CN113969268B - Glu/Leu/Phe/Val dehydrogenase mutant and application thereof in preparation of L-glufosinate - Google Patents

Abstract

The application relates to a Glu/Leu/Phe/Val dehydrogenase mutant and application thereof in preparation of L-glufosinate. The amino acid sequence of the Glu/Leu/Phe/Val dehydrogenase mutant comprises substitutions corresponding to amino acid residues at positions 91 and/or 168, the positions 91 and 168 being defined with reference to SEQ ID NO.5, and the amino acid sequence of the Glu/Leu/Phe/Val dehydrogenase mutant having at least 90% identity to the sequence shown in SEQ ID NO.5 when aligned with the amino acid sequence of a Glu/Leu/Phe/Val dehydrogenase comprising the sequence shown in SEQ ID NO. 5.

Description

Glu/Leu/Phe/Val dehydrogenase mutant and application thereof in preparation of L-glufosinate

Technical Field

The application relates to the field of biotechnology; in particular, the application relates to Glu/Leu/Phe/Val dehydrogenase mutants, the use of the Glu/Leu/Phe/Val dehydrogenase mutants in the preparation of L-glufosinate, and a method for preparing L-glufosinate using the Glu/Leu/Phe/Val dehydrogenase mutants.

Background

Glufosinate (also known as bialaphos, glufosinate, trade names including pilot, baiston, etc., and the english name phosphinothricin (abbreviated PPT), the chemical name 2-amino-4- [ hydroxy (methyl) phosphono ] butanoic acid) is a low-toxicity, high-efficiency, nonselective contact-type organophosphorus herbicide developed by german helrst corporation (now belonging to bayer corporation) in the 80 s of the 20 th century. The glufosinate can inhibit glutamine synthetase after acting on plants, so that the reversible reaction of glutamic acid in the plants is interrupted, metabolic disorder is caused, the plants accumulate excessive ammonia to poison, and meanwhile, the plants cannot synthesize chlorophyll, so that photosynthesis is inhibited, and the plants die. The glufosinate is mainly used for orchards, potato fields, non-cultivated lands and the like, and is used for preventing and controlling annual and perennial grassy and dicotyledonous weeds, such as crabgrass, green bristlegrass and wild wheat; perennial grassy weeds and nutgrass flatsedge, such as fescue, duck buds, etc.

The market for biocidal herbicides is enormous. At present, three herbicides in the world are paraquat, glyphosate and glufosinate respectively. In the aspect of market use, glyphosate is the only chelating head, but because of the long-term use, a large number of weeds generate resistance, and glyphosate also tends to lose efficacy; paraquat is forbidden or limited in more and more countries worldwide because of its extremely toxic property, and the Chinese department of agriculture has issued bulletin that paraquat stops production in 7 months 1 of 2014, and is forbidden in 7 months 1 of 2016; at present, the glufosinate has small yield, but has excellent weeding performance and small phytotoxicity and side effects, so that the glufosinate has great market potential in a future period of time.

The glufosinate has two optical isomers, namely L-glufosinate and D-glufosinate, but only L-form has herbicidal activity, is easy to decompose in soil, has small toxicity to human beings and animals, has wide herbicidal spectrum and has small damage to the environment.

Currently, glufosinate is commercially available as a racemic mixture. If the glufosinate-ammonium product can be used in the form of pure optical isomer with L-configuration, the use amount of the glufosinate-ammonium can be obviously reduced, which has important significance for improving the atom economy, reducing the use cost and relieving the environmental pressure.

The main preparation method of chiral pure L-glufosinate mainly comprises three steps: chiral resolution, chemical synthesis and biocatalysis.

Chiral resolution requires the use of expensive chiral resolving agents (e.g., quinine), the resolution steps are very cumbersome (e.g., undergo salt formation, induced crystallization, salt decomposition, etc.), and the theoretical yield of resolution is only 50%, which results in a route with relatively low industrial value.

Chemical synthesis methods include asymmetric synthesis methods, natural amino acid chiral source methods, and the like, and disadvantages include the need to use expensive noble metals and ligands or starting materials, or the need for highly toxic substances in the reaction route, or the longer reaction synthesis route, and the like.

The biocatalytic method for producing glufosinate has the advantages of strict stereoselectivity, mild reaction condition, high yield and the like, and is an advantageous method for producing L-glufosinate. Mainly comprises the following two types: (1) The L-glufosinate derivative is used as a substrate and is obtained by direct hydrolysis through an enzymatic method, and the method has the main advantages of high conversion rate and higher ee value of a product, but expensive and difficult-to-obtain chiral raw materials are needed as precursors; (2) The method takes the precursor of the racemic glufosinate-ammonium as a substrate, and is obtained through selective resolution of enzyme, and has the main advantages that the raw materials are relatively easy to obtain, the activity of the catalyst is high, but the theoretical yield can only reach 50%, and the raw materials are wasted.

In addition to these two traditional biocatalytic processes, the de-racemization synthesis process using D, L-glufosinate-ammonium as the starting material stands out a great cost advantage. As the commercial glufosinate-ammonium is D, L-glufosinate-ammonium, the industrial production technology is mature, the racemization synthesis method directly takes the D, L-glufosinate-ammonium as the raw material, is simple and easy to obtain, has lower cost, and can better match the existing glufosinate-ammonium industrial production system.

Disclosure of Invention

The application is based on the identification of Glu/Leu/Phe/Val dehydrogenase mutants which can be used for improving the biological enzymatic production of L-glufosinate.

In a first aspect, the present application relates to a Glu/Leu/Phe/Val dehydrogenase mutant having Glu/Leu/Phe/Val dehydrogenase activity, wherein the amino acid sequence of the Glu/Leu/Phe/Val dehydrogenase mutant comprises a substitution corresponding to amino acid residues at position 91 and/or 168, the position 91 and 168 being defined with reference to SEQ ID NO.5, and the amino acid sequence of the Glu/Leu/Phe/Val dehydrogenase mutant has at least 90% identity with the sequence shown in SEQ ID NO.5 when aligned with the amino acid sequence of a Glu/Leu/Phe/Val dehydrogenase comprising the sequence shown in SEQ ID NO. 5.

In some embodiments, the substitution of the amino acid residue at position 91 is V91I, i.e., the amino acid residue at position 91 is substituted with V to I. In some embodiments, the substitution of the amino acid residue at position 168 is N168G, i.e., the amino acid residue at position 168 is substituted with G by N.

In some embodiments, the Glu/Leu/Phe/Val dehydrogenase mutant comprises a V91I amino acid substitution when aligned with an amino acid sequence of a Glu/Leu/Phe/Val dehydrogenase comprising the sequence shown in SEQ ID NO.5, wherein the position of the amino acid is defined with reference to SEQ ID NO. 5. In some embodiments, the Glu/Leu/Phe/Val dehydrogenase mutant comprises an N168G amino acid substitution when aligned with the amino acid sequence of Glu/Leu/Phe/Val dehydrogenase comprising the sequence shown in SEQ ID NO.5, wherein the position of the amino acid is defined with reference to SEQ ID NO. 5. In some embodiments, the Glu/Leu/Phe/Val dehydrogenase mutant comprises V91I and N168G amino acid substitutions when aligned with the amino acid sequence of Glu/Leu/Phe/Val dehydrogenase comprising the sequence shown in SEQ ID NO.5, wherein the positions of the amino acids are defined with reference to SEQ ID NO. 5.

The amino acid sequence of Glu/Leu/Phe/Val dehydrogenase comprising the sequence shown in SEQ ID No.5 may be referred to herein as a wild-type enzyme having Glu/Leu/Phe/Val dehydrogenase activity. The nucleotide sequence of the wild-type enzyme may be the nucleotide sequence shown as SEQ ID NO. 10.

The Glu/Leu/Phe/Val dehydrogenase mutant has Glu/Leu/Phe/Val dehydrogenase activity, namely activity of converting an alpha-keto acid precursor of glutamic acid/leucine/phenylalanine/valine and other structurally similar amino acids into L-amino acid, and particularly, the Glu/Leu/Phe/Val dehydrogenase or the mutant thereof has activity of converting 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butyric acid (PPO for short) into L-glufosinate.

"Amino acid position" is defined with reference to SEQ ID NO.5 "means that the amino acid in the Glu/Leu/Phe/Val dehydrogenase mutant is aligned with a particular amino acid position (e.g., position 91, 168) when aligned with the amino acid sequence of SEQ ID NO. 5.

The Glu/Leu/Phe/Val dehydrogenase mutants of the application may have improved activity compared to wild-type enzymes having Glu/Leu/Phe/Val dehydrogenase activity, e.g.having a higher catalytic efficiency in the catalytic reaction of 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid into L-glufosinate, and optionally having a better stability etc. for use in biocatalytic processes for the production of L-glufosinate, in particular in the biocatalytic processes described herein.

The term "catalytic efficiency" as used in the present application refers to the property of Glu/Leu/Phe/Val dehydrogenase to allow its conversion of 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid to L-glufosinate. In one embodiment, the catalytic efficiency of the Glu/Leu/Phe/Val dehydrogenase mutants of the application is enhanced compared to wild-type or reference Glu/Leu/Phe/Val dehydrogenase. Preferably, the catalytic efficiency of the Glu/Leu/Phe/Val dehydrogenase mutant of the application is at least 1.1, 1.2 or 1.3 times that of the wild-type or reference Glu/Leu/Phe/Val dehydrogenase.

In some embodiments, the Glu/Leu/Phe/Val dehydrogenase mutant is derived from Delftia acidovorans.

In some embodiments, the amino acid sequence of the Glu/Leu/Phe/Val dehydrogenase mutant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the nucleotide sequence of the Glu/Leu/Phe/Val dehydrogenase mutant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID NO. 10. In some embodiments, the Glu/Leu/Phe/Val dehydrogenase mutant comprises an amino acid sequence having one or several (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, deletions, and/or insertions compared to the wild-type enzyme and/or one or more truncated amino acid sequences compared to the wild-type enzyme.

In some embodiments, the Glu/Leu/Phe/Val dehydrogenase mutant is an amino acid substitution at amino acid residues 91 and/or 168 when aligned with an amino acid sequence of Glu/Leu/Phe/Val dehydrogenase comprising the sequence shown in SEQ ID NO. 5.

In a second aspect, the application provides a nucleic acid or polynucleotide sequence comprising a sequence encoding the Glu/Leu/Phe/Val dehydrogenase mutant described above. The nucleic acid or polynucleotide sequence may be isolated.

The term "nucleic acid" or "polynucleotide" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA produced using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

The features, definitions and preferences described in the first aspect apply equally to the second aspect.

In a third aspect, the application provides an expression vector comprising a nucleic acid or polynucleotide sequence as described above. The expression vector may comprise an operative linkage to one or more control sequences capable of directing expression of the Glu/Leu/Phe/Val dehydrogenase mutants described above in a suitable expression host.

The term "operably linked" refers to the linkage of polynucleotide elements (or coding sequences or nucleic acid sequences) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. In some embodiments, the control sequences may include promoters, enhancers, terminators, and the like.

The expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can cause the expression of a polynucleotide of the Glu/Leu/Phe/Val dehydrogenase mutant. The choice of expression vector will generally depend on the compatibility of the vector with the cell into which the vector is to be introduced. The expression vector may exist as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. Alternatively, the expression vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

More than one copy (e.g., 2, 3, or 4) of the expression vector of the application may be inserted into a host cell to increase the production (overexpression) of the Glu/Leu/Phe/Val dehydrogenase mutant encoded by the nucleic acid sequence contained within the expression vector.

In some embodiments, the expression vectors of the application may also comprise nucleic acid sequences encoding formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase to effect expression of these dehydrogenases with mutants, preferably co-expression with L-amino acid dehydrogenase mutants.

The features, definitions and preferences described in the first and second aspects apply equally to the third aspect.

In a fourth aspect, the application provides a recombinant host cell comprising a nucleic acid according to the application or an expression vector according to the application.

In some embodiments, the recombinant host cell may be a prokaryotic or eukaryotic cell. In some embodiments, the host cell belongs to the genus Saccharomyces (Saccharomyces), aspergillus (Aspergillus), pichia (Pichia), kluyveromyces (Kluyveromyces), candida (Candida), hansenula (Hansenula), humicola (Humicola), issatchenkia (ISSATCHENKIA), trichosporon (Trichosporon), brettanomyces (Brettanomyces), pachysolenosis (Pachysolen), yarrowia (Yarrowia), actinomyces (Actinomies), streptomyces (Streptomyces), bacillus (Bacillus) or Escherichia (Escherichia); preferably one of Saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolitica), candida krusei (Candida krusei), issatchenkia orientalis (ISSATCHENKIA ORIENTALIS), bacillus subtilis (Bacillus subtilis) or Escherichia coli (ESCHERICHIA COLI).

The expression vectors of the application may be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into host cells known to those of skill in the art. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al (Molecular Cloning: A Laboratory Manual, 2 nd edition .Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,1989),Davis et al, basic Methods in Molecular Biology (1986) and other laboratory manuals.

In some embodiments, the recombinant host cell is a host cell that co-expresses (a) an L-amino acid dehydrogenase having 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid activity and (b) a dehydrogenase selected from formate dehydrogenase, glucose dehydrogenase, or alcohol dehydrogenase.

The features, definitions and preferences described in the first, second, third aspects apply equally to the fourth aspect.

In a fifth aspect, the application provides the use of a Glu/Leu/Phe/Val dehydrogenase mutant, nucleic acid, expression vector or recombinant host cell according to the application in the preparation of L-glufosinate.

The features, definitions and preferences described in the first, second, third, fourth aspects apply equally to the fifth aspect.

In a sixth aspect, the present application provides a method for preparing L-glufosinate comprising reacting D-glufosinate in the presence of an enzyme catalytic system to produce L-glufosinate, wherein the enzyme catalytic system comprises Glu/Leu/Phe/Val dehydrogenase mutants for converting 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid into L-glufosinate.

In some embodiments, the D-glufosinate is initially present in a racemic mixture of D-and L-glufosinate or salts thereof. The racemic glufosinate starting material may be provided in a variety of forms. Various salts of racemic glufosinate may be used, such as ammonium salts and hydrochloride salts, or zwitterionic.

In some embodiments, the enzyme catalytic system further comprises a D-amino acid oxidase for converting D-glufosinate of D, L-glufosinate to 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid. The D-amino acid oxidase may be any enzyme known in the art having D-amino acid oxidase activity or a variant thereof. For example, the D-amino acid oxidase described in CN107502647B, CN111019916B, CN 111321193B.

In some embodiments, catalase is also included in the enzyme catalysis system. The catalase is used to remove byproduct hydrogen peroxide because hydrogen peroxide accumulation can have a deleterious effect on the enzyme catalyst. The catalase may be any enzyme known in the art having catalase activity, such as catalase commercially available from Ningxia-Shangsheng Industrial group Co., ltd.

In some embodiments, the enzyme catalytic system further comprises a coenzyme circulation system selected from at least one of the following:

(1) Formate dehydrogenase coenzyme circulatory system: including formate dehydrogenase, formate and coenzyme;

(2) Glucose dehydrogenase coenzyme circulatory system: including glucose dehydrogenase, glucose and coenzyme;

(3) Alcohol dehydrogenase coenzyme circulatory system: including alcohol dehydrogenase, isopropanol and coenzyme.

In some preferred embodiments, the coenzyme is NADH.

The Formate Dehydrogenase (FDH) according to the application can be any enzyme or enzyme variant known in the art having formate dehydrogenase activity. In some embodiments, the formate dehydrogenase is derived from Lactobacillus buchneri. In some embodiments, the amino acid sequence of the formate dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID No. 2. In some embodiments, the nucleotide sequence of the formate dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 7.

The Glucose Dehydrogenase (GDH) of the present application may be any enzyme or enzyme variant known in the art having glucose dehydrogenase activity. In some embodiments, the glucose dehydrogenase is derived from Exiguobacterium sibiricum. In some embodiments, the amino acid sequence of the glucose dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID No. 3. In some embodiments, the nucleotide sequence of the glucose dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 8.

The Alcohol Dehydrogenase (ADH) of the present application may be any enzyme or enzyme variant known in the art having alcohol dehydrogenase activity. In some embodiments, the alcohol dehydrogenase is derived from Lactobacillus brevis. In some embodiments, the amino acid sequence of the alcohol dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID No. 4. In some embodiments, the nucleotide sequence of the alcohol dehydrogenase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 9.

The enzyme of the application (e.g., glu/Leu/Phe/Val dehydrogenase mutant, D-amino acid oxidase, catalase, formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase) may be in the form of a purified enzyme; a partially purified enzyme; cell-free or crude cell extract; liquid, powder or fixed form; permeabilized cells, whole cells, or whole fermentation broths containing enzymes, or any other suitable form. In some embodiments, each enzyme in the enzyme catalytic system is in a form that is each independently selected from: free enzyme and recombinant host cell expressing the enzyme.

In some embodiments, wherein the recombinant host cells expressing the enzyme are each independently selected from the group consisting of: saccharomyces (Saccharomyces), aspergillus (Pichia), kluyveromyces (Kluyveromyces), candida (Candida), hansenula (Hansenula), humicola (Humicola), issatchenkia (ISSATCHENKIA), massa (Trichosporon), brettanomyces (Brettanomyces), pachysolen (Pachysolen), yarrowia (Yarrowia), actinomyces (Actinomyces), streptomyces (Streptomyces), bacillus (Bacillus) or Escherichia (Escherichia); for example selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolitica), candida krusei (Candida krusei), issatchenkia orientalis (ISSATCHENKIA ORIENTALIS), bacillus subtilis (Bacillus subtilis) or Escherichia coli (ESCHERICHIA COLI).

In some embodiments, the conversion reaction is performed in a reaction solution. Preferably, the pH of the reaction solution is 7-10, preferably 8-9. Among the reaction liquids having a pH of 7 to 10, preferable reaction efficiency can be obtained when the reaction is carried out in a reaction liquid having a pH of 8 to 9.

The method of the application can comprise the following steps: step a) in which an oxidation reaction catalyzed by D-amino acid oxidase takes place and step b) in which a reductive amination reaction catalyzed by Glu/Leu/Phe/Val dehydrogenase mutant takes place.

In some embodiments, the temperature of the oxidation reaction of step a) is 25-45 ℃, e.g., 30-45 ℃,35-45 ℃, etc.; the time is 6-24 hours, e.g., 6-12 hours, 12-24 hours, e.g., 6 hours, 12 hours, etc.

In step b), the PPO produced in step a) is catalytically reduced by an L-amino acid dehydrogenase to L-glufosinate, thereby achieving in situ racemization of D, L-glufosinate to obtain L-glufosinate with an ee value of greater than 99%. In some embodiments, the reaction system of step b) further comprises a coenzyme NADH. In some embodiments, the molar ratio of NADH to substrate is from 1:10 to 1:5000. In some embodiments, NADH is added in a molar concentration of 0.1-2mM; more preferably 0.5mM.

In some embodiments, the temperature of the reductive amination reaction of step b) is 25-45 ℃, e.g., 30-45 ℃,35-45 ℃, etc.; the time is 6-24 hours, e.g., 6-12 hours, 12-24 hours, e.g., 6 hours, 12 hours, etc.

In some embodiments, in step b), the molar ratio of inorganic ammonium donor to substrate at the start of the reaction is from 1:1 to 10:1.

In some embodiments, in step b), the inorganic ammonium donor may be ammonium phosphate, ammonium chloride, ammonium sulfate, ammonium formate, ammonium acetate, aqueous ammonia; preferably, the inorganic ammonium donor may be ammonium phosphate, ammonium formate, aqueous ammonia; more preferably, the inorganic ammonium donor may be aqueous ammonia.

The process of the present application may be carried out in one or more reaction vessels. Preferably, the process of the present application is carried out in one reaction vessel (i.e. "one pot two step process").

In some preferred embodiments, the D-amino acid oxidase used in step a) is expressed by a first recombinant microorganism. Thus, step a) may comprise: and (3) oxidizing the D-glufosinate-ammonium in the presence of the first recombinant microorganism and oxygen to obtain 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid. The use of said first recombinant microorganism enables to confer a higher catalytic efficiency on the process of the application. The first recombinant microorganism may be constructed using any method known in the art. For example, the first recombinant microorganism may be constructed as follows: constructing a recombinant expression vector containing the D-amino acid oxidase gene, converting the recombinant expression vector into microorganisms, performing induction culture on the obtained recombinant microorganisms, and separating culture solution to obtain a first recombinant microorganism containing the D-amino acid oxidase gene. Preferably, the addition amount of the first recombinant microorganism is 1g/L to 200g/L of the reaction solution according to the wet weight of the thalli after centrifugation at 10000rpm for 10 min; more preferably, 10g/L to 100g/L of the reaction liquid; most preferably 30g/L of reaction solution.

In some preferred embodiments, the Glu/Leu/Phe/Val dehydrogenase mutant used in step b) and the enzyme for coenzyme cycling are co-expressed by a second recombinant microorganism. Thus, step b) may comprise: subjecting the 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid obtained in step a) to reductive amination in the presence of a second recombinant microorganism co-expressing a Glu/Leu/Phe/Val dehydrogenase mutant and an enzyme for coenzyme circulation (e.g.formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase) and an inorganic ammonium salt to obtain L-glufosinate. The use of the second recombinant microorganism can confer a higher catalytic efficiency on the process of the application. The second recombinant microorganism may be constructed using any method known in the art. For example, the second recombinant microorganism can be constructed as follows: constructing a recombinant expression vector containing the Glu/Leu/Phe/Val dehydrogenase mutant and genes of enzymes for coenzyme circulation, converting the recombinant expression vector into microorganisms, performing induction culture on the obtained recombinant microorganisms, and separating culture solution to obtain second recombinant microorganisms containing the Glu/Leu/Phe/Val dehydrogenase mutant and genes of enzymes for coenzyme circulation. Preferably, the addition amount of the second recombinant microorganism is 1g/L to 200g/L of the reaction solution according to the thallus wet weight after centrifugation at 10000rpm for 10 min; more preferably, 3g/L to 100g/L of the reaction solution; most preferably 30g/L of reaction solution.

The first and second recombinant microorganisms may be any engineered bacteria suitable for enzyme expression. In some embodiments, the first and second recombinant microorganisms each independently belong to one of the following genera: saccharomyces (Saccharomyces), aspergillus (Pichia), kluyveromyces (Kluyveromyces), candida (Candida), hansenula (Hansenula), humicola (Humicola), issatchenkia (ISSATCHENKIA), massa (Trichosporon), brettanomyces (Brettanomyces), pachysolen (Pachysolen), yarrowia (Yarrowia), actinomyces (Actinomyces), streptomyces (Streptomyces), bacillus (Bacillus) or Escherichia (Escherichia). In some preferred embodiments, the first and second recombinant microorganisms are each independently selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolitica), candida krusei (Candida krusei), issatchenkia orientalis (ISSATCHENKIA ORIENTALIS), bacillus subtilis (Bacillus subtilis), or Escherichia coli (ESCHERICHIA COLI). In some more preferred embodiments, the first and second recombinant microorganisms are both E.coli.

The yield of the process of the application may be measured by any method known in the art. For example, the two conformational contents of the resulting glufosinate-ammonium product can be measured by chiral HPLC. In some embodiments, the resulting L-glufosinate product has an enantiomeric excess (e.e.) of at least 99% (relative to D-glufosinate, supra). In some embodiments, the yield of the L-glufosinate product obtained is at least 95%, 96% or 97%.

The capital english letters of the present application represent amino acids as known to those skilled in the art, and according to the present application, the corresponding amino acid residues are represented herein.

The experimental methods in the invention are all conventional methods unless otherwise specified, and the gene cloning operation can be specifically found in the "molecular cloning Experimental guidelines" by J.Sam Broker et al.

Description of the sequence Listing:

SEQ ID NO.1 is an amino acid sequence annotated as D-amino acid oxidase (DAAO) derived from Microbotryumintermedium.

SEQ ID NO.2 is an amino acid sequence annotated as Formate Dehydrogenase (FDH) derived from Lactobacillus buchneri.

SEQ ID NO.3 is an amino acid sequence annotated as Glucose Dehydrogenase (GDH) derived from Exiguobacterium sibiricum.

SEQ ID NO.4 is an amino acid sequence derived from Lactobacillus brevis annotated as Alcohol Dehydrogenase (ADH).

SEQ ID NO.5 is an amino acid sequence derived from Delftia acidovorans annotated as Glu/Leu/Phe/Val dehydrogenase.

SEQ ID NO.6 is a nucleotide sequence annotated as D-amino acid oxidase (DAAO) derived from Microbotryumintermedium.

SEQ ID NO.7 is a nucleotide sequence annotated as Formate Dehydrogenase (FDH) derived from Lactobacillus buchneri.

SEQ ID NO.8 is a nucleotide sequence annotated as Glucose Dehydrogenase (GDH) derived from Exiguobacterium sibiricum.

SEQ ID NO.9 is a nucleotide sequence derived from Lactobacillus brevis annotated as Alcohol Dehydrogenase (ADH).

SEQ ID NO.10 is a nucleotide sequence derived from Delftia acidovorans A. Glu/Leu/Phe/Val dehydrogenase.

The Glu/Leu/Phe/Val dehydrogenase mutant provided by the application has better catalytic efficiency, and when the PPO is used as a substrate for catalytic reaction, the conversion rate is far higher than that of a wild type, and the yield of L-glufosinate is also greatly improved.

Drawings

FIG. 1 schematically shows the reaction scheme for the production of L-glufosinate by the multi-enzyme system resolution method employed in the process of the present application.

FIG. 2 schematically shows a reaction scheme (glucose dehydrogenase coenzyme cycle) for the racemization of L-glufosinate.

FIG. 3 schematically shows a reaction scheme (formate dehydrogenase coenzyme cycle) for the racemization of L-glufosinate.

FIG. 4 shows an exemplary reaction sequence for preparing L-glufosinate by racemization with a two-bacteria multi-enzyme one-pot two-step method.

Detailed Description

Examples

Materials and methods

Reagents for upstream genetic engineering: the genome extraction kit, plasmid extraction kit, DNA purification recovery kit used in the examples were purchased from corning life sciences (Wu Jiang) inc; one-step cloning kit was purchased from nuuzan limited; e.coli BL21 (DE 3), plasmid pET-28a (+) and the like were purchased from Shanghai Xueguan Biotech development Co., ltd; DNA markers, low molecular weight standard proteins, protein pre-gels were purchased from Beijing GenStar Co., ltd; clonExpress IIOne Step Cloning Kit seamless cloning kit was purchased from Nanjinouzan Biotech Co., ltd; pfu DNA polymerase and Dpn I endonuclease were purchased from Semer Feishmania technology (China); primer synthesis and sequence sequencing are completed by catalpa in Hangzhou, and total gene synthesis is completed by Shanghai, inc. The above methods of reagent use are referred to in the commercial specifications.

The reagent 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid (PPO) used in the downstream catalytic process is available from Yu Yongnong biosciences inc; d, L-glufosinate was from Yu Yongnong biosciences Inc.; other commonly used reagents are purchased from national pharmaceutical group chemical reagent limited.

The progress of the reaction was detected by High Performance Liquid Chromatography (HPLC) in the examples and the PPO was analyzed. The HPLC analysis method comprises the following steps: a chromatographic column PBR; column temperature/30 ℃; flow rate/1 mL/min; detection wavelength/210 nm; mobile phase: 5mM (NH ₄)₂HPO₄).

The content of two configurations of glufosinate-ammonium is detected by chiral HPLC analysis method, which is: chromatographic column/OA-5000L; mobile phase/0.5 g/L copper ammonium sulfate pentahydrate solution, and 0.3% v/v acetonitrile; detection wavelength/254 nm; flow rate/1 mL/min; column temperature/35 ℃.

Example 1: construction of genetically engineered bacteria

The gene sequence of D-amino acid oxidase (DAAO, genBank: FMSP01000004.1, amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO. 6) derived from Microbotryumintermedium is subjected to total gene synthesis, and then inserted into an expression plasmid pET-28a (+) to obtain pET-28a-DAAO. After the sequencing verification, pET-28a-daao is transferred into an expression host E.coli BL21 (DE 3) for the expression of subsequent recombinant enzyme.

The sequence derived from Lactobacillus buchneri of Formate Dehydrogenation (FDH) (the amino acid sequence is shown as SEQ ID NO.2, the nucleotide sequence is shown as SEQ ID NO. 7) is subjected to total gene synthesis, and then inserted into an expression plasmid pET-28a (+) to obtain pET-28a-FDH. After the sequencing verification, pET-28a-fdh is transferred into an expression host E.coli BL21 (DE 3) for the expression of subsequent recombinant enzymes.

The sequence derived from Exiguobacterium sibiricum Glucose Dehydrogenase (GDH) (with the amino acid sequence shown as SEQ ID NO.3 and the nucleotide sequence shown as SEQ ID NO. 8) is subjected to total gene synthesis, and then inserted into an expression plasmid pET-28a (+), thus obtaining pET-28a-GDH. After the sequencing verification, pET-28a-gdh is transferred into an expression host E.coli BL21 (DE 3) for the expression of subsequent recombinant enzyme.

The sequence derived from Lactobacillus brevis Alcohol Dehydrogenase (ADH) (the amino acid sequence of which is shown as SEQ ID NO.4, and the nucleotide sequence of which is shown as SEQ ID NO. 9) is subjected to total gene synthesis, and then inserted into an expression plasmid pET-28a (+) to obtain pET-28a-ADH. After the sequencing verification, pET-28a-adh is transferred into an expression host E.coli BL21 (DE 3) for the expression of the subsequent recombinase.

The gene sequence derived from Delftia acidovorans Glu/Leu/Phe/Val dehydrogenase (GenBank: WP-012202150.1, amino acid sequence shown as SEQ ID No.5, nucleotide sequence shown as SEQ ID No. 10) was subjected to total gene synthesis and inserted into expression plasmid pET-28a (+) to obtain plasmid pET-28a-laadh. After the sequencing verification, the recombinant DNA is transferred into an expression host E.coli BL21 (DE 3) for the expression of the subsequent recombinant enzyme.

Example 2: culturing of engineering bacteria

After recombinant E.coli E.coli BL21(DE3)/pET-28a-DAAO、E.coli BL21(DE3)/pET-28a-LAADH、E.coli BL21(DE3)/pET-28a-FDH、E.coli BL21(DE3)/pET-28a-GDH and E.coli BL21 (DE 3)/pET-28 a-ADH were streaked and activated on a plate, single colonies were picked and inoculated into 10mL LB liquid medium containing 50. Mu.g/mL kanamycin, and shake-cultured at 37℃for 10h. Transfer to 50mL of LB liquid medium containing 50. Mu.g/mL kanamycin at 2% inoculum size, shake culture at 37℃until OD600 reaches about 0.8, adding IPTG at a final concentration of 0.1mM, shake culture at 25℃for 12h. After the culture is finished, centrifuging the culture solution at 8000rpm for 10min, discarding the supernatant, collecting thalli, and storing in an ultralow temperature refrigerator at-80 ℃ for later use.

Example 3: construction of D-amino acid oxidase (DAAO) mutant (at position 62, 226)

The 62 nd and/or 226 th position (in particular F62K, M226T) was mutated on the basis of the wild-type DAAO sequence described in example 1. The primer sequences of PCR were designed for the mutants mutated at positions 62 and 226 of the mutated D-amino acid oxidase sequence, as shown in Table 1:

TABLE 1

Sequence number	Primer name	Primer sequences
			1	F62KF	gattcttgcgggtccaccttggggcaccagttcgctc
2	F62KR	gagcgaactggtgccccaaggtggacccgcaagaatc
			3	M226TF	ggggtctgacgcatcggtagtgcacagcttgac
4	M226TR	gtcaagctgtgcactaccgatgcgtcagacccc

The PCR (25. Mu.L) amplification system was as follows:

Pfu buffer 12.5. Mu.L, primer 2. Mu.L, template plasmid 1. Mu.L, dNTP 0.5. Mu.L, pfu 1. Mu.L, ddH ₂ O was added to make up to 25. Mu.L.

PCR amplification conditions:

(1) pre-denaturation at 95℃for 3min, (2) denaturation at 95℃for 30 sec, (3) annealing at 65℃for 30 sec, (4) elongation at 72℃for 5min,20 cycles, (5) elongation at 72℃for 10min, and (6) preservation at4 ℃.

After PCR, 5. Mu.L of the amplified product was subjected to nucleic acid gel electrophoresis analysis to obtain a clear target band, and the remaining product was added with 0.5. Mu.L of Dpn I endonuclease and digested at 37℃for 3 hours.

The reaction was completed and transformed into BL21 competent cells, which were plated on LB solid medium containing 50. Mu.g/mL kanamycin, and incubated overnight at 37 ℃. Single colonies were picked and mutant transformants were obtained. Cells were obtained as described in example 2.

Example 4: construction of Glu/Leu/Phe/Val dehydrogenase mutant (position 91, 168)

Positions 91 and 168 (specifically V91I, N168G) were mutated on the basis of the wild-type LAADH sequence described in example 1. Primer sequences were designed for mutant PCR in which mutations were made at positions 91 and 168 of the LAADH sequence, as shown in Table 2:

TABLE 2

Sequence number	Primer name	Primer sequences
			1	V91IF	cctggtggaaacggatgccgcccttgccg
2	V91IR	cggcaagggcggcatccgtttccaccagg
			3	N168GF	gggtccgaagaatcggtcgtgcagcgcttgc
4	N168GR	gcaagcgctgcacgaccgattcttcggaccc

The PCR amplification system and conditions were the same as those described in example 3.

The reaction was completed and transformed into BL21 competent cells, which were plated on LB solid medium containing 50. Mu.g/mL kanamycin, and incubated overnight at 37 ℃. Single colonies were picked and mutant transformants were obtained. Cells were obtained as described in example 2.

Example 5: comparison of Glu/Leu/Phe/Val dehydrogenase mutant enzyme Activity

The catalytic efficiency of L-amino acid dehydrogenase was compared with that of its mutant by measuring the consumption of PPO. When only L-amino acid dehydrogenase and mutant are used, the reaction system is as follows: 250mM PPO,100mM pH8.0 phosphate buffer, 300mM glucose, 10g/L L-amino acid dehydrogenase or mutant lyophilized cells and 10g/L glucose dehydrogenase lyophilized cells. After 24 hours of reaction, a sample of the reaction mixture was taken and treated, and the concentration of L-PPT was measured by HPLC and the conversion (concentration of product L-PPT/concentration of initial substrate PPO. Times.100%) was calculated.

TABLE 3 Table 3

As can be seen from Table 3, the resulting mutants all had higher conversion than the wild type LAADH. Wherein, the highest conversion rate is LAADH mutant 4, the V at the 91 st position of the mutation site is mutated into I, and the N at the 168 th position is mutated into G.

Example 6: construction of LAADH expression Strain

1. Construction of expression Strain containing glucose dehydrogenase coenzyme circulation System

On a vector pET-28a-LAADH V91I-N168G, a glucose dehydrogenase gene fragment is connected to a multiple cloning site through a seamless cloning kit, the enzyme cutting site is HindIII, a plasmid pET-28a-LAADH V91I-N168G-GDH is constructed, and an expression strain E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-GDH is constructed.

2. Construction of expression Strain containing formate dehydrogenase coenzyme circulatory System

On a vector pET-28a-LAADH V91I-N168G, a formate dehydrogenase gene fragment is connected to a multiple cloning site through a seamless cloning kit, the enzyme cutting site is HindIII, a plasmid pET-28a-LAADH V91I-N168G-FDH is constructed, and an expression strain E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-FDH is constructed.

3. Construction of an expression Strain of the alcohol dehydrogenase coenzyme circulation System

On a vector pET-28a-LAADH V91I-N168G, connecting an alcohol dehydrogenase gene fragment to a multiple cloning site through a seamless cloning kit, and constructing and obtaining a plasmid pET-28a-LAADH V91I-N168G-ADH by using a Hind III enzyme cutting site, and constructing and obtaining an expression strain E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-ADH.

Example 7: preparation of L-glufosinate (glucose dehydrogenase-containing GDH coenzyme circulation system) by deracemizing with double-bacteria multienzyme

The strain E.coli BL21 (DE 3)/pET-28 a-DAAO F62K-M226T capable of expressing D-amino acid oxidase and the strain E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-GDH capable of expressing L-amino acid dehydrogenase and glucose dehydrogenase were cultured as in example 2, and the somatic cells were collected by centrifugation and lyophilized.

600ML of ammonium phosphate buffer (pH 8.0, 100 mM) containing 400mM D, L-PPT,8000U/L catalase, 5% (v/v) defoamer, 20G/L E.coli BL21 (DE 3)/pET-28 a-DAAO F62K-M226T cells, 20G/L E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-GDH cells, 0.5mM NADH and 250mM glucose was added, air was introduced, aeration was 2L/min, and ammonia was added to control pH8, temperature 30℃and reaction was carried out for 24 hours. After the reaction is finished, the L-PPT is 388mM by liquid phase detection, the e.e. value of the product L-glufosinate is more than 99%, and the conversion yield of the L-PPT is 97%.

Example 8: preparation of L-glufosinate (formate dehydrogenase-containing FDH coenzyme circulation system) by deracemizing with double bacteria multienzyme

The strain E.coli BL21 (DE 3)/pET-28 a-DAAO F62K-M226T capable of expressing D-amino acid oxidase and the strain E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-FDH capable of expressing L-amino acid dehydrogenase and formate dehydrogenase were cultured as in example 2, and the somatic cells were collected by centrifugation and lyophilized.

600ML of ammonium phosphate buffer (pH 8.0, 100 mM) containing 400mM D, L-PPT,8000U/L catalase, 5% (v/v) defoamer, 20G/L E.coli BL21 (DE 3)/pET-28 a-DAAO F62K-M226T freeze-dried cells, 20G/L E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-FDH freeze-dried cells, 0.5mM NADH and 250mM ammonium formate were added, air was introduced, aeration was 2L/min, pH was controlled at 8 with ammonia water at 30℃and the reaction was carried out for 24 hours. After the reaction is finished, the L-PPT is 382mM by liquid phase detection, the e.e. value of the product L-glufosinate is more than 99%, and the conversion yield of the L-PPT is 95.5%.

Example 9: preparation of L-glufosinate (alcohol dehydrogenase ADH coenzyme circulation system) by despination of double-bacterial multienzyme

The strain E.coli BL21 (DE 3)/pET-28 a-DAAO F62K-M226T capable of expressing D-amino acid oxidase and the expression strain E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-ADH capable of expressing L-amino acid dehydrogenase and alcohol dehydrogenase were cultured as in example 2, and the somatic cells were collected by centrifugation and lyophilized.

600ML of the reaction solution (ammonia water was adjusted to pH 8.0), containing 400mM of D, L-PPT,8000U/L catalase, 0.5% (v/v) of an antifoaming agent, 20G/L of E.coli BL21 (DE 3)/pET-28 a-DAAO F62K-M226T freeze-dried cells, 20G/L of E.coli BL21 (DE 3)/pET-28 a-LAADH V91I-N168G-ADH freeze-dried cells, 0.5mM of NADH and 250mM of isopropyl alcohol, air was introduced, aeration was 2L/min, ammonia water was used to control pH8, temperature was 30℃and reaction was carried out for 24 hours. The liquid phase detection method shown in the example was used to detect the consumption of D-glufosinate and the formation of L-glufosinate during the reaction, and the reaction progress curve is shown in FIG. 4. The graph shows that the concentration of D-glufosinate gradually decreases and the concentration of L-glufosinate gradually increases over time. After the reaction is finished, the L-PPT is 380mM by liquid phase detection, the e.e. value of the product L-glufosinate is more than 99%, and the conversion yield of the L-PPT is 95%.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Sequence listing

<110> Yongnong biosciences Co., ltd

University of Industry of China

Ningxia Yongnong bioscience Co.Ltd

<120> Glu/Leu/Phe/Val dehydrogenase mutant and application thereof in preparation of L-glufosinate

<130> PD210084N

<160> 10

<170> Patent in version 3.5

<210> 1

<211> 384

<212> PRT

<213> Microbotryum intermedium

<400> 1

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

1 5 10 15

Gly Val Ile Gly Leu Thr Ser Ala Leu Val Leu Ala Gln Ser Asn His

20 25 30

Asn Val Thr Leu Val Ala Arg Asp Leu Pro Ser Asp Val Ser Ser Gln

35 40 45

Ala Phe Ala Ser Pro Trp Ala Gly Ala Asn Trp Cys Pro Phe Val Asp

50 55 60

Pro Gln Glu Ser Val Lys Asn Lys Arg Ile Cys Asp Trp Glu Thr Gln

65 70 75 80

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

85 90 95

Leu Val Met Arg Leu Pro Ala Arg Arg Tyr Ala Glu Asn Glu Lys Ala

100 105 110

Leu Leu Gly His Trp Tyr Lys Ser Val Val Pro Arg Tyr Ser Thr Leu

115 120 125

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

130 135 140

Ile Ser Val Asn Ala Pro Leu Tyr Cys Gln Trp Leu Glu Ala Gln Leu

145 150 155 160

Leu Ser His Asn Ala Thr Ile Ile Arg Arg Ser Leu Asn Ser Leu Asp

165 170 175

Glu Ala Leu Ser Leu Ala Pro Ser Cys Ser Val Ile Val Asn Ala Thr

180 185 190

Gly Leu Gly Ala Lys Ser Leu Gly Gly Val Glu Asp Gln Thr Val Thr

195 200 205

Pro Ile Arg Gly Gln Thr Val Leu Ile Lys Thr Asp Val Lys Leu Cys

210 215 220

Thr Met Asp Ala Ser Asp Pro Thr Lys Pro Ser Tyr Ile Ile Pro Arg

225 230 235 240

Pro Gly Gly Glu Ala Val Cys Gly Gly Cys Tyr Gly Leu Gly Glu Trp

245 250 255

Asn Leu Ser Thr Asp Thr Glu Leu Ala Lys Leu Ile Leu Glu Arg Cys

260 265 270

Leu Val Leu Asp Pro Arg Ile Ser Ser Asn Gly Ala Leu Asp Gly Ile

275 280 285

Glu Val Leu Arg His Asn Val Gly Leu Arg Pro Ser Arg Gly Thr Asn

290 295 300

Glu Pro Arg Leu Glu Ala Glu Arg Val Val Leu Pro Ser Tyr Ser Leu

305 310 315 320

Asn Pro His Arg Arg His Ala Leu Gly Ala Glu Gly Asn Ala Ala Thr

325 330 335

Val Ile His Ala Tyr Gly Val Gly Pro Ala Gly Tyr Gln Val Ser Trp

340 345 350

Gly Val Ala Asn Glu Val Lys Ala Leu Val Asp Glu His Phe Ala Lys

355 360 365

Phe Asp Thr Arg Thr Thr Gln Asp Gly Val His Arg Asp Ile Lys Leu

370 375 380

<210> 2

<211> 398

<212> PRT

<213> Lactobacillus buchneri

<400> 2

Met Thr Leu Val Leu Ala Val Leu Thr Pro Ala Pro Val Ala Gly Pro

1 5 10 15

Pro Pro Leu Thr Val Ala Ala Ala Ile Pro Leu Ile Thr His Thr Pro

20 25 30

Ala Gly Ser Thr Val Pro Thr Pro Gly Gly Ile Ala Pro Leu Pro Gly

35 40 45

Gly Leu Leu Gly Ser Val Ser Gly Gly Leu Gly Leu Leu Leu Thr Leu

50 55 60

Gly Ser Leu Gly Val Gly Pro Val Val Thr Ser Ala Leu Gly Gly Pro

65 70 75 80

Ala Ser Val Pro Gly Leu Gly Leu Pro Thr Ala Ala Val Val Ile Ser

85 90 95

Gly Pro Pro Thr Pro Ala Thr Leu Thr Ala Ala Leu Ile Ala Leu Ala

100 105 110

Leu Leu Leu Leu Leu Ala Ile Thr Ala Gly Ile Gly Ser Ala His Val

115 120 125

Ala Leu Ala Ala Ala Ala Gly His Ala Ile Thr Val Ala Gly Val Thr

130 135 140

Thr Ser Ala Ser Val Ser Val Ala Gly Ala Gly Val Met Gly Leu Leu

145 150 155 160

Ala Leu Val Ala Ala Pro Ile Pro Ala His Ala Ile Val Leu Ala Gly

165 170 175

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

180 185 190

Met Thr Val Gly Val Ile Gly Ala Gly Ala Ile Gly Ala Ala Val Leu

195 200 205

Gly Ala Leu Leu Pro Pro Gly Val Leu Leu Val Thr Ala Gly Ala His

210 215 220

Gly Leu Pro Ala Gly Val Gly Ala Gly Leu Gly Leu Thr Thr Pro Pro

225 230 235 240

Ala Val His Gly Met Val Leu Val Val Ala Ala Val Val Leu Ala Ala

245 250 255

Pro Leu His Ala Gly Thr Thr His Leu Pro Ala Ala Gly Val Leu Ala

260 265 270

Thr Met Leu Ala Gly Ala Thr Ile Val Ala Ala Ser Ala Gly Gly Gly

275 280 285

Val Ala Ala Ala Ala Ile Val Ala Ala Leu Ala Ser Gly Gly Ile Gly

290 295 300

Gly Thr Ser Gly Ala Val Thr Thr Pro Gly Pro Ala Pro Leu Ala His

305 310 315 320

Pro Thr Ala Thr Met Pro Ala Gly Ala Met Thr Pro His Met Ser Gly

325 330 335

Thr Thr Leu Ser Ala Gly Ala Ala Thr Ala Ala Gly Ala Ala Gly Ile

340 345 350

Leu Gly Ala Pro Leu Gly Ala Leu Pro Ile Ala Pro Gly Thr Leu Ile

355 360 365

Ala Gly Gly Gly Ser Leu Ala Gly Thr Gly Ala Leu Ser Thr Thr Val

370 375 380

Leu Leu Gly Gly Gly Thr Pro Gly Ser Gly Gly Ala Gly Leu

385 390 395

<210> 3

<211> 262

<212> PRT

<213> Exiguobacterium sibiricum

<400> 3

Met Gly Thr Ala Ser Leu Leu Gly Leu Val Ala Ile Val Thr Gly Gly

1 5 10 15

Ser Met Gly Ile Gly Gly Ala Ile Ile Ala Ala Thr Ala Gly Gly Gly

20 25 30

Met Ala Val Val Ile Ala Thr Ala Ser His Pro Gly Gly Ala Leu Leu

35 40 45

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

50 55 60

Gly Ala Val Ser Leu Gly Gly Ala Met Ile Ala Leu Val Leu Gly Thr

65 70 75 80

Val Ala His Pro Gly Gly Leu Ala Val Pro Val Ala Ala Ala Gly Val

85 90 95

Gly Met Pro Ser Pro Ser His Gly Met Ser Leu Gly Ala Thr Gly Leu

100 105 110

Val Ile Ala Val Ala Leu Thr Gly Ala Pro Leu Gly Ala Ala Gly Ala

115 120 125

Leu Leu Thr Pro Val Gly His Ala Val Leu Gly Ala Ile Ile Ala Met

130 135 140

Ser Ser Val His Gly Ile Ile Pro Thr Pro Thr Pro Val His Thr Ala

145 150 155 160

Ala Ser Leu Gly Gly Val Leu Leu Met Thr Gly Thr Leu Ala Met Gly

165 170 175

Thr Ala Pro Leu Gly Ile Ala Ile Ala Ala Ile Gly Pro Gly Ala Ile

180 185 190

Ala Thr Pro Ile Ala Ala Gly Leu Pro Gly Ala Pro Leu Gly Ala Ala

195 200 205

Ala Val Gly Ser Met Ile Pro Met Gly Ala Ile Gly Leu Pro Gly Gly

210 215 220

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

225 230 235 240

Thr Gly Ile Thr Leu Pro Ala Ala Gly Gly Met Thr Leu Thr Pro Ser

245 250 255

Pro Gly Ala Gly Ala Gly

260

<210> 4

<211> 252

<212> PRT

<213> Lactobacillus brevis

<400> 4

Met Ser Ala Ala Leu Ala Gly Leu Val Ala Ile Ile Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Ile Ala Thr Leu Pro Val Gly Gly Gly Ala

20 25 30

Leu Val Met Ile Thr Gly Ala His Ser Ala Val Gly Gly Leu Ala Ala

35 40 45

Leu Ser Val Gly Thr Pro Ala Gly Ile Gly Pro Pro Gly His Ala Ser

50 55 60

Ser Ala Gly Ala Gly Thr Thr Leu Leu Pro Ala Ala Thr Gly Leu Ala

65 70 75 80

Pro Gly Pro Val Ser Thr Leu Val Ala Ala Ala Gly Ile Ala Val Ala

85 90 95

Leu Ser Val Gly Gly Thr Thr Thr Ala Gly Thr Ala Leu Leu Leu Ala

100 105 110

Val Ala Leu Ala Gly Val Pro Pro Gly Thr Ala Leu Gly Ile Gly Ala

115 120 125

Met Leu Ala Leu Gly Leu Gly Ala Ser Ile Ile Ala Met Ser Ser Ile

130 135 140

Gly Gly Pro Val Gly Ala Pro Ser Leu Gly Ala Thr Ala Ala Ser Leu

145 150 155 160

Gly Ala Val Ala Ile Met Ser Leu Ser Ala Ala Leu Ala Cys Ala Leu

165 170 175

Leu Ala Thr Ala Val Ala Val Ala Thr Val His Pro Gly Thr Ile Leu

180 185 190

Thr Pro Leu Val Ala Ala Leu Pro Gly Ala Gly Gly Ala Met Ser Gly

195 200 205

Ala Thr Leu Thr Pro Met Gly His Ile Gly Gly Pro Ala Ala Ile Ala

210 215 220

Thr Ile Cys Val Thr Leu Ala Ser Ala Gly Ser Leu Pro Ala Thr Gly

225 230 235 240

Ser Gly Pro Val Val Ala Gly Gly Thr Thr Ala Gly

245 250

<210> 5

<211> 434

<212> PRT

<213> Delftia acidovorans

<400> 5

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

1 5 10 15

Tyr Leu Gln Ala Asp His Leu Gly Pro Trp Gly Asn Tyr Leu Gln Gln

20 25 30

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

35 40 45

Thr Leu Lys Arg Pro Lys Arg Ile Leu Ile Val Asp Val Pro Ile Glu

50 55 60

Leu Asp Asn Gly Thr Ile Ala His Tyr Glu Gly Tyr Arg Val Gln His

65 70 75 80

Asn Leu Ser Arg Gly Pro Gly Lys Gly Gly Val Arg Phe His Gln Asp

85 90 95

Val Thr Leu Ser Glu Val Met Ala Leu Ser Ala Trp Met Ser Val Lys

100 105 110

Asn Ala Ala Val Asn Val Pro Tyr Gly Gly Ala Lys Gly Gly Ile Arg

115 120 125

Val Asp Pro Lys Thr Leu Ser Arg Gly Glu Leu Glu Arg Leu Thr Arg

130 135 140

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

145 150 155 160

Pro Ala Pro Asp Val Asn Thr Asn Gly Gln Ile Met Ala Trp Met Met

165 170 175

Asp Thr Tyr Ser Met Asn Thr Gly Ala Thr Ala Thr Gly Val Val Thr

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Gly Thr Ala Gly Lys Leu Phe Ala Asp Val Gly Ala Lys Val Val

245 250 255

Ala Val Gln Asp His Thr Gly Thr Ile His Asn Ala Asn Gly Leu Asp

260 265 270

Val Pro Ala Leu Leu Ala His Val Ala Ala Lys Gly Gly Val Gly Gly

275 280 285

Phe Asp Gly Ala Glu Ala Met Asp Ala Ala Asp Phe Trp Ser Val Asp

290 295 300

Cys Asp Ile Leu Ile Pro Ala Ala Leu Glu Gly Gln Ile Thr Lys Glu

305 310 315 320

Asn Ala Gly Lys Ile Lys Ala Lys Met Val Ile Glu Gly Ala Asn Gly

325 330 335

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

340 345 350

Val Leu Pro Asp Val Leu Ala Asn Ala Gly Gly Val Thr Val Ser Tyr

355 360 365

Phe Glu Trp Val Gln Asp Phe Ser Ser Phe Phe Trp Ser Glu Asp Glu

370 375 380

Ile Asn Ala Arg Leu Val Arg Ile Met Gln Asp Ala Phe Ala Ala Ile

385 390 395 400

Trp Gln Val Ala Gln Gln His Gly Val Thr Leu Arg Thr Ala Thr Phe

405 410 415

Ile Val Ala Cys Gln Arg Ile Leu His Ala Arg Glu Met Arg Gly Leu

420 425 430

Tyr Pro

<210> 6

<211> 1155

<212> DNA

<213> Microbotryum intermedium

<400> 6

atgtcgtcaa gcacttcatc cgacaagcaa gtcgtcgtca ttggtgctgg tgttattggc 60

ctcacgtcgg cgctcgttct cgcgcagtcg aaccacaacg tcaccctcgt cgctcgggat 120

ctcccctcgg atgtatcgtc ccaagcgttt gcctcacctt gggccggagc gaactggtgc 180

ccctttgtgg acccgcaaga atcggtcaag aacaagagga tctgcgactg ggagacgcag 240

tcgttcgcaa acttccagca actcataaga gaacacggcg atggcaaact cgtcatgagg 300

cttccggcga ggagatacgc cgagaacgaa aaagccctcc tggggcattg gtacaaatca 360

gtcgtgccta gatactcgac cttgccctcg tccgaggtcc ccaacaacgg cgtcggcgtc 420

gaattcgaga ccatctcggt taacgcgccg ctctactgcc aatggctcga ggctcaactc 480

ttgtctcaca acgccaccat catccgccgc tcgctcaact ccctcgacga ggccttgtcg 540

ctcgcacctt cttgctcggt catcgtcaac gccaccgggc tcggcgccaa atcactcgga 600

ggagtcgagg atcagacggt cacccccatc cgagggcaga ccgtcttgat caagaccgac 660

gtcaagctgt gcactatgga tgcgtcagac cccaccaaac cgtcctatat cattccgagg 720

ccagggggcg aggccgtttg tggtggttgc tacggcctcg gggaatggaa tctctccacc 780

gatacggaac tggccaagct gattctcgaa cgatgcctgg tgctcgaccc ccgcatctca 840

tccaatggtg cgcttgacgg catcgaagtg cttcgacaca atgtcgggct gcggccatca 900

cgaggcacga atgaacccag gctagaggcc gaacgagtcg tccttccttc ctattctttg 960

aaccctcatc gaaggcatgc gctcggtgca gagggcaacg ccgcgacggt cattcacgcc 1020

tacggggtcg ggccggcagg atatcaagtc agctgggggg tcgcgaacga ggtgaaagcg 1080

ctagtcgacg aacacttcgc caagtttgac actcgaacga cccaagacgg cgtccaccgg 1140

gacattaaac tctag 1155

<210> 7

<211> 1197

<212> DNA

<213> Lactobacillus buchneri

<400> 7

atgaccaaag ttctggccgt gctgtatccg gatccggtgg atggttttcc gccgaaatat 60

gttcgtgatg atattccgaa aatcacccat tatccggatg gcagtaccgt tccgaccccg 120

gaaggcattg attttaaacc gggtgaactg ctgggtagcg ttagtggcgg tctgggcctg 180

aaaaaatatc tggaaagtaa aggtgtggaa tttgttgtta ccagtgataa agaaggcccg 240

gatagtgtgt ttgaaaaaga actgccgacc gccgatgtgg ttattagtca gccgttttgg 300

ccggcctatc tgaccgcaga tctgattgat aaagcaaaaa agctgaaact ggcaattacc 360

gccggtattg gcagcgatca tgtggatctg aatgccgcca atgaacataa tattaccgtt 420

gcagaagtga cctatagcaa tagtgttagt gttgcagaag cagaagtgat gcagctgctg 480

gccctggtgc gtaattttat tccggcacat gatattgtga aagccggtgg ctggaatatt 540

gcagatgcag ttagccgtgc ctatgatctg gaaggtatga ccgttggtgt gattggtgca 600

ggccgcattg gtcgtgccgt tctggaacgt ctgaaaccgt ttggcgttaa actggtgtat 660

aatcagcgcc atcagctgcc ggatgaagtt gaaaatgaac tgggcctgac ctattttccg 720

gatgttcatg aaatggtgaa agttgtggat gccgttgttc tggcagcacc gctgcatgca 780

cagacctatc atctgtttaa tgatgaagtt ctggccacca tgaaacgtgg cgcctatatt 840

gtgaataata gccgcggcga agaagttgat cgcgatgcaa ttgttcgcgc actgaatagc 900

ggtcagattg gcggttatag tggcgatgtt tggtatccgc agccggcacc gaaagatcat 960

ccgtggcgta ccatgccgaa tgaagcaatg accccgcata tgagtggcac caccctgagt 1020

gcccaggcac gctatgccgc aggtgcacgt gaaattctgg aagattttct ggaagataaa 1080

ccgattcgtc cggaatatct gattgcccag ggtggtagtc tggccggtac cggtgccaaa 1140

agttataccg tgaaaaaagg cgaagaaacc ccgggtagcg gcgaagcaga aaaataa 1197

<210> 8

<211> 789

<212> DNA

<213> Exiguobacterium sibiricum

<400> 8

atgggttata attctctgaa aggcaaagtc gcgattgtta ctggtggtag catgggcatt 60

ggcgaagcga tcatccgtcg ctatgcagaa gaaggcatgc gcgttgttat caactatcgt 120

agccatccgg aggaagccaa aaagatcgcc gaagatatta aacaggcagg tggtgaagcc 180

ctgaccgtcc agggtgacgt ttctaaagag gaagacatga tcaacctggt gaaacagact 240

gttgatcact tcggtcagct ggacgtcttt gtgaacaacg ctggcgttga gatgccttct 300

ccgtcccacg aaatgtccct ggaagactgg cagaaagtga tcgatgttaa tctgacgggt 360

gcgttcctgg gcgctcgtga agctctgaaa tacttcgttg aacataacgt gaaaggcaac 420

attatcaata tgtctagcgt ccacgaaatc atcccgtggc ctactttcgt acattacgct 480

gcttctaagg gtggcgttaa actgatgacc cagactctgg ctatggaata tgcaccgaaa 540

ggtatccgca ttaacgctat cggtccaggc gcgatcaaca ctccaattaa tgcagaaaaa 600

ttcgaggatc cgaaacagcg tgcagacgtg gaaagcatga tcccgatggg caacatcggc 660

aagccagagg agatttccgc tgtcgcggca tggctggctt ctgacgaagc gtcttacgtt 720

accggcatca ccctgttcgc agatggtggc atgaccctgt acccgagctt tcaggctggc 780

cgtggttga 789

<210> 9

<211> 759

<212> DNA

<213> Lactobacillus brevis

<400> 9

atgagcaacc gtctggacgg caaggtggcg atcattaccg gtggcaccct gggtattggt 60

ctggcgattg cgaccaagtt cgtggaggaa ggtgcgaaag ttatgatcac cggccgtcac 120

agcgacgtgg gcgagaaggc ggcgaaaagc gttggcaccc cggaccagat tcaattcttt 180

cagcacgata gcagcgacga ggatggttgg accaagctgt tcgatgcgac cgaaaaagcg 240

tttggcccgg ttagcaccct ggttaacaac gcgggtattg cggtgaacaa gagcgttgag 300

gaaaccacca ccgcggagtg gcgtaaactg ctggcggtga acctggatgg tgttttcttt 360

ggcacccgtc tgggtatcca acgtatgaag aacaaaggtc tgggcgcgag catcattaac 420

atgagcagca ttgaaggttt cgttggtgac ccgagcctgg gtgcgtacaa cgcgagcaag 480

ggtgcggttc gtatcatgag caaaagcgcg gcgctggatt gcgcgctgaa ggactacgat 540

gtgcgtgtta acaccgtgca cccgggctat attaaaaccc cgctggttga cgatctgccg 600

ggtgcggagg aagcgatgag ccagcgtacc aagaccccga tgggtcacat cggcgaaccg 660

aacgacatcg cgtacatttg cgtttatctg gcgagcaacg agagcaaatt cgcgaccggt 720

agcgaatttg tggttgatgg tggctatacc gcgcaataa 759

<210> 10

<211> 1305

<212> DNA

<213> Delftia acidovorans

<400> 10

atgcagcaac ccgcttcggc cggcgttacc aaccacgcca tcccttccta cctgcaggcc 60

gatcacctcg gcccctgggg caactacctg cagcaggtcg atcgcgtcac gccctacctg 120

ggccatctcg cccgctgggt cgaaaccctc aagcgcccca agcgcatcct gatcgtcgat 180

gtgccgatcg agctggacaa cggcaccatc gcccactacg aaggctaccg cgtgcagcac 240

aacctgagcc gcggtcccgg caagggcggc gtgcgtttcc accaggacgt gaccctgtcc 300

gaagtcatgg ccctgtcggc ctggatgtcg gtcaagaacg cggccgtcaa cgtgccctat 360

ggtggcgcca agggcggcat ccgtgtcgat cccaagacgc tgtcgcgcgg tgagctggag 420

cgcctgacgc gccgctacac cagcgagatc ggcctgctga tcggcccctc caaggacatc 480

cccgcgcctg acgtcaacac caatggccag atcatggcct ggatgatgga cacgtactcc 540

atgaacaccg gcgccaccgc caccggcgtg gtcacgggca agcccgtgga cctgggcggc 600

tcgctgggcc gcgtcgaggc caccggccgc ggcgtgttca ccgtgggcgt ggaagcggcc 660

aagctgaccg gcctgtcggt ccagggcgcg cgcatcgccg tgcagggctt cggcaacgtg 720

ggcggcacgg cgggcaagct gttcgccgac gtgggcgcca aggtcgtggc cgtgcaggac 780

cacaccggca ccatccacaa cgccaatggc ctggacgtgc cggccctgct ggcccacgtg 840

gctgccaagg gcggcgtggg cggctttgac ggcgccgagg ccatggacgc tgccgacttc 900

tggagcgtgg actgcgacat cctgatcccc gccgcactgg aaggccagat caccaaggaa 960

aacgccggca agatcaaggc caagatggtg atcgagggcg ccaacggccc caccaccacc 1020

gaggccgacg acatcctgac cgaaaagggc gtgctggtgc tgcccgatgt gctggccaat 1080

gccggcggcg tgacggtgag ctacttcgaa tgggtgcagg acttctccag cttcttctgg 1140

agcgaggacg agatcaacgc ccgcctggtg cgcatcatgc aggacgcctt cgcggccatc 1200

tggcaggtcg cccagcagca cggcgtgacg ctgcgcaccg ccaccttcat cgtggcctgc 1260

cagcgcatcc tgcatgcgcg cgagatgcgg ggactgtatc cctga 1305

Claims (29)

1. A Glu/Leu/Phe/Val dehydrogenase mutant is obtained by mutating the 91 st amino acid residue of Glu/Leu/Phe/Val dehydrogenase with an amino acid sequence shown as SEQ ID NO. 5 into I and/or mutating the 168 th amino acid residue into G.

2. A nucleic acid encoding the Glu/Leu/Phe/Val dehydrogenase mutant of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A recombinant host cell comprising the nucleic acid of claim 2 or the expression vector of claim 3.

5. The recombinant host cell of claim 4, wherein the host cell belongs to one of saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolitica), candida krusei (Candida krusei), isaccharomyces orientalis (ISSATCHENKIA ORIENTALIS), actinomycetes (Actinomycetes), streptomyces (Streptomyces), bacillus subtilis (Bacillus subtilis), or escherichia coli (ESCHERICHIA COLI).

6. Use of the Glu/Leu/Phe/Val dehydrogenase mutant of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, or the recombinant host cell of any one of claims 4-5 in the preparation of L-glufosinate.

7. A method of preparing L-glufosinate comprising converting 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid to L-glufosinate in the presence of an enzyme catalytic system comprising the Glu/Leu/Phe/Val dehydrogenase mutant of claim 1 for converting 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid to L-glufosinate.

8. The method of claim 7, wherein the enzyme catalytic system further comprises a D-amino acid oxidase that converts D-glufosinate to 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid.

9. The method of claim 7 or 8, wherein the enzyme catalytic system further comprises catalase.

10. The method of claim 7 or 8, wherein the enzyme catalytic system further comprises a coenzyme circulation system selected from at least one of the following:

(1) Formate dehydrogenase coenzyme circulatory system: including formate dehydrogenase, formate and coenzyme;

(2) Glucose dehydrogenase coenzyme circulatory system: including glucose dehydrogenase, glucose and coenzyme;

(3) Alcohol dehydrogenase coenzyme circulatory system: including alcohol dehydrogenase, isopropanol and coenzyme.

11. The method of claim 9, wherein the enzyme catalytic system further comprises a coenzyme circulatory system selected from at least one of:

(1) Formate dehydrogenase coenzyme circulatory system: including formate dehydrogenase, formate and coenzyme;

(2) Glucose dehydrogenase coenzyme circulatory system: including glucose dehydrogenase, glucose and coenzyme;

(3) Alcohol dehydrogenase coenzyme circulatory system: including alcohol dehydrogenase, isopropanol and coenzyme.

12. The method of claim 7 or 8, wherein each enzyme in the enzyme catalytic system is in a form independently selected from the group consisting of: free enzyme and recombinant host cell expressing the enzyme.

13. The method of claim 12, wherein the recombinant host cells expressing the enzyme are each independently selected from the group consisting of: saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolitica), candida krusei (Candida krusei), issatchenkia orientalis (ISSATCHENKIA ORIENTALIS), actinomyces, streptomyces, bacillus subtilis (Bacillus subtilis) or Escherichia coli (ESCHERICHIA COLI).

14. The method of claim 10, wherein each enzyme in the enzyme catalytic system is in a form independently selected from the group consisting of: free enzyme and recombinant host cell expressing the enzyme.

15. The method of claim 14, wherein the recombinant host cells expressing the enzyme are each independently selected from the group consisting of: saccharomyces cerevisiae (Saccharomyces cerevisiae), yarrowia lipolytica (Yarrowia lipolitica), candida krusei (Candida krusei), issatchenkia orientalis (ISSATCHENKIA ORIENTALIS), actinomyces, streptomyces, bacillus subtilis (Bacillus subtilis) or Escherichia coli (ESCHERICHIA COLI).

16. The method of claim 12, wherein the total amount of the recombinant host cells added is 1-200 g/L of the reaction solution based on the weight of the wet bacteria.

17. The method according to any one of claims 13 to 15, wherein the total added amount of the recombinant host cells is 1 to 200 g/L of the reaction solution based on the weight of the wet bacteria.

18. The method according to claim 7 or 8, wherein the conversion reaction is carried out in a reaction liquid having a pH of 7 to 10.

19. The method of claim 18, wherein the reaction solution is a reaction solution having a pH of 8-9.

20. The process according to claim 10, wherein the conversion reaction is carried out in a reaction liquid having a pH of 7-10.

21. The method of claim 20, wherein the reaction solution is a reaction solution having a pH of 8-9.

22. The method according to claim 7 or 8, wherein in the Glu/Leu/Phe/Val dehydrogenase mutant catalyzed reductive amination reaction, the molar ratio of inorganic ammonium donor to substrate at the start of the reaction is 1:1-10:1.

23. The method of claim 10, wherein in the Glu/Leu/Phe/Val dehydrogenase mutant-catalyzed reductive amination reaction, the molar ratio of inorganic ammonium donor to substrate at the start of the reaction is 1:1-10:1.

24. The method according to claim 7 or 8, wherein the Glu/Leu/Phe/Val dehydrogenase mutant catalyzes a reductive amination reaction at a reaction temperature of 25-45 ℃ for a period of 6-24h.

25. The method of claim 10, wherein the Glu/Leu/Phe/Val dehydrogenase mutant catalyzes a reductive amination reaction at a reaction temperature of 25-45 ℃ for a period of 6-24 hours.

26. The method of claim 7 or 8, wherein the Glu/Leu/Phe/Val dehydrogenase mutant-catalyzed reductive amination reaction is performed in the presence of coenzyme NADH.

27. The method of claim 26, wherein the molar ratio of NADH to substrate is 1:10-1:5000.

28. The method of claim 10, wherein the Glu/Leu/Phe/Val dehydrogenase mutant-catalyzed reductive amination reaction is performed in the presence of coenzyme NADH.

29. The method of claim 28, wherein the molar ratio of NADH to substrate is 1:10-1:5000.