Glutamate dehydrogenase mutant and application thereof

Abstract

The invention provides a glutamate dehydrogenase mutant and application thereof. The glutamate dehydrogenase variant is obtained by performing single-point mutation or multi-point combined mutation on the 22th site, the 99th site, the 185th site, the 197th site, the 200th site, the 201th site, the 202 site, the 204 site, the 357th site, the 369th site, the 370th site, the 374th site, the 376th site, the 378th site, the 379th site, the 380th site, the 381th site, the 382th site, the 383th site, the 386th site, the 387th site, the 390th site, the 391th site, the 392th site, the 394th site, the 395th site, the 395th site, the 398th site, the According to the invention, a glutamate dehydrogenase V143G / A145G variant derived from Clostridium difficile is taken as a basis, and molecular modification is carried out on a hinge region of the variant, so that the catalytic efficiency of the variant on a non-natural substrate is improved, the catalytic reaction time is shortened, mutants capable of catalytically preparing a plurality of L-amino acids except a natural product L-glutamic acid are obtained, and the mutant can be used for preparing L-glutamic acid. The raw material conversion rate and the product ee value are both greater than 99%.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a glutamate dehydrogenase mutant and an application thereof. Background Art

[0002] Chiral amino acids are a class of organic compounds containing carboxyl (-COOH) and amino (-NH2) groups as well as side chain functional groups (R groups) that are unique to each amino acid. Chiral amino acids are widely used in the fields of medicine, chemical industry, and pesticides. In the field of medicine, chiral amino acids are key intermediates for many drugs, such as L-tert-leucine, which is used to synthesize the anti-AIDS drug atazanavir, and L-homoalanine (L-2-aminobutyric acid), which is a key intermediate for the anti-tuberculosis ethambutol and the anti-epileptic drugs levetiracetam and brivaracetam. In the chemical industry, L-homoserine and its derivative O-succinyl-L-homoserine can be used to synthesize a variety of C4 compounds. In the field of pesticides, L-phosphinothricin ammonium is a potential herbicide. In the food field, L-hydroxyproline can be used as a flavor enhancer and nutritional enhancer. In addition, by introducing non-protein amino acids, the limitation of 20 natural protein amino acids can be broken through, and the additional properties and functions of proteins or polypeptides can be regulated and expanded.

[0003] There are four common methods for synthesizing amino acids: protein hydrolysis, fermentation, chemical synthesis and biocatalysis. Protein hydrolysis and fermentation are limited to the production of protein chiral amino acids. Only a few non-protein amino acids such as ornithine and citrulline that exist in organisms can be produced by fermentation. At present, chiral amino acids are mainly synthesized and prepared by chemical synthesis and biocatalysis. Among them, the bioenzyme catalysis method has the characteristics of mild reaction conditions, high stereoselectivity, and green and environmentally friendly process. It is a potential advantageous method for preparing chiral non-protein amino acids. Enzyme production can avoid the shortcomings of high pressure, high temperature, high energy consumption and high pollution of traditional chemical methods. It is an advanced manufacturing model that conforms to the concept of green production.

[0004] Glutamate dehydrogenase (EC 1.4.1.2-4) is a key enzyme in the synthesis of glutamate. It participates in the synthesis and catabolism of glutamate and is widely present in all living organisms. It can be divided into three categories according to coenzyme specificity: NAD(H), NADP(H) and dual coenzyme specificity. Due to its wide source and strict stereoselectivity, glutamate dehydrogenase has great application value and potential in the field of chiral amino acid synthesis. However, wild glutamate dehydrogenase has strong substrate specificity and has almost no enzyme activity or extremely low enzyme activity for other keto acids except the natural substrate α-ketoglutarate, which cannot meet the requirements of industrial applications. Therefore, improving the catalytic ability of wild-type glutamate dehydrogenase for non-natural substrates through molecular modification will help it to be better applied in industry.

[0005] Patent application PCT / CN2018 / 105158 improves the production capacity of glutamate dehydrogenase from multiple sources for L-phosphinothricin and other L-amino acids by modifying the binding pocket. Among them, the mutant LsGluDH-A175G has an enzyme activity of 34.47U / mL for 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO), and the mutants PpGluDH-A167G and PpGluDH-V378A have an enzyme activity of 14.85U / mL and 13.3U / mL for PPO, respectively. The patent application with the patent authorization announcement number CN114196642B is for the modification of the binding pocket of glutamate dehydrogenase from Clostridium difficile, and the best enzyme activities of different mutants for L-phosphinothricin, L-norvaline, and L-homophenylalanine are 10.09U / mg DCW, 18.58U / mgDCW, and 10.69U / mg DCW, respectively. Among the glutamate dehydrogenases reported so far, the activity of glutamate dehydrogenase specific for the cheaper coenzyme NAD(H) is still lower than that of NAD(P)H-specific glutamate dehydrogenase. Its catalytic efficiency for PPO and other α-keto acids is limited, and there is room for further improvement.

[0006] In view of this, further in-depth research is needed to further improve the enzymatic activity of NAD(H)-specific glutamate dehydrogenase towards non-natural substrates through molecular modification. Summary of the invention

[0007] In order to solve the problem that the NAD(H)-specific glutamate dehydrogenase variant derived from Clostridium difficile has low enzymatic activity on non-natural substrates, the present invention provides a variety of glutamate dehydrogenase mutants and applications of these mutants in preparing different L-amino acids.

[0008] The specific technical solutions are as follows:

[0009] The present invention provides a plurality of glutamate dehydrogenase mutants, wherein the glutamate dehydrogenase variants are obtained by performing single-point mutation or multi-point combination mutation at positions 22, 99, 185, 197, 200, 201, 202, 204, 357, 369, 370, 374, 376, 378, 379, 380, 381, 382, ​​383, 386, 387, 390, 391, 392, 394, 395, 397, 398, 399, 401 and 409 of the amino acid sequence shown in SEQ ID NO.1.

[0010] The present invention performs homology modeling on the glutamate dehydrogenase variant V143G / A145G derived from Clostridium difficile (obtained by modifying the glutamate dehydrogenase with NCBI accession number: YP_001086649.1 to glycine at positions 143 and 145, and the amino acid sequence is shown in SEQ ID NO.1), and selects the α-helix (S369-N396) in the hinge region and its Amino acids within the range (K22, L23, V98, T99, Y103, G104, S184, L185, G186, A190, F193, G194, V197, T198, R200, E201, A202, A204, K205, L346, T347, A349, G350, G351, T353, V354, F357, Q361, W368, V397, T398, M399, E401, A402, A403, M405, H406, K409) were transformed, and the above-mentioned positive sites were combined to produce the precursor keto acid 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid (PPO) of L-phosphinothricin ammonium as a model substrate to screen glutamate dehydrogenase positive mutants.

[0011] Finally, some positive mutants were selected to test the activity of precursor keto acids of various amino acids in order to screen out the best mutants for each.

[0012] Furthermore, the glutamate dehydrogenase variant is obtained by the following single point mutations, and the single letter abbreviations of each mutation site and the amino acids before and after the mutation are:

[0013] K22L, K22H, K22I, K22T, T99V, T99M, L185K, L185W, L185Y, L185R, L185H, L185M, L185Q, V197T , V197L, V197M, R200N, R200Q, E201L, E201R, E201K, E201C, A202T, A202M, A202V, A204I, F357 Y, S369A, S369K, S369P, S369N, E370L, E370R, E370Q, E370F, E374T, E374I, K376L, E378H, E37 8Y, E378L, E378T, E378R, I379E, A380R, M381L, V382F, V382L, V382H, K383V, K383T, K383E, E38 6F, E386W, S387K, S387M, S387H, S387L, S387T, S387R, S387I, S387V, K390W, K390A, I391Q, K3 92Q, K392M, K392S, K392H, K392I, E394C, Y395A, Y395C, Y395D, Y395E, Y395F, Y395G, Y395H, Y 395K, Y395M, Y395N, Y395Q, Y395R, Y395S, Y395T, Y395V, Y395W, Y395L, V397Y, V397C, V397G, V397L, T398L, M399A, M399P, M399I, E401L, E401V, E401I, E401T, E401M, E401Y, K409V, K409Q.

[0014] Furthermore, the glutamate dehydrogenase variant is obtained by multi-point combined mutation, and the multi-point combined mutation form is one of the following forms:

[0015] <h2 style=";text-align:left;direction:ltr">(I)K22I / Y395N,K22I / E394A,K22I / K409Q,K22I / E394T,K22I / K409T,K22I / Y395H,K22I / K392T,K22I / K392Q,K22I / Y395S,K22I / Y395A,K22I / E394D,K22I / E394D / Y395M,K22 K22I / E3 I / E394D / K409S、K22I / E394D / Y395V、K22I / E394D / Y395T、K22I / E394D / Y395N 94D / E401T、K22I / Y395A / E401T、K22I / Y395A / E401T / E394Q、K22I / Y395A / E401T / T398S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0016] <h2 style=";text-align:left;direction:ltr"> (II)K383T / E386R,K383T / I379V,K383T / S387L,K383T / E378H,K383T / K390Y,K383T / K390H,K383T / E386Y,K383T / E378F,K383T / S387T,K383T / E378Y,K383T / S387I,K383T / E378Y / S387F,K383 T / E378Y / S387I、K383T / E378Y / K390T、K383T / E378Y / S387M、K383T / E378Y / S387T、K383T / E378Y / I179V、K383T / E378Y / I179S、K383T / E378Y / I179H、K383T / E378Y / I179T、K383T / E378Y / I179A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0017] <h2 style=";text-align:left;direction:ltr">(III)V197L / R200K,V197L / K205R,V197L / A204V,V197L / E201I,V197L / E201R,V197L / E201L,V197L / E201V,V197L / A204R,V197L / A204I,V197L / E201K,V197L / E201K / R200G,V197L / E201K / A204E,V197L / E201K / T9 9I、V197L / E201K / T99M、V197L / E201K / T99V、V197L / E201K / T99V / R200N、V197L / E201K / T99V / R200H、V197 L / E201K / T99V / A204L、V197L / E201K / T99V / R200G、V197L / E201K / T99V / R200A、V197L / E201K / T99V / R200S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0018] <h2 style=";text-align:left;direction:ltr"> (IV)F357Y / S369G, F357Y / E374I, F357Y / E374S, F357Y / E374V, F357Y / S369P, F357Y / E370Y, F357Y / L185K, F357Y / E370L, F357Y / E374C, F357Y / E374T<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0019] <h2 style=";text-align:left;direction:ltr"> F357Y / E374T / S369R, F357Y / E374T / E370L;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0020] (V)K22I / Y395A / E401T / T398S / S369P, K22I / Y395A / E401T / T398S / E370M, K22I / Y395A / E401T / T398S / E370Y, K22I / Y395A / E401T / T398S / A204L, K22I / Y395A / E401T / T398S / T99I, K22I / Y395A / E401T / T398S / K205R, K22I / Y395A / E401T / T398S / T99V, K22I / Y395A / E401T / T398S / I379Q, K2 2I / Y395A / E401T / T398S / S369T, K22I / Y395A / E401T / T398S / K390N, K22I / Y395A / E401T / T398S / V197I, K22I / Y395A / E401T / T398S / S387R, K22I / Y395A / E401T / T398S / S387L, K22I / Y395A / E40 1T / T398S / E201N, K22I / Y395A / E401T / T398S / I379S, K22I / Y395A / E401T / T398S / E370L, K22I / Y 395A / E401T / T398S / T99M, K22I / Y395A / E401T / T398S / T99M / K205R, K22I / Y395A / E401T / T398S / T99M / A202S, K22I / Y395A / E401T / T398S / T99M / E201I, K22I / Y395A / E401T / T398S / T99M / E201C.

[0021] The “ / ” in the mutant description above means “and,” that is, the two sites before and after the “ / ” are mutated at the same time.

[0022] The present invention also provides a gene encoding the glutamate dehydrogenase variant as described in any one of the above items.

[0023] The present invention also provides an expression vector comprising the coding gene as described above. Preferably, the original expression vector is pET-28a(+).

[0024] The present invention also provides a genetically engineered bacterium comprising the above-mentioned coding gene. Preferably, the host cell of the genetically engineered bacterium is E. coli BL21 (DE3).

[0025] The present invention also provides the use of the glutamate dehydrogenase variant or the genetically engineered bacteria in the preparation of L-norvaline and L-phosphinothricin ammonium.

[0026] The present invention also provides a method for preparing L-phosphinothricin ammonium, comprising: using 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, using a biocatalyst to carry out a catalytic reaction in a buffer solution to obtain L-phosphinothricin ammonium; the biocatalyst is such as the glutamate dehydrogenase variant or its immobilized enzyme; or, such as the genetically engineered bacteria.

[0027] The coenzyme used in the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH (oxidized coenzyme NAD + ).

[0028] Furthermore, the coenzyme regeneration system uses alcohol dehydrogenase as the coenzyme regeneration enzyme and isopropanol as the coenzyme regeneration substrate.

[0029] The present invention also provides a method for preparing L-norvaline, comprising: using 2-oxopentanoic acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, using a biocatalyst to carry out a catalytic reaction in a buffer solution to obtain L-norvaline (i.e., 2-aminopentanoic acid); the biocatalyst is such as the glutamate dehydrogenase variant or its immobilized enzyme; or, such as the genetically engineered bacteria.

[0030] The coenzyme used in the coenzyme regeneration system is alcohol dehydrogenase, glucose dehydrogenase or formate dehydrogenase; the reduced coenzyme is NADH (oxidized coenzyme NAD + ).

[0031] The present invention also provides a method for preparing L-2-aminobutyric acid, comprising: using 2-ketobutyric acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, using a biocatalyst to carry out a catalytic reaction in a buffer solution to obtain L-2-aminobutyric acid; the biocatalyst is such as the glutamate dehydrogenase variant or its immobilized enzyme; or, such as the genetically engineered bacteria.

[0032] The present invention also provides a method for preparing L-norleucine, comprising: using 2-oxohexanoic acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, using a biocatalyst to carry out a catalytic reaction in a buffer solution to obtain L-norleucine; the biocatalyst is such as the glutamate dehydrogenase variant or its immobilized enzyme; or, such as the genetically engineered bacteria.

[0033] Furthermore, the coenzyme regeneration system uses alcohol dehydrogenase as the coenzyme regeneration enzyme and isopropanol as the coenzyme regeneration substrate;

[0034] In the preparation of L-phosphinothricin ammonium, the temperature of the catalytic reaction is 30 to 50° C., and the time of the catalytic reaction is 30 to 50 minutes;

[0035] In the preparation of L-norvaline, the catalytic reaction temperature is 30-50°C, and the catalytic reaction time is 160-200 minutes;

[0036] In the preparation of L-2-aminobutyric acid, the catalytic reaction temperature is 30-50°C, and the catalytic reaction time is 300-340 minutes;

[0037] In the preparation of L-norleucine, the temperature of the catalytic reaction is 30-50° C., and the time of the catalytic reaction is 220-260 min.

[0038] Furthermore, in the method for preparing L-phosphinothricin, the glutamate dehydrogenase variant is obtained by the following mutations, and the single-letter abbreviations of each mutation site and the amino acids before and after the mutation are: V143G / A145G / K22I / Y395A / E401T / T398S / T99M / E201C;

[0039] In the method for preparing L-norvaline, the glutamate dehydrogenase variant is obtained by the following mutations, and the single-letter abbreviations of each mutation site and the amino acids before and after the mutation are: V143G / A145G / V197L / E201K / T99V / R200S;

[0040] In the method for preparing L-2-aminobutyric acid or L-norleucine, the glutamate dehydrogenase variant is obtained by the following mutations, and the single-letter abbreviations of each mutation site and the amino acids before and after the mutation are: V197L / E201K / T99V / R200S.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The present invention is based on the glutamate dehydrogenase variant V143G / A145G derived from Clostridium difficile. Through a rationally designed molecular modification method, the catalytic activity of the variant on non-natural substrates is further improved, the catalytic reaction time is shortened, and a mutant that can efficiently catalyze the preparation of a variety of L-amino acids other than the natural product L-glutamate is obtained.

[0043] (2) The semi-rational design method used in the present invention transforms the hinge region site of glutamate dehydrogenase to obtain a glutamate dehydrogenase mutant with higher catalytic activity.

[0044] (3) The present invention uses the corresponding keto acid as a substrate, and generates L-amino acid by glutamate dehydrogenase-catalyzed reductive amination in the presence of inorganic ammonia and coenzyme. The process is simple, and the raw material conversion rate and product chiral purity are both greater than 99%.

[0045] (4) The method of the present invention uses NAD(H) as a coenzyme, which is cheaper than NADP(H), thus greatly reducing the production cost of L-amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 HPLC chromatography of glufosinate and PPO in the reaction sample prepared for the coupling of the mutant CdGluDH-V143G / A145G / K22I / Y395A / E401T / T398S / T99M / E201C with alcohol dehydrogenase.

[0047] Figure 2 This is the HPLC chromatogram of D,L-phosphinothricin standard, L-phosphinothricin standard, and pre-column derivatization of the reaction sample. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0049] The experimental methods in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

[0050] Reagents used in upstream genetic engineering: DPN I used in the examples of the present invention was purchased from TaKaRa, Takara Biotechnology (Dalian) Co., Ltd.; plasmid extraction kits and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21 (DE3), plasmid pET-28a (+), etc. were purchased from Novagen; DNA marker, low molecular weight standard protein, agarose electrophoresis reagents were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; primer synthesis and sequence sequencing were completed by Qingke Biotechnology Co., Ltd. The use of the above reagents refers to the product instructions.

[0051] Reagents used in the downstream catalytic process: PPO and L-glufosinate were synthesized in the laboratory, pyruvic acid, 2-ketobutyric acid, 2-oxohexanoic acid, and benzoylformic acid were purchased from Shanghai McLean Biochemical Technology Co., Ltd., 2-ketopentanoic acid was purchased from Shanghai Qiaoyi Biotechnology Co., Ltd., 3-methyl 2-oxobutyric acid was purchased from Acros organics, 3,3-dimethyl 2-oxobutyric acid and 4-methyl 2-oxopentanoic acid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., 2-oxo-4-phenylbutyric acid was purchased from Shanghai Bid Pharmaceutical, D,L-glufosinate was purchased from Sigma-Aldrich; 2-ketoglutaric acid and other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0052] Glutamate dehydrogenase activity standard detection system (HPLC method): appropriate amount of enzyme solution, 80mM substrate, 40mM NADH, 2M NH4 + ((NH4)2SO4), the total system is 500μL, the reaction medium is pH 7.5 0.1M phosphate buffer. The reaction is carried out at 40℃ for 10min, and 500μL 1M NaOH or 1M HCl is added to terminate the reaction. The L-amino acids generated in the sample are quantitatively analyzed by HPLC.

[0053] Definition of enzyme activity unit (U): the amount of enzyme required to generate 1 μmol L-amino acid per minute under standard reaction conditions.

[0054] HPLC chiral analysis method for aliphatic amino acids: pre-column derivatization is used for determination. Derivatization reagent: weigh 0.03g of o-phthalaldehyde and 0.1g of N-acetyl-L-cysteine ​​respectively, dissolve them with 400μL of ethanol, then add 4mL of 0.2mol / borate buffer (pH 9.8), shake to fully dissolve, and store in a refrigerator at 4℃ for use (no more than 4 days). Derivatization reaction and determination: take 100μL of sample and add 100μL of derivatization reagent, mix well and keep warm at 25℃ for 5min. Chromatographic conditions: chromatographic column / QS-C18; detection wavelength / 334nm; column temperature / 30℃; injection volume / 20μL; mobile phase A: 50mM sodium acetate aqueous solution, mobile phase B: acetonitrile, mobile phase C: methanol, mobile phase ratio (V / V) is shown in Table 1.

[0055] Table 1 HPLC mobile phase ratio

[0056]

[0057] HPLC analysis method of PPO: chromatographic column / QS-C18; detection wavelength / UV 205nm; column temperature / 40℃; injection volume / 20μL; flow rate / 1mL / min; mobile phase / 50mM (NH4)2HPO4 aqueous solution, add 1% of 10% tetrabutylammonium hydroxide aqueous solution, adjust the pH to 3.6 with 50% phosphoric acid, and add 8% acetonitrile.

[0058] HPLC analysis method of 2-oxopentanoic acid: chromatographic column / QS-C18; detection wavelength / UV 205nm; column temperature / 40℃; injection volume / 20μL; flow rate / 1mL / min; mobile phase / 50mM (NH4)2HPO4 aqueous solution:acetonitrile=95:5.

[0059] Standard detection system for glutamate dehydrogenase activity (enzyme reader method): The specific enzyme activity of CdGluDH starting enzyme and mutants on different ketoacid substrates was determined by monitoring the absorbance change of NADH at 340nm during the reduction amination process by the enzyme reader method. During the detection, 100μL of 2× reaction solution (see Table 2 for detailed system) was first added to the enzyme plate, and then 100μL of purified enzyme protein diluted to an appropriate concentration was added, and the plate was placed in the enzyme reader, and a time scan was performed at 340nm for a total of 3min, and the absorbance value data was taken every 15s. In addition, the standard curve of NADH needs to be determined for enzyme activity calculation.

[0060] Enzyme activity unit (U) definition: the amount of enzyme required to oxidize 1 μmol NADH per minute under standard reaction conditions.

[0061] Table 2 2× reaction solution system

[0062]

[0063] Example 1 Construction and screening of single point mutation library

[0064] The 22nd, 23rd, 98th, 99th, 103rd, 104th, 184th, 185th, 186th, 190th, 193rd, 194th, 197th, 198th, 200th, 201st, 202nd, 204th, 205th, 346th, 347th, 349th, 350th, 351st, 353rd, 354th, 357th, 361st, 368th, 369th, 370th, 371st, Single point saturation mutation at position 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 401, 402, 403, 405, 406, 409.

[0065] Among them, CdGluDH-V143G / A145G is based on glutamate dehydrogenase from Clostridium difficile (CdGluDH, NCBI accession number: YP_001086649.1), and is obtained by mutating valine at position 143 to glycine and alanine at position 145 to glycine.

[0066] The amino acid sequence of CdGluDH-V143G / A145G is shown in SEQ ID NO.1; the nucleotide sequence of CdGluDH-V143G / A145G is shown in SEQ ID NO.2.

[0067] SEQ ID NO.1: MSGKDVNVFEMAQSQVKNACDKLGMEPAVYELLKEPMRVIEVSIPVKMDDGSIKTFKGFRSQHNDAVGPTKGGIRFHQNVSRDEVKALSIWMTFKCSVTGIPYGGGKGGIIVDPSTLSQGELERLSRGYIDGIYKLIGEKVDGPGPDVNTNGQIMSWMVDEYNKLTGQSSIGVITGKPVEFGGSLGRTAATGFGVAVTAREAAAKLGIDMKKAKIAVQGIGNVGSYTVLNCEKLGGTVVAMAEWCKSEGSYAIYNENGLDGQAMLDYMKEHGNLLNFPGAKRISLEEFWASDVDIVIPAALENSITKEVAESIKAKLVCEAANGPTTPEADEVFAERGIVLTPDILTNAGGVTVSYFEWVQNLYGYYWSEEEVEQKEEIAMVKAFESIWKIKEEYNVTMREAAYMHSIKKVAEAMKLRGWY.

[0068]

[0069] The specific method is as follows:

[0070] 1. Whole plasmid PCR

[0071] Using pET-28a(+)-CdGluDH plasmid as a template, upstream and downstream primers covering the mutation point were designed (Appendix Table 1) for whole plasmid PCR.

[0072] PCR amplification system:

[0073]

[0074] PCR amplification conditions:

[0075] 1) Pre-denaturation: 95℃ for 5min;

[0076] 2) Denaturation: 98°C for 10 s; annealing: 58°C for 15 s; extension: 72°C for 10 s; 30 cycles in total;

[0077] 3) Post-extension: 72°C for 10 min;

[0078] 4) Store at 4℃.

[0079] 2. Template digestion:

[0080] The PCR product was subjected to agarose gel electrophoresis and then digested with Dpn1 enzyme. The plasmid template digestion system was as follows: 1 μL Dpn1 enzyme, 17 μL PCR product, and 2 μL Buffer. The template digestion was completed at 37°C for 2 hours.

[0081] 3. Conversion:

[0082] The digested product was verified by nucleic acid agarose gel electrophoresis and then transformed into E. coli BL21 (DE3) competent cells using the 42°C heat shock method. The specific process is as follows:

[0083] (1) Thaw the competent cells on ice for 15 min;

[0084] (2) Add 10 μL DNA to 100 μL competent cells under sterile conditions and mix gently.

[0085] Place on for 30 minutes;

[0086] (3) The EP tube was placed in a 42°C metal bath for 90 seconds for heat shock and then cooled on ice for 2 minutes.

[0087] (4) Add 800 μL of LB medium to the EP tube and mix well with a pipette tip, then incubate at 37°C in a shaker at 200 rpm for 40-60 min;

[0088] (5) After concentration, take an appropriate volume and apply it to the corresponding resistance plate. Incubate in a 37°C incubator for 12-16 hours until colonies appear.

[0089] 4. High-throughput screening assay

[0090] The recombinant bacteria of site-directed saturation mutagenesis were cultured in 96-well plates.

[0091] First, take a sterilized 96-well plate as plate A, add 200 μL of LB medium with kanamycin resistance to each well, pick a single colony into each well with a sterilized toothpick, and culture plate A at 37°C and 200 rpm for 8 hours.

[0092] Take another sterilized 96-well plate as plate B, add 400 μL of LB medium with kanamycin resistance to each well, take 50 μL of culture solution from plate A to plate B, add 20% glycerol to plate A and store at -80°C. After transfer, plate B is placed at 37°C and 200 rpm for 3 hours, add 50 μL of LB containing 5 mM IPTG to each well, and induce at 18°C ​​and 200 rpm for 16-18 hours.

[0093] After the culture was completed, plate B was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the plate was washed once with phosphate buffer. The collected bacteria were stored at -80°C for later use.

[0094] Cell disruption: Place plate B at -80℃ for more than 12 hours, take it out, dilute the 50× lysate to 1× with phosphate buffer, add 300μL to each well, shake to completely resuspend the cells, and incubate in a shaker at 37℃ and 200rpm for 1 hour. After the end, centrifuge at 4℃ and 4000rpm for 20 minutes, discard the precipitate, and take the supernatant as the crude enzyme solution.

[0095] Reaction solution preparation: Prepare 2× reaction solution as shown in Table 1.

[0096] Enzyme activity determination: Add 200 μL of 2× reaction solution to each well of a new 96-well plate, and then dilute the crude enzyme solution to an appropriate concentration in another 96-well plate. After keeping the two 96-well plates at 37°C for 10 minutes, take 200 μL of enzyme solution and add it to the reaction solution, place it on a shaker at 37°C and 200 rpm for 20 minutes, take a sample of 100 μL and add it to an ELISA plate pre-filled with 100 μL of deionized water to dilute it by one half, and use an ELISA reader to measure the absorbance at a wavelength of 340 nm. The lower the absorbance, the higher the enzyme activity. Compare the control group in the plate, and select the points with increased enzyme activity for subsequent rescreening.

[0097] 5. Rescreening enzyme activity assay

[0098] According to the results of the microplate reader, samples were taken from the corresponding wells of plate A for streaking, and a single colony was inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking for 12 h. A 2% inoculum was transferred to 50 mL LB liquid medium also containing 50 μg / mL kanamycin, and cultured at 37°C with shaking until OD 600 When the concentration reaches about 0.6-0.8, add IPTG to a final concentration of 0.5mM, and induce the culture at 18℃ for 16h. After the culture is completed, centrifuge the culture solution at 12000g for 10min at 4℃, discard the supernatant, and collect the bacteria. Wash the collected bacteria twice with 50mM pH 7.5 phosphate buffer, resuspend in phosphate buffer, and ultrasonically disrupt 30 times at 400W power, each ultrasonication lasting 3s, with a rest of 7s. Centrifuge the cell disrupted solution at 12000g for 10min at 4℃ to remove the precipitate, and the supernatant obtained is the crude enzyme solution.

[0099] Table 3 Primer sequences

[0100]

[0101]

[0102] Table 3 Primer sequences (continued)

[0103]

[0104]

[0105] Table 3 Primer sequences (continued)

[0106]

[0107]

[0108] Table 3 Primer sequences (continued)

[0109]

[0110]

[0111] Table 3 Primer sequences (continued)

[0112]

[0113] The enzyme activity of the mutant library against PPO was determined by HPLC analysis. The results of the positive mutation point determination are shown in Table 4:

[0114] Table 4 Determination of the enzyme activity of glutamate dehydrogenase V143G / A145G and its positive single point mutants on PPO

[0115]

[0116]

[0117] Example 2 Group Iterative Saturation Mutation Library Construction and Screening

[0118] The 31 sites with positive mutations were divided into 4 groups according to spatial position, interaction and other factors, and iterative saturation mutation was performed within the group according to the greedy algorithm.

[0119] The specific operation steps are: select the mutant with the highest enzyme activity in the group as the starting enzyme for the next round, perform saturation mutation on all other mutation sites in the group, then select the mutant with the highest enzyme activity from the saturated mutation sites as the starting enzyme, and perform saturation mutation on the remaining sites in the group again, and repeat this process until all sites have been screened or a new round of saturation mutation does not produce a positive mutant with increased enzyme activity.

[0120] The detailed PCR operation, transformation, primary screening and secondary screening steps are the same as those in Example 1.

[0121] The enzyme activity of the mutant library against PPO was determined by HPLC analysis. The results of the positive mutation point determination are shown in Table 5:

[0122] Table 5 Determination of PPO enzyme activity of positive mutants in the iterative saturation mutation library of glutamate dehydrogenase

[0123]

[0124]

[0125]

[0126] In order to better perform inter-regional synergistic mutations and further obtain higher enzyme activity, the screening results within the group, this section will start from K22I / Y395A / E401T / T398S with the highest enzyme activity at present, and iterate the other three groups according to the greedy algorithm. The detailed PCR operation, transformation, primary screening, and rescreening steps are the same as those in Example 1. The enzyme activity of the above mutant library on PPO was determined by HPLC analysis, and the results of positive mutation point determination are shown in Table 6:

[0127] Table 6 Determination of enzyme activity of positive mutants of glutamate dehydrogenase intergroup iterative saturation mutation library against PPO

[0128]

[0129] Subsequently, the mutant with the highest enzyme activity in each round was subjected to comparative enzyme activity determination. The detailed steps for obtaining the crude enzyme solution were the same as those in Example 1. The pure enzyme solution was obtained by nickel affinity chromatography, and the enzyme activity was determined by HPLC. The results are shown in Table 7:

[0130] Table 7 Specific enzyme activity of mutants against PPO

[0131]

[0132] Example 4 Substrate spectrum determination

[0133] The catalytic efficiency of the mutant with the highest enzyme activity in each round in Example 3 for different α-keto acids was determined by the ELISA method. The results are shown in Table 8:

[0134] Table 8 Catalytic efficiency of different starting strains for different α-keto acids

[0135]

[0136]

[0137] Unit: U / mg protein

[0138] Example 5 Preparation of L-phosphinothricin by coupling of glutamate dehydrogenase mutant (CdGluDH-V143G / A145G / K22I / Y395A / E401T / T398S / T99M / E201C) and glucose dehydrogenase

[0139] Culture of bacteria and preparation of crude enzyme solution: After glycerol tubes of engineered bacteria expressing CdGluDH-A145G / V143A / P144G glutamate dehydrogenase mutant gene and glucose dehydrogenase gene (NCBI accession number: WP_087960837.1, base sequence cloned into expression plasmid pET-28a(+), inserted with restriction sites BamHⅠ and HindⅢ, transferred into expression host E.coli BL21(DE3)) were activated by streaking on a plate, single colonies were picked and inoculated into 50mL LB liquid medium containing 50μg / mL kanamycin, and cultured at 37℃ with shaking for 12h. Transferred to 1L fresh LB liquid medium containing 50μg / ml Kan at a 2% inoculum, and cultured at 37℃ with shaking until OD 600 When the pH reached about 0.6, IPTG was added to a final concentration of 0.5 mM, and the culture was induced for 16 h at 18° C. After the culture was completed, the culture solution was centrifuged at 12000 g for 10 min at 4° C., the cells were collected, and the cells were broken by ultrasonic wave to prepare the crude enzyme solution.

[0140] The reaction system is 50 mL, containing 200 mM substrate PPO, 300 mM glucose, 200 mM (NH4)2SO4 and 0.2 mM NAD +; The concentration of glutamate dehydrogenase bacteria (wet weight) was 1 g / L, and the concentration of glucose dehydrogenase bacteria (wet weight) was 2 g / L. The reaction temperature was controlled at 40°C by a water bath, and the pH was controlled at 8.0 by dropping ammonia water during the reaction. After 1.5 hours of reaction, the residual concentration of PPO was detected by HPLC, and the generation amount and ee value of L-phosphinothricin were detected by pre-column derivatization high performance liquid chromatography.

[0141] The data at the end of the reaction are as follows: the raw material PPO is below the detection limit, and the conversion rate is >99%; the generated concentration of L-glufosinate is 32.3 g / L, and the ee value is >99%.

[0142] Example 6 Preparation of L-phosphinothricin by coupling glutamate dehydrogenase starting strains and mutants with alcohol dehydrogenase

[0143] The engineered bacteria expressing the glutamate dehydrogenase mutant genes of CdGluDH-V143G / A145G and CdGluDH-V143G / A145G / K22I / Y395A / E401T / T398S / T99M / E201C and alcohol dehydrogenase (NCBI accession number: NZ_JYNW01000069.1, the base sequence was cloned into the expression plasmid pET-28a(+), the restriction sites were BamHI and HindIII, and the cells were transferred into the expression host E.coli BL21(DE3)) were cultured in the same manner as in Example 5, the cells were collected by centrifugation, and the cells were ruptured by ultrasound to prepare a crude enzyme solution.

[0144] The reaction system is 50 mL, containing 200 mM substrate PPO, 300 mM isopropanol, 200 mM (NH4)2SO4 and 0.2 mM NAD + ; The concentration of glutamate dehydrogenase bacteria (wet weight) was 2 g / L, and the concentration of alcohol dehydrogenase bacteria (wet weight) was 4 g / L. The reaction temperature was controlled at 40°C by a water bath, and the pH was controlled at 8.0 by dropping ammonia water during the reaction. Samples were taken at regular intervals, and the residual concentration of PPO was detected by HPLC. At the same time, the final production amount and ee value of L-phosphinothricin were detected by pre-column derivatization high performance liquid chromatography.

[0145] CdGluDH-V143G / A145G catalyzed the reaction for 75 min: the raw material PPO was below the detection limit, and the conversion rate was >99%; the generated concentration of L-glufosinate was 31.6 g / L, and the ee value was >99%.

[0146] CdGluDH-V143G / A145G / K22I / Y395A / E401T / T398S / T99M / E201C catalyzed the reaction for 40 min: the raw material PPO was below the detection limit, and the conversion rate was >99%; the generated concentration of L-glufosinate was 31.8 g / L, and the ee value was >99%;

[0147] Example 7 Preparation of L-norvaline by dual enzyme coupling of wild-type and mutant glutamate dehydrogenase and alcohol dehydrogenase

[0148] The expression of CdGluDH-V143G / A145G and

[0149] The engineered bacteria expressing the mutant gene of glutamate dehydrogenase CdGluDH-V143G / A145G / V197L / E201K / T99V / R200S and alcohol dehydrogenase (NCBI accession number: NZ_JYNW01000069.1) were collected by centrifugation and the cells were ruptured by ultrasonication to prepare a crude enzyme solution.

[0150] The reaction system is 50 mL, containing 200 mM substrate 2-oxopentanoic acid, 300 mM isopropanol, 200 mM (NH4)2SO4 and 0.2 mM NAD + ; The concentration of glutamate dehydrogenase bacteria (wet weight) was 2 g / L, and the concentration of alcohol dehydrogenase bacteria (wet weight) was 4 g / L. The reaction temperature was controlled at 40°C by a water bath, and the pH was controlled at 8.0 by adding ammonia water during the reaction. Samples were taken at regular intervals to detect the residual concentration of 2-oxopentanoic acid and the final production amount and ee value of L-norvaline.

[0151] CdGluDH-V143G / A145G catalyzed the reaction for 280 min: the raw material 2-oxopentanoic acid was below the detection limit, and the conversion rate was >99%; the generated concentration of L-norvaline was 22.0 g / L, and the ee value was >99%;

[0152] CdGluDH-V143G / A145G / V197L / E201K / T99V / R200S catalyzed the reaction for 180 min: the raw material 2-oxopentanoic acid was below the detection limit, and the conversion rate was >99%; the generated concentration of L-norvaline was 21.7 g / L, and the ee value was >99%.

[0153] Example 8 Preparation of L-2-aminobutyric acid by dual enzyme coupling of wild-type and mutant glutamate dehydrogenase and alcohol dehydrogenase

[0154] The engineered bacteria expressing the CdGluDH-V197L / E201K / T99V / R200S glutamate dehydrogenase mutant gene and alcohol dehydrogenase (NCBI accession number: NZ_JYNW01000069.1) were cultured in the same manner as in Example 5, the cells were collected by centrifugation, and the cells were disrupted by ultrasound to prepare a crude enzyme solution.

[0155] The reaction system is 50 mL, containing 100 mM substrate 2-butyric acid, 300 mM isopropanol, 200 mM (NH4)2SO4 and 0.2 mM NAD +; The concentration of glutamate dehydrogenase bacteria (wet weight) was 10 g / L, and the concentration of alcohol dehydrogenase bacteria (wet weight) was 4 g / L. The reaction temperature was controlled at 40°C by a water bath, and the pH was controlled at 8.0 by adding ammonia water during the reaction. Samples were taken at regular intervals to detect the residual concentration of 2-ketobutyric acid and the final production amount and ee value of L-2-aminobutyric acid.

[0156] CdGluDH-V197L / E201K / T99V / R200S catalyzed the reaction for 320 min: the raw material 2-butyric acid was below the detection limit, the conversion rate exceeded 99%, the generated concentration of L-2-aminobutyric acid was 92 mM, and the ee value was >99%.

[0157] Example 9 Preparation of L-norleucine by dual enzyme coupling of wild-type and mutant glutamate dehydrogenase and alcohol dehydrogenase

[0158] The engineered bacteria expressing the CdGluDH-V197L / E201K / T99V / R200S glutamate dehydrogenase mutant gene and alcohol dehydrogenase (NCBI accession number: NZ_JYNW01000069.1) were cultured in the same manner as in Example 5, the cells were collected by centrifugation, and the cells were disrupted by ultrasound to prepare a crude enzyme solution.

[0159] The reaction system is 50 mL, containing 100 mM substrate 2-oxohexanoic acid, 300 mM isopropanol, 200 mM (NH4)2SO4 and 0.2 mM NAD + ; The concentration of glutamate dehydrogenase bacteria (wet weight) was 2 g / L, and the concentration of alcohol dehydrogenase bacteria (wet weight) was 4 g / L. The reaction temperature was controlled at 40°C by a water bath, and the pH was controlled at 8.0 by adding ammonia water during the reaction. Samples were taken at regular intervals to detect the residual concentration of 2-oxohexanoic acid and the final production amount and ee value of L-norleucine.

[0160] CdGluDH-V197L / E201K / T99V / R200S catalyzed the reaction for 240 min: the raw material 2-oxohexanoic acid was below the detection limit, the conversion rate exceeded 99%, the generated concentration of L-norleucine was 93.8 mM, and the ee value was >99%.

Claims

1. A glutamate dehydrogenase mutant, characterized in that The glutamate dehydrogenase variant is obtained by performing single point mutation or multi-point combination mutation at position 22, position 99, position 185, position 197, position 200, position 201, position 202, position 204, position 357, position 369, position 370, position 374, position 376, position 378, position 379, position 380, position 381, position 382, ​​position 383, position 386, position 387, position 390, position 391, position 392, position 394, position 395, position 397, position 398, position 399, position 401, and position 409 of the amino acid sequence shown in SEQ ID NO.

1.

2. The glutamate dehydrogenase mutant according to claim 1, characterized in that The glutamate dehydrogenase variant is obtained by the following single point mutations, and the single letter abbreviations of each mutation site and the amino acids before and after the mutation are: K22L, K22H, K22I, K22T, T99V, T99M, L185K, L185W, L185Y, L185R, L185H, L185M, L185Q, V197T , V197L, V197M, R200N, R200Q, E201L, E201R, E201K, E201C, A202T, A202M, A202V, A204I, F357 Y, S369A, S369K, S369P, S369N, E370L, E370R, E370Q, E370F, E374T, E374I, K376L, E378H, E37 8Y, E378L, E378T, E378R, I379E, A380R, M381L, V382F, V382L, V382H, K383V, K383T, K383E, E38 6F, E386W, S387K, S387M, S387H, S387L, S387T, S387R, S387I, S387V, K390W, K390A, I391Q, K3 92Q, K392M, K392S, K392H, K392I, E394C, Y395A, Y395C, Y395D, Y395E, Y395F, Y395G, Y395H, Y 395K, Y395M, Y395N, Y395Q, Y395R, Y395S, Y395T, Y395V, Y395W, Y395L, V397Y, V397C, V397G, V397L, T398L, M399A, M399P, M399I, E401L, E401V, E401I, E401T, E401M, E401Y, K409V, K409Q.

3. The glutamate dehydrogenase mutant according to claim 1, characterized in that The glutamate dehydrogenase variant is one of the following multiple point mutations: <h2 style=";text-align:left;direction:ltr">(I)K22I / Y395N,K22I / E394A,K22I / K409Q,K22I / E394T,K22I / K409T,K22I / Y395H,K22I / K392T,K22I / K392Q,K22I / Y395S,K22I / Y395A,K22I / E394D,K22I / E394D / Y395M,K22 K22I / E3 I / E394D / K409S、K22I / E394D / Y395V、K22I / E394D / Y395T、K22I / E394D / Y395N 94D / E401T、K22I / Y395A / E401T、K22I / Y395A / E401T / E394Q、K22I / Y395A / E401T / T398S; <h2 style=";text-align:left;direction:ltr"> (II)K383T / E386R,K383T / I379V,K383T / S387L,K383T / E378H,K383T / K390Y,K383T / K390H,K383T / E386Y,K383T / E378F,K383T / S387T,K383T / E378Y,K383T / S387I,K383T / E378Y / S387F,K383 T / E378Y / S387I、K383T / E378Y / K390T、K383T / E378Y / S387M、K383T / E378Y / S387T、K383T / E378Y / I179V、K383T / E378Y / I179S、K383T / E378Y / I179H、K383T / E378Y / I179T、K383T / E378Y / I179A; <h2 style=";text-align:left;direction:ltr"> (III)V197L / R200K,V197L / K205R,V197L / A204V,V197L / E201I,V197L / E201R,V197L / E201L,V197L / E201V,V197L / A204R,V197L / A204I,V197L / E201K,V197L / E201K / R200G,V197L / E201K / A204E,V197L / E201K / T9 9I、V197L / E201K / T99M、V197L / E201K / T99V、V197L / E201K / T99V / R200N、V197L / E201K / T99V / R200H、V197 L / E201K / T99V / A204L、V197L / E201K / T99V / R200G、V197L / E201K / T99V / R200A、V197L / E201K / T99V / R200S; (IV)F357Y / S369G, F357Y / E374I, F357Y / E374S, F357Y / E374V, F357Y / S369P, F357Y / E370Y, F357 Y / L185K, F357Y / E370L, F357Y / E374C, F357Y / E374T, F357Y / E374T / S369R, F357Y / E374T / E370L; (V)K22I / Y395A / E401T / T398S / S369P, K22I / Y395A / E401T / T398S / E370M, K22I / Y395A / E401T / T398S / E370Y, K22I / Y395A / E401T / T398S / A204L, K22I / Y395A / E401T / T398S / T99I, K22I / Y395A / E401T / T398S / K205R, K22I / Y395A / E401T / T398S / T99V, K22I / Y395A / E401T / T398S / I379Q, K2 2I / Y395A / E401T / T398S / S369T, K22I / Y395A / E401T / T398S / K390N, K22I / Y395A / E401T / T398S / V197I, K22I / Y395A / E401T / T398S / S387R, K22I / Y395A / E401T / T398S / S387L, K22I / Y395A / E40 1T / T398S / E201N, K22I / Y395A / E401T / T398S / I379S, K22I / Y395A / E401T / T398S / E370L, K22I / Y 395A / E401T / T398S / T99M, K22I / Y395A / E401T / T398S / T99M / K205R, K22I / Y395A / E401T / T398S / T99M / A202S, K22I / Y395A / E401T / T398S / T99M / E201I, K22I / Y395A / E401T / T398S / T99M / E201C.

4. A gene encoding the glutamate dehydrogenase variant according to any one of claims 1 to 3.

5. An expression vector or genetically engineered bacteria comprising the coding gene according to claim 4.

6. Use of the glutamate dehydrogenase variant according to any one of claims 1 to 3 or the genetically engineered bacteria according to claim 7 in the preparation of L-norvaline and L-phosphinothricin.

7. A method for preparing L-phosphinothricin ammonium, characterized in that: include: Using 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, a biocatalyst is used to carry out a catalytic reaction in a buffer solution to obtain L-phosphinothricin ammonium; The biocatalyst is the glutamate dehydrogenase variant or immobilized enzyme thereof according to any one of claims 1 to 3; Or, the genetically engineered bacteria as described in claim 7.

8. A method for preparing L-norvaline, characterized in that: include: Using 2-oxopentanoic acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, a biocatalyst is used to carry out a catalytic reaction in a buffer solution to obtain L-norvaline; The biocatalyst is the glutamate dehydrogenase variant or immobilized enzyme thereof according to any one of claims 1 to 3; Or, the genetically engineered bacteria as described in claim 7.

9. A method for preparing L-2-aminobutyric acid, characterized in that: include: Using 2-ketobutyric acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, a biocatalyst is used to carry out a catalytic reaction in a buffer solution to obtain L-2-aminobutyric acid; The biocatalyst is the glutamate dehydrogenase variant or immobilized enzyme thereof according to any one of claims 1 to 3; Or, the genetically engineered bacteria as described in claim 7.

10. A method for preparing L-norleucine, characterized in that: include: Using 2-oxohexanoic acid as a substrate, under the action of an amino donor and a coenzyme regeneration system, a biocatalyst is used to carry out a catalytic reaction in a buffer solution to obtain L-norleucine; The biocatalyst is the glutamate dehydrogenase variant or immobilized enzyme thereof according to any one of claims 1 to 3; Or, the genetically engineered bacteria as described in claim 7.

11. The method according to any one of claims 7 to 10, characterized in that: The coenzyme regeneration system uses alcohol dehydrogenase as the coenzyme regeneration enzyme and isopropanol as the coenzyme regeneration substrate; In the preparation of L-phosphinothricin, the catalytic reaction temperature is 30-50°C, and the catalytic reaction time is 30-50min; In the preparation of L-norvaline, the catalytic reaction temperature is 30-50°C, and the catalytic reaction time is 160-200 minutes; In the preparation of L-2-aminobutyric acid, the catalytic reaction temperature is 30-50°C, and the catalytic reaction time is 300-340 minutes; In the preparation of L-norleucine, the temperature of the catalytic reaction is 30-50° C., and the time of the catalytic reaction is 220-260 min.

12. The method according to any one of claims 7 to 10, characterized in that: In the method of claim 7, the glutamate dehydrogenase variant is obtained by the following mutations, and the single-letter abbreviations of each mutation site and the amino acids before and after the mutation are: V143G / A145G / K22I / Y395A / E401T / T398S / T99M / E201C; In the method of claim 8, the glutamate dehydrogenase variant is obtained by the following mutations, and the single-letter abbreviations of each mutation site and the amino acids before and after the mutation are: V143G / A145G / V197L / E201K / T99V / R200S; In the method according to claim 9 or claim 10, the glutamate dehydrogenase variant is obtained by the following mutations, and the single-letter abbreviations of each mutation site and the amino acids before and after the mutation are: V197L / E201K / T99V / R200S.

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