CN109825538B - Synthesis method of chiral 2-amino-1-butanol - Google Patents

Abstract

The invention discloses a method for synthesizing chiral 2-amino-1-butanol. The method comprises the following steps: using 1, 2-butanediol as a substrate, and generating 2-ketone-1-butanol through catalytic reaction of enzyme A and coenzyme thereof; 2-keto-1-butanol is used as a substrate, and chiral 2-amino-1-butanol is generated through catalytic reaction of enzyme B and coenzyme thereof; the enzyme A is selected from alcohol dehydrogenase, carbonyl reductase or a mutant of the two enzymes; the enzyme B is selected from amino acid dehydrogenase, transaminase or mutants of both enzymes. The invention provides a brand-new green biosynthesis route, which takes cheap 1, 2-butanediol as a raw material to synthesize chiral 2-amino-1-butanol, namely (S) -2-amino-1-butanol and (R) -2-amino-1-butanol, through multi-enzyme co-expression or cascade or step-by-step catalysis.

Description

Synthesis method of chiral 2-amino-1-butanol

Technical Field

The invention belongs to the technical field of biology, and relates to a method for synthesizing chiral 2-amino-1-butanol, in particular to a method for synthesizing chiral 2-amino-1-butanol by using the catalysis of a biological enzyme.

Background

In recent years green chemistry processes centered on the application of biocatalysts have received increasing attention, especially cascaded catalytic reactions using natural or novel engineered enzymes in combination as multienzyme molecular machines are favored by researchers and by the industry (Fischer et al, ACS Catal.,2016,6(1): 23-30; France et al, ACS Catal.,2016,6(6):3753- > 3759; Li et al.,2016, J Agr Food chem.,64(46):8927- > 8934.). By adopting a new cascade reaction path constructed by a multienzyme molecular machine, the loss of intermediates can be reduced, the conversion efficiency can be improved, and the process production cost is greatly reduced (Schritwieser et al, Curr Opin Chem biol.,2011,15(2): 249-256; Wheeldon et al, Nat Chem.,2016,8(4): 299-309.).

Chiral 2-amino-1-butanol, especially (S) -2-amino-1-butanol, has wide application in organic synthesis and pharmaceutical production, and can be used as an important intermediate for preparing compounds with optical activity. The structural formula is shown in figure 1. (S) -2-Amino-1-butanol ((S) -2-Amino-1-butanol) is an important pharmaceutical intermediate, and chemical methods are currently used for production, such as: using butyraldehyde, dibenzyl azodicarboxylate and D-proline as raw materials, adding NaBH₄、H₂Synthesizing (S) -2-aminobutanol (Kotkar) under the catalytic action of nickel and the like&Sudalai, Tetrahedron: Asymmetry,2006,17(11): 1738-; reduction of 2-aminobutyric acid with lithium aluminum hydride to the corresponding chiral amino alcohol product (Doherty) has also been reported&Shapira, J Org Chem,1963,28(5): 1339-; by means of H₂And nickel reduction of L-2-aminobutyric acid under high pressure conditions to obtain (S) -2-aminobutanol (CN 105481703A); or using NaHB₄At H₂SO₄Reduction of the corresponding alpha-amino acid under THF conditions gave (Li et al, J Agr Food Chem, 2016,64(46): 8927-8934). Chemical resolution methods of aminobutanol, such as: dibenzoyl tartaric acid resolution (Periasamy et al, Synthesis-Stuttgart,2003(13): 1965-1967); l-tartaric acid resolution (Zhao et al, J Heterocyclic Chem, 2012,49(4): 943-946). Enzymatic resolution has also been reported, as: in the case of protection of the amino group, the ester bond formed by the hydroxyl group is selectively hydrolyzed by a lipase to obtain a chiral monomer (Francalanci et al, J Org Chem, 1987,52(23): 5079-5082); the immobilized penicillin G acylase selectively hydrolyzes (S) -configuration in the racemic mixture of N-phenylacetyl-derived 2-aminobutanol to obtain (S) -2-aminobutanol with ee value of 99% (Fadnavis et al, Tetrahedron: Asymmetry,1999,10(23): 4495-4500); the continuous resolution is realized by acylating and fixing immobilized penicillin G on a fixed bed, the conversion rate reaches 39.3 percent, and the ee value reaches 98.2 percent.

The chemical method of the synthesis method of (S) -2-amino-1-butanol requires high temperature and high pressure and is obtained by hydrogenation reduction of a metal catalyst, so that the pollution is large and the safety coefficient is low. The chemical resolution method also needs a large amount of acid, alkali and other chemical reagents, and although the enzymatic resolution reaction condition is mild and the stereoselectivity is good, the conversion rate of the resolution method is only 50%, and the yield is low.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for synthesizing chiral 2-amino-1-butanol using bio-enzyme catalysis, which may include the following steps (fig. 2):

(A) using 1, 2-butanediol as a substrate, and generating 2-ketone-1-butanol through catalytic reaction of enzyme A and coenzyme thereof;

(B) taking the 2-ketone-1-butanol generated in the step (A) as a substrate, and generating chiral 2-amino-1-butanol through catalytic reaction of an enzyme B and a coenzyme thereof;

the enzyme A can be selected from any one of the following: alcohol dehydrogenase, carbonyl reductase, a mutant of said alcohol dehydrogenase, a mutant of said carbonyl reductase;

the enzyme B can be selected from any one of the following: amino acid dehydrogenases, transaminases, mutants of said amino acid dehydrogenases, mutants of said transaminases.

The method provided by the invention is realized by a method of multi-enzyme co-expression or cascade or step-by-step catalysis.

Further, both the alcohol dehydrogenase and the carbonyl reductase may be derived from any of the following microorganisms: bacillus stearothermophilus (Geobacillus stearothermophilus), Lactobacillus kefir (Lactobacillus kefir), Lactobacillus brevis (Lactobacillus brevis), Bacillaceae (Bacillus), Thermoanaerobacter Thermoanaerobacter (Thermoanaerobacter brockii), Lymphacter species (Leifsonia sp.), Thermoanaerobacter virginiae (Thermoanaerobacter wiegelii), Saccharomyces ochraceus (Sporobolomyces salmonicola).

Preference is given to the alcohol dehydrogenase LBADH from Lactobacillus brevis (Lactobacillus brevis), the alcohol dehydrogenase TbSADH from Thermoanaerobacter brevis (Thermoanaerobacter brevis), and the alcohol dehydrogenase from Bacillus (Bacillus stearothermophilus), Lactobacillus kefir (Geobacillus stearothermophilus), Lactobacillus kefir (Lactobacillus kefir), Lactobacillus lisi (Leifsonia sp.), anaerobacter virginiana (Thermoanaerobacter wiegelii) and the carbonyl reductase from Sporobolomyces salmonicola (Sporobolomyces salmonicola).

Further, the amino acid dehydrogenase may be derived from: bacillus stearothermophilus (Geobacillus stearothermophilus).

Further, the transaminase may be derived from any of the following microorganisms: bacillus megaterium (Bacillus megaterium), Pseudomonas aeruginosa (P. aeruginosa), Aspergillus terreus (Aspergillus terreus), Aspergillus fumigatus (Aspergillus fumigatus), Fusarium fischeri (Neosartorya fischeri), Gibberella zeae (Gibberella zeae), Mycobacterium (Mycobacterium vanbaaleni), or ATA-117 transaminase by Codexis.

Further, the alcohol dehydrogenase may specifically be any one of the following (a1) - (a 8):

(a1) alcohol dehydrogenase derived from Lactobacillus brevis (Lactobacillus brevis) and having an amino acid sequence of SEQ ID No. 2;

(a2) alcohol dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus), and the amino acid sequence of the alcohol dehydrogenase is SEQ ID No. 4;

(a3) alcohol dehydrogenase derived from Thermoanaerobacter braskii (Thermoanaerobacter braskii), the amino acid sequence of which is SEQ ID No. 6;

(a4) alcohol dehydrogenase derived from Lactobacillus kefir (Lactobacillus kefir), the amino acid sequence of which is SEQ ID No. 8;

(a5) an alcohol dehydrogenase derived from Bacillaceae (Bacillaceae), having an amino acid sequence of SEQ ID No. 10;

(a6) an alcohol dehydrogenase derived from lysenina sp having an amino acid sequence of SEQ ID No. 12;

(a7) an alcohol dehydrogenase derived from Thermoanaerobacter virginiae (Thermoanaerobacter wiegelii) having an amino acid sequence of SEQ ID No. 14;

(a8) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (a1) to (a 7).

Further, the carbonyl reductase may specifically be (b1) or (b2) as follows:

(b1) carbonyl reductase derived from Sporobolomyces salmonicolor, having an amino acid sequence of SEQ ID No. 16;

(b2) and (b1) attaching a tag to the N-terminus and/or C-terminus of the protein defined in (b 1).

Further, the amino acid dehydrogenase may specifically be any one of the following (c1) to (c 2):

(c1) leucine dehydrogenase derived from bacillus stearothermophilus (Geobacillus stearothermophilus), SEQ ID No. 18;

(c2) and (C1) attaching a tag to the N-terminus and/or C-terminus of the protein defined in (C1).

Further, the transaminase may specifically be any one of the following (d1) to (d 9):

(d1) ATA-117 transaminase of Codexis corporation, with amino acid sequence of SEQ ID No. 20;

(d2) a transaminase derived from Aspergillus terreus (Aspergillus terreus) having an amino acid sequence of SEQ ID No. 22;

(d3) a transaminase derived from Aspergillus fumigatus (Aspergillus fumigatus) having an amino acid sequence of SEQ ID No. 24;

(d3) a transaminase derived from Fusarium fischer (Neosartorya fischeri) having an amino acid sequence of SEQ ID No. 26;

(d5) a transaminase derived from Gibberella zeae (Gibberella zeae) having an amino acid sequence of SEQ ID No. 28;

(d6) a transaminase derived from Mycobacterium (Mycobacterium vanbalenii) having an amino acid sequence of SEQ ID No. 30;

(d7) a transaminase derived from Bacillus megaterium (Bacillus megaterium) having an amino acid sequence of SEQ ID No. 32;

(d8) a transaminase derived from pseudomonas aeruginosa (p. aeruginosa) having an amino acid sequence of SEQ ID No. 34;

(d9) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (d1) to (d 8).

Further, the mutant of the alcohol dehydrogenase may specifically be (e1) or (e2) as follows:

(e1) compared with the alcohol dehydrogenase derived from Lactobacillus brevis (Lactobacillus brevis) shown in SEQ ID No.2, at least one of the following mutations is present or present: I11V, G37D;

(e2) and (e1) attaching a tag to the N-terminus and/or C-terminus of the protein defined in (e 1).

Further, the mutant of the amino acid dehydrogenase may specifically be any one of the following (f1) to (f 2):

(f1) compared with the leucine dehydrogenase derived from the bacillus stearothermophilus (Geobacillus stearothermophilus) shown in SEQ ID No.18, at least one of the following mutations exists or exists only: K68X, N261X, wherein X represents 19 amino acids other than the wild-type amino acid (presented in accepted single letter abbreviations including F, L, I, V, S, P, T, a, Y, H, Q, N, D, E, C, R, G, M, W). Preferably, X is a polar amino acid (e.g., S, T, Y, H, Q, N, D, E, C, R, etc.). Further preferably, X is S, Y, L or C. Still further preferably, the amino acid dehydrogenase mutant is a mutant in which the following mutations are present or present, as compared with the leucine dehydrogenase derived from bacillus stearothermophilus (Geobacillus stearothermophilus) shown in SEQ ID No. 18: K68S/N261L

Or K68Y/N261C.

(f2) And (f1) attaching a tag to the N-terminus and/or C-terminus of the protein defined in (f 1).

In the present invention, for amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, the substitution of valine (V) for isoleucine (I) originally present at position 11 of SEQ ID No.2 is designated as "I11V". Variants containing multiple changes are separated by a slash symbol ("/").

Further, in the method, the enzyme A and the enzyme B can be catalyzed in the form of crude enzyme solution, crude enzyme solution freeze-dried powder, pure enzyme or whole cells.

Further, the crude enzyme solution freeze-dried powder and the pure enzyme can be prepared by the method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell; cracking the recombinant cells to obtain crude enzyme solution, crude enzyme solution freeze-dried powder or pure enzyme of the enzyme A and/or the enzyme B. The whole cell can be prepared according to a method comprising the following steps: expressing the enzyme A and/or the enzyme B in host cells, and obtaining recombinant cells, namely the whole cells of the enzyme A and/or the enzyme B.

Still further, the recombinant cell can be prepared according to a method comprising the following steps: introducing a nucleic acid molecule capable of expressing the enzyme A and/or the enzyme B into the host cell, and obtaining the recombinant cell expressing the enzyme A and/or the enzyme B after induction culture.

Further, the "nucleic acid molecule capable of expressing the enzyme A and/or the enzyme B" may be introduced into the host cell in the form of a recombinant vector. Wherein, the recombinant vector can be a bacterial plasmid (such as an expression vector based on T7 promoter expressed in bacteria, such as pET-28a and the like), a bacteriophage, a yeast plasmid (such as YEp series vector and the like) or a retrovirus packaging plasmid, wherein the bacterial plasmid carries the gene coding for the enzyme A and/or the enzyme B.

In one embodiment of the invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a small fragment between enzyme cutting sites Nde I and Xho I of pET22B vector with the gene encoding the enzyme A or the enzyme B.

In another embodiment of the invention, the recombinant vector is a recombinant plasmid obtained by inserting the coding gene of the enzyme A into the enzyme cutting sites EcoRI and Hind III of the pETDuet-1 vector and inserting the coding gene of the enzyme B into the enzyme cutting sites Nde I and Xho I of the pETDuet-1 vector.

Further, the host cell may be a prokaryotic cell or a lower eukaryotic cell.

Further, the prokaryotic cell may specifically be a bacterium; the lower eukaryotic cell may specifically be a yeast cell.

In one embodiment of the invention, the host cell is specifically e.coli, more specifically e.coli BL21(DE 3). Correspondingly, the induction culture is to add IPTG to the culture system to a final concentration of 0.1-0.5mM (specifically 0.1mM), and induce culture at 20-37 ℃ for 12-24h (specifically 16 h).

The sequence of the coding gene of the alcohol dehydrogenase from Lactobacillus brevis is SEQ ID No.1 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the Geobacillus stearothermophilus is SEQ ID No.3 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the sequence or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the high temperature anaerobic bacillus (Thermoanaerobacter brockii) is SEQ ID No.5 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the Lactobacillus kefiri is SEQ ID No.7 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from Bacillaceae (Bacillaceae) is SEQ ID No.9 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the lysine (Leifsonia sp) is SEQ ID No.11 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the thermoanaerobacterium virginiae (Thermoanaerobacter wiegelii) is SEQ ID No.13 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has functions and codes the same protein.

The sequence of the coding gene of the carbonyl reductase derived from the Sporobolomyces salmonicolor is SEQ ID No.15 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a label coding sequence or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the leucine dehydrogenase derived from the bacillus stearothermophilus is SEQ ID No.17 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene are connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of ATA-117 transaminase of Codexis company is SEQ ID No.19 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from Aspergillus terreus (Aspergillus terreus) is SEQ ID No.21 or a fusion sequence obtained by connecting tag coding sequences at the 5 'end and/or 3' end thereof or a random or/and site-directed mutagenesis sequence which retains the function and encodes the same protein.

The sequence of the coding gene of the transaminase derived from Aspergillus fumigatus is SEQ ID No.23 or a fusion sequence obtained by connecting tag coding sequences to the 5 'end and/or 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which retains the function and encodes the same protein.

The sequence of the coding gene of the transaminase derived from the fisher-seft (Neosartorya fischeri) is SEQ ID No.25 or a fusion sequence obtained by connecting tag coding sequences at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from the Gibberella zeae is SEQ ID No.27 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from the mycobacteria is SEQ ID No.29 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from Bacillus megaterium is SEQ ID No.31 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from the pseudomonas aeruginosa is SEQ ID No.33 or a fusion sequence obtained by connecting tag coding sequences at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has functions and codes the same protein.

The sequence of the encoding gene of the mutant of the alcohol dehydrogenase derived from Lactobacillus brevis (Lactobacillus brevis) is any one of the following (g1) - (g 3): (g1) compared to SEQ ID No.1, at least one of the following mutations is present or present only: A31G/T33G, G110A/C111T; (g2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (g 1); (g3) random or/and site-directed mutagenesis sequences which retain the function and encode the same protein as the sequences defined in (g1) or (g 2).

The sequence of the coding gene of the mutant of leucine dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus) is any one of the following (h1) to (h 3): (h1) compared to SEQ ID No.17, at least one of the following mutations is present or present only: A203G/A204C, A202T/A204T, A781C/A782T/C783G, A781T/A782G; (h2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (h 1); (h3) random or/and site-directed mutagenesis sequences which retain function and encode the same protein as compared to the sequences defined in (h1) or (h 2).

Further, the (h1) is: in comparison to SEQ ID No.17, any of the following mutations is present or only present: A203G/A204C/A781C/A782T/C783G, A202T/A204T/A781T/A782G.

In the present invention, for the base substitution, the following nomenclature is used: the original base, position (i.e. the position in the nucleotide sequence of W1 or W2 or W3), is substituted for the base. Accordingly, substitution of the original G with A at position 31 of SEQ ID No.1 was designated "A31G". Variants containing multiple changes are separated by a slash symbol ("/").

In the step (A) and the step (B), the temperature of the catalytic reaction can be 25-37 ℃, such as 30-37 ℃, and specifically such as 30 ℃ or 37 ℃.

In the step (A) and the step (B), the time of the catalytic reaction can be 4-48 h, such as 24 h.

When the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A), the catalytic reaction is carried out in a buffer solution shown as the following (k 1); in the step (B), the catalytic reaction is carried out in a buffer solution shown as any one of (k2) to (k3) below; when the enzyme A and the enzyme B are catalyzed in the form of whole cells co-expressing the enzyme A and the enzyme B, the catalytic reactions of step (A) and step (B) are carried out in a buffer as shown below (k 1);

(k1) a phosphate buffer solution having a concentration of 50 to 100mM and a pH of 6.5 to 8.0;

the method specifically comprises the following steps: phosphate buffer at a concentration of 100mM, pH 8.0.

(k2) NH with a concentration of 100 mM-2M and a pH of 8.0-9.6₃·H₂O or NH₄A Cl buffer solution;

the method specifically comprises the following steps: NH at a concentration of 1M and a pH of 8.7₃·H₂O or NH₄And (4) Cl buffer solution.

(k3) A phosphate buffer solution with a concentration of 50-100 mM, an isopropylamine concentration of 250mM-1M and a pH value of 7.5-8.5;

the method specifically comprises the following steps: phosphate buffer with 100mM concentration, 500mM concentration of isopropylamine and pH 8.0.

When the enzyme A and the enzyme B are catalyzed by crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A) and the step (B), the concentration of the enzyme A and the concentration of the enzyme B in respective reaction systems can be 0.1-10 g/L, such as 10 g/L. When the enzyme A and the enzyme B are catalyzed in the form of whole cells co-expressing the enzyme A and the enzyme B, the step (A) and the step (B) are completed in one reaction system in which the concentration of the whole cells (co-expressing the enzyme A and the enzyme B) is 100g/L (100 g of wet weight of the whole cells is contained per liter of the reaction system).

In the present invention, the coenzyme of the enzyme A is in particular the oxidized cofactor II, NADP⁺Or an oxidized cofactor I, i.e. NAD⁺. When the enzyme B is an amino acid dehydrogenase or a mutant of said amino acid dehydrogenase, the coenzyme of the enzyme B is in particular oxidized cofactor I, NAD⁺(ii) a When the enzyme B is a transaminase or a mutant of the transaminase, the coenzyme of the enzyme B is in particular pyridoxal phosphate (PLP).

In the invention, when the enzyme A and the enzyme B are catalyzed by crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, the concentrations of the coenzymes of the enzyme A and the enzyme B in respective reaction systems can be 0.5-3 mM (specifically 1 mM).

When the enzyme A and the enzyme B are catalyzed by crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A), a reaction system of the catalytic reaction contains acetone besides 1, 2-butanediol, the enzyme A and the coenzyme thereof.

Specifically, in the step (a), the reaction system of the catalytic reaction comprises: phosphate buffered at a concentration of 100mM, pH 8.0, 1, 2-butanediol to a final concentration of 20mM, and oxidized cofactor II, NADP, to a final concentration of 1mM⁺Or an oxidized cofactor I, i.e. NAD⁺Acetone (v/v) with the final concentration of 5%, and crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme of the enzyme A with the final concentration of 10 g/L.

When the enzyme A and the enzyme B are subjected to catalytic action in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (B), when the enzyme B is amino acid dehydrogenase or a mutant of the amino acid dehydrogenase, a reaction system of the catalytic reaction contains 2-keto-1-butanol, the enzyme B and a coenzyme thereof, and also contains glucose dehydrogenase and glucose. Further, the reaction system also contains calcium carbonate.

Specifically, in the step (B), when the enzyme B is an amino acid dehydrogenase or a mutant of the amino acid dehydrogenase, the composition of the reaction system for catalyzing the reaction may be as follows: NH at a concentration of 1M and a pH of 8.7₃·H₂O or NH₄A Cl buffer solution; oxidized cofactor I, NAD, at a final concentration of 1mM⁺(ii) a Glucose dehydrogenase at a final concentration of 1 g/L; glucose at a final concentration of 100 mM; crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme of the enzyme B with the final concentration of 10 g/L; calcium carbonate at a final concentration of 5 g/L. When the enzyme B is a transaminase or a mutant of the transaminase, the composition of the reaction system for the catalytic reaction may be as follows: phosphate buffer solution with the concentration of 50-100 mM, the concentration of isopropylamine of 500mM and the pH value of 8.0; pyridoxal phosphate (PLP) at a final concentration of 1 mM; crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme of the enzyme B with the final concentration of 10 g/L.

When the enzyme a and the enzyme B are catalyzed by a whole cell co-expressing the enzyme a and the enzyme B, no coenzyme may be added to the reaction system, and further, glucose may be contained in the reaction system.

Specifically, the composition of the reaction system may be as follows: phosphate buffer at a concentration of 100mM and pH 8.0; 1, 2-butanediol at a final concentration of 50 mM; 100mM glucose; the whole cells were contained at a final concentration of 100g/L (i.e., 100g of wet weight per liter of the reaction system).

In the method, the chiral 2-amino-1-butanol is (S) -2-amino-1-butanol and/or (R) -2-amino-1-butanol.

The amino acid dehydrogenases and mutants thereof as described above, as well as ATA-117 transaminase derived from Codexis, transaminase derived from Aspergillus terreus (Aspergillus terreus), transaminase derived from Aspergillus fumigatus (Aspergillus fumigatus), transaminase derived from Fusarium fischeri (Neosartorya fischeri), transaminase derived from Gibberella zeae (Gibberella zeae), transaminase derived from Mycobacterium (Mycobalalii), for the synthesis of (S) -2-amino-1-butanol; the transaminase derived from Bacillus megaterium (Bacillus megaterium) and the transaminase derived from pseudomonas aeruginosa (p. aeruginosa) described above are used for the synthesis of (R) -2-amino-1-butanol.

The invention also provides an enzyme system and a related product thereof.

The enzyme system provided by the invention comprises the enzyme A and the enzyme B. Of course, the respective coenzymes of the enzyme A and the enzyme B may be included.

The related products are nucleic acid molecules capable of expressing each enzyme in the enzyme system, or expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules.

The use of the enzyme system or the related product in the synthesis of chiral 2-amino-1-butanol also falls within the scope of the present invention.

In the method for synthesizing chiral 2-amino-1-butanol provided by the invention, a cofactor regeneration system exists. The cofactor regeneration system is used for catalyzing glucose oxidation by glucose dehydrogenase, or catalyzing acetone reduction or isopropanol oxidation by alcohol dehydrogenase to promote cofactor regeneration. In the process of the invention for the synthesis of chiral 2-amino-1-butanol, an alcohol dehydrogenase or a carboreductase catalyzes the oxidation of 1, 2-butanediol to 2-keto-1-butanol, NAD (P)⁺Is reduced to NAD (P) H, simultaneously, alcohol dehydrogenase catalyzes the reduction of acetone to isopropanol, NAD (P) H is re-oxidized to NAD (P)⁺NAD (P) produced⁺The oxidation of the 1, 2-butanediol into the 2-keto-1-butanol is participated in again. Amino acid dehydrogenase and its mutant catalyze the reduction of 2-keto-1-butanol to 1, 2-butanediol, NADH is oxidized to NAD⁺Simultaneously, Glucose Dehydrogenase (GDH) catalyzes the oxidation of glucose to gluconic acid, NAD⁺Is reduced to NADH again, and the generated NADH participates in the oxidation of the 2-ketone-1-butanol to generate the (S) -2-amino-1-butanol again.

The invention provides a brand-new green biosynthesis route, which takes cheap 1, 2-butanediol as a raw material to synthesize chiral 2-amino-1-butanol, namely (S) -2-amino-1-butanol and/or (R) -2-amino-1-butanol, through multi-enzyme co-expression or cascade or step-by-step catalysis.

Drawings

FIG. 1 shows the structural formulae of (S) -2-amino-1-butanol and (R) -2-amino-1-butanol.

FIG. 2 is a reaction scheme of the biological preparation method of chiral 2-amino-1-butanol of the present invention. A: the (S) -2-amino-1-butanol is prepared by coupling alcohol dehydrogenase or carbonyl reductase with amino acid dehydrogenase. B: alcohol dehydrogenase or carbonyl reductase coupled with transaminase to prepare (S) -2-amino-1-butanol or (R) -2-amino-1-butanol.

FIG. 3 shows the results of Gas Chromatography (GC) identification of 2-keto-1-butanol.

FIG. 4 is a graph showing the separation effect of 2-amino-1-butanol after derivatization with o-phthalaldehyde. A is racemic type 2-amino-1-butanol; b: (S) -2-amino-1-butanol standard; c: (R) -2-amino-1-butanol standard.

FIG. 5 is a liquid chromatogram of the reaction solution. A: the liquid chromatography result of the reaction liquid of the negative control group; b: negative control + (S) -2-amino-1-butanol standard liquid chromatography result; c: negative control + (R) -2-amino-1-butanol standard liquid chromatography result; d: experimental group 1 liquid chromatography of the reaction solution (product is (S) -2-amino-1-butanol); e: experimental group 2 liquid chromatography of the reaction solution (product is (R) -2-amino-1-butanol). Wherein, the negative control reaction system only contains the expression host crude enzyme powder or enzyme liquid or whole cells of the empty expression vector, and other components are the same as those of the experimental group.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of (S) -2-amino-1-Butanol by coupling alcohol dehydrogenase or carbonyl reductase with leucine dehydrogenase of Bacillus stearothermophilus (Geobacillus stearothermophilus) or mutant thereof

Preparation of engineering bacteria of recombinant alcohol dehydrogenase or mutant thereof and amino acid dehydrogenase or mutant thereof

The coding genes of related enzymes are respectively subjected to whole-gene synthesis (codon optimization is carried out by taking escherichia coli as a host according to needs), and the synthesized genes are connected to various expression vectors to construct the recombinant expression vector. The expression vector is any vector conventionally used in the art. The vector is specifically pET22b (+), and the coding gene of related enzyme after whole gene synthesis is inserted between enzyme cutting sites Nde I and Xho I of pET22b (+), and the recombinant vector is obtained after the coding gene is verified to be correct through sequencing. And obtaining related gene mutants by using a site-directed mutagenesis method.

The recombinant expression vector with the correct sequencing verification is transformed into a suitable microbial host. The host microorganism is any host microorganism which is conventional in the art, as long as the recombinant expression vector can stably replicate itself and the carried alcohol dehydrogenase and amino acid dehydrogenase genes can be efficiently expressed. Among them, the preferred host microorganism is Escherichia coli (Escherichia coli), preferably Escherichia coli BL21(DE3), and the above recombinant expression plasmid is transformed into E.coli BL21(DE3) to obtain the genetically engineered strain of the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably an electro-transformation method or a chemical transformation method.

The enzymes and their mutants referred to in this example are detailed in table 1.

TABLE 1 enzymes and mutants thereof referred to in example 1

Note: w1 represents an alcohol dehydrogenase LBADH derived from Lactobacillus brevis (Lactobacillus brevis); w2 represents an alcohol dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus); w3 represents an alcohol dehydrogenase TbSADH derived from Thermoanaerobacter braskii; w4 represents an alcohol dehydrogenase derived from Lactobacillus kefir; w5 represents an alcohol dehydrogenase derived from Bacillaceae (Bacillaceae); w6 represents an alcohol dehydrogenase derived from lysine (Leifsonia sp.); w7 represents an alcohol dehydrogenase derived from Thermoanaerobacter wegiae (Thermoanaerobacter wiegelii); w8 represents carbonyl reductase SSCR derived from Sporobolomyces salmonicolor; w9 represents leucine dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus). Wn-Mn represents a mutant of Wn (n is a natural number). The numbering of the protein substitution is from the N-terminus of the wild-type amino acid sequence as indicated by Wn (N is a natural number); the numbering of the gene substitution is from the 5' end of the wild-type nucleotide sequence represented by Wn (n is a natural number). In the table, for amino acid substitutions, the following nomenclature is used: original amino acid, position (i.e., position in the Wn amino acid sequence), substituted amino acid. Accordingly, the substitution of valine (V) for isoleucine (I) originally present at position 11 of SEQ ID No.2 was designated as "I11V". For base substitutions, the following nomenclature is used: original base, position (i.e., position in Wn nucleotide sequence), substituted base. Accordingly, substitution of the original G with A at position 31 of SEQ ID No.1 was designated "A31G". Variants containing multiple changes are separated by a slash symbol ("/").

Expression of recombinant alcohol dehydrogenase or its mutant, amino acid dehydrogenase or its mutant and preparation of crude enzyme

Transferring the recombinant expression vector constructed in the step one into competent cells of escherichia coli BL21(DE3), culturing for 12-16h at 37 ℃, inoculating a single transformant into 5mL LB culture medium containing corresponding antibiotics after the transformant grows out, and culturing overnight (12-16h) at 37 ℃ and 220 rmp. Then inoculating the cells into a TB culture medium according to the proportion of 1 percent (volume percentage content), culturing the cells at 37 ℃ and 220rmp until the OD600 is about 0.6, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mM, carrying out induced culture at 20-37 ℃ for 16h, then centrifuging the cells at 4000rpm and 4 ℃ for 10min to collect the cells, carrying out resuspension and washing once by using phosphate buffer with the pH value of 8.0 and carrying out ultrasonic bacteria breaking and preparing enzyme freeze-dried powder.

Preparation of tris, chiral 2-amino-1-butanol

The first step of reaction: phosphate buffer with a concentration of 100mM and a pH value of 8.0, 1, 2-butanediol with a final concentration of 20mM, and oxidized cofactor II, i.e., NADP, with a final concentration of 1mM are sequentially added to the reaction system⁺Or an oxidized cofactor I, i.e. NAD⁺And acetone (v/v) with the final concentration of 5 percent, and alcohol dehydrogenase or carbonyl reductase freeze-dried powder or enzyme solution with the final concentration of 10 g/L. After the reaction system was reacted at 30 ℃ for 24 hours, the product was subjected to Gas Chromatography (GC).

The detection conditions for Gas Chromatography (GC) were as follows: sample introduction amount: 2 mu L of the solution; a chromatographic column: HP-5; the split ratio is as follows: 20: 1; flow splitting: 40 mL/min; temperature rising procedure: 5 minutes at 40 ℃; heating to 60 ℃ at the speed of 5 ℃/min for 2 minutes; the temperature is raised to 200 ℃ at the rate of 30 ℃/min for 2.333 minutes. Operating time: for 18 minutes.

Results referring to FIG. 3, it was confirmed that 2-keto-1-butanone was obtained by this reaction.

The second step of reaction: 2-ketone-1-butanol generated by the reaction in the previous step is taken as a substrate, NH with the pH of 8.7 and the final concentration of 1M is sequentially added into a reaction system₃·H₂O or NH₄Cl buffer, oxidized cofactor I, NAD, at a final concentration of 1mM⁺The reaction system consists of glucose dehydrogenase with the final concentration of 1g/L, glucose with the final concentration of 100mM, leucine dehydrogenase mutant freeze-dried powder or enzyme liquid with the final concentration of 10g/L of bacillus stearothermophilus and calcium carbonate with the final concentration of 5 g/L. The reaction system is reacted for 24 hours at the temperature of 30 ℃, and then (S) -2-amino-1-butanol is obtained. The reaction solution is detected by liquid chromatography after derivation of o-phthalaldehyde.

The HPLC detection conditions were as follows: an Agilent SB-Aq C18 column (4.6mm 250mm,5 um); the detection wavelength is 334 nm; column temperature: 35 ℃; flow rate: 1 mL/min; the gradient elution procedure is shown in table 2.

TABLE 2 gradient elution procedure for HPLC

Note: (1) the% in the table indicates the volume percentage.

ee＝(A_S-A_R)/(A_S+A_R)×100％；A_S: analyzing the peak area value of the obtained (S) -2-amino-1-butanol by liquid chromatography; a. the_R: the peak area value of the obtained (R) -2-amino-1-butanol was analyzed by liquid chromatography.

Substrate conversion efficiency ═ C_{Rotating shaft}/C_{General assembly}×100％；C_{Rotating shaft}: the number of moles of the substrate converted into (S) or (R) -2-amino-1-butanol in the reaction system; c_{General assembly}: the total mole number of the substrate in the reaction system.

HPLC detection results refer to D in FIGS. 4 and 5, substrate conversion efficiency is 45-55%, and ee value is greater than 99%. See table 3 for specific results.

TABLE 3 results of preparation of (S) -2-amino-1-butanol by coupling of alcohol dehydrogenase or carbonyl reductase with leucine dehydrogenase of Bacillus stearothermophilus (Geobacillus stearothermophilus) or its mutant

Note: in the table, Wn and Wn-Mn (n is a natural number) in the first reaction and the second reaction have the same meanings as in Table 1.

Example 2 preparation of (S) -2-amino-1-butanol by coupling alcohol dehydrogenase or carbonyl reductase with transaminase

Preparation of engineering bacteria of recombinant alcohol dehydrogenase or carbonyl reductase and transaminase

The process is carried out according to the first step of example 1.

The enzymes and their mutants referred to in this example are detailed in Table 4.

TABLE 4 enzymes and mutants thereof referred to in example 2

Note: W1-W8 have the same meanings as in Table 1. W10 represents ATA-117 transaminase by Codexis; w11 represents a transaminase derived from Aspergillus terreus (Aspergillus terreus); w12 represents a transaminase derived from Aspergillus fumigatus (Aspergillus fumigatus); w13 represents a transaminase derived from Fusarium fischeri (Neosartorya fischeri); w14 represents a transaminase derived from Gibberella zeae (Gibberella zeae); w15 represents a transaminase derived from Mycobacterium (Mycobacterium vanbaaleni). Wn-Mn represents a mutant of Wn (n is a natural number). The numbering and specific nomenclature of the substitutions of proteins and genes are as in Table 1.

Expression of recombinant alcohol dehydrogenase or carbonyl reductase, transaminase and preparation of crude enzyme

Reference is made to example 1, step two.

Preparation of tris, chiral 2-amino-1-butanol

The first step of reaction: phosphate buffer with a concentration of 100mM and a pH value of 8.0, 1, 2-butanediol with a final concentration of 20mM, and oxidized cofactor II, i.e., NADP, with a final concentration of 1mM are sequentially added to the reaction system⁺Or an oxidized cofactor I, i.e. NAD⁺Acetone (v/v) with the final concentration of 5 percent and alcohol dehydrogenase or carbonyl reductase enzyme freeze-dried powder or enzyme liquid with the final concentration of 10g/L form a reaction system; after the reaction system was reacted at 30 ℃ for 24 hours, the product was subjected to Gas Chromatography (GC). The detection conditions for Gas Chromatography (GC) are as shown in step three of example 1. Results referring to FIG. 3, it was confirmed that 2-keto-1-butanone was obtained by this reaction.

The second step of reaction: the 2-keto-1-butanol generated by the above-mentioned one-step reaction is used as a substrate, phosphate buffer solution with the concentration of 50-100 mM, isopropylamine concentration of 500mM and pH value of 8.0, pyridoxal phosphate (PLP) with the final concentration of 1mM, ATA-117 transaminase of Codexes company with the final concentration of 10g/L, transaminase of Aspergillus terreus (Aspergillus terreus), transaminase of Aspergillus fumigatus (Aspergillus fumigatus), transaminase of Fusarium fischeri (Neosarya fischeri), transaminase of Gibberella zeae, transaminase freeze-dried powder of Mycobacterium vanmbaaleni or transaminase liquid are sequentially added into a reaction system to form the reaction system, and the reaction system is reacted at 30 ℃ for 24 hours to obtain the (S) -2-amino-1-butanol. The reaction solution is detected by liquid chromatography after derivation of o-phthalaldehyde. HPLC detection conditions are as shown in step three of example 1.

The concrete calculation methods of ee value and substrate conversion efficiency are the same as those of the third step in example 1.

HPLC detection results refer to D in FIGS. 4 and 5, substrate conversion efficiency is 45-55%, and ee value is greater than 99%. See table 5 for specific results.

TABLE 5 results of preparing (S) -2-amino-1-butanol by coupling alcohol dehydrogenase or its mutant with transaminase

Note: in the table, Wn and Wn-Mn (n is a natural number) in the first reaction and the second reaction have the same meanings as in Table 4.

Example 3 preparation of (R) -2-amino-1-butanol by coupling alcohol dehydrogenase or carbonyl reductase with transaminase

Preparation of engineering bacteria of recombinant alcohol dehydrogenase and transaminase

The process is carried out according to the first step of example 1.

The enzymes and their mutants referred to in this example are detailed in Table 6.

TABLE 6 enzymes and mutants thereof referred to in example 3

Note: W1-W8 have the same meanings as in Table 1. W16 represents a transaminase derived from Bacillus megaterium (Bacillus megaterium); w17 represents a transaminase derived from pseudomonas aeruginosa (p. Wn-Mn represents a mutant of Wn (n is a natural number). The numbering and specific nomenclature of the substitutions of proteins and genes are as in Table 1.

Secondly, expression of recombinant alcohol dehydrogenase and transaminase and preparation of crude enzyme

Reference is made to example 1, step two.

Preparation of tris, chiral 2-amino-1-butanol

The first step of reaction: adding phosphate buffer solution with concentration of 100mM and pH value of 8.0, 1, 2-butanediol with final concentration of 20mM, and oxidation type cofactor II with final concentration of 1mM, i.e. NADP into the reaction system in sequence⁺Or an oxidized cofactor I, i.e. NAD⁺Acetone (v/v) with the final concentration of 5 percent, alcohol dehydrogenase or carbonyl reductase freeze-dried powder with the final concentration of 10g/L or enzyme solution to form a reaction system; after the reaction system was reacted at 30 ℃ for 24 hours, the product was subjected to Gas Chromatography (GC). The detection conditions for Gas Chromatography (GC) are as shown in step three of example 1. Results referring to FIG. 3, it was confirmed that 2-keto-1-butanone was obtained by this reaction.

The second step of reaction: the 2-keto-1-butanol generated by the reaction in the previous step is taken as a substrate, phosphate buffer solution with the concentration of 100mM, the concentration of isopropylamine of 500mM and the pH value of 8.0, pyridoxal phosphate (PLP) with the final concentration of 1mM and transaminase of Bacillus megaterium (Bacillus megaterium) with the final concentration of 10g/L or transaminase freeze-dried powder or enzyme liquid of pseudomonas aeruginosa PAO2 are sequentially added into a reaction system to form the reaction system, and the reaction system is reacted for 24 hours at the temperature of 30 ℃ to obtain the (S) -2-amino-1-butanol. The reaction solution is detected by liquid chromatography after derivation of o-phthalaldehyde. The HPLC detection conditions are as shown in example step three.

The HPLC detection results refer to E in FIGS. 4 and 5, the substrate conversion efficiency is 45-50%, and the ee value is greater than 99%. See table 7 for specific results.

TABLE 7 results of alcohol dehydrogenase coupling transaminase production of (R) -2-amino-1-butanol

Note: in the table, Wn and Wn-Mn (n is a natural number) in the first reaction and the second reaction have the same meanings as in Table 6.

Example 4 Co-expression of enzyme A and enzyme B Whole cell preparation of (S) -2-amino-1-butanol and (R) -2-amino-1-butanol

Preparation of engineering bacteria co-expressed by enzyme A and enzyme B

The coding genes of related enzymes are respectively subjected to whole-gene synthesis (codon optimization is carried out by taking escherichia coli as a host according to needs), and the synthesized genes are connected to various expression vectors to construct the recombinant expression vector. The expression vector is any vector conventionally used in the art. The vector of the invention specifically contains pETDuet-1, the DNA fragment of enzyme A after the whole gene synthesis is inserted between enzyme cutting sites EcoRI and Hind III of pETDuet-1, and the DNA fragment of enzyme B after the whole gene synthesis is inserted between enzyme cutting sites Nde I and Xho I of pETDuet-1. Transferring the recombinant vector into escherichia coli DH5 alpha competent cells; and (4) selecting a positive transformant, sequencing and identifying to obtain a correct recombinant expression vector.

The recombinant expression vector with the correct sequencing verification is transformed into a suitable microbial host. The host microorganism is any host microorganism which is conventional in the art, as long as the recombinant expression vector can stably replicate itself and the carried alcohol dehydrogenase and amino acid dehydrogenase genes can be efficiently expressed. Wherein the host microorganism is preferably: coli (Escherichia coli), preferably E.coli BL21(DE3), transformed into E.coli BL21(DE3) to obtain the genetically engineered strain of the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably an electro-transformation method or a chemical transformation method.

The enzymes and their mutants referred to in this example are detailed in Table 8.

TABLE 8 enzymes and mutants thereof referred to in example 4

Note: W1-W17 have the same meanings as in tables 1, 4 and 6. Wn-Mn represents a mutant of Wn (n is a natural number). The numbering and specific nomenclature of the substitutions of proteins and genes are as in Table 1.

Co-expression of enzyme A and enzyme B

Transferring the recombinant expression vector constructed in the step one into competent cells of escherichia coli BL21(DE3), culturing for 12-16h at 37 ℃, inoculating a single transformant into 5mL LB culture medium containing corresponding antibiotics after the transformant grows out, and culturing overnight (12-16h) at 37 ℃ and 220 rmp. Then inoculating into TB medium according to the proportion of 1% (volume percentage content), culturing at 37 deg.C and 220rmp until OD600 is about 0.6, adding IPTG with final concentration of 0.1mM, inducing and culturing at 20-37 deg.C for 16h, and centrifuging at 4000rpm and 4 deg.C for 10min to collect cells.

Preparation of tris, chiral 2-amino-1-butanol

A phosphate buffer solution with a concentration of 100mM and a pH value of 8.0, 1, 2-butanediol with a final concentration of 50mM, 100mM glucose, and a whole cell (wet bacterial weight) capable of co-expressing the enzyme A and the enzyme B with a final concentration of 100g/L are sequentially added to the reaction system to constitute the reaction system. Reacting the reaction system at 30 ℃ for 24h to obtain (R) or (S) -2-amino-1-butanol. And (3) performing liquid chromatography detection on the fermentation liquor after derivation by using o-phthalaldehyde. HPLC detection conditions are as shown in step three of example 1.

The concrete calculation methods of ee value and substrate conversion efficiency are the same as those of the third step in example 1.

The HPLC detection results refer to D-E in FIGS. 4 and 5, the substrate conversion efficiency is 15-30%, and the ee value is greater than 99%. See table 9 for specific results.

TABLE 9 results of Whole-cell catalyzed preparation of chiral 2-amino-1-butanol by coexpression of enzymes A and B

Note: in the table, Wn and Wn-Mn (n is a natural number) have the same meanings as in Table 8.

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> synthetic method of chiral 2-amino-1-butanol

<130> GNCLN171921

<160> 34

<170> PatentIn version 3.5

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Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys

195 200 205

Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu

210 215 220

Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile

225 230 235 240

Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val

245 250 255

Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu

260 265 270

Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys

275 280 285

Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg

290 295 300

Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val

305 310 315 320

Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu

325 330 335

Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala

340 345 350

<210> 7

<211> 759

<212> DNA

<213> Lactobacillus kefiri yogurt (Lactobacillus kefiri)

<400> 7

atgactgatc gtttaaaagg caaagtagca attgtaactg gcggtacctt gggaattggc 60

ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120

gctgatgtag gtgaaaaagc tgccaaatca atcggcggca cagacgttat ccgttttgtc 180

caacacgatg cttctgatga agccggctgg actaagttgt ttgatacgac tgaagaagca 240

tttggcccag ttaccacggt tgtcaacaat gccggaattg cggtcagcaa gagtgttgaa 300

gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttggatgg tgtcttcttc 360

ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420

atgtcatcta tcgaaggttt tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480

ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540

gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcttgaa 600

ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660

aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720

gcagaattcg ttgtcgatgg tggatacact gctcaataa 759

<210> 8

<211> 252

<212> PRT

<213> Lactobacillus kefiri yogurt (Lactobacillus kefiri)

<400> 8

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser

85 90 95

Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 9

<211> 1023

<212> DNA

<213> Bacillaceae (Bacillus)

<400> 9

atgaaagctg cagtagtaga gcaatttaag gaaccattaa aaattaaaga agtggaaaag 60

ccatccattt catatggcga agtattagtc cgcattaaag catgcggtgt atgccatacg 120

gacttgcacg ccgctcatgg cgattggcca gtaaaaccaa aacttccttt aatccctggc 180

catgaaggag tcggaattgt tgaagaagtc ggtccggggg taacccattt aaaagtggga 240

gaccgcgttg gaattccttg gttatattct gcttgcggcc attgcgaata ttgtttaagc 300

ggacaagaga cattatgtga acatcaagaa aacgccggct actcagtcga cgggggttat 360

gcagaatatt gcagagctgc ggcagactat gtggtgaaaa ttcctgacaa cttgtcgttt 420

gaagaagctg ctcctatttt ctgcgccgga gttactactt ataaagcgtt aaaagtcaca 480

ggtacaaaac cgggagaatg ggtagcgatc tatggcatcg gcggccttgg acatgttgcc 540

gtccagtatg cgaaagcgat ggggcttcat gttgttgcag tggatatcgg cgatgagaaa 600

ctggaacttg caaaagagct tggcgccgat cttgttgtaa atcctgcaaa agaaaatgcg 660

gcacaattta tgaaagagaa agtcggcgga gtacacgcgg ctgttgtgac agctgtatct 720

aaacctgctt ttcaatctgc gtacaattct atccgcagag gcggcacgtg cgtgcttgtc 780

ggattaccgc cggaagaaat gcctattcca atctttgata cggtattaaa cggaattaaa 840

attatcggtt ccattgtcgg cacgcggaaa gacttgcaag aagcgcttca gttcgctgca 900

gaaggtaaag taaaaaccat tattgaagtg caacctcttg aaaaaattaa cgaagtattt 960

gacagaatgc taaaaggaga aattaacgga cgggttgttt taacgttaga aaataataat 1020

taa 1023

<210> 10

<211> 340

<212> PRT

<213> Bacillaceae (Bacillus)

<400> 10

Met Lys Ala Ala Val Val Glu Gln Phe Lys Glu Pro Leu Lys Ile Lys

1 5 10 15

Glu Val Glu Lys Pro Ser Ile Ser Tyr Gly Glu Val Leu Val Arg Ile

20 25 30

Lys Ala Cys Gly Val Cys His Thr Asp Leu His Ala Ala His Gly Asp

35 40 45

Trp Pro Val Lys Pro Lys Leu Pro Leu Ile Pro Gly His Glu Gly Val

50 55 60

Gly Ile Val Glu Glu Val Gly Pro Gly Val Thr His Leu Lys Val Gly

65 70 75 80

Asp Arg Val Gly Ile Pro Trp Leu Tyr Ser Ala Cys Gly His Cys Glu

85 90 95

Tyr Cys Leu Ser Gly Gln Glu Thr Leu Cys Glu His Gln Glu Asn Ala

100 105 110

Gly Tyr Ser Val Asp Gly Gly Tyr Ala Glu Tyr Cys Arg Ala Ala Ala

115 120 125

Asp Tyr Val Val Lys Ile Pro Asp Asn Leu Ser Phe Glu Glu Ala Ala

130 135 140

Pro Ile Phe Cys Ala Gly Val Thr Thr Tyr Lys Ala Leu Lys Val Thr

145 150 155 160

Gly Thr Lys Pro Gly Glu Trp Val Ala Ile Tyr Gly Ile Gly Gly Leu

165 170 175

Gly His Val Ala Val Gln Tyr Ala Lys Ala Met Gly Leu His Val Val

180 185 190

Ala Val Asp Ile Gly Asp Glu Lys Leu Glu Leu Ala Lys Glu Leu Gly

195 200 205

Ala Asp Leu Val Val Asn Pro Ala Lys Glu Asn Ala Ala Gln Phe Met

210 215 220

Lys Glu Lys Val Gly Gly Val His Ala Ala Val Val Thr Ala Val Ser

225 230 235 240

Lys Pro Ala Phe Gln Ser Ala Tyr Asn Ser Ile Arg Arg Gly Gly Thr

245 250 255

Cys Val Leu Val Gly Leu Pro Pro Glu Glu Met Pro Ile Pro Ile Phe

260 265 270

Asp Thr Val Leu Asn Gly Ile Lys Ile Ile Gly Ser Ile Val Gly Thr

275 280 285

Arg Lys Asp Leu Gln Glu Ala Leu Gln Phe Ala Ala Glu Gly Lys Val

290 295 300

Lys Thr Ile Ile Glu Val Gln Pro Leu Glu Lys Ile Asn Glu Val Phe

305 310 315 320

Asp Arg Met Leu Lys Gly Glu Ile Asn Gly Arg Val Val Leu Thr Leu

325 330 335

Glu Asn Asn Asn

340

<210> 11

<211> 747

<212> DNA

<213> Lysidium (Leifsonia sp.)

<400> 11

atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60

gggcgcgccg tggcgctcac tctcgcggcg agcggcgcag ccgtcctcgt caccgacctg 120

aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg taaggccgcc 180

gcgctcgcgg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cggggcgaac 240

gctctcgcgc ccctcaagat cgcggtcaac aacgcgggca tcggcggcga ggccgccacg 300

gtcggcgact actcgctcga cagctggcgc acggtgatcg aggtcaacct caacgccgtg 360

ttctacggga tgcagccgca gctgaaggcc atggccgcca acggcggcgg tgcgatcgtc 420

aacatggcgt ccatcctggg aagcgtcggc ttcgccaact cgtcggccta cgtcacggcc 480

aagcacgcgc tgctcggtct cacccagaac gccgcgctcg agtacgccgc cgacaaggtg 540

cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600

gccgacgcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggaa 660

gaggtcgcct cgctggtcgc gttcctcgcc tccgacgccg cgagcttcat caccggcagc 720

taccacctgg tggacggcgg ctacacc 747

<210> 12

<211> 249

<212> PRT

<213> Lysidium (Leifsonia sp.)

<400> 12

Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly

1 5 10 15

Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly

20 25 30

Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val

35 40 45

Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly

50 55 60

Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Gly Ala Asn

65 70 75 80

Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly

85 90 95

Glu Ala Ala Thr Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Thr Val

100 105 110

Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu

115 120 125

Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser

130 135 140

Ile Leu Gly Ser Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala

145 150 155 160

Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala

165 170 175

Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr

180 185 190

Pro Leu Val Glu Ala Asn Leu Ser Ala Asp Ala Leu Ala Phe Leu Glu

195 200 205

Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser

210 215 220

Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser

225 230 235 240

Tyr His Leu Val Asp Gly Gly Tyr Thr

245

<210> 13

<211> 1059

<212> DNA

<213> Thermoanaerobacter virginiae (Thermoanaerobacter wiegelii)

<400> 13

atgaaaggtt ttgcaatgct cagtatcggt aaggttggct ggattgaggt agaaaagcct 60

aatccaggac cctttgatgc tatcgtaaga cccctagctg tggccccttg ctcttcggac 120

attcacactg tttttgaagg aggccttggt gaacttcaca acgcagtgct aggtcacgaa 180

gctgtaggtg aagtagtcga agtcggtagt gaagtaaaag actttaaacc tggtgataag 240

gtggtcattc ctgctatcac tcctgattgg agaacgttag atgttcaacg tggttatcat 300

cagcagtccg gaggtatgct tgctggttac aagttcacag cccagaaacc tggtgtgttc 360

gccgagtaca tctacgttaa cgatgcagac atgaatcttg ctcatttacc tgacggcatc 420

tctttagaag cggccgtcat gatcacagat atgatgacta ccggttttca cggagccgaa 480

ctggcagaaa tagaattagg tgcaacagta gcggttttgg gtattggtcc agtaggtctt 540

atggcagtcg ctggtgccaa attgcggggt gctggaagaa ttattgcagt aggcagtaga 600

cctgtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaaaat 660

ggtcctatcg acagtcagat tatggattta acgaaaggca aaggtgttga tgctgccatc 720

atcgctggag gaaatgttga catcatggct acagcagtta agattgttaa acctggtggc 780

accattgcta atgtaaatta ctttggcgaa ggagatgttt tgcctgttcc tcgtcttgaa 840

tggggttgcg gcatggctca taaagctata aaaggcggtt tatgccctgg tggacgtcta 900

agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc caaactcgtc 960

actcatgttt ttcaaggatt tgataatatt gaaaaagctc taatgctgat gaaagataaa 1020

ccaaaggacc taatcaaacc tgttgtaata ttagcataa 1059

<210> 14

<211> 352

<212> PRT

<213> Thermoanaerobacter virginiae (Thermoanaerobacter wiegelii)

<400> 14

Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu

1 5 10 15

Val Glu Lys Pro Asn Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu

20 25 30

Ala Val Ala Pro Cys Ser Ser Asp Ile His Thr Val Phe Glu Gly Gly

35 40 45

Leu Gly Glu Leu His Asn Ala Val Leu Gly His Glu Ala Val Gly Glu

50 55 60

Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Lys

65 70 75 80

Val Val Ile Pro Ala Ile Thr Pro Asp Trp Arg Thr Leu Asp Val Gln

85 90 95

Arg Gly Tyr His Gln Gln Ser Gly Gly Met Leu Ala Gly Tyr Lys Phe

100 105 110

Thr Ala Gln Lys Pro Gly Val Phe Ala Glu Tyr Ile Tyr Val Asn Asp

115 120 125

Ala Asp Met Asn Leu Ala His Leu Pro Asp Gly Ile Ser Leu Glu Ala

130 135 140

Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu

145 150 155 160

Leu Ala Glu Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly

165 170 175

Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly

180 185 190

Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys

195 200 205

Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Pro Ile Asp

210 215 220

Ser Gln Ile Met Asp Leu Thr Lys Gly Lys Gly Val Asp Ala Ala Ile

225 230 235 240

Ile Ala Gly Gly Asn Val Asp Ile Met Ala Thr Ala Val Lys Ile Val

245 250 255

Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Asp

260 265 270

Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys

275 280 285

Ala Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg

290 295 300

Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val

305 310 315 320

Thr His Val Phe Gln Gly Phe Asp Asn Ile Glu Lys Ala Leu Met Leu

325 330 335

Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala

340 345 350

<210> 15

<211> 1035

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 15

atggcgaaga tcgacaacgc ggtgctgccg gaaggtagcc tggtgctggt taccggtgcg 60

aacggtttcg tggcgagcca cgtggttgag cagctgctgg aacacggtta caaggttcgt 120

ggcaccgcgc gtagcgcgag caaactggcg aacctgcaaa agcgttggga cgcgaaatac 180

ccgggtcgtt ttgagaccgc ggtggttgaa gacatgctga agcagggcgc gtatgatgaa 240

gtgatcaagg gtgcggcggg cgttgcgcac attgcgagcg tggttagctt cagcaacaag 300

tatgatgagg tggttacccc ggcgatcggt ggcaccctga acgcgctgcg tgctgcggcg 360

gcgaccccga gcgtgaaacg ttttgttctg accagcagca ccgtgagcgc gctgatcccg 420

aagccgaacg ttgagggtat ttacctggat gagaagagct ggaacctgga gagcattgac 480

aaggcgaaaa ccctgccgga aagcgatccg caaaagagcc tgtgggtgta tgcggcgagc 540

aaaaccgagg cggaactggc ggcgtggaag ttcatggacg agaacaaacc gcactttacc 600

ctgaacgcgg ttctgccgaa ctacaccatc ggcaccattt tcgatccgga aacccagagc 660

ggtagcacca gcggctggat gatgagcctg tttaacggcg aggtgtctcc ggcgctggcg 720

ctgatgccgc cgcagtacta tgtgagcgcg gttgacatcg gtctgctgca cctgggttgc 780

ctggtgctgc cgcaaattga gcgtcgtcgt gtttacggca ccgcgggcac cttcgattgg 840

aacaccgttc tggcgacctt tcgtaagctg tatccgagca aaaccttccc ggcggacttt 900

ccggatcagg gtcaagacct gagcaagttc gataccgcgc cgagcctgga aattctgaaa 960

agcctgggtc gtccgggttg gcgtagcatc gaggaaagca ttaaagacct ggttggcagc 1020

gagaccgcgc actaa 1035

<210> 16

<211> 344

<212> PRT

<213> Ochragma Sporobolomyces salmonicolor)

<400> 16

Met Ala Lys Ile Asp Asn Ala Val Leu Pro Glu Gly Ser Leu Val Leu

1 5 10 15

Val Thr Gly Ala Asn Gly Phe Val Ala Ser His Val Val Glu Gln Leu

20 25 30

Leu Glu His Gly Tyr Lys Val Arg Gly Thr Ala Arg Ser Ala Ser Lys

35 40 45

Leu Ala Asn Leu Gln Lys Arg Trp Asp Ala Lys Tyr Pro Gly Arg Phe

50 55 60

Glu Thr Ala Val Val Glu Asp Met Leu Lys Gln Gly Ala Tyr Asp Glu

65 70 75 80

Val Ile Lys Gly Ala Ala Gly Val Ala His Ile Ala Ser Val Val Ser

85 90 95

Phe Ser Asn Lys Tyr Asp Glu Val Val Thr Pro Ala Ile Gly Gly Thr

100 105 110

Leu Asn Ala Leu Arg Ala Ala Ala Ala Thr Pro Ser Val Lys Arg Phe

115 120 125

Val Leu Thr Ser Ser Thr Val Ser Ala Leu Ile Pro Lys Pro Asn Val

130 135 140

Glu Gly Ile Tyr Leu Asp Glu Lys Ser Trp Asn Leu Glu Ser Ile Asp

145 150 155 160

Lys Ala Lys Thr Leu Pro Glu Ser Asp Pro Gln Lys Ser Leu Trp Val

165 170 175

Tyr Ala Ala Ser Lys Thr Glu Ala Glu Leu Ala Ala Trp Lys Phe Met

180 185 190

Asp Glu Asn Lys Pro His Phe Thr Leu Asn Ala Val Leu Pro Asn Tyr

195 200 205

Thr Ile Gly Thr Ile Phe Asp Pro Glu Thr Gln Ser Gly Ser Thr Ser

210 215 220

Gly Trp Met Met Ser Leu Phe Asn Gly Glu Val Ser Pro Ala Leu Ala

225 230 235 240

Leu Met Pro Pro Gln Tyr Tyr Val Ser Ala Val Asp Ile Gly Leu Leu

245 250 255

His Leu Gly Cys Leu Val Leu Pro Gln Ile Glu Arg Arg Arg Val Tyr

260 265 270

Gly Thr Ala Gly Thr Phe Asp Trp Asn Thr Val Leu Ala Thr Phe Arg

275 280 285

Lys Leu Tyr Pro Ser Lys Thr Phe Pro Ala Asp Phe Pro Asp Gln Gly

290 295 300

Gln Asp Leu Ser Lys Phe Asp Thr Ala Pro Ser Leu Glu Ile Leu Lys

305 310 315 320

Ser Leu Gly Arg Pro Gly Trp Arg Ser Ile Glu Glu Ser Ile Lys Asp

325 330 335

Leu Val Gly Ser Glu Thr Ala His

340

<210> 17

<211> 1104

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 17

atggaactgt ttaagtatat ggaaacctat gattatgaac aagtgctgtt ttgccaggac 60

aaggaaagcg gtctgaaggc gattattgcc attcatgata ccacgctggg tccggcactg 120

ggcggtaccc gtatgtggat gtataacagc gaagaagaag cgctggaaga tgccctgcgt 180

ctggcacgcg gtatgaccta caaaaacgca gcagcaggtc tgaatctggg cggtggcaag 240

acggtgatta tcggtgatcc gcgcaaagac aagaacgaag ctatgtttcg tgcgttcggc 300

cgctttatcc agggtctgaa tggccgttat attaccgcgg aagatgtggg taccacggtt 360

gccgatatgg acattatcta tcaagaaacc gactacgtga cgggcatttc accggaattt 420

ggtagctctg gtaacccgtc gccggcaacg gcctatggtg tttaccgtgg catgaaagct 480

gcggccaagg aagcatttgg tagtgattcc ctggaaggca aagtggttgc agttcagggt 540

gtcggcaatg tggcttatca cctgtgccgc catctgcacg aagaaggcgc caaactgatt 600

gtgaccgata tcaacaagga agtcgtggca cgtgctgttg aagaatttgg tgcgaaagcc 660

gtcgatccga atgacatcta cggcgtggaa tgcgatattt ttgcgccgtg tgccctgggt 720

ggcattatca acgaccagac catcccgcaa ctgaaagcga aggttattgc aggtagtgct 780

aacaatcagc tgaaagaacc gcgtcatggc gatattatcc acgaaatggg catcgtctat 840

gccccggact acgtgattaa cgcaggtggc gttatcaatg tcgctgatga actgtatggc 900

tacaatcgtg aacgcgcgat gaaaaagatt gaacaaatct atgacaacat cgaaaaagtt 960

ttcgcaatcg ctaagcgtga taatattccg acctacgtcg cagctgaccg tatggccgaa 1020

gaacgcattg aaacgatgcg taaagcccgt tcccagttcc tgcaaaatgg tcatcatatt 1080

ctgagccgcc gtcgtgcccg ctaa 1104

<210> 18

<211> 367

<212> PRT

<213> Bacillus stearothermophilus (Geobacillus stearothermophilus)

<400> 18

Met Glu Leu Phe Lys Tyr Met Glu Thr Tyr Asp Tyr Glu Gln Val Leu

1 5 10 15

Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala Ile His

20 25 30

Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Met Tyr

35 40 45

Asn Ser Glu Glu Glu Ala Leu Glu Asp Ala Leu Arg Leu Ala Arg Gly

50 55 60

Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys

65 70 75 80

Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe

85 90 95

Arg Ala Phe Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr

100 105 110

Ala Glu Asp Val Gly Thr Thr Val Ala Asp Met Asp Ile Ile Tyr Gln

115 120 125

Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Glu Phe Gly Ser Ser Gly

130 135 140

Asn Pro Ser Pro Ala Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala

145 150 155 160

Ala Ala Lys Glu Ala Phe Gly Ser Asp Ser Leu Glu Gly Lys Val Val

165 170 175

Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Arg His Leu

180 185 190

His Glu Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Val

195 200 205

Val Ala Arg Ala Val Glu Glu Phe Gly Ala Lys Ala Val Asp Pro Asn

210 215 220

Asp Ile Tyr Gly Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly

225 230 235 240

Gly Ile Ile Asn Asp Gln Thr Ile Pro Gln Leu Lys Ala Lys Val Ile

245 250 255

Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Pro Arg His Gly Asp Ile

260 265 270

Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala

275 280 285

Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu

290 295 300

Arg Ala Met Lys Lys Ile Glu Gln Ile Tyr Asp Asn Ile Glu Lys Val

305 310 315 320

Phe Ala Ile Ala Lys Arg Asp Asn Ile Pro Thr Tyr Val Ala Ala Asp

325 330 335

Arg Met Ala Glu Glu Arg Ile Glu Thr Met Arg Lys Ala Arg Ser Gln

340 345 350

Phe Leu Gln Asn Gly His His Ile Leu Ser Arg Arg Arg Ala Arg

355 360 365

<210> 19

<211> 993

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 19

atggcatttt ctgcagatac cagcgaaatt gtttataccc atgataccgg tctggattat 60

attacctata gcgattatga actggaccct gccaatccgc tggccggcgg tgctgcttgg 120

attgaaggtg cctttgtgcc gccgagtgaa gcacgcatta gtatttttga tcagggttat 180

ctgcatagtg atgtgaccta taccgtgttt catgtttgga atggtaatgc ctttcgtctg 240

gatgatcata ttgaacgtct gtttagcaat gccgaaagca tgcgcattat tccgccgctg 300

acccaggatg aagttaaaga aattgcactg gaactggttg caaaaaccga actgcgtgaa 360

gcatttgtta gtgttagcat tacccgtggc tatagcagca ccccgggtga acgtgatatt 420

accaaacatc gcccgcaggt ttatatgtat gcagttccgt atcagtggat tgttccgttt 480

gatcgcattc gtgatggtgt gcatgcaatg gttgcacaga gtgttcgtcg caccccgcgt 540

agcagcattg atccgcaggt taaaaatttt cagtggggcg atctgattcg cgccgttcag 600

gaaacccatg atcgcggctt tgaagcaccg ctgctgctgg atggcgatgg tctgctggcc 660

gaaggtagcg gctttaatgt ggtggttatt aaggatggcg ttgttcgcag cccgggtcgt 720

gcagcactgc cgggtattac ccgcaaaacc gtgctggaaa ttgcagaaag cctgggccat 780

gaagccattc tggcagatat taccctggca gaactgctgg atgcagatga agttctgggt 840

tgtaccaccg ccggtggcgt gtggccgttt gttagtgtgg atggtaatcc gattagcgat 900

ggcgtgccgg gtccggttac ccagagtatt attcgccgtt attgggaact gaatgtggaa 960

agcagcagtc tgctgacccc ggttcagtat taa 993

<210> 20

<211> 330

<212> PRT

<213> ATA-117

<400> 20

Met Ala Phe Ser Ala Asp Thr Ser Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp

50 55 60

Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Met Arg Ile

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Ser Val Ser Ile Thr

115 120 125

Arg Gly Tyr Ser Ser Thr Pro Gly Glu Arg Asp Ile Thr Lys His Arg

130 135 140

Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

Asp Arg Ile Arg Asp Gly Val His Ala Met Val Ala Gln Ser Val Arg

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

Gly Asp Leu Ile Arg Ala Val Gln Glu Thr His Asp Arg Gly Phe Glu

195 200 205

Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Leu Ala Glu Leu

260 265 270

Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly

290 295 300

Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210> 21

<211> 975

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 21

atggctagta tggataaggt gttcgccggc tatgccgcac gtcaagcaat tctggaaagt 60

accgaaacca ccaatccgtt tgcaaaaggt attgcctggg ttgaaggtga actggtgccg 120

ttagccgaag cacgtattcc gctgctggat cagggcttta tgcatagtga tctgacctat 180

gatgtgccga gtgtttggga tggtcgcttt ttccgcctgg atgatcatat tacccgcctg 240

gaagcaagct gcaccaaact gcgtctgcgc ttaccgctgc ctcgtgacca ggtgaaacag 300

attctggtgg aaatggttgc aaagagcggt attcgcgatg cctttgtgga actgattgtt 360

acccgcggcc tgaaaggtgt gcgcggtacg cgtcctgaag atattgtgaa taatctgtac 420

atgttcgtgc agccgtatgt ttgggttatg gaaccggata tgcagcgtgt gggtggcagt 480

gcagtggttg caagaaccgt gcgtcgcgtt cctcctggtg caattgatcc gaccgttaaa 540

aatctgcagt ggggtgacct ggttcgcggt atgtttgaag ccgccgatcg tggtgccacc 600

tatccttttc tgaccgatgg tgacgcacat ctgaccgaag gcagcggttt taatattgtg 660

ctggttaaag acggcgtgct gtataccccg gatcgcggtg tgttacaggg cgtgaccaga 720

aaaagtgtta ttaatgcagc cgaggccttt ggcattgaag tgcgtgtgga atttgtgccg 780

gtggaactgg cctatcgctg cgacgagatt tttatgtgca ccaccgccgg tggtattatg 840

ccgattacca ccctggatgg tatgccggtg aatggcggtc agattggccc tattaccaaa 900

aagatttggg acggttactg ggccatgcat tatgatgcag catatagctt tgagatcgac 960

tataacgagc gtaat 975

<210> 22

<211> 325

<212> PRT

<213> Aspergillus terreus (Aspergillus terreus)

<400> 22

Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala

1 5 10 15

Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala

20 25 30

Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu

35 40 45

Leu Asp Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser

50 55 60

Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu

65 70 75 80

Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp

85 90 95

Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg

100 105 110

Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg

115 120 125

Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln

130 135 140

Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser

145 150 155 160

Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp

165 170 175

Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe

180 185 190

Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp

195 200 205

Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp

210 215 220

Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg

225 230 235 240

Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val

245 250 255

Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met

260 265 270

Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met

275 280 285

Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp

290 295 300

Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp

305 310 315 320

Tyr Asn Glu Arg Asn

325

<210> 23

<211> 969

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 23

atggcaagta tggataaggt gttcagtggt tattacgccc gccagaaact gctggaacgt 60

agcgataatc cgtttagcaa aggtattgca tacgtggaag gcaaactggt tctgccgagc 120

gatgcacgta ttccgctgtt agatgaaggt tttatgcaca gtgacctgac ctatgatgtt 180

attagcgtgt gggatggtcg ctttttccgc ctggatgatc atctgcagcg tattctggaa 240

agttgtgata aaatgcgcct gaaattcccg ctggccctgt caagtgttaa aaatattctg 300

gcagagatgg tggccaaaag tggcattcgt gatgcatttg ttgaggttat tgtgacccgc 360

ggtctgaccg gtgtgagagg tagcaaaccg gaagatttgt ataacaacaa catctacctg 420

ctggtgctgc cgtatatttg ggttatggca ccggaaaatc agctgcatgg tggtgaagcc 480

attattaccc gcaccgtgcg ccgtacaccg cctggtgcat tcgaccctac aattaagaat 540

ctgcagtggg gtgacctgac caaaggtctg tttgaagcaa tggatcgcgg cgccacctat 600

ccttttctga ccgatggtga caccaatctg accgaaggta gcggttttaa tatcgttctg 660

gttaagaacg gcatcatcta taccccggat cgtggtgtgc tgcgtggtat tacccgcaaa 720

agtgtgattg atgttgcacg tgcaaacagt attgacattc gtctggaagt tgtgccggtt 780

gaacaggcct atcatagcga tgaaattttc atgtgcacca ccgccggtgg cattatgcct 840

attaccctgc tggatggcca gccggttaat gatggtcagg tgggtccgat taccaaaaag 900

atttgggatg gttactggga aatgcattac aatccggcat atagcttccc ggtggattat 960

ggtagtggt 969

<210> 24

<211> 323

<212> PRT

<213> Aspergillus fumigatus (Aspergillus fumigatus)

<400> 24

Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr Tyr Ala Arg Gln Lys

1 5 10 15

Leu Leu Glu Arg Ser Asp Asn Pro Phe Ser Lys Gly Ile Ala Tyr Val

20 25 30

Glu Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Ile Pro Leu Leu Asp

35 40 45

Glu Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Ile Ser Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Gln Arg Ile Leu Glu

65 70 75 80

Ser Cys Asp Lys Met Arg Leu Lys Phe Pro Leu Ala Leu Ser Ser Val

85 90 95

Lys Asn Ile Leu Ala Glu Met Val Ala Lys Ser Gly Ile Arg Asp Ala

100 105 110

Phe Val Glu Val Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ser

115 120 125

Lys Pro Glu Asp Leu Tyr Asn Asn Asn Ile Tyr Leu Leu Val Leu Pro

130 135 140

Tyr Ile Trp Val Met Ala Pro Glu Asn Gln Leu His Gly Gly Glu Ala

145 150 155 160

Ile Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Lys Gly Leu Phe Glu

180 185 190

Ala Met Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asn Gly

210 215 220

Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Arg Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Val Ala Arg Ala Asn Ser Ile Asp Ile Arg Leu Glu

245 250 255

Val Val Pro Val Glu Gln Ala Tyr His Ser Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Leu Leu Asp Gly Gln Pro

275 280 285

Val Asn Asp Gly Gln Val Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly

290 295 300

Tyr Trp Glu Met His Tyr Asn Pro Ala Tyr Ser Phe Pro Val Asp Tyr

305 310 315 320

Gly Ser Gly

<210> 25

<211> 969

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 25

atggctagta tggataaggt gttcagcggc tatcatgccc gccagaaact gctggaacgt 60

agtgataatc cgtttagtaa gggcattgcc tatgtggaag gtaaactggt gctgccgagt 120

gatgcccgta ttcctctgct ggatgaaggc tttatgcatg gtgacctgac ctatgatgtt 180

accaccgtgt gggatggtcg ctttttccgt ctggatgatc acatgcagcg tattctggaa 240

agctgcgata aaatgcgtct gaaattcccg ctggccccga gtacagttaa aaatattctg 300

gcagagatgg tggcaaagag cggcattcgc gatgcctttg ttgaagtgat tgttacccgt 360

ggtctgaccg gtgttcgtgg tagtaaaccg gaagatttgt ataacaacaa catctacctg 420

ctggtgctgc cttatgtgtg ggttatggca ccggaaaatc agctgctggg cggttcagca 480

attattaccc gcaccgtgcg ccgtacccct cctggtgcat tcgaccctac aattaagaat 540

ctgcagtggg gcgatctgac caaaggctta tttgaagcaa tggatcgcgg cgccacctat 600

ccttttctga ccgatggtga caccaatctg accgaaggta gcggctttaa tattgttctg 660

gtgaaaaacg gcatcatcta caccccggat cgcggtgttc tgcgtggtat tacccgcaaa 720

agtgttattg atgtggcccg cgcaaataat attgatattc gtctggaggt ggtgccggtt 780

gaacaggttt atcatagtga tgaaatcttc atgtgcacca ccgccggcgg tattatgcct 840

attaccctgc tggatggtca gccggttaat gatggtcagg ttggcccgat taccaaaaag 900

atttgggatg gctattggga aatgcattac aatccggcat acagctttcc ggttgattat 960

ggtagcggc 969

<210> 26

<211> 323

<212> PRT

<213> Fishchito bacterium (Neosartorya fischeri)

<400> 26

Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr His Ala Arg Gln Lys

1 5 10 15

Leu Leu Glu Arg Ser Asp Asn Pro Phe Ser Lys Gly Ile Ala Tyr Val

20 25 30

Glu Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Ile Pro Leu Leu Asp

35 40 45

Glu Gly Phe Met His Gly Asp Leu Thr Tyr Asp Val Thr Thr Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Met Gln Arg Ile Leu Glu

65 70 75 80

Ser Cys Asp Lys Met Arg Leu Lys Phe Pro Leu Ala Pro Ser Thr Val

85 90 95

Lys Asn Ile Leu Ala Glu Met Val Ala Lys Ser Gly Ile Arg Asp Ala

100 105 110

Phe Val Glu Val Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ser

115 120 125

Lys Pro Glu Asp Leu Tyr Asn Asn Asn Ile Tyr Leu Leu Val Leu Pro

130 135 140

Tyr Val Trp Val Met Ala Pro Glu Asn Gln Leu Leu Gly Gly Ser Ala

145 150 155 160

Ile Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Lys Gly Leu Phe Glu

180 185 190

Ala Met Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asn Gly

210 215 220

Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Arg Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Val Ala Arg Ala Asn Asn Ile Asp Ile Arg Leu Glu

245 250 255

Val Val Pro Val Glu Gln Val Tyr His Ser Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Leu Leu Asp Gly Gln Pro

275 280 285

Val Asn Asp Gly Gln Val Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly

290 295 300

Tyr Trp Glu Met His Tyr Asn Pro Ala Tyr Ser Phe Pro Val Asp Tyr

305 310 315 320

Gly Ser Gly

<210> 27

<211> 975

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 27

atgagcacaa tggataagat ttttgcaggc catgcacagc gccaggcaac attagtggcc 60

agtgataata ttttcgcgaa cggcattgca tggattcagg gtgaactggt tccgctgaat 120

gaagcacgta ttccgctgat ggatcagggc tttatgcatg gtgacctgac ctatgatgtt 180

ccggcagttt gggatggccg ctttttccgt ctggatgatc atctggatcg cctggaagca 240

agtgttaaaa agatgcgtat gcagttcccg attccgcgtg atgaaattcg catgaccctg 300

ctggatatgc tggcaaaaag tggcattaag gatgcctttg tggaactgat tgtgacccgc 360

ggtctgaaac cggtgcgtga ggcaaaaccg ggtgaagttc tgaataatca tctgtatctg 420

atcgtgcagc cgtatgtttg ggttatgagc ccggaagccc agtatgttgg cggtaatgcc 480

gtgattgcac gcaccgttcg tcgtattccg ccgggtagca tggaccctac aattaagaat 540

ctgcagtgga gcgatttcac ccgcggcatg tttgaagcct atgatcgcgg cgcccagtat 600

ccttttctga ccgatggcga taccaatatt accgaaggta gcggttttaa cgtggttttt 660

gttaagaaca acgtgatcta caccccgaat cgtggtgttc tgcagggtat tacccgtaaa 720

agtgttattg acgccgcaaa atggtgtggt catgaagtgc gtgtggaata tgttccggtt 780

gaaatggcct atgaagcaga tgaaatcttc atgtgcacca ccgcaggcgg cattatgcct 840

attaccacaa tggatggtaa accggtgaaa gatggtaaag tgggtccggt taccaaagca 900

atttgggatc gttattgggc catgcattgg gaagatgaat tttcattcaa gatcgactac 960

cagaagctga aactg 975

<210> 28

<211> 325

<212> PRT

<213> Gibberella zeae

<400> 28

Met Ser Thr Met Asp Lys Ile Phe Ala Gly His Ala Gln Arg Gln Ala

1 5 10 15

Thr Leu Val Ala Ser Asp Asn Ile Phe Ala Asn Gly Ile Ala Trp Ile

20 25 30

Gln Gly Glu Leu Val Pro Leu Asn Glu Ala Arg Ile Pro Leu Met Asp

35 40 45

Gln Gly Phe Met His Gly Asp Leu Thr Tyr Asp Val Pro Ala Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Asp Arg Leu Glu Ala

65 70 75 80

Ser Val Lys Lys Met Arg Met Gln Phe Pro Ile Pro Arg Asp Glu Ile

85 90 95

Arg Met Thr Leu Leu Asp Met Leu Ala Lys Ser Gly Ile Lys Asp Ala

100 105 110

Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Pro Val Arg Glu Ala

115 120 125

Lys Pro Gly Glu Val Leu Asn Asn His Leu Tyr Leu Ile Val Gln Pro

130 135 140

Tyr Val Trp Val Met Ser Pro Glu Ala Gln Tyr Val Gly Gly Asn Ala

145 150 155 160

Val Ile Ala Arg Thr Val Arg Arg Ile Pro Pro Gly Ser Met Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Ser Asp Phe Thr Arg Gly Met Phe Glu

180 185 190

Ala Tyr Asp Arg Gly Ala Gln Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Ile Thr Glu Gly Ser Gly Phe Asn Val Val Phe Val Lys Asn Asn

210 215 220

Val Ile Tyr Thr Pro Asn Arg Gly Val Leu Gln Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Ala Ala Lys Trp Cys Gly His Glu Val Arg Val Glu

245 250 255

Tyr Val Pro Val Glu Met Ala Tyr Glu Ala Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Met Asp Gly Lys Pro

275 280 285

Val Lys Asp Gly Lys Val Gly Pro Val Thr Lys Ala Ile Trp Asp Arg

290 295 300

Tyr Trp Ala Met His Trp Glu Asp Glu Phe Ser Phe Lys Ile Asp Tyr

305 310 315 320

Gln Lys Leu Lys Leu

325

<210> 29

<211> 1011

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 29

atgggtatcg acaccggtac aagcaatctg gtggccgtgg aaccgggtgc aattagagaa 60

gataccccgg ccggtagcgt gattcagtat agcgattatg aaatcgacta cagcagcccg 120

tttgcaggtg gtgtggcttg gattgaaggc gaatatctgc cggccgaaga tgccaaaatt 180

agcatttttg acaccggttt cggccatagc gatctgacct ataccgttgc acatgtttgg 240

catggcaata ttttccgcct gggcgatcat ctggatcgtc tgttagatgg cgcacgtaaa 300

ctgcgtctgg atagtggcta taccaaagat gaactggcag atattaccaa gaagtgcgtg 360

agcctgagcc agctgcgtga atcatttgtg aatctgacca ttacccgcgg ttatggtaaa 420

cgcaaaggtg aaaaagacct gagtaagctg acccatcagg tgtatatcta tgccattccg 480

tatctgtggg cctttccgcc tgccgagcaa atttttggca ccaccgccgt tgtgccgcgt 540

cacgtgcgtc gtgcaggtcg taacacagtt gatccgacca ttaagaatta ccagtggggt 600

gacctgaccg cagccagctt cgaggcaaaa gatcgcggtg ctcgcaccgc aattctgatg 660

gatgccgata attgtgtggc agaaggcccg ggttttaatg tgtgcattgt taaagacggc 720

aagctggcaa gcccgagtcg taatgcactg cctggtatta cccgtaaaac cgtgtttgaa 780

atcgccggtg caatgggcat tgaagccgca ttacgtgatg ttaccagtca tgaactgtac 840

gatgcagatg aaatcatggc agttaccacc gccggcggtg ttacacctat taataccctg 900

gatggcgttc cgattggtga cggtgaaccg ggtcctgtta ccgttgctat tcgtgatcgc 960

ttttgggcac tgatggatga accgggtccg ttaattgaag ccattcagta t 1011

<210> 30

<211> 337

<212> PRT

<213> Mycobacterium (Mycobacterium vanbaaleni)

<400>30

Met Gly Ile Asp Thr Gly Thr Ser Asn Leu Val Ala Val Glu Pro Gly

1 5 10 15

Ala Ile Arg Glu Asp Thr Pro Ala Gly Ser Val Ile Gln Tyr Ser Asp

20 25 30

Tyr Glu Ile Asp Tyr Ser Ser Pro Phe Ala Gly Gly Val Ala Trp Ile

35 40 45

Glu Gly Glu Tyr Leu Pro Ala Glu Asp Ala Lys Ile Ser Ile Phe Asp

50 55 60

Thr Gly Phe Gly His Ser Asp Leu Thr Tyr Thr Val Ala His Val Trp

65 70 75 80

His Gly Asn Ile Phe Arg Leu Gly Asp His Leu Asp Arg Leu Leu Asp

85 90 95

Gly Ala Arg Lys Leu Arg Leu Asp Ser Gly Tyr Thr Lys Asp Glu Leu

100 105 110

Ala Asp Ile Thr Lys Lys Cys Val Ser Leu Ser Gln Leu Arg Glu Ser

115 120 125

Phe Val Asn Leu Thr Ile Thr Arg Gly Tyr Gly Lys Arg Lys Gly Glu

130 135 140

Lys Asp Leu Ser Lys Leu Thr His Gln Val Tyr Ile Tyr Ala Ile Pro

145 150 155 160

Tyr Leu Trp Ala Phe Pro Pro Ala Glu Gln Ile Phe Gly Thr Thr Ala

165 170 175

Val Val Pro Arg His Val Arg Arg Ala Gly Arg Asn Thr Val Asp Pro

180 185 190

Thr Ile Lys Asn Tyr Gln Trp Gly Asp Leu Thr Ala Ala Ser Phe Glu

195 200 205

Ala Lys Asp Arg Gly Ala Arg Thr Ala Ile Leu Met Asp Ala Asp Asn

210 215 220

Cys Val Ala Glu Gly Pro Gly Phe Asn Val Cys Ile Val Lys Asp Gly

225 230 235 240

Lys Leu Ala Ser Pro Ser Arg Asn Ala Leu Pro Gly Ile Thr Arg Lys

245 250 255

Thr Val Phe Glu Ile Ala Gly Ala Met Gly Ile Glu Ala Ala Leu Arg

260 265 270

Asp Val Thr Ser His Glu Leu Tyr Asp Ala Asp Glu Ile Met Ala Val

275 280 285

Thr Thr Ala Gly Gly Val Thr Pro Ile Asn Thr Leu Asp Gly Val Pro

290 295 300

Ile Gly Asp Gly Glu Pro Gly Pro Val Thr Val Ala Ile Arg Asp Arg

305 310 315 320

Phe Trp Ala Leu Met Asp Glu Pro Gly Pro Leu Ile Glu Ala Ile Gln

325 330 335

Tyr

<210> 31

<211> 1428

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 31

atgagcctga ccgtgcaaaa aattaactgg gaacaggtta aggagtggga tcgtaaatat 60

ctgatgcgta cctttagcac ccagaatgaa tatcagccgg ttccgattga aagtaccgaa 120

ggcgattatc tgatcatgcc ggatggtaca cgcctgctgg atttctttaa tcagctgtat 180

tgcgtgaacc tgggtcagaa aaatcagaaa gttaacgcag ccatcaagga agcactggat 240

cgctatggct ttgtttggga tacctatgcc accgattata aagccaaagc agcaaaaatc 300

atcatcgagg atattctggg tgacgaagat tggccgggca aagtgcgttt tgtgagtacc 360

ggcagcgaag ccgtggaaac agctttaaat attgcacgcc tgtacaccaa tcgcccgctg 420

gtggtgacac gtgaacatga ttatcatggc tggaccggcg gcgcagcaac cgtgacccgt 480

ctgcgtagct atcgtagcgg tctggtgggt gaaaatagcg aaagttttag tgcccagatc 540

ccgggcagta gctataatag cgcagtgctg atggccccga gccctaacat gtttcaggat 600

agcgatggta atctgctgaa agatgaaaac ggcgaactgc tgagcgttaa atatacccgc 660

cgcatgattg aaaactacgg tccggaacag gtggcagcag ttattaccga agttagccag 720

ggtgccggta gtgctatgcc tccttatgaa tatatcccgc agattcgcaa aatgaccaaa 780

gaactgggcg tgctgtggat taatgatgaa gtgctgaccg gttttggccg caccggtaaa 840

tggtttggtt atcagcatta cggtgtgcag ccggatatta ttacaatggg taaaggtctg 900

agcagcagca gtctgccggc tggtgcagtg ttagtgagca aagaaattgc agcattcatg 960

gataagcacc gttgggaaag cgtgagtacc tatgccggtc atccggttgc aatggctgcc 1020

gtgtgtgcaa atctggaagt gatgatggaa gaaaacttcg ttgagcaggc aaaagatagt 1080

ggtgaatata tccgtagcaa gctggaactg ctgcaggaaa aacataaaag catcggtaac 1140

ttcgacggct atggcctgct gtggattgtt gatattgtta atgccaagac caagaccccg 1200

tatgttaaac tggatcgcaa ttttacccac ggtatgaatc cgaatcagat tccgacccag 1260

attattatga agaaggccct ggaaaagggc gtgctgattg gtggtgtgat gccgaatacc 1320

atgcgcattg gtgcaagcct gaatgtgagt cgcggcgata ttgataaagc aatggatgca 1380

ctggactacg ccctggatta tctggaaagt ggtgaatggc agcagagc 1428

<210> 32

<211> 476

<212> PRT

<213> Bacillus megaterium (Bacillus megaterium)

<400> 32

Met Ser Leu Thr Val Gln Lys Ile Asn Trp Glu Gln Val Lys Glu Trp

1 5 10 15

Asp Arg Lys Tyr Leu Met Arg Thr Phe Ser Thr Gln Asn Glu Tyr Gln

20 25 30

Pro Val Pro Ile Glu Ser Thr Glu Gly Asp Tyr Leu Ile Met Pro Asp

35 40 45

Gly Thr Arg Leu Leu Asp Phe Phe Asn Gln Leu Tyr Cys Val Asn Leu

50 55 60

Gly Gln Lys Asn Gln Lys Val Asn Ala Ala Ile Lys Glu Ala Leu Asp

65 70 75 80

Arg Tyr Gly Phe Val Trp Asp Thr Tyr Ala Thr Asp Tyr Lys Ala Lys

85 90 95

Ala Ala Lys Ile Ile Ile Glu Asp Ile Leu Gly Asp Glu Asp Trp Pro

100 105 110

Gly Lys Val Arg Phe Val Ser Thr Gly Ser Glu Ala Val Glu Thr Ala

115 120 125

Leu Asn Ile Ala Arg Leu Tyr Thr Asn Arg Pro Leu Val Val Thr Arg

130 135 140

Glu His Asp Tyr His Gly Trp Thr Gly Gly Ala Ala Thr Val Thr Arg

145 150 155 160

Leu Arg Ser Tyr Arg Ser Gly Leu Val Gly Glu Asn Ser Glu Ser Phe

165 170 175

Ser Ala Gln Ile Pro Gly Ser Ser Tyr Asn Ser Ala Val Leu Met Ala

180 185 190

Pro Ser Pro Asn Met Phe Gln Asp Ser Asp Gly Asn Leu Leu Lys Asp

195 200 205

Glu Asn Gly Glu Leu Leu Ser Val Lys Tyr Thr Arg Arg Met Ile Glu

210 215 220

Asn Tyr Gly Pro Glu Gln Val Ala Ala Val Ile Thr Glu Val Ser Gln

225 230 235 240

Gly Ala Gly Ser Ala Met Pro Pro Tyr Glu Tyr Ile Pro Gln Ile Arg

245 250 255

Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Asn Asp Glu Val Leu

260 265 270

Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly

275 280 285

Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser

290 295 300

Leu Pro Ala Gly Ala Val Leu Val Ser Lys Glu Ile Ala Ala Phe Met

305 310 315 320

Asp Lys His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val

325 330 335

Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn

340 345 350

Phe Val Glu Gln Ala Lys Asp Ser Gly Glu Tyr Ile Arg Ser Lys Leu

355 360 365

Glu Leu Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Tyr

370 375 380

Gly Leu Leu Trp Ile Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro

385 390 395 400

Tyr Val Lys Leu Asp Arg Asn Phe Thr His Gly Met Asn Pro Asn Gln

405 410 415

Ile Pro Thr Gln Ile Ile Met Lys Lys Ala Leu Glu Lys Gly Val Leu

420 425 430

Ile Gly Gly Val Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn

435 440 445

Val Ser Arg Gly Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala

450 455 460

Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Gln Ser

465 470 475

<210> 33

<211> 1344

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 33

atgaaccagc cgctgaatgt ggccccgccg gttagcagcg aactgaatct gcgtgcccat 60

tggatgccgt ttagcgcaaa tcgtaatttt cagaaagatc cgcgtattat tgttgccgca 120

gaaggtagtt ggctgaccga tgataaaggc cgcaaagtgt atgatagtct gagtggcctg 180

tggacctgcg gtgcaggcca tagccgtaaa gaaattcagg aagcagtggc acgccagctg 240

ggcaccctgg attatagccc gggttttcag tatggccatc cgctgagttt tcagctggca 300

gaaaaaattg ccggtctgct gccgggtgaa ctgaatcatg ttttctttac cggtagtggc 360

agcgaatgcg ccgataccag cattaagatg gcccgtgcat attggcgcct gaaaggtcag 420

ccgcagaaaa ccaaactgat tggccgtgca cgcggttatc atggcgtgaa tgttgccggc 480

accagcctgg gcggcattgg tggtaatcgc aaaatgtttg gtcagctgat ggatgtggat 540

catctgccgc ataccctgca gccgggcatg gcattcactc gtggtatggc acagaccggc 600

ggcgttgaac tggcaaatga actgctgaaa ctgattgaac tgcatgatgc cagtaatatt 660

gccgcagtga ttgtggaacc gatgagtggc agtgcaggtg ttctggtgcc gccggtgggt 720

tatctgcagc gtctgcgtga aatttgtgat cagcataata ttctgctgat ttttgatgaa 780

gtgatcaccg catttggccg tctgggtaca tatagcggtg ccgaatattt tggtgtgacc 840

ccggatctga tgaatgtggc aaaacaggtg accaatggtg ccgtgccgat gggcgcagtt 900

attgcaagca gcgaaatcta tgataccttt atgaatcagg ccctgccgga acatgccgtg 960

gaattttctc atggttatac ctatagtgca catccggttg cctgtgccgc cggcctggca 1020

gcactggata ttctggcccg tgataatctg gtgcagcaga gtgcagaact ggcaccgcat 1080

tttgaaaaag gtctgcatgg tctgcagggc gccaaaaatg ttattgatat tcgtaattgc 1140

ggcctggccg gcgccattca gattgcaccg cgtgatggtg acccgaccgt tcgcccgttt 1200

gaagccggca tgaaactgtg gcagcagggt ttttatgtgc gctttggcgg cgataccctg 1260

cagtttggtc cgacctttaa tgcacgcccg gaagaactgg atcgcctgtt tgatgcagtg 1320

ggtgaagcac tgaatggtat tgcc 1344

<210> 34

<211> 448

<212> PRT

<213> Pseudomonas aeruginosa (P. aeruginosa)

<400> 34

Met Asn Gln Pro Leu Asn Val Ala Pro Pro Val Ser Ser Glu Leu Asn

1 5 10 15

Leu Arg Ala His Trp Met Pro Phe Ser Ala Asn Arg Asn Phe Gln Lys

20 25 30

Asp Pro Arg Ile Ile Val Ala Ala Glu Gly Ser Trp Leu Thr Asp Asp

35 40 45

Lys Gly Arg Lys Val Tyr Asp Ser Leu Ser Gly Leu Trp Thr Cys Gly

50 55 60

Ala Gly His Ser Arg Lys Glu Ile Gln Glu Ala Val Ala Arg Gln Leu

65 70 75 80

Gly Thr Leu Asp Tyr Ser Pro Gly Phe Gln Tyr Gly His Pro Leu Ser

85 90 95

Phe Gln Leu Ala Glu Lys Ile Ala Gly Leu Leu Pro Gly Glu Leu Asn

100 105 110

His Val Phe Phe Thr Gly Ser Gly Ser Glu Cys Ala Asp Thr Ser Ile

115 120 125

Lys Met Ala Arg Ala Tyr Trp Arg Leu Lys Gly Gln Pro Gln Lys Thr

130 135 140

Lys Leu Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Val Ala Gly

145 150 155 160

Thr Ser Leu Gly Gly Ile Gly Gly Asn Arg Lys Met Phe Gly Gln Leu

165 170 175

Met Asp Val Asp His Leu Pro His Thr Leu Gln Pro Gly Met Ala Phe

180 185 190

Thr Arg Gly Met Ala Gln Thr Gly Gly Val Glu Leu Ala Asn Glu Leu

195 200 205

Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile

210 215 220

Val Glu Pro Met Ser Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly

225 230 235 240

Tyr Leu Gln Arg Leu Arg Glu Ile Cys Asp Gln His Asn Ile Leu Leu

245 250 255

Ile Phe Asp Glu Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Tyr Ser

260 265 270

Gly Ala Glu Tyr Phe Gly Val Thr Pro Asp Leu Met Asn Val Ala Lys

275 280 285

Gln Val Thr Asn Gly Ala Val Pro Met Gly Ala Val Ile Ala Ser Ser

290 295 300

Glu Ile Tyr Asp Thr Phe Met Asn Gln Ala Leu Pro Glu His Ala Val

305 310 315 320

Glu Phe Ser His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala

325 330 335

Ala Gly Leu Ala Ala Leu Asp Ile Leu Ala Arg Asp Asn Leu Val Gln

340 345 350

Gln Ser Ala Glu Leu Ala Pro His Phe Glu Lys Gly Leu His Gly Leu

355 360 365

Gln Gly Ala Lys Asn Val Ile Asp Ile Arg Asn Cys Gly Leu Ala Gly

370 375 380

Ala Ile Gln Ile Ala Pro Arg Asp Gly Asp Pro Thr Val Arg Pro Phe

385 390 395 400

Glu Ala Gly Met Lys Leu Trp Gln Gln Gly Phe Tyr Val Arg Phe Gly

405 410 415

Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Ala Arg Pro Glu Glu

420 425 430

Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Ala Leu Asn Gly Ile Ala

435 440 445

Claims (16)

1. A method for synthesizing chiral 2-amino-1-butanol comprises the following steps:

(A) using 1, 2-butanediol as a substrate, and generating 2-ketone-1-butanol through catalytic reaction of enzyme A and coenzyme thereof;

(B) taking the 2-ketone-1-butanol generated in the step (A) as a substrate, and generating chiral 2-amino-1-butanol through catalytic reaction of an enzyme B and a coenzyme thereof;

the enzyme A and the enzyme B are any one of the following three combinations:

a first combination of:

the enzyme A is selected from any one of the following:

(a1) derived from Lactobacillus brevis (Lactobacillus brevis) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 2;

(a2) derived from Bacillus stearothermophilus (B.) (Geobacillus stearothermophilus) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 4;

(a3) derived from Thermoanaerobacter (b) ((b))Thermoanaerobacter brockii) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 6;

(a4) from Lactobacillus kefir (L.) sour milk: (Lactobacillus kefiri) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 8;

(a5) derived from the family Bacillaceae (Bacillaceae) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 10;

(a6) derived from the genus lysergia (Leifsonia sp.) with the amino acid sequence of SEQ ID No. 12;

(a7) from Thermoanaerobacter virginiae (A)Thermoanaerobacter wiegelii) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 14;

(a8) compared with the alcohol dehydrogenase derived from Lactobacillus brevis shown in SEQ ID No.2, only the following mutation I11V exists;

(a9) compared with the alcohol dehydrogenase derived from Lactobacillus brevis shown in SEQ ID No.2, only the following mutations exist: G37D;

(a10) carbonyl reductase derived from Sporobolomyces ochracea, the amino acid sequence of which is SEQ ID No. 16;

(a11) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of a protein defined in any one of (a1) to (a 10);

the enzyme B is selected from any one of the following:

(b1) leucine dehydrogenase derived from Bacillus stearothermophilus, and the amino acid sequence of the leucine dehydrogenase is SEQ ID No. 18;

(b2) compared with the leucine dehydrogenase shown in SEQ ID No.18 and derived from the Bacillus stearothermophilus, the leucine dehydrogenase only has the following mutations: K68S/N261L;

(b3) compared with the leucine dehydrogenase shown in SEQ ID No.18 and derived from the Bacillus stearothermophilus, the leucine dehydrogenase only has the following mutations: K68Y/N261C;

(b4) ATA-117 transaminase of Codexis corporation, with an amino acid sequence of SEQ ID No. 20;

(b5) derived from Aspergillus terreus (Aspergillus terreus) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 22;

(b6) derived from Aspergillus fumigatus (Aspergillus fumigatus) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 24;

(b7) derived from Fusarium fischeri (Neosartorya fischeri) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 26;

(b8) derived from gibberella zeae (Gibberella zeae) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 28;

(b9) derived from mycobacteria (Mycobacterium vanbaalenii) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 30;

(b10) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of a protein defined in any one of (b1) to (b 9);

a second combination of:

the enzyme A is selected from any one of the following:

(a1) derived from Lactobacillus brevis (Lactobacillus brevis) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 2;

(a2) derived from Bacillus stearothermophilus (B.) (Geobacillus stearothermophilus) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 4;

(a3) derived from Thermoanaerobacter (b) ((b))Thermoanaerobacter brockii) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 6;

(a4) from Lactobacillus kefir (L.) sour milk: (Lactobacillus kefiri) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 8;

(a5) derived from the family Bacillaceae (Bacillaceae) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 10;

(a6) derived from the genus lysergia (Leifsonia sp.) with the amino acid sequence of SEQ ID No. 12;

(a8) compared with the alcohol dehydrogenase derived from Lactobacillus brevis shown in SEQ ID No.2, only the following mutation I11V exists;

(a9) compared with the alcohol dehydrogenase derived from Lactobacillus brevis shown in SEQ ID No.2, only the following mutations exist: G37D;

(a12) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of a protein defined in any one of (a1) - (a6) and (a8) - (a 9);

the enzyme B is selected from any one of the following:

(b9) derived from mycobacteria (Mycobacterium vanbaalenii) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 30;

(b11) derived from Bacillus megaterium (Bacillus megaterium) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 32;

(b12) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (b 9) or (b 11);

in a third combination:

the enzyme A is selected from any one of the following:

(a7) from Thermoanaerobacter virginiae (A)Thermoanaerobacter wiegelii) The alcohol dehydrogenase of (1), the amino acid sequence of which is SEQ ID No. 14;

(a10) carbonyl reductase derived from Sporobolomyces ochracea, the amino acid sequence of which is SEQ ID No. 16;

(a13) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (a7) or (a 10);

the enzyme B is selected from any one of the following:

(b11) derived from Bacillus megaterium (Bacillus megaterium) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 32;

(b13) derived from Pseudomonas aeruginosa (P. aeruginosa) The transaminase of (1), the amino acid sequence of which is SEQ ID No. 34;

(b14) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (b 11) or (b 13);

the chiral 2-amino-1-butanol used for the synthesis of the enzymes of the first combination is: (S) -2-amino-1-butanol; the chiral 2-amino-1-butanol used for the synthesis by the enzymes in the second and third combinations is: (R) -2-amino-1-butanol.

2. The method of claim 1, wherein: in the method, the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder, pure enzyme or whole cells.

3. The method of claim 2, wherein: the crude enzyme solution, the crude enzyme solution freeze-dried powder and the pure enzyme are prepared according to the method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell; and cracking the recombinant cells to obtain the crude enzyme solution, the crude enzyme solution freeze-dried powder or the pure enzyme.

4. The method of claim 2, wherein: the whole cells are prepared according to a method comprising the following steps: expressing the enzyme A and the enzyme B in host cells, and obtaining recombinant cells, namely the whole cells.

5. The method of claim 4, wherein: the recombinant cell is prepared according to a method comprising the following steps: introducing a nucleic acid molecule capable of expressing the enzyme A and the enzyme B into the host cell, and obtaining the recombinant cell expressing the enzyme A and the enzyme B after induction culture.

6. The method of claim 5, wherein: the nucleic acid molecules capable of expressing said enzyme a and said enzyme B are introduced into said host cell in the form of a recombinant vector; the recombinant vector is a bacterial plasmid, a bacteriophage, a yeast plasmid or a retrovirus packaging plasmid carrying the coding genes of the enzyme A and the enzyme B.

7. The method of claim 5, wherein: the host cell is a prokaryotic cell or a lower eukaryotic cell.

8. The method of claim 7, wherein: the prokaryotic cell is a bacterium; the lower eukaryotic cell is a yeast cell.

9. The method of claim 8, wherein: the bacterium is Escherichia coli.

10. The method of claim 1, wherein: the sequence of the coding gene of the alcohol dehydrogenase from the Lactobacillus brevis is SEQ ID No.1 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the alcohol dehydrogenase from the bacillus stearothermophilus is SEQ ID No.3 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the sequence;

the sequence of the coding gene of the alcohol dehydrogenase from the thermophilic anaerobacterium is SEQ ID No.5 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence;

the sequence of the coding gene of the alcohol dehydrogenase from the lactobacillus gasseri is SEQ ID No.7 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the alcohol dehydrogenase from the Baciliaceae is SEQ ID No.9 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the alcohol dehydrogenase from the lysine is SEQ ID No.11 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence;

the sequence of the coding gene of the alcohol dehydrogenase from the anaerobic bacillus virginiae is SEQ ID No.13 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the carbonyl reductase derived from the rhodosporidium ochraceus is SEQ ID No.15 or a fusion sequence obtained by connecting a tag coding sequence to the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the leucine dehydrogenase from the bacillus stearothermophilus is SEQ ID No.17 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of ATA-117 transaminase of Codexis company is SEQ ID No.19 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the aminotransferase from the aspergillus terreus is SEQ ID No.21 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the transaminase derived from the aspergillus fumigatus is SEQ ID No.23 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the transaminase coding gene from Fischer-Tropsch is SEQ ID No.25 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the transaminase coding gene;

the sequence of the transaminase coding gene derived from the gibberella zeae is SEQ ID No.27 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the transaminase coding gene;

the sequence of the coding gene of the transaminase derived from the mycobacteria is SEQ ID No.29 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the transaminase derived from the bacillus megaterium is SEQ ID No.31 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the transaminase derived from the pseudomonas aeruginosa is SEQ ID No.33 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene;

the sequence of the coding gene of the mutant of the alcohol dehydrogenase derived from the lactobacillus brevis is any one of the following (g1) to (g 3): (g1) compared with SEQ ID No.1, only the following mutations are present: A31G/T33G; (g2) compared with SEQ ID No.1, only the following mutations are present: G110A/C111T; (g3) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or the 3' end of the sequence defined in (g1) or (g 2);

the sequence of the coding gene of the leucine dehydrogenase mutant derived from the bacillus stearothermophilus is any one of the following (h1) to (h 3): (h1) compared with SEQ ID No.17, only the following mutations are present: A203G/A204C/A781C/A782T/C783G; (h2) compared with SEQ ID No.17, only the following mutations are present: A202T/A204T/A781T/A782G; (h3) and (h) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or the 3' end of the sequence defined in (h1) or (h 2).

11. The method according to any one of claims 1-10, wherein: in the step (A) and the step (B), the temperature of the catalytic reaction is 25-37 ℃; the time of the catalytic reaction is 4-48 h.

12. The method according to any one of claims 1-10, wherein: when the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A) and the step (B), the concentration of the enzyme A and the concentration of the enzyme B in respective reaction systems are both 0.1 g/L-10 g/L; when the enzyme A and the enzyme B are catalyzed in the form of whole cells co-expressing the enzyme A and the enzyme B, the wet weight of the whole cells contained in each liter of the reaction system is 100 g.

13. The method according to any one of claims 1-10, wherein: when the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A), the catalytic reaction is carried out in a buffer solution shown as the following (k 1); in the step (B), the catalytic reaction is carried out in a buffer solution as shown in any one of (k2) to (k3) below; when the enzyme A and the enzyme B are catalytically effected in the form of whole cells co-expressing the enzyme A and the enzyme B, the catalytic reactions of step (A) and step (B) are each carried out in a buffer as shown in (k1) below;

(k1) a phosphate buffer solution having a concentration of 50 to 100mM and a pH of 6.5 to 8.0; (k2) NH with a concentration of 100 mM-2M and a pH of 8.0-9.6₃·H₂O or NH₄A Cl buffer solution; (k3) a phosphate buffer solution with a concentration of 50-100 mM, a concentration of isopropylamine of 250mM-1M and a pH value of 7.5-8.5.

14. The method according to any one of claims 1-10, wherein: when the enzyme A and the enzyme B are catalyzed by crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A), a reaction system of the catalytic reaction contains acetone besides 1, 2-butanediol, the enzyme A and coenzyme thereof.

15. The method according to any one of claims 1-10, wherein: when the enzyme A and the enzyme B are subjected to catalytic action in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (B), when the enzyme B is amino acid dehydrogenase or a mutant of the amino acid dehydrogenase, a reaction system of the catalytic reaction contains 2-ketone-1-butanol, the enzyme B and a coenzyme thereof, and also contains glucose dehydrogenase and glucose.

16. The application of the enzyme system or the related products thereof in synthesizing chiral 2-amino-1-butanol;

the chiral 2-amino-1-butanol is (S) -2-amino-1-butanol or (R) -2-amino-1-butanol;

when the chiral 2-amino-1-butanol is (S) -2-amino-1-butanol, said enzyme system is designated enzyme system I and said related product is designated related product I; the enzyme system I comprising the enzyme A and the enzyme B in the first combination of claim 1; the related product I is a nucleic acid molecule capable of expressing each enzyme in the enzyme system I, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

when the chiral 2-amino-1-butanol is (R) When 2-amino-1-butanol, the enzyme system is denoted as enzyme system II and the related product is denoted as related product II; the enzyme system II comprises the enzyme A and the enzyme B in the second combination or the third combination of claim 1; the related product II is a nucleic acid molecule capable of expressing each enzyme in the enzyme system II, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

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