CN114196642A - Glutamate dehydrogenase variants and their use in the preparation of L-amino acids - Google Patents

Glutamate dehydrogenase variants and their use in the preparation of L-amino acids Download PDF

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CN114196642A
CN114196642A CN202111324471.4A CN202111324471A CN114196642A CN 114196642 A CN114196642 A CN 114196642A CN 202111324471 A CN202111324471 A CN 202111324471A CN 114196642 A CN114196642 A CN 114196642A
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glutamate dehydrogenase
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amino acid
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杨立荣
王子渊
周海胜
张红玉
吴坚平
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ZJU Hangzhou Global Scientific and Technological Innovation Center
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Abstract

The invention discloses a glutamate dehydrogenase variant and application thereof in preparing L-amino acid, wherein the glutamate dehydrogenase variant is obtained by carrying out single-point mutation or multi-point combined mutation on the 71 th, 92 th, 143 th, 144 th, 145 th, 146 th, 175 th, 187 th, 352 th and 355 th positions of an amino acid sequence shown in SEQ ID NO. 1. The invention solves the problem that the mutant has no or low enzyme activity to non-natural substrates by a rationally designed molecular modification method on the basis of glutamic acid dehydrogenase derived from Clostridium difficile, obtains mutants which can catalyze and prepare various L-amino acids except natural products of L-glutamic acid, and has the raw material conversion rate and the product ee value of more than 99 percent.

Description

Glutamate dehydrogenase variants and their use in the preparation of L-amino acids
Technical Field
The invention relates to the technical field of biology, in particular to a glutamate dehydrogenase variant and application thereof in preparing L-amino acid.
Background
Amino acids (amino acids) are a generic name for a class of organic compounds containing both amino and carboxyl groups. Wherein, the alpha-amino acid is the basic composition unit of the biological functional macromolecular protein and forms the basic skeleton (primary structure) of the protein molecule. It has been found that 22 amino acids can constitute protein, including selenocysteine (Sec) found in 1986 and pyrrolysine (pyrrolysine, Pyl) reported in 2002, which are both alpha-amino acids and are commonly referred to as protein amino acids. Amino acids other than the 22 protein amino acids are called non-protein amino acids (non-proteinogenic amino acids).
The protein amino acid can be applied to industries closely related to human life, such as medicines, foods, feeds and the like, for example, the protein amino acid can be used as a nutriment to maintain nitrogen balance of a human body, can be used as a flavor additive of foods, can be used as a feed additive to be used in the breeding industry and the like. Non-protein amino acids, due to their structural and functional diversity, have a wider range of applications in the fields of chemical industry, medicine, agricultural chemicals, etc., such as phenylisoserine (β -amino acid, side chain moiety of taxol, an anticancer drug), L-norvaline (intermediate of perindopril, a hypotensive drug), L-homophenylalanine (intermediate of enalapril, a hypotensive drug), L-glufosinate (broad-spectrum herbicide), etc.
Protein amino acids are usually produced by fermentation or extraction from cheap raw materials, which is cost-effective, but enzymatic production also has its advantages, for example higher concentrations of the product can be obtained by enzymatic methods and the subsequent separation process is relatively simple. In terms of non-protein amino acids, unlike protein amino acids, they have not been industrially produced by fermentation methods, and are now mainly produced by organic synthesis methods. The organic synthesis method for preparing chiral non-protein amino acid has the disadvantages of harsh reaction conditions, low product yield and optical purity, environment-friendly process and the like, and has almost no related industrial reports. The bio-enzyme catalysis method has the characteristics of mild reaction conditions, high stereoselectivity, green and environment-friendly process and the like, and is a potential dominant method for preparing the chiral non-protein amino acid. For example, glutamate dehydrogenase catalyzes reductive amination of a keto acid to produce the corresponding amino acid in the presence of a coenzyme, and has high stereoselectivity. The enzyme method can avoid the defects of high pressure, high temperature, high energy consumption and high pollution of the traditional chemical method, and is an advanced manufacturing mode which accords with the green production concept.
Glutamate dehydrogenase (EC 1.4.1.2-4) is a key enzyme for synthesizing glutamate, participates in the synthesis and catabolism of glutamate, is widely present in all living bodies, and can be classified into NAD (H), NADP (H) and double-coenzyme specificity according to coenzyme specificity. The glutamate dehydrogenase has the characteristics of wide source, strict stereoselectivity and the like, and has great application value and potential in the field of chiral amino acid synthesis. However, the wild glutamate dehydrogenase has very strong substrate specificity, almost has no enzyme activity or extremely low enzyme activity for other ketoacids except natural substrate alpha-ketoglutarate, and cannot meet industrial application. Therefore, the catalytic capability of wild-type glutamate dehydrogenase on non-natural substrates is improved through molecular modification, and industrial application of the wild-type glutamate dehydrogenase is facilitated.
The patent PCT/CN2018/105158 aims at the glutamate dehydrogenase with multiple sources, changes alanine at a specific position in a substrate binding pocket into glycine and/or changes valine into alanine, and improves the production capacity of the glutamate dehydrogenase with multiple sources on L-glufosinate-ammonium and other L-amino acids. Wherein the enzyme activity of the mutant LsGluDH-A175G on 2-carbonyl-4- (hydroxymethylphosphono) butyric acid (PPO) reaches 34.47U/mL, and the enzyme activities of the mutants PpGluDH-A167G and PpGluDH-V378A on PPO respectively reach 14.85U/mL and 13.3U/mL. However, all of the glutamate dehydrogenases are NADP (H) -specific, and the cost of coenzyme NADP (H) is 2-3 times that of coenzyme NAD (H) in the industrial production, and the glutamate dehydrogenase with NADP (H) -specific does not have production advantages. Although some nad (h) -specific glutamate dehydrogenases have also been reported in the patent, the catalytic efficiency for PPO and other alpha-keto acids is not ideal.
In view of the above, further intensive research is needed to solve the problem that nad (h) -specific glutamate dehydrogenase has no or low enzymatic activity on non-natural substrates through molecular modification.
Disclosure of Invention
The invention aims to solve the problem that original glutamate dehydrogenase (amino acid sequence is shown as SEQ ID NO. 1) derived from Clostridium difficile has no enzyme activity or low enzyme activity on non-natural substrates, thereby providing a plurality of glutamate dehydrogenase mutants and application of the mutants in preparing different L-amino acids.
The specific technical scheme is as follows:
the invention provides a glutamate dehydrogenase variant, which is obtained by carrying out single-point mutation or multi-point combined mutation on the 71 th position, the 92 th position, the 143 th position, the 144 th position, the 145 th position, the 146 th position, the 175 th position, the 187 th position, the 352 th position and the 355 th position of an amino acid sequence shown in SEQ ID NO. 1.
The invention carries out homologous modeling on glutamate dehydrogenase (CdGluDH, NCBI accession number: YP-001086649.1, amino acid sequence shown as SEQ ID NO.1 and nucleotide sequence shown as SEQ ID NO. 2) derived from Clostridium difficile, uses natural substrate alpha-ketoglutaric acid to carry out molecular docking, selects alpha-ketoglutaric acid side chain group
Figure BDA0003346501720000021
Mutating 10 key amino acid residues in the range into alanine or smaller glycine to enlarge the pocket volume or change the pocket electrostatic property, constructing a glutamate dehydrogenase mutant library by means of single-point mutation, sequential iterative mutation based on the distance between sites and catalytic active centers and dominant site combined mutation, and screening the glutamate dehydrogenase mutant to catalytically prepare L-glufosinate, L-norvaline, L-glycine, L-alanine, L-glycine, L-glutamate dehydrogenase mutant, L-glycine, L-lysine, L-glycine, L-lysine, L-beta-lysine, L-glycine, L-lysine, L-lysine, L-lysine, L-lysine, L,A positive mutant strain of 3 amino acids of L-homophenylalanine.
Finally, optimizing mutation sites for specific unnatural amino acid products; performing back mutation on a mutation point which possibly plays a negative role in the optimal mutant obtained after the sequential iteration aiming at the L-homophenylalanine to obtain the optimal combination of the mutation points; aiming at L-glufosinate-ammonium, iterative saturation mutation is carried out on mutation points with remarkable catalytic effect on substrate PPO, so as to obtain the most suitable combined mutant by screening.
Further, the glutamate dehydrogenase variant is obtained by single point mutation, and the mutation sites and the amino acid single letter abbreviations before and after the mutation are respectively as follows: K71A, M92A, V143A, P144A, A145G, P146A, T175A, R187A, V352A, S355A.
Wherein K71A represents: the 71 th amino acid is mutated from lysine to alanine; M92A denotes: the amino acid at the 92 th position is mutated from methionine to alanine; V143A denotes: the amino acid at the 143 st position is mutated from valine to alanine; P144A denotes: the 144 th amino acid is mutated from proline to alanine; a145G denotes: the 145 th amino acid is mutated from alanine to glycine; P146A denotes: the 146 th amino acid is mutated from proline to alanine; T175A denotes: the 175 th amino acid is mutated from threonine to alanine; R187A represents: the 187 th amino acid is mutated from arginine to alanine; V352A denotes: the 352 th amino acid is mutated from valine to alanine; S355A denotes: the amino acid at position 355 was mutated from serine to alanine.
Further, the glutamate dehydrogenase variant is obtained by a multiple point combinatorial mutation in the form of one of the following forms:
(1) sequentially carrying out iterative mutation on two or more adjacent sites according to the arrangement sequence of 145 th, 355 th, 352 th, 71 th, 187 th, 175 th, 92 th, 146 th, 144 th and 143 th;
wherein, each mutation site and the amino acid single letter abbreviations before and after the mutation are respectively: A145G, S355A, V352A, K71A, R187A, T175A, M92A, P146A, P144A, V143A;
(2) carrying out combined mutation on two or more sites of 71 th site, 145 th site, 143 th site and 144 th site;
wherein, each mutation site and the amino acid single letter abbreviations before and after the mutation are respectively: K71A, V143A, V143C, V143G, V143I, V143M, V143S, P144M, P144G and P144A.
Specifically, V143C represents: the amino acid at the 143 st position is mutated from valine to cysteine; V143G denotes: the amino acid at the 143 st position is mutated from valine to glycine; V143I denotes: the amino acid at the 143 st position is mutated from valine to isoleucine; V143M denotes: the amino acid at the 143 st position is mutated from valine to methionine; V143S denotes: the amino acid at the 143 st position is mutated from valine to serine; P144M denotes: the 144 th amino acid is mutated from proline to methionine; P144G denotes: the amino acid at position 144 was mutated from proline to glycine.
Still further, the glutamate dehydrogenase variant is one of the following multiple point mutations:
(I)A145G/S355A，A145G/S355A/V352A，A145G/S355A/V352A/K71A，A145G/S355A/V352A/K71A/R187A，A145G/S355A/V352A/K71A/R187A/T175A，A145G/S355A/V352A/K71A/R187A/T175A/M92A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A/V143A；
(II)A145G/K71A，A145G/P144A，A145G/V143A，K71A/P144A，K71A/V143A，V143A/P144A，A145G/K71A/P144A，A145G/K71A/V143A，A145G/V143A/P144A，K71A/V143A/P144A，A145G/K71A/V143A/P144A；
(III)S355A/V352A/K71A/R187A/T175A，A145G/V352A/K71A/R187A/T175A，A145G/S355A/K71A/R187A/T175A，V352A/K71A/R187A/T175A，S355A/K71A/R187A/T175A，A145G/K71A/R187A/T175A，K71A/R187A/T175A；
(IV)A145G/V143A，A145G/V143C，A145G/V143G，A145G/V143I，A145G/V143M，A145G/V143S，A145G/V143A/P144M，A145G/V143A/P144G，A145G/V143A/P144A。
the "/" above means "and" i.e./"both before and after the site are mutated simultaneously; for example: A145G/S355A shows that alanine at position 145 is mutated to glycine and the amino acid at position 355 is mutated from serine to alanine; A145G/S355A/V352A/K71A/R187A shows that alanine at position 145 is mutated into glycine, and amino acid at position 355 is mutated from serine into alanine; the 352 th amino acid is mutated from valine to alanine; the 71 th amino acid is mutated from lysine to alanine; the amino acid at position 187 was mutated from arginine to alanine.
The present invention also provides a gene encoding the glutamate dehydrogenase variant as described in any one of the above.
The invention also provides an expression vector containing the coding gene. Preferably, the original expression vector is pET-28a (+).
The invention also provides a genetic engineering bacterium containing the coding gene. Preferably, the host cell of the genetically engineered bacterium is e.coli BL21(DE 3).
The invention also provides the application of the glutamate dehydrogenase variant in preparing 2-aminopentanoic acid, L-glufosinate-ammonium or L-homophenylalanine.
The invention also provides application of the genetic engineering bacteria in preparation of 2-aminopentanoic acid, L-glufosinate-ammonium or L-homophenylalanine.
The invention also provides a method for preparing L-norvaline, which comprises the following steps: taking 2-oxovaleric acid as a substrate, and carrying out catalytic reaction in a buffer solution by using a biocatalyst under the action of an amino donor and a coenzyme regeneration system to obtain L-norvaline (namely 2-aminovaleric acid);
the biocatalyst is a glutamate dehydrogenase variant or immobilized enzyme thereof, or a genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase variant; the coenzyme adopted by the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH (oxidized coenzyme NAD)+)；
The glutamate dehydrogenase variant is obtained by carrying out single-point mutation or multi-point combined mutation on an amino acid sequence shown in SEQ ID NO.1, and specifically comprises one of the following mutations:
(a)K71A、M92A、V143A、P144A、A145G、P146A、T175A、R187A、V352A、S355A；
(b)A145G/S355A，A145G/S355A/V352A，A145G/S355A/V352A/K71A，A145G/S355A/V352A/K71A/R187A，A145G/S355A/V352A/K71A/R187A/T175A，A145G/S355A/V352A/K71A/R187A/T175A/M92A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A/V143A；
(c)A145G/K71A，A145G/P144A，A145G/V143A，K71A/P144A，K71A/V143A，V143A/P144A，A145G/K71A/P144A，A145G/K71A/V143A，A145G/V143A/P144A，K71A/V143A/P144A，A145G/K71A/V143A/P144A。
the invention also provides a method for preparing L-glufosinate-ammonium, which comprises the following steps: taking 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid as a substrate, and carrying out catalytic reaction in a buffer solution by using a biocatalyst under the action of an amino donor and a coenzyme regeneration system to obtain L-glufosinate-ammonium;
the biocatalyst is a glutamate dehydrogenase variant or immobilized enzyme thereof, or a genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase variant; the coenzyme adopted by the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH (oxidized coenzyme NAD)+)；
The glutamate dehydrogenase variant is obtained by carrying out single-point mutation or multi-point combined mutation on an amino acid sequence shown in SEQ ID NO.1, and specifically comprises one of the following mutations: A145G, A145G/S355A, A145G/S355A/V352A, A145G/P144A, A145G/V143A, A145G/V143A/P144A, A145G/V143A, A145G/V143C, A145G/V143G, A145G/V143I, A145G/V143M, A145G/V143S, A145G/V143A/P144M, A145G/V143A/P144G, A145G/V143A/P144A.
The invention also provides a method for preparing L-homophenylalanine, which comprises the following steps: 2-oxo-4-phenylbutyric acid is taken as a substrate, and a biological catalyst is utilized to perform catalytic reaction in a buffer solution under the action of an amino donor and a coenzyme regeneration system to obtain L-homophenylalanine;
the biocatalyst is a glutamate dehydrogenase variant or immobilized enzyme thereof, or a genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase variant; the coenzyme adopted by the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH (oxidized coenzyme NAD)+)；
The glutamate dehydrogenase variant is obtained by carrying out single-point mutation or multi-point combined mutation on an amino acid sequence shown in SEQ ID NO.1, and specifically comprises one of the following mutations:
(i)V143A，A145G；
(ii)A145G/S355A，A145G/S355A/V352A/K71A，A145G/S355A/V352A/K71A/R187A，A145G/S355A/V352A/K71A/R187A/T175A，A145G/S355A/V352A/K71A/R187A/T175A/M92A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A/V143A；
(iii)A145G/V352A/K71A/R187A/T175A，A145G/S355A/K71A/R187A/T175A，A145G/K71A/R187A/T175A；A145G/K71A，A145G/V143A，V143A/P144A，A145G/K71A/P144A，A145G/K71A/V143A，A145G/V143A/P144A，A145G/K71A/V143A/P144A。
compared with the prior art, the invention has the following beneficial effects:
(1) the invention solves the problem that the mutant has no or low enzyme activity to non-natural substrates by taking glutamic acid dehydrogenase (CdGluDH, NCBI accession number: YP-001086649.1) derived from Clostridium difficile as a base through a rational design molecular modification method, and obtains the mutant which can catalyze and prepare various L-amino acids except natural product L-glutamic acid.
(2) The rational design method used by the invention can quickly obtain the glutamate dehydrogenase mutant with higher catalytic activity aiming at the non-natural substrate by screening a smaller mutation library.
(3) The invention takes corresponding keto acid as a substrate, and generates L-amino acid by catalytic reductive amination of glutamate dehydrogenase in the presence of inorganic ammonia and coenzyme, the process is simple, and the conversion rate of raw materials and the chiral purity of the product are both more than 99 percent.
(4) In the method, NAD (H) is used as coenzyme, and compared with NADP (H), the price is low, so that the production cost of the L-amino acid is greatly reduced.
Drawings
FIG. 1 shows high performance liquid chromatography spectra of the pre-column derivatization of D, L-glufosinate-L and reaction samples.
FIG. 2 is a high performance liquid chromatography of 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid (PPO) standard.
FIG. 3 is a High Performance Liquid Chromatography (HPLC) of the pre-column derivatization of the reaction samples of D, L-norvaline standard and L-norvaline standard.
FIG. 4 is a high performance liquid chromatography of 2-oxopentanoic acid standard.
FIG. 5 shows high performance liquid chromatography maps of a chiral column of a reaction sample, a standard product of D, L-homophenylalaninase and a standard product of L-homophenylalaninase.
FIG. 6 is a high performance liquid chromatography of the homophenylalanine and 2-oxo-4-phenylbutyric acid standards.
FIG. 7 is a graph showing the reaction equation and the reaction process for asymmetrically synthesizing L-glufosinate-ammonium by glutamate dehydrogenase wild type and mutant in example 8.
FIG. 8 is a graph showing the reaction scheme and the course of asymmetric synthesis of L-norvaline by the wild-type and mutant glutamate dehydrogenase in example 9.
FIG. 9 is a graph showing the reaction formula and the reaction process for asymmetrically synthesizing L-homophenylalanine using the wild type and the mutant of glutamate dehydrogenase in example 10.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning procedures can be specifically described in molecular cloning protocols, compiled by J. Sambruka et al.
Reagents used in upstream genetic engineering: DPN I used in the examples of the present invention was purchased from TaKaRa, Takara Bio Inc.; the plasmid extraction kit and the DNA recovery and purification kit are purchased from Axygen Hangzhou limited company; coli BL21(DE3), plasmid pET-28a (+) and the like from Novagen; DNA marker, low molecular weight standard protein and agarose electrophoresis reagent are purchased from Beijing all-style gold biotechnology limited; the primer synthesis and sequence sequencing work is completed by the Oncomen bioengineering company Limited. The method of using the above reagent is referred to the commercial specification.
Reagents used in the downstream catalytic process: PPO and L-glufosinate-ammonium are synthesized in a laboratory; 2-oxopentanoic acid was purchased from Tokyo Chemical Industry, 2-oxo-4-phenylbutyric acid was purchased from Shanghai Biao pharmaceutical science and technology, Inc., L-norvaline, D-norvaline, L-homophenylalanine, and D-homophenylalanine were purchased from Beijing Bailingwei science and technology, Inc., and D, L-glufosinate was purchased from Sigma-Aldrich; alpha-ketoglutaric acid, L-glutamic acid, D, L-glutamic acid and other commonly used reagents were purchased from national pharmaceutical group chemical reagents, Inc.
Glutamate dehydrogenase enzyme activity standard detection system (HPLC method): appropriate amount of enzyme solution, 80mM substrate, 40mM NADH, 2M NH4 +((NH4)2SO4) The total volume was 500. mu.L, and the reaction medium was phosphate buffer pH 7.50.1M. The reaction was carried out at 40 ℃ for 10min, and 500. mu.L of 1M NaOH or 1M HCl was added to terminate the reaction. The L-amino acid formed in the sample was quantitatively analyzed by HPLC.
Definition of enzyme activity unit (U): the amount of enzyme required to produce 1. mu. mol L-amino acid per minute under standard reaction conditions.
Aliphatic amino acid HPLC chiral analysis method: pre-column derivatization was used. Derivatization reagent: 0.03g of o-phthalaldehyde and 0.1g of 0.1g N-acetyl-L-cysteine are weighed respectively, dissolved with 400 mu L of ethanol, added with 4mL of 0.2 mol/boric acid buffer solution (pH 9.8), shaken to be fully dissolved, and stored in a refrigerator at 4 ℃ for standby (no more than 4 days). Derivatization reaction and determination: 100 mul of sample is taken and added with 100 mul of derivatization reagent, and after being mixed evenly, the mixture is kept at 25 ℃ for 5 min. Chromatographic conditions are as follows: chromatographic column-
Figure BDA0003346501720000071
QS-C18; detection wavelength/334 nm; column temperature/30 ℃; sample injection amount/20 mu L; mobile phase A: 50mM aqueous sodium acetate, mobile phase B: acetonitrile, mobile phase C: the methanol and mobile phase ratio (V/V) are shown in Table 1.
Table 1 HPLC mobile phase ratio
Figure BDA0003346501720000081
HPLC chiral analysis method of homophenylalanine: chromatography column/CHIRALPAK ZWIX (-); detection wavelength/225 nm; flow rate/0.5 mL/min; column temperature/40 ℃; sample injection amount/20 mu L; mobile phase/methanol with 1.9mL/L formic acid and 2mL/L ethylenediamine.
HPLC analysis method for PPO: chromatographic column-
Figure BDA0003346501720000082
QS-C18; detection wavelength/UV 205 nm; column temperature/40 ℃; sample injection amount/20 mu L; flow rate/1 mL/min; mobile phase/50 mM (NH)4)2HPO4Aqueous solution, 1% aqueous 10% tetrabutylammonium hydroxide solution, pH adjusted to 3.6 with 50% phosphoric acid, and 8% acetonitrile was added.
HPLC analytical method for 2-oxopentanoic acid: chromatographic column-
Figure BDA0003346501720000083
QS-C18; detection wavelength/UV 205 nm; column temperature/40 ℃; sample injection amount/20 mu L; flow rate/1 mL/min; mobile phase/50 mM (NH)4)2HPO4Acetonitrile 95:5 as aqueous solution.
HPLC analysis method of homophenylalanine achiral and 2-oxo-4 phenylbutyric acid: chromatographic column-
Figure BDA0003346501720000085
QS-C18; detection wavelength/UV 205 nm; column temperature/40 ℃; sample injection amount/20 mu L; flow rate/1 mL/min; mobile phase/50 mM (NH)4)2HPO4Acetonitrile 85:15 as aqueous solution.
Example 1 construction and screening of Single Point mutation library
CdGluDH was subjected to single point mutation of K71A, M92A, V143A, P144A, A145G, P146A, T175A, R187A, V352A and S355A. The specific method comprises the following steps:
1. whole plasmid PCR
Using pET-28a (+) -CdGluDH plasmid as template, designing upstream and downstream primers (Table 2) covering mutation points to perform whole plasmid PCR:
TABLE 2 primers used for Single-Point mutation library construction
Figure BDA0003346501720000084
Figure BDA0003346501720000091
PCR amplification System:
Figure BDA0003346501720000092
PCR amplification conditions:
1) pre-denaturation: 5min at 95 ℃;
2) denaturation: 10s at 98 ℃; annealing: 15s at 58 ℃; extension: 10s at 72 ℃; circulating for 30 times;
3) and (3) post-extension: 10min at 72 ℃;
4) storing at 4 ℃.
2. Template digestion:
carrying out agarose gel electrophoresis on the PCR product, and digesting the plasmid template in the PCR product by using Dpn I enzyme after recovery as follows: 1 mu L of Dpn I enzyme, 17 mu L of PCR product and 2 mu L of Buffer. Digestion of the template was completed at 37 ℃ for 2 hours.
3. Transformation and verification:
after the digestion products are verified by nucleic acid agarose gel electrophoresis, escherichia coli BL21(DE3) competent cells are transformed by a heat shock method at 42 ℃. The specific process is as follows:
(1) thawing the competent cells on ice for 15 min;
(2) add 10. mu.L of DNA to 100. mu.L of competent cells in sterile conditions and mix gently, ice
Standing for 30 min;
(3) standing the EP tube in a metal bath at 42 ℃ for heat shock for 90s, and cooling on ice for 2min after the heat shock is finished;
(4) adding 800 μ L LB culture medium into EP tube, mixing with tip, placing in 200rpm shaker, incubating at 37 deg.C for 40-60 min;
(5) after concentration, a proper volume of the extract is coated on a corresponding resistant plate, and bacterial colonies can appear after the extract is cultured in a 37 ℃ incubator for 12-16 h. 3-4 single colonies are picked out from each plate for culture, and sequencing is performed to verify whether mutation is successful.
4. Mutant strain culture and protein expression
After the successfully sequenced mutant strain is activated by plate streaking, a single colony is selected and inoculated into 5mL LB liquid culture medium containing 50 ug/mL kanamycin, and shake culture is carried out at 37 ℃ for 12 h. Inoculating the strain at 2% into 50mL LB liquid medium containing 50. mu.g/mL kanamycin, and shake-culturing at 37 ℃ to OD600When the concentration reaches about 0.6-0.8, IPTG is added to the final concentration of 0.5mM, and the induction culture is carried out for 16h at the temperature of 18 ℃. After the completion of the culture, 12000g of the culture solution was centrifuged at 4 ℃ for 10min, and the supernatant was discarded to collect the cells. The collected cells were washed twice with 50mM phosphate buffer pH7.5, resuspended in phosphate buffer, and sonicated at 400W power for 30 cycles, each for 3 seconds with 7 seconds intervals. The cell disruption solution was centrifuged at 12000g at 4 ℃ for 10min to remove the precipitate, and the supernatant was obtained as a crude enzyme solution.
5. Enzyme activity assay
The enzyme activities of the mutant library on natural substrate alpha-ketoglutaric acid and 3 non-natural substrates are determined by an HPLC analysis method, and the determination results are shown in Table 3:
TABLE 3 determination of the enzymatic activities of glutamate dehydrogenase and its single-site mutants on different substrates
Figure BDA0003346501720000101
Note: N/A indicates that no enzyme activity was detected.
Example 2 construction and screening of sequential iterative mutation library based on site-to-catalytic activity center distance
Based on the sequence of the distance from the catalytic active center, the sequential iterative mutation is carried out on 10 amino acid residue positions of 71, 92, 143, 144, 145, 146, 175, 187, 352 and 355, and specifically, the following mutants are designed:
2X：A145G/S355A；
3X：A145G/S355A/V352A；
4X：A145G/S355A/V352A/K71A；
5X：A145G/S355A/V352A/K71A/R187A；
6X：A145G/S355A/V352A/K71A/R187A/T175A；
7X：A145G/S355A/V352A/K71A/R187A/T175A/M92A；
8X：A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A；
9X：A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A；
10X：A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A /P144A/V143A。
the specific experimental steps are as follows:
pET-28a (+) -CdGluDH-A145G plasmid is used as a template, upstream and downstream primers 2X-F and 2X-R (table 3) covering mutation points are designed, whole plasmid PCR is carried out to obtain a mutant 2X, then 2X is used as a template, 3X-F and 3X-R are used as upstream and downstream primers, whole plasmid PCR is carried out to obtain a mutant 3X, and the above nine combined mutants are obtained by analogy, wherein the used primers are shown in table 4, the detailed PCR operation, transformation, strain culture and protein expression steps are the same as those in example 1.
TABLE 4 primers used for the construction of the sequentially iterative mutation library
Figure BDA0003346501720000111
The enzyme activities of the mutant library on natural substrates alpha-ketoglutaric acid and 3 non-natural substrates are determined by an HPLC analysis method, and the determination results are shown in Table 5.
TABLE 5 determination of enzymatic Activity of glutamate dehydrogenase mutants on Natural substrates alpha-ketoglutarate and 3 non-Natural substrates
Figure BDA0003346501720000121
Note: N/A indicates that no enzyme activity was detected.
Example 3 construction and screening of combinatorial mutation libraries based on Positive Single mutation Point
Based on the screening results of example 1, 4 mutants K71A, V143A, P144A and A145G having positive catalytic effects on one or more substrates were combined at two points, three points and four points to construct a new combinatorial mutation library. The specific construction method is that A145G, K71A and V143A plasmids are used as templates, corresponding primers are used for constructing two-point mutant strains such as A145G/K71A and A145G/P144A, then the two-point mutant strains are used as templates, corresponding primers are used for constructing three-point mutant strains, finally the three-point mutant strains are used as templates for constructing four-point mutant strains, related primers are shown in a table 6, and other detailed steps are the same as those in the example 1.
TABLE 6 primers used for construction of positive single-mutation-point combinatorial mutation library
Figure BDA0003346501720000122
The enzyme activities of the mutant library on natural substrate alpha-ketoglutaric acid and 3 non-natural substrates were determined by HPLC analysis, and the results are shown in Table 7.
TABLE 7 determination of enzyme Activity on different substrates based on Positive Single mutation Point combination mutants
Figure BDA0003346501720000131
Note: N/A indicates that no enzyme activity was detected.
Example 4 construction and screening of a library of Return mutations for the substrate 2-oxo-4-phenylbutyric acid
From the results of example 2, the optimal mutations for catalyzing the substrate 2-oxo-4-phenylbutyric acid to prepare L-homophenylalanine are A145G/S355A/V352A/K71A/R187A/T175A (6X), but the enzyme activities of the first 3 points A145G, S355A and V352A on homophenylalanine are not obviously improved in the sequential iteration process, and the three mutations are presumed to have no effect or a negative effect on improving the enzyme activity, so that the three points are subjected to single-point or combined back mutation starting from 6-point mutation, and 6 mutants are constructed to screen the optimal mutation point combination, and the constructed mutants are as follows:
6X-A：S355A/V352A/K71A/R187A/T175A；
6X-B：A145G/V352A/K71A/R187A/T175A；
6X-C：A145G/S355A/K71A/R187A/T175A；
6X-AB：V352A/K71A/R187A/T175A；
6X-AC：S355A/K71A/R187A/T175A；
6X-BC：A145G/K71A/R187A/T175A；
6X-ABC：K71A/R187A/T175A。
the specific construction method is to use 6X or 6X-A or 6X-B mutant as template, and primer shown in Table 8 to perform whole plasmid PCR, and the detailed steps are shown in example 1
TABLE 8 primers used for the construction of the revertant library
Figure BDA0003346501720000141
The enzyme activity of the mutant on 2-oxo-4-phenylbutyric acid was measured by HPLC analysis, and the measurement results are shown in Table 9.
TABLE 9 determination of enzyme Activity of glutamate dehydrogenase Return mutants on 2-oxo-4-phenylbutyric acid
Figure BDA0003346501720000142
Note: N/A indicates that no enzyme activity was detected.
Example 5 construction and screening of iterative saturated mutation library for substrate PPO
And (3) carrying out iterative saturation mutation on the 143 th site, the 144 th site and the 145 th site by taking PPO as a model substrate, and screening out the optimal combined mutant.
The method comprises the following steps: single point saturation mutation of 143, 144 and 145
CdGluDH was subjected to single-point saturation mutagenesis at positions 143, 144 and 145, respectively. The specific method comprises the following steps:
1. saturated mutation library construction
Plasmid-wide PCR was performed using pET-28a (+) -CdGluDH plasmid as template and upstream and downstream primers (Table 10) covering the mutation site were designed, and the detailed procedures are as described in example 1:
TABLE 10 primers used for construction of Single-Point saturated mutant pools
Figure BDA0003346501720000151
2. Preliminary screening
To a sterilized 96-deep well plate, 200. mu.L of LB medium (containing 50. mu.g/mL of kanamycin) was added, and a single colony was picked up to the 96-deep well plate using a sterilized tip. The deep-well plate was then incubated at 37 ℃ for 8h at 200rpm, and was referred to as a primary plate. In another sterilized 96-well plate, 400. mu.L of LB medium (containing 50. mu.g/mL kanamycin) was added as a secondary plate, 50. mu.L of the bacterial solution was aspirated from the primary plate to the secondary plate, and the primary plate was placed in a refrigerator at-80 ℃ for long-term storage with 20% glycerol added thereto. The secondary plate was then incubated at 37 ℃ for 3h with shaking, induced by the addition of IPTG to a final concentration of 0.5mM, and then incubated at 18 ℃ for a further 18h at 200 rpm.
The secondary plate was centrifuged at 4000rpm at 4 ℃ for 20min to collect cells, which were then frozen at-80 ℃ for more than 3 h. The secondary plate was removed from-80 ℃ and left to stand at room temperature for 0.5h for thawing, after which 300. mu.L of a lysate (10mM phosphate buffer pH7.5, 750mg/L lysozyme, 10mg/L DNase I) was added to each well, the cells were suspended by shaking and placed in a shaker at 37 ℃ and incubated at 200rpm for 1 h. After the incubation is finished, centrifuging at 4000rpm and 4 ℃ for 20min, and taking the supernatant to perform enzyme activity determination.
Preparing enzyme activity determination solution for screening: pH7.5 phosphate buffer (0.1M) containing 2mM NADH, 10mM substrate PPO, 1M NH4 +. To each well of a new 96-well plate (reaction plate), 200. mu.L of an enzyme activity measuring solution was added and juxtaposedIncubate at 37 ℃ for 15 min. The reaction was started by sucking 200. mu.L of the enzyme solution and adding it to the reaction plate, and 100. mu.L of the enzyme solution was sampled at 20min, 40min and 60min and added to the microplate to which 100. mu.L of a phosphate buffer solution (0.1M) of pH7.5 had been added in advance, and the absorbance at 340nm was measured with a microplate reader. The lower the absorbance value represents the higher the catalytic activity, and the mutant strain with the absorbance value obviously lower than that of a control (wild type) is selected as a candidate strain for secondary screening.
3. Double sieve
And (4) re-screening the initially screened mutant with obviously improved enzyme activity by using HPLC. After the corresponding mutant strain on the primary plate is activated by plate streaking, a single colony is selected and inoculated into 5mL LB liquid culture medium containing 50 ug/mL kanamycin, and shake culture is carried out at 37 ℃ for 12 h. Inoculating the strain at 2% into 50mL LB liquid medium containing 50. mu.g/mL kanamycin, and shake-culturing at 37 ℃ to OD600When the concentration reaches about 0.6-0.8, IPTG is added to the final concentration of 0.5mM, and the induction culture is carried out for 16h at the temperature of 18 ℃. After the completion of the culture, 12000g of the culture medium was centrifuged at 4 ℃ for 10min, and the supernatant was discarded to collect cells. The collected cells, with 50mM pH7.5 phosphate buffer washing twice, heavy suspension in phosphate buffer, 400W power ultrasonic disruption 30 times, each ultrasonic duration for 3s, pause for 7 s. The cell disruption solution was centrifuged at 12000g at 4 ℃ for 10min to remove the precipitate, and the supernatant was obtained as a crude enzyme solution. And (3) measuring the crude enzyme liquid enzyme activity of each mutant strain according to a standard enzyme activity detection system (with PPO as a substrate). Finally, the screened A145G mutant has obviously improved enzyme activity, and the enzyme activity reaches 1.06 +/-0.04U/mg of dry cell weight.
Step two: iterative saturation mutation of 143 locus based on A145G
Iterative saturation mutagenesis was performed on position 143 of CdGluDH-A145G. The primers were designed as follows:
A145G-143-F:AGTTGATnnkCCGGGGCCGGATGTGAATACCA
A145G-143-R:GCCCCGGmnnATCAACTTTTTCGCCAATCAGTT
constructing and screening a mutation library according to the method of the step one, and finally obtaining 6 mutants with obviously improved enzyme activity, wherein the results are shown in Table 11.
TABLE 11A 145G/143 iterative saturated mutation library screening results
Figure BDA0003346501720000161
Step three: iterative saturation mutation is carried out on position 144 on the basis of A145G/V143A, A145G/V143G and A145G/V143M
Iterative saturation mutation is carried out on 144 bits by taking CdGluDH-A145G/V143A, CdGluDH-A145G/V143G and CdGluDH-A145G/V143M as templates, and primers are shown in Table 12.
TABLE 12 primers used for construction of Single-Point saturated mutation library
Figure BDA0003346501720000162
Constructing and screening a mutation library according to the method of the first step, and finally screening to obtain 3 mutants with obviously improved enzyme activity (Table 13):
positive mutation screening results of 13144 iterative saturation mutation library
Figure BDA0003346501720000171
EXAMPLE 6 preparation of L-Glufosinate-ammonium by glucose dehydrogenase Bienzyme coupling of glutamate dehydrogenase mutant (CdGluDH-A145G/V143A/P144G)
Culturing thallus and preparing a crude enzyme solution: the glycerol tubes of the engineering bacteria expressing the CdGluDH-A145G/V143A/P144G glutamate dehydrogenase mutant gene and glucose dehydrogenase gene (NCBI accession number: WP _087960837.1, base sequence cloned to expression plasmid pET-28a (+), inserted with BamH I and HindIII sites, transferred to expression host E.coli BL21(DE3) were streaked and activated, and then single colonies were selected and inoculated into 50mL LB liquid medium containing 50. mu.g/mL kanamycin and shake cultured at 37 ℃ for 12 h. Inoculating 2% of the strain into 1L of fresh LB liquid medium containing 50. mu.g/ml Kan, and shake-culturing at 37 ℃ to OD600When the concentration reached about 0.6, IPTG was added to a final concentration of 0.5mM, and the cells were induced at 18 ℃ for 16 hours. After the culture is finished, the culture medium is culturedThe culture solution is centrifuged at 12000g and 4 ℃ for 10min, thalli are collected, and the cells are broken by ultrasonic waves to prepare a crude enzyme solution.
The reaction system is 50mL, and contains 200mM substrate PPO, 300mM glucose, 200mM (NH)4)2SO4With 0.2mM NAD+(ii) a The concentration of glutamate dehydrogenase cells (wet weight) was 2g/L, and the concentration of glucose dehydrogenase cells (wet weight) was 4 g/L. The reaction temperature was controlled to 40 ℃ by water bath, and the pH was controlled to 8.0 by dropwise addition of ammonia water during the reaction. After reacting for 2h, detecting the residual concentration of PPO by using HPLC, and simultaneously detecting the generation amount and ee value of L-glufosinate-ammonium by using pre-column derivatization high performance liquid chromatography.
The reaction end data are as follows: PPO remained at 0mM, conversion 100%. The L-glufosinate-ammonium is produced at a concentration of 32.3g/L and an ee value of > 99.9%.
Example 7 preparation of L-Glufosinate-ammonium by coupling of glutamate dehydrogenase mutant (CdGluDH-A145G/V143A/P144M) with Formate dehydrogenase Bizyme
An engineering bacterium expressing the CdGluDH-A145G/V143A/P144M glutamate dehydrogenase mutant gene and formate dehydrogenase (NCBI accession number: P33160.3, base sequence cloned to expression plasmid pET-28a (+) with BamH I and HindIII inserted into expression host E.coli BL21(DE 3)) was cultured in the same manner as in example 6, cells were collected by centrifugation, and the cells were disrupted by sonication to prepare a crude enzyme solution.
The reaction system is 50mL and contains 200mM substrate PPO, 600mM ammonium formate, 100mM (NH)4)2SO4With 0.2mM NAD+(ii) a The concentration of glutamate dehydrogenase cells (wet weight) was 2g/L, and the concentration of formate dehydrogenase cells (wet weight) was 4 g/L. The reaction temperature was controlled to 40 ℃ by water bath, and the pH was controlled to 8.0 by dropwise addition of ammonia water during the reaction. After reacting for 2h, detecting the residual concentration of PPO by using HPLC, and simultaneously detecting the generation amount and ee value of L-glufosinate-ammonium by using pre-column derivatization high performance liquid chromatography.
The reaction end data are as follows: PPO remained at 0mM, conversion 100%. The L-glufosinate-ammonium is produced in a concentration of 32.1g/L and an ee value of > 99.9%.
EXAMPLE 8 preparation of L-Glufosinate-ammonium by coupling of glutamate dehydrogenase wild-type and mutant with alcohol dehydrogenase Dual enzyme
Wild-type (CdGluDH-WT) and mutant (CdGluDH-A145G, CdGluDH-A145G/V143A/P144A) expressing glutamate dehydrogenase and alcohol dehydrogenase (NCBI accession No.: NZ _ JYNW01000069.1) were cultured in the same manner as in example 6, the base sequences were cloned on expression plasmid pET-28a (+) with BamH I and HindIII inserted into the engineered bacteria of expression host E.coli BL21(DE3), the cells were collected by centrifugation, and the crude enzyme solutions were prepared by sonication.
The reaction system is 50mL and contains 200mM substrate PPO, 300mM isopropanol, 200mM (NH)4)2SO4With 0.2mM NAD+(ii) a The concentration of glutamate dehydrogenase cells (wet weight) was 2g/L, and the concentration of alcohol dehydrogenase cells (wet weight) was 4 g/L. The reaction temperature was controlled to 40 ℃ by water bath, and the pH was controlled to 8.0 by dropwise addition of ammonia water during the reaction. Samples were taken periodically and the residual concentration of PPO was determined by HPLC, and the data for conversion as a function of time are shown in FIG. 7. And simultaneously detecting the final generation amount and ee value of the L-glufosinate-ammonium by using pre-column derivatization high performance liquid chromatography.
CdGluDH-WT catalyzed reaction: the residual PPO is 205.3mM, the conversion rate is 0%, and the generation concentration of L-glufosinate-ammonium is 0 g/L;
CdGluDH-A145G catalyzed reaction for 330 min: the residual PPO is 0mM, the conversion rate is 100 percent, the generation concentration of L-glufosinate-ammonium is 32.8g/L, and the ee value is more than 99.9 percent;
CdGluDH-A145G/V143A/P144A catalyzes reaction for 75 min: the residual PPO is 0mM, the conversion rate is 100 percent, the generation concentration of the L-glufosinate-ammonium is 30.9g/L, and the ee value is more than 99.9 percent.
EXAMPLE 9 Dual enzymatic coupling of glutamate dehydrogenase wild-type and mutant with alcohol dehydrogenase to produce L-norvaline
Engineered bacteria expressing wild-type glutamate dehydrogenase (CdGluDH-WT), mutant (CdGluDH-A145G/K71A/V143A, CdGluDH-A145G/V352A/K71A/R187A/T175A) and alcohol dehydrogenase (NCBI accession No.: NZ-JYNW 01000069.1) were cultured in the same manner as in example 6, and cells were collected by centrifugation and disrupted by sonication to prepare a crude enzyme solution.
The reaction system was 50mL, containing 200mM substrate 2-oxopentanoic acid, 300mM isopropanol, 200mM (NH)4)2SO4With 0.2mM NAD+(ii) a Glutamate dehydrogenaseThe concentration of the bacterial cells (wet weight) was 2g/L, and the concentration of the alcohol dehydrogenase bacterial cells (wet weight) was 4 g/L. The reaction temperature was controlled to 40 ℃ by water bath, and the pH was controlled to 8.0 by dropwise addition of ammonia water during the reaction. The samples were taken periodically and the residual concentration of 2-oxopentanoic acid was determined by HPLC, the conversion data as a function of time are shown in FIG. 8. Meanwhile, the final production amount and ee value of the L-norvaline are detected by using pre-column derivatization high performance liquid chromatography.
CdGluDH-WT catalyzed reaction for 270 min: the 2-oxovaleric acid remained at 0mM, the conversion rate was 100%, the L-norvaline production concentration was 21.8g/L, and the ee value was > 99.5%;
CdGluDH-A145G/K71A/V143A catalyzes reaction for 150 min: the 2-oxovaleric acid remained at 0mM, the conversion rate was 100%, the L-norvaline production concentration was 22.1g/L, and the ee value was > 99.6%;
CdGluDH-A145G/V352A/K71A/R187A/T175A catalyzed reaction for 360 min: the residue of 2-oxopentanoic acid was 81.2mM, conversion was 59.4%, L-norvaline was formed at a concentration of 12.7g/L, and ee value was > 99.3%.
EXAMPLE 10 preparation of L-homophenylalanine by coupling of glutamate dehydrogenase wild type and mutant with alcohol dehydrogenase double enzymes
Engineered bacteria expressing wild-type glutamate dehydrogenase (CdGluDH-WT), mutants (CdGluDH-A145G/S355A/V352A/K71A/R187A/T175A, CdGluDH-A145G/V352A/K71A/R187A/T175A) and alcohol dehydrogenase (NCBI accession number: NZ _ JYNW01000069.1) were cultured in the same manner as in example 6, and cells were collected by centrifugation and disrupted by sonication to prepare a crude enzyme solution.
The reaction system was 50mL, containing 50mM substrate 2-oxo-4-phenylbutyric acid, 300mM isopropanol, 200mM (NH)4)2SO4With 0.2mM NAD+(ii) a The concentration of glutamate dehydrogenase cells (wet weight) was 2g/L, and the concentration of alcohol dehydrogenase cells (wet weight) was 4 g/L. The reaction temperature was controlled to 40 ℃ by water bath, and the pH was controlled to 8.0 by dropwise addition of ammonia water during the reaction. The sample was taken out periodically, the residual concentration of 2-oxo-4-phenylbutyric acid was measured by HPLC, and the data of the conversion with time are shown in FIG. 9. And simultaneously detecting the final generation amount and ee value of the L-homophenylalanine by using pre-column derivatization high performance liquid chromatography.
CdGluDH-WT catalyzed reaction: 2-oxo-4-phenylbutyric acid remained 52.8mM, the conversion rate was 0%, and the L-homophenylalanine formation concentration was 0 g/L;
CdGluDH-A145G/S355A/V352A/K71A/R187A/T175A catalyzed reaction for 180 min: the 2-oxo-4-phenylbutyric acid is remained at 0mM, the conversion rate is 100 percent, the generation concentration of the L-homophenylalanine is 8.8g/L, and the ee value is more than 99.9 percent;
CdGluDH-A145G/V352A/K71A/R187A/T175A catalyzed reaction for 105 min: the 2-oxo-4-phenylbutyric acid remained at 0mM, the conversion rate was 100%, the L-homophenylalanine formation concentration was 8.7g/L, and the ee value was > 99.9%.
Sequence listing
<110> Hangzhou international scientific center of Zhejiang university
<120> glutamate dehydrogenase variants and their use for the preparation of L-amino acids
<160> 70
<170> SIPOSequenceListing 1.0
<210> 1
<211> 421
<212> PRT
<213> Clostridium difficile (Clostridium difficile)
<400> 1
Met Ser Gly Lys Asp Val Asn Val Phe Glu Met Ala Gln Ser Gln Val
1 5 10 15
Lys Asn Ala Cys Asp Lys Leu Gly Met Glu Pro Ala Val Tyr Glu Leu
20 25 30
Leu Lys Glu Pro Met Arg Val Ile Glu Val Ser Ile Pro Val Lys Met
35 40 45
Asp Asp Gly Ser Ile Lys Thr Phe Lys Gly Phe Arg Ser Gln His Asn
50 55 60
Asp Ala Val Gly Pro Thr Lys Gly Gly Ile Arg Phe His Gln Asn Val
65 70 75 80
Ser Arg Asp Glu Val Lys Ala Leu Ser Ile Trp Met Thr Phe Lys Cys
85 90 95
Ser Val Thr Gly Ile Pro Tyr Gly Gly Gly Lys Gly Gly Ile Ile Val
100 105 110
Asp Pro Ser Thr Leu Ser Gln Gly Glu Leu Glu Arg Leu Ser Arg Gly
115 120 125
Tyr Ile Asp Gly Ile Tyr Lys Leu Ile Gly Glu Lys Val Asp Val Pro
130 135 140
Ala Pro Asp Val Asn Thr Asn Gly Gln Ile Met Ser Trp Met Val Asp
145 150 155 160
Glu Tyr Asn Lys Leu Thr Gly Gln Ser Ser Ile Gly Val Ile Thr Gly
165 170 175
Lys Pro Val Glu Phe Gly Gly Ser Leu Gly Arg Thr Ala Ala Thr Gly
180 185 190
Phe Gly Val Ala Val Thr Ala Arg Glu Ala Ala Ala Lys Leu Gly Ile
195 200 205
Asp Met Lys Lys Ala Lys Ile Ala Val Gln Gly Ile Gly Asn Val Gly
210 215 220
Ser Tyr Thr Val Leu Asn Cys Glu Lys Leu Gly Gly Thr Val Val Ala
225 230 235 240
Met Ala Glu Trp Cys Lys Ser Glu Gly Ser Tyr Ala Ile Tyr Asn Glu
245 250 255
Asn Gly Leu Asp Gly Gln Ala Met Leu Asp Tyr Met Lys Glu His Gly
260 265 270
Asn Leu Leu Asn Phe Pro Gly Ala Lys Arg Ile Ser Leu Glu Glu Phe
275 280 285
Trp Ala Ser Asp Val Asp Ile Val Ile Pro Ala Ala Leu Glu Asn Ser
290 295 300
Ile Thr Lys Glu Val Ala Glu Ser Ile Lys Ala Lys Leu Val Cys Glu
305 310 315 320
Ala Ala Asn Gly Pro Thr Thr Pro Glu Ala Asp Glu Val Phe Ala Glu
325 330 335
Arg Gly Ile Val Leu Thr Pro Asp Ile Leu Thr Asn Ala Gly Gly Val
340 345 350
Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Leu Tyr Gly Tyr Tyr Trp
355 360 365
Ser Glu Glu Glu Val Glu Gln Lys Glu Glu Ile Ala Met Val Lys Ala
370 375 380
Phe Glu Ser Ile Trp Lys Ile Lys Glu Glu Tyr Asn Val Thr Met Arg
385 390 395 400
Glu Ala Ala Tyr Met His Ser Ile Lys Lys Val Ala Glu Ala Met Lys
405 410 415
Leu Arg Gly Trp Tyr
420
<210> 2
<211> 1266
<212> DNA
<213> Clostridium difficile (Clostridium difficile)
<400> 2
atgagtggta aagatgttaa cgttttcgaa atggcacaga gccaggttaa aaatgcctgt 60
gataaactgg gtatggaacc ggccgtttat gaactgctga aagaaccgat gcgtgtgatt 120
gaagtgagca ttccggtgaa aatggatgat ggcagcatta agacctttaa aggttttcgt 180
agtcagcata atgatgccgt gggtccgacc aaaggtggca ttcgctttca tcagaatgtg 240
agtcgcgatg aagttaaagc actgagtatt tggatgacct ttaaatgcag tgtgaccggc 300
attccgtatg gtggcggcaa aggtggcatc attgttgatc cgagtaccct gagtcagggc 360
gaactggaac gtctgagccg cggttatatt gatggcatct ataaactgat tggcgaaaaa 420
gttgatgtgc cggccccgga tgtgaatacc aatggtcaga ttatgagttg gatggtggat 480
gaatataata agctgaccgg tcagagtagt attggcgtga ttaccggcaa accggtggaa 540
tttggcggca gcctgggccg caccgccgca accggttttg gtgttgccgt taccgcacgt 600
gaagcagccg ccaaactggg tattgatatg aaaaaagcaa agattgcagt gcagggtatt 660
ggcaatgtgg gtagctatac cgttctgaat tgtgaaaaac tgggcggcac cgtggttgca 720
atggccgaat ggtgcaaaag tgaaggtagc tatgccatct ataatgaaaa tggtctggat 780
ggccaggcaa tgctggatta tatgaaagaa catggcaatc tgctgaattt tccgggtgca 840
aaacgtatta gcctggaaga attttgggca agcgatgttg atattgtgat tccggccgcc 900
ctggaaaata gtattaccaa agaagtggcc gaaagcatta aggcaaaact ggtgtgtgaa 960
gcagcaaatg gcccgaccac cccggaagca gatgaagtgt ttgccgaacg tggcattgtg 1020
ctgaccccgg atattctgac caatgccggt ggtgttaccg tgagttattt tgaatgggtt 1080
cagaatctgt atggttatta ttggagcgaa gaagaagtgg aacagaaaga agaaattgcc 1140
atggtgaaag catttgaaag catttggaaa attaaggaag agtataacgt gaccatgcgt 1200
gaagccgcct atatgcatag cattaagaaa gtggcagaag caatgaaact gcgtggctgg 1260
tattaa 1266
<210> 3
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcaggtggca ttcgctttca tcagaatgtg ag 32
<210> 4
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aagcgaatgc cacctgcggt cggacccacg gcatc 35
<210> 5
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gagtatttgg gcaaccttta aatgcagtgt gaccgg 36
<210> 6
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aggttgccca aatactcagt gctttaactt catc 34
<210> 7
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aaagttgatg caccggcccc ggatgtgaat ac 32
<210> 8
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gccggtgcat caactttttc gccaatcagt tt 32
<210> 9
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aagttgatgt ggcagccccg gatgtgaata cca 33
<210> 10
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggctgccaca tcaacttttt cgccaatcag tt 32
<210> 11
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gggtccggat gtgaatacca atggtcagat ta 32
<210> 12
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tattcacatc cggacccggc acatcaactt tttcgc 36
<210> 13
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ggccgcagat gtgaatacca atggtcagat tatg 34
<210> 14
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tattcacatc tgcggccggc acatcaactt tt 32
<210> 15
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgtgattgca ggcaaaccgg tggaatttgg cg 32
<210> 16
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gtttgcctgc aatcacgcca atactactct gaccg 35
<210> 17
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
agcctgggcg caaccgccgc aaccggtttt gg 32
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gcggttgcgc ccaggctgcc gccaaattcc ac 32
<210> 19
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gtgccaccgt gagttatttt gaatgggttc ag 32
<210> 20
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ataactcacg gtggcaccac cggcattggt caga 34
<210> 21
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gtgttaccgt ggcatatttt gaatgggttc agaatctgta t 41
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atatgccacg gtaacaccac cggcattggt ca 32
<210> 23
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gtgttaccgt ggcatatttt gaatgggttc agaatctgta t 41
<210> 24
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
atatgccacg gtaacaccac cggcattggt ca 32
<210> 25
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gtgccaccgt ggcatatttt gaatgggttc ag 32
<210> 26
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
atatgccacg gtggcaccac cggcattggt caga 34
<210> 27
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gcaggtggca ttcgctttca tcagaatgtg ag 32
<210> 28
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aagcgaatgc cacctgcggt cggacccacg gcatc 35
<210> 29
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
agcctgggcg caaccgccgc aaccggtttt gg 32
<210> 30
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gcggttgcgc ccaggctgcc gccaaattcc ac 32
<210> 31
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cgtgattgca ggcaaaccgg tggaatttgg cg 32
<210> 32
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gtttgcctgc aatcacgcca atactactct gaccg 35
<210> 33
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gagtatttgg gcaaccttta aatgcagtgt gaccgg 36
<210> 34
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
aggttgccca aatactcagt gctttaactt catc 34
<210> 35
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gggtgcagat gtgaatacca atggtcagat tatg 34
<210> 36
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tattcacatc tgcacccggc acatcaactt tt 32
<210> 37
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
aagttgatgt ggcagcagat gatgtgaata cca 33
<210> 38
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
acctgccaca tcaacttttt cgccaatcag tt 32
<210> 39
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
aaagttgatg cagcagcaga tgatgtgaat ac 32
<210> 40
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cctgctgcat caactttttc gccaatcagt tt 32
<210> 41
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gcaggtggca ttcgctttca tcagaatgtg ag 32
<210> 42
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
aagcgaatgc cacctgcggt cggacccacg gcatc 35
<210> 43
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
aaagttgatg caccggcccc ggatgtgaat ac 32
<210> 44
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gccggtgcat caactttttc gccaatcagt tt 32
<210> 45
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
aagttgatgt ggcagccccg gatgtgaata cca 33
<210> 46
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ggctgccaca tcaacttttt cgccaatcag tt 32
<210> 47
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
aaagttgcag caccggcccc ggatgtgaat ac 32
<210> 48
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gccggtgctg caactttttc gccaatcagt tt 32
<210> 49
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ggccccggat gtgaatacca atggtcagat ta 32
<210> 50
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tattcacatc cggggccggc acatcaactt tttcgc 36
<210> 51
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tggtgttacc gtggcatatt ttgaatgggt tc 32
<210> 52
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
atgccacggt aacaccaccg gcattggtca ga 32
<210> 53
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gtgcaaccgt gagttatttt gaatgggttc agaatctgt 39
<210> 54
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
ataactcacg gttgcaccac cggcattggt ca 32
<210> 55
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gtggtgttac cgtgagttat tttgaatggg ttcagaatc 39
<210> 56
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
aactcacggt aacaccaccg gcattggtca gaa 33
<210> 57
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 57
agttgatnnk ccggccccgg atgtgaatac ca 32
<210> 58
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 58
gggccggmnn atcaactttt tcgccaatca gtt 33
<210> 59
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 59
ttgatgtgnn kgccccggat gtgaatacca at 32
<210> 60
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 60
cggggcmnnc acatcaactt tttcgccaat ca 32
<210> 61
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 61
atgtgccgnn kccggatgtg aataccaatg gtc 33
<210> 62
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 62
atccggmnnc ggcacatcaa ctttttcgcc aa 32
<210> 63
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 63
agttgatnnk ccggggccgg atgtgaatac ca 32
<210> 64
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 64
gccccggmnn atcaactttt tcgccaatca gtt 33
<210> 65
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 65
tgatatgnnk gggccggatg tgaataccaa tg 32
<210> 66
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 66
ccggcccmnn catatcaact ttttcgccaa tcag 34
<210> 67
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 67
ttgatggtnn kgggccggat gtgaatacca at 32
<210> 68
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 68
cggcccmnna ccatcaactt tttcgccaat ca 32
<210> 69
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (8)..(9)
<223> n is a, c, g, or t
<400> 69
tgatgcannk gggccggatg tgaataccaa tg 32
<210> 70
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(10)
<223> n is a, c, g, or t
<400> 70
ccggcccmnn tgcatcaact ttttcgccaa tc 32

Claims (10)

1. A glutamate dehydrogenase variant, wherein said glutamate dehydrogenase variant comprises a single point mutation or a multiple point combination mutation at position 71, 92, 143, 144, 145, 146, 175, 187, 352, 355 of the amino acid sequence of SEQ ID NO. 1.
2. The glutamate dehydrogenase variant according to claim 1, wherein said glutamate dehydrogenase variant is obtained by single point mutation, and the amino acids at each mutation site and before and after the mutation are respectively: K71A, M92A, V143A, P144A, A145G, P146A, T175A, R187A, V352A, S355A.
3. The glutamate dehydrogenase variant of claim 1, wherein said glutamate dehydrogenase variant is obtained by a combination of mutations in multiple points, in the form of one of the following forms:
(1) sequentially carrying out iterative mutation on two or more adjacent sites according to the arrangement sequence of 145 th, 355 th, 352 th, 71 th, 187 th, 175 th, 92 th, 146 th, 144 th and 143 th;
wherein, each mutation site and the amino acid single letter abbreviations before and after the mutation are respectively: A145G, S355A, V352A, K71A, R187A, T175A, M92A, P146A, P144A, V143A;
(2) carrying out combined mutation on two or more sites of 71 th site, 145 th site, 143 th site and 144 th site;
wherein, each mutation site and the amino acid single letter abbreviations before and after the mutation are respectively: K71A, V143A, V143C, V143G, V143I, V143M, V143S, P144M, P144G and P144A.
4. The glutamate dehydrogenase variant of claim 1, wherein said glutamate dehydrogenase variant is one of the following multiple point mutations:
(I)A145G/S355A，A145G/S355A/V352A，A145G/S355A/V352A/K71A，A145G/S355A/V352A/K71A/R187A，A145G/S355A/V352A/K71A/R187A/T175A，A145G/S355A/V352A/K71A/R187A/T175A/M92A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A/V143A；
(II)A145G/K71A，A145G/P144A，A145G/V143A，K71A/P144A，K71A/V143A，V143A/P144A，A145G/K71A/P144A，A145G/K71A/V143A，A145G/V143A/P144A，K71A/V143A/P144A，A145G/K71A/V143A/P144A；
(III)S355A/V352A/K71A/R187A/T175A，A145G/V352A/K71A/R187A/T175A，A145G/S355A/K71A/R187A/T175A，V352A/K71A/R187A/T175A，S355A/K71A/R187A/T175A，A145G/K71A/R187A/T175A，K71A/R187A/T175A；
(IV)A145G/V143A，A145G/V143C，A145G/V143G，A145G/V143I，A145G/V143M，A145G/V143S，A145G/V143A/P144M，A145G/V143A/P144G，A145G/V143A/P144A。
5. the glutamate dehydrogenase variant of any one of claims 1 to 4, encoding a gene.
6. An expression vector or a genetically engineered bacterium comprising the coding gene of claim 5.
7. Use of the glutamate dehydrogenase variant according to any of claims 1 to 4 or the genetically engineered bacterium according to claim 7 for the preparation of L-norvaline, L-glufosinate-ammonium or L-homophenylalanine.
8. A process for producing L-norvaline, which comprises: taking 2-oxovaleric acid as a substrate, and carrying out catalytic reaction in a buffer solution by using a biocatalyst under the action of an amino donor and a coenzyme regeneration system to obtain L-norvaline;
the biocatalyst is a glutamate dehydrogenase variant or immobilized enzyme thereof, or a genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase variant; the coenzyme adopted by the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH;
the glutamate dehydrogenase variant is obtained by carrying out single-point mutation or multi-point combined mutation on an amino acid sequence shown in SEQ ID NO.1, and specifically comprises one of the following mutations:
(a)K71A、M92A、V143A、P144A、A145G、P146A、T175A、R187A、V352A、S355A；
(b)A145G/S355A，A145G/S355A/V352A，A145G/S355A/V352A/K71A，A145G/S355A/V352A/K71A/R187A，A145G/S355A/V352A/K71A/R187A/T175A，A145G/S355A/V352A/K71A/R187A/T175A/M92A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A/V143A；
(c)A145G/K71A，A145G/P144A，A145G/V143A，K71A/P144A，K71A/V143A，V143A/P144A，A145G/K71A/P144A，A145G/K71A/V143A，A145G/V143A/P144A，K71A/V143A/P144A，A145G/K71A/V143A/P144A。
9. a method of preparing L-glufosinate, comprising: taking 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid as a substrate, and carrying out catalytic reaction in a buffer solution by using a biocatalyst under the action of an amino donor and a coenzyme regeneration system to obtain L-glufosinate-ammonium;
the biocatalyst is a glutamate dehydrogenase variant or immobilized enzyme thereof, or a genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase variant; the coenzyme adopted by the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH;
the glutamate dehydrogenase variant is obtained by carrying out single-point mutation or multi-point combined mutation on an amino acid sequence shown in SEQ ID NO.1, and specifically comprises one of the following mutations: A145G, A145G/S355A, A145G/S355A/V352A, A145G/P144A, A145G/V143A, A145G/V143A/P144A, A145G/V143A, A145G/V143C, A145G/V143G, A145G/V143I, A145G/V143M, A145G/V143S, A145G/V143A/P144M, A145G/V143A/P144G, A145G/V143A/P144A.
10. A method for producing L-homophenylalanine, comprising: 2-oxo-4-phenylbutyric acid is taken as a substrate, and a biological catalyst is utilized to perform catalytic reaction in a buffer solution under the action of an amino donor and a coenzyme regeneration system to obtain L-homophenylalanine;
the biocatalyst is a glutamate dehydrogenase variant or immobilized enzyme thereof, or a genetically engineered bacterium comprising a gene encoding the glutamate dehydrogenase variant; the coenzyme adopted by the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH, the oxidized coenzyme NAD+；
The glutamate dehydrogenase variant is obtained by carrying out single-point mutation or multi-point combined mutation on an amino acid sequence shown in SEQ ID NO.1, and specifically comprises one of the following mutations:
(i)V143A，A145G；
(ii)A145G/S355A，A145G/S355A/V352A/K71A，A145G/S355A/V352A/K71A/R187A，A145G/S355A/V352A/K71A/R187A/T175A，A145G/S355A/V352A/K71A/R187A/T175A/M92A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A，A145G/S355A/V352A/K71A/R187A/T175A/M92A/P146A/P144A/V143A；
(iii)A145G/V352A/K71A/R187A/T175A，A145G/S355A/K71A/R187A/T175A，A145G/K71A/R187A/T175A；A145G/K71A，A145G/V143A，V143A/P144A，A145G/K71A/P144A，A145G/K71A/V143A，A145G/V143A/P144A，A145G/K71A/V143A/P144A。
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