 210 215 220 Arg Arg Glu Val His Thr Pro Val Gln Glu Thr Ser Asp Ser Ala Gln 225 230 235 240 Val Ser Ile Gly Trp Pro Gly Lys Val Ile Ala Ala Gly Ser Val Ile 245 250 255 Glu Ile Gly Glu Ser Phe Ala Val Val His Pro Gly Asp Tyr Tyr Asn 260 265 270 Gly Leu Arg Gly Tyr Lys Asn Ala Met Asp His Leu Gly Val Ile Met 275 280 285 Pro Ala Pro Gly Asp Ile Pro Asp Ser Ser Tyr Asp Leu Arg Trp Glu 290 295 300 Ser Trp Gly Trp Gly Phe Asn Trp Thr Ile Asp Leu Ile Ile Gly Lys 305 310 315 320 Leu Asp Glu Leu Gln Ala Ala Gly Val Lys Gln Ile Thr Leu Asp Asp 325 330 335 Gly Trp Tyr Thr Asn Ala Gly Asp Trp Ala Leu Asn Pro Glu Lys Phe 340 345 350 Pro Asn Gly Ala Ser Asp Ala Leu Arg Leu Thr Asp Ala Ile His Glu 355 360 365 His Gly Met Thr Ala Leu Leu Trp Trp Arg Pro Cys Asp Gly Gly Ile 370 375 380 Asp Ser Ile Leu Tyr Gln Gln His Pro Glu Tyr Phe Val Met Asp Ala 385 390 395 400 Asp Gly Arg Pro Ala Arg Leu Pro Thr Pro Gly Gly Gly Thr Asn Pro 405 410 415 Ser Leu Gly Tyr Ala Leu Cys Pro Met Ala Asp Gly Ala Ile Ala Ser 420 425 430 Gln Val Asp Phe Val Asn Arg Ala Met Asn Asp Trp Gly Phe Asp Gly 435 440 445 Phe Lys Gly Asp Tyr Val Trp Ser Met Pro Glu Cys Tyr Asn Pro Ala 450 455 460 His Asn His Ala Ser Pro Glu Glu Ser Thr Glu Lys Gln Ser Glu Ile 465 470 475 480 Tyr Arg Val Ser Tyr Glu Ala Met Val Ala Asn Asp Pro Asn Val Phe 485 490 495 Asn Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu Pro 500 505 510 Tyr Met Thr Gln Ile Ala Thr Ala Asp Pro Thr Ser Val Asp Gln Thr 515 520 525 Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Met Gly Asp Tyr Phe Pro 530 535 540 Val Thr Ala Asp His Asn Asn Ile Trp Tyr Pro Ser Ala Val Gly Thr 545 550 555 560 Gly Ser Val Leu Ile Glu Lys Arg Asp Leu Ser Gly Thr Ala Lys Glu 565 570 575 Glu Tyr Glu Lys Trp Leu Gly Ile Ala Asp Thr Val Gln Leu Gln Lys 580 585 590 Gly Arg Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Phe Asp Pro Tyr Glu 595 600 605 Thr Tyr Val Val Glu Lys Asp Gly Val Met Tyr Tyr Ala Phe Tyr Lys 610 615 620 Asp Gly Ser Lys Tyr Ser Pro Thr Gly Tyr Pro Asp Ile Glu Leu Lys 625 630 635 640 Gly Leu Asp Pro Asn Lys Met Tyr Arg Ile Val Asp Tyr Val Asn Asp 645 650 655 Arg Val Val Ala Thr Asn Leu Met Gly Asp Asn Ala Val Phe Asn Thr 660 665 670 Arg Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile Ser Glu Pro 675 680 685 Asp Pro Glu Pro Val Asp Pro Asp Tyr Gly Phe Thr Ser Val Asp Asp 690 695 700 Arg Asp Glu Ala Leu Ile Tyr Thr Gly Thr Trp His Asp Asp Asn Asn 705 710 715 720 Ala Ser Phe Ser Glu Gly Thr Ala Arg Tyr Thr Asn Ser Thr Asp Ala 725 730 735 Ser Val Val Phe Ser Phe Thr Gly Thr Ser Ile Arg Trp Tyr Gly Gln 740 745 750 Arg Asp Thr Asn Phe Gly Thr Ala Glu Val Tyr Leu Asp Asp Glu Leu 755 760 765 Lys Thr Thr Val Asp Ala Asn Gly Ala Ala Glu Ala Gly Val Cys Leu 770 775 780 Phe Glu Ala Leu Asp Leu Pro Ala Ala Glu His Thr Ile Lys Ile Val 785 790 795 800 Cys Lys Ser Gly Val Ile Asp Ile Asp Arg Phe Ala Tyr Glu Ala Ala 805 810 815 Thr Leu Glu Pro Ile Tyr Glu Lys Val Asp Ala Leu Ser Asp Arg Ile 820 825 830 Thr Tyr Val Gly Asn Trp Glu Glu Tyr His Asn Ser Glu Phe Tyr Met 835 840 845 Gly Asn Ala Met Arg Thr Asp Glu Ala Gly Ala Tyr Ala Glu Leu Thr 850 855 860 Phe Arg Gly Thr Ala Val Arg Leu Tyr Ala Glu Met Ser Phe Asn Phe 865 870 875 880 Gly Thr Ala Asp Val Tyr Leu Asp Gly Glu Leu Val Glu Asn Ile Ile 885 890 895 Leu Tyr Gly Gln Glu Ala Thr Gly Gln Leu Met Phe Glu Arg Thr Gly 900 905 910 Leu Glu Glu Gly Glu His Thr Ile Arg Leu Val Gln Asn Ala Trp Asn 915 920 925 Ile Asn Leu Asp Tyr Ile Ser Tyr Leu Pro Glu Gln Asp Gln Pro Thr 930 935 940 Pro Pro Glu Thr Thr Val Thr Val Asp Ala Met Asp Ala Gln Leu Val 945 950 955 960 Tyr Thr Gly Val Trp Asn Asp Asp Tyr His Asp Val Phe Gln Glu Gly 965 970 975 Thr Ala Arg Tyr Ala Ser Ser Ala Gly Ala Ser Val Glu Phe Glu Phe 980 985 990 Thr Gly Ser Glu Ile Arg Trp Tyr Gly Gln Asn Asp Ser Asn Phe Gly 995 1000 1005 Val Ala Ser Val Tyr Ile Asp Asn Glu Phe Val Gln Gln Val Asn 1010 1015 1020 Val Asn Gly Ala Ala Ala Val Gly Lys Leu Leu Phe Gln Lys Ala 1025 1030 1035 Asp Leu Pro Ala Gly Ser His Thr Ile Arg Ile Val Cys Asp Thr 1040 1045 1050 Pro Val Ile Asp Leu Asp Tyr Leu Thr Tyr Thr Thr Asn Ala 1055 1060 1065 <210> 11 <211> 3963 <212> DNA <213> Clostridium tertium <400> 11 atgaaaaaaa gaattttagc tacttttatt acagctatgt gtggactggg atttttttca 60 aactggactt caagtaatgc ttataattta attgataata ttagtgttga aaaattagat 120 actgatattt cacaagcaaa tgaaaatgtt tttttgaatg gaaatggaat tgctttagaa 180 gtagataata gaggcgctac atgtatttat ctagtagatg aaaatggagt taaaacaaaa 240 gctacgactt ctttagatac agcagatttt tcaggttatc caataatagg tggacaaaag 300 atagagatt ttgtattat atcaaaaat ctagaagaaa acataaactc gatattaggt 360 gttggaaata gacttactat tatactaaa agttcatcta ctaatctgat aaagaata 420 gtatttgaaa catctaacag caatccagga gcaatatt cacagtaag tttaaagca 480 gaagtaacg attattagt agatagcttt catgaaaatg agtatacaat gagtttaggg 540 caggacctt ttctgcata tcaagggtgt gcagatcac aaggagcaa tactatcgtt 600 aatgttacta atggatataa ccatatagt ggacaaaata attattctgt aggagttcca 660 tttagttatg ttataacc tgtgggggga attggaatag gtgatgcatc aacttcaaga 720 agagaattta agttgcctat tataggaaa gatacag ttcattagg atggagtgg 780 aatggacaaa ctttaaaaaa aggtgctgaa actgctatag gtacaagtgt tataactaca 840 acaaatggtg attattattc tgggctaaag agttacgcag aagttatgaa agataggga 900 atatctgcac cagcttcaat acctgatata gcatatgatt ctagatggga aagttgggga 960 ttcgaatttg attttacaat agaaaaaata gttaataat tagatgact taagcgatg 1020 gggataaaac aaattactct agatgatggg tggtacactt atgctggtga ttggaaatta 1080 agtcctcaaa agtttccaaa tggaaatgca gacatgaaat atcttacaga tgaaatccat 1140 aaaagaggaa tgacagctat tttatggtgg agaccagtag acggagggat aaatagcaaa 1200 ttagtatctg aacatccaga gtggtttatt aagaactcac aagggaatat ggtaggtta 1260 ccagggcctg gaggtggaaa tggaggaaca gcaggatatg cattatgtcc aaattcagaa 1320 ggttcaattc aacatcataa agattttgta actgtggcat tagaagaatg gggatttgat 1380 ggattcaaag aagattatgt atggggaata cctaaatgct atgatagttc tcataaacac 1440 tcaagtttat cagatacatt agaaaatcaa tataaattct atgaagccat atatgaacag 1500 tccatagcga taaatccaga tactttata gaattatgta attgcggaac acctcaggat 1560 ttttattcaa caccatatgt gaaccatgca ccaacagcag atccaatttc gagagtacaa 1620 acaagaacaa gagtgaaagc atttaaagct atatttggag atgattttcc agtaacaaca 1680 gatcataatt cagtttggtt accgtcagca ttaggtacag gatcagttat gattactaaaa 1740 catacacat tagtagttc agatagagaa cataata atacttcgg acttgcaga 1800 gatttagaat tagcaaggg agaatttatta ggaacttat ataatacgg atagatcca 1860 ttagagtcat atgttattaag aaaaaaaaaaaaaggttttcattcta 1920s tctagttatt caggaatt agaataag gggttagaca gtaacgccac atatagaatt 1980 gagattatg ttacatag agttattgct aggagtaa aggaccac agcgactata 2040 atacaagct ttactgata tttattagtt agagcaatac attacaagct ttactgatac 2100 gttactacat ttgatgttgg aaatataca attackcat caacagatg tggaaatttct 2160 aaatatta atgctgtttc tactacatta gaaagacag cacaataga tagtttagt 2220 atttatatag gaaatattc agaaaatggc aaactacaaa ttgctattta tgacgataat 2280 aaacgggaaac ctggtactaa aaagcttac gtagaagagt ttgttcctac taaaatagt 2340 tggaatacaa agaaggttgt aaattctgtt acattacctt caggcaata tggttagtt 2400 ttccaacctg ataacgatgt actacaaca aaaactaatc catcatccat gaacaagt 2460 gctaacaata atccatataa ttataatata ttaccaaatt catttcctat tggaacagga 2520 tataatgctt ataaaggcga tgtatctttc tatgcaacct ttaaagaagc aagcagtcaa 2580 gcaattcctc aaaattcttg ggctctaaaa tatgtagata gtgaagaaac tacaggcgaa 2640 aatggaagag ctacaaatgc ttttgatggt aataataata ctatttggca cacaaaatat 2700 agtggcggaa acgctgcacc aatgccgcat gagattcaaa ttgatttaag aggagtatat 2760 aatataaatc aaattaatta tctaccaaga caagatggag gaaccaatgg tacaataaag 2820 gactatgaag tttatttaag tttagatgga gtgaactggg gacaacctat atcaaaagga 2880 acctttgaat caaacctac agaaaaata gtaaaattca acgaaacaaa atctaggtat 2940 gtaaaactta aagctctgtc agaaattaat aataaacaat ttactacagt agctgattta 3000 aaggtatttg gatgggagat atccaaaata gaaaaaccat tacaaaatgc tgaaacttat 3060 ttgaatac caacttatga tggattaaat caaagtactc atccagatgt caaatatttt 3120 aaaaatggtt ggaatggata taaatattgg atgataatga ctccaaatag aacaggtagc 3180 tcagttgctg aaaatccttc ataactagca tctgatgatg gaataaattg ggaggttcct 3240 gcaggtgtta caaatcctat agctccaatg ccacaagtag gacataattg tgatgttgat 3300 atgatatata atgaagcaac tgatgagtta tgggtgtact gggtagaatc agatgatata 3360 acaaaaggat gggttaaatt aataaaatca aaggatggag taaattggag ttctcagcaa 3420 gtggtagttg atgataatag ggcaaaatat agtactttat caccatctat aatattcaaa 3480 gataataaat actatatgtg gtcagttaat acaggaaata gtggttggaa caatcaaagt 3540 aataaagttg attaagaga atcaagtgac ggagtaaatt ggtcaaatcc aacagttgta 3600 aacacattag ctcaagatgg ttctcaaata tggcatgtaa atgtagaata tataccatca 3660 aaaaacgaat attgggctat atatccagca tataaaaatg gaacaggtag cgataaaaca 3720 gaattgtatt atgcgaaatc aagtgatgga gtaaattgga caacttataa gaatcctata 3780 tttcaaaag gaacatctgg taaatgggat gatatggaga tatatagaag ttgttttgtg 3840 tacgatgaag atacaaatat gataaaggtt tggtatggag ctgtgagtca aaatccacaa 3900 atatggaaaa taggttttac tgaaaatgat tatgataagt ttattgaggg tttaacacaa 3960 taa 3963 <210> 12 <211> 1320 <212> PRT <213> Clostridium tertium <400> 12 Met Lys Lys Arg Ile Leu Ala Thr Phe Ile Thr Ala Met Cys Gly Leu 1 5 10 15 Gly Phe Phe Ser Asn Trp Thr Ser Ser Asn Ala Tyr Asn Leu Ile Asp 20 25 30 Asn Ile Ser Val Glu Lys Leu Asp Thr Asp Ile Ser Gln Ala Asn Glu 35 40 45 Asn Val Phe Leu Asn Gly Asn Gly Ile Ala Leu Glu Val Asp Asn Arg 50 55 60 Gly Ala Thr Cys Ile Tyr Leu Val Asp Glu Asn Gly Val Lys Thr Lys 65 70 75 80 Ala Thr Thr Ser Leu Asp Thr Ala Asp Phe Ser Gly Tyr Pro Ile Ile 85 90 95 Gly Gly Gln Lys Ile Arg Asp Phe Val Ile Ile Ser Lys Asn Leu Glu 100 105 110 Glu Asn Ile Asn Ser Ile Leu Gly Val Gly Asn Arg Leu Thr Ile Ile 115 120 125 Ser Lys Ser Ser Ser Thr Asn Leu Ile Arg Lys Ile Val Phe Glu Thr 130 135 140 Ser Asn Ser Asn Pro Gly Ala Ile Tyr Ser Thr Val Ser Tyr Lys Ala 145 150 155 160 Glu Ser Asn Asp Leu Leu Val Asp Ser Phe His Glu Asn Glu Tyr Thr 165 170 175 Met Ser Leu Gly Gln Gly Pro Phe Leu Ala Tyr Gln Gly Cys Ala Asp 180 185 190 Gln Gln Gly Ala Asn Thr Ile Val Asn Val Thr Asn Gly Tyr Asn His 195 200 205 Asn Ser Gly Gln Asn Asn Tyr Ser Val Gly Val Pro Phe Ser Tyr Val 210 215 220 Tyr Asn Ser Val Gly Gly Ile Gly Ile Gly Asp Ala Ser Thr Ser Arg 225 230 235 240 Arg Glu Phe Lys Leu Pro Ile Ile Gly Lys Asp Asn Thr Val Ser Leu 245 250 255 Gly Met Glu Trp Asn Gly Gln Thr Leu Lys Lys Gly Ala Glu Thr Ala 260 265 270 Ile Gly Thr Ser Val Ile Thr Thr Thr Asn Gly Asp Tyr Tyr Ser Gly 275 280 285 Leu Lys Ser Tyr Ala Glu Val Met Lys Asp Lys Gly Ile Ser Ala Pro 290 295 300 Ala Ser Ile Pro Asp Ile Ala Tyr Asp Ser Arg Trp Glu Ser Trp Gly 305 310 315 320 Phe Glu Phe Asp Phe Thr Ile Glu Lys Ile Val Asn Lys Leu Asp Glu 325 330 335 Leu Lys Ala Met Gly Ile Lys Gln Ile Thr Leu Asp Asp Gly Trp Tyr 340 345 350 Thr Tyr Ala Gly Asp Trp Lys Leu Ser Pro Gln Lys Phe Pro Asn Gly 355 360 365 Asn Ala Asp Met Lys Tyr Leu Thr Asp Glu Ile His Lys Arg Gly Met 370 375 380 Thr Ala Ile Leu Trp Trp Arg Pro Val Asp Gly Gly Ile Asn Ser Lys 385 390 395 400 Leu Val Ser Glu His Pro Glu Trp Phe Ile Lys Asn Ser Gln Gly Asn 405 410 415 Met Val Arg Leu Pro Gly Pro Gly Gly Gly Asn Gly Gly Thr Ala Gly 420 425 430 Tyr Ala Leu Cys Pro Asn Ser Glu Gly Ser Ile Gln His His Lys Asp 435 440 445 Phe Val Thr Val Ala Leu Glu Glu Trp Gly Phe Asp Gly Phe Lys Glu 450 455 460 Asp Tyr Val Trp Gly Ile Pro Lys Cys Tyr Asp Ser Ser His Lys His 465 470 475 480 Ser Ser Leu Ser Asp Thr Leu Glu Asn Gln Tyr Lys Phe Tyr Glu Ala 485 490 495 Ile Tyr Glu Gln Ser Ile Ala Ile Asn Pro Asp Thr Phe Ile Glu Leu 500 505 510 Cys Asn Cys Gly Thr Pro Gln Asp Phe Tyr Ser Thr Pro Tyr Val Asn 515 520 525 His Ala Pro Thr Ala Asp Pro Ile Ser Arg Val Gln Thr Arg Thr Arg 530 535 540 Val Lys Ala Phe Lys Ala Ile Phe Gly Asp Asp Phe Pro Val Thr Thr 545 550 555 560 Asp His Asn Ser Val Trp Leu Pro Ser Ala Leu Gly Thr Gly Ser Val 565 570 575 Met Ile Thr Lys His Thr Thr Leu Ser Ser Ser Asp Arg Glu Gln Tyr 580 585 590 Asn Lys Tyr Phe Gly Leu Ala Arg Asp Leu Glu Leu Ala Lys Gly Glu 595 600 605 Phe Ile Gly Asn Leu Tyr Lys Tyr Gly Ile Asp Pro Leu Glu Ser Tyr 610 615 620 Val Ile Arg Lys Gly Glu Asp Ile Tyr Tyr Ser Phe Tyr Lys Asp Asn 625 630 635 640 Ser Ser Tyr Ser Gly Asn Ile Glu Ile Lys Gly Leu Asp Ser Asn Ala 645 650 655 Thr Tyr Arg Ile Glu Asp Tyr Val Asn Asn Arg Val Ile Ala Arg Gly 660 665 670 Val Lys Gly Pro Thr Ala Thr Ile Asn Thr Ser Phe Thr Asp Asn Leu 675 680 685 Leu Val Arg Ala Ile Pro Asp Asp Thr Pro Ala Glu Val Thr Thr Phe 690 695 700 Asp Val Gly Asn Asn Thr Ile Leu Ser Ser Thr Asp Ser Gly Asn Ser 705 710 715 720 Lys Tyr Leu Asn Ala Val Ser Thr Thr Leu Glu Lys Thr Ala Thr Ile 725 730 735 Asp Ser Leu Ser Ile Tyr Ile Gly Asn Asn Ser Glu Asn Gly Lys Leu 740 745 750 Gln Ile Ala Ile Tyr Asp Asp Asn Asn Gly Lys Pro Gly Thr Lys Lys 755 760 765 Ala Tyr Val Glu Glu Phe Val Pro Thr Lys Asn Ser Trp Asn Thr Lys 770 775 780 Lys Val Val Asn Ser Val Thr Leu Pro Ser Gly Gln Tyr Trp Leu Val 785 790 795 800 Phe Gln Pro Asp Asn Asp Val Leu Gln Thr Lys Thr Asn Pro Ser Ser 805 810 815 Met Lys Gln Ser Ala Asn Asn Asn Pro Tyr Asn Tyr Asn Ile Leu Pro 820 825 830 Asn Ser Phe Pro Ile Gly Thr Gly Tyr Asn Ala Tyr Lys Gly Asp Val 835 840 845 Ser Phe Tyr Ala Thr Phe Lys Glu Ala Ser Ser Gln Ala Ile Pro Gln 850 855 860 Asn Ser Trp Ala Leu Lys Tyr Val Asp Ser Glu Glu Thr Thr Gly Glu 865 870 875 880 Asn Gly Arg Ala Thr Asn Ala Phe Asp Gly Asn Asn Asn Thr Ile Trp 885 890 895 His Thr Lys Tyr Ser Gly Gly Asn Ala Ala Pro Met Pro His Glu Ile 900 905 910 Gln Ile Asp Leu Arg Gly Val Tyr Asn Ile Asn Gln Ile Asn Tyr Leu 915 920 925 Pro Arg Gln Asp Gly Gly Thr Asn Gly Thr Ile Lys Asp Tyr Glu Val 930 935 940 Tyr Leu Ser Leu Asp Gly Val Asn Trp Gly Gln Pro Ile Ser Lys Gly 945 950 955 960 Thr Phe Glu Ser Asn Ser Thr Glu Lys Ile Val Lys Phe Asn Glu Thr 965 970 975 Lys Ser Arg Tyr Val Lys Leu Lys Ala Leu Ser Glu Ile Asn Asn Lys 980 985 990 Gln Phe Thr Thr Val Ala Asp Leu Lys Val Phe Gly Trp Glu Ile Ser 995 1000 1005 Lys Ile Glu Lys Pro Leu Gln Asn Ala Glu Thr Tyr Leu Asn Ile 1010 1015 1020 Pro Thr Tyr Asp Gly Leu Asn Gln Ser Thr His Pro Asp Val Lys 1025 1030 1035 Tyr Phe Lys Asn Gly Trp Asn Gly Tyr Lys Tyr Trp Met Ile Met 1040 1045 1050 Thr Pro Asn Arg Thr Gly Ser Ser Val Ala Glu Asn Pro Ser Ile 1055 1060 1065 Leu Ala Ser Asp Asp Gly Ile Asn Trp Glu Val Pro Ala Gly Val 1070 1075 1080 Thr Asn Pro Ile Ala Pro Met Pro Gln Val Gly His Asn Cys Asp 1085 1090 1095 Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu Leu Trp Val Tyr 1100 1105 1110 Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val Lys Leu Ile 1115 1120 1125 Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln Val Val Val 1130 1135 1140 Asp Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro Ser Ile Ile 1145 1150 1155 Phe Lys Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn Thr Gly Asn 1160 1165 1170 Ser Gly Trp Asn Asn Gln Ser Asn Lys Val Glu Leu Arg Glu Ser 1175 1180 1185 Ser Asp Gly Val Asn Trp Ser Asn Pro Thr Val Val Asn Thr Leu 1190 1195 1200 Ala Gln Asp Gly Ser Gln Ile Trp His Val Asn Val Glu Tyr Ile 1205 1210 1215 Pro Ser Lys Asn Glue Tyr Trp Ala Ile Tyr Pro Ala Tyr Lys Asn 1220 1225 1230 Gly Thr Gly Ser Asp Lys Thr Glu Leu Tyr Tyr Ala Lys Ser Ser 1235 1240 1245 Asp Gly Val Asn Trp Thr Thr Tyr Lys Asn Pro Ile Leu Ser Lys 1250 1255 1260 Gly Thr Ser Gly Lys Trp Asp Asp Met Glu Ile Tyr Arg Ser Cys 1265 1270 1275 Phe Val Tyr Asp Glu Asp Thr Asn Met Ile Lys Val Trp Tyr Gly 1280 1285 1290 Only Gln Asn Pro Gln Ile Trp Lys Ile Gly Phe Thr Glu 1295 1300 1305 Asn Asp Tyr Asp Lys Phe Ile Glu Gly Leu Thr Gln 1310 1315 1320 <210> 13 <211> 3882 <212> DNA <213> Clostridium tertium (Clostridium tertium) <400> 13 tataatttaa ttgatatat tagtgttgaa aattagata ctgatatttc acagcaaat 60 gaaaatgtttt tttgaatgg aaatggaatt gctttagaag tagatatag aggcgctaca 120 tgtatttatc tagtagatga aaatggagtt aaaaaaaag ctacgacttc tttagataca 180 gcagattttt caggttatcc ataataggt ggacaaaga taagagattt tgtaattata 240 tcaaaaaatc tagaaaaa cataaactcg atattaggtg ttggaatag acttactatt 300 atactaaaa gttcatctac taatctgata agaagatag tatttgaac atctacagc 360 aatccaggag caatattc aacagtagt tataaagcag aaagtaacga tttagta 420 gatagctttc atgaaaatga gtatacaatg agtttaggc aaggacctttt tcttgcatat 480 caagggtgtg cagatcaca aggagcaat actatcgtta atgttacta tggatataac 540 cataatagtg gataaaata ttattctgta ggagttccat ttagttatgt ttataactct 600 gtggggggaa ttggaatagg tgatgcatca acttcagaa gagaatttaa gttgcctatt 660 ataggaaag atatacagt ttcattagga atggagtgga atggacaac tttaaaaaaa 720 ggtgctgaaa ctgctatagg tacaagtgtt ataactacaa caatggtga ttattattct 780 gggctaaga gttacgcaga agttatgaa gatagggaa tatctgcacc agcttcaata 840 cctgatatag catatgattc tagagggaa agttgggat tcgaatttga ttttacaata 900 gaaaaatag ttaataattt agatgaactt aaagcgatgg ggaaaaca attackcta 960 gatgatgggt ggtacactta tgctggtgat tggaaattaa gtcctcaaa gtttccaaat 1020 ggaaatgcag acatgaata tcttacagat gaatccata aagaggaat gagactatt 1080 ttatggtgga gaccagtaga cggagggata atagcaat tagtatctga acatccagag 1140 tggtttatta agaaccaca agggaatatg gttaggttac caggggctgg aggtggaat 1200 ggaggaacag caggatatgc attatgtcca attcagaag gttcaatca acatcataaa 1260 gattttgtaa ctgtggcatt agagaatgg ggatttgatg gattcaaga agattgta 1320 tggggaatac ctaaatgcta tgatagttct cataacact caagtttatc agatacatta 1380 gaaaatcaat ataattcta tgaagccata tatgaacagt ccatagcgat aaatccagat 1440 acttttatag aattatgtaa ttgcggaca cctcaggatt tttattcaac accatatgtg 1500 aaccatgcac storm tccaatttcg agagtacciaa storm storm 1560 tttaaagcta tatttggaga tgatttcca gtaacaacag atcaatattc agttttggtta 1620 ccgtcagcat taggtacagg atcagttg attackaac atacacatt aagtagttca 1680 gagagaac attaata atacttcgga cttgcagag atttagaatt agcaagga 1740 gatttattag gaacttata taaatacgga atagatccat taggtcata tgttataga 1800 aaaggaag attattatta ttcattctac aagaatatt ctagttattc aggaatta 1860 gaaataagg ggttagacag taacgccaca tatagaattg aagatttagt glassataga 1920 gttattgcta gaggagtaa gggaccaca gcgacttaa atacaagctt tactgataat 1980 ttattagtta gagcaatacc agatgataca ccagcagagg ttactacatt tgatgttga 2040 aaatacaa tattatcatc aacagatagt ggaaatttcta atattttaa tgctgttttct 2100 actacattag aaagacagc aaacatagat agtttaagta tttatatagg aaatattca 2160 gaaaatggca aactacaaat tgctatttat gacgatata acgggaaacc tgtactaaa 2220 aaagcttacg tagagagtt tgttcctact aaaatagtt ggaatacaa gaaggttgta 2280 aattctgtta cattaccttc agggcaatat tggttagttt tccaacctga taacgatgta 2340 ctacaaacaa aaactaatcc atcatccatg aaacaaagtg ctaacaataa tccatataat 2400 tataatatat taccaaattc atttcctatt ggaacaggat ataatgctta taaaggcgat 2460 gtatctttct atgcaacctt taagaagca agcagtcaag caattcctca aaattcttgg 2520 gctctaaaat atgtagatag tgaagaaact acaggcgaaa atggaagagc tacaaatgct 2580 tttgatggta ataataatac tatttggcac acaaaatata gtggcggaaa cgctgcacca 2640 atgccgcatg agattcaaat tgatttaaga ggagtata atataaatca attaattat 2700 ctaccaagac aagatggagg aaccaatggt acaataaagg actatgaagt ttatttaagt 2760 ttagatggag tgaactgggg acaacctata tcaaaaggaa cctttgaatc aaactctaca 2820 gaaaaatag taaaattcaa cgaaacaaaa tctaggtatg taaaacttaa agctctgtca 2880 gaaattaata ataaacaatt tactacagta gctgatttaa aggtatttgg atgggagata 2940 tccaaatag aaaaaccatt acaaaatgct gaaacttatt tgaatatacc aacttatgat 3000 ggattaaatc aaagtactca tccagatgtc aaatatttta aaaatggttg gaatggatat aaatattgga tgataatgac tccaataga acaggtagct cagttgctga aaatccttca atactagcat ctgatgatgg aataaattgg gaggttcctg caggtgttac aaatcctata gctccaatgc cacaagtagg acataattgt gatgttgata tgatatata tgaagcaact gatgagttat gggtgtactg ggtagaatca gatgatata caaaaggatg ggttaatta ataaaatcaa aggatggagt aaattggagt tctcagcaag tggtagttga tgataatagg gcaaatata gtactttatc accatctata atattcaaag father ctatatgtgg tcagttaata caggaaatag tggttggaac aatcaaagta ataaagttga attaagaaga tcaagtgacg gagtaaattg gtcaaatcca acagttgtaa acacattagc tcaagatggt tctcaatat ggcatgtaaa tgtagaatat attack aaaacgaata ttgggctata 3660. 3660. 3660. 3660. 3660. 3660. 3660. 3660. 3660. 3660 agtgatggag taaattggac aacttataag aatcctatat tatcaaaagg aacatctggt aaatgggatg atatggagat atatagaagt tgttttgtgt acgatgaaga tacaaatatg 3780 ataaaggttt ggtatggagc tgtgagtcaa aatccacaaa tatggaaaat aggttttact 3840 gaaaatgatt atgataagtt tattgagggt ttaacacaat aa 3882 <210> 14 <211> 1293 <212> PRT <213> Clostridium tertium <400> 14 Tyr Asn Leu Ile Asp Asn Ile Ser Val Glu Lys Leu Asp Thr Asp Ile 1 5 10 15 Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn Gly Ile Ala Leu 20 25 30 Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu Val Asp Glu Asn 35 40 45 Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr Ala Asp Phe Ser 50 55 60 Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp Phe Val Ile Ile 65 70 75 80 Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu Gly Val Gly Asn 85 90 95 Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn Leu Ile Arg Lys 100 105 110 Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala Ile Tyr Ser Thr 115 120 125 Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val Asp Ser Phe His 130 135 140 Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro Phe Leu Ala Tyr 145 150 155 160 Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile Val Asn Val Thr 165 170 175 Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr Ser Val Gly Val 180 185 190 Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile Gly Ile Gly Asp 195 200 205 Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile Ile Gly Lys Asp 210 215 220 Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln Thr Leu Lys Lys 225 230 235 240 Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr Thr Thr Asn Gly 245 250 255 Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val Met Lys Asp Lys 260 265 270 Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala Tyr Asp Ser Arg 275 280 285 Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile Glu Lys Ile Val 290 295 300 Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys Gln Ile Thr Leu 305 310 315 320 Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys Leu Ser Pro Gln 325 330 335 Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu Thr Asp Glu Ile 340 345 350 His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg Pro Val Asp Gly 355 360 365 Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu Trp Phe Ile Lys 370 375 380 Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro Gly Gly Gly Asn 385 390 395 400 Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser Glu Gly Ser Ile 405 410 415 Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu Glu Trp Gly Phe 420 425 430 Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro Lys Cys Tyr Asp 435 440 445 Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu Glu Asn Gln Tyr 450 455 460 Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala Ile Asn Pro Asp 465 470 475 480 Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln Asp Phe Tyr Ser 485 490 495 Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro Ile Ser Arg Val 500 505 510 Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile Phe Gly Asp Asp 515 520 525 Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu Pro Ser Ala Leu 530 535 540 Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr Leu Ser Ser Ser 545 550 555 560 Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala Arg Asp Leu Glu 565 570 575 Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys Tyr Gly Ile Asp 580 585 590 Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp Ile Tyr Tyr Ser 595 600 605 Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile Glu Ile Lys Gly 610 615 620 Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr Val Asn Asn Arg 625 630 635 640 Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr Ile Asn Thr Ser 645 650 655 Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp Asp Thr Pro Ala 660 665 670 Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile Leu Ser Ser Thr 675 680 685 Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser Thr Thr Leu Glu 690 695 700 Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile Gly Asn Asn Ser 705 710 715 720 Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp Asn Asn Gly Lys 725 730 735 Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val Pro Thr Lys Asn 740 745 750 Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr Leu Pro Ser Gly 755 760 765 Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val Leu Gln Thr Lys 770 775 780 Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn Asn Pro Tyr Asn 785 790 795 800 Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr Gly Tyr Asn Ala 805 810 815 Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys Glu Ala Ser Ser 820 825 830 Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr Val Asp Ser Glu 835 840 845 Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala Phe Asp Gly Asn 850 855 860 Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly Asn Ala Ala Pro 865 870 875 880 Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val Tyr Asn Ile Asn 885 890 895 Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr Asn Gly Thr Ile 900 905 910 Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val Asn Trp Gly Gln 915 920 925 Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr Glu Lys Ile Val 930 935 940 Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu Lys Ala Leu Ser 945 950 955 960 Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp Leu Lys Val Phe 965 970 975 Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro Leu Gln Asn Ala Glu Thr 980 985 990 Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu Asn Gln Ser Thr His Pro 995 1000 1005 Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn Gly Tyr Lys Tyr Trp 1010 1015 1020 Met Ile Met Thr Pro Asn Arg Thr Gly Ser Ser Val Ala Glu Asn 1025 1030 1035 Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile Asn Trp Glu Val Pro 1040 1045 1050 Ala Gly Val Thr Asn Pro Ile Ala Pro Met Pro Gln Val Gly His 1055 1060 1065 Asn Cys Asp Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu Leu 1070 1075 1080 Trp Val Tyr Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val 1085 1090 1095 Lys Leu Ile Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln 1100 1105 1110 Val Val Val Asp Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro 1115 1120 1125 Ser Ile Ile Phe Lys Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn 1130 1135 1140 Thr Gly Asn Ser Gly Trp Asn Asn Gln Ser Asn Lys Val Glu Leu 1145 1150 1155 Arg Glu Ser Ser Asp Gly Val Asn Trp Ser Asn Pro Thr Val Val 1160 1165 1170 Asn Thr Leu Ala Gln Asp Gly Ser Gln Ile Trp His Val Asn Val 1175 1180 1185 Glu Tyr Ile Pro Ser Lys Asn Glu Tyr Trp Ala Ile Tyr Pro Ala 1190 1195 1200 Tyr Lys Asn Gly Thr Gly Ser Asp Lys Thr Glu Leu Tyr Tyr Ala 1205 1210 1215 Lys Ser Ser Asp Gly Val Asn Trp Thr Thr Tyr Lys Asn Pro Ile 1220 1225 1230 Leu Ser Lys Gly Thr Ser Gly Lys Trp Asp Asp Met Glu Ile Tyr 1235 1240 1245 Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr Asn Met Ile Lys Val 1250 1255 1260 Trp Tyr Gly Ala Val Ser Gln Asn Pro Gln Ile Trp Lys Ile Gly 1265 1270 1275 Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile Glu Gly Leu Thr Gln 1280 1285 1290 <210> 15 <211> 1313 <212> PRT <213> Clostridium tertium <400> 15 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Tyr Asn Leu Ile Asp Asn Ile Ser Val Glu Lys Leu 20 25 30 Asp Thr Asp Ile Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn 35 40 45 Gly Ile Ala Leu Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu 50 55 60 Val Asp Glu Asn Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr 65 70 75 80 Ala Asp Phe Ser Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp 85 90 95 Phe Val Ile Ile Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu 100 105 110 Gly Val Gly Asn Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn 115 120 125 Leu Ile Arg Lys Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala 130 135 140 Ile Tyr Ser Thr Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val 145 150 155 160 Asp Ser Phe His Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro 165 170 175 Phe Leu Ala Tyr Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile 180 185 190 Val Asn Val Thr Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr 195 200 205 Ser Val Gly Val Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile 210 215 220 Gly Ile Gly Asp Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile 225 230 235 240 Ile Gly Lys Asp Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln 245 250 255 Thr Leu Lys Lys Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr 260 265 270 Thr Thr Asn Gly Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val 275 280 285 Met Lys Asp Lys Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala 290 295 300 Tyr Asp Ser Arg Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile 305 310 315 320 Glu Lys Ile Val Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys 325 330 335 Gln Ile Thr Leu Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys 340 345 350 Leu Ser Pro Gln Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu 355 360 365 Thr Asp Glu Ile His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg 370 375 380 Pro Val Asp Gly Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu 385 390 395 400 Trp Phe Ile Lys Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro 405 410 415 Gly Gly Gly Asn Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser 420 425 430 Glu Gly Ser Ile Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu 435 440 445 Glu Trp Gly Phe Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro 450 455 460 Lys Cys Tyr Asp Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu 465 470 475 480 Glu Asn Gln Tyr Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala 485 490 495 Ile Asn Pro Asp Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln 500 505 510 Asp Phe Tyr Ser Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro 515 520 525 Ile Ser Arg Val Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile 530 535 540 Phe Gly Asp Asp Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu 545 550 555 560 Pro Ser Ala Leu Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr 565 570 575 Leu Ser Ser Ser Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala 580 585 590 Arg Asp Leu Glu Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys 595 600 605 Tyr Gly Ile Asp Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp 610 615 620 Ile Tyr Tyr Ser Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile 625 630 635 640 Glu Ile Lys Gly Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr 645 650 655 Val Asn Asn Arg Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr 660 665 670 Ile Asn Thr Ser Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp 675 680 685 Asp Thr Pro Ala Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile 690 695 700 Leu Ser Ser Thr Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser 705 710 715 720 Thr Thr Leu Glu Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile 725 730 735 Gly Asn Asn Ser Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp 740 745 750 Asn Asn Gly Lys Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val 755 760 765 Pro Thr Lys Asn Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr 770 775 780 Leu Pro Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val 785 790 795 800 Leu Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn 805 810 815 Asn Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr 820 825 830 Gly Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys 835 840 845 Glu Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr 850 855 860 Val Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala 865 870 875 880 Phe Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly 885 890 895 Asn Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val 900 905 910 Tyr Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr 915 920 925 Asn Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val 930 935 940 Asn Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr 945 950 955 960 Glu Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu 965 970 975 Lys Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp 980 985 990 Leu Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro Leu Gln 995 1000 1005 Asn Ala Glu Thr Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu Asn 1010 1015 1020 Gln Ser Thr His Pro Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn 1025 1030 1035 Gly Tyr Lys Tyr Trp Met Ile Met Thr Pro Asn Arg Thr Gly Ser 1040 1045 1050 Ser Val Ala Glu Asn Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile 1055 1060 1065 Asn Trp Glu Val Pro Ala Gly Val Thr Asn Pro Ile Ala Pro Met 1070 1075 1080 Pro Gln Val Gly His Asn Cys Asp Val Asp Met Ile Tyr Asn Glu 1085 1090 1095 Ala Thr Asp Glu Leu Trp Val Tyr Trp Val Glu Ser Asp Asp Ile 1100 1105 1110 Thr Lys Gly Trp Val Lys Leu Ile Lys Ser Lys Asp Gly Val Asn 1115 1120 1125 Trp Ser Ser Gln Gln Val Val Val Asp Asp Asn Arg Ala Lys Tyr 1130 1135 1140 Ser Thr Leu Ser Pro Ser Ile Ile Phe Lys Asp Asn Lys Tyr Tyr 1145 1150 1155 Met Trp Ser Val Asn Thr Gly Asn Ser Gly Trp Asn Asn Gln Ser 1160 1165 1170 Asn Lys Val Glu Leu Arg Glu Ser Ser Asp Gly Val Asn Trp Ser 1175 1180 1185 Asn Pro Thr Val Val Asn Thr Leu Ala Gln Asp Gly Ser Gln Ile 1190 1195 1200 Trp His Val Asn Val Glu Tyr Ile Pro Ser Lys Asn Glu Tyr Trp 1205 1210 1215 Ala Ile Tyr Pro Ala Tyr Lys Asn Gly Thr Gly Ser Asp Lys Thr 1220 1225 1230 Glu Leu Tyr Tyr Ala Lys Ser Ser Asp Gly Val Asn Trp Thr Thr 1235 1240 1245 Tyr Lys Asn Pro Ile Leu Ser Lys Gly Thr Ser Gly Lys Trp Asp 1250 1255 1260 Asp Met Glu With Tyr Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr 1265 1270 1275 Asn With Lys Val Trp Tyr Gly Only Will Be Gln Asn Pro Gln 1280 1285 1290 Ile Trp Lys Ile Gly Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile 1295 1300 1305 Glu Gly Leu Thr Gln 1310 <210> 16 <211> 1584 <212> DNA <213> Clostridium tertium (Clostridium tertium) <400> 16 tcagggcaat attggttagt ttcaacct gataacgatg tactacaaac aaaaactaat 60 ccatcatcca tgaacaag tgctaacaat aatccatata attatatat attaccaat 120 tcatttccta ttggaacagg atataatgct tataaaggcg atgtatcttt ctatgcaacc 180 tttaagaag caagcagtca agcaatcct caaatttctt gggctctaaa atatgtagat 240 agtgagaaa ctacaggcga aaatggaag gctacaaatg cttttgatgg taataat 300 actatttggc acacaaata tagtggcgga aacgctgcac caatgccgca tgagattca 360 attgattaa gaggatta tattaat slowly atctaccag ahaatgga 420 ggaaccaatg gtacaataaa ggactgaa gtttatttaa gtttagatgg agtgaactgg 480 ggacaaccta tatcaaagg aacctttgaa tcaactcta cagaaaaaat agtaaaatttc 540 aacgaaaaaaatctaggta tgtaaaactt aaagctctgt cagaaatta taaaaca 600 ttactacag tagctgatt aaaggtattt ggatgggaga tatccaaaat agaaaaacca 660 ttacaaaatg ctgaaactta ttgaatata ccaacttatg atggattaaa tcaagtact 720 catccagatg tcaatattt taaaaatggt tggaatggat aataatattg gatgataatg 780 actccaaata gaacaggtag ctcagttgct gaaaatcctt caatactagc atctgatgat 840 ggataaatt gggaggttcc tgcaggtgtt acaaatccta tagctccaat gccacaagta 900 ggacataatt gtgatgttga tatgatatat atgaagcaa ctgatgagtt atgggtgtac 960 tgggttagaat cagatgat aaaaagga tgggttaat taaaaatc aaaggatgga 1020 gtaaattgga gttctcagca agtggtagtt gatgataata gggcaaata tagtacttta 1080 tcaccatcta taatattca agatataaa tactatatgt ggtcagttaa tacaggaat 1140 agtggttgga acatcaag taadaagtt gattaagg atcaagtga cggagtaat 1200 tggtcaaatc cacagttgt aaacacatta gctcaatg gttctcaat atggcatgta 1260 aatgtagaat ataccatc aaaaaacgaa tattgggcta tatatccagc atataaaaat 1320 ggaacaggta gcgataaaac agaattgtat tatgcgaat caagtgatgg agtaaattgg 1380 acaacttata agaatcctat atttacaaa ggaacatctg gtaaatggga tgatatggag 1440 atatatagaa gttgttttgt gtacgatgaa gatacaata tgataaggt ttggtatgga 1500 gctgtgagtc aaatccaca atatggaaa ataggtttta ctgaaaatga ttatgataag 1560 1584 <210> 17 <211> 547 <212> PRT <213> Clostridium tertium (Clostridium tertium) <400> 17 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn 20 25 30 Asp Val Leu Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala 35 40 45 Asn Asn Asn Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile 50 55 60 Gly Thr Gly Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr 65 70 75 80 Phe Lys Glu Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu 85 90 95 Lys Tyr Val Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr 100 105 110 Asn Ala Phe Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser 115 120 125 Gly Gly Asn Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg 130 135 140 Gly Val Tyr Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly 145 150 155 160 Gly Thr Asn Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp 165 170 175 Gly Val Asn Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn 180 185 190 Ser Thr Glu Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val 195 200 205 Lys Leu Lys Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val 210 215 220 Ala Asp Leu Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro 225 230 235 240 Leu Gln Asn Ala Glu Thr Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu 245 250 255 Asn Gln Ser Thr His Pro Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn 260 265 270 Gly Tyr Lys Tyr Trp Met Ile Met Thr Pro Asn Arg Thr Gly Ser Ser 275 280 285 Val Ala Glu Asn Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile Asn Trp 290 295 300 Glu Val Pro Ala Gly Val Thr Asn Pro Ile Ala Pro Met Pro Gln Val 305 310 315 320 Gly His Asn Cys Asp Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu 325 330 335 Leu Trp Val Tyr Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val 340 345 350 Lys Leu Ile Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln Val 355 360 365 Val Val Asp Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro Ser Ile 370 375 380 Ile Phe Lys Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn Thr Gly Asn 385 390 395 400 Ser Gly Trp Asn Asn Gln Ser Asn Lys Val Glu Leu Arg Glu Ser Ser 405 410 415 Asp Gly Val Asn Trp Ser Asn Pro Thr Val Val Asn Thr Leu Ala Gln 420 425 430 Asp Gly Ser Gln Ile Trp His Val Asn Val Glu Tyr Ile Pro Ser Lys 435 440 445 Asn Glu Tyr Trp Ala Ile Tyr Pro Ala Tyr Lys Asn Gly Thr Gly Ser 450 455 460 Asp Lys Thr Glu Leu Tyr Tyr Ala Lys Ser Ser Asp Gly Val Asn Trp 465 470 475 480 Thr Thr Tyr Lys Asn Pro Ile Leu Ser Lys Gly Thr Ser Gly Lys Trp 485 490 495 Asp Asp Met Glu Ile Tyr Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr 500 505 510 Asn Met Ile Lys Val Trp Tyr Gly Ala Val Ser Gln Asn Pro Gln Ile 515 520 525 Trp Lys Ile Gly Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile Glu Gly 530 535 540 Leu Thr Gln 545 <210> 18 <211> 2958 <212> DNA <213> Clostridium tertium (Clostridium tertium) <400> 18 tataatttaa ttgataatat tagtgttgaa aaattagata ctgatatttc acaagcaaat 60 gaaaatgttt ttttgaatgg aaatggaatt gctttagaag tagataatag aggcgctaca 120 tgtatttatc tagtagatga aaatggagtt aaaacaaaag ctacgacttc tttagataca 180 gcagattttt caggttatcc aataataggt ggacaaaaga taagagatttt tgtaattata 240 tcaaaaaatc tagaagaaaa cataaactcg atattaggtg ttggaaatag acttactatt 300 atatctaaaa gttcatctac taatctgata agaaagatag tatttgaaac atctaacagc 360 aatccaggag caatattc aacagtagt tataaagcag aaagtaacga tttagta 420 gatagctttc atgaaaatga gtatacaatg agtttaggc aaggacctttt tcttgcatat 480 caagggtgtg cagatcaca aggagcaat actatcgtta atgttacta tggatataac 540 cataatagtg gataaaata ttattctgta ggagttccat ttagttatgt ttataactct 600 gtggggggaa ttggaatagg tgatgcatca acttcagaa gagaatttaa gttgcctatt 660 ataggaaag atatacagt ttcattagga atggagtgga atggacaac tttaaaaaaa 720 ggtgctgaaa ctgctatagg tacaagtgtt ataactacaa caatggtga ttattattct 780 gggctaaga gttacgcaga agttatgaa gatagggaa tatctgcacc agcttcaata 840 cctgatatag catatgattc tagagggaa agttgggat tcgaatttga ttttacaata 900 gaaaaatag ttaataattt agatgaactt aaagcgatgg ggaaaaca attackcta 960 gatgatgggt ggtacactta tgctggtgat tggaaattaa gtcctcaaa gtttccaaat 1020 ggaaatgcag acatgaata tcttacagat gaatccata aagaggaat gagactatt 1080 ttatggtgga gaccagtaga cggagggata atagcaat tagtatctga acatccagag 1140 tggtttatta agaaccaca agggaatatg gttaggttac caggggctgg aggtggaat 1200 ggaggaacag caggatatgc attatgtcca attcagaag gttcaatca acatcataaa 1260 gattttgtaa ctgtggcatt agagaatgg ggatttgatg gattcaaga agattgta 1320 tggggaatac ctaaatgcta tgatagttct cataacact caagtttatc agatacatta 1380 gaaaatcaat ataattcta tgaagccata tatgaacagt ccatagcgat aaatccagat 1440 acttttatag aattatgtaa ttgcggaca cctcaggatt tttattcaac accatatgtg 1500 aaccatgcac storm tccaatttcg agagtacciaa storm storm 1560 tttaaagcta tatttggaga tgatttcca gtaacaacag atcaatattc agttttggtta 1620 ccgtcagcat taggtacagg atcagttg attackaac atacacatt aagtagttca 1680 gagagaac attaata atacttcgga cttgcagag atttagaatt agcaagga 1740 gatttattag gaacttata taaatacgga atagatccat taggtcata tgttataga 1800 aaaggaag attattatta ttcattctac aagaatatt ctagttattc aggaatta 1860 gaaataagg ggttagacag taacgccaca tatagaattg aagatttagt glassataga 1920 gttattgcta gaggagtaa gggaccaca gcgacttaa atacaagctt tactgataat 1980 ttattagtta gagcaatacc agatgataca ccagcagagg ttactacatt tgatgttga 2040 aaatacaa tattatcatc aacagatagt ggaaatttcta atattttaa tgctgttttct 2100 actacattag aaagacagc aaacatagat agtttaagta tttatatagg aaatattca 2160 gaaaatggca aactacaaat tgctatttat gacgatata acgggaaacc tgtactaaa 2220 aaagcttacg tagagagtt tgttcctact aaaatagtt ggaatacaa gaaggttgta 2280 aattctgtta cattaccttc agggcaat tggttagtt tccaacctga taacgatgta 2340 ctacaaaaaaactaatcc atcatccatg aaaaaaagtg ctaacaataa tccatataat 2400 tataatatat taccaatttc atttccttatt ggaacaggat ataatgctta taaaggcgat 2460 gtatctttct atgcacctt taagaagca agcagtcaag caatttcctca aaatttctgg 2520 gctctaaaat atgtagatag tgagaact acaggcgaaa atggaagc tacaatgct 2580 tttgatggta ataataatac tattttggcac acaaaata gtggcggaaa cgctgcacca 2640 atgccgcatg agatcaat tgatttaga ggagtatata ataataatca attaattat 2700 ctaccaagac aagatggagg aaccaatggt acataagg actagaagt ttatttaagt 2760 ttagatggag tgaactgggg acaactata tcaaaggaa ccttgaatc aaactctaca 2820 gaaaaaatag taaaattcaa cgaaaaaaaa tctaggtag taaaacttaa agctctgtca 2880 gaaattaata aaacatt tactacagta gctgatttaa aggtatttgg atgggata 2940 tccaaaatag aaaaata 2958 <210> 19 <211> 1005 <212> PRT <213> Clostridium tertium (Clostridium tertium) <400> 19 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Serves His Tyr Asn To Asp Asn Serves Val Lys Glu 20 25 30 Asp Thr Asp Ile Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn 35 40 45 Gly Ile Ala Leu Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu 50 55 60 Val Asp Glu Asn Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr 65 70 75 80 Ala Asp Phe Ser Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp 85 90 95 Phe Val Ile Ile Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu 100 105 110 Gly Val Gly Asn Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn 115 120 125 Leu Ile Arg Lys Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala 130 135 140 Ile Tyr Ser Thr Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val 145 150 155 160 Asp Ser Phe His Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro 165 170 175 Phe Leu Ala Tyr Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile 180 185 190 Val Asn Val Thr Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr 195 200 205 Ser Val Gly Val Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile 210 215 220 Gly Ile Gly Asp Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile 225 230 235 240 Ile Gly Lys Asp Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln 245 250 255 Thr Leu Lys Lys Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr 260 265 270 Thr Thr Asn Gly Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val 275 280 285 Met Lys Asp Lys Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala 290 295 300 Tyr Asp Ser Arg Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile 305 310 315 320 Glu Lys Ile Val Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys 325 330 335 Gln Ile Thr Leu Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys 340 345 350 Leu Ser Pro Gln Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu 355 360 365 Thr Asp Glu Ile His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg 370 375 380 Pro Val Asp Gly Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu 385 390 395 400 Trp Phe Ile Lys Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro 405 410 415 Gly Gly Gly Asn Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser 420 425 430 Glu Gly Ser Ile Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu 435 440 445 Glu Trp Gly Phe Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro 450 455 460 Lys Cys Tyr Asp Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu 465 470 475 480 Glu Asn Gln Tyr Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala 485 490 495 Ile Asn Pro Asp Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln 500 505 510 Asp Phe Tyr Ser Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro 515 520 525 Ile Ser Arg Val Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile 530 535 540 Phe Gly Asp Asp Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu 545 550 555 560 Pro Ser Ala Leu Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr 565 570 575 Leu Ser Ser Ser Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala 580 585 590 Arg Asp Leu Glu Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys 595 600 605 Tyr Gly Ile Asp Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp 610 615 620 Ile Tyr Tyr Ser Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile 625 630 635 640 Glu Ile Lys Gly Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr 645 650 655 Val Asn Asn Arg Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr 660 665 670 Ile Asn Thr Ser Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp 675 680 685 Asp Thr Pro Ala Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile 690 695 700 Leu Ser Ser Thr Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser 705 710 715 720 Thr Thr Leu Glu Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile 725 730 735 Gly Asn Asn Ser Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp 740 745 750 Asn Asn Gly Lys Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val 755 760 765 Pro Thr Lys Asn Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr 770 775 780 Leu Pro Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val 785 790 795 800 Leu Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn 805 810 815 Asn Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr 820 825 830 Gly Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys 835 840 845 Glu Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr 850 855 860 Val Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala 865 870 875 880 Phe Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly 885 890 895 Asn Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val 900 905 910 Tyr Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr 915 920 925 Asn Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val 930 935 940 Asn Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr 945 950 955 960 Glu Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu 965 970 975 Lys Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp 980,985,990 Leu Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys 995 1000 1005 <210> 20 <211> 3786 <212> DNA <213> Robinsoniella peoriensis <400> 20 gggaacggat tagaggtgaa agcctcgcca agggaggtgg cacaaataac cggaaacggg 60 gtatcggtga cgttttttca ggaagatggc acggtgcagt tatcctgtat agaggatgat 120 ggcaatactg cttttatgac caggaactca gaggtctctt atccggtggt gggtggggag 180 gaagtaacag acttttcaga ctttcaatgt gaagtacagg aaaacgtaac cggagctgcg 240 ggagccggca gccggatgac aatcacctcc atttccagcg gcagggggat tcagcggtcg 300 gtagtcattg agacggtaga tgaggtaaaaa ggcctgctcc atatcagcag ttcttatagg 360 gcagaagaag aggtagatgc agacgaattt attgacagca gattcagcct ggataatccc 420 tcagatacag tctggagtta caatggcggc ggtgaggggg cccagagccg atacgatact 480 ctacagaaaa tagatctgtc ggatggtgaa agcttctata gggagaactt acagaatcaa 540 actgcggcag gtattccggt ggcggatatc tacgggaaag acgggggtat tacggtgggt 600 gatgccagtg tgacccggcg acagctttcc actccggtaa acgagaggaa tggtaccgct 660 tatgtgtccg tgaaacatcc aggtgcagtt attacccaaa gggaaacaga aatcagccag 720 agctttgtca atgtacacag aggcgactat tattcggggc tgcggggtta tgccgatggt 780 atgaagcaga taggatttac cacactctcc cgggaacaga ttcctgaaag cagctatgat 840 ctccgctggg agagctgggg atgggaattt gactggacag tggaactgat tatcaataag 900 ctggacgagt taaaagagat gggaatcaaa cagattaccc tggatgacgg ctggtataat 960 gccgcaggag aatgggggct gaacaactgg aagcttccta atggtgcttt ggacatgcgg 1020 catctgactg atgcaattca tgaaaggggg atgactgcag tattgtggtg gcgtccctgt 1080 gacggtggaa gggaagacag cgcattattt aaagagcatc cagagtattt tataaaaaac 1140 caggacggaa gctttgggaa gctggcagga ccgggacagt ggaacagttt tctgggaagc 1200 tgcggttatg cgctgtgtcc ttgtcagaa ggggcagtac agagccaggt tgattttatt 1260 aaccgtgcta tgaatgaatg gggatttgat ggatttaaaa gtgattgt atggagcctt 1320 ccaaagtgct acagtcagga ccatcaccat gatacccgg aagaatccac agacagcag 1380 gctgtgttct accggcagt ttgaggct atgacagaca atgacccgaa tgcattcac 1440 cttctatgca actgcggaac gccacaggat tattattc tgcctagt aacccaggtg 1500 cctactgccg atcccactc tgtggatcag aaggagaa gggtaaggc atataaagca 1560 ctatgcggtg attatttccc tgttacgaca gatcataatg aagtctggta tccttcaacc 1620 ataggaacgg gagccatact gattgaaaaa cgtgacttgt caggctggga agaggagg 1680 tatgcaaat ggcttaaaat tgctcaggaa aaccaattgc ataagggac atttattggg 1740 gatttgtaca gttacggata tgaccttat gaaacctata cggtgtataa agacggaatc 1800 atgtattatg cattctataa agacggaac cggtaccgtc cgtccggtaa cccggatatc 1860 gattaaag ggctggaag cggaagctg taccgcatcg tagattagt aaataatcag 1920 gtagttgcca caatgtaac cagtagcaat gctgtattt cttacccttt cagcgattac 1980 ttgctggtaa aagcagtaga aatcagcgaa ccggatacgg atggactgg acctgtaccg 2040 gatcctgagg gggcggtaac agtagaggaa atgatcctg aactggtata tacagggat 2100 tgggtaaggg aaaaaatga cggataccat ggaggagg cccgttatac aaagaagca 2160 gaagctctg tagaattggc attctatgga acaggtgctg cctggtatgg acagcacgac 2220 gttaactttg gtagtgcacg gatatatata gacggaacct atgtcaagac cgtatcatgc 2280 atgggagaac ctggaataaa tattaaattg tttgaatca gcggcttgga cttggcttcc 2340 cacaggatta aaatagaatg tgagacaccg gtattgata tgacaggct gacttacac 2400 aaaggagaag aagttcctgc taaagtaatg acggcggacc tccggcttt gactgttata 2460 gcaaccaat acgatatgaa cagttttgca gatggcaatt acagacca gctgggggta 2520 tccttagttc gtgccaacca gcttctggca gcggatg taacccaggg ggctgtaaat 2580 gagaacaga ataccttct gatgccatg ctgaaaata gaaaaaagt tgataagagt 2640 tggatcgggc ttcccggacc aatcccgcag gatatacaga cagaaaatat cagcagagat 2700 aaccttgcta aagtaatatc ttatactggg cagttggaca gagatgagat tattcctgcc 2760 aaaagaac agctgaacga ttctgat aaggctgtct ccatagcaga acgccaggat 2820 gcatcccagc cggaataga cagagcgtgg gcagagttaa tgaatgcagt gcaatagc 2880 agctatatca ggggatcaa agaggactg ttcactc tggaatgaata cggaaggta 2940 gataccaccg tttataaaga cgctgcttta tttatagaat ccttagaagc cgctaaaag 3000 gtgtatcagg atgaaaatgc aatggatggg gagatcagtg attgtatcaa acaattgcga 3060 gatgcaaag atcagctaca actaaaggat ccggtagatc cgccgaaacc cgatccggac 3120 cccgatccaa agcctgatcc aacaccagac ccgggaccag atccaagcc cgatccaaca 3180 cctgacccga cgccagaccc aaagcccaat ccacaccga cgcccgatcc aacaccagag 3240 ccagctctaa aaagccgga acaggtatct ggtttgaagt cgaagcgga gactgattat 3300 ctgacggttt cctggaagaa attgaataat gctgaatcct aataaggtgta tattataaa 3360 agcggcaaat ggcgcctggc tggaaaact aaagacat ccataagat aaaaaactg 3420 gtttcgggaa cgaatacac cgtaaaagtt gctgcggtca ataagcagg gcaggggaaa 3480 tattcatcac aggtgtatac ggcagcaag cccaaaaag tcaattaa atccgtcagc 3540 aggtaccgca catchaaagt aaagttaaac tatggaaag taaaagcagg cggatatgaa 3600 attggatga agaatggaa gggttcttat aagaaggcag ccaccagtac gagacaca 3660 gccataaga gcggattaaa aaaggaaaa acatattact ttaaagtcag ggcttatgtt 3720 aaaaaayaa atcaggtgat ttacggcagc ttttccaata tAAagaata caaatgta 3780 page 3786 <210> 21 <211> 1281 <212> PRT <213> Robinsoniella peoriensis <400> 21 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Gly Asn Gly Leu Glu Val Lys Ala Ser Pro Arg Glu 20 25 30 Val Ala Gln Ile Thr Gly Asn Gly Val Ser Val Thr Phe Phe Gln Glu 35 40 45 Asp Gly Thr Val Gln Leu Ser Cys Ile Glu Asp Asp Gly Asn Thr Ala 50 55 60 Phe Met Thr Arg Asn Ser Glu Val Ser Tyr Pro Val Val Gly Gly Glu 65 70 75 80 Glu Val Thr Asp Phe Ser Asp Phe Gln Cys Glu Val Gln Glu Asn Val 85 90 95 Thr Gly Ala Ala Gly Ala Gly Ser Arg Met Thr Ile Thr Ser Ile Ser 100 105 110 Ser Gly Arg Gly Ile Gln Arg Ser Val Val Ile Glu Thr Val Asp Glu 115 120 125 Val Lys Gly Leu Leu His Ile Ser Ser Ser Tyr Arg Ala Glu Glu Glu 130 135 140 Val Asp Ala Asp Glu Phe Ile Asp Ser Arg Phe Ser Leu Asp Asn Pro 145 150 155 160 Ser Asp Thr Val Trp Ser Tyr Asn Gly Gly Gly Glu Gly Ala Gln Ser 165 170 175 Arg Tyr Asp Thr Leu Gln Lys Ile Asp Leu Ser Asp Gly Glu Ser Phe 180 185 190 Tyr Arg Glu Asn Leu Gln Asn Gln Thr Ala Ala Gly Ile Pro Val Ala 195 200 205 Asp Ile Tyr Gly Lys Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Val 210 215 220 Thr Arg Arg Gln Leu Ser Thr Pro Val Asn Glu Arg Asn Gly Thr Ala 225 230 235 240 Tyr Val Ser Val Lys His Pro Gly Ala Val Ile Thr Gln Arg Glu Thr 245 250 255 Glu Ile Ser Gln Ser Phe Val Asn Val His Arg Gly Asp Tyr Tyr Ser 260 265 270 Gly Leu Arg Gly Tyr Ala Asp Gly Met Lys Gln Ile Gly Phe Thr Thr 275 280 285 Leu Ser Arg Glu Gln Ile Pro Glu Ser Ser Tyr Asp Leu Arg Trp Glu 290 295 300 Ser Trp Gly Trp Glu Phe Asp Trp Thr Val Glu Leu Ile Ile Asn Lys 305 310 315 320 Leu Asp Glu Leu Lys Glu Met Gly Ile Lys Gln Ile Thr Leu Asp Asp 325 330 335 Gly Trp Tyr Asn Ala Ala Gly Glu Trp Gly Leu Asn Asn Trp Lys Leu 340 345 350 Pro Asn Gly Ala Leu Asp Met Arg His Leu Thr Asp Ala Ile His Glu 355 360 365 Arg Gly Met Thr Ala Val Leu Trp Trp Arg Pro Cys Asp Gly Gly Arg 370 375 380 Glu Asp Ser Ala Leu Phe Lys Glu His Pro Glu Tyr Phe Ile Lys Asn 385 390 395 400 Gln Asp Gly Ser Phe Gly Lys Leu Ala Gly Pro Gly Gln Trp Asn Ser 405 410 415 Phe Leu Gly Ser Cys Gly Tyr Ala Leu Cys Pro Leu Ser Glu Gly Ala 420 425 430 Val Gln Ser Gln Val Asp Phe Ile Asn Arg Ala Met Asn Glu Trp Gly 435 440 445 Phe Asp Gly Phe Lys Ser Asp Tyr Val Trp Ser Leu Pro Lys Cys Tyr 450 455 460 Ser Gln Asp His His His Glu Tyr Pro Glu Glu Ser Thr Glu Gln Gln 465 470 475 480 Ala Val Phe Tyr Arg Ala Val Tyr Glu Ala Met Thr Asp Asn Asp Pro 485 490 495 Asn Ala Phe His Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr 500 505 510 Ser Leu Pro Tyr Val Thr Gln Val Pro Thr Ala Asp Pro Thr Ser Val 515 520 525 Asp Gln Thr Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Cys Gly Asp 530 535 540 Tyr Phe Pro Val Thr Thr Asp His Asn Glu Val Trp Tyr Pro Ser Thr 545 550 555 560 Ile Gly Thr Gly Ala Ile Leu Ile Glu Lys Arg Asp Leu Ser Gly Trp 565 570 575 Glu Glu Glu Glu Tyr Ala Lys Trp Leu Lys Ile Ala Gln Glu Asn Gln 580 585 590 Leu His Lys Gly Thr Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Tyr Asp 595 600 605 Pro Tyr Glu Thr Tyr Thr Val Tyr Lys Asp Gly Ile Met Tyr Tyr Ala 610 615 620 Phe Tyr Lys Asp Gly Asn Arg Tyr Arg Pro Ser Gly Asn Pro Asp Ile 625 630 635 640 Glu Leu Lys Gly Leu Glu Asp Gly Lys Leu Tyr Arg Ile Val Asp Tyr 645 650 655 Val Asn Asn Gln Val Val Ala Thr Asn Val Thr Ser Ser Asn Ala Val 660 665 670 Phe Ser Tyr Pro Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile 675 680 685 Ser Glu Pro Asp Thr Asp Gly Pro Gly Pro Val Pro Asp Pro Glu Gly 690 695 700 Ala Val Thr Val Glu Glu Asn Asp Pro Glu Leu Val Tyr Thr Gly Asp 705 710 715 720 Trp Val Arg Glu Glu Asn Asp Gly Tyr His Gly Gly Gly Ala Arg Tyr 725 730 735 Thr Lys Glu Ala Glu Ala Ser Val Glu Leu Ala Phe Tyr Gly Thr Gly 740 745 750 Ala Ala Trp Tyr Gly Gln His Asp Val Asn Phe Gly Ser Ala Arg Ile 755 760 765 Tyr Ile Asp Gly Thr Tyr Val Lys Thr Val Ser Cys Met Gly Glu Pro 770 775 780 Gly Ile Asn Ile Lys Leu Phe Glu Ile Ser Gly Leu Asp Leu Ala Ser 785 790 795 800 His Arg Ile Lys Ile Glu Cys Glu Thr Pro Val Ile Asp Ile Asp Arg 805 810 815 Leu Thr Tyr Ile Lys Gly Glu Glu Val Pro Ala Lys Val Met Thr Ala 820 825 830 Asp Leu Arg Ala Leu Thr Val Ile Ala Asn Gln Tyr Asp Met Asn Ser 835 840 845 Phe Ala Asp Gly Asn Tyr Lys Asp Gln Leu Gly Val Ser Leu Val Arg 850 855 860 Ala Asn Gln Leu Leu Ala Ala Asp Asp Val Thr Gln Gly Ala Val Asn 865 870 875 880 Glu Glu Gln Lys Tyr Leu Leu Asn Ala Met Leu Lys Ile Arg Lys Lys 885 890 895 Val Asp Lys Ser Trp Ile Gly Leu Pro Gly Pro Ile Pro Gln Asp Ile 900 905 910 Gln Thr Glu Asn Ile Ser Arg Asp Asn Leu Ala Lys Val Ile Ser Tyr 915 920 925 Thr Gly Gln Leu Asp Arg Asp Glu Ile Ile Pro Ala Ile Lys Glu Gln 930 935 940 Leu Asn Asp Ser Tyr Asp Lys Ala Val Ser Ile Ala Glu Arg Gln Asp 945 950 955 960 Ala Ser Gln Pro Glu Ile Asp Arg Ala Trp Ala Glu Leu Met Asn Ala 965 970 975 Val Gln Tyr Ser Ser Tyr Ile Arg Gly Ser Lys Glu Glu Leu Leu Ser 980 985 990 Leu Leu Asp Glu Tyr Gly Lys Val Asp Thr Thr Val Tyr Lys Asp Ala 995 1000 1005 Ala Leu Phe Ile Glu Ser Leu Glu Ala Ala Lys Lys Val Tyr Gln 1010 1015 1020 Asp Glu Asn Ala Met Asp Gly Glu Ile Ser Asp Cys Ile Lys Gln 1025 1030 1035 Leu Arg Asp Ala Lys Asp Gln Leu Gln Leu Lys Asp Pro Val Asp 1040 1045 1050 Pro Pro Lys Pro Asp Pro Asp Pro Asp Pro Lys Pro Asp Pro Thr 1055 1060 1065 Pro Asp Pro Gly Pro Asp Pro Lys Pro Asp Pro Thr Pro Asp Pro 1070 1075 1080 Thr Pro Asp Pro Lys Pro Asn Pro Thr Pro Thr Pro Asp Pro Thr 1085 1090 1095 Pro Glu Pro Ala Leu Lys Lys Pro Glu Gln Val Ser Gly Leu Lys 1100 1105 1110 Ser Lys Ala Glu Thr Asp Tyr Leu Thr Val Ser Trp Lys Lys Leu 1115 1120 1125 Asn Asn Ala Glu Ser Tyr Lys Val Tyr Ile Tyr Lys Ser Gly Lys 1130 1135 1140 Trp Arg Leu Ala Gly Lys Thr Thr Lys Thr Ser Ile Lys Ile Lys 1145 1150 1155 Lys Leu Val Ser Gly Thr Lys Tyr Thr Val Lys Val Ala Ala Val 1160 1165 1170 Asn Lys Ala Gly Gln Gly Lys Tyr Ser Ser Gln Val Tyr Thr Ala 1175 1180 1185 Ala Lys Pro Lys Lys Val Lys Leu Lys Ser Val Ser Arg Tyr Arg 1190 1195 1200 Thr Ser Lys Val Lys Leu Asn Tyr Gly Lys Val Lys Ala Gly Gly 1205 1210 1215 Tyr Glu Ile Trp Met Lys Asn Gly Lys Gly Ser Tyr Lys Lys Ala 1220 1225 1230 Ala Thr Ser Thr Lys Thr Thr Ala Ile Lys Ser Gly Leu Lys Lys 1235 1240 1245 Gly Lys Thr Tyr Tyr Phe Lys Val Arg Ala Tyr Val Lys Asn Lys 1250 1255 1260 Asn Gln Val Ile Tyr Gly Ser Phe Ser Asn Ile Lys Lys Tyr Lys 1265 1270 1275 Met Val Leu 1280 <210> 22 <211> 1347 <212> DNA <213> Ruthenibacterium lactatiformans <400> 22 gaagaaaccg atttgcttgt aaacggaggt tttgagaccg gcgacagcac cggatggaat 60 tggttcaata acgccgttgt tgacagcgct gctccgcata gcggaaacta ttgtgctaaa 120 gtagccaaaa acagcagtta tgagcaagtt gttacggtat ctccggatac gaaatatgtt 180 ttaacagggt gggcaaaatc tgaggggcagt tccgttatga cgctggggcgt aaaaaattac 240 ggtgggcagg aaactttttc ggctacgctt tcagccgact atcagcagct ggcggttact 300 ttcacaaccg ggcccaatgc gcaaacagcg actatatatg gatatcgaca gaatagtggt 360 tccggtgcag gctatttcga cgatgtagaa cttacagcgg tgcaagattt tgctccatat 420 cagccgttgg caaatgccat agcgcctcaa gcaattccta cctatgacgg cgccaaccag 480 cctacacatc cctcggtggt gaaatttgaa cagccttgga atggttatct gtattggatg 540 gcaatgacac cttatccctt caatgatggg agctacgaaa acccatcgat tgttgcgtca 600 aacgatggag aaaattggat tgtgccagaa ggggtctcga atcctttggc cggcacgcca 660 agtccgggcc acaattgtga cgtggatctt gtatatgttc cagcctcgga tgaattgcgg 720 atgtactacg tagaggcaga tgatatcatc agctcaaggg taaaaatgat aagttcccgt 780 gacggtgtac actggagcga gccgcaggtc gtaatgcagg atctggtaag gaaatacagt 840 attctatcgc cgtctattga gattctgcca gatggcacct atatgatgtg gtatgtggat 900 acgggggaatg caggatggaa tagccagaat aaccaagtaa aatatcgtac atctgcggat 960 ggaatcaaat ggtcaggcgc agtcacctgt acggattttg tacaacctgg atatcaaata 1020 tggcacatcg atgtacatta tgacacatca agcggagctt actatgcagt ttatccggct 1080 tatccgaatg gcaccgattg cgaccactgc aatttgtttt tcgcagtgaa tcggacagga 1140 aaacagtggg aaacttttag ccggccaatt ttgaagccgt caacggaagg cggctgggat 1200 gatttctgca tttaccggtc ctctatgctg attgacgacg gaatgttgaa agtgtggtac 1260 ggagcaaaaa agcaagagga ttcttcctgg catactgggc taaccatgcg tgatttttct 1320 gaatttatga aaatattgga acgctaa 1347 <210> 23 <211> 468 <212> PRT <213> Ruthenibacterium lactatiformans <400> 23 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Glu Glu Thr Asp Leu Leu Val Asn Gly Gly Phe Glu 20 25 30 Thr Gly Asp Ser Thr Gly Trp Asn Trp Phe Asn Asn Ala Val Val Asp 35 40 45 Ser Ala Ala Pro His Ser Gly Asn Tyr Cys Ala Lys Val Ala Lys Asn 50 55 60 Ser Ser Tyr Glu Gln Val Val Thr Val Ser Pro Asp Thr Lys Tyr Val 65 70 75 80 Leu Thr Gly Trp Ala Lys Ser Glu Gly Ser Ser Val Met Thr Leu Gly 85 90 95 Val Lys Asn Tyr Gly Gly Gln Glu Thr Phe Ser Ala Thr Leu Ser Ala 100 105 110 Asp Tyr Gln Gln Leu Ala Val Thr Phe Thr Thr Gly Pro Asn Ala Gln 115 120 125 Thr Ala Thr Ile Tyr Gly Tyr Arg Gln Asn Ser Gly Ser Gly Ala Gly 130 135 140 Tyr Phe Asp Asp Val Glu Leu Thr Ala Val Gln Asp Phe Ala Pro Tyr 145 150 155 160 Gln Pro Leu Ala Asn Ala Ile Ala Pro Gln Ala Ile Pro Thr Tyr Asp 165 170 175 Gly Ala Asn Gln Pro Thr His Pro Ser Val Val Lys Phe Glu Gln Pro 180 185 190 Trp Asn Gly Tyr Leu Tyr Trp Met Ala Met Thr Pro Tyr Pro Phe Asn 195 200 205 Asp Gly Ser Tyr Glu Asn Pro Ser Ile Val Ala Ser Asn Asp Gly Glu 210 215 220 Asn Trp Ile Val Pro Glu Gly Val Ser Asn Pro Leu Ala Gly Thr Pro 225 230 235 240 Ser Pro Gly His Asn Cys Asp Val Asp Leu Val Tyr Val Pro Ala Ser 245 250 255 Asp Glu Leu Arg Met Tyr Tyr Val Glu Ala Asp Asp Ile Ile Ser Ser 260 265 270 Arg Val Lys Met Ile Ser Ser Arg Asp Gly Val His Trp Ser Glu Pro 275 280 285 Gln Val Val Met Gln Asp Leu Val Arg Lys Tyr Ser Ile Leu Ser Pro 290 295 300 Ser Ile Glu Ile Leu Pro Asp Gly Thr Tyr Met Met Trp Tyr Val Asp 305 310 315 320 Thr Gly Asn Ala Gly Trp Asn Ser Gln Asn Asn Gln Val Lys Tyr Arg 325 330 335 Thr Ser Ala Asp Gly Ile Lys Trp Ser Gly Ala Val Thr Cys Thr Asp 340 345 350 Phe Val Gln Pro Gly Tyr Gln Ile Trp His Ile Asp Val His Tyr Asp 355 360 365 Thr Ser Ser Gly Ala Tyr Tyr Ala Val Tyr Pro Ala Tyr Pro Asn Gly 370 375 380 Thr Asp Cys Asp His Cys Asn Leu Phe Phe Ala Val Asn Arg Thr Gly 385 390 395 400 Lys Gln Trp Glu Thr Phe Ser Arg Pro Ile Leu Lys Pro Ser Thr Glu 405 410 415 Gly Gly Trp Asp Asp Phe Cys Ile Tyr Arg Ser Ser Met Leu Ile Asp 420 425 430 Asp Gly Met Leu Lys Val Trp Tyr Gly Ala Lys Lys Gln Glu Asp Ser 435 440 445 Ser Trp His Thr Gly Leu Thr Met Arg Asp Phe Ser Glu Phe Met Lys 450 455 460 The Glu Arg 465 <210> 24 <211> 5277 <212> DNA <213> Robinsoniella peoriensis <400> 24 tcaccattga gcgctgcggc agaagtggc acaggaacca gattagtgaa agggcaacg 60 gggtatttga cagaggaaca ggctatccgg aaccaggagc agacaccga agaagggag 120 cagaagttaa ccggggaga gagacagag gttttgatgg aaggtacaa agacagcgggg 180 attgtacaga cagagaagt acagacaaaa gaatgcaga cagagatgc gcagacaga 240 gaagtacaga cagagaaat gcagacaga gaggcgcaga caaagaagt acagaagaat 300 gaatgcaga cagagatgc gcagacaga gaagtacaga CAAAAG accggcagaa 360 gaacacaca tgaaagaat acagacgcaa ggacaaga aagcgtcaga taggacgga 420 aaggcaaggg taactgaat tctggagat gcccaggatc cagcaaccg gattgtgtat 480 ctgtcagacc tgcaatggaa gtcagaaaat catacagtag atagcgagct gcctaccaga 540 aaggataagt cctttggcgg cggaaaaatt acgctaaaag tggatggaac ggtaacagaa 600 tttgataagg ggattggaac acagacagat tccaccattg tgtacgatct ggagggaaag 660 ggatatacaa agtttgaaac ttacgtgggt gtagactaca gccagaaaga aaacattccg 720 ggggaagtct gcgacgtaaa attcagggtg aaaattgatg acaagattgt atcagaaacc 780 ggtgtactgg atccgctttc gaatgcggtt aagatttctg ttaacatacc cgatacagcc 840 aaaactttaa cattatacgc ggataaagta acggaaactt ggtctgatca cgccaattgg 900 gcagatgcaa aattttatca ggcactgccg gaacccgaaa atgttgcatt caaaaaaacg 960 gtagtgacac gaaagacatc agataattcg gaggctcctg ttaatccgga ttcagcagtt 1020 aacagttcta aggctgttga cggtgttatt gacagctcca gttattttga ttttggagat 1080 caggcaaata gcggagccgt aagggagtca ctctatatgg aggtagattt aaaagggagc 1140 tattactgt ccgatataca actgtggaga tactggaaag atggcagaac ttatgcagct 1200 actgcaattg tagtagctga ggatgagaac tttgaaatg cagcagttat ctataactcg 1260 gatacgacgg gagaataca tcacctggga gcaggaagtg atatgctcta tgcagaaaca 1320 gaagtggca agacattcc ggtaccggaa atacaaag caagtatat cagagtttat 1380 acatagtg ttaatgggac atcaggcgta acaatcaca tgtagaatt aaggtgaat 1440 gcttacgtat ttggagatga aatcttaccg gaaaagccgg atgacagca gatttccca 1500 aatgcagtta atccgctgaa gctacaggga ccggcacga atgatcaggt aacccaccg 1560 gatgttacgg tgttgatga gccgtggaat gggtataaat actggatggc atacaccg 1620 aaaaccgg gaagttccta ttttgaaat ccctgtatag ctgcatccaa cgatggcgta 1680 aactgggagt ttcctgccca gaaccctgta cagccgcgct atgacagtga atagaaaat 1740 CAAAATGAAC atactgtga taccgatatt gtatatgacc cggtaaatga ccggttgatt 1800 atgtactggg aatgggcaca ggatgaggcg gttaatggta aaacacatcg ttctgaatc 1860 agataccgtg tttcttga tgggattaac tggggagtgg agacaaac tggtgttttg 1920s atgactggac caacggatca tggctgcgcc attgccacag aaggcgaaag atattcagac 1980 cttctccaa ccgtagtata tgataaaaca gaaaaaatct acaaaatgtg ggcaaatgat 2040 gccggagatg taggatatga aaacaaacag aataacaaag tatggtatcg gacatcccaa 2100 gacgggatca gcaattggtc ggataagact tacgtggaga atttcttgg agtaaatgaa 2160 gacgggctgc agatgtatcc atggcaccag gatatccagt gggtagagga atttcaggaa 2220 tattgggcac ttcagcaggc atttccggca ggaagcggac cggataattc ttccctgcgt 2280 ttctcgaaat ccaaagatgg tcttcattgg gagccggtat ctgaaaaagc tttaattaca 2340 gtaggggcac ccgggacctg ggatgcagga cagatatacc gttctacttt ctggtatgag 2400 ccaggtgggg caaaaggaaa cggaacattc catatctggt atgctgcatt ggcggaaggc 2460 cagtctcact gggatatagg atatacatct gcaaactatg cagatgccat gtacaaatta 2520 acgggaagca gaccggaagt ggaaaaaaga atagaggtaa ataatgaaaa tcctctgctg 2580 attatgccgc tttacggaaa gtcttacagt gaatcaggaa gtaccctgga ttggggagat 2640 gatctggttt cacgctggaa acaggttccg gaagatttaa aagaaaacgc agttattgaa 2700 attcatctgg gtggcaagat tggcttaaat gaaagtgatt cccacacggc aaaagcgttt 2760 tatgagcagc agctggcaat tgcccaggaa ataacatcc cggtaatgat ggtggtagct 2820 acggcaggcc agcagaacta ctggacggga acagcgaatc tggatgctga gtggattgac 2880 cggatgttca agcagcacag tgtgttaaaa ggaattatgt ccactgaaaa ttatggact 2940 gactacaata aggttgctac tatgggtgcc gattatctgc gggttgcagc tgaaaacggc 3000 ggatattttg tatggagcga gcaccaggag ggtgttattg aaaatgtaat agcaaatgag 3060 aaattaatg aagcattgaa actttacggt aataatttta ttttcacctg gaagaacagg 3120 cctgccggta ctaactccaa tgcaggaaca gccagctata tgcagggcct ctggctaacc 3180 ggaatttgtg caaatgggg cggtctggct gatacctgga aatggtatga aaaaggattt 3240 ggtaaattat ttgatggtca gtattcttat aatccgggtg gggaagaagc aagaccggtt 3300 3360 3420. tgtgtctaca actttgagca cccggcgtat gtatatggtt cttataacca gaattcacct tgctttgaaa atgtaattgc agagttcatg cgctatgcga ttaagaatcc ggcaccaggt aaagaggaag tgcttgctga tacaaaagca gtgttctatg gaaattaag ttctttaag agtgcagga acttactgca aaaaggtttg aactgggag atgccacact gccaacccag actacgggtc price aatacctgca gtcccggagg price aaaaaactgta aaagcagtat tcggcgatat tgagatattg aatcaatcca gtgcacagct tgcgaataaa gatgcgaaaa aagcatattt tgaagaaaaa tatccggaac agtataccgg tacggcattt ggacagctat tgaatgatac ctggtattta tacaacagta atgtgaatgt ggatggggtg caaaatgcaa aacttccgtt aagaggtaat aaatccgtag attack gacaccgcat acttatgtga tcctggatga tcaggatggt gagcttcaga ttaaactgaa caattatcgt gtggataaag acagtatctg ggaggatac ggcaccacgg tgacggaccg ctgggatacg gaccacaata ccaaacttca ggactggata cgggatgagt atattccaaa tccggacgat gataccttca gagatacaac ctttgactg gttggactgg aaagtgagcc ggaggtaaat 4140 gtaactaatg gcttaagga tcagtatcag gaaccggttg tggaatatga tgccgctgca 4200 ggtacggcta tgattactgt atccggaat ggctgggtag atctgacaat tgacacgac 4260 acggcagaag taccccaggt tgataaagca aagttaaatt ccaaatagc agaagctaaa 4320 gggatcagac agggaacta tacggatgaa tcctacaag ctctcagga agagattgga 4380 aaatcccagg cggtatcaa caaacagat gccacacagg aggagtaa tgcacagtta 4440 agcaggttag aaagtgcaat agccagatta aaagaaaaac cggcggtggt atccaaaacg 4500 gcattaaatg caaaatagc tgaggcaaa gggatcagac aaggaaacta tacggatga 4560 tcctacaaag ccctgcaaaa tgcaatagta aaagctcagg agttacaaa caaacagat 4620 gccacacagc agcaggtaaa tgatctgta tcagcattaa caatgcaat taaaaattta 4680 aaatagatg cagataagct ggcagcagag tcagcaag aagtagcggc agttaaggtt 4740 gccgtaaag cagtaccta taaatcaaa gagatttaat tatcctggaa aacggtagca 4800 gatgcggacg gatatgtaat ccgtgtaag acaggcaaa agtggagtac ggagagacc 4860 attagaaca accgcataat cacttatact tatagaag gtactcccgg windows 4920 gtatttgag t'aagcttt t'agagta aatgaaag cgacctatag t'aatacaa 4980 acagccacta aaaaagttgt gccgcaaacg gtgaccgcaa agggcaaagc ttctaaaaat 5040 aatgtagtgg taaaatggaa aaagtgtct ggcgcatccg gatatgttgt tatgaaaag 5100 aaagggaaaa catgggtaaa ggctgcgcag gtaaatgcaa agaaactata ctttacggat 5160 aagaaggtca aaaaaggaaa agtatattca tachaagtaaagcttaca agtatattaa 5220 ggtaaaaag tatatggaag ctatagcaag tctgtaaatg ttaaaacaa gtcataa 5277 <210> 25 <211> 1778 <212> PRT <213> Robinsoniella peoriensis <400> 25 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Ser Pro Leu Ser Ala Ala Ala Glu Ser Gly Thr Gly 20 25 30 Thr Arg Leu Val Lys Gly Gln Thr Gly Tyr Leu Thr Glu Glu Gln Ala 35 40 45 Ile Arg Asn Gln Glu Gln Thr Thr Glu Glu Arg Glu Gln Lys Leu Thr 50 55 60 Gly Glu Glu Thr Ala Glu Val Leu Met Glu Gly Thr Lys Asp Ser Gly 65 70 75 80 Ile Val Gln Thr Glu Glu Val Gln Thr Lys Glu Met Gln Thr Glu Asp 85 90 95 Ala Gln Thr Glu Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala 100 105 110 Gln Thr Lys Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln 115 120 125 Thr Glu Glu Val Gln Thr Lys Glu Glu Pro Ala Glu Glu Thr His Met 130 135 140 Lys Glu Ile Gln Thr Gln Gly Thr Lys Lys Ala Ser Asp Arg Asn Gly 145 150 155 160 Lys Ala Arg Val Thr Glu Ile Leu Glu Asp Ala Gln Asp Pro Ala Asn 165 170 175 Arg Ile Val Tyr Leu Ser Asp Leu Gln Trp Lys Ser Glu Asn His Thr 180 185 190 Val Asp Ser Glu Leu Pro Thr Arg Lys Asp Lys Ser Phe Gly Gly Gly 195 200 205 Lys Ile Thr Leu Lys Val Asp Gly Thr Val Thr Glu Phe Asp Lys Gly 210 215 220 Ile Gly Thr Gln Thr Asp Ser Thr Ile Val Tyr Asp Leu Glu Gly Lys 225 230 235 240 Gly Tyr Thr Lys Phe Glu Thr Tyr Val Gly Val Asp Tyr Ser Gln Lys 245 250 255 Glu Asn Ile Pro Gly Glu Val Cys Asp Val Lys Phe Arg Val Lys Ile 260 265 270 Asp Asp Lys Ile Val Ser Glu Thr Gly Val Leu Asp Pro Leu Ser Asn 275 280 285 Ala Val Lys Ile Ser Val Asn Ile Pro Asp Thr Ala Lys Thr Leu Thr 290 295 300 Leu Tyr Ala Asp Lys Val Thr Glu Thr Trp Ser Asp His Ala Asn Trp 305 310 315 320 Ala Asp Ala Lys Phe Tyr Gln Ala Leu Pro Glu Pro Glu Asn Val Ala 325 330 335 Phe Lys Lys Thr Val Val Thr Arg Lys Thr Ser Asp Asn Ser Glu Ala 340 345 350 Pro Val Asn Pro Asp Ser Ala Val Asn Ser Ser Lys Ala Val Asp Gly 355 360 365 Val Ile Asp Ser Ser Ser Tyr Phe Asp Phe Gly Asp Gln Ala Asn Ser 370 375 380 Gly Ala Val Arg Glu Ser Leu Tyr Met Glu Val Asp Leu Lys Gly Ser 385 390 395 400 Tyr Leu Leu Ser Asp Ile Gln Leu Trp Arg Tyr Trp Lys Asp Gly Arg 405 410 415 Thr Tyr Ala Ala Thr Ala Ile Val Val Ala Glu Asp Glu Asn Phe Glu 420 425 430 Asn Ala Ala Val Ile Tyr Asn Ser Asp Thr Thr Gly Glu Ile His His 435 440 445 Leu Gly Ala Gly Ser Asp Met Leu Tyr Ala Glu Thr Glu Ser Gly Lys 450 455 460 Thr Phe Pro Val Pro Glu Asn Thr Lys Ala Arg Tyr Ile Arg Val Tyr 465 470 475 480 Thr Tyr Gly Val Asn Gly Thr Ser Gly Val Thr Asn His Ile Val Glu 485 490 495 Leu Lys Val Asn Ala Tyr Val Phe Gly Asp Glu Ile Leu Pro Glu Lys 500 505 510 Pro Asp Asp Ser Lys Ile Phe Pro Asn Ala Val Asn Pro Leu Lys Leu 515 520 525 Gln Gly Pro Gly Thr Asn Asp Gln Val Thr His Pro Asp Val Thr Val 530 535 540 Phe Asp Glu Pro Trp Asn Gly Tyr Lys Tyr Trp Met Ala Tyr Thr Pro 545 550 555 560 Asn Lys Pro Gly Ser Ser Tyr Phe Glu Asn Pro Cys Ile Ala Ala Ser 565 570 575 Asn Asp Gly Val Asn Trp Glu Phe Pro Ala Gln Asn Pro Val Gln Pro 580 585 590 Arg Tyr Asp Ser Glu Ile Glu Asn Gln Asn Glu His Asn Cys Asp Thr 595 600 605 Asp Ile Val Tyr Asp Pro Val Asn Asp Arg Leu Ile Met Tyr Trp Glu 610 615 620 Trp Ala Gln Asp Glu Ala Val Asn Gly Lys Thr His Arg Ser Glu Ile 625 630 635 640 Arg Tyr Arg Val Ser Tyr Asp Gly Ile Asn Trp Gly Val Glu Asp Lys 645 650 655 Thr Gly Val Leu Met Thr Gly Pro Thr Asp His Gly Cys Ala Ile Ala 660 665 670 Thr Glu Gly Glu Arg Tyr Ser Asp Leu Ser Pro Thr Val Val Tyr Asp 675 680 685 Lys Thr Glu Lys Ile Tyr Lys Met Trp Ala Asn Asp Ala Gly Asp Val 690 695 700 Gly Tyr Glu Asn Lys Gln Asn Asn Lys Val Trp Tyr Arg Thr Ser Gln 705 710 715 720 Asp Gly Ile Ser Asn Trp Ser Asp Lys Thr Tyr Val Glu Asn Phe Leu 725 730 735 Gly Val Asn Glu Asp Gly Leu Gln Met Tyr Pro Trp His Gln Asp Ile 740 745 750 Gln Trp Val Glu Glu Phe Gln Glu Tyr Trp Ala Leu Gln Gln Ala Phe 755 760 765 Pro Ala Gly Ser Gly Pro Asp Asn Ser Ser Leu Arg Phe Ser Lys Ser 770 775 780 Lys Asp Gly Leu His Trp Glu Pro Val Ser Glu Lys Ala Leu Ile Thr 785 790 795 800 Val Gly Ala Pro Gly Thr Trp Asp Ala Gly Gln Ile Tyr Arg Ser Thr 805 810 815 Phe Trp Tyr Glu Pro Gly Gly Ala Lys Gly Asn Gly Thr Phe His Ile 820 825 830 Trp Tyr Ala Ala Leu Ala Glu Gly Gln Ser His Trp Asp Ile Gly Tyr 835 840 845 Thr Ser Ala Asn Tyr Ala Asp Ala Met Tyr Lys Leu Thr Gly Ser Arg 850 855 860 Pro Glu Val Glu Lys Arg Ile Glu Val Asn Asn Glu Asn Pro Leu Leu 865 870 875 880 Ile Met Pro Leu Tyr Gly Lys Ser Tyr Ser Glu Ser Gly Ser Thr Leu 885 890 895 Asp Trp Gly Asp Asp Leu Val Ser Arg Trp Lys Gln Val Pro Glu Asp 900 905 910 Leu Lys Glu Asn Ala Val Ile Glu Ile His Leu Gly Gly Lys Ile Gly 915 920 925 Leu Asn Glu Ser Asp Ser His Thr Ala Lys Ala Phe Tyr Glu Gln Gln 930 935 940 Leu Ala Ile Ala Gln Glu Asn Asn Ile Pro Val Met Met Val Val Ala 945 950 955 960 Thr Ala Gly Gln Gln Asn Tyr Trp Thr Gly Thr Ala Asn Leu Asp Ala 965 970 975 Glu Trp Ile Asp Arg Met Phe Lys Gln His Ser Val Leu Lys Gly Ile 980 985 990 Met Ser Thr Glu Asn Tyr Trp Thr Asp Tyr Asn Lys Val Ala Thr Met 995 1000 1005 Gly Ala Asp Tyr Leu Arg Val Ala Ala Glu Asn Gly Gly Tyr Phe 1010 1015 1020 Val Trp Ser Glu His Gln Glu Gly Val Ile Glu Asn Val Ile Ala 1025 1030 1035 Asn Glu Lys Phe Asn Glu Ala Leu Lys Leu Tyr Gly Asn Asn Phe 1040 1045 1050 Ile Phe Thr Trp Lys Asn Thr Pro Ala Gly Thr Asn Ser Asn Ala 1055 1060 1065 Gly Thr Ala Ser Tyr Met Gln Gly Leu Trp Leu Thr Gly Ile Cys 1070 1075 1080 Ala Gln Trp Gly Gly Leu Ala Asp Thr Trp Lys Trp Tyr Glu Lys 1085 1090 1095 Gly Phe Gly Lys Leu Phe Asp Gly Gln Tyr Ser Tyr Asn Pro Gly 1100 1105 1110 Gly Glu Glu Ala Arg Pro Val Ala Thr Glu Pro Glu Ala Leu Leu 1115 1120 1125 Gly Ile Glu Met Met Ser Ile Tyr Thr Asn Gly Gly Cys Val Tyr 1130 1135 1140 Asn Phe Glu His Pro Ala Tyr Val Tyr Gly Ser Tyr Asn Gln Asn 1145 1150 1155 Ser Pro Cys Phe Glu Asn Val Ile Ala Glu Phe Met Arg Tyr Ala 1160 1165 1170 Ile Lys Asn Pro Ala Pro Gly Lys Glu Glu Val Leu Ala Asp Thr 1175 1180 1185 Lys Ala Val Phe Tyr Gly Lys Leu Ser Ser Leu Lys Ser Ala Gly 1190 1195 1200 Asn Leu Leu Gln Lys Gly Leu Asn Trp Glu Asp Ala Thr Leu Pro 1205 1210 1215 Thr Gln Thr Thr Gly Arg Tyr Gly Leu Ile Pro Ala Val Pro Glu 1220 1225 1230 Ala Val Asp Glu Lys Thr Val Lys Ala Val Phe Gly Asp Ile Glu 1235 1240 1245 Ile Leu Asn Gln Ser Ser Ala Gln Leu Ala Asn Lys Asp Ala Lys 1250 1255 1260 Lys Ala Tyr Phe Glu Glu Lys Tyr Pro Glu Gln Tyr Thr Gly Thr 1265 1270 1275 Ala Phe Gly Gln Leu Leu Asn Asp Thr Trp Tyr Leu Tyr Asn Ser 1280 1285 1290 Asn Val Asn Val Asp Gly Val Gln Asn Ala Lys Leu Pro Leu Glu 1295 1300 1305 Gly Asn Lys Ser Val Asp Ile Thr Met Thr Pro His Thr Tyr Val 1310 1315 1320 Ile Leu Asp Asp Gln Asp Gly Glu Leu Gln Ile Lys Leu Asn Asn 1325 1330 1335 Tyr Arg Val Asp Lys Asp Ser Ile Trp Glu Gly Tyr Gly Thr Thr 1340 1345 1350 Val Thr Asp Arg Trp Asp Thr Asp His Asn Thr Lys Leu Gln Asp 1355 1360 1365 Trp Ile Arg Asp Glu Tyr Ile Pro Asn Pro Asp Asp Asp Thr Phe 1370 1375 1380 Arg Asp Thr Thr Phe Glu Leu Val Gly Leu Glu Ser Glu Pro Glu 1385 1390 1395 Val Asn Val Thr Asn Gly Leu Lys Asp Gln Tyr Gln Glu Pro Val 1400 1405 1410 Val Glu Tyr Asp Ala Ala Ala Gly Thr Ala Met Ile Thr Val Ser 1415 1420 1425 Gly Asn Gly Trp Val Asp Leu Thr Ile Asp Thr Asn Thr Ala Glu 1430 1435 1440 Val Pro Gln Val Asp Lys Ala Lys Leu Asn Ser Lys Ile Ala Glu 1445 1450 1455 Ala Lys Gly Ile Arg Gln Gly Asn Tyr Thr Asp Glu Ser Tyr Lys 1460 1465 1470 Ala Leu Gln Glu Glu Ile Gly Lys Ser Gln Ala Val Ser Asn Lys 1475 1480 1485 Thr Asp Ala Thr Gln Glu Glu Val Asn Ala Gln Leu Ser Arg Leu 1490 1495 1500 Glu Ser Ala Ile Ala Arg Leu Lys Glu Lys Pro Ala Val Val Ser 1505 1510 1515 Lys Thr Ala Leu Asn Ala Lys Ile Ala Glu Ala Lys Gly Ile Arg 1520 1525 1530 Gln Gly Asn Tyr Thr Asp Glu Ser Tyr Lys Ala Leu Gln Asn Ala 1535 1540 1545 Ile Val Lys Ala Gln Glu Leu Ser Asn Lys Thr Asp Ala Thr Gln 1550 1555 1560 Gln Gln Val Asn Asp Leu Val Ser Ala Leu Thr Asn Ala Ile Lys 1565 1570 1575 Asn Leu Lys Ile Asp Ala Asp Lys Leu Ala Ala Glu Ser Ala Lys 1580 1585 1590 Light Val Ala Ala Val Light Val Ala Val Light Ala Val Ser Tyr Light 1595 1600 1605 Ser Lys Glu Ile Lys Leu Ser Trp Lys Thr Val Ala Asp Ala Asp 1610 1615 1620 Gly Tyr Val Ile Arg Val Lys Thr Gly Lys Lys Trp Ser Thr Glu 1625 1630 1635 Light Thr Ile Light Asn Asn Arg Ile Ile Thr Tyr Thr Tyr Light Light 1640 1645 1650 Gly Thr Pro Gly Lys Lys Tyr Val Phe Glu Val Lys Ala Phe Lys 1655 1660 1665 Lys Val Asn Gly Lys Thr Thr Tyr Ser Lys Tyr Lys Thr Ala Thr 1670 1675 1680 Light Light Val Val Pro Gln Thr Val Thr Ala Light Ala Light Ala Ser 1685 1690 1695 Lys Asn Asn Val Val Val Lys Trp Asn Lys Val Ser Gly Ala Ser 1700 1705 1710 Gly Tyr Val Val Met Lys Lys Lys Gly Lys Thr Trp Val Lys Ala 1715 1720 1725 Ala Gln Val Asn Ala Lys Lys Leu Tyr Phe Thr Asp Lys Lys Val 1730 1735 1740 Lys Lys Gly Lys Val Tyr Ser Tyr Lys Val Lys Ala Tyr Lys Val 1745 1750 1755 Tyr Lys Gly Lys Lys Val Tyr Gly Ser Tyr Ser Lys Ser Val Asn 1760 1765 1770 Val Lys Thr Lys Ser 1775 <210> 26 <211> 7899 <212> DNA <213> Robinsoniella peoriensis <400> 26 gctgagactg caacagaaga aaatgcggcg ctggaaaaaa cagttacatt gcataagagc 60 gatggaacag aactgccgga ggattatcga aatccccaaa gaccagctac catggcggta 120 gatggtatta ttgacgatac aggagagtac aactattgcg atttcggtaa agacggtgat 180 aaagcagccc tgtatatgca ggtggacctt ggaggtctgt atgatttaag cagagtcaat 240 atgtggagat actggaaaga cagcagaact tacgatgcaa cagtaattac cacatctgag 300 agcggcgatt tcacagatga agcagtcata tataattcag acaggtcgaa tgtacatgga 360 tttggggcag gaggagatga acgctacgca gagactgcct ccggacatga attcccagta 420 ccggacggta caaaggcaca ggcagtacgc gtatatgtat ttggcagcca aaacggtact 480 acaaaccaca tcaatgaatt gcaggtctgg ggaactcccc atacagagaa tccggatgta 540 aattcttatc aggtgacaat tccacaggga aatggatatc aggtaatacc ttatgaaaat 600 gacccgacga cagtggaaga aggcggttct ttccgttttc aggtactgat tgactccgat 660 aatggttaca gcgcaaccag tgcggtaaaa gcaaatggag taagtctgga ggcagttgac 720 agtgtttata ccattgagaa cattactgaa gatcaggtaa tcaccattga aggcgtacat 780 aaagcacagt atgaagtgaa attcccggaa aatccacagg gatacagtgt tgagattcag 840 aatgaaggaa gtacaacggt agactataat ggttctgtca gttttaagct tattatagac 900 gaagcttata atgaatccgt accggttgta aaagcaaacg gcggtgcagc tttgggaaaa 960 gatgagctcg gtgtatatac aattgcaaat atccaggacg atattacggt tacagttgag 1020 ggtatccagg aaaataccgt agtaaagaca aaaacaatgt acttgtctga tatggattgg 1080 aagagtgctg aaatgcagt aggtgcaca gggaaaag acacccac aaggacctg 1140 atcatttac agcagcagat gaattattg gtaaacggag cagagaagtc tttgataaa 1200 ggaattggag tcagacgga ttctctatc gtttatgatc tggaagacaa aggctacact 1260 tctttccaca ccctggcagg cgttgatt tcagcaatgg atatgtaga cggagaggc 1320 tgtgatatcc agtttaaagt atatctggat gatgtcgtag tatttgacag cggagtagtt 1380 catcatctg atgaggctca ggagttaat gttgctataa catcagagaa window 1440 aaactggaag ctaaaatggt taagagcct tataatgact ggggaactg ggcagatgcc 1500 agctttgaaa tggcttatcc cgaaccgtct aatgtggctt taataaac agttaccgtt 1560 aagaaaacag cggatactc agactctgaa gtaaattcca gcagaccggg atcaatggct 1620 gtagatggaa tcattggacc tacatcagat tctaactatt gtgattttgg acaggatgggg 1680 gataactt cccgttatct gcaggtagat ttagggatg ttatgact taccagatt 1740 atatgttta gatactggggc agatggcaga gtatatatatg gtactgtaat tgcagttttcc 1800 gaaaacgcag actttagtaa tccaactttt attaatatt cagataaagc agacaacac 1860 ggacttggcg caggcagtga tgacacttat ggagaaccc agagtggaaa attattcga 1920 gttccggcgg gaaccatggg acagtatgtc cgtgtgtata tggctggttc cacaaggt 1980 acaacgaacc attcgctga attacaggta atgggttata atttcaatac agaaccaaaa 2040 ccatatgaag caaatgcatt tgaaatgca gaagtttatt tagatatgcc aactcattc 2100 caggatctgg attccataa aaacgacgat ggaagcttaa agcacattgg cggacaggtg 2160 acacatcctg atatccaggt atttgaccaa ccgtggaacg gttataata ctggatgatt 2220 tacacaccaa atacaatgat cactcccag tatgaaaatc catatatcgt agcatctgaa 2280 catggacaga catgggtaga accggaaggg atttccaatc cattgaacc agaaccgcca 2340 tcaccagat ttcatactg tgatgcagat ctgttatacg actctgtcaa tgaccgttta 2400 cttgcttact ggaactgggc agatgacggc ggcggaattg atgacgaatt aaagatcag 2460 aactgtcaga ttcgtctgag aatttcttat gatggaatta actgggagt tccttacgac 2520 aaagacggca atttgccac aacagctgat actgtagtaa gatggaac aggataag 2580 gatttcattc ctgcaatcag cgaaaaagac cgttatgta tgctttcccc aacatttacc 2640 tatgacgatt tccgcggcat atacaatg tgggcacaaa actcgggtga tgcgggatac 2700 aaccagtccg gaagttcat cgaatgaga tggtctgagg atggataaa ctggtctgaa 2760 ccacaaaag tgaataattt ccttggaaaa gatgagaatg gcagacagct ttggccatgg 2820 catcaggata ttcagtatat ccctgagcta caggaatatt ggggactgtc ccagtgtttc 2880 tctacatcta atcccgatgg atccgtatta tacctgacca agtccagaga tggtgtcaac 2940 tgggagcagg caggacaca gccggtatta agggcaggaaatcaggtac ctgggatgat 3000 ttccagattt accgttctac cttctattat gataatcagt cagacagccc tactggtggg 3060 aaatttagaa tctggtacag tgcactgcag gcaatactt caggcagac cgttttggct 3120 cctgatggaa cagtgtctct tcaggttgga agccaggata ccaggatctg gcgtatcggg 3180 tatacagaaa atgactacat ggaagtcatg aaagctctga cccagaataa aaactacat 3240 gaaccggaat tagtagacgc agtttcctta aatctgtcaa tggataaaac aagcatttca 3300 gtaggtgaag aagcaacggt aagcactgct ttcgtaccgg aaaatgctac cgaccgcatt 3360 gtaaaatata catctcagga tccggaaatt gcagttattg atccaacagg cattgttaca 3420 ggggttaagg atggaaccac aactattgtt gcagaaacaa aatcgggcgc aaaaggtgaa 3480 ttatccgtaa cggttggtga gcttcaaaga ggtgaaattc gatttgaggt cagcaatgac 3540 catccgatgt atctggagaa ttactattgg agtgatgatg caccaaaaaa agacggctta 3600 gacgcaaaca agaactacta tggggatgaa cgtgtcgaca gtccggtaat gctgtataat 3660 accgttcctg aagaattgaa ggataataca gtcatcctgt taattgcaga gagaagctta 3720 aacagcacag atgcagtaag ggattggatt aaaaagaatg ttgaattatg taatgaaaat 3780 aagattccat gtgcagttca gattgcaaat ggagaaacaa atgtaaatac aaccattcca 3840 ttatcgttct ggaatgagct ggcaacgaac aatgaatacc tggttggatt taatgcagcc 3900 gagatgtata accgttttgc aggtgacaac cgcagctatg ttatggatat gatccgttta 3960 ggggtatccc acggcgtatg catgatgtgg accgatacca atatttttgg tacaaacggt 4020 gtgttgtatg actggctgac tcaggatgaa aaactgtccg gtcttatgcg ggaatacaaa 4080 gagtatatct ctctgatgac aaaagagtct tacggcagtg aggcagcaaa tacagatgct 4140 ctgtttaagg gcctgtggat gacagactac tgcgagaact ggggaatcgc ctccgactgg 4200 tggcattggc agttagacag caatggagca ctctttgatg caggcagcgg cggagatgca 4260 tggaaacagt gtctgacatg gccggaaaat atgtatacgc aggacgttgt gcgtgcagta 4320 agccagggtg caacctgctt taaatcagaa gcacagtggt attcaaatgc tacaaaaggc 4380 atgcgtacac cgacatatca gtattccatg attccgttcc tggagaaact ggtaagcaaa 4440 gaggtaaaaa ttcctacaaa agaagagatg ctggaaagaa caaaagcaat tgttgtaggg 4500 gcagaaaact ggaataactt taattataat actacttatt caaatctgta tccaagcaca 4560 ggacaatatg gaatcgtacc ttatgtacct tcaaattgtc cggaagaaga actggcaggc 4620 tatgatctgg tagtaaggga aaaccttggc aaagcaggac tgaagtctgc acttgatacg 4680 gtatacccgg ttcagaatc agaggaacc gcatactgtg aaacctttgg agatacctgg 4740 tactggatga attcctcgga agacaaaac gtaagccagt acactgaatt tacaaccgca 4800 atcaatggag ctgaagtgt aaagatagcc ggcgaacccc atgtatttgg tattataaa 4860 gaaaatccgg gatctttaaa tgtatactta agcaactacc gcctggataa aacagaactc 4920 tgggatggta caatccccgg aggattaagc gatcagggct gctatatta tgtatggcag 4980 atgtgtgagc gcatgaagaa tggaacaggg ctggatacac agctcgtga caccgttatt 5040 accgttaaaa atgcagtaga accgaagta aactttgtaa cagaatctcc ggcagacaga 5100 agttttgcag agaatatta tgtaagacca acaata cggttgcaca aaagaaggc 5160 acaacgatg aatgggtgat tacggtcagc cacaatggta tgtggaatt caatattgta 5220 acaggcgatg aaaagtgcc ggcaacaagt gtggattt acatgataa agttgatgta 5280 atccgtaacc ggacagcagt tgtaaggca acggtattgc cgcagaatgc aggaataaa 5340 cagttaacat ggacaatcgc cgatcctgag attgctctg tagacaaca aggaaccgta 5400 accggactaa aagaaggaaa aaccgtatta cgtgcagcta tttctggcag tgtttataaa 5460 gaatgcgaag taaatgtaat tgaccgaaaa gtaacggaag taaacttaaa caaaacagag 5520 ttgtctctta gtgcagggga ttctgcgaaa ctggaagcat catagcacc ggaagacccg 5580 tctgacagca gcattacctg gacttccaca aatgaaaatg ttgcaacggt tgcatcaaac 5640 ggtaccgtta cagctcataa agcaggtgta gctcagatta tcgcccagtc tgcttaccag 5700 gcaaagggta tcgcaactgt taccgttaat tatgcggctt ccgtaaaatt agaccgtaca 5760 ggaatgacgg ctacagccaa cagcgaacag tctaaatcag gtggagaagg acctgcttcc 5820 aacgtactgg acggtaagca ggacacaatg tggcatacaa gctggacaga taaacctgaa 5880 ttacatcctc actggattaa aattgattta aacggaacaa aaacaattaa caaatttgct 5940 tatacaccaa gaaccggagc atctaacgga acaatttata attatgttct gattatcacc 6000 gatctggaag gaaatgaaaa acaggttgca aagggcgtat gggcagcaaa tgcagatgta 6060 aaattgctg aatttgacgc agttgaagct acggcgatca agctgcaggt agacggcaac 6120 gatgacaagg catcaaagg aggatatggt tccgcggcag aaatcaat tttgaagtg 6180 gcacagaaac cttccgcaaa tgagcttgcc gaaatatta aagtaattgc acctgtaaaa 6240 gcagaagata caaagtatc tatcccagtc attactggat ttgatacgt atcagtaat 6300 tccagcaatc cggacgtaat tggtattgat ggcagcatca ccagaccgga aaatgataca 6360 gttgtaactt taacattaaa agtaaagaa acagacgcaa agagtgtaaa ggcagcagga 6420 actgaagcaa ccacaatgt ggatgttctg gttaccggta caagacatc tgatgtagag 6480 gcagaaagcg ttacgttaga tcagacatca gctgatttaa cagttggagg cgaactttta 6540 ttaaatgccg tgtgaagcc ggacattgca actaatagg ctgttaccctg gagctcagat 6600 aagccgggaa cgctactgt tgaaaatggc agggtaaag cgttagcggc aggaggca 6660 cgtattacag cagcaactgc aaatggaaag acagcagact gcgtcattaa cgtaaggaa 6720 aaagaggagc cggagtaat tctcccggca gaagtgcgct taacattcc atcagctgaa 6780 tttacagtag gagatcagat tcagttaac gcttctgtac tgccggcaa tgcagcaat 6840 aagacaatta cctggaaatc agacaacct gaagtggcaa ctgtcgcaaa tggatgggta 6900 aaaggtattg cagccggaac tgctaagatt acagcacat cagtcaatgg aaaaacggct 6960 gtatgtgtga tcacagtca agcacagcca cagaatctac caacggtgt ttcactgaac 7020 aagaaaacag caagtgtaaa actgaataaa acccttacac ttccgctgt agtacagcct 7080 tccaatgcgg ataataagac cgttaaatgg acgtctgaca atacgtatgt tgcaacagtt 7140 gagaatggag tcgtaaaagc agttaatgca ggaacagcca gatcactgc agctaccgta 7200 aacggacata agcaacttg tactataca gtaccgggca caagatttc aaggcaaaa 7260 gtaagccttg catcatcaa aacacataca ggcaagcat taaaaccac tgtaaagta 7320 acttacggta agaatacatt aaagaaaaat actgattata ccgtatctta caaaataat 7380 aaatcctg gaactgcatc tgttacgatt acgggcaagg gtaattatta tgtaccatc 7440 aaaaaactt ttgcaatcaa ggcagcagaa ggaagacct acacggttgg taaggaaaa 7500 tataaagtta ctgatgcttc agcaagaac aaaacagtaa cctttatggc tcctgtaaag 7560 aagacctaca gctcattcag cgtaccttct aaggttaaga tcgggaatga tacttacaaa 7620 gtaactgcag ttgcaaaaaa tgcattcaa aagaatacaa agcttacaa gttaaccatt 7680 ggttcgaatg taaaaacaat tggttctt gcattttatg gcgctccca attaaaaacg 7740 cttaccttaa aaactaccgg acttacagt gtaggcaaga atgcatttaa gaaaacaat 7800 gcaaagctga ctgtaaggt tccaagtca aaattagcag attataagaa gctgttaaaa 7860 ggaaaaggt tatctggcaa ggcaaaatt summer 7899 <210> 27 <211> 2652 <212> PRT <213> Robinsoniella peoriensis <400> 27 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Ala Glu Thr Ala Thr Glu Glu Asn Ala Ala Ala Leu Glu 20 25 30 Lys Thr Val Thr Leu His Lys Ser Asp Gly Thr Glu Leu Pro Glu Asp 35 40 45 Tyr Arg Asn Pro Gln Arg Pro Ala Thr Met Ala Val Asp Gly Ile Ile 50 55 60 Asp Asp Thr Gly Glu Tyr Asn Tyr Cys Asp Phe Gly Lys Asp Gly Asp 65 70 75 80 Lys Ala Ala Leu Tyr Met Gln Val Asp Leu Gly Gly Leu Tyr Asp Leu 85 90 95 Ser Arg Val Asn Met Trp Arg Tyr Trp Lys Asp Ser Arg Thr Tyr Asp 100 105 110 Ala Thr Val Ile Thr Thr Ser Glu Ser Gly Asp Phe Thr Asp Glu Ala 115 120 125 Val Ile Tyr Asn Ser Asp Arg Ser Asn Val His Gly Phe Gly Ala Gly 130 135 140 Gly Asp Glu Arg Tyr Ala Glu Thr Ala Ser Gly His Glu Phe Pro Val 145 150 155 160 Pro Asp Gly Thr Lys Ala Gln Ala Val Arg Val Tyr Val Phe Gly Ser 165 170 175 Gln Asn Gly Thr Thr Asn His Ile Asn Glu Leu Gln Val Trp Gly Thr 180 185 190 Pro His Thr Glu Asn Pro Asp Val Asn Ser Tyr Gln Val Thr Ile Pro 195 200 205 Gln Gly Asn Gly Tyr Gln Val Ile Pro Tyr Glu Asn Asp Pro Thr Thr 210 215 220 Val Glu Glu Gly Gly Ser Phe Arg Phe Gln Val Leu Ile Asp Ser Asp 225 230 235 240 Asn Gly Tyr Ser Ala Thr Ser Ala Val Lys Ala Asn Gly Val Ser Leu 245 250 255 Glu Ala Val Asp Ser Val Tyr Thr Ile Glu Asn Ile Thr Glu Asp Gln 260 265 270 Val Ile Thr Ile Glu Gly Val His Lys Ala Gln Tyr Glu Val Lys Phe 275 280 285 Pro Glu Asn Pro Gln Gly Tyr Ser Val Glu Ile Gln Asn Glu Gly Ser 290 295 300 Thr Thr Val Asp Tyr Asn Gly Ser Val Ser Phe Lys Leu Ile Ile Asp 305 310 315 320 Glu Ala Tyr Asn Glu Ser Val Pro Val Val Lys Ala Asn Gly Gly Ala 325 330 335 Ala Leu Gly Lys Asp Glu Leu Gly Val Tyr Thr Ile Ala Asn Ile Gln 340 345 350 Asp Asp Ile Thr Val Thr Val Glu Gly Ile Gln Glu Asn Thr Val Val 355 360 365 Lys Thr Lys Thr Met Tyr Leu Ser Asp Met Asp Trp Lys Ser Ala Ala 370 375 380 Asn Ala Val Gly Ala Thr Gly Glu Lys Asp Thr Pro Thr Lys Asp Leu 385 390 395 400 Asn His Leu Gln Gln Gln Met Lys Leu Leu Val Asn Gly Ala Glu Lys 405 410 415 Ser Phe Asp Lys Gly Ile Gly Val Gln Thr Asp Ser Ser Ile Val Tyr 420 425 430 Asp Leu Glu Asp Lys Gly Tyr Thr Ser Phe His Thr Leu Ala Gly Val 435 440 445 Asp Tyr Ser Ala Met Glu Tyr Val Asp Gly Glu Gly Cys Asp Ile Gln 450 455 460 Phe Lys Val Tyr Leu Asp Asp Val Val Val Phe Asp Ser Gly Val Val 465 470 475 480 Asp Ala Ser Asp Glu Ala Gln Glu Val Asn Val Ala Ile Thr Ser Glu 485 490 495 Asn Lys Glu Leu Lys Leu Glu Ala Lys Met Val Lys Glu Pro Tyr Asn 500 505 510 Asp Trp Gly Asn Trp Ala Asp Ala Ser Phe Glu Met Ala Tyr Pro Glu 515 520 525 Pro Ser Asn Val Ala Leu Asn Lys Thr Val Thr Val Lys Lys Thr Ala 530 535 540 Asp Asn Ser Asp Ser Glu Val Asn Ser Ser Arg Pro Gly Ser Met Ala 545 550 555 560 Val Asp Gly Ile Ile Gly Pro Thr Ser Asp Ser Asn Tyr Cys Asp Phe 565 570 575 Gly Gln Asp Gly Asp Asn Thr Ser Arg Tyr Leu Gln Val Asp Leu Gly 580 585 590 Asp Val Tyr Glu Leu Thr Gln Ile Asn Met Phe Arg Tyr Trp Ala Asp 595 600 605 Gly Arg Val Tyr Asn Gly Thr Val Ile Ala Val Ser Glu Asn Ala Asp 610 615 620 Phe Ser Asn Pro Thr Phe Ile Tyr Asn Ser Asp Lys Ala Asp Lys His 625 630 635 640 Gly Leu Gly Ala Gly Ser Asp Asp Thr Tyr Gly Glu Thr Gln Ser Gly 645 650 655 Lys Leu Phe Glu Val Pro Ala Gly Thr Met Gly Gln Tyr Val Arg Val 660 665 670 Tyr Met Ala Gly Ser Asn Lys Gly Thr Thr Asn His Ile Ala Glu Leu 675 680 685 Gln Val Met Gly Tyr Asn Phe Asn Thr Glu Pro Lys Pro Tyr Glu Ala 690 695 700 Asn Ala Phe Glu Asn Ala Glu Val Tyr Leu Asp Met Pro Thr His Phe 705 710 715 720 Gln Asp Leu Asp Ser Asn Lys Asn Asp Asp Gly Ser Leu Lys His Ile 725 730 735 Gly Gly Gln Val Thr His Pro Asp Ile Gln Val Phe Asp Gln Pro Trp 740 745 750 Asn Gly Tyr Lys Tyr Trp Met Ile Tyr Thr Pro Asn Thr Met Ile Thr 755 760 765 Ser Gln Tyr Glu Asn Pro Tyr Ile Val Ala Ser Glu Asp Gly Gln Thr 770 775 780 Trp Val Glu Pro Glu Gly Ile Ser Asn Pro Ile Glu Pro Glu Pro Pro 785 790 795 800 Ser Thr Arg Phe His Asn Cys Asp Ala Asp Leu Leu Tyr Asp Ser Val 805 810 815 Asn Asp Arg Leu Leu Ala Tyr Trp Asn Trp Ala Asp Asp Gly Gly Gly 820 825 830 Ile Asp Asp Glu Leu Lys Asp Gln Asn Cys Gln Ile Arg Leu Arg Ile 835 840 845 Ser Tyr Asp Gly Ile Asn Trp Gly Val Pro Tyr Asp Lys Asp Gly Asn 850 855 860 Ile Ala Thr Thr Ala Asp Thr Val Val Arg Met Glu Thr Gly Asp Lys 865 870 875 880 Asp Phe Ile Pro Ala Ile Ser Glu Lys Asp Arg Tyr Gly Met Leu Ser 885 890 895 Pro Thr Phe Thr Tyr Asp Asp Phe Arg Gly Ile Tyr Thr Met Trp Ala 900 905 910 Gln Asn Ser Gly Asp Ala Gly Tyr Asn Gln Ser Gly Lys Phe Ile Glu 915 920 925 Met Arg Trp Ser Glu Asp Gly Ile Asn Trp Ser Glu Pro Gln Lys Val 930 935 940 Asn Asn Phe Leu Gly Lys Asp Glu Asn Gly Arg Gln Leu Trp Pro Trp 945 950 955 960 His Gln Asp Ile Gln Tyr Ile Pro Glu Leu Gln Glu Tyr Trp Gly Leu 965 970 975 Ser Gln Cys Phe Ser Thr Ser Asn Pro Asp Gly Ser Val Leu Tyr Leu 980 985 990 Thr Lys Ser Arg Asp Gly Val Asn Trp Glu Gln Ala Gly Thr Gln Pro 995 1000 1005 Val Leu Arg Ala Gly Lys Ser Gly Thr Trp Asp Asp Phe Gln Ile 1010 1015 1020 Tyr Arg Ser Thr Phe Tyr Tyr Asp Asn Gln Ser Asp Ser Pro Thr 1025 1030 1035 Gly Gly Lys Phe Arg Ile Trp Tyr Ser Ala Leu Gln Ala Asn Thr 1040 1045 1050 Ser Gly Lys Thr Val Leu Ala Pro Asp Gly Thr Val Ser Leu Gln 1055 1060 1065 Val Gly Ser Gln Asp Thr Arg Ile Trp Arg Ile Gly Tyr Thr Glu 1070 1075 1080 Asn Asp Tyr Met Glu Val Met Lys Ala Leu Thr Gln Asn Lys Asn 1085 1090 1095 Tyr Glu Glu Pro Glu Leu Val Asp Ala Val Ser Leu Asn Leu Ser 1100 1105 1110 Met Asp Lys Thr Ser Ile Ser Val Gly Glu Glu Ala Thr Val Ser 1115 1120 1125 Thr Ala Phe Val Pro Glu Asn Ala Thr Asp Arg Ile Val Lys Tyr 1130 1135 1140 Thr Ser Gln Asp Pro Glu Ile Ala Val Ile Asp Pro Thr Gly Ile 1145 1150 1155 Val Thr Gly Val Lys Asp Gly Thr Thr Thr Ile Val Ala Glu Thr 1160 1165 1170 Lys Ser Gly Ala Lys Gly Glu Leu Ser Val Thr Val Gly Glu Leu 1175 1180 1185 Gln Arg Gly Glu Ile Arg Phe Glu Val Ser Asn Asp His Pro Met 1190 1195 1200 Tyr Leu Glu Asn Tyr Tyr Trp Ser Asp Asp Ala Pro Lys Lys Asp 1205 1210 1215 Gly Leu Asp Ala Asn Lys Asn Tyr Tyr Gly Asp Glu Arg Val Asp 1220 1225 1230 Ser Pro Val Met Leu Tyr Asn Thr Val Pro Glu Glu Leu Lys Asp 1235 1240 1245 Asn Thr Val Ile Leu Leu Ile Ala Glu Arg Ser Leu Asn Ser Thr 1250 1255 1260 Asp Ala Val Arg Asp Trp Ile Lys Lys Asn Val Glu Leu Cys Asn 1265 1270 1275 Glu Asn Lys Ile Pro Cys Ala Val Gln Ile Ala Asn Gly Glu Thr 1280 1285 1290 Asn Val Asn Thr Thr Ile Pro Leu Ser Phe Trp Asn Glu Leu Ala 1295 1300 1305 Thr Asn Asn Glu Tyr Leu Val Gly Phe Asn Ala Ala Glu Met Tyr 1310 1315 1320 Asn Arg Phe Ala Gly Asp Asn Arg Ser Tyr Val Met Asp Met Ile 1325 1330 1335 Arg Leu Gly Val Ser His Gly Val Cys Met Met Trp Thr Asp Thr 1340 1345 1350 Asn Ile Phe Gly Thr Asn Gly Val Leu Tyr Asp Trp Leu Thr Gln 1355 1360 1365 Asp Glu Lys Leu Ser Gly Leu Met Arg Glu Tyr Lys Glu Tyr Ile 1370 1375 1380 Ser Leu Met Thr Lys Glu Ser Tyr Gly Ser Glu Ala Ala Asn Thr 1385 1390 1395 Asp Ala Leu Phe Lys Gly Leu Trp Met Thr Asp Tyr Cys Glu Asn 1400 1405 1410 Trp Gly Ile Ala Ser Asp Trp Trp His Trp Gln Leu Asp Ser Asn 1415 1420 1425 Gly Ala Leu Phe Asp Ala Gly Ser Gly Gly Asp Ala Trp Lys Gln 1430 1435 1440 Cys Leu Thr Trp Pro Glu Asn Met Tyr Thr Gln Asp Val Val Arg 1445 1450 1455 Ala Val Ser Gln Gly Ala Thr Cys Phe Lys Ser Glu Ala Gln Trp 1460 1465 1470 Tyr Ser Asn Ala Thr Lys Gly Met Arg Thr Pro Thr Tyr Gln Tyr 1475 1480 1485 Ser Met Ile Pro Phe Leu Glu Lys Leu Val Ser Lys Glu Val Lys 1490 1495 1500 Ile Pro Thr Lys Glu Glu Met Leu Glu Arg Thr Lys Ala Ile Val 1505 1510 1515 Val Gly Ala Glu Asn Trp Asn Asn Phe Asn Tyr Asn Thr Thr Tyr 1520 1525 1530 Ser Asn Leu Tyr Pro Ser Thr Gly Gln Tyr Gly Ile Val Pro Tyr 1535 1540 1545 Val Pro Ser Asn Cys Pro Glu Glu Glu Leu Ala Gly Tyr Asp Leu 1550 1555 1560 Val Val Arg Glu Asn Leu Gly Lys Ala Gly Leu Lys Ser Ala Leu 1565 1570 1575 Asp Thr Val Tyr Pro Val Gln Lys Ser Glu Gly Thr Ala Tyr Cys 1580 1585 1590 Glu Thr Phe Gly Asp Thr Trp Tyr Trp Met Asn Ser Ser Glu Asp 1595 1600 1605 Lys Asn Val Ser Gln Tyr Thr Glu Phe Thr Thr Ala Ile Asn Gly 1610 1615 1620 Ala Glu Ser Val Lys Ile Ala Gly Glu Pro His Val Phe Gly Ile 1625 1630 1635 Ile Lys Glu Asn Pro Gly Ser Leu Asn Val Tyr Leu Ser Asn Tyr 1640 1645 1650 Arg Leu Asp Lys Thr Glu Leu Trp Asp Gly Thr Ile Pro Gly Gly 1655 1660 1665 Leu Ser Asp Gln Gly Cys Tyr Asn Tyr Val Trp Gln Met Cys Glu 1670 1675 1680 Arg Met Lys Asn Gly Thr Gly Leu Asp Thr Gln Leu Arg Asp Thr 1685 1690 1695 Val Ile Thr Val Lys Asn Ala Val Glu Pro Lys Val Asn Phe Val 1700 1705 1710 Thr Glu Ser Pro Ala Asp Arg Ser Phe Ala Glu Asp Asn Tyr Val 1715 1720 1725 Arg Pro Tyr Lys Tyr Thr Val Ala Gln Lys Glu Gly Thr Thr Asp 1730 1735 1740 Glu Trp Val Ile Thr Val Ser His Asn Gly Ile Val Glu Phe Asn 1745 1750 1755 Ile Val Thr Gly Asp Glu Lys Val Pro Ala Thr Ser Val Glu Leu 1760 1765 1770 Ser Thr Asp Lys Val Asp Val Ile Arg Asn Arg Thr Ala Val Val 1775 1780 1785 Lys Ala Thr Val Leu Pro Gln Asn Ala Gly Asn Lys Gln Leu Thr 1790 1795 1800 Trp Thr Ile Ala Asp Pro Glu Ile Ala Ser Val Asp Asn Lys Gly 1805 1810 1815 Thr Val Thr Gly Leu Lys Glu Gly Lys Thr Val Leu Arg Ala Ala 1820 1825 1830 Ile Ser Gly Ser Val Tyr Lys Glu Cys Glu Val Asn Val Ile Asp 1835 1840 1845 Arg Lys Val Thr Glu Val Asn Leu Asn Lys Thr Glu Leu Ser Leu 1850 1855 1860 Ser Ala Gly Asp Ser Ala Lys Leu Glu Ala Ser Ile Ala Pro Glu 1865 1870 1875 Asp Pro Ser Asp Ser Ser Ile Thr Trp Thr Ser Thr Asn Glu Asn 1880 1885 1890 Val Ala Thr Val Ala Ser Asn Gly Thr Val Thr Ala His Lys Ala 1895 1900 1905 Gly Val Ala Gln Ile Ile Ala Gln Ser Ala Tyr Gln Ala Lys Gly 1910 1915 1920 Ile Ala Thr Val Thr Val Asn Tyr Ala Ala Ser Val Lys Leu Asp 1925 1930 1935 Arg Thr Gly Met Thr Ala Thr Ala Asn Ser Glu Gln Ser Lys Ser 1940 1945 1950 Gly Gly Glu Gly Pro Ala Ser Asn Val Leu Asp Gly Lys Gln Asp 1955 1960 1965 Thr Met Trp His Thr Ser Trp Thr Asp Lys Pro Glu Leu His Pro 1970 1975 1980 His Trp Ile Lys Ile Asp Leu Asn Gly Thr Lys Thr Ile Asn Lys 1985 1990 1995 Phe Ala Tyr Thr Pro Arg Thr Gly Ala Ser Asn Gly Thr Ile Tyr 2000 2005 2010 Asn Tyr Val Leu Ile Ile Thr Asp Leu Glu Gly Asn Glu Lys Gln 2015 2020 2025 Val Ala Lys Gly Val Trp Ala Ala Asn Ala Asp Val Lys Tyr Ala 2030 2035 2040 Glu Phe Asp Ala Val Glu Ala Thr Ala Ile Lys Leu Gln Val Asp 2045 2050 2055 Gly Asn Asp Asp Lys Ala Ser Lys Gly Gly Tyr Gly Ser Ala Ala 2060 2065 2070 Glu Ile Asn Ile Phe Glu Val Ala Gln Lys Pro Ser Ala Asn Glu 2075 2080 2085 Leu Ala Glu Asn Ile Lys Val Ile Ala Pro Val Lys Ala Glu Asp 2090 2095 2100 Thr Lys Val Ser Ile Pro Val Ile Thr Gly Phe Asp Ile Val Ile 2105 2110 2115 Ser Asn Ser Ser Asn Pro Asp Val Ile Gly Ile Asp Gly Ser Ile 2120 2125 2130 Thr Arg Pro Glu Asn Asp Thr Val Val Thr Leu Thr Leu Lys Val 2135 2140 2145 Lys Glu Thr Asp Ala Lys Ser Val Lys Ala Ala Gly Thr Glu Ala 2150 2155 2160 Thr Thr Asn Val Asp Val Leu Val Thr Gly Thr Lys Thr Ser Asp 2165 2170 2175 Val Glu Ala Glu Ser Val Thr Leu Asp Gln Thr Ser Ala Asp Leu 2180 2185 2190 Thr Val Gly Gly Glu Leu Leu Leu Asn Ala Val Val Lys Pro Asp 2195 2200 2205 Ile Ala Thr Asn Lys Ala Val Thr Trp Ser Ser Asp Lys Pro Gly 2210 2215 2220 Thr Ala Thr Val Glu Asn Gly Arg Val Lys Ala Leu Ala Ala Gly 2225 2230 2235 Glu Ala Arg Ile Thr Ala Ala Thr Ala Asn Gly Lys Thr Ala Asp 2240 2245 2250 Cys Val Ile Asn Val Lys Glu Lys Glu Glu Pro Glu Val Ile Leu 2255 2260 2265 Pro Ala Glu Val Arg Leu Asn Ile Pro Ser Ala Glu Phe Thr Val 2270 2275 2280 Gly Asp Gln Ile Gln Leu Thr Ala Ser Val Leu Pro Ala Asn Ala 2285 2290 2295 Ala Asp Lys Thr Ile Thr Trp Lys Ser Asp Lys Pro Glu Val Ala 2300 2305 2310 Thr Val Ala Asn Gly Trp Val Lys Gly Ile Ala Ala Gly Thr Ala 2315 2320 2325 Lys Ile Thr Ala Thr Ser Val Asn Gly Lys Thr Ala Val Cys Val 2330 2335 2340 Ile Thr Val Lys Ala Gln Pro Gln Asn Leu Pro Thr Gly Val Ser 2345 2350 2355 Leu Asn Lys Lys Thr Ala Ser Val Lys Leu Asn Lys Thr Leu Thr 2360 2365 2370 Leu Ser Ala Val Val Gln Pro Ser Asn Ala Asp Asn Lys Thr Val 2375 2380 2385 Lys Trp Thr Ser Asp Asn Thr Tyr Val Ala Thr Val Glu Asn Gly 2390 2395 2400 Val Val Lys Ala Val Asn Ala Gly Thr Ala Arg Ile Thr Ala Ala 2405 2410 2415 Thr Val Asn Gly His Lys Ala Thr Cys Thr Ile Thr Val Pro Gly 2420 2425 2430 Thr Lys Ile Ser Lys Ala Lys Val Ser Leu Ala Ser Ser Lys Thr 2435 2440 2445 His Thr Gly Lys Ala Leu Lys Pro Ser Val Lys Val Thr Tyr Gly 2450 2455 2460 Lys Asn Thr Leu Lys Lys Asn Thr Asp Tyr Thr Val Ser Tyr Lys 2465 2470 2475 Asn Asn Ile Asn Pro Gly Thr Ala Ser Val Thr Ile Thr Gly Lys 2480 2485 2490 Gly Lys Tyr Tyr Gly Thr Ile Asn Lys Thr Phe Ala Ile Lys Ala 2495 2500 2505 Ala Glu Gly Lys Thr Tyr Thr Val Gly Lys Gly Lys Tyr Lys Val 2510 2515 2520 Thr Asp Ala Ser Ala Lys Asn Lys Thr Val Thr Phe Met Ala Pro 2525 2530 2535 Val Light Light Thr Tyr Ser Ser Phe Ser Val Pro Ser Light Val Light 2540 2545 2550 Ile Gly Asn Asp Thr Tyr Lys Val Thr Ala Val Ala Lys Asn Ala 2555 2560 2565 Phe Lys Lys Asn Thr Lys Leu Thr Lys Leu Thr Ile Gly Ser Asn 2570 2575 2580 Val Lys Thr Ile Gly Ser Tyr Ala Phe Tyr Gly Ala Ser Gln Leu 2585 ​​2590 2595 Lys Thr Leu Thr Leu Lys Thr Thr Gly Leu Asn Ser Val Gly Lys 2600 2605 2610 Asn Ala Phe Lys Lys Thr Asn Ala Lys Leu Thr Val Lys Val Pro 2615 2620 2625 Lys Ser Lys Leu Ala Asp Tyr Lys Lys Leu Leu Lys Gly Lys Gly 2630 2635 2640 Leu Ser Gly Lys Ala Lys Ile Gln Lys 2645 2650 <210> 28 <211> 2535 <212> DNA <213> Robinsoniella peoriensis <400> 28 tcaccattga gcgctgcggc agaaagtggc acaggaacca gattagtgaa agggcaaacg 60 gggtatttga cagaggaaca ggctatccgg aaccaggagc agacaaccga agaaagggag 120 cagaagttaa ccgggggaaga gacagcagag gttttgatgg aaggtacaaa agacagcggg 180 attgtacaga cagaaagt acagacaaaa gaaatgcaga cagaagtgc cagacagaa 240 gaagtacaga cagagaaat cagacagaa gatgcgcaga caaaaagaagt acagacagaa 300 gaaatgcaga cagaagatgc cagacagaa gaagtacaca caaaaagaa accggcagaa 360 gaacacaca tgaaagaaat acagacgcaa gggacaaaga aagcgtcaga taggaacgga 420 aaggcaaggg taactgaaat tctggaagat gcccaggatc cagcaaaccg gattgtgtat 480 ctgtcagacc tgcaatggaa gtcagaaaat catacagtag atagcgagct gcctaccaga 540 aaggataagt cctttggcgg cggaaaaatt acgctaaaaag tggatggaac ggtaacagaa 600 tttgataagg ggattggaac agacagat tccaccattg tgtacgatct ggagggaaag 660 720. ggatatacaa agtttgaaac ttacgtgggt gccagaaga aaacattccg ggggaagtct gcgacgtaaa attcagggtg aaaattgatg acaagattgt atcagaaacc 780 ggtgtactgg atccgctttc gaatgcggtt aagattctg ttaacatacc cgatacagcc 840 aaaactttaa cattatacgc ggataaagta acggaaactt ggtctgatca cgccaattgg gcagatgca aattttatca ggcactgccg gaacccgaa atgttgcatt caaaaaaacg gtagtgacac gaaagacatc agataattcg gaggctcctg ttaatccgga ttcagcagtt aacagttcta aggctgttga cggtgttatt gacagctcca gttattttga ttttggagat caggcaata gcggagccgt aagggagtca ctctatatgg aggtagattt aaaagggagc tatttactgt ccgatataca actgtggaga tactggaaag atggcagaac ttatgcagct actgcaattg tagtagctga ggatgagaac tttgaaaatg cagcagttat ctataactcg gatacgacgg gagaaataca tcacctggga gcaggaagtg atatgctcta tgcagaaaca gaaagtggca agacatttcc ggtaccgga aatacaaaag caaggtatat cagagtttat acatatggtg ttaatgggac atcaggcgta acaaatcaca ttgtagaatt aaaggtgaat 1440 gcttacgtat ttggagatga aatcttaccg gaaaagccgg atgacagcaa gattttccca 1500 aatgcagtta atccgctgaa gctacaggga ccgggcacga atgatcaggt aacccacccg 1560 gatgttacgg tgtttgatga gccgtggaat gggtataaat actggatggc atatacaccg 1620 aataaaccgg gaagttccta ttttgaaaat ccctgtatag ctgcatccaa cgatggcgta 1680 aactgggagt ttcctgccca gaaccctgta cagccgcgct atgacagtga aatagaaaat 1740 caaaatgaac ataactgtga taccgatatt gtatatgacc cggtaaatga ccggttgatt 1800 atgtactggg aatgggcaca ggatgaggcg gttaatggta aaacacatcg ttctgaaatc 1860 agataccgtg tttcttatga tgggattaac tggggagtgg aagacaaaac tggtgttttg 1920 atgactggac caacggatca tggctgcgcc attgccacag aaggcgaaag atattcagac 1980 cttctccaa ccgtagtata tgataaaaca gaaaaaatct acaaaatgtg ggcaaatgat 2040 gccggagatg taggatatga aaacaaacag aataacaaag tatggtatcg gacatcccaa 2100 gacgggatca gcaattggtc ggataagact tacgtggaga atttcttgg agtaaatgaa 2160 gacgggctgc agatgtatcc atggcaccag gatatccagt gggtagagga atttcaggaa 2220 tattgggcac ttcagcaggc atttccggca ggaagcggac cggataattc ttccctgcgt 2280 ttctcgaaat ccaaagatgg tcttcattgg gagccggtat ctgaaaaagc tttaattaca 2340 gtaggggcac ccgggacctg ggatgcagga cagatatacc gttctacttt ctggtatgag 2400 ccaggtgggg caaaaggaaa cggaacattc catatctggt atgctgcatt ggcggaaggc 2460 cagtctcact gggatatagg atatacatct gcaaactatg cagatgccat gtacaaatta 2520 acgggaagca gatga 2535 <210> 29 <211> 864 <212> PRT <213> Robinsoniella peoriensis <400> 29 Met Gly Ser Ser His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Ser Pro Leu Ser Ala Ala Ala Glu Ser Gly Thr Gly 20 25 30 Thr Arg Leu Val Lys Gly Gln Thr Gly Tyr Leu Thr Glu Glu Gln Ala 35 40 45 Ile Arg Asn Gln Glu Gln Thr Thr Glu Glu Arg Glu Gln Lys Leu Thr 50 55 60 Gly Glu Glu Thr Ala Glu Val Leu Met Glu Gly Thr Lys Asp Ser Gly 65 70 75 80 Ile Val Gln Thr Glu Glu Val Gln Thr Lys Glu Met Gln Thr Glu Asp 85 90 95 Ala Gln Thr Glu Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala 100 105 110 Gln Thr Lys Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln 115 120 125 Thr Glu Glu Val Gln Thr Lys Glu Glu Pro Ala Glu Glu Thr His Met 130 135 140 Lys Glu Ile Gln Thr Gln Gly Thr Lys Lys Ala Ser Asp Arg Asn Gly 145 150 155 160 Lys Ala Arg Val Thr Glu Ile Leu Glu Asp Ala Gln Asp Pro Ala Asn 165 170 175 Arg Ile Val Tyr Leu Ser Asp Leu Gln Trp Lys Ser Glu Asn His Thr 180 185 190 Val Asp Ser Glu Leu Pro Thr Arg Lys Asp Lys Ser Phe Gly Gly Gly 195 200 205 Lys Ile Thr Leu Lys Val Asp Gly Thr Val Thr Glu Phe Asp Lys Gly 210 215 220 Ile Gly Thr Gln Thr Asp Ser Thr Ile Val Tyr Asp Leu Glu Gly Lys 225 230 235 240 Gly Tyr Thr Lys Phe Glu Thr Tyr Val Gly Val Asp Tyr Ser Gln Lys 245 250 255 Glu Asn Ile Pro Gly Glu Val Cys Asp Val Lys Phe Arg Val Lys Ile 260 265 270 Asp Asp Lys Ile Val Ser Glu Thr Gly Val Leu Asp Pro Leu Ser Asn 275 280 285 Ala Val Lys Ile Ser Val Asn Ile Pro Asp Thr Ala Lys Thr Leu Thr 290 295 300 Leu Tyr Ala Asp Lys Val Thr Glu Thr Trp Ser Asp His Ala Asn Trp 305 310 315 320 Ala Asp Ala Lys Phe Tyr Gln Ala Leu Pro Glu Pro Glu Asn Val Ala 325 330 335 Phe Lys Lys Thr Val Val Thr Arg Lys Thr Ser Asp Asn Ser Glu Ala 340 345 350 Pro Val Asn Pro Asp Ser Ala Val Asn Ser Ser Lys Ala Val Asp Gly 355 360 365 Val Ile Asp Ser Ser Ser Tyr Phe Asp Phe Gly Asp Gln Ala Asn Ser 370 375 380 Gly Ala Val Arg Glu Ser Leu Tyr Met Glu Val Asp Leu Lys Gly Ser 385 390 395 400 Tyr Leu Leu Ser Asp Ile Gln Leu Trp Arg Tyr Trp Lys Asp Gly Arg 405 410 415 Thr Tyr Ala Ala Thr Ala Ile Val Val Ala Glu Asp Glu Asn Phe Glu 420 425 430 Asn Ala Ala Val Ile Tyr Asn Ser Asp Thr Thr Gly Glu Ile His His 435 440 445 Leu Gly Ala Gly Ser Asp Met Leu Tyr Ala Glu Thr Glu Ser Gly Lys 450 455 460 Thr Phe Pro Val Pro Glu Asn Thr Lys Ala Arg Tyr Ile Arg Val Tyr 465 470 475 480 Thr Tyr Gly Val Asn Gly Thr Ser Gly Val Thr Asn His Ile Val Glu 485 490 495 Leu Lys Val Asn Ala Tyr Val Phe Gly Asp Glu Ile Leu Pro Glu Lys 500 505 510 Pro Asp Asp Ser Lys Ile Phe Pro Asn Ala Val Asn Pro Leu Lys Leu 515 520 525 Gln Gly Pro Gly Thr Asn Asp Gln Val Thr His Pro Asp Val Thr Val 530 535 540 Phe Asp Glu Pro Trp Asn Gly Tyr Lys Tyr Trp Met Ala Tyr Thr Pro 545 550 555 560 Asn Lys Pro Gly Ser Ser Tyr Phe Glu Asn Pro Cys Ile Ala Ala Ser 565 570 575 Asn Asp Gly Val Asn Trp Glu Phe Pro Ala Gln Asn Pro Val Gln Pro 580 585 590 Arg Tyr Asp Ser Glu Ile Glu Asn Gln Asn Glu His Asn Cys Asp Thr 595 600 605 Asp Ile Val Tyr Asp Pro Val Asn Asp Arg Leu Ile Met Tyr Trp Glu 610 615 620 Trp Ala Gln Asp Glu Ala Val Asn Gly Lys Thr His Arg Ser Glu Ile 625 630 635 640 Arg Tyr Arg Val Ser Tyr Asp Gly Ile Asn Trp Gly Val Glu Asp Lys 645 650 655 Thr Gly Val Leu Met Thr Gly Pro Thr Asp His Gly Cys Ala Ile Ala 660 665 670 Thr Glu Gly Glu Arg Tyr Ser Asp Leu Ser Pro Thr Val Val Tyr Asp 675 680 685 Lys Thr Glu Lys Ile Tyr Lys Met Trp Ala Asn Asp Ala Gly Asp Val 690 695 700 Gly Tyr Glu Asn Lys Gln Asn Asn Lys Val Trp Tyr Arg Thr Ser Gln 705 710 715 720 Asp Gly Ile Ser Asn Trp Ser Asp Lys Thr Tyr Val Glu Asn Phe Leu 725 730 735 Gly Val Asn Glu Asp Gly Leu Gln Met Tyr Pro Trp His Gln Asp Ile 740 745 750 Gln Trp Val Glu Glu Phe Gln Glu Tyr Trp Ala Leu Gln Gln Ala Phe 755 760 765 Pro Ala Gly Ser Gly Pro Asp Asn Ser Ser Leu Arg Phe Ser Lys Ser 770 775 780 Lys Asp Gly Leu His Trp Glu Pro Val Ser Glu Lys Ala Leu Ile Thr 785 790 795 800 Val Gly Ala Pro Gly Thr Trp Asp Ala Gly Gln Ile Tyr Arg Ser Thr 805 810 815 Phe Trp Tyr Glu Pro Gly Gly Ala Lys Gly Asn Gly Thr Phe His Ile 820 825 830 Trp Tyr Ala Ala Leu Ala Glu Gly Gln Ser His Trp Asp Ile Gly Tyr 835 840 845 Thr Ser Ala Asn Tyr Ala Asp Ala Met Tyr Lys Leu Thr Gly Ser Arg 850 855 860 <210> 30 <211> 3246 <212> DNA <213> Robinsoniella peoriensis <400> 30 gctgagactg caacagaaga aaatgcggcg ctggaaaaaa cagttacatt gcataagagc 60 gatggaacag aactgccgga ggattatcga aatccccaaa gaccagctac catggcggta 120 gatggtatta ttgacgatac aggagagtac aactattgcg atttcggtaa agacggtgat 180 aaagcagccc tgtatatgca ggtggacctt ggaggtctgt atgatttaag cagagtcaat 240 atgtggagat actggaaaga cagcagaact tacgatgcaa cagtaattac cacatctgag 300 agcggcgatt tcacagatga agcagtcata tataattcag acaggtcgaa tgtacatgga 360 tttggggcag gaggagatga acgctacgca gagactgcct cccggacatga attcccagta 420 ccggacggta caaaggcaca ggcagtacgc gtatatgtat ttggcagcca aaacggtact 480 acaaaccaca tcaatgaatt gcaggtctgg ggaactcccc atacagagaa tccggatgta 540 aattcttatc aggtgacaat tccacaggga aatggatatc aggtaatacc ttatgaaaat 600 gacccgacga cagtggaaga aggcggttct ttccgttttc aggtactgat tgactccgat 660 aatggttaca gcgcaaccag tgcggtaaaa gcaaatggag taagtctgga ggcagttgac 720 agtgtttata ccattgagaa cattactgaa gatcaggtaa tcaccattga aggcgtacat 780 aaagcacagt atgaagtgaa attcccggaa aatccacagg gatacagtgt tgagattcag 840 aatgaaggaa gtacaacggt agactataat ggttctgtca gttttaagct tattatagac 900 gaagcttata atgaatccgt accggttgta aaagcaaacg gcggtgcagc tttgggaaaa 960 gatgagctcg gtgtatatac aattgcaaat atccaggacg atattacggt tacagttgag 1020 ggtatccagg aaaataccgt agtaaagaca aaaacaatgt acttgtctga tatggattgg 1080 aagagtgctg caaatgcagt aggtgcaaca ggagaaaaag acactccaac aaaggacctg 1140 atcatttac agcagcagat gaattattg gtaaacggag cagagaagtc tttgataaa 1200 ggaattggag tcagacgga ttctctatc gtttatgatc tggaagacaa aggctacact 1260 tctttccaca ccctggcagg cgttgatt tcagcaatgg atatgtaga cggagaggc 1320 tgtgatatcc agtttaaagt atatctggat gatgtcgtag tatttgacag cggagtagtt 1380 catcatctg atgaggctca ggagttaat gttgctataa catcagagaa window 1440 aaactggaag ctaaaatggt taagagcct tataatgact ggggaactg ggcagatgcc 1500 agctttgaaa tggcttatcc cgaaccgtct aatgtggctt taataaac agttaccgtt 1560 aagaaaacag cggatactc agactctgaa gtaaattcca gcagaccggg atcaatggct 1620 gtagatggaa tcattggacc tacatcagat tctaactatt gtgattttgg acaggatgggg 1680 gataactt cccgttatct gcaggtagat ttagggatg ttatgact taccagatt 1740 atatgttta gatactggggc agatggcaga gtatatatatg gtactgtaat tgcagttttcc 1800 gaaaacgcag actttagtaa tccaactttt attaatatt cagataaagc agacaacac 1860 ggacttggcg caggcagtga tgacacttat ggagaaccc agagtggaaa attattcga 1920 gttccggcgg gaaccatggg acagtatgtc cgtgtgtata tggctggttc cacaaggt 1980 acaacgaacc attcgctga attacaggta atgggttata atttcaatac agaaccaaaa 2040 ccatatgaag caaatgcatt tgaaatgca gaagtttatt tagatatgcc aactcattc 2100 caggatctgg attccataa aaacgacgat ggaagcttaa agcacattgg cggacaggtg 2160 acacatcctg atatccaggt atttgaccaa ccgtggaacg gttataata ctggatgatt 2220 tacacaccaa atacaatgat cactcccag tatgaaaatc catatatcgt agcatctgaa 2280 catggacaga catgggtaga accggaaggg atttccaatc cattgaacc agaaccgcca 2340 tcaccagat ttcatactg tgatgcagat ctgttatacg actctgtcaa tgaccgttta 2400 cttgcttact ggaactgggc agatgacggc ggcggaattg atgacgaatt aaagatcag 2460 aactgtcaga ttcgtctgag aatttcttat gatggaatta actgggagt tccttacgac 2520 aaagacggca atttgccac aacagctgat actgtagtaa gatggaac aggataag 2580 gatttcattc ctgcaatcag cgaaaaagac cgttatgta tgctttcccc aacatttacc 2640 tatgacgatt tccgcggcat atacaatg tgggcacaaa actcgggtga tgcgggatac 2700 aaccagtccg gaagttcat cgaatgaga tggtctgagg atggataaa ctggtctgaa 2760 ccacaaaag tgaataattt ccttggaaaa gatgagaatg gcagacagct ttggccatgg 2820 catcaggata ttcagtatat ccctgagcta caggaatatt ggggactgtc ccagtgtttc 2880 tctacatcta atcccgatgg atccgtatta tacctgacca agtccagaga tggtgtcaac 2940 tgggagcagg caggacaca gccggtatta agggcaggaaatcaggtac ctgggatgat 3000 ttccagattt accgttctac cttctattat gataatcagt cagacagccc tactggtggg 3060 aaatttagaa tctggtacag tgcactgcag gcaatactt caggcagac cgttttggct 3120 cctgatggaa cagtgtctct tcaggttgga agccaggata ccaggatctg gcgtatcggg 3180 tatacagaaa atgactacat ggaagtcatg aaagctctga cccagaataa aaactacat 3240 gatga 3246 <210> 31 <211> 1101 <212> PRT <213> Robinsoniella peoriensis <400> 31 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Ala Glu Thr Ala Thr Glu Glu Asn Ala Ala Leu Glu 20 25 30 Lys Thr Val Thr Leu His Lys Ser Asp Gly Thr Glu Leu Pro Glu Asp 35 40 45 Tyr Arg Asn Pro Gln Arg Pro Ala Thr Met Ala Val Asp Gly Ile Ile 50 55 60 Asp Asp Thr Gly Glu Tyr Asn Tyr Cys Asp Phe Gly Lys Asp Gly Asp 65 70 75 80 Lys Ala Ala Leu Tyr Met Gln Val Asp Leu Gly Gly Leu Tyr Asp Leu 85 90 95 Ser Arg Val Asn Met Trp Arg Tyr Trp Lys Asp Ser Arg Thr Tyr Asp 100 105 110 Ala Thr Val Ile Thr Thr Ser Glu Ser Gly Asp Phe Thr Asp Glu Ala 115 120 125 Val Ile Tyr Asn Ser Asp Arg Ser Asn Val His Gly Phe Gly Ala Gly 130 135 140 Gly Asp Glu Arg Tyr Ala Glu Thr Ala Ser Gly His Glu Phe Pro Val 145 150 155 160 Pro Asp Gly Thr Lys Ala Gln Ala Val Arg Val Tyr Val Phe Gly Ser 165 170 175 Gln Asn Gly Thr Thr Asn His Ile Asn Glu Leu Gln Val Trp Gly Thr 180 185 190 Pro His Thr Glu Asn Pro Asp Val Asn Ser Tyr Gln Val Thr Ile Pro 195 200 205 Gln Gly Asn Gly Tyr Gln Val Ile Pro Tyr Glu Asn Asp Pro Thr Thr 210 215 220 Val Glu Glu Gly Gly Ser Phe Arg Phe Gln Val Leu Ile Asp Ser Asp 225 230 235 240 Asn Gly Tyr Ser Ala Thr Ser Ala Val Lys Ala Asn Gly Val Ser Leu 245 250 255 Glu Ala Val Asp Ser Val Tyr Thr Ile Glu Asn Ile Thr Glu Asp Gln 260 265 270 Val Ile Thr Ile Glu Gly Val His Lys Ala Gln Tyr Glu Val Lys Phe 275 280 285 Pro Glu Asn Pro Gln Gly Tyr Ser Val Glu Ile Gln Asn Glu Gly Ser 290 295 300 Thr Thr Val Asp Tyr Asn Gly Ser Val Ser Phe Lys Leu Ile Ile Asp 305 310 315 320 Glu Ala Tyr Asn Glu Ser Val Pro Val Val Lys Ala Asn Gly Gly Ala 325 330 335 Ala Leu Gly Lys Asp Glu Leu Gly Val Tyr Thr Ile Ala Asn Ile Gln 340 345 350 Asp Asp Ile Thr Val Thr Val Glu Gly Ile Gln Glu Asn Thr Val Val 355 360 365 Lys Thr Lys Thr Met Tyr Leu Ser Asp Met Asp Trp Lys Ser Ala Ala 370 375 380 Asn Ala Val Gly Ala Thr Gly Glu Lys Asp Thr Pro Thr Lys Asp Leu 385 390 395 400 Asn His Leu Gln Gln Gln Met Lys Leu Leu Val Asn Gly Ala Glu Lys 405 410 415 Ser Phe Asp Lys Gly Ile Gly Val Gln Thr Asp Ser Ser Ile Val Tyr 420 425 430 Asp Leu Glu Asp Lys Gly Tyr Thr Ser Phe His Thr Leu Ala Gly Val 435 440 445 Asp Tyr Ser Ala Met Glu Tyr Val Asp Gly Glu Gly Cys Asp Ile Gln 450 455 460 Phe Lys Val Tyr Leu Asp Asp Val Val Val Phe Asp Ser Gly Val Val 465 470 475 480 Asp Ala Ser Asp Glu Ala Gln Glu Val Asn Val Ala Ile Thr Ser Glu 485 490 495 Asn Lys Glu Leu Lys Leu Glu Ala Lys Met Val Lys Glu Pro Tyr Asn 500 505 510 Asp Trp Gly Asn Trp Ala Asp Ala Ser Phe Glu Met Ala Tyr Pro Glu 515 520 525 Pro Ser Asn Val Ala Leu Asn Lys Thr Val Thr Val Lys Lys Thr Ala 530 535 540 Asp Asn Ser Asp Ser Glu Val Asn Ser Ser Arg Pro Gly Ser Met Ala 545 550 555 560 Val Asp Gly Ile Ile Gly Pro Thr Ser Asp Ser Asn Tyr Cys Asp Phe 565 570 575 Gly Gln Asp Gly Asp Asn Thr Ser Arg Tyr Leu Gln Val Asp Leu Gly 580 585 590 Asp Val Tyr Glu Leu Thr Gln Ile Asn Met Phe Arg Tyr Trp Ala Asp 595 600 605 Gly Arg Val Tyr Asn Gly Thr Val Ile Ala Val Ser Glu Asn Ala Asp 610 615 620 Phe Ser Asn Pro Thr Phe Ile Tyr Asn Ser Asp Lys Ala Asp Lys His 625 630 635 640 Gly Leu Gly Ala Gly Ser Asp Asp Thr Tyr Gly Glu Thr Gln Ser Gly 645 650 655 Lys Leu Phe Glu Val Pro Ala Gly Thr Met Gly Gln Tyr Val Arg Val 660 665 670 Tyr Met Ala Gly Ser Asn Lys Gly Thr Thr Asn His Ile Ala Glu Leu 675 680 685 Gln Val Met Gly Tyr Asn Phe Asn Thr Glu Pro Lys Pro Tyr Glu Ala 690 695 700 Asn Ala Phe Glu Asn Ala Glu Val Tyr Leu Asp Met Pro Thr His Phe 705 710 715 720 Gln Asp Leu Asp Ser Asn Lys Asn Asp Asp Gly Ser Leu Lys His Ile 725 730 735 Gly Gly Gln Val Thr His Pro Asp Ile Gln Val Phe Asp Gln Pro Trp 740 745 750 Asn Gly Tyr Lys Tyr Trp Met Ile Tyr Thr Pro Asn Thr Met Ile Thr 755 760 765 Ser Gln Tyr Glu Asn Pro Tyr Ile Val Ala Ser Glu Asp Gly Gln Thr 770 775 780 Trp Val Glu Pro Glu Gly Ile Ser Asn Pro Ile Glu Pro Glu Pro Pro 785 790 795 800 Ser Thr Arg Phe His Asn Cys Asp Ala Asp Leu Leu Tyr Asp Ser Val 805 810 815 Asn Asp Arg Leu Leu Ala Tyr Trp Asn Trp Ala Asp Asp Gly Gly Gly 820 825 830 Ile Asp Asp Glu Leu Lys Asp Gln Asn Cys Gln Ile Arg Leu Arg Ile 835 840 845 Ser Tyr Asp Gly Ile Asn Trp Gly Val Pro Tyr Asp Lys Asp Gly Asn 850 855 860 Ile Ala Thr Thr Ala Asp Thr Val Val Arg Met Glu Thr Gly Asp Lys 865 870 875 880 Asp Phe Ile Pro Ala Ile Ser Glu Lys Asp Arg Tyr Gly Met Leu Ser 885 890 895 Pro Thr Phe Thr Tyr Asp Asp Phe Arg Gly Ile Tyr Thr Met Trp Ala 900 905 910 Gln Asn Ser Gly Asp Ala Gly Tyr Asn Gln Ser Gly Lys Phe Ile Glu 915 920 925 Met Arg Trp Ser Glu Asp Gly Ile Asn Trp Ser Glu Pro Gln Lys Val 930 935 940 Asn Asn Phe Leu Gly Lys Asp Glu Asn Gly Arg Gln Leu Trp Pro Trp 945 950 955 960 His Gln Asp Ile Gln Tyr Ile Pro Glu Leu Gln Glu Tyr Trp Gly Leu 965 970 975 Ser Gln Cys Phe Ser Thr Ser Asn Pro Asp Gly Ser Val Leu Tyr Leu 980 985 990 Thr Lys Ser Arg Asp Gly Val Asn Trp Glu Gln Ala Gly Thr Gln Pro 995 1000 1005 Val Leu Arg Ala Gly Lys Ser Gly Thr Trp Asp Asp Phe Gln Ile 1010 1015 1020 Tyr Arg Ser Thr Phe Tyr Tyr Asp Asn Gln Ser Asp Ser Pro Thr 1025 1030 1035 Gly Gly Lys Phe Arg Ile Trp Tyr Ser Ala Leu Gln Ala Asn Thr 1040 1045 1050 Ser Gly Lys Thr Val Leu Ala Pro Asp Gly Thr Val Ser Leu Gln 1055 1060 1065 Val Gly Ser Gln Asp Thr Arg Ile Trp Arg Ile Gly Tyr Thr Glu 1070 1075 1080 Asn Asp Tyr Met Glu Val Met Lys Ala Leu Thr Gln Asn Lys Asn 1085 1090 1095 Tyr Glu Glu 1100 <210> 32 <211> 528 <212> PRT <213> Clostridium tertium <400> 32 His Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val Leu 1 5 10 15 Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn Asn 20 25 30 Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr Gly 35 40 45 Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys Glu 50 55 60 Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr Val 65 70 75 80 Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala Phe 85 90 95 Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly Asn 100 105 110 Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val Tyr 115 120 125 Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr Asn 130 135 140 Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val Asn 145 150 155 160 Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr Glu 165 170 175 Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu Lys 180 185 190 Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp Leu 195 200 205 Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro Leu Gln Asn 210 215 220 Ala Glu Thr Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu Asn Gln Ser 225 230 235 240 Thr His Pro Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn Gly Tyr Lys 245 250 255 Tyr Trp Met Ile Met Thr Pro Asn Arg Thr Gly Ser Ser Val Ala Glu 260 265 270 Asn Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile Asn Trp Glu Val Pro 275 280 285 Ala Gly Val Thr Asn Pro Ile Ala Pro Met Pro Gln Val Gly His Asn 290 295 300 Cys Asp Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu Leu Trp Val 305 310 315 320 Tyr Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val Lys Leu Ile 325 330 335 Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln Val Val Val Asp 340 345 350 Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro Ser Ile Ile Phe Lys 355 360 365 Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn Thr Gly Asn Ser Gly Trp 370 375 380 Asn Asn Gln Ser Asn Lys Val Glu Leu Arg Glu Ser Ser Asp Gly Val 385 390 395 400 Asn Trp Ser Asn Pro Thr Val Val Asn Thr Leu Ala Gln Asp Gly Ser 405 410 415 Gln Ile Trp His Val Asn Val Glu Tyr Ile Pro Ser Lys Asn Glu Tyr 420 425 430 Trp Ala Ile Tyr Pro Ala Tyr Lys Asn Gly Thr Gly Ser Asp Lys Thr 435 440 445 Glu Leu Tyr Tyr Ala Lys Ser Ser Asp Gly Val Asn Trp Thr Thr Tyr 450 455 460 Lys Asn Pro Ile Leu Ser Lys Gly Thr Ser Gly Lys Trp Asp Asp Met 465 470 475 480 Glu Ile Tyr Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr Asn Met Ile 485 490 495 Lys Val Trp Tyr Gly Ala Val Ser Gln Asn Pro Gln Ile Trp Lys Ile 500 505 510 Gly Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile Glu Gly Leu Thr Gln 515 520 525 <210> 33 <211> 449 <212> PRT <213> Ruthenibacterium lactatiformans <400> 33 His Glu Glu Thr Asp Leu Leu Val Asn Gly Gly Phe Glu Thr Gly Asp 1 5 10 15 Ser Thr Gly Trp Asn Trp Phe Asn Asn Ala Val Val Asp Ser Ala Ala 20 25 30 Pro His Ser Gly Asn Tyr Cys Ala Lys Val Ala Lys Asn Ser Ser Tyr 35 40 45 Glu Gln Val Val Thr Val Ser Pro Asp Thr Lys Tyr Val Leu Thr Gly 50 55 60 Trp Ala Lys Ser Glu Gly Ser Ser Val Met Thr Leu Gly Val Lys Asn 65 70 75 80 Tyr Gly Gly Gln Glu Thr Phe Ser Ala Thr Leu Ser Ala Asp Tyr Gln 85 90 95 Gln Leu Ala Val Thr Phe Thr Thr Gly Pro Asn Ala Gln Thr Ala Thr 100 105 110 Ile Tyr Gly Tyr Arg Gln Asn Ser Gly Ser Gly Ala Gly Tyr Phe Asp 115 120 125 Asp Val Glu Leu Thr Ala Val Gln Asp Phe Ala Pro Tyr Gln Pro Leu 130 135 140 Ala Asn Ala Ile Ala Pro Gln Ala Ile Pro Thr Tyr Asp Gly Ala Asn 145 150 155 160 Gln Pro Thr His Pro Ser Val Val Lys Phe Glu Gln Pro Trp Asn Gly 165 170 175 Tyr Leu Tyr Trp Met Ala Met Thr Pro Tyr Pro Phe Asn Asp Gly Ser 180 185 190 Tyr Glu Asn Pro Ser Ile Val Ala Ser Asn Asp Gly Glu Asn Trp Ile 195 200 205 Val Pro Glu Gly Val Ser Asn Pro Leu Ala Gly Thr Pro Ser Pro Gly 210 215 220 His Asn Cys Asp Val Asp Leu Val Tyr Val Pro Ala Ser Asp Glu Leu 225 230 235 240 Arg Met Tyr Tyr Val Glu Ala Asp Asp Ile Ile Ser Ser Arg Val Lys 245 250 255 Met Ile Ser Ser Arg Asp Gly Val His Trp Ser Glu Pro Gln Val Val 260 265 270 Met Gln Asp Leu Val Arg Lys Tyr Ser Ile Leu Ser Pro Ser Ile Glu 275 280 285 Ile Leu Pro Asp Gly Thr Tyr Met Met Trp Tyr Val Asp Thr Gly Asn 290 295 300 Ala Gly Trp Asn Ser Gln Asn Asn Gln Val Lys Tyr Arg Thr Ser Ala 305 310 315 320 Asp Gly Ile Lys Trp Ser Gly Ala Val Thr Cys Thr Asp Phe Val Gln 325 330 335 Pro Gly Tyr Gln Ile Trp His Ile Asp Val His Tyr Asp Thr Ser Ser 340 345 350 Gly Ala Tyr Tyr Ala Val Tyr Pro Ala Tyr Pro Asn Gly Thr Asp Cys 355 360 365 Asp His Cys Asn Leu Phe Phe Ala Val Asn Arg Thr Gly Lys Gln Trp 370 375 380 Glu Thr Phe Ser Arg Pro Ile Leu Lys Pro Ser Thr Glu Gly Gly Trp 385 390 395 400 Asp Asp Phe Cys Ile Tyr Arg Ser Ser Met Leu Ile Asp Asp Gly Met 405 410 415 Leu Lys Val Trp Tyr Gly Ala Lys Lys Gln Glu Asp Ser Ser Trp His 420 425 430 Thr Gly Leu Thr Met Arg Asp Phe Ser Glu Phe Met Lys Ile Leu Glu 435 440 445 Arg <210> 34 <211> 845 <212> PRT <213> Robinsoniella peoriensis <400> 34 His Ser Pro Leu Ser Ala Ala Ala Glu Ser Gly Thr Gly Thr Arg Leu 1 5 10 15 Val Lys Gly Gln Thr Gly Tyr Leu Thr Glu Glu Gln Ala Ile Arg Asn 20 25 30 Gln Glu Gln Thr Thr Glu Glu Arg Glu Gln Lys Leu Thr Gly Glu Glu 35 40 45 Thr Ala Glu Val Leu Met Glu Gly Thr Lys Asp Ser Gly Ile Val Gln 50 55 60 Thr Glu Glu Val Gln Thr Lys Glu Met Gln Thr Glu Asp Ala Gln Thr 65 70 75 80 Glu Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln Thr Lys 85 90 95 Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln Thr Glu Glu 100 105 110 Val Gln Thr Lys Glu Glu Pro Ala Glu Glu Thr His Met Lys Glu Ile 115 120 125 Gln Thr Gln Gly Thr Lys Lys Ala Ser Asp Arg Asn Gly Lys Ala Arg 130 135 140 Val Thr Glu Ile Leu Glu Asp Ala Gln Asp Pro Ala Asn Arg Ile Val 145 150 155 160 Tyr Leu Ser Asp Leu Gln Trp Lys Ser Glu Asn His Thr Val Asp Ser 165 170 175 Glu Leu Pro Thr Arg Lys Asp Lys Ser Phe Gly Gly Gly Lys Ile Thr 180 185 190 Leu Lys Val Asp Gly Thr Val Thr Glu Phe Asp Lys Gly Ile Gly Thr 195 200 205 Gln Thr Asp Ser Thr Ile Val Tyr Asp Leu Glu Gly Lys Gly Tyr Thr 210 215 220 Lys Phe Glu Thr Tyr Val Gly Val Asp Tyr Ser Gln Lys Glu Asn Ile 225 230 235 240 Pro Gly Glu Val Cys Asp Val Lys Phe Arg Val Lys Ile Asp Asp Lys 245 250 255 Ile Val Ser Glu Thr Gly Val Leu Asp Pro Leu Ser Asn Ala Val Lys 260 265 270 Ile Ser Val Asn Ile Pro Asp Thr Ala Lys Thr Leu Thr Leu Tyr Ala 275 280 285 Asp Lys Val Thr Glu Thr Trp Ser Asp His Ala Asn Trp Ala Asp Ala 290 295 300 Lys Phe Tyr Gln Ala Leu Pro Glu Pro Glu Asn Val Ala Phe Lys Lys 305 310 315 320 Thr Val Val Thr Arg Lys Thr Ser Asp Asn Ser Glu Ala Pro Val Asn 325 330 335 Pro Asp Ser Ala Val Asn Ser Ser Lys Ala Val Asp Gly Val Ile Asp 340 345 350 Ser Ser Ser Tyr Phe Asp Phe Gly Asp Gln Ala Asn Ser Gly Ala Val 355 360 365 Arg Glu Ser Leu Tyr Met Glu Val Asp Leu Lys Gly Ser Tyr Leu Leu 370 375 380 Ser Asp Ile Gln Leu Trp Arg Tyr Trp Lys Asp Gly Arg Thr Tyr Ala 385 390 395 400 Ala Thr Ala Ile Val Val Ala Glu Asp Glu Asn Phe Glu Asn Ala Ala 405 410 415 Val Ile Tyr Asn Ser Asp Thr Thr Gly Glu Ile His His Leu Gly Ala 420 425 430 Gly Ser Asp Met Leu Tyr Ala Glu Thr Glu Ser Gly Lys Thr Phe Pro 435 440 445 Val Pro Glu Asn Thr Lys Ala Arg Tyr Ile Arg Val Tyr Thr Tyr Gly 450 455 460 Val Asn Gly Thr Ser Gly Val Thr Asn His Ile Val Glu Leu Lys Val 465 470 475 480 Asn Ala Tyr Val Phe Gly Asp Glu Ile Leu Pro Glu Lys Pro Asp Asp 485 490 495 Ser Lys Ile Phe Pro Asn Ala Val Asn Pro Leu Lys Leu Gln Gly Pro 500 505 510 Gly Thr Asn Asp Gln Val Thr His Pro Asp Val Thr Val Phe Asp Glu 515 520 525 Pro Trp Asn Gly Tyr Lys Tyr Trp Met Ala Tyr Thr Pro Asn Lys Pro 530 535 540 Gly Ser Ser Tyr Phe Glu Asn Pro Cys Ile Ala Ala Ser Asn Asp Gly 545 550 555 560 Val Asn Trp Glu Phe Pro Ala Gln Asn Pro Val Gln Pro Arg Tyr Asp 565 570 575 Ser Glu Ile Glu Asn Gln Asn Glu His Asn Cys Asp Thr Asp Ile Val 580 585 590 Tyr Asp Pro Val Asn Asp Arg Leu Ile Met Tyr Trp Glu Trp Ala Gln 595 600 605 Asp Glu Ala Val Asn Gly Lys Thr His Arg Ser Glu Ile Arg Tyr Arg 610 615 620 Val Ser Tyr Asp Gly Ile Asn Trp Gly Val Glu Asp Lys Thr Gly Val 625 630 635 640 Leu Met Thr Gly Pro Thr Asp His Gly Cys Ala Ile Ala Thr Glu Gly 645 650 655 Glu Arg Tyr Ser Asp Leu Ser Pro Thr Val Val Tyr Asp Lys Thr Glu 660 665 670 Lys Ile Tyr Lys Met Trp Ala Asn Asp Ala Gly Asp Val Gly Tyr Glu 675 680 685 Asn Lys Gln Asn Asn Lys Val Trp Tyr Arg Thr Ser Gln Asp Gly Ile 690 695 700 Ser Asn Trp Ser Asp Lys Thr Tyr Val Glu Asn Phe Leu Gly Val Asn 705 710 715 720 Glu Asp Gly Leu Gln Met Tyr Pro Trp His Gln Asp Ile Gln Trp Val 725 730 735 Glu Glu Phe Gln Glu Tyr Trp Ala Leu Gln Gln Ala Phe Pro Ala Gly 740 745 750 Ser Gly Pro Asp Asn Ser Ser Leu Arg Phe Ser Lys Ser Lys Asp Gly 755 760 765 Leu His Trp Glu Pro Val Ser Glu Lys Ala Leu Ile Thr Val Gly Ala 770 775 780 Pro Gly Thr Trp Asp Ala Gly Gln Ile Tyr Arg Ser Thr Phe Trp Tyr 785 790 795 800 Glu Pro Gly Gly Ala Lys Gly Asn Gly Thr Phe His Ile Trp Tyr Ala 805 810 815 Ala Leu Ala Glu Gly Gln Ser His Trp Asp Ile Gly Tyr Thr Ser Ala 820 825 830 Asn Tyr Ala Asp Ala Met Tyr Lys Leu Thr Gly Ser Arg 835 840 845 <210> 35 <211> 1082 <212> PRT <213> Robinsoniella peoriensis <400> 35 His Ala Glu Thr Ala Thr Glu Glu Asn Ala Ala Leu Glu Lys Thr Val 1 5 10 15 Thr Leu His Lys Ser Asp Gly Thr Glu Leu Pro Glu Asp Tyr Arg Asn 20 25 30 Pro Gln Arg Pro Ala Thr Met Ala Val Asp Gly Ile Ile Asp Asp Thr 35 40 45 Gly Glu Tyr Asn Tyr Cys Asp Phe Gly Lys Asp Gly Asp Lys Ala Ala 50 55 60 Leu Tyr Met Gln Val Asp Leu Gly Gly Leu Tyr Asp Leu Ser Arg Val 65 70 75 80 Asn Met Trp Arg Tyr Trp Lys Asp Ser Arg Thr Tyr Asp Ala Thr Val 85 90 95 Ile Thr Thr Ser Glu Ser Gly Asp Phe Thr Asp Glu Ala Val Ile Tyr 100 105 110 Asn Ser Asp Arg Ser Asn Val His Gly Phe Gly Ala Gly Gly Asp Glu 115 120 125 Arg Tyr Ala Glu Thr Ala Ser Gly His Glu Phe Pro Val Pro Asp Gly 130 135 140 Thr Lys Ala Gln Ala Val Arg Val Tyr Val Phe Gly Ser Gln Asn Gly 145 150 155 160 Thr Thr Asn His Ile Asn Glu Leu Gln Val Trp Gly Thr Pro His Thr 165 170 175 Glu Asn Pro Asp Val Asn Ser Tyr Gln Val Thr Ile Pro Gln Gly Asn 180 185 190 Gly Tyr Gln Val Ile Pro Tyr Glu Asn Asp Pro Thr Thr Val Glu Glu 195 200 205 Gly Gly Ser Phe Arg Phe Gln Val Leu Ile Asp Ser Asp Asn Gly Tyr 210 215 220 Ser Ala Thr Ser Ala Val Lys Ala Asn Gly Val Ser Leu Glu Ala Val 225 230 235 240 Asp Ser Val Tyr Thr Ile Glu Asn Ile Thr Glu Asp Gln Val Ile Thr 245 250 255 Ile Glu Gly Val His Lys Ala Gln Tyr Glu Val Lys Phe Pro Glu Asn 260 265 270 Pro Gln Gly Tyr Ser Val Glu Ile Gln Asn Glu Gly Ser Thr Thr Val 275 280 285 Asp Tyr Asn Gly Ser Val Ser Phe Lys Leu Ile Ile Asp Glu Ala Tyr 290 295 300 Asn Glu Ser Val Pro Val Val Lys Ala Asn Gly Gly Ala Ala Leu Gly 305 310 315 320 Lys Asp Glu Leu Gly Val Tyr Thr Ile Ala Asn Ile Gln Asp Asp Ile 325 330 335 Thr Val Thr Val Glu Gly Ile Gln Glu Asn Thr Val Val Lys Thr Lys 340 345 350 Thr Met Tyr Leu Ser Asp Met Asp Trp Lys Ser Ala Ala Asn Ala Val 355 360 365 Gly Ala Thr Gly Glu Lys Asp Thr Pro Thr Lys Asp Leu Asn His Leu 370 375 380 Gln Gln Gln Met Lys Leu Leu Val Asn Gly Ala Glu Lys Ser Phe Asp 385 390 395 400 Lys Gly Ile Gly Val Gln Thr Asp Ser Ser Ile Val Tyr Asp Leu Glu 405 410 415 Asp Lys Gly Tyr Thr Ser Phe His Thr Leu Ala Gly Val Asp Tyr Ser 420 425 430 Ala Met Glu Tyr Val Asp Gly Glu Gly Cys Asp Ile Gln Phe Lys Val 435 440 445 Tyr Leu Asp Asp Val Val Val Phe Asp Ser Gly Val Val Asp Ala Ser 450 455 460 Asp Glu Ala Gln Glu Val Asn Val Ala Ile Thr Ser Glu Asn Lys Glu 465 470 475 480 Leu Lys Leu Glu Ala Lys Met Val Lys Glu Pro Tyr Asn Asp Trp Gly 485 490 495 Asn Trp Ala Asp Ala Ser Phe Glu Met Ala Tyr Pro Glu Pro Ser Asn 500 505 510 Val Ala Leu Asn Lys Thr Val Thr Val Lys Lys Thr Ala Asp Asn Ser 515 520 525 Asp Ser Glu Val Asn Ser Ser Arg Pro Gly Ser Met Ala Val Asp Gly 530 535 540 Ile Ile Gly Pro Thr Ser Asp Ser Asn Tyr Cys Asp Phe Gly Gln Asp 545 550 555 560 Gly Asp Asn Thr Ser Arg Tyr Leu Gln Val Asp Leu Gly Asp Val Tyr 565 570 575 Glu Leu Thr Gln Ile Asn Met Phe Arg Tyr Trp Ala Asp Gly Arg Val 580 585 590 Tyr Asn Gly Thr Val Ile Ala Val Ser Glu Asn Ala Asp Phe Ser Asn 595 600 605 Pro Thr Phe Ile Tyr Asn Ser Asp Lys Ala Asp Lys His Gly Leu Gly 610 615 620 Ala Gly Ser Asp Asp Thr Tyr Gly Glu Thr Gln Ser Gly Lys Leu Phe 625 630 635 640 Glu Val Pro Ala Gly Thr Met Gly Gln Tyr Val Arg Val Tyr Met Ala 645 650 655 Gly Ser Asn Lys Gly Thr Thr Asn His Ile Ala Glu Leu Gln Val Met 660 665 670 Gly Tyr Asn Phe Asn Thr Glu Pro Lys Pro Tyr Glu Ala Asn Ala Phe 675 680 685 Glu Asn Ala Glu Val Tyr Leu Asp Met Pro Thr His Phe Gln Asp Leu 690 695 700 Asp Ser Asn Lys Asn Asp Asp Gly Ser Leu Lys His Ile Gly Gly Gln 705 710 715 720 Val Thr His Pro Asp Ile Gln Val Phe Asp Gln Pro Trp Asn Gly Tyr 725 730 735 Lys Tyr Trp Met Ile Tyr Thr Pro Asn Thr Met Ile Thr Ser Gln Tyr 740 745 750 Glu Asn Pro Tyr Ile Val Ala Ser Glu Asp Gly Gln Thr Trp Val Glu 755 760 765 Pro Glu Gly Ile Ser Asn Pro Ile Glu Pro Glu Pro Pro Ser Thr Arg 770 775 780 Phe His Asn Cys Asp Ala Asp Leu Leu Tyr Asp Ser Val Asn Asp Arg 785 790 795 800 Leu Leu Ala Tyr Trp Asn Trp Ala Asp Asp Gly Gly Gly Ile Asp Asp 805 810 815 Glu Leu Lys Asp Gln Asn Cys Gln Ile Arg Leu Arg Ile Ser Tyr Asp 820 825 830 Gly Ile Asn Trp Gly Val Pro Tyr Asp Lys Asp Gly Asn Ile Ala Thr 835 840 845 Thr Ala Asp Thr Val Val Arg Met Glu Thr Gly Asp Lys Asp Phe Ile 850 855 860 Pro Ala Ile Ser Glu Lys Asp Arg Tyr Gly Met Leu Ser Pro Thr Phe 865 870 875 880 Thr Tyr Asp Asp Phe Arg Gly Ile Tyr Thr Met Trp Ala Gln Asn Ser 885 890 895 Gly Asp Ala Gly Tyr Asn Gln Ser Gly Lys Phe Ile Glu Met Arg Trp 900 905 910 Ser Glu Asp Gly Ile Asn Trp Ser Glu Pro Gln Lys Val Asn Asn Phe 915 920 925 Leu Gly Lys Asp Glu Asn Gly Arg Gln Leu Trp Pro Trp His Gln Asp 930 935 940 Ile Gln Tyr Ile Pro Glu Leu Gln Glu Tyr Trp Gly Leu Ser Gln Cys 945 950 955 960 Phe Ser Thr Ser Asn Pro Asp Gly Ser Val Leu Tyr Leu Thr Lys Ser 965 970 975 Arg Asp Gly Val Asn Trp Glu Gln Ala Gly Thr Gln Pro Val Leu Arg 980 985 990 Ala Gly Lys Ser Gly Thr Trp Asp Asp Phe Gln Ile Tyr Arg Ser Thr 995 1000 1005 Phe Tyr Tyr Asp Asn Gln Ser Asp Ser Pro Thr Gly Gly Lys Phe 1010 1015 1020 Arg Ile Trp Tyr Ser Ala Leu Gln Ala Asn Thr Ser Gly Lys Thr 1025 1030 1035 Val Leu Ala Pro Asp Gly Thr Val Ser Leu Gln Val Gly Ser Gln 1040 1045 1050 Asp Thr Arg Ile Trp Arg Ile Gly Tyr Thr Glu Asn Asp Tyr Met 1055 1060 1065 Glu Val Met Lys Ala Leu Thr Gln Asn Lys Asn Tyr Glu Glu 1070 1075 1080 <210> 36 <211> 986 <212> PRT <213> Clostridium tertium <400> 36 His Tyr Asn Leu Ile Asp Asn Ile Ser Val Glu Lys Leu Asp Thr Asp 1 5 10 15 Ile Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn Gly Ile Ala 20 25 30 Leu Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu Val Asp Glu 35 40 45 Asn Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr Ala Asp Phe 50 55 60 Ser Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp Phe Val Ile 65 70 75 80 Ile Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu Gly Val Gly 85 90 95 Asn Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn Leu Ile Arg 100 105 110 Lys Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala Ile Tyr Ser 115 120 125 Thr Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val Asp Ser Phe 130 135 140 His Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro Phe Leu Ala 145 150 155 160 Tyr Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile Val Asn Val 165 170 175 Thr Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr Ser Val Gly 180 185 190 Val Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile Gly Ile Gly 195 200 205 Asp Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile Ile Gly Lys 210 215 220 Asp Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln Thr Leu Lys 225 230 235 240 Lys Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr Thr Thr Asn 245 250 255 Gly Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val Met Lys Asp 260 265 270 Lys Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala Tyr Asp Ser 275 280 285 Arg Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile Glu Lys Ile 290 295 300 Val Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys Gln Ile Thr 305 310 315 320 Leu Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys Leu Ser Pro 325 330 335 Gln Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu Thr Asp Glu 340 345 350 Ile His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg Pro Val Asp 355 360 365 Gly Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu Trp Phe Ile 370 375 380 Lys Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro Gly Gly Gly 385 390 395 400 Asn Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser Glu Gly Ser 405 410 415 Ile Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu Glu Trp Gly 420 425 430 Phe Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro Lys Cys Tyr 435 440 445 Asp Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu Glu Asn Gln 450 455 460 Tyr Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala Ile Asn Pro 465 470 475 480 Asp Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln Asp Phe Tyr 485 490 495 Ser Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro Ile Ser Arg 500 505 510 Val Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile Phe Gly Asp 515 520 525 Asp Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu Pro Ser Ala 530 535 540 Leu Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr Leu Ser Ser 545 550 555 560 Ser Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala Arg Asp Leu 565 570 575 Glu Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys Tyr Gly Ile 580 585 590 Asp Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp Ile Tyr Tyr 595 600 605 Ser Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile Glu Ile Lys 610 615 620 Gly Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr Val Asn Asn 625 630 635 640 Arg Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr Ile Asn Thr 645 650 655 Ser Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp Asp Thr Pro 660 665 670 Ala Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile Leu Ser Ser 675 680 685 Thr Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser Thr Thr Leu 690 695 700 Glu Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile Gly Asn Asn 705 710 715 720 Ser Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp Asn Asn Gly 725 730 735 Lys Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val Pro Thr Lys 740 745 750 Asn Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr Leu Pro Ser 755 760 765 Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val Leu Gln Thr 770 775 780 Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn Asn Pro Tyr 785 790 795 800 Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr Gly Tyr Asn 805 810 815 Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys Glu Ala Ser 820 825 830 Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr Val Asp Ser 835 840 845 Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala Phe Asp Gly 850 855 860 Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly Asn Ala Ala 865 870 875 880 Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val Tyr Asn Ile 885 890 895 Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr Asn Gly Thr 900 905 910 Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val Asn Trp Gly 915 920 925 Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr Glu Lys Ile 930 935 940 Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu Lys Ala Leu 945 950 955 960 Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp Leu Lys Val 965 970 975 Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys 980 985 <210> 37 <211> 1262 <212> PRT <213> Robinsoniella peoriensis <400> 37 His Gly Asn Gly Leu Glu Val Lys Ala Ser Pro Arg Glu Val Ala Gln 1 5 10 15 Ile Thr Gly Asn Gly Val Ser Val Thr Phe Phe Gln Glu Asp Gly Thr 20 25 30 Val Gln Leu Ser Cys Ile Glu Asp Asp Gly Asn Thr Ala Phe Met Thr 35 40 45 Arg Asn Ser Glu Val Ser Tyr Pro Val Val Gly Gly Glu Glu Val Thr 50 55 60 Asp Phe Ser Asp Phe Gln Cys Glu Val Gln Glu Asn Val Thr Gly Ala 65 70 75 80 Ala Gly Ala Gly Ser Arg Met Thr Ile Thr Ser Ile Ser Ser Gly Arg 85 90 95 Gly Ile Gln Arg Ser Val Val Ile Glu Thr Val Asp Glu Val Lys Gly 100 105 110 Leu Leu His Ile Ser Ser Ser Tyr Arg Ala Glu Glu Glu Val Asp Ala 115 120 125 Asp Glu Phe Ile Asp Ser Arg Phe Ser Leu Asp Asn Pro Ser Asp Thr 130 135 140 Val Trp Ser Tyr Asn Gly Gly Gly Glu Gly Ala Gln Ser Arg Tyr Asp 145 150 155 160 Thr Leu Gln Lys Ile Asp Leu Ser Asp Gly Glu Ser Phe Tyr Arg Glu 165 170 175 Asn Leu Gln Asn Gln Thr Ala Ala Gly Ile Pro Val Ala Asp Ile Tyr 180 185 190 Gly Lys Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Val Thr Arg Arg 195 200 205 Gln Leu Ser Thr Pro Val Asn Glu Arg Asn Gly Thr Ala Tyr Val Ser 210 215 220 Val Lys His Pro Gly Ala Val Ile Thr Gln Arg Glu Thr Glu Ile Ser 225 230 235 240 Gln Ser Phe Val Asn Val His Arg Gly Asp Tyr Tyr Ser Gly Leu Arg 245 250 255 Gly Tyr Ala Asp Gly Met Lys Gln Ile Gly Phe Thr Thr Leu Ser Arg 260 265 270 Glu Gln Ile Pro Glu Ser Ser Tyr Asp Leu Arg Trp Glu Ser Trp Gly 275 280 285 Trp Glu Phe Asp Trp Thr Val Glu Leu Ile Ile Asn Lys Leu Asp Glu 290 295 300 Leu Lys Glu Met Gly Ile Lys Gln Ile Thr Leu Asp Asp Gly Trp Tyr 305 310 315 320 Asn Ala Ala Gly Glu Trp Gly Leu Asn Asn Trp Lys Leu Pro Asn Gly 325 330 335 Ala Leu Asp Met Arg His Leu Thr Asp Ala Ile His Glu Arg Gly Met 340 345 350 Thr Ala Val Leu Trp Trp Arg Pro Cys Asp Gly Gly Arg Glu Asp Ser 355 360 365 Ala Leu Phe Lys Glu His Pro Glu Tyr Phe Ile Lys Asn Gln Asp Gly 370 375 380 Ser Phe Gly Lys Leu Ala Gly Pro Gly Gln Trp Asn Ser Phe Leu Gly 385 390 395 400 Ser Cys Gly Tyr Ala Leu Cys Pro Leu Ser Glu Gly Ala Val Gln Ser 405 410 415 Gln Val Asp Phe Ile Asn Arg Ala Met Asn Glu Trp Gly Phe Asp Gly 420 425 430 Phe Lys Ser Asp Tyr Val Trp Ser Leu Pro Lys Cys Tyr Ser Gln Asp 435 440 445 His His His Glu Tyr Pro Glu Glu Ser Thr Glu Gln Gln Ala Val Phe 450 455 460 Tyr Arg Ala Val Tyr Glu Ala Met Thr Asp Asn Asp Pro Asn Ala Phe 465 470 475 480 His Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu Pro 485 490 495 Tyr Val Thr Gln Val Pro Thr Ala Asp Pro Thr Ser Val Asp Gln Thr 500 505 510 Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Cys Gly Asp Tyr Phe Pro 515 520 525 Val Thr Thr Asp His Asn Glu Val Trp Tyr Pro Ser Thr Ile Gly Thr 530 535 540 Gly Ala Ile Leu Ile Glu Lys Arg Asp Leu Ser Gly Trp Glu Glu Glu 545 550 555 560 Glu Tyr Ala Lys Trp Leu Lys Ile Ala Gln Glu Asn Gln Leu His Lys 565 570 575 Gly Thr Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Tyr Asp Pro Tyr Glu 580 585 590 Thr Tyr Thr Val Tyr Lys Asp Gly Ile Met Tyr Tyr Ala Phe Tyr Lys 595 600 605 Asp Gly Asn Arg Tyr Arg Pro Ser Gly Asn Pro Asp Ile Glu Leu Lys 610 615 620 Gly Leu Glu Asp Gly Lys Leu Tyr Arg Ile Val Asp Tyr Val Asn Asn 625 630 635 640 Gln Val Val Ala Thr Asn Val Thr Ser Ser Asn Ala Val Phe Ser Tyr 645 650 655 Pro Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile Ser Glu Pro 660 665 670 Asp Thr Asp Gly Pro Gly Pro Val Pro Asp Pro Glu Gly Ala Val Thr 675 680 685 Val Glu Glu Asn Asp Pro Glu Leu Val Tyr Thr Gly Asp Trp Val Arg 690 695 700 Glu Glu Asn Asp Gly Tyr His Gly Gly Gly Ala Arg Tyr Thr Lys Glu 705 710 715 720 Ala Glu Ala Ser Val Glu Leu Ala Phe Tyr Gly Thr Gly Ala Ala Trp 725 730 735 Tyr Gly Gln His Asp Val Asn Phe Gly Ser Ala Arg Ile Tyr Ile Asp 740 745 750 Gly Thr Tyr Val Lys Thr Val Ser Cys Met Gly Glu Pro Gly Ile Asn 755 760 765 Ile Lys Leu Phe Glu Ile Ser Gly Leu Asp Leu Ala Ser His Arg Ile 770 775 780 Lys Ile Glu Cys Glu Thr Pro Val Ile Asp Ile Asp Arg Leu Thr Tyr 785 790 795 800 Ile Lys Gly Glu Glu Val Pro Ala Lys Val Met Thr Ala Asp Leu Arg 805 810 815 Ala Leu Thr Val Ile Ala Asn Gln Tyr Asp Met Asn Ser Phe Ala Asp 820 825 830 Gly Asn Tyr Lys Asp Gln Leu Gly Val Ser Leu Val Arg Ala Asn Gln 835 840 845 Leu Leu Ala Ala Asp Asp Val Thr Gln Gly Ala Val Asn Glu Glu Gln 850 855 860 Lys Tyr Leu Leu Asn Ala Met Leu Lys Ile Arg Lys Lys Val Asp Lys 865 870 875 880 Ser Trp Ile Gly Leu Pro Gly Pro Ile Pro Gln Asp Ile Gln Thr Glu 885 890 895 Asn Ile Ser Arg Asp Asn Leu Ala Lys Val Ile Ser Tyr Thr Gly Gln 900 905 910 Leu Asp Arg Asp Glu Ile Ile Pro Ala Ile Lys Glu Gln Leu Asn Asp 915 920 925 Ser Tyr Asp Lys Ala Val Ser Ile Ala Glu Arg Gln Asp Ala Ser Gln 930 935 940 Pro Glu Ile Asp Arg Ala Trp Ala Glu Leu Met Asn Ala Val Gln Tyr 945 950 955 960 Ser Ser Tyr Ile Arg Gly Ser Lys Glu Glu Leu Leu Ser Leu Leu Asp 965 970 975 Glu Tyr Gly Lys Val Asp Thr Thr Val Tyr Lys Asp Ala Ala Leu Phe 980 985 990 Ile Glu Ser Leu Glu Ala Ala Lys Lys Val Tyr Gln Asp Glu Asn Ala 995 1000 1005 Met Asp Gly Glu Ile Ser Asp Cys Ile Lys Gln Leu Arg Asp Ala 1010 1015 1020 Lys Asp Gln Leu Gln Leu Lys Asp Pro Val Asp Pro Pro Lys Pro 1025 1030 1035 Asp Pro Asp Pro Asp Pro Lys Pro Asp Pro Thr Pro Asp Pro Gly 1040 1045 1050 Pro Asp Pro Lys Pro Asp Pro Thr Pro Asp Pro Thr Pro Asp Pro 1055 1060 1065 Lys Pro Asn Pro Thr Pro Thr Pro Asp Pro Thr Pro Glu Pro Ala 1070 1075 1080 Leu Lys Lys Pro Glu Gln Val Ser Gly Leu Lys Ser Lys Ala Glu 1085 1090 1095 Thr Asp Tyr Leu Thr Val Ser Trp Lys Lys Leu Asn Asn Ala Glu 1100 1105 1110 Ser Tyr Lys Val Tyr Ile Tyr Lys Ser Gly Lys Trp Arg Leu Ala 1115 1120 1125 Gly Lys Thr Thr Lys Thr Ser Ile Lys Ile Lys Lys Leu Val Ser 1130 1135 1140 Gly Thr Lys Tyr Thr Val Lys Val Ala Ala Val Asn Lys Ala Gly 1145 1150 1155 Gln Gly Lys Tyr Ser Ser Gln Val Tyr Thr Ala Ala Lys Pro Lys 1160 1165 1170 Lys Val Lys Leu Lys Ser Val Ser Arg Tyr Arg Thr Ser Lys Val 1175 1180 1185 Lys Leu Asn Tyr Gly Lys Val Lys Ala Gly Gly Tyr Glu Ile Trp 1190 1195 1200 Met Lys Asn Gly Lys Gly Ser Tyr Lys Lys Ala Ala Thr Ser Thr 1205 1210 1215 Lys Thr Thr Ala Ile Lys Ser Gly Leu Lys Lys Gly Lys Thr Tyr 1220 1225 1230 Tyr Phe Lys Val Arg Ala Tyr Val Lys Asn Lys Asn Gln Val Ile 1235 1240 1245 Tyr Gly Ser Phe Ser Asn Ile Lys Lys Tyr Lys Met Val Leu 1250 1255 1260 <210> 38 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNAcDeAc_no signal P_fw <400> 38 atggtctcgc catgcagact ccagcgagtc cg 32 <210> 39 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNAcDeAc_D1min_rv <400> 39 atggtctcga ttcttacgtc gtgtagccgg ggtc 34 <210> 40 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNAcDeAc_D1ext_rv <400> 40 atggtctcga ttcttaatca ctggaggtat atttcacgac c 41 <210> 41 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNAcDeAc_D1+2_rv <400> 41 atggtctcga ttcttacgca ggctcgattg gaccatac 38 <210> 42 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNAcDeAc_D2ext_fw <400> 42 atggtctcgc catgatgtgg cgacggtgga tgag 34 <210> 43 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNAcDeAc_rv <400> 43 atggtctcga ttcttattct cccacatacg aaaaatagtc g 41 <210> 44 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNase_no signal P_fw <400> 44 atggtctcgc catcgtggta aaaagttcat atcactcac 39 <210> 45 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNase_truncated_rv <400> 45 atggtctcga ttctttatgcg ttagtggtat aagtcaaata gtc 43 <210> 46 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer FpGalNase_rv <400> 46 atggtctcga ttcttattcc gaaatttcca ccgctttaac 40 <210> 47 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primer Ct5757_fw <400> 47 atggtctcgc cattataatt taattgataa tattagtgtt gaaaaattag 50 <210> 48 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer Ct5757_rv <400> 48 atggtctcga ttcttattgt gttaaaccct caataaac 38 <210> 49 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer Ct5757_GalNase_rv <400> 49 atggtctcga ttcttaatga gtactttgat ttaatccatc ataag 45 <210> 50 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer Ct5757_DeAcase_fw <400> 50 atggtctcgc cattcagggc aatattggtt agttttc 37 <210> 51 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer Rp1021_fw <400> 51 atggtctcgc catgggaacg gattagaggt gaaag 35 <210> 52 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer Rp1021_rv <400> 52 atggtctcga ttctcataat accattttgt atttctttat attgg 45 <210> 53 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer R18755_fw <400> 53 atggtctcgc catgaagaaa ccgatttgct tgtaaac 37 <210> 54 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer R18755_rv <400> 54 atggtctcga ttcttagcgt tccaatattt tcataaattc ag 42 <210> 55 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer Rp3671_fw <400> 55 atggtctcgc cattcaccat tgagcgctgc gg 32 <210> 56 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primer Rp3671_rv <400> 56 atggtctcga ttctttatgac tttgttttaa catttacaga cttg 44 <210> 57 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer Rp3672_fw <400> 57 atggtctcgc catgctgaga ctgcaacaga agaaaatg 38 <210> 58 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer Rp3672_rv <400> 58 atggtctcga ttcttatttc tgaatttttg ccttgccag 39 <210> 59 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence AU1 epitope <400> 59 Asp Thr Tyr Arg Tyr Ile 1 5 <210> 60 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence AU5 epitope <400> 60 Thr Asp Phe Tyr Leu Lys 1 5 <210> 61 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Avi tag <400> 61 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 1 5 10 15 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence T7 tag <400> 62 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly 1 5 10 <210> 63 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence V5 tag <400> 63 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 64 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence B tag <400> 64 Gln Tyr Pro Ala Leu Thr 1 5 <210> 65 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Calmodulin tag <400> 65 Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg 1 5 10 15 Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu 20 25 <210> 66 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence C tag <400> 66 Glu Pro Glu Ala 1 <210> 67 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence DogTag <400> 67 Asp Ile Pro Ala Thr Tyr Glu Phe Thr Asp Gly Lys His Tyr Ile Thr 1 5 10 15 Asn Glu Pro Ile Pro Pro Lys 20 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence E2 epitope <400> 68 Ser Ser Thr Ser Ser Asp Phe Arg Asp Arg 1 5 10 <210> 69 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Affinity tag sequence E tag <400> 69 Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 1 5 10 <210> 70 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Affinity tag sequence FLAG tag <400> 70 Asp Tyr Lys Asp Asp Asp Lys 1 5 <210> 71 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence EE tag (1) <400> 71 Glu Tyr Met Pro Met Glu 1 5 <210> 72 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence EE tag (2) <400> 72 Glu Phe Met Pro Met Glu 1 5 <210> 73 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence HA tag <400> 73 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 74 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence HAT <400> 74 Lys Asp His Leu Ile His Asn Val His Lys Glu Phe His Ala His Ala 1 5 10 15 His Asn Lys <210> 75 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence HQ tag <400> 75 His Gln His Gln His Gln 1 5 <210> 76 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence HN tag <400> 76 His Asn His Asn His Asn His Asn His Asn His Asn 1 5 10 <210> 77 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence HSV epitope <400> 77 Gln Pro Glu Leu Ala Pro Glu Asp 1 5 <210> 78 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Isopep tag <400> 78 Thr Asp Lys Asp Met Thr Ile Thr Phe Thr Asn Lys Lys Asp Ala Glu 1 5 10 15 <210> 79 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence KT3 epitope <400> 79 Lys Pro Pro Thr Pro Pro Pro Glu Pro Glu Thr 1 5 10 <210> 80 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Myc epitope <400> 80 Cys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 81 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Myc tag <400> 81 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 82 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence NE tag <400> 82 Thr Lys Glu Asn Pro Arg Ser Asn Gln Glu Glu Ser Tyr Asp Asp Asn 1 5 10 15 Glu Ser <210> 83 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Arg tag <400> 83 Arg Arg Arg Arg Arg 1 5 <210> 84 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Asp tag <400> 84 Asp Asp Asp Asp Asp 1 5 <210> 85 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Cys tag <400> 85 Cys Cys Cys Cys 1 <210> 86 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Glu tag <400> 86 Glu Glu Glu Glu Glu Glu 1 5 <210> 87 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence His tag <400> 87 His His His His His His 1 5 <210> 88 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Phe tag <400> 88 Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe 1 5 10 <210> 89 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Rho1D4 tag <400> 89 Thr Glu Thr Ser Gln Val Ala Pro Ala 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence S1 tag <400> 90 Asn Ala Asn Asn Pro Asp Trp Asp Phe 1 5 <210> 91 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence S tag <400> 91 Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser 1 5 10 15 <210> 92 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Softtag 1 <400> 92 Ser Leu Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser 1 5 10 <210> 93 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Softtag 3 <400> 93 Thr Gln Asp Pro Ser Arg Val Gly 1 5 <210> 94 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Spy tag <400> 94 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 <210> 95 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence SBP tag <400> 95 Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 1 5 10 15 Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro 20 25 30 Gln Gly Gln Arg Glu Pro 35 <210> 96 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Strep tag (1) <400> 96 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Strep tag (2) <400> 97 Ala Trp Ala His Pro Gln Pro Gly Gly 1 5 <210> 98 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Strep tag II <400> 98 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 99 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Sdy tag <400> 99 Asp Pro Ile Val Met Ile Asp Asn Asp Lys Pro Ile Thr 1 5 10 <210> 100 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> SnoopTag <400> 100 Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys 1 5 10 <210> 101 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence SnoopTagJr <400> 101 Lys Leu Gly Ser Ile Glu Phe Ile Lys Val Asn Lys 1 5 10 <210> 102 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Spot tag <400> 102 Pro Asp Arg Val Arg Ala Val Ser His Trp Ser Ser 1 5 10 <210> 103 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence TC tag <400> 103 Cys Cys Pro Gly Cys Cys 1 5 <210> 104 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence Ty tag <400> 104 Glu Val His Thr Asn Gln Asp Pro Leu Asp 1 5 10 <210> 105 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence universal <400> 105 His Thr Thr Pro His His 1 5 <210> 106 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence VSV tag <400> 106 Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 1 5 10 <210> 107 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Protein tag sequence V5 tag <400> 107 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 <210> 108 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Protein Tag Sequence Xpress Tag <400> 108 Asp Leu Tyr Asp Asp Asp Asp Lys 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Schematic diagram of the carbohydrate structures of type A, H, and B cell surface antigens terminating in α-1,3-linked N-acetylgalactosamine (GalNAc) or galactose (Gal), with triangles marking the cleavage sites for α-N-acetylgalactosaminidase EmGH109 and α-galactosidase BfGal110.

[0052] Figure 2 The deacetylase pathway for A antigen cleavage is shown, whereby GalNAc deacetylase from Fusobacterium prausnitzii (Fp) cleaves the terminal α-N-acetylgalactosamine (-42 m / z ) of the acetyl group, and then the galactosamine intermediate is converted to galactosaminidase (-161 m / z ) and analyzed by corresponding mass spectrometry (MS).

[0053] Figure 3 A treated with different concentrations of EmGH109 or GalNAc deacetylase from Fusobacterium prausnitzii (FpGalNAc deacetylase) plus galactosamidase from Fusobacterium prausnitzii (FpGalNAc deacetylase) is shown. + RBC or A treated at 37℃ for 1h + FACS analysis of RBCs using anti-H antibodies (plus a secondary FITC-labeled antibody) and APC-labeled anti-A antibodies for visualization, with the region where the H antigen appears in the box in the upper left corner. Rows A to D compare EmGH109 and FpGalNAcDeAc+FpGalNase at 5 μg / ml (A); 10 μg / ml (B); 50 μg / ml (C); and 50 μg / ml + dextran 40k (D).

[0054] Figure 4 Shown is a comparison of EmGH109 and FpGalNAcDeAc+FpGalNase at various enzyme concentrations with (■) and without (◆) dextran at different temperatures (ie, 4°C, room temperature (RT), and 37°C).

[0055] Figure 5 Shown are HPAE-PAD analysis of A+B+ and O+ erythrocyte cleavage products, and a comparison of the full-length F. prausnitzii GalNAc deacetylase (FpGalNAcDeAc) + F. prausnitzii galactosamidase (FpGalNase) enzyme with the truncated FpGalNAcDeAc + FpGalNase enzymes on A+ erythrocytes.

[0056] Figure 6 The pH profile of each of (A) FpGalNAc deacetylase and (B) Fpgalactosaminidase is shown.

[0057] Figure 7 Shown are the conversion of A antigen to H antigen on A RBCs analyzed by FACS for (A) A+ RBC control, (B) GalNAc deacetylase from Fusobacterium prausnitzii (FpGalNAcDeAc) + galactosamidase from Fusobacterium prausnitzii (FpGalNase) (10 μg / mL), (C) FpGalNAcDeAc + Clostridium tertiotrophoside (Ct) Ct57577_GalNase (10 ug / mL), and (D) FpGalNAcDeAc + galactosamidase from Robinsoniella peoriensis (Rp) (Rp1021)GalNase (10 ug / mL).

[0058] Details

[0059] The following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings show embodiments of the invention. However, the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0060] Any terms not directly defined herein should be understood to have the meaning commonly associated therewith as understood in the field of the present invention.

[0061] As used herein, "immobilized enzyme" is an enzyme attached to a surface, which may be an inert, insoluble material. Immobilization of enzymes can provide enhanced resistance to changes in conditions (such as pH, temperature, etc.) and facilitate their removal after use and reuse of the enzyme.

[0062] Enzyme immobilization can be achieved in various ways (e.g., affinity tag binding, surface adsorption on glass, resin, alginate beads or matrices, entrapment by beads, fibers or microspheres, crosslinking to surfaces or other enzymes and covalent binding to surfaces).

[0063] As used herein, "affinity tag binding" refers to the immobilization of an enzyme to a surface (e.g., a porous material using a non-covalent or covalent protein tag). Affinity tag binding has been used for protein purification and has recently been used for EziG TM (ENGINZYME AB TM, Sweden - e.g., PCT / US1992 / 010113 and PCT / SE2015 / 050108) for biocatalytic applications. Alternative systems for attaching active enzymes to surfaces are known in the art (see, for example, US4088538; US4141857; US4206259; US4218363; US4229536; US4239854; US4619897; US4748121; US4749653; US4897352; US4954444; US4978619; US5154808; US5914367; US5962279; US6030933; US6291582; ​​US6254645; US10,016,490 and US10,041,055).

[0064] Protein tag is the peptide sequence that gene is transplanted on the recombinant protein, is usually by chemical reagent or by enzymatic method removable, and is connected on the protein for various purposes.The protein tag listed in Table A is intended as example and is not intended to be restricted in any way.One type of protein tag is affinity tag, and it is added in protein or peptide sequence, makes them can use affinity technology (for example, from expression system organism) to be purified from crude biological source, or promotes " marked " protein to be fixed on the surface.Some examples of affinity tag include chitin binding domain (CBD), maltose binding protein (MBP), Strep tag, glutathione-S-transferase (GST) and the polyhistidine (His tag) combined with metal matrix.Another type of protein tag is epitope tag (for example, including V5 tag, Myc tag, HA tag, Spot tag and NE tag), they are short peptide sequences selected for being easy to produce high-affinity antibody, and are usually derived from viral gene sequence to improve immunoreactivity. Epitope tags are particularly useful in Western blotting, immunofluorescence, and immunoprecipitation experiments, although they are also used for purification and immobilization of proteins to surfaces. Another type of protein tag is a chromatographic tag (e.g., a polyanionic amino acid, such as a FLAG tag), which can be used to change the chromatographic properties of a protein to help separation and purification or immobilization. Other protein tags are solubilizing tags (e.g., maltose binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), and poly-(NANP)) and fluorescent tags (e.g., green fluorescent protein (GFP)). Protein tags can allow specific enzymatic modification, chemical modification, or protein to be connected to other components. However, depending on the type or quantity of the tag added to the protein sequence, the natural function of the protein, in this case, the enzymatic function may be impaired by the tag. Therefore, it is necessary to select a protein tag to ensure that the activity of the enzyme is not impaired, or alternatively, the protein tag can be cut from the protein before use.

[0065] Table A: Exemplary protein tags

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] In this application, the use of protein tags is exemplified by using polyhistidine protein tags (His tags) as shown in SEQ ID NOs: 5, 10, 15, 17, 19, 21, 23, 25, 27, 29 and 31, but those skilled in the art will readily appreciate that any number of other protein tags can be used to purify the enzyme and / or to attach the enzyme to a surface as described herein, depending on the purification method used and / or the surface to which the enzyme is attached. Such protein tags can be selected from any one or more of the protein tags listed in Table A, but other such protein tags are known in the art.

[0072] In addition, one or more cleavage sites (e.g., thrombin cleavage sites as used in SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, and 31) can be used to release a protein tag from an enzyme or otherwise cleave the enzyme. The cleavage site can be used to remove an N-terminal methionine, a signal peptide, and / or convert an inactive or non-functional protein into an active protein (i.e., zymogens / proenzymes). Alternatively, the cleavage site can be used to separate two or more enzymes expressed in the same reading frame. Examples of enzymes capable of cleaving proteins or peptides and having sequence-specific cleavage sites may be selected from one or more of the following: Arg-C protease; Asp-N endopeptidase; Asp-N endopeptidase + N-terminal GluBNPS-skatole; Caspase 1; Caspase 2; Caspase 3; Caspase 4; Caspase 5; Caspase 6; Caspase 7; Caspase 8; Caspase 9; Caspase 10; Chymotrypsin - high specificity (C-terminal to [FYW], not before P); Chymotrypsin - low specificity Isotropic (C-terminal to [FYWML], not before P); clostripain (Clostridium peptidase B); CNBr; enterokinase; factor Xa; formic acid; glutamyl endopeptidase; granzyme B; hydroxylamine; iodosobenzoic acid; LysC; LysN; NTCB (2-nitro-5-thiocyanatobenzoic acid); neutrophil elastase; pepsin (pH 1.3); pepsin (pH > 2); proline-endopeptidase; proteinase K; staphylococcal peptidase I; tobacco etch virus protease; thermolysin; thrombin and trypsin.

[0073] Those skilled in the art will appreciate that the combination of an active galactosamidase and an active GalNAc deacetylase that are capable of effectively cleaving the A antigen as described herein is important, and those skilled in the art will also appreciate that the addition of one or more cleavage sites and / or one or more protein tags is optional, and such modifications can be selected based on specific expression systems, purification systems, and possible surface attachment strategies. In addition, other modifications to the galactosamidase and GalNAc deacetylase sequences are also possible, as long as the cleavage activity of the A antigen is not significantly impaired. In addition, modifications to the galactosamidase and GalNAc deacetylase sequences are possible, as long as the cleavage activity of the A antigen is not significantly impaired. Modifications to the galactosamidase and GalNAc deacetylase sequences can be deletions, insertions, and / or substitutions. Substitutions can be conservative substitutions or neutral substitutions. For example, the galactosamidase and GalNAc deacetylase sequences may share 90% or higher sequence identity with the mature enzyme. For example, the galactosamidase and GalNAc deacetylase sequences may share 85% or higher sequence identity with the mature enzyme. For example, the galactosamidase and GalNAc deacetylase sequences may share 75% or greater sequence identity with the mature enzyme. Alternatively, the galactosamidase and GalNAc deacetylase sequences may be modified in 5%, 10%, 13%, 15%, 20% or up to 25% of the amino acids.

[0074] As used herein, "adsorbed on glass, alginate beads or a matrix" refers to attaching an enzyme to the exterior of an inert material. Typically, this type of immobilization is not caused by a chemical reaction, and the active sites of the immobilized enzyme may be blocked by the surface to which it is adsorbed, which may reduce the activity of the adsorbed enzyme.

[0075] As used herein, "entrapment" refers to the capture of an enzyme within an insoluble bead or microsphere. However, entrapment can hinder the arrival of the substrate and the departure of the product. An example is the use of calcium alginate beads, which can be produced by reacting a mixture of a sodium alginate solution and an enzyme solution with calcium chloride.

[0076] As used herein, "cross-linking" refers to the covalent bonding of enzymes to each other to produce a matrix consisting almost entirely of enzymes. When designing a cross-linked enzyme reaction, the binding site ideally does not overlap the active site of the enzyme, so that the activity of the enzyme is only affected by the immobilization, rather than by the closure of the enzyme active site. However, spacer molecules (such as poly (ethylene glycol)) can be used to reduce the steric hindrance of the substrate.

[0077] As used herein, "covalent bonding" refers to the bonding of the enzyme to an insoluble support or surface (e.g., silica gel) via a covalent bond. Due to the strength of the covalent bond between the enzyme and the support or surface, the enzyme is much less likely to detach from the support or surface.

[0078] As used herein, "crowding agent" refers to any polymer or protein that promotes crowding of macromolecules by concentrating enzymes on the cell surface to improve enzyme activity. Crowding agents can be, for example, dextran, dextran sulfate, dextrin, pullulan, poly(ethylene glycol), polysucrose, TM , hyperbranched glycerol and inert proteins. (Kuznetsova, IM et al., Int J Mol Sci. (2014) “What Macromolecular Crowding Can Do to a Protein” 15(12): 23090–23140).

[0079] As used herein, "dextran" refers to a polysaccharide with a molecular weight ≥1,000 daltons and a linear backbone of d-glucopyranose repeating units connected in an α-linked manner. Dextran can be divided into three structural categories (i.e., Class 1-3) based on the pyranose ring structure, which contains five carbon atoms and one oxygen atom. Class 1 dextran contains an α (1 → 6) connected d-glucopyranose main chain modified with small side chains of d-glucose branches with α (1 → 2), α (1 → 3), and α (1 → 4) linkages. Class 1 dextran varies in their molecular weight, spatial arrangement, branching type and degree, and branch length, with 3-5 depending on the microbial production strain and culture conditions. Isomaltose and isomaltotriose are oligosaccharides with a Class 1 dextran backbone structure. Class 2 dextran (alternating) contains a backbone structure of d-glucopyranose units connected in alternating α (1 → 3) and α (1 → 6) with branches connected in an α (1 → 3) manner. Class 3 dextrans (mutans) have a backbone structure of consecutive α(1→3) linked d-glucopyranosyl units with α(1→6) linked branches.

[0080] As used herein, "pullulan" is a structural polysaccharide produced primarily from starch by the fungus Aureobasidium pullulans and consists of repeating α(1→6)-linked maltotriose (D-glucopyranosyl-α(1→4)-D-glucopyranosyl-α(1→4)-D-glucose) units with occasional maltotetraose units.

[0081] As used herein, "dextrin" refers to a chain length of D-glucopyranosyl units shorter than dextran, which begins with a single α(1→6) linkage but has a linear continuation of α(1→4) linked D-glucopyranosyl units.

[0082] As used herein, "dextran sulfate" is derived from dextran via sulfation.

[0083] As used herein, "polysucrose TM" is a neutral, highly branched, high-quality hydrophilic polysaccharide that is easily soluble in aqueous solutions.

[0084] Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

[0085] Materials and methods

[0086] Unless otherwise stated, chemicals and commercial enzymes used in this study were purchased from Sigma-Aldrich. TM The monosaccharide methylumbelliferyl glycoside was a generous gift from Dr. Hongming Chen, and the subtype 1 A antigen 五 -MU was a generous gift from Dr. David Kwan (Kwan et al., 2015).

[0087] Human fecal metagenomic library

[0088] To generate human metagenomic fosmid libraries, + Fresh human fecal samples were collected from healthy Asian male volunteers with different blood types. Direct DNA extraction and fosmid library creation were performed according to the procedures described in the MoE protocol (Armstrong et al., 2017).

[0089] Fosmid library screening

[0090] Place 51×384 holes AB + The blood Fosmid library plate was thawed at room temperature and replicated into a 384-well plate containing 50 μL of screening LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO4). The plate was incubated at 37°C for 18 hours in a sealed container containing a water reservoir to prevent excessive evaporation. The plate was then plated using QFill TM Instruments [Genetix TM ] 45 μl of reaction mixture (100 mM NaH2PO4, pH 7.4, 2% (v / v) Triton-X 100, 100 μM GalNAc-α-MU, 100 μM Galα-MU) was added to the growing screening plate. The plate was then incubated at 37°C in a sealed container for 24 h and read at 1, 2, 4, 8 and 24 h using a Synergy H1 plate reader [BioTek TM ] The fluorescence of each plate was measured (Ex: 365 nm, Em: 435 nm, scanning mode, gain 80). For all wells, a Z score was calculated, which was given by the following formula: Z score = (fluorescence value - median) / standard deviation.

[0091] All positive hits above a certain threshold were re-arrayed in a new 384-well plate, referred to as the “simple substrate hit” plate, and stored at −70° C. Two replicate screening plates were created from the “simple substrate hit” plate and re-screened for GalNAc-α-MU or Gal-α-MU activity to validate and deconvolute the previously detected activity.

[0092] To determine which hits could cleave the A or B antigen structures, a coupled enzyme assay was used to determine their activity against 50 μM of subtype 1 A antigen. 四 -MU or 50 μM subtype 1 B antigen 四 -MU activity. A version of this coupled assay was previously described by Kwan (Kwan et al., 2015). Our assay was modified to also detect cleavage of subtype I A antigen by using BgaC (Jeong 2009) instead of BgaA (Singh 2014) as the coupling enzyme. Potential α-N-acetylgalactosaminidase or α-galactosidase will cleave the terminal sugar, releasing the subtype IH antigen. 三 -MU. Subsequently, α-fucosidase (AfcA (Katayarna 2004)), β-galactosidase (BgaC (Jeong 2009)), and β-hexosaminidase (SpHex (Williams 2002)) cleave the remaining sugars in an outward manner until 4-methylumbelliferyl alcohol is released; this is detectable as an increase in fluorescence. To achieve this, 50 μg / mL of each enzyme is added to the reaction mixture. All positive hits above a certain threshold are rescreened in triplicate, and a host cell line containing a vector lacking any insert is used as a negative control. All validated hits are stored separately at -70°C in LB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 15% (v / v) glycerol, 0.2% (v / v) maltose, 10 mM MgSO4).

[0093] Fosmid hit sequencing

[0094] To isolate fosmid DNA for sequencing, 5 mL of TB medium (12.5 μg / mL chloramphenicol, 25 μg / mL kanamycin, 100 μg / mL arabinose, 0.2% (v / v) maltose, 10 mM MgSO4) was incubated overnight at 37°C and 220 rpm using GeneJet. TM Plasmid miniprep kit (Thermo Fisher TM ) for Fosmid separation. Use Plasmid-SafeTM ATP-dependent DNA enzyme (Epicentre TM ) Fosmid was purified from contaminated linear E. coli DNA and then isolated using GeneJet TM PCR purification kit (Thermo Fisher TM ) for another round of purification. TM Fluorometer (ThermoFisher TM ) using Quant-iT TM dsDNA HS Assay Kit (Invitrogen TM ) The concentration was calculated. The expected DNA size was verified on a 1% agarose gel. For complete fosmid sequencing, 2 ng of each fosmid was sent to the UBC Sequencing Center (Vancouver, BC, Canada). TM Each fosmid is individually barcoded and sequenced by the system.

[0095] Use on GitHub TM The python script available on (https: / / github.com / hallamlab / FabFos) is used for all Illumina MiSeq TM The raw sequence data were trimmed and assembled. Briefly, Trimmomatic was used to remove adapters and low-quality sequences from the reads (Bolger 2014). These reads were filtered for vector and host sequences using BWA (Li 2013) and then analyzed using Samtools. TM The high-quality and purified reads were assembled by MEGAHIT with k-mer values ​​ranging from 71 to 241 in increments of 10 (Li 2015). Since these libraries typically have over 20,000-fold coverage and to prevent the accumulation of sequencing errors that would interfere with the assembly of correct sequences, a minimum k-mer multiplicity was calculated using 1% of the estimated coverage of the fosmid. More than one contig was then generated using the minimus2 (Treangen 2011) python script in addition to the python script assembly. Parameterized commands can be found on GitHub. TM page and the documentation for the Python script itself.

[0096] Fosmid ORF prediction and hit validation

[0097] Using Prodigal TMFosmid ORFs were identified using the metagenomic version of (Hyatt 2010) and used as a MetaPathway TM BLASTP as part of the v2.5 package (Konwar 2015) TM CAZy TM MetaPathway TM Parameters: length>60, BLAST score>20, blast score ratio>0.4, E 值 <1×10-6.

[0098] Using Golden Gate TM Cloning strategy (Engler 2008) All predicted ORFs with annotations for GH or CBM family members (with known or suspected α-galactosidase and / or α-N-acetylgalactosaminidase activity) were cloned into the pET16b plasmid. The primer sequences are listed in Table B. Proteins were expressed in BL21 (DE3) cells cultured at 37°C and 220 rpm for 20 h in 10 mL of ZY5052 autoinduction medium (Studier 2005). Cells were harvested by centrifugation (4000 × g, 4°C, 10 min) and resuspended in 1 mL of lysis buffer (100 mM NaH2PO4, pH 7.4, 2% (v / v) Triton-X). TM 100, 1× protease inhibitor without EDTA [Pierce TM ]). The cells were stained with 50 μL of assay buffer (100 mM NaH2PO4, pH 7.4, 50 μg / mL Sphex, 50 μg / mL AfcA, 50 μg / mL BgaC, 100 μM subtype 1 A antigen). 四 -MU or 100 μM subtype 1B antigen 四 The coupling assay was performed with 50 μL of crude cell lysate of the candidate (-MU) mixed and incubated at 37°C (Kwan 2015). All reactions were performed in triplicate on a black 96-well plate. TM H1 plate reader [BioTek TM ] Fluorescence (365 / 435 nm) was continuously monitored for 4 hours. The assay was repeated from crude extracts showing A or B antigen cleavage activity, this time without the coupled enzyme, and the reaction products were separated via HF Bond Elut C18 columns and analyzed by LC-MS and / or TLC. TLC silica gel 60 F254 TLC plates [EMD Millipore Corp. TM , Billerica, Ma, USA] for TLC.

[0099] Table B: Primer sequences

[0100]

[0101]

[0102] HPAE-PAD assay

[0103] Analysis of enzymatic release of galactosamine was performed on HPAE-PAD (Dionex TM The cleavage activity of the different proteins was tested on the following substrates: 7.5 μg / μL type II mucin from porcine stomach in 100 mM NaH2PO4 (pH 7.4); 5 mM subtype 1 A antigen in 100 mM NaH2PO4 (pH 7.4); 五 -MU and RBCs from A+ donors, B+ donors, and O donors (50% hematocrit) in 1× PBS (pH 7.4). Samples containing 10 μg / mL enzyme were incubated at 37°C for 2 hours and then stored at -80°C for further analysis. A small aliquot of the reaction (10 μl) was diluted in H2O (100 μl) and analyzed on a HPAE-PAD instrument. The reaction was performed on a CarboPac PA200 with a guard column. TM The separation was performed on a (150 mm) column and detected using disposable gold and four potential waveforms on polytetrafluoroethylene (PTFE) electrodes. The separation conditions were as follows: a 100 mM sodium hydroxide and sodium acetate gradient from 70 to 300 mM within the first 10 minutes of separation. The eluent was maintained at the final gradient conditions for 1 minute and then returned to the starting conditions within the next minute. The flow rate was 1.0 ml / min and injections were performed every 27 minutes. Standards of the free sugars GalNAc, Gal1 and GalN (10 μM) were also applied to the HPAE-PAD to determine the peak elution time for reference.

[0104] Kinetic assay

[0105] All kinetic determinations using 4-methylumbelliferone as leaving group were performed by fluorescence measurements. In order to avoid measurement errors based on inner filter effects (Palmier 2007), a standard curve was used to verify the linear range of the fluorophore.

[0106] Fp galactosamidase

[0107] Subtype 1 GalN antigen was determined in 100 mM NaH2PO4 (pH 7.4) at 37°C. 五 -MU and subtype 1 A antigens 五-MU Michaelis-Menten parameters. Reactions were performed in 100 μL with 3.4 nM Fp galactosaminidase (5.31 nM FpGalNase_truncated) and 0.1 mg / mL Sphex, AfcA, 0.2 mg / mL BgaC and varying concentrations of substrate (5 μM-2 mM). Reactions were run in a series of four replicates, with the control (no Fp galactosaminidase) performed in duplicate. The reaction was performed by Synergy H1 TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a MU standard concentration curve determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM The kinetic parameters were determined by plotting the graphs.

[0108] Determine subtype 1 / 2 / 4 GalN antigen at pH 7.4 and 37°C 四 -MU and subtype 1 B antigens 四 -MU's K cat / K M Parameters. Reactions were performed in black 96-well plates (total volume of 100 μL) and were performed as coupled assays in 100 mM NaH2PO4 (pH 7.4) with 8.63 nM Fp-galactosaminidase, 0.1 mg / mL Sphex, BgaC (BgaA of isoform 2), AfcA, and varying concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM). Reactions were run in a series of four replicates, with the control (no Fp-galactosaminidase) performed in duplicate. The assay was performed by Synergy H1 TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a MU standard concentration curve determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM Make a graph to determine k cat / K M (s -1 *mM -1 )parameter.

[0109] The Michaelis-Menten parameters of GalN-α-pNP were determined in a volume of 100 μl in a transparent 96-well plate at 37°C in the presence of 863.2 nM Fp galactosaminidase (in 100 mM NaH2PO4, pH 7.4) or 369.9 nM FpGH4 (in 50 mM Tris / HCl (pH 7.4), 100 μM NAD+, 1 mM MnCl2) and different concentrations of substrate (10 μM-5 mM). The reactions were run in a series of three replicates, with two controls (no enzyme). The assay was performed by Synergy H1 TM Plate reader [BioTek TM The absorption (at 405 nm) generated by the hydrolysis of pNP was monitored and converted to concentration using a standard concentration curve of p-nitrophenol determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM The kinetic parameters were determined by plotting the graphs.

[0110] FpGalNAc deacetylase

[0111] Subtype 1 A antigen was determined using a previously described coupled assay (Kwan 2015) at 37°C in 100 mM NaH2PO4 (pH 7.4). 五 -Michaelis-Menten parameters for MU. The assay was modified to allow detection of cleavage of subtype 1 (and later 4) by using BgaC (Jeong 2009) instead of BgaA (Singh 2014) as β-galactosidase. In addition, since subtype 1 A antigen 五 -MU contains additional galactose, and the concentration of BgaC is increased to 0.2 mg / mL to compensate for its need to cleave both Gal-β-1,3-β-GlcNAc-β-1,3-Gal-β-MU and Gal-β-MU. In addition, Fp galactosaminidase is included to allow cleavage of galactosamine-containing intermediates. The reaction setup in 100 μm is 3 nM FpGalNAc deacetylase (4.52 nM FpGalNacDeAc_D1ext, 3.55 nM FpGalNacDeAc_D1+2) and 0.01 mg / mL Fp galactosaminidase, 0.1 mg / mL Sphex, AfcA, 0.2 mg / mL BgaC and different concentrations of substrate (5 μM–2.5 mM). The reaction was performed in a series of four replicates, with the control (no FpGalNac deacetylase) being performed in duplicate. The reaction was performed by Synergy H1 TM Plate reader [BioTek TMThe fluorescence signal (365 / 435 nm) generated by the hydrolytic release of MU was monitored and converted to concentration using a standard MU concentration curve determined under the same reaction conditions. The initial rate (μM / s) was determined and plotted in Grafit 7.0 to determine the kinetic parameters.

[0112] Determine subtype 1 / 2 / 4A antigens at pH 7.4 and 37°C 四 -MU's K cat / K M Parameters. Reactions were performed in black 96-well plates (total volume of 100 μL) and were performed as coupled assays in 100 mM NaH2PO4 (pH 7.4) with 12 nM FpGalNac deacetylase, 0.1 mg / mL Sphex, BgaC (BgaA of subtype II), AfcA, and different concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM). Reactions were run in a series of four replicates, with the control (no FpGalNac deacetylase) performed in duplicate. The assay was performed by Synergy H1 TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a MU standard concentration curve determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM Make a graph to determine k cat / K M (s -1 *mM -1 )parameter.

[0113] Dynamics of GH109 isoforms

[0114] Determine subtype 1 / 2 / 4A antigens at pH 7.4 and 37°C 四 -MU's K cat / K MParameters. Reactions were performed in black 96-well plates (total volume of 100 μL) and were performed as coupled assays in 100 mM NaH2PO4 (pH 7.4) with 86.02 nM BvGH109_1 / 100.49 nM EmGH109 / 80.52 nM BvGH109_2 / 87.4 nM BsGH109 and 5 μM NAD+, each 0.1 mg / mL of Sphex, BgaC (BgaA of subtype 2), AfcA, and different concentrations of substrate (25 μM, 20 μM, 15 μM, 10 μM, 7.5 μM, 5 μM). Reactions were run in a series of four replicates, with the control (no α-N-galactosaminidase) performed in duplicate. The assay was performed by Synergy H1 TM Plate reader [BioTek TM The fluorescence signal (365 / 435 nm) generated by the release of MU by hydrolysis was monitored and converted to concentration using a MU standard concentration curve determined under the same reaction conditions. The initial rate (μM / s) was determined and analyzed in Grafit7.0. TM Make a graph to determine k cat / K M (s -1 *mM -1 )parameter.

[0115] Crystallography

[0116] Prior to crystallization, thrombin (Novagen TM ) was digested with FpGalNAcDeAc_D1ext overnight. The protein was then purified by HisTrap FF column and the flow-through was collected, buffer exchanged into 10 mM Tris (pH 8.0) + 75 mM NaCl, and concentrated to 12 mg / mL.

[0117] crystallization

[0118] Use hanging drop diffusion method, use by 0.2M CaCl , 0.1M MES (pH 6), 18% PEG 4000 and 20mM MnCl the storage solution of composition with 1: 1 protein: storage solution ratio, make FpGalNAcDeAc_D1ext (12mg / mL) crystallization.Use fast bromide soaking to derive the crystal for phase determination, and by crystal is transferred to 1M NaBr, 25% glycerol, 18% PEG4000, 20mM CaCl with 0.1M Mes pH solution 30 seconds and prepare in liquid nitrogen fast freezing.Under the same condition as above but omit MnCl before setting droplet, there is the crystal complex of B blood type antigen trisaccharide (B_ three) by by albumen (12mg / mL) and 10mM B_ three pre-incubation 2 hours prepare.Crystal is cryoprotected with the storage solution that is supplemented with 25% glycerol.

[0119] Data acquisition, phasing, and structure determination

[0120] Canadian Light Source TM (Canadian Light Source TM ) were collected. The data were integrated using XDS (Kabsch 2010) and Aimless TM (Evans 2013) Determine the ratio using CRANK2 TM (Skubak 2013) in CCP4I2 TM Phase determination and automated structure solution were performed using the Coot TM (Emsley 2004) and Refmac TM (Vagin 2004) by alternating cycles of checking and refining the structure. The B_tri-structure complex was solved by the difference Fourier method, and the ligand was TM Manually constructed in Coot, water and metal ions are also TM The difference density map confirmed the presence of Mn in the apo structure. 2+ and the presence of Ca in the ligand structure 2+ By Coot TM and Molprobity TM The model was validated by (Chen 2010). The atomic coordinates and structure factors of the apo and B_tri complex have been deposited in the Protein Data Bank (PDB) with accession numbers:

[0121] SEQ ID NO: WP_009260926.1 of the GalNAc deacetylase protein of Fusobacterium prausnitzii; and

[0122] SEQ ID NO: WP_044942952.1 of the galactosamidase protein of Fusobacterium prausnitzii

[0123] Active site mutagenesis

[0124] Based on structural information (not shown) and sequence alignment (not shown), the Quickchange TM Protocol (Zhang 2004), using the primers shown in Table B, FpGalNAcDeAc_D1min and FpGalNase_Truncate were mutated. Mutants were purified via NiNTA and HIC columns as described above. The structural integrity of all mutants was checked via CD spectroscopy; all tested enzymes were structurally similar to their wild-type counterparts. For mutants with relatively low activity, reactions were performed under the same conditions used for the full kinetic assay; however, kcat / KM values ​​were determined using the substrate consumption method as previously described (Vocadlo 2002). Briefly: [substrate] <K M (equivalent to K m At low substrate concentrations (~1 / 5-1 / 10 of the cat / K M Values ​​can be approximated by nonlinearly fitting the reaction time course to a first-order curve and dividing by the enzyme concentration.

[0125] GH36 phylogeny

[0126] Using Saccharis TM cazy_extract.pl script (Jones 2018) from CAZy TM The reference sequence of GH36 was downloaded from the database. The protein analysis software TreeSAPP based on phylogeny was used. TM (available at https: / / github.com / hallamlab / TreeSAPP) to build reference trees and map sequences to these trees. Briefly, HMMs from dbCAN were used to map sequences from CAZy TM (Yin 2012) All full-length sequences were downloaded to extract protein family domains. Then, UCLUST was used to TM The sequences were clustered at 70% sequence similarity to remove redundant sequence space and reduce the size of the tree (Edgar 2010). TM 8.2.0 version to build reference trees with "--autoMRE" to decide when to exit bootstrapping before 1000 repetitions, and PROTGAMMAAUTO TM The best protein model was selected (Stamatakis 2006 and Stamatakis 2008).

[0127] Then use TreeSAPP TM Map the query sequence onto these reference trees. Briefly, use hmmsearch TM Align protein sequences with HMMs and extract aligned regions (Eddy 1998). hmmalign TM is used to include new query sequences in the reference multiple alignment, and then TrimA1 TM Non-conserved positions were removed from the alignment file (Capella-Gutierrez 2009). TM The query sequence is sorted by insertion in the reference tree. The position of each query sequence is filtered and concatenated into a single query sequence. TM Jplace before visualization TM documents (Matsen 2012 and Letunic 2016).

[0128] RBC determination

[0129] Whole blood from healthy consenting donors was collected into citrate vacuum blood collection tubes using a protocol approved by the Clinical Ethics Committee of the University of British Columbia. The tube was spun at 1000 × g for 4 min at RT to separate RBCs and washed 3 times with 1 × PBS (pH 7.4). For the assay in the presence of dextran 40k, washed RBCs (200 μL, 10% hematocrit) were placed in a tube, the supernatant was partially removed, and replaced with 1 × PBS (pH 7.4) containing or without dextran 40k (final concentration of 300 mg / mL). In addition, some assays were performed in 1 × PBS (pH 7.4) + 25% plasma or 100% plasma. The RBCs were carefully mixed and placed on an orbital shaker for 30 s. The diluted enzyme solution was then added to a final volume of 200 μL. The tube was vortexed very lightly and placed on an orbital shaker for a defined time at a set temperature.

[0130] MTS card

[0131] After the reaction, RBCs were washed three times with excess 1× PBS (pH 7.4) and lysed using a microprinting system. TM (MicroTyping System TM ,MTS) card [MTS TM, Florida, USA] were analyzed. RBCs (12 μl, 5% hematocrit) suspended in diluent [MTS, Florida, USA] were carefully added to the microgel column, leaving space between the blood and the contents of the microgel. A Beckman Coulter AllegraX-22R with a modified sample holder was used as recommended. TM Centrifuge the MTS card at 156 × g for 6 min at RT. Assess the extent of antigen removal from the RBC surface based on the position of the RBCs in the mini-gel after spinning according to the manufacturer's instructions. RBCs with high surface antigen concentrations aggregate and become impermeable when interacting with the monoclonal antibodies present in the gel column (MTS TM RBCs without surface antigens did not agglutinate and migrated to the bottom of the minigel (MTS score of 0). RBCs with partially depleted surface antigens that migrated to a position between these antigens were assigned a score of 0 (absent) to 4 (present) according to the manufacturer's instructions.

[0132] H antigen agglutination assay

[0133] To analyze the conversion of A antigen to H antigen after enzymatic treatment, washed A-ECO-RBC were incubated in equal parts with 2 μg / mL of anti-H antibody (anti-H ab blood group antigen antibody [97-1]: catalog number: ab24213 (Abcam TM )) were mixed and the agglutination phenomenon was monitored over a period of 30 minutes. RBCs agglutinated with anti-H antibodies were given a score of 0 (no agglutination within 1800 sec) to 5 (agglutination within 120 sec).

[0134] FACS

[0135] The enzyme-treated RBCs were washed twice with 1×PBS (pH 7.4), and ECO-RBCs with a 1% hematocrit were incubated with 1 / 100 APC-anti-A antibody (Alexa Fluor TM 647 Mouse Anti-Human Blood Type A: Catalog Number: 565384 (BD Pharmingen TM )) and / or anti-H antibody (anti-H ab blood group antigen antibody [97-1]: Catalog number: ab24213 (Abcam TM )) were treated at RT for 30 minutes and then washed twice with 1× PBS (pH 7.4). To detect anti-H antibodies, a secondary FITC-labeled antibody (goat F(ab')2 anti-mouse IgM mu chain (FITC)) was used at a concentration of 1 / 500: Catalog number: ab5926 (Abcam TM )). Using flow cytometer (CytoFLEX TM(Beckman Coulter TM )) After reconstitution into 1×PBS (pH 7.4) (1% hematocrit), the data were evaluated.

[0136] Enzyme adsorption and antigenicity

[0137] To test whether the enzymes could be easily removed from RBCs after treatment, potential adsorption was assessed. Pacific Blue labeled FpGalNAc deacetylase and FpGalNase (F / P = 1) were incubated with RBCs individually at 37°C for 1 h and after several washing steps, the enzymes were analyzed using a flow cytometer (CytoFLEX). TM (Beckman Coulter TM )) to measure the residual fluorescence.

[0138] Antigenicity was tested by incubating RBCs with 50 μg / mL of each enzyme and mixing the enzyme-treated RBCs with allogeneic or autologous serum to observe potential agglutination. In addition, to assess potential anti-IgG, -C3d exposure, anti-IgG, -C3d MTS TM Card [MTS TM , Florida, USA]. The treated RBCs were tested on a 37°C incubation time of 30 minutes.

[0139] Antigen subtype synthesis

[0140] Subtype 1 / 2 / 4A and B antigens 四 -MU synthesis was performed using a modified protocol as described by Kwan (Kwan et al., 2015).

[0141] Two-step subtype 1 / 2 / 4H antigen 三 -MU synthesis

[0142] All three syntheses were performed on a 20 mg GalNAc-α-MU / GlcNAc-α-MU scale in 10 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl, 50 U alkaline phosphatase, 1.5 equivalents of UDP-Gal, and 1.2 equivalents of GDP-Fuc (ratio determined by LacNAc-MU product). Depending on the desired product, different glycosyltransferases were added at a concentration of 100 μg / mL: isoform I CgtB S42 and Te2FT, isoform II HP0826 and WbgL, and isoform IV LgtD and Te2FT. Reactions were performed at 37° C. and progress was monitored by TLC (mobile phase, EtAc:MeOH:H2O, 6:2:1 ratio). 4-Methylumbelliferone was hydrolyzed from the compound by 10% H2SO4 and detected by UV (360 nm). After no further product increase was observed, the reaction was applied to a HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product eluted with 25% methanol. The solvent was then removed in vacuo.

[0143] Subtype 1 / 2 / 4 A antigen 四 -MU synthesis

[0144] The final synthesis step was performed at 37°C with 10 mg of subtype 1 / 2 / 4 H antigen in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 10 mM MnCl2, 25 U of alkaline phosphatase, 1.5 equivalents of UDP-Gal, and 100 μg / mL of BgtA. 三 The reaction was carried out on a 1.5-MU scale. Progress was followed by TLC. After no further product increase was observed, the reaction was applied to an HF Bond Elut C18 column, washed with several column volumes of 5% methanol, and the product eluted with 25% methanol. The solvent was then removed in vacuo. The final product was further purified on a 1.5 x 46 cm HW-40F size exclusion column and then freeze-dried.

[0145] Subtype 1 / 2 / 4 B antigen 四 -MU synthesis

[0146] The final synthesis step was carried out at 37°C with 10 mg of subtype 1 / 2 / 4H antigen in 5 mL of 50 mM Tris / HCl, 200 mM NaCl, pH 7.4, 25 U of alkaline phosphatase, 1.5 equivalents of UDP-Gal and 100 μg / mL of BoGT6a. 三The reaction was carried out on a 1.5 x 46 cm HW-40F size exclusion column and then freeze-dried.

[0147] Subtype 1 GalN antigen 五 -MU synthesis

[0148] 10 mg of subtype 1 A antigen 五 -MU was incubated with 1 μg / mL FpGalNAc deacetylase in 5 mL of 100 mM NaH2PO4 at 37°C for 30 min, then stopped by adding 1 mM EDTA. Complete conversion of the substrate was checked by TLC, and the reaction was applied to a HF Bond Elut C18 column, washed with several column volumes of 2% methanol, and the product was eluted with 10% methanol. The solvent was then removed in vacuo.

[0149] Protein purification

[0150] Via Golden Gate TM All proteins and their truncations were cloned into pET16b or pET28a by cloning (Engler 2008) or PIPE cloning (Klock 2008). Primer sequences are listed in Table B.

[0151] Proteins for extended characterization were produced in BL21 (DE3) cells in 200 mL of ZY5052 auto-induction medium (Studier 2005) at 37°C, 220 rpm for 20 h and inoculated with 100 μl of an overnight LB culture. Cells were harvested by centrifugation (4000 × g, 40°C, 10 min) and resuspended in 10 mL of lysis buffer (50 mM Tris / HCl, 150 mM NaCl, 1% (v / v) glycerol, 40 mM imidazole, pH 7.4, 2 mM DTT, 1× EDTA-free protease inhibitors (Pierce TM ), 2U Benzonase (Novagen TM ), 0.3 mg / mL lysozyme, 10 mM MgCl2), and then sonicated on ice (3 min pulse time; 5 sec pulse, 10 sec pause, 35% amplitude). After removing cell debris by centrifugation (14000 × g, 4 ° C, 30 min), the supernatant was collected and loaded onto a nickel affinity chromatography column (5 mL HisTrap HP TM Column (GETM )) on AEKTApurifier TM System (GE TM Elution was performed and monitored using a 10-75% gradient of 50 mM Tris / HCl, 400 mM imidazole, pH 7.4, 2 mM DTT, and protein-containing fractions were identified by SDS-PAGE and then pooled. TM MWCO 10kDa (Millipore TM ), the buffer was exchanged into 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, 2 mM DTT, and concentrated.

[0152] FpGalNAc deacetylase, Fpgalactosaminidase and their truncations had to undergo a second round of purification before the proteins were loaded onto a hydrophobic interaction chromatography column (10 mL of Phenyl Sepharose High Performance Column (Pharmacia Biotech TM )) Before use, use Amicon Ultra-15 centrifugal filter device TM MWCO 10kDa (Millipore TM ) exchange buffer. The column was loaded, washed and eluted (gradient 0-100%) by AEKTApurifier TM System (GE TM ) were treated using the following buffer conditions: FpGalNAc deacetylase; binding 1×PBS, 800mM NH2PO4, pH 7.4 and elution 1×PBS (pH 7.4) and Fpgalactosaminidase; binding 25mM Tris / HCl, 1M NaCl, pH 7.4 and elution 25mM Tris / HCl (pH 7.4). Protein-containing fractions were identified by SDS-PAGE and then pooled. The protein was isolated and purified in an Amicon Ultra-15 centrifugal filter device. TM MWCO10kDa(Millipore TM ), the buffer was exchanged into 50 mM Tris / HCl, 150 mM NaCl, pH 7.4, and concentrated.

[0153] Protein characterization

[0154] Optimal pH

[0155] For subtype 1 A antigen 五 -MU and subtype 1 GalN antigens 五The typical pH ranges for the activities of MU, FpGalNAc deacetylase, and Fpgalactosaminidase were determined by the appearance of products on TLC plates that responded to changes in pH. Reactions were performed at 37°C using 50 μM substrate and 1 μg / mL enzyme in the appropriate buffer system at a 100 μl scale. The buffer for pH 4-6 was based on 50 mM citric acid / sodium citrate buffer, the buffer for pH 6-8 was based on 50 mM sodium phosphate buffer, and the buffer for pH 8-10 was based on 50 mM glycine / sodium hydroxide buffer.

[0156] To determine the optimal pH, 5 μg / mL of Fp-galactosaminidase was incubated in 100 μL of 50 mM sodium phosphate buffer with different pH ranges (5.8-8.0) and 200 μM of GalN-α-pNP. The absorption (at 405 nm) generated by the release of pNP was measured by Synergy H1 TM Plate reader (BioTek TM ) were monitored at 37°C for 1 h.

[0157] 5 μg / mL FpGalNAc deacetylase and 50 μM subtype IA antigen 五 -MU was pre-incubated in 25 mM sodium phosphate buffer with different pH ranges (5.8-10.0) at 37 ° C for 10 min. The reaction was quenched with 100 mM sodium phosphate buffer (pH 7.5), 100 μM EDTA, 5 μg / mL Fp galactosaminidase, 50 μg / mL Sphex, 50 μg / mL AfcA and 50 μg / mL BgaC (final volume of 100 μl). The fluorescence signal (365 / 435 nm) generated by the hydrolysis of MU was detected by Synergy H1 TM Plate reader (BioTek TM ) were monitored at 37°C for 30 min.

[0158] Protein stability

[0159] FpGalNAc deacetylase and FpGalNase were stored in 1× PBS buffer (pH 7.4) at 4° C. After 2 and 12 weeks, subtype IA antigens were detected in the coupled enzyme reaction for FpGalNAc deacetylase. 五 The enzyme activity was tested according to the optimal pH conditions for the coupled enzyme reaction of GalN-α-pNP with FpGalNase.

[0160] Inhibition of FpGalNAc deacetylase

[0161] FpGalNAc deacetylase was tested against various potential inhibitors as a coupled assay in a 96-well plate format. Reactions were performed at 37°C on a 100 μL scale with 50 μM subtype 1 A antigen in 100 mM NaH2PO4 (pH 7.4) with 10 μg / mL FpGalNAc deacetylase, 50 μg / mL Sphex, 50 μg / mL AfcA, and 50 μg / mL BgaC. 五 As inhibitors, EDTA (1, 10, 100 μM), Marimastat (1, 10, 100, 1000 μM), DMSO (2%, 4%), EDTA-free protease inhibitor cocktail (Pierce TM )(1×, 2× and 4×). Using Synergy H1 TM Plate reader (BioTek TM ) Fluorescence (365 / 435 nm) was monitored continuously for 1 hour. Additives showing strong effects were run again without the coupled enzyme and analyzed for product formation via TLC.

[0162] Limited proteolysis

[0163] To investigate whether a smaller, stable subdomain of Fp galactosaminidase exists, limited proteolysis was performed. Fp galactosaminidase was treated with thermolysin (10:1 protein:protease mass ratio) for 1.5 hours at various temperatures (20°C, 37°C, 42°C, 50°C, and 65°C). The samples were then run on SDS-PAGE gels, and a stable fragment running at approximately 70 kDa (down from an initial 118 kDa) was identified, with nearly complete digestion achieved at an incubation temperature of 50°C. This fragment was sent to the UBC Proteomics Core Facility for peptide identification and was determined to be a C-terminal truncated form of the full-length protein with a cleavage site between amino acids 690-700.

[0164] Glycan array screening

[0165] For glycan array screening, use Fluorotag TM FITC conjugation kit (Sigma TM ) 500 μg of FpGalNAcDeAc_D2ext was labeled with fluorescein isothiocyanate (FITC) at an F / P ratio of 1. Screening was performed at the CFG Protein-Glycan Interaction Core Facility TM (the CFG's Protein-Glycan Interaction Core Facility TM) was performed in a 5.3-well plate using the 5.3-well plate, consisting of 600 glycans in six replicate samples at protein concentrations of 5 μg / mL and 50 μg / mL. Binding motif analysis was performed using a web-based tool from Emory University (https: / / glycopattern.emory.edu / ). Example

[0166] Example 1: Metagenomic library construction and screening

[0167] We constructed a metagenomic library containing + Large (35-65 kb) DNA fragments extracted from fecal samples provided by male donors with different blood types. Such libraries contain multiple genes per bacterium, which increases the probability that at least some of these genes are expressed and allows the expression of small "pathways" of multiple genes. Our library contains ~19,500 clones in 51×384 well plates, and potentially ~800,000 genes, so initial screening of such libraries with expensive A antigen substrates is impractical. Instead, we first screened with simple, sensitive, fluorescent substrates - galactose and methylumbelliferyl α-glycosides of N-acetyl-galactosamine (Gal-α-MU and GalNAc-α-MU). This initial screen produced a subset of 226 hits with a mixture of the two substrates. These substrates were rescreened against each individual substrate, identifying 44 with GalNACase and 166 with galactosidase activity. Using Figure 1 A second round of screening of these hits was performed using a coupled enzyme assay (Kwan 2015) with the tetrasaccharide glycoside substrates A and B antigens shown in , along with a no-substrate control: the coupled enzyme can function and release MU only when the initial Gal or GalNAc is cleaved. Eleven of these hits contained A antigen cleavage activity, one of which also cleaved B antigen, while six produced fluorescence in the absence of substrate, thus encoding pathways that produce unrelated fluorescent products.

[0168] Example 2: Sequencing and initial analysis of hits

[0169] On Illumina MiSeq TM Eleven fosmids were sequenced and analyzed using Metapathway TM Software (Konwar 2015) identified the presence of CAZy TMORFs in the CAZy database (http: / / www.cazy.org / ) (Lombard 2014). Due to the considerable depth of human microbiome sequencing now available, the organisms from which all fosmids originated can be identified. Their sequences can be divided into five clusters, because eight of the eleven are derived from overlapping fragments of the genomes of just two Bacteroides sp. The only gene common to all fosmids in cluster B is the GH109 enzyme (B. vulgatus); cluster A also contains GH109 (B. stercoris), which is the only CAZy gene found in a fosmid derived from another Bacteroides genus (B. vulgatus). Fosmid No. 8 from the obligate anaerobe Fusobacterium prausnitzii (Li 2015) contains three ORFs found in CAZy: the apparent carbohydrate binding module CBM32 and two potential glycoside hydrolases - GH36 and GH4. Finally, fosmid K05 from Collinsella sp. (probably Collinsella tanakaei) does not contain a CAZy-associated ORF. Here, generation of a sublibrary of fosmid K05 allowed identification of an ORF with A-cleavage activity, subsequently identified as GH36 (not shown).

[0170] Example 3: Analysis of GH109 enzyme

[0171] The GH109 family was established based on the A antigen cleavage activity of several of its members. These enzymes use the unusual NAD + The GH109-dependent mechanism was first discovered in an enzyme from GH4 Add Yip Ref (2004) J. Amer. Chem. Soc., 126, 8354-8355, as this is the one that shows the mechanism (Varrot 2005; and Liu 2007). After removing the signal peptide, the three GH109 genes identified here were cloned with a His tag and expressed in Escherichia coli BL21 (DE3). The three proteins (BsGH109, ​​BvGH109_1 and BvGH109_2) were purified (not shown), as well as the classic GH109 from Elizabethkingia menosepticum (EmGH109) (Liu 2007) as a standard, and the kinetic parameters of each protein were determined. The three new enzymes showed similar catalytic efficiency in each of the three subtype A substrates tested, which largely reflected the kinetic parameters of the EmGH109 standard. In contrast, when using an approved MTS card in A +When their A antigen removal activity was tested on RBCs, disappointingly, only EmGH109 was significantly active. When tested in the presence of dextran 40K as a crowding agent, we have shown that activity is increased by concentrating the enzyme on the cell surface (Chapanian 2014). In its absence, even 150 μg / mL of EmGH109 was ineffective, whereas in the presence of 300 mg / mL dextran 40K, 15 μg / mL of enzyme was sufficient (see Figure 3 and Figure 4 ). Previous studies have shown that low ionic strength also enhances the activity of EmGH109 on cells (Liu 2007). Therefore, EmGH109 is ineffective in whole blood.

[0172] Example 4: Analysis of GH36 from Fosmid K05 of Collinsella

[0173] The GH36 protein identified in fosmid K05 (designated K05GH36) exhibits activity against GalNAc-α-MU and the A antigen tetrasaccharide. This is consistent with membership in the GH36 family, which primarily contains α-galactosidases and α-N-acetylgalactosaminidase enzymes and hydrolyzes via a double displacement mechanism involving a covalent β-glycosylase intermediate (Comfort 2007). Phylogenetic analysis places its sequence within cluster 4 of the GH36 subfamily (Fredslund 2011). Interestingly, this cluster also contains the closely related characterized GH36 from Clostridium perfringens, which is also known to cleave the A antigen structure (Calcutt 2002). However, when K05GH36 was tested for its ability to remove A antigen from red blood cells, its activity was disappointing, with a score of only 3 even when used in combination with a crowding agent.

[0174] Example 5: Analysis of Fosmid No. 8 from Fusobacterium prausnitzii

[0175] Since these new enzymes offered no advantages, our attention turned to the No8 fosmid from Fusobacterium plautii (F. plautii), particularly because its gene product cleaves both A and B antigens. Three CAZy-related genes were cloned, their signal peptide sequences removed, expressed in Escherichia coli BL21(DE3), and the resulting enzymes purified with yields as high as 140 mg / L. Surprisingly, when we tested the purified proteins individually against the A and B tetrasaccharide substrates, the only cleavage observed was by No8GH36 on the B antigen, while none of them cleaved the A antigen. We therefore tested combinations of these enzymes in pairs and surprisingly found that a mixture of No8CBM32 and No8GH36 rapidly cleaved the A antigen tetrasaccharide. TLC analysis of the reaction mixtures using the individual enzymes showed that No8CBM32 catalyzed the conversion of the A antigen to a more polar but still UV-active product, while the subsequent addition of No8GH36 released a sugar product that comigrated with galactosamine, as well as the H antigen trisaccharide. MS analysis of the reaction mixture showed that No8CBM32 is an A antigen deacetylase, resulting in a lower m / z of 42 and a more polar species, while No8GH36 is a galactosaminidase, a new activity in this family ( Figure 2 This was further confirmed by high performance anion exchange chromatography (HPAE-PAD) analysis of the reaction ( Figure 5 ), which showed that treatment of the A antigen with both enzymes released galactosamine, whereas the individual enzymes did not. Similar results were obtained with a gastric mucin substrate, for which the enzymes were presumed to release galactosamine. Therefore, these two enzymes are hereafter referred to as FpGalNAc deacetylase (FpGalNAcDeAc) and Fpgalactosaminidase (FpGalNase).

[0176] Although this pathway for degradation of A antigens has not been previously characterized, it is fascinating that an explanation was proposed over 50 years ago to explain the so-called “acquired” B phenomenon, in which the blood type of type A patients infected with Clostridium difficile serovars changed significantly to type B (Gerbal 1975), as did forensic samples of human tissue submerged in the Thames (Ref Judd and Annesley https: / / doi.org / 10.1016 / S0887-7963(96)80087-3, Transfusion medicine reviews (1996) 10, 111-117). This was presumably because the anti-B antibodies used for typing could not distinguish between terminal Gal and GalN.

[0177] Studies of the third enzyme in fosmid GH4 have shown that, although it hydrolyzes Gal-α-pNP, GalN-α-pNP, and GlcN-α-pNP, it does not cleave any A-antigen-based substrates. Therefore, it does not appear to play a direct role in the conversion of A-antigen. However, these glycosaminidase enzymes do represent novel activities within the GH4 family.

[0178] Example 6: Characterization of FpGalNAc deacetylase

[0179] Using Phyre2 TM (Kelley 2015) Closer bioinformatics analysis of this gene revealed a previously unknown function domain of ~308 amino acids at the N-terminus and a ~145 amino acid CBM32 near the C-terminus, with a linker region between them. Truncation analysis confirmed this basic structure, as all constructs containing the complete deacetylase domain were catalytically active (Table 2). Therefore, this protein was classified as a founding member of a new carbohydrate esterase family, CExx.

[0180] Acetamidosugar deacetylase has been shown to be a metalloenzyme that requires divalent metal ions (Blair 2005). Accordingly, treatment with 100 μM EDTA largely abolished the enzyme activity, while addition of Mn 2+ 、Co 2+ 、Ni 2+ or Zn 2+ Increased enzyme activity. Other (non-metallic) amidase inhibitors had no effect. The enzyme has a wide pH distribution with an optimum around pH 8 ( Figure 6 ) and has narrow substrate specificity, limited to the different A isoforms and their shorter forms. However, within those isoforms, it is not very discriminatory, with the specific activity between all of these isoforms varying only ~2-fold (Table 2). This pH dependence and specificity profile is ideal for RBC conversion, as all isoforms of A are deacetylated, but no other.

[0181] The specificity of the CBM portion of the protein was explored using glycan arrays from the Consortium for Functional Glycomics (CFG). Preferred targets were glycans with repetitive N-acetyllactosamine (LacNAc) structures, which are found in the founding member of the CBM32 family and also in the N-acetylglucosaminidase of Clostridium perfringens (Ficko-Blean 2006). However, unlike this CBM, ours did not show high-affinity binding to blood antigen structures. Repetitive LacNAc structures are common components of cell surfaces (Cohen 2009), serving as universal components of complex and hybrid N-glycans, as well as some O-glycans and glycolipids. In our case, they likely serve as anchors for the deacetylase domain. This would bring the catalytic domain into close proximity with the A antigen without competing for its own substrate. Supporting this model, domain removal resulted in reduced RBC activity without affecting the cleavage rate of soluble substrates (Table 2).

[0182] Example 7: Crystallization Analysis of FpGalNAc Deacetylase

[0183] To provide structural insight into the activity of this novel enzyme, crystallization experiments were conducted on a truncated protein, resulting in crystals diffracting to the finest resolution. Solution of this structure revealed a catalytic domain adopting a five-fold β-propeller structure, with an active site containing a divalent metal ion coordinated by D100 and H252. Co-crystallization of the enzyme with the B-antigen trisaccharide, a close analog of the reaction product, revealed its binding mode. Within the active site pocket, the non-reducing terminal galactosyl moiety serves as a distinguishing group between the A and B antigens, forming hydrogen-bonding interactions with H97, E64, and both metal-coordinated waters. The remainder of the ligand is surface-exposed, and polar interactions are identified between the fucosyl group and the side chains of S61 and D121. The C1-OH group of the reducing terminal galactosyl moiety is solvent-exposed, making the enzyme readily adaptable to substrate extension (i.e., with GlcNAc). Mimicking the N-acetyl group of the A-trisaccharide in this structure allowed for rational mutation of nearby amino acids potentially involved in substrate deacetylation. The present invention relates to the enzyme that the FpGalNAc deacetylase of the present invention.Because two mutants are all inactive, therefore residue E64 is proved to be key for activity, and this shows the direct effect (table 1) that may be in the activation of nucleophilic water molecule.The residue D100, Y315 and the H252 of coordination divalent metal are also proved to be important, and wherein any sudden change causes~5000 times of speed reduction, consistent with their apparent effect in conjunction with divalent metal ion.Similar to other acetylamino sugar deacetylases, we propose that FpGalNAc deacetylase is hydrolyzed by following mechanism, and wherein metal is used for polarized carbonyl and activated water molecule forms tetrahedral intermediate with nucleophilic attack carbonyl.Proton supply to sugar nitrogen atom by His 100 promotes the decomposition of this intermediate.

[0184] Table 1 | FpGalNAcDeAc_D1min and its mutants cleave type 2 A antigen 四 -MU specific activity

[0185]

[0186] ND = no detectable activity

[0187] Example 8: Characterization of FpGalNAcDeAc and FpGalNase

[0188] Phylogenetic analysis of the sequence placed FpGalNase in a new subgroup of the GH36 family (5) (Fredslund 2011). The 390 amino acid catalytic domain is located in the center of this large (1079 amino acid) protein, with a potential carbohydrate binding domain at the C-terminus. Removal of this C-terminal domain had no effect on the kinetic parameters of the enzyme with soluble substrates (Table 2), but resulted in a decrease in the deacetylated A +The cleavage efficiency of RBC was reduced. The enzyme is specific for galactosamine-containing sugars and will not cleave GalNAc residues in any context tested. However, it has a fairly broad specificity for cleavage of de-N-acetylated galactosamines from simple aryl glycosides GalN-α-pNP upwards. In fact (Table 2) the k cat / K M values ​​with each other and with those of the deacetylase k cat / K M The values ​​are similar. cat / K M The value is more than 2000-fold lower than that of the corresponding GalN antigen, but still sufficient to generate a positive hit in the original screen. This specificity for deacetylated α-galactose conformation substrates, combined with its pH optimum of ~6.5-7.0, is well suited for blood group conversion in conjunction with deacetylase ( Figure 6 ).

[0189] Table 2 | Kinetic parameters of FpGalNAcDeAc and FpGalNase constructs with different antigenic substrates

[0190]

[0191] Example 9: Cleavage of A antigen from RBCs

[0192] A + 、B + and O + RBCs were incubated with FpGalNAcDeAc and FpGalNase individually and in mixtures, and the released sugars were analyzed on HPAE-PAD ion chromatograms. None of the enzymes released any sugar products individually. However, when a mixture of the two was used, galactosamine was clearly released from A + Type RBC release, not from B + or O + The release of GalNAc is highly specific only for the A antigen. This is very important because it shows that GalNAc is not released from the RBC surface under any other circumstances. A truncated form of FpGalNase is also effective, but slightly less active.

[0193] We then continue to use the industry standard MTS TMCard test removes antigen from RBC.The post of these antibody conjugates is loaded with RBC, and rotates in centrifuge.Antigen-free RBC migrates to the bottom of post and is scored as 0, while untreated RBC carries corresponding antigen rod at the top and is scored as 4, and intermediate score is then ranked the degree of antigen removal.Separately, with FpGalNase processing, can not remove A or B antigenicity (table 3) under the concentration adopted, and this is consistent with its inactivity to GalNAc substrate and the low activity to Gal.Hatched with FpGalNAcDeAc, removed the antigenicity that causes because acetamide is transformed into amine, thereby impaired the combination of adopted anti-A antibody.In the absence and presence of 300mg / ml dextran as crowding agent, assessed the minimum amount of enzyme required for the complete antigen deacetylation of independent FpGalNAcDeAc and combined with FpGalNase. Without the aid of dextran, amounts of FpGalNase down to 3 μg / ml were sufficient, while the inclusion of 300 mg / ml of dextran reduced the required loading to 0.5 μg / ml (Table 3). By comparison, the best previous enzyme, EmGH109, ​​was ineffective in the absence of dextran unless a low-salt buffer was used, while the minimum effective concentration in the presence of dextran was 15 μg / ml (30-fold higher loading). The FpGalNAcDeAc form lacking a CBM was much less efficient.

[0194] Table 3 | Treatment of A with EmGH109, ​​FpGalNAcDeAc, and FpGalNase + 、B + and AB + MTS card results from RBC.

[0195]

[0196]

[0197] Due to the MTS TM The card test does not assess complete conversion of the A antigen, and since no antibodies are available to detect the GalN antigen, we focused on detecting the newly formed H antigen on the treated RBCs. FpGalNase is functional at a concentration of only 5 μg / ml, which results in an increase in H antigen levels consistent with loss of the A antigen, as shown in Figure 2. Figure 3 By measuring the aggregation time in the presence of anti-H-antibody, we demonstrated that both enzymes are active against several A + The functionality of the RBC donor was also achieved under whole blood reaction conditions, which was previously unattainable by other blood conversion enzymes. As a result, this pair of enzymes used a much lower enzyme loading than the best previous enzymes required to convert A +RBCs are converted to O-type "universal donor" RBCs. However, before these RBCs are delivered to patients, it is recommended to remove all traces of the enzymes used in the conversion to avoid adverse immune responses, most preferably by washing the cells after centrifugation. To confirm that this is achievable, we treated A with fluorescently labeled FpGalNAcDeAc and FpGalNase samples. + RBCs were then analyzed using FACS to confirm that simple washing was indeed effective ( Figure 3 ).

[0198] Further characterization of the A-ECO RBCs produced can be used to assess their full viability for use in transfusion medicine, but the possibility of including the enzyme directly in the plasma when collecting donated blood could allow for easy and cost-effective separation of the process from existing automated procedures for blood collection and storage. Specifically, the stability of the enzymes was tested as shown in Table 4.

[0199] Table 4: Storage stability of galactosaminidase and GalNAc deacetylase

[0200]

[0201] Substrate: 100 μM GalN-α-pNP

[0202]

[0203]

[0204] Substrate: 100uM T1 A antigen 四 -MU

[0205] Example 10: Fusion of GalNac deacetylase and galactosaminidase from Clostridium tertiotrophicum

[0206] In the search for similar enzymes, a new natural fusion of a galactosaminidase and a GalNAc deacetylase linked by a CBM (GH36_domain-CBM-deacetylating_domain) was identified. Initial tests showed that this enzyme cleaves the A antigen of red blood cells (same mechanism, first deacetylation, then galactosamine cleavage), but not very efficiently (i.e., similar to EmGH109). The deacetylation domain of C. difficile is not as effective as the GalNAc deacetylase of F. prausnitzii, but if supplemented with the GalNAc deacetylase of F. prausnitzii, the galactosaminidase domain of C. difficile shows activity on red blood cells similar to that of the F. prausnitzii galactosaminidase.

[0207] Example 11: Alternative GalNAc deacetylase and galactosaminidase

[0208] The data showed that the galactosaminidase of C. difficile (Ct5757_GalNAse) and Rp1021 did have comparable enzymatic activities for the conversion of GalN antigen to H antigen (the second reaction step).

[0209] The data of alternative GalNAc deacetylase and galactosaminidase have also been collected, and alternative enzyme is compared with the GalNAc deacetylase of Fusobacterium prausnitzii and the galactosaminidase of Fusobacterium prausnitzii.As shown in Table 5, the MTS scoring of the anti-A antibody of the natural fusion of the 3rd clostridial bacillus of galactosidase and GalNAc deacetylase on processed A RBC is shown, this needs the existence of dextran to effectively cut A antigen, and when combined with the galactosaminidase (FpGalNase) of Fusobacterium prausnitzii, also demonstrates the good activity of the GalNAc deacetylase (Ct5757_DeAcase) of the 3rd clostridial bacillus.Equally in Table 6, data show that Rp3672 and Rp3671 of Robinsoniella peoriensis (Rp) can make the A antigen deacetylation on RBC, but efficiency is lower than FpGalNAcDeAase, and only when there is crowding agent (i.e. dextran 40k), just realize activity.

[0210] Table 5: MTS Scores of Anti-A Antibodies on Treated A RBCs

[0211]

[0212] Table 6: MTS scores of 3671 and 3672 of Robinsoniella peoriensis (Rp)

[0213] sample Anti-A MTS score A RBC control 4 Rp3671 (50 μg / mL) + dextran 40k 3 Rp3672 (50 μg / mL) + dextran 40k 1

[0214] Figure 7Shown are the conversion of A antigen to H antigen on ARBCs analyzed by FACS sorting for (A) A+RBC control, (B) GalNAc deacetylase from Fusobacterium prausnitzii (FpGalNAcDeAc) + galactosamidase from Fusobacterium prausnitzii (FpGalNase) (10 μg / mL), (C) FpGalNAcDeAc + Ct57577_GalNase from Clostridium tertiotrophoside (Ct) (10 ug / mL), and (D) FpGalNAcDeAc + galactosamidase (Rp1021)GalNase from Robinsoniella peoriensis (Rp) (10 ug / mL). The data showed that galactosamidase (Rp1021) GalNase from Clostridium tertiary (Ct) Ct5757_GalNase and Robinsoniella peoriensis (Rp) had enzyme activities comparable to that of galactosamidase (FpGalNase) from Fusobacterium prausnitzii for converting GalN antigen to H antigen (second reaction step).

[0215] Although various embodiments of the present invention are disclosed herein, many adaptations and modifications can be made within the scope of the present invention according to the common knowledge of those skilled in the art. Such modifications include replacing any aspect of the present invention with known equivalents so as to obtain the same results in substantially the same manner. Numerical ranges include the numbers defining the ranges. The word "comprising" is used as an open term in this article, which is substantially equivalent to the phrase "including but not limited to", and the word "comprise" has a corresponding meaning. As used herein, the singular forms "a / an" and "the" include plural indicators unless the context clearly dictates otherwise. Therefore, for example, mentioning "things" includes more than one such thing. The references cited herein are not intended to admit that these references are prior art to embodiments of the present invention. The present invention includes all embodiments and modifications substantially as described above and with reference to the examples and drawings.

[0216] sequence

[0217] The DNA sequence of F. prausnitzii was modified from the naturally occurring DNA sequence (GalNAc deacetylase 2311 / 2319 nt / galactosamidase 3228 / 3237 nt). In particular, the length of the sequence used for protein purification was varied, thereby removing the signal peptide and adding an N-terminal His tag via the vector backbone.

[0218] Informal Sequence Listing

[0219] SEQ ID NO: 2

[0220] Description: GalNAc deacetylase from Fusobacterium prausnitzii (protein sequence)

[0221] MRNRRKAVSLLTGLLVTAQLFPTAALAADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVAD GEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKE FVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAK SEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTN GIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE

[0222] SEQ ID NO: 4

[0223] Description: GalNAc deacetylase from Fusobacterium prausnitzii (protein sequence that removes the signal peptide)

[0224] ADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLE...

Claims

1. A composition comprising: (a) purified GalNAc deacetylase protein; and (b) purified galactosaminidase protein, The purified GalNAc deacetylase protein and the purified galactosaminidase protein are derived from Fusobacterium prausnitzii ( Flavonifractor plautii ), and where: The purified GalNAc deacetylase protein is selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; and the purified galactosaminidase protein is selected from one or more of the following: SEQ ID NO: 7; SEQ ID NO: 9 and SEQ ID NO:

10.

2. The composition according to claim 1, wherein: (a) the purified GalNAc deacetylase protein is a purified Fusobacterium prausnitzii GalNAc deacetylase protein, the amino acid sequence of which is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; and / or (b) The purified galactosamidase protein is a purified galactosamidase protein of Fusobacterium prausnitzii, and its amino acid sequence is shown in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:

10.

3. The composition according to claim 1 or 2, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving A antigen at 1 μg / ml or below.

4. The composition according to claim 1 or 2, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5.

5. The composition according to claim 3, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5. 6 . The composition according to claim 1 , wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4° C. to 37° C. 7 . The composition according to claim 3 , wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4° C. to 37° C.

8. The composition according to claim 4, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

9. The composition according to claim 1 or 2, wherein (a) the purified GalNAc deacetylase protein and the purified galactosaminidase protein are immobilized; (b) the purified GalNAc deacetylase protein is immobilized; or (c) The purified galactosamidase protein is immobilized.

10. The composition according to claim 3, wherein (a) the purified GalNAc deacetylase protein and the purified galactosaminidase protein are immobilized; (b) the purified GalNAc deacetylase protein is immobilized; or (c) The purified galactosamidase protein is immobilized.

11. The composition according to claim 4, wherein (a) the purified GalNAc deacetylase protein and the purified galactosaminidase protein are immobilized; (b) the purified GalNAc deacetylase protein is immobilized; or (c) The purified galactosamidase protein is immobilized.

12. The composition according to claim 6, wherein (a) the purified GalNAc deacetylase protein and the purified galactosaminidase protein are immobilized; (b) the purified GalNAc deacetylase protein is immobilized; or (c) The purified galactosamidase protein is immobilized.

13. The composition of claim 9, wherein the immobilized purified GalNAc deacetylase protein and / or purified galactosamidase protein is attached to a surface selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) pipe; (d) a column; or (e) Matrix.

14. The composition of claim 1 or 2, wherein the composition further comprises a crowding agent.

15. The composition of claim 3, wherein the composition further comprises a crowding agent.

16. The composition of claim 4, wherein the composition further comprises a crowding agent.

17. The composition of claim 6, wherein the composition further comprises a crowding agent.

18. The composition of claim 13, wherein the composition further comprises a crowding agent.

19. The composition of claim 14, wherein the crowding agent is selected from one or more of the following: dextran, dextran sulfate, dextrin, pullulan, polyethylene glycol, polysucrose, and inert protein.

20. An ex vivo method for enzymatic cleavage of A antigen from red blood cells, the method comprising: (a) combining a GalNAc deacetylase protein and a galactosamidase protein with: (i) blood containing type A antigen; or (ii) type A or AB red blood cells; (b) incubating the enzyme with (i) the blood; or (ii) the type A or type AB red blood cells for a period of time sufficient for the enzyme to cleave the A antigen from the blood or the red blood cells, wherein the GalNAc deacetylase is a purified protein selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; and the galactosaminidase is a purified protein selected from one or more of the following: SEQ ID NO: 7; SEQ ID NO: 9 and SEQ ID NO:

10.

21. An ex vivo method for enzymatic cleavage of A antigen from red blood cells or donor organs, the method comprising: (a) adding a GalNAc deacetylase protein and a galactosaminidase protein to: (i) blood containing a type A antigen; (ii) type A or type AB red blood cells; or (iii) donor organs displaying type A antigens; (b) incubating the enzyme with (i) the blood; (ii) the type A or type AB red blood cells; or (iii) the donor organ for a period of time sufficient for the enzyme to cleave the A antigen from the red blood cells or the donor organ, wherein both the GalNAc deacetylase protein and the galactosamidase protein are derived from Fusobacterium prausnitzii, wherein the GalNAc deacetylase is a purified protein selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; and the galactosamidase is a purified protein selected from one or more of the following: SEQ ID NO: 7; SEQ ID NO: 9 and SEQ ID NO:

10.

22. The method according to claim 20 or 21, wherein the GalNAc deacetylase is a purified GalNAc deacetylase protein of Fusobacterium prausnitzii, whose amino acid sequence is shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5; and / or the galactosamidase is a purified galactosamidase protein of Fusobacterium prausnitzii, whose amino acid sequence is shown in SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:

10.

23. The method of claim 20 or 21 further comprising adding a crowding agent.

24. The method of claim 22, further comprising adding a crowding agent.

25. The method of claim 23, wherein the crowding agent is selected from one or more of the following: dextran; dextran sulfate; dextrin; pullulan; polyethylene glycol; polysucrose; hyperbranched glycerol and inert protein.

26. The method of claim 20 or 21, further comprising washing the blood, red blood cells or donor organ to remove the GalNAc deacetylase protein and the galactosamidase protein.

27. The method of claim 22, further comprising washing the blood, red blood cells or donor organ to remove the GalNAc deacetylase protein and the galactosamidase protein.

28. The method of claim 23, further comprising washing the blood, red blood cells or donor organ to remove the GalNAc deacetylase protein and the galactosamidase protein.

29. The method of claim 25, further comprising washing the blood, red blood cells or donor organ to remove the GalNAc deacetylase protein and the galactosamidase protein.

30. The method of claim 20 or 21, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving the A antigen at or below 1 μg / ml.

31. The method of claim 22, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving the A antigen at or below 1 μg / ml.

32. The method of claim 23, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving the A antigen at or below 1 μg / ml.

33. The method of claim 25, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving the A antigen at or below 1 μg / ml.

34. The method of claim 26, wherein the GalNAc deacetylase and galactosaminidase are capable of cleaving the A antigen at or below 1 μg / ml.

35. The method of claim 20 or 21, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5.

36. The method of claim 22, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5.

37. The method of claim 23, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5.

38. The method of claim 25, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5.

39. The method of claim 26, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5.

40. The method of claim 30, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a pH of 6.5 to 7.

5.

41. The method according to claim 20 or 21, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

42. The method of claim 22, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

43. The method of claim 23, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

44. The method of claim 25, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

45. The method of claim 26, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

46. ​​The method of claim 30, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

47. The method of claim 35, wherein the GalNAc deacetylase and galactosaminidase have A antigen cleavage activity at a temperature of 4°C to 37°C.

48. A blood collection and storage system comprising: (a) purified GalNAc deacetylase protein; and (b) purified galactosaminidase protein; wherein the purified GalNAc deacetylase protein and the purified galactosaminidase protein are both derived from Fusobacterium prausnitzii, and wherein the purified GalNAc deacetylase is selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; and the purified galactosaminidase is selected from one or more of the following: SEQ ID NO: 7; SEQ ID NO: 9 and SEQ ID NO:

10.

49. The system of claim 48, wherein the amino acid sequence of the purified GalNAc deacetylase protein is shown in one of SEQ ID NOs: 2, 4, and 5; and / or the amino acid sequence of the purified galactosaminidase protein is shown in one of SEQ ID NOs: 7, 9, and 10.

50. The system of claim 48 or 49, wherein the system further comprises a surface on which the purified GalNAc protein and the purified galactosamidase protein are immobilized, the surface being selected from one or more of the following: (a) beads or microspheres; (b) containers; (c) pipe; (d) a column; or (e) Matrix.

51. A blood collection and storage device, the device comprising: (a) Surface; (b) purified GalNAc deacetylase protein immobilized on the surface; and (c) purified galactosamidase protein immobilized on the surface; wherein the purified GalNAc deacetylase protein and the purified galactosaminidase protein are both derived from Fusobacterium prausnitzii, and wherein the purified GalNAc deacetylase is selected from one or more of the following: SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; and the purified galactosaminidase is selected from one or more of the following: SEQ ID NO: 7; SEQ ID NO: 9 and SEQ ID NO:

10.

52. The apparatus according to claim 51, wherein the amino acid sequence of the purified GalNAc deacetylase protein is shown in one of SEQ ID NOs: 2, 4, and 5; and / or the amino acid sequence of the purified galactosaminidase protein is shown in one of SEQ ID NOs: 7, 9, and 10.

53. The apparatus of claim 51 or 52, wherein the purified GalNAc protein and the purified galactosamidase protein are immobilized on a surface selected from one or more of: (a) beads or microspheres; (b) containers; (c) pipe; (d) a column; or (e) Matrix.

54. Apparatus according to claim 53, wherein said container is a bag.

55. The composition of claim 13, wherein the container is a bag.

56. The system of claim 50, wherein the container is a bag.

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