Process for the enzymatic synthesis of 3-hydroxybutyrate

Abstract

Provided is a method for enzymatic synthesis of 3-hydroxybutyrate, which uses acetoacetate as a substrate and uses alcohol dehydrogenase SEQ ID NO: 1 or a mutant thereof SEQ ID NOs: 3-27 to catalyze a reduction reaction to obtain 3-hydroxybutyrate.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme catalysis technology, specifically, it relates to a method for the enzymatic synthesis of 3-hydroxybutyrate. Background Technology

[0002] In recent years, the ketogenic diet has gradually become a recognized healthy lifestyle. By consuming foods containing ketones, the body can be replenished with ketones, which are then used for ketone metabolism. Acetoacetic acid, 3-hydroxybutyrate, and acetone are the three ketone body forms required by the human body. Among them, ketone body supplements with 3-hydroxybutyrate (3-HB) as the main raw material have been successfully commercialized, and market demand is increasing year by year.

[0003] The preparation of 3-hydroxybutyric acid mainly includes chemical synthesis, enzymatic conversion, and microbial fermentation. Current enzymatic processes for producing 3-hydroxybutyrate esters primarily use methyl acetoacetate or ethyl acetoacetate as substrates. Catalyzed by alcohol dehydrogenase (EC 1.1.1.1) or carbonyl reductase (EC 1.1.1.148), and with NADPH or NADH as coenzymes, the ketone group is reduced to a hydroxyl group, producing methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate. Further ester hydrolysis of methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate yields 3-hydroxybutyric acid.

[0004] However, the enzyme activities of alcohol dehydrogenases and carbonyl reductases currently used in industry are generally low, resulting in high production costs. Moreover, due to their high substrate specificity, these alcohol dehydrogenases and carbonyl reductases have a very narrow range of applicable substrates. Summary of the Invention

[0005] To improve the enzymatic production process of 3-hydroxybutyrate, this invention conducted extensive screening of alcohol dehydrogenases and carbonyl reductases, studied their catalytic performance on methyl acetoacetate and ethyl acetoacetate, and modified the alcohol dehydrogenase (SEQ ID NO: 1) derived from Lactobacillus kefiri DSM 20587 (a substrate with a wide applicability range) using random mutagenesis and combinatorial mutagenesis techniques, obtaining mutants with significantly enhanced enzyme activity to efficiently catalyze the production of 3-hydroxybutyrate from acetoacetate. Specifically, this invention includes the following technical solutions.

[0006] A method for the enzymatic synthesis of 3-hydroxybutyrate, characterized in that acetoacetate is used as a substrate, and an alcohol dehydrogenase SEQ ID NO: 1 or its mutant is used to catalyze a reduction reaction to obtain 3-hydroxybutyrate.

[0007] ,

[0008] Wherein R is a C1-C4 alkyl group, selected from methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl. That is, the 3-hydroxybutyrate ester is selected from methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, propyl 3-hydroxybutyrate, isopropyl 3-hydroxybutyrate, butyl 3-hydroxybutyrate, sec-butyl 3-hydroxybutyrate, isobutyl 3-hydroxybutyrate, and tert-butyl 3-hydroxybutyrate.

[0009] The above-mentioned alcohol dehydrogenase mutant is a polypeptide that has the function of alcohol dehydrogenase SEQ ID NO: 1, formed by mutation (including but not limited to substitution, deletion or addition) of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1; or a polypeptide that has more than 85% homology with the amino acid sequence of SEQ ID NO: 1, preferably more than 90% homology, more preferably more than 95% homology, and has the function of alcohol dehydrogenase SEQ ID NO: 1.

[0010] The function of the alcohol dehydrogenase SEQ ID NO: 1 mentioned above refers to its ability to catalyze the reduction of methyl acetoacetate to methyl 3-hydroxybutyrate and the reduction of ethyl acetoacetate to ethyl 3-hydroxybutyrate.

[0011] Preferably, the enzyme activity of the above-mentioned alcohol dehydrogenase mutant is higher than that of SEQ ID NO: 1.

[0012] Preferably, the substrate acetoacetate is methyl acetoacetate or ethyl acetoacetate, and correspondingly, the product 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate.

[0013] The aforementioned 3-hydroxybutyrates include methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate, and particularly refer to R-configured 3-hydroxybutyrates, including (R)-3-hydroxybutyrate methyl 3-hydroxybutyrate or (R)-3-hydroxybutyrate ethyl 3-hydroxybutyrate.

[0014] In a preferred embodiment, isopropanol and coenzyme NADP+ (nicotinamide adenine dinucleotide phosphate, coenzyme II) are also added to the enzyme-catalyzed reaction system. The role of NADP+ is to act as an oxidant to steal electrons, and alcohol dehydrogenase uses isopropanol to reduce NADP+ to NADPH, generating sufficient NADPH as a reducing agent for biosynthesis, thereby promoting the reduction reaction.

[0015] The pH of the enzyme-catalyzed reaction system of the present invention can be 7.0-8.0, preferably 7.2-7.8, and more preferably 7.4-7.5.

[0016] The enzyme-catalyzed reaction temperature is 25-45℃, preferably 28-40℃, and more preferably 30-35℃.

[0017] In the above method, the mutation site in the alcohol dehydrogenase mutant can be a site selected from the group consisting of the amino acid sequence of SEQ ID NO: 1: position 6, position 19, position 25, position 57, position 77, position 89, position 97, position 123, position 147, position 149, position 151, position 155, position 190, position 197, position 202, position 220, position 221, position 235, or a combination of two or more of them.

[0018] A second aspect of the present invention provides an alcohol dehydrogenase mutant, which is the alcohol dehydrogenase mutant described above. For example, it is a mutant formed by mutation at the following sites in the amino acid sequence of SEQ ID NO: 1: position 6, position 19, position 25, position 57, position 77, position 89, position 97, position 123, position 147, position 149, position 151, position 155, position 190, position 197, position 202, position 220, position 221, position 235, or a combination of two or more of them.

[0019] In a preferred embodiment, the mutations in the above-mentioned alcohol dehydrogenase mutants are selected from the group consisting of: K6N, I19L, D25G, I57N or I57T, T77M or T77S, N89T or N89K, K97R or K97N, R123S or R123H, F147I or F147C, G149D or G149R, P151L, A155D, Y190F or Y190G, D197E, A202V or A202T, P220Q, N221T or N221I or N221V, S235Y, or combinations of two or more of them.

[0020] For example, the alcohol dehydrogenase mutants mentioned above are selected from the following group:

[0021] SEQ ID NO: 3, which is a mutant of the amino acid sequence A202T and K97R of SEQ ID NO: 1;

[0022] SEQ ID NO: 4 is a mutant of the amino acid sequence A202V, K97R, Y190G of SEQ ID NO: 1;

[0023] SEQ ID NO: 5, which is a mutant of the amino acid sequence A202T, K97R, F147I, K6N of SEQ ID NO: 1;

[0024] SEQ ID NO: 6 is a mutant of the amino acid sequence A202T, K97R, N89T, R123H, N221T of SEQ ID NO: 1;

[0025] SEQ ID NO: 7, which is a mutant of the amino acid sequence A202T and D25G of SEQ ID NO: 1;

[0026] SEQ ID NO: 8 is a mutant of the amino acid sequence A202T, K97R, S235Y, I57N, R123H of SEQ ID NO: 1;

[0027] SEQ ID NO: 9, which is a mutant of the amino acid sequence A202V, K97R, N221I, Y190F of SEQ ID NO: 1;

[0028] SEQ ID NO: 10 is a mutant of the amino acid sequence A202V, K97R, N221I, Y190F, D25G, K6N, R123S of SEQ ID NO: 1;

[0029] SEQ ID NO: 11 is a mutant of the amino acid sequence A202V, N221I, Y190F, G149D, D25G of SEQ ID NO: 1;

[0030] SEQ ID NO: 12 is a mutant of the amino acid sequence A202V, Y190F, D25G of SEQ ID NO: 1;

[0031] SEQ ID NO: 13 is a mutant of the amino acid sequence A202V, Y190F, D25G, I57T of SEQ ID NO: 1;

[0032] SEQ ID NO: 14 is a mutant of the amino acid sequence A202V, N221I, Y190F, and F147I of SEQ ID NO: 1;

[0033] SEQ ID NO: 15 is a mutant of the amino acid sequence K97R, N221I, Y190F, and F147I of SEQ ID NO: 1;

[0034] SEQ ID NO: 16 is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, D197E, P151L of SEQ ID NO: 1;

[0035] SEQ ID NO: 17 is a mutant of the amino acid sequence A202V, N221V, Y190F, F147I, I19L, G149R of SEQ ID NO: 1;

[0036] SEQ ID NO: 18 is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S of SEQ ID NO: 1;

[0037] SEQ ID NO: 19 is a mutant of the amino acid sequence A202T, N221I, Y190F, K6N of SEQ ID NO: 1;

[0038] SEQ ID NO: 20 is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, A155D, T77M of SEQ ID NO: 1;

[0039] SEQ ID NO: 21 is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, T77S, G149R, P151L of SEQ ID NO: 1;

[0040] SEQ ID NO: 22 is a mutant of the amino acid sequence Y190F of SEQ ID NO: 1;

[0041] SEQ ID NO: 23 is a mutant of the amino acid sequence K97R of SEQ ID NO: 1;

[0042] SEQ ID NO: 24 is a mutant of the amino acid sequence P220Q and F147C of SEQ ID NO: 1;

[0043] SEQ ID NO: 25, which is a mutant of the amino acid sequence I57N of SEQ ID NO: 1;

[0044] SEQ ID NO: 26 is a mutant of the amino acid sequence G149D of SEQ ID NO: 1;

[0045] SEQ ID NO: 27 is a mutant of the amino acid sequence R123S of SEQ ID NO: 1.

[0046] The alcohol dehydrogenase mutants described above are particularly preferred to be SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 21.

[0047] A third aspect of the present invention provides a microorganism expressing an alcohol dehydrogenase SEQ ID NO: 1 or one of the above-mentioned alcohol dehydrogenase mutants SEQ ID NOs: 3-27.

[0048] The microorganisms mentioned above are selected from Escherichia coli, Pichia pastoris, and Bacillus subtilis, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred. When the microorganism is Escherichia coli, the encoding gene of wild-type alcohol dehydrogenase SEQ ID NO: 1 can be the nucleotide sequence SEQ ID NO: 2.

[0049] The aforementioned microorganisms can be used directly as a natural immobilized form of alcohol dehydrogenase for the production of 3-hydroxybutyrate.

[0050] The wild-type alcohol dehydrogenase SEQ ID NO: 1 screened from numerous alcohol dehydrogenases and carbonyl reductases 1#-23#, and the mutants SEQ ID NOs: 3-27 constructed based on it, can catalyze the reduction of methyl acetoacetate to methyl 3-hydroxybutyrate and ethyl acetoacetate to ethyl 3-hydroxybutyrate when applied in the enzymatic synthesis of 3-hydroxybutyrate. This broadens the substrate range of acetoacetate and has promising prospects for industrial application. Detailed Implementation

[0051] The wild-type alcohol dehydrogenase SEQ ID NO: 1 screened in this invention is derived from Lactobacillus skefiri DSM 20587, and is numbered 19# in the examples. It requires the participation of coenzyme NADPH when catalyzing the reduction of acetoacetate to 3-hydroxybutyrate.

[0052] The amino acid sequence of SEQ ID NO: 1 is as follows:

[0053] MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQR MKNKGLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ (SEQ ID NO: 1).

[0054] By performing multiple rounds of mutations on SEQ ID NO: 1, a series of mutation points were discovered, and several mutants with the mentioned enzyme activities were constructed, including SEQ ID NOs: 3-27. They can all catalyze the production of methyl acetoacetate and ethyl acetoacetate, respectively, using methyl acetoacetate and ethyl acetoacetate as substrates.

[0055] Some mutations are not single mutations. For example, the mutation at position 202 can be either A202T or A202V. The A202T mutation refers to the mutation in which the alanine (A or Ala) residue at position 202 of the amino acid sequence of SEQ ID NO: 1 is replaced by a threonine (T or Thr), and the A202V mutation refers to the mutation in which the alanine (A or Ala) residue at position 202 is replaced by a valine (V or Val).

[0056] In the embodiments, the terms "wild-type", "wild-enzyme", and "wild-type enzyme" have the same meaning and all refer to the wild-type sequence SEQ ID NO: 1 of alcohol dehydrogenase. For the purpose of distinguishing it from mutants (mutant enzymes) and for ease of description, wild-type alcohol dehydrogenase may be referred to as "wild-type alcohol dehydrogenase" or "wild-type enzyme" in this invention.

[0057] Since the function of the alcohol dehydrogenase mutant SEQ ID NOs: 3-27 remains unchanged, for the sake of convenience, the term "alcohol dehydrogenase mutant" is sometimes simply referred to as "alcohol dehydrogenase," which is easily understood by those skilled in the art.

[0058] To express alcohol dehydrogenase SEQ ID NO: 1 in *E. coli*, the most commonly used gene in genetic engineering, this invention optimized the codons of its expression gene, using this as the basic template for constructing alcohol dehydrogenase mutants. The encoding gene of wild-type alcohol dehydrogenase SEQ ID NO: 1 can be the nucleotide sequence SEQ ID NO: 2.

[0059] (SEQ ID NO: 2).

[0060] Multiple mutant sequences were obtained using a multi-round error-prone PCR random mutagenesis technique, namely the mutants with amino acid sequences SEQ ID NOs: 3-27 in this invention.

[0061] The alcohol dehydrogenase mutant of the present invention has only 252 amino acids and a well-defined sequence. Therefore, those skilled in the art can easily obtain its encoding gene, expression cassettes and plasmids containing these genes, and transformants containing the plasmids.

[0062] To achieve optimal expression of protein SEQ ID NOs: 3-27 in different microorganisms, codon optimization can be performed for specific microorganisms, such as *E. coli*. Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of the gene of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in rapidly growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Therefore, in rapidly growing microorganisms, low-frequency codons for amino acids can be replaced with high-frequency codons for the same amino acid. Thus, the expression of the optimized DNA sequence is improved in rapidly growing microorganisms.

[0063] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0064] The host for the transformant can be any microorganism suitable for expressing polyphosphokinases, including bacteria and fungi. Preferred microorganisms are *Escherichia coli*, *Pichia pastoris*, *Saccharomyces cerevisiae*, or *Bacillus subtilis*, with *Escherichia coli* being the most preferred, and *Escherichia coli* BL21(DE3) being more preferred.

[0065] In this reaction system, alcohol dehydrogenase can exist in either enzyme form or cell form. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, and enzymes immobilized on carriers; the cell form includes live cells and dead cells.

[0066] The aforementioned bacterial cell form itself is a natural immobilized enzyme, and it can be used as an enzyme preparation for catalytic reactions without the need for disruption or even extraction and purification. Since both the reaction substrate and product are small molecule compounds, they can easily cross the bacterial cell membrane—the biological barrier—eliminating the need for cell disruption, which is economically advantageous.

[0067] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0068] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0069] Example

[0070] Materials and methods

[0071] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium with an additional 20 g / L agar powder.)

[0072] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K₂HPO₄·3H₂O, 2.31 g / L KH₂PO₄, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium is supplemented with 20 g / L agar powder.)

[0073] ZYM medium: Prepare the following stock solutions according to the formula: ZY medium, 50×M medium, 50×5052 medium, 1M MgSO4, 1000×trace elements, 1000×antibiotics.

[0074] ZY medium: 10g peptone, 5g yeast extract, add water to a final volume of 950ml, sterilize at 121℃ for 20min.

[0075] 50×M medium: 223g Na2HPO4·12H2O, 85g KH2PO4, 66.88g NH4Cl, 17.7g Na2SO4, add water to a final volume of 500ml, sterilize at 121℃ for 20min.

[0076] 50×5052 medium: 125g glycerol, 12.5g glucose, 50g α-lactose, add water to a final volume of 500ml, sterilize at 121℃ for 20min.

[0077] 1M MgSO4: 24.65g MgSO4·7H2O, bring the volume to 100ml, sterilize at 121℃ for 20min.

[0078] 1000× Trace Elements: Dissolve 1.35g FeCl3·6H2O in 50ml of 0.12M HCl, then add 0.32g CaCl2·2H2O, 0.2g MnCl2·4H2O, 0.3g ZnSO4·7H2O, 0.05g CoCl2·6H2O, 0.04g CuCl2·2H2O, 0.05g NiCl2·6H2O, 0.05g Na2MoO4·2H2O, 0.04g Na2SeO3, and 0.02g H3BO3 respectively. Add water to a final volume of 100ml and filter to sterilize.

[0079] 1000× Antibiotic: 500mg kanamycin, add water to a final volume of 10ml, filter to sterilize.

[0080] Mix the sterilized stock solutions thoroughly with 950ml ZY medium, 20ml 50×M, 20ml 50×5052, 2ml 1MmgSO4, 2ml 1000× trace elements, and 1ml 1000× antibiotics to obtain ZYM self-induction medium.

[0081] The whole genome synthesis in this embodiment was performed by Suzhou Genewiz Biotechnology Co., Ltd., and loaded into the vector pET24a. Primer synthesis and sequencing were also performed by Suzhou Genewiz Biotechnology Co., Ltd.

[0082] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, and culture medium preparation, etc., mainly referring to *Molecular Cloning: A Laboratory Manual* (3rd edition), edited by J. Sambrook and DW. Russell, translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions can be determined through simple experiments if necessary.

[0083] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0084] It should be noted that, for ease of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share the same number. This is easily understood by those skilled in the art, meaning that the same number can refer to different biological forms in different environments. For example, 19# can represent the strain Lactobacillus kefiri DSM 20587, or it can represent the plasmid pET24a-19# number, the enzyme SEQ ID NO: 1 number, and the enzyme-encoding gene SEQ ID NO: 2 number.

[0085] Example 1: Screening for enzymes used to synthesize 3-hydroxybutyrate

[0086] This study investigated microbial enzymes for the reduction of methyl acetoacetate / ethyl acetoacetate to methyl 3-hydroxybutyrate / ethyl 3-hydroxybutyrate. A total of 23 enzyme genes were retrieved from the NCBI database, as shown in Table 1.

[0087] Table 1. List of wild-type enzyme libraries constructed from microorganisms

[0088] serial number category Species origin Accession number Coenzyme 1# alcohol dehydrogenase Lactobacillus acidifarinae DSM 19394 KRK96696.1 NADPH 2# alcohol dehydrogenase Lactobacillus similis DSM 23365 KRN21727.1 NADPH 3# alcohol dehydrogenase Oenococcus alcoholitolerans KGO31568.1 NADPH 4# alcohol dehydrogenase Thermococcus guaymasensis ADV18977.1 NADPH 5# alcohol dehydrogenase Clostridium botulinum WP_003399463.1 NADPH 6# alcohol dehydrogenase Thermoanaerobacter ethanolicus WP_003868131.1 NADPH 7# alcohol dehydrogenase Thermosinus carboxydivorans WP_007290608.1 NADPH 8# alcohol dehydrogenase Thermoanaerobacter mathranii WP_013150923.1 NADPH 9# alcohol dehydrogenase Thermovirga lienii WP_014162331.1 NADPH 10# alcohol dehydrogenase Firmicutes bacterium CAG:137 CDB31037.1 NADPH 11# alcohol dehydrogenase Treponema lecithinolyticum ATCC700332 ERJ92146.1 NADPH 12# alcohol dehydrogenase Methanosarcina thermophila WP_048166386.1 NADPH 13# alcohol dehydrogenase uncultured Clostridium sp. SCI81347.1 NADPH 14# alcohol dehydrogenase Desulfovibrio litoralis WP_072695399.1 NADPH 15# alcohol dehydrogenase Desulfotomaculum putei WP_073236284.1 NADPH 16# alcohol dehydrogenase Clostridium botulinum WP_096043277.1 NADPH 17# alcohol dehydrogenase Thermoanaerobacter brockii WP_041589967.1 NADPH 18# alcohol dehydrogenase Lactobacillus brevis CAD66648.1 NADPH 19# alcohol dehydrogenase Lactobacillus kefiri DSM 20587 AY267012 NADPH 20# carbonyl reductase Leifsonia sp. Strain S749 AB213459 NADH 21# carbonyl reductase Rhodococcus erythropolis WZ010 KX827723 NADH 22# carbonyl reductase Acetobacter sp. CCTCC M209061 MF419650 NADH 23# carbonyl reductase Lactobacillus parabuchneri WP057909612.1 NADPH

[0089] Construction of engineered bacteria for enzyme expression: using the codon optimization tool Codon Adaptation Tool ( http: / / www.jcat.de / Twenty-three enzymes underwent codon optimization to adapt to *E. coli*, excluding NdeI / XhoI site features to obtain the corresponding coding gene base sequences. For example, the coding gene for alcohol dehydrogenase #19 (SEQ ID NO: 1) could be the nucleotide sequence (SEQ ID NO: 2). The genes for these 23 enzymes were synthesized by Suzhou Genewise Biotechnology Co., Ltd., and the synthesized gene fragments were loaded into the NdeI / XhoI sites of the *E. coli* expression plasmid system pET24a vector as required, resulting in 23 expression plasmids: pET24a-1#, pET24a-2#, pET24a-3#, pET24a-4#, pET24a-5#, pET24a-6#, pET24a-7#, pET24a-8#, ...2#, pET24a-2#, pET24a-2#, pET24a-2#, pET24a-2#, p T24a-9#, pET24a-10#, pET24a-11#, pET24a-12#, pET24a-13#, pET24a-14#, pET24a-15#, pET24a-16#, pET24a-17#, pET24a-18#, pET24a-19#, pET24a-20#, pET24a-21#, pET24a-22#, and pET24a-23# are used for subsequent protein expression.

[0090] Example 2: Detection of enzyme activity in engineered bacteria

[0091] The above 23 expression plasmids pET24a-1#, pET24a-2#, pET24a-3#, pET24a-4#, pET24a-5#, pET24a-6#, pET24a-7#, pET24a-8#, pET24a-9#, pET24a-10#, pET24a-11#, pET24a-12#, pET24a-13#, and pET2 were converted by electroporation. Cells 4a-14#, pET24a-15#, pET24a-16#, pET24a-17#, pET24a-18#, pET24a-19#, pET24a-20#, pET24a-21#, pET24a-22#, and pET24a-23# were transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing kanamycin, and incubated overnight at 37°C. Two single colonies from each colony were selected and inoculated into test tubes containing LB agar, incubated overnight, centrifuged to collect the cells, extracted plasmids, and sequenced to confirm correctness, yielding recombinant strains. Select single clones from the genetically engineered bacterial strain on a plate, inoculate them into 5 mL of LB medium, and incubate at 37°C. Inoculate them at 1% v / v into a 250 mL shake flask containing 20 mL of TB medium and incubate for 4-6 hours. After the OD600 reaches 1.2-1.5, add 0.2 mM IPTG to induce incubation, cool to 25°C and incubate for 10-16 hours. Centrifuge to obtain bacterial cells and freeze at -80°C for 24 hours for later use.

[0092] Enzyme activity was determined using the following method: 5 mL of reaction solution (100 g / L methyl acetoacetate or ethyl acetoacetate, 100 mL / L isopropanol, 3 mM NADP or NAD, pH=7.5) was added, and 0.05 g of accurately weighed wet bacterial cells were added. The mixture was incubated in a 30°C water bath for 30 min, and the reaction was terminated with 1 M hydrochloric acid. A sample of the reaction solution was taken, and the concentration of the product methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate was determined by HPLC.

[0093] HPLC detection conditions: injection volume 10 μL; column: SB-AQ; mobile phase A: 0.3% phosphoric acid; mobile phase B: acetonitrile, A:B = 80:20; flow rate: 1 mL / min; column temperature: 40 °C; detection wavelength: 210 nm; detection time: 15 min. Calculate the enzyme activity per unit cell based on the product concentration.

[0094] Enzyme activity definition: The amount of bacterial cells required to generate 1 μM of product per unit time (min) is defined as one enzyme activity unit (U).

[0095] The enzyme activities of 23 wild-type engineered bacteria were compared, and the results are shown in Table 2.

[0096] Table 2. Results of wild-type enzyme activity assessment

[0097]

[0098] *Note: All activity test data are calculated with the enzyme activity of enzyme #1 catalyzing ethyl acetoacetate substrate as 00%.

[0099] The activity of enzyme #19 was relatively balanced for the two substrates. Based on this result, the pET24a-19# plasmid was subsequently used to construct and screen an error-prone PCR mutant library (hereinafter referred to as the error-prone mutant library).

[0100] Example 3: Construction and Screening of the First-Round Mutant Library

[0101] Using pET24a-19# plasmid as a template, the following primer pairs were designed for amplification:

[0102] Forward 19#-F: 5'-CATATGACCGACCGTCTGAAAGG-3',

[0103] Reverse 19#-R: 5'-CTCGAG TTACTGAGCGGTGTAACCACCG-3'.

[0104] A random mutant library was constructed using error-prone PCR technology. The 50 μL error-prone PCR reaction system consisted of: 500 ng plasmid template, 500 pmol of 19#-F primer, 500 pmol of 19#-R primer, 1x PCR buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, 0.1 mM MnCl2, and 2.5 units of Taq enzyme (Invitrogen™).

[0105] Error-prone PCR reaction conditions were: 95 ℃ for 5 min; 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 2 min / kbp, 30 cycles; 72 ℃ for 10 min. The randomly mutated fragments were recovered from the gel and used as the megaprimer for the next round of PCR. Megaprimer PCR was performed using KOD FX DNA polymerase (TOYOBO). The reaction mixture consisted of 50 μl of 1X PCR buffer, 2 mM dNTPs, 250 ng of megaprimer, 50 ng of pET24a-19# plasmid, and 1 unit of KOD FX. The PCR program was: 94 ℃ for 5 min; 98 ℃ for 10 s, 60 ℃ for 30 s, 68 ℃ for 1 min, 25 cycles; 68 ℃ for 10 min.

[0106] After PCR products were digested with DpnI for 10 h, they were electrotransformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing kanamycin, and cultured overnight at 37°C to obtain random mutant library clones.

[0107] After obtaining the mutant library clones, clone selection, culture, and reaction screening were performed. In a sterile 96-well plate, 400 μl of LB medium (containing 50 μg / ml kanamycin) was added to each well. Single clones of the mutant library were picked using sterile toothpicks, with BL21(DE3) engineered bacteria transformed with pET24a or pET24a-19# plasmid serving as blanks and negative controls, respectively. The above-mentioned plates were incubated at 37℃ on a plate shaker at 280 rpm for 20 h to serve as seed culture. A new 96-well plate was prepared, and 400 μl of ZYM medium (containing 50 μg / ml kanamycin) was added to each well. 50 μl of bacterial culture was inoculated from the seed plate and incubated at 30℃ at 280 rpm for 24 h. The remaining seed culture was diluted with 15% glycerol and stored at -80℃ for later use. After the ZYM plate culture was completed, the bacterial concentration OD600 was measured. Simultaneously, 50 μL of the bacterial cells from each well was transferred to a new 96-well plate, centrifuged at 4000 rpm for 10 min, and the precipitated bacterial cells were frozen and stored for 16 h. The frozen bacterial cells in the 96-well plate were thawed at room temperature for 20 min, and 200 μL of the prepared reaction solution (100 g / L ethyl acetoacetate, 100 mL / L isopropanol, 3 mM NADP, pH=7.5) was added. The plate was thoroughly shaken to resuspend the cells and incubated on a shaker at 30℃ and 280 rpm for 40 min. After the reaction was complete, the plate was placed on ice, and 200 μL of dilute hydrochloric acid was added to the 96-well plate using a multi-channel pipette to terminate the reaction. The plate was centrifuged at 4000 rpm for 10 min to obtain the supernatant. The supernatant was diluted 5-50 times with pure water, and the OD240 value was measured using a microplate reader. By comparing the absorbance values ​​of each well, the higher the enzyme activity, the lower the OD240 reading.

[0108] After the first round of screening, dominant clones were transferred from seed plates to TB shake flasks. 200 μl of each clone was inoculated into a 250 mL shake flask containing 20 mL of TB medium and cultured at 37°C for 4-6 hours. Once the OD600 reached 1.2-1.5, 0.2 mM IPTG was added for induction, and the culture was cooled to 25°C and incubated for 10-16 hours. Cells were obtained by centrifugation; one portion was frozen at -80°C for 24 hours for later use, while the other portion underwent plasmid extraction and sequencing to determine mutation sites. Enzyme activities of the corresponding mutants on two substrates were measured, and the results are shown in Table 3.

[0109] Table 3. Enzyme protein sequencing and enzyme activity of different clones

[0110] strain number amino acid sequence number Enzyme mutation site Ethyl acetoacetate substrate relative enzyme activity Methyl acetoacetate substrate relative enzyme activity 19# 1 none 100.00%* 105.01% The following are the mutation sites compared to #19. 785 22 Y190F 203.05% 206.44% 1024 3 A202T, K97R 245.12% 234.55% 1938 23 K97R 157.68% 148.23% 3453 24 P220Q, F147C 143.68% 172.87% 7766 25 I57N 175.01% 183.23% 7942 26 G149D 159.85% 136.68% 8174 27 R123S 136.68% 178.61%

[0111] *Note: All activity test data are calculated with the enzyme activity of enzyme 19# catalyzing ethyl acetoacetate substrate as 100%.

[0112] As shown in Table 3, enzyme 1024 (SEQ ID NO: 3) exhibits the highest and relatively balanced catalytic activity for both substrates.

[0113] Based on the above comparison results, and referring to the method in Example 1, the encoding gene of enzyme No. 1024 was designed and the pET24a-1024 plasmid was constructed. Subsequently, the pET24a-1024 plasmid was used to construct and screen an error-prone PCR mutant library.

[0114] Example 4: Construction and Screening of the Second Round of Mutant Libraries

[0115] Using plasmid pET24a-1024 as a template, the second round of error-prone PCR mutant library construction and screening was carried out according to the method in Example 3. BL21(DE3) engineered bacteria transformed with pET24a or pET24a-1024 plasmids respectively were used as blanks and negative controls. The results are shown in Table 4.

[0116] Table 4. Enzyme protein sequencing and enzyme activity of different strains in the second-round mutant library.

[0117] strain number amino acid sequence number mutation site Ethyl acetoacetate substrate relative enzyme activity Methyl acetoacetate substrate relative enzyme activity 1024 3 A202T, K97R 100% 98% The following are the mutation sites compared to 1024. 10859 4 T202V, Y190G 142% 129% 14578 5 F147I, K6N 128% 122% 15793 6 N89T, R123H, N221T 145% 158% 23458 7 R97K, D25G 137% 142% 27894 8 S235Y, I57N, R123H 144% 145% 30231 9 N221I, Y190F, T202V 175% 188%

[0118] As can be seen from Table 4, enzyme 30231 (SEQ ID NO: 9) exhibits relatively high and balanced catalytic activity for both substrates.

[0119] Based on the above comparison results, and referring to the method in Example 1, the encoding gene of enzyme No. 30231 was designed and the pET24a-30231 plasmid was constructed. Subsequently, the pET24a-30231 plasmid was used to construct and screen an error-prone PCR mutant library.

[0120] Example 5: Construction and Screening of the Third-Round Mutant Library

[0121] Using plasmid pET24a-30231 as a template, the third round of error-prone PCR mutant library construction and screening was carried out according to the method in Example 3. BL21(DE3) engineered bacteria transformed with pET24a or pET24a-30231 plasmids were used as blanks and negative controls, respectively. The results are shown in Table 5.

[0122] Table 5. Enzyme protein sequencing and enzyme activity of different strains in the third-round mutant library.

[0123] strain number amino acid sequence number mutation site Ethyl acetoacetate substrate relative enzyme activity Methyl acetoacetate substrate relative enzyme activity 30231 9 A202V, K97R, N221I, Y190F 100% 107% The following are the mutation sites compared to 30231. 40231 10 D25G, K6N, R123S 158% 150% 48751 11 G149D, R97K, D25G 146% 142% 49667 12 I221N, D25G, R97K 133% 148% 52368 13 I221N, D25G, R97K, I57T 159% 149% 55786 14 R97K, F147I 186% 182% 59753 15 V202A, F147I 165% 158%

[0124] As can be seen from Table 5, enzyme 55786 (SEQ ID NO: 14) exhibits relatively high and balanced catalytic activity for both substrates.

[0125] Based on the above comparison results, and referring to the method in Example 1, the encoding gene of enzyme No. 55786 was designed and the pET24a-55786 plasmid was constructed. Subsequently, the pET24a-55786 plasmid was used to construct and screen an error-prone PCR mutant library.

[0126] Example 6: Construction and Screening of the Fourth Round of Mutant Libraries

[0127] Using plasmid pET24a-55786 as a template, the fourth round of error-prone PCR mutant library construction and screening was carried out according to the method in Example 3. BL21(DE3) engineered bacteria transformed with pET24a or pET24a-55786 plasmids were used as blanks and negative controls, respectively. The results are shown in Table 6.

[0128] Table 6. Enzyme protein sequencing and enzyme activity of different strains in the fourth round of mutant library

[0129] strain number amino acid sequence number mutation site Ethyl acetoacetate substrate relative enzyme activity Methyl acetoacetate substrate relative enzyme activity 55786 14 A202V, N221I, Y190F, F147I 100% 100% The following are the mutation sites compared to 55786. 60278 16 D197E, P151L 116% 121% 64231 17 I19L, I221V, G149R 112% 112% 65781 18 K97N, N89K, R123S 132% 128% 66713 19 I147F, V202T, K6N 118% 100%

[0130] As can be seen from Table 6, enzyme 65781 (SEQ ID NO: 18) exhibits relatively high and balanced catalytic activity for both substrates.

[0131] Based on the above comparison results, and referring to the method in Example 1, the encoding gene of enzyme No. 65781 was designed and the pET24a-65781 plasmid was constructed. Subsequently, the pET24a-65781 plasmid was used to construct and screen an error-prone PCR mutant library.

[0132] Example 7: Construction and Screening of the Fifth Round of Mutant Libraries

[0133] Using plasmid pET24a-65781 as a template, the fourth round of error-prone PCR mutant library construction and screening was carried out according to the method in Example 3. BL21(DE3) engineered bacteria transformed with pET24a or pET24a-65781 plasmids were used as blanks and negative controls, respectively. The results are shown in Table 7.

[0134] Table 7. Enzyme protein sequencing and enzyme activity of different strains in the fifth round of mutant library

[0135] strain number amino acid sequence number mutation site Ethyl acetoacetate substrate relative enzyme activity Methyl acetoacetate substrate relative enzyme activity 65781 18 A202V, N221I, Y190F, F147I, K97N, N89K, R123S 100% 100% The following are mutation sites compared to 65781. 76789 20 A155D, T77M 137% 141% 78932 21 T77S, G149R, P151L 134% 151%

[0136] As can be seen from Table 7, enzymes 76789 (SEQ ID NO: 20) and 78932 (SEQ ID NO: 21) both showed a certain degree of improvement in the catalytic activity of the two substrates, and the improvement was relatively balanced.

[0137] Following the method described in Example 1, the encoding genes for enzymes 76789 and 78932 were designed, and plasmids pET24a-76789 and pET24a-78932 were constructed. Following the method described in Example 2, these two plasmids were transformed into *E. coli* BL21(DE3) competent cells by electroporation to obtain the genetically engineered bacteria pET24a-76789 / BL21(DE3) and pET24a-78932 / BL21(DE3), which were used to catalyze the reaction of methyl acetoacetate and ethyl acetoacetate to prepare 3-hydroxybutyrate.

[0138] Example 8: Preparation of 3-hydroxybutyrate ester catalyzed by a mutant enzyme

[0139] The engineered bacteria pET24a-76789 / BL21(DE3) and pET24a-78932 / BL21(DE3), which were transformed with the mutant plasmids pET24a-76789 and pET24a-78932 respectively, were inoculated into test tubes containing LB medium and cultured overnight at 37°C. Then, they were inoculated into 500 mL shake flasks containing 100 mL of TB medium at a ratio of 1% v / v and cultured at 37°C for 4-6 hours. After the OD600 reached 1.2-1.5, 0.2 mM IPTG was added for induction, and the temperature was lowered to 25°C for 10-16 hours. The bacterial cells were obtained by centrifugation and stored at -80°C for 24 hours for later use.

[0140] The catalytic reaction used a 1L reaction system: methyl acetoacetate or ethyl acetoacetate 50g / L, isopropanol 80ml / L, NADP cofactor 10mM, pH 7.5, and wet bacterial cells 1.5%. The reaction was carried out at 30℃ and 230rpm for 15h, then terminated with hydrochloric acid. The product and substrate concentrations were quantitatively determined, and the substrate conversion rate was calculated. Simultaneously, samples were taken for 3-hydroxybutyrate chirality detection. The conversion solution was centrifuged at 12000rpm for 3min, and the supernatant was collected. Ethyl acetate was added to the supernatant, and the mixture was vortexed for 5min. After centrifugation at 12000rpm for 3min, the ethyl acetate phase was collected, and 0.2g of anhydrous sodium sulfate was added. The mixture was shaken overnight. After drying, the ethyl acetate phase was centrifuged at high speed for 10min, and the supernatant was collected for GC analysis.

[0141] GC detection conditions were as follows: column: Gamma DEXTM 225 Capillary Column, 30m*0.25nm*0.25μm film thickness; injection volume: 0.1μL; injector temperature: 250℃; split ratio: 190:1; carrier gas pressure: 10.795psi; flow rate: 1mL / min; temperature program: initial temperature 40℃, hold for 5min, increase to 170℃ at a rate of 10℃ / min, hold for 2min; run time: 20min; detector: FID, 300℃; air flow rate: 400mL / min; hydrogen flow rate: 30mL / min; make-up gas (N2): 25mL / min.

[0142] The experimental results of the preparation of 3-hydroxybutyrate by catalysis of methyl acetoacetate / ethyl acetoacetate by two strains, pET24a-76789 and pET24a-78932, are listed in Table 8.

[0143] Table 8. Results of enzyme-catalyzed preparation of 3-hydroxybutyrate

[0144]

[0145] Table 8 shows that both mutant enzymes, enzyme 76789 (SEQ ID NO: 20) and enzyme 78932 (SEQ ID NO: 21), can catalyze the reaction of the two substrates to obtain R-configuration 3-hydroxybutyrate. The conversion rate of both substrates can reach more than 90%, and the chiral purity of the products is higher than 99%, which shows promising prospects for industrial application. SEQUENCE LISTING <110> Zhejiang Huarui Biotechnology Co., Ltd. <120> Enzymatic synthesis of 3-hydroxybutyrate <130> SHPCT2010388 <160> 27 <170> PatentIn version 3.3 <210> 1 <211> 252 <212> PRT <213> Lactobacillus kefiri DSM 20587 <400> 1 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> 2 <211> 759 <212> DNA <213> ˹ <400> 2 atgaccgacc gtctgaaagg taaagttgct atcgttaccg gtggtaccct gggtatcggt 60 ctggctatcg ctgacaaatt cgttgaagaa ggtgctaaag ttgttatcac cggtcgtcac 120 gctgacgttg gtgaaaaagc tgctaaatct atcggtggta ccgacgttat ccgtttcgtt 180 cagcacgacg cttctgacga agctggttgg accaaactgt tcgacaccac cgaagaagct 240 ttcggtccgg ttaccaccgt tgttaacaac gcgggcatcg ctgtaagcaa atctgttgaa 300 gacaccacca ccgaagaatg gcgtaaactg ctgtctgtta acctggacgg tgttttcttc 360 ggtacccgtc tgggtatcca gcgtatgaaa aaaaaggtc tggtgcttc tatcatcaac 420 atgtctagta tagaaggctt cgttggtgac ccaaccctgg gtgcttacaa cgcttctaaa 480 ggtgctgttc gtatcatgtc taaatctgct gctctggact gcgctctgaa agactacgac 540 gttcgtgtta acaccgttca cccgggttac atcaaaaccc cgctggttga cgacctggaa 600 ggtgctgaag aaatgatgtc tcagcgtacc aaaccccga tgggtcacat cggtgaaccg 660 aacgacatcg cgtggatctg cgtctacctg gcgtctgacg aatctaaatt cgctaccggt 720 gctgaattcg ttgttgacgg tggttacacc gctcagtaa 759 <210> 3 <211> 252 <212> PRT <213> ˹ <400> 3 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 Arg 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 Thr 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> 4 <211> 252 <212> PRT <213> ˹ <400> 4 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 Arg 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 Gly Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val 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> 5 <211> 252 <212> PRT <213> ˹ <400> 5 Met Thr Asp Arg Leu Asn 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 Arg 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 Ile 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 Thr 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> 6 <211> 252 <212> PRT <213> ˹ <400> 6 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 Thr Asn Ala Gly Ile Ala Val Ser 85 90 95 Arg 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 His 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 Thr Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Thr 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> 7 <211> 252 <212> PRT <213> ˹ <400> 7 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 Gly 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 Thr 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> 8 <211> 252 <212> PRT <213> ˹ <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 Asn 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 Arg 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 His 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 Thr 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 Tyr 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> 252 <212> PRT <213> ˹ <400> 9 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 Arg 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 10 <211> 252 <212> PRT <213> ˹ <400> 10 Met Thr Asp Arg Leu Asn Gly Lys Val Ala Ile Val Thr Gly Gly Thr 1 5 10 15 Leu Gly Ile Gly Leu Ala Ile Ala Gly 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 Arg 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 Ser 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 11 <211> 252 <212> PRT <213> ˹ <400> 11 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 Gly 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 Asp 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 12 <211> 252 <212> PRT <213> ˹ <400> 12 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 Gly 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val 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> 13 <211> 252 <212> PRT <213> ˹ <400> 13 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 Gly 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 Thr 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val 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> 14 <211> 252 <212> PRT <213> ˹ <400> 14 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 Ile 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 15 <211> 252 <212> PRT <213> ˹ <400> 15 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 Arg 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 Ile 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 Phe 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 Ile 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> 16 <211> 252 <212> PRT <213> ˹ <400> 16 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 Ile Val Gly Asp Leu 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Glu Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 17 <211> 252 <212> PRT <213> ˹ <400> 17 Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr 1 5 10 15 Leu Gly Leu 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 Ile Val Arg 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Val 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> 18 <211> 252 <212> PRT <213> ˹ <400> 18 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 Lys Asn Ala Gly Ile Ala Val Ser 85 90 95 Asn 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 Ser 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 Ile 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 19 <211> 252 <212> PRT <213> ˹ <400> 19 Met Thr Asp Arg Leu Asn 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Thr Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 20 <211> 252 <212> PRT <213> ˹ <400> 20 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 Met Glu Glu Ala 65 70 75 80 Phe Gly Pro Val Thr Thr Val Val Lys Asn Ala Gly Ile Ala Val Ser 85 90 95 Asn 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 Ser 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 Ile Val Gly Asp Pro Thr Leu Gly Asp 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 21 <211> 252 <212> PRT <213> ˹ <400> 21 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 Ser Glu Glu Ala 65 70 75 80 Phe Gly Pro Val Thr Thr Val Val Lys Asn Ala Gly Ile Ala Val Ser 85 90 95 Asn 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 Ser 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 Ile Val Arg Asp Leu 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 Phe Ile Lys 180 185 190 Thr Pro Leu Val Asp Asp Leu Glu Gly Val Glu Glu Met Met Ser Gln 195 200 205 Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Ile 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> 22 <211> 252 <212> PRT <213> ˹ <400> 22 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 Phe 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> 23 <211> 252 <212> PRT <213> ˹ <400> 23 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 Arg 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> 24 <211> 252 <212> PRT <213> ˹ <400> 24 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 Cys 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 Gln 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> 25 <211> 252 <212> PRT <213> ˹ <400> 25 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 Asn 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> 26 <211> 252 <212> PRT <213> ˹ <400> 26 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 Asp 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> 27 <211> 252 <212> PRT <213> ˹ <400> 27 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 Ser 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

Claims

1. An alcohol dehydrogenase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO: 20 or SEQ ID NO: 21, where SEQ ID NO: 20 is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, A155D, T77M of alcohol dehydrogenase SEQ ID NO: 1; and SEQ ID NO: 21 is a mutant of the amino acid sequence A202V, N221I, Y190F, F147I, K97N, N89K, R123S, T77S, G149R, P151L of alcohol dehydrogenase SEQ ID NO:

1.

2. A microorganism, characterized in that, It expresses the alcohol dehydrogenase mutant as described in claim 1.

3. The microorganism as described in claim 2, characterized in that, The microorganisms were selected from Escherichia coli, Pichia pastoris, and Bacillus subtilis.

4. Use of the alcohol dehydrogenase mutant of claim 1 or the microorganism of claim 2 in the production of 3-hydroxybutyrate.

5. The use as described in claim 4, characterized in that, Using acetoacetate as a substrate, 3-hydroxybutyrate is obtained by using the alcohol dehydrogenase mutant as described in claim 1 or the microbial catalytic reduction reaction as described in claim 2.

6. The use as described in claim 5, characterized in that, The acetoacetate is methyl acetoacetate or ethyl acetoacetate, and correspondingly, the 3-hydroxybutyrate is methyl 3-hydroxybutyrate or ethyl 3-hydroxybutyrate.

7. The use as described in claim 5, characterized in that, Isopropanol and coenzyme NADP+ were added to the reaction system.

Images