L-GLUTAMATE DEHYDROGENASE MUTANT AND APPLICATION THEREOF

Abstract

Disclosed in the present invention is an L-glutamate dehydrogenase mutant, the sequence of the L-glutamate dehydrogenase mutant being a sequence in which amino acid residue A at position 175 in SEQ ID NO: 1 is mutated to be G, and amino acid residue V at position 386 is mutated to be an amino acid residue having less steric hindrance. Further disclosed in the present invention is an application of the described L-amino acid dehydrogenase mutant in the preparation of L-glufosinate-ammonium or a salt thereof. When the L-glutamate dehydrogenase mutant of the present invention is used to prepare L-glufosinate-ammonium or a salt thereof, compared to an L-glutamate dehydrogenase mutant in which only position 175 or 386 is mutated, the specific enzyme activity is higher. Therefore, the action efficiency of the enzyme is improved, reaction costs are reduced, and industrial production is facilitated.

Claims

1. An L-glutamate dehydrogenase mutant, wherein the L-glutamate dehydrogenase mutant has a sequence obtained by mutating amino acid residue A at position 175 of SEQ ID NO: 1 to G and mutating amino acid residue V at position 386 to an amino acid residue with smaller steric hindrance; wherein the L-glutamate dehydrogenase mutant has catalytic activity on 2-oxo-4-(hydroxymethylphosphinyl)-butyric acid or salts thereof.

2. The L-glutamate dehydrogenase mutant of claim 1, wherein the amino acid sequence of L-glutamate dehydrogenase mutant is set forth as SEQ ID NO: 7 or SEQ ID NO: 9.

3. An isolated nucleic acid, wherein the nucleic acid encodes the L-glutamate dehydrogenase mutant of claim 1.

4. A recombinant expression vector comprising the nucleic acid of claim 3.

5. A transformant comprising the nucleic acid of claim 3.

6. A method for preparing an L-glufosinate salt, comprising: subjecting 2-oxo-4-(hydroxymethylphosphinyl) butyrate to an amination reaction to obtain an L-glufosinate salt in the presence of a reaction solvent, an L-glutamate dehydrogenase mutant, an inorganic amino donor and a reduced coenzyme NADPH; wherein the L-glutamate dehydrogenase mutant is the L-glutamate dehydrogenase mutant of claim 1.

7. The method of claim 6, further comprising: subjecting a D-glufosinate salt to an oxidation reaction to obtain the 2-oxo-4-(hydroxymethylphosphinyl) butyrate in the presence of a D-amino acid oxidase.

8. The method of claim 6, wherein the L-glutamate dehydrogenase mutant has a concentration of 0.05-3 U/m; and/or, the inorganic amino donor has a concentration of 100-2000 mM; and/or, the 2-oxo-4-(hydroxymethylphosphinyl) butyrate has a concentration of 100-600 mM; and/or, the mass ratio of the reduced coenzyme NADPH to the 2-oxo-4-(hydroxymethylphosphinyl) butyrate is 1 :100-1 :20000; and/or, the inorganic amino donor is one or more of ammonia, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, ammonium acetate, ammonium formate and ammonium bicarbonate; and/or, the reaction solvent is water; and/or, the amination reaction has a reaction system with a pH of 7-9; and/or, the amination reaction has a reaction system with a temperature of 20-50° C.

9. The method of claim 6, further comprising: subjecting an oxidized coenzyme NADP.sup.+ to a reduction reaction to obtain the reduced coenzyme NADPH in the presence of a dehydrogenase and a hydrogen donor.

10. A method for preparing an L-glufosinate, comprising: (a) preparing an L-glufosinate salt according to the method of claim 6; and (b) subjecting the L-glufosinate prepared in step (a) to an acidification reaction to obtain an L-glufosinate.

11. A method for preparing an L-glufosinate salt, comprising: (a) subjecting a D-glufosinate salt is subjected to an oxidation reaction to obtain the 2-oxo-4-(hydroxymethylphosphinyl) butyrate in the presence of a D-amino acid oxidase; and (b) subjecting 2-oxo-4-(hydroxymethylphosphinyl) butyrate of step (a) to an amination reaction to obtain an L-glufosinate salt in the presence of a reaction solvent, an L-glutamate dehydrogenase or a mutant thereof, an inorganic amino donor and a reduced coenzyme NADPH.

12. The method of claim 11 said mutant is an L-glutamate dehydrogenase mutant, wherein the L-glutamate dehydrogenase mutant has a sequence obtained by mutating amino acid residue A at position 175 of SEQ ID NO: 1 to G and mutating amino acid residue V at position 386 to an amino acid residue with smaller steric hindrance; wherein the L-glutamate dehydrogenase mutant has catalytic activity on 2-oxo-4-(hydroxymethylphosphinyl)-butyric acid or salts thereof.

13. (canceled)

14. The method of claim 12, wherein the D-glufosinate salt has a form of existing alone, or, coexisting with L-glufosinate salt; the form of coexisting with L-glufosinate salt is a D-type enriched glufosinate salt, an L-type enriched glufosinate salt or a racemic glufosinate salt.

15. A composition comprising 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid, L-glufosinate and D-glufosinate, wherein 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid is more than 40% (in mole) of the total amount of L-glufosinate, D-glufosinate and 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid.

16. The composition of claim 15, wherein the L-glufosinate is 50% (in mole) of the total amount of L-glufosinate, D-glufosinate and 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid; 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid is more than 40% (in mole) of the total amount of L-glufosinate, D-glufosinate and 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid; the D-glufosinate is less than 10% (in mole) of the total amount of L-glufosinate, D-glufosinate and 2-oxo-4-(hydroxymethyl phosphinyl) butyric acid.

17. The composition of claim 15, wherein the composition further comprises a D-amino acid oxidase.

18. The composition of claim 17 , wherein the composition further comprises a catalase.

19. A solution comprising the composition of claim 15.

20. The solution of claim 19, wherein the solution is a water solution.

21. The method of claim 1, wherein the nucleotide sequence of L-glutamate dehydrogenase mutant is set forth as SEQ ID NO: 8 or SEQ ID NO: 10.

22. A transformant comprising the recombinant expression vector of claim 4.

23. The method of claim 7, wherein: the D-glufosinate salt has a form of existing alone, or, coexisting with L-glufosinate salt; the form of coexisting with L-glufosinate salt is a D-type enriched glufosinate salt, an L-type enriched glufosinate salt or a racemic glufosinate salt; and/or, the D-amino acid oxidase has a concentration of 0.6-6 U/mL; and/or, the oxidation reaction is performed under a condition of ventilation; and/or, the oxidation reaction is performed in the presence of catalase; and/or, the D-glufosinate salt has a concentration of 100-600 mM; and/or, the oxidation reaction has a reaction system with a pH of 7-9; and/or, the oxidation reaction has a reaction system with a temperature of 20-50° C.

24. The method of claim 9, wherein: the dehydrogenase is a glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase; and/or, the hydrogen donor is a glucose, isopropanol or formate; and/or, the dehydrogenase has a concentration of 0.6-6 U/mL; and/or, the mass ratio of the oxidized coenzyme NADP.sup.+ to the 2-oxo-4-(hydroxymethylphosphinyl) butyrate is 1 :100-1 :20000; and/or, the hydrogen donor has a concentration of 100-1000 mM; and/or, the reduction reaction has a reaction system with a pH of 7-9; and/or, the reduction reaction has a reaction system with a temperature of 20-50° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 shows Marfey’s reagent pre-column derivatization HPLC analysis result of racemic glufosinate standard, wherein the last two peaks are the peaks of the Marfey’s reagent itself.

[0079] FIG. 2 shows Marfey’s reagent pre-column derivatization HPLC analysis result of the D-glufosinate and the L-glufosinate in the prepared product.

[0080] FIG. 3 shows ion-pair HPLC analysis result of racemic glufosinate standard.

[0081] FIG. 4 shows ion-pair HPLC analysis result of PPO standard.

[0082] FIG. 5 shows ion-pair HPLC analysis result of reaction mixture after reaction.

[0083] FIG. 6 shows mass spectrum of PPO standard.

DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of examples thereto. The experimental methods for which specific conditions are not indicated in the following examples shall be selected according to conventional methods and conditions, or according to the specification of commodity.

[0085] Unless otherwise specified, the experimental methods of the present invention are conventional methods, and specific gene cloning operations can be found in the “Molecular Cloning: A Laboratory Manual” compiled by J. Sambrook et al.

[0086] Unless otherwise specified, the abbreviations of amino acids in the present invention are conventional in the art, and the amino acids corresponding to the specific abbreviations are shown in Table 1.

TABLE-US-00001 Name of Amino Acid Three-Letter Code Single Letter Code Name of Amino Acid Three-Letter Code Single Letter Code alanine Ala A leucine Leu L arginine Arg R lysine Lys K asparagine Asn N methionine Met M aspartic acid Asp D phenylalanine Phe F cysteine Cys C proline Pro P glutanine G1n Q serine Ser S glutamic acid Glu E threonine Thr T glycine Gly G tryptophan Trp W histidine His H tyrosine Tyr Y isoleucine Ile I valine Val V

[0087] The codons corresponding to the amino acids are also conventional in the art, and the corresponding relationships between specific amino acids and codons are shown in Table 2.

TABLE-US-00002 First Nucleo tide Second Nucleotide Third Nucle otide T C A G T F(Phenylalanine) S(Serine) Y(Tyrosine) C(Cysteine) T F(Phenylalanine) S(Serine) Y(Tyrosine) C(Cysteine) C L(Leucine) S(Serine) Stop Codon Stop Codon A L(Leucine) S(Serine) Stop Codon W (Tryptophan) G C L(Leucine) P(Proline) H(Histidine) R(Arginine) T L(Leucine) P(Proline) H(Histidine) R(Arginine) C L(Leucine) P(Proline) Q(Glutamine) R(Arginine) A L(Leucine) P(Proline) Q(Glutamine) R(Arginine) G A I(Isoleucine) T(Threonine) N(Asparagine) S(Serine) T I(Isoleucine) T(Threonine) N(Asparagine) S(Serine) C I(Isoleucine) T(Threonine) K(Lysine) R(Arginine) A M(Methionine) T(Threonine) K(Lysine) R(Arginine) G G V(Valine) A(Alanine) D(Aspartic acid) G(Glycine) T V(Valine) A(Alanine) D(Aspartic acid) G(Glycine) C V(Valine) A(Alanine) E(Glutamate) G(Glycine) A V(Valine) A(Alanine) E(Glutamate) G(Glycine) G

[0088] pET28a was purchased from Novagen; NdeI and HindIII were purchased from Thermo Fisher, and E.coli BL21 (DE3) competent cells were purchased From Beijing Dingguo Changsheng Biotechnology Co., Ltd.; the catalase was purchased from Shandong Fengtai Biotechnology Co., Ltd.; NADPH was purchased from Shenzhen Bontac Bio-engineering Co., Ltd.; NH.sub.4Cl was purchased from Shanghai Titan Technology Co., Ltd.

[0089] The chiral analysis of the product was performed by pre-column derivatization high performance liquid chromatography (HPLC), and the specific analysis method is as follows: [0090] (1) Chromatographic conditions: Agilent ZORBAX Eclipse plus C18, 3.5 .Math.m, 150*4.6 mm. Mobile phase A: 0.1% TFA+H.sub.2O, mobile phase B: 0.1% TFA+CAN. Detection wavelength: 340 nm, flow rate: 1.0 mL/min, column temperature: 30° C. [0091] (2) Derivatization reagent: Marfey’s reagent. 50 mg of N-α-(2,4-dinitro-5-fluorophenyl)-L-alaninamide was weighed accurately, and dissolved with acetonitrile to prepare 25 mL solution for later use. [0092] (3) Derivatization reaction: The reaction solution was diluted 100 times and added with equal volume of Marfey’s reagent for derivatization. 10 .Math.L of mixture was injected for analysis.

[0093] Conversion rate=(reactant-remaining reactant)/reactantxl00%

[0094] 2-oxo-4-(hydroxymethylphosphinyl) butyric acid (PPO for short) was analyzed by ion-pair high performance liquid chromatography (HPLC). The specific analysis method is as follows: Chromatographic conditions: ULtimate AQ-C18, 5 .Math.m, 4.6*250 mm; mobile phase: 0.05 mol/L diammonium hydrogen phosphate PH=3.6: 10% tetrabutylammonium hydroxide aqueous solution: acetonitrile=91:1:8; detection wavelength: 205 nm; flow rate: 1.0 mL/min; column temperature: 25° C.In the following examples, the terms described are all “glufosinate”, since “glufosinate” is in the reaction system, a person skilled in the art refer to “glufosinate ammonium salt” as “glufosinate” by default. Therefore, “glufosinate” refers to “glufosinate ammonium salt” actually, and the corresponding glufosinate standard also refer to glufosinate ammonium salt standard, and the corresponding PPO is also PPO ammonium salt. Before the optical rotation of glufosinate is tested, the resulting glufosinate ammonium salt is subjected to the acidification reaction to obtain glufosinate.

Example 1 Acquisition of Mutant Enzymes of L-glutamate Dehydrogenase

[0095] The sequence of a glutamate dehydrogenase (referred to as LsGluDH for short hereinafter) derived from Lysinibacillus sphaericus was searched out on NCBI, shown as SEQ ID NO: 1, with Genbank Accession No.: WP_012293812.1. Genes were synthesized according to the nucleotide sequences of the genes of mutants as SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 in Table 3, and the genes were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. (Building C3, Bionano Technology Park, Xinghu Street 218, Suzhou Industrial Park).

[0096] Then, the genes of mutants were enzyme-ligated to pET28a with NdeI and HindIII as restriction sites. The vector enzyme-ligated was transformed into E.coli BL21 competent cells. The constructed bacterial seed was inoculated on TB medium at 37° C., shaking with 200 rpm, and induced with 0.1 mM IPTG overnight. The bacteria was then harvested, and the engineered bacteria containing the gene of glutamate dehydrogenase were obtained.

[0097] After the engineered bacteria containing the gene of glutamate dehydrogenase were activated by plate-streaking, single colony was selected and inoculated into 5 mL LB liquid medium containing 50 .Math.g/mL kanamycin, and cultured with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 50 mL fresh LB liquid medium containing 50 .Math.g/mL kanamycin the same, shook at 37° C. until OD600 value reached about 0.8. IPTG was added to a final concentration of 0.5 mM for induced culturing at 18° C. for 16 h. After cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacteria was collected and stored in an ultra-low temperature refrigerator at -80° C. for later use.

[0098] 5 g of bacteria collected above was washed twice with 50 mM Tris-HCl buffer solution, pH 8.5, resuspended in 30 mL of Tris-HCl buffer solution with pH 8.5, and lysed homogeneously. The lysis liquid was centrifuged at 12,000 rpm for 10 min to remove precipitation, and a supernatant crude enzyme solution containing a recombinant glutamate dehydrogenase was obtained.

TABLE-US-00003 Mutant No. Mutation Site SEQ ID NO. of amino acid sequence SEQ ID NO. of nucleotide sequence 1 LsGluDH-WT (WP_012293812.1) 1 2 1-1 LsGluDH-A175G 3 4 1-2 LsGluDH-V386A 5 6 1-3 LsGluDH-A175G-V386S 7 8 1-4 LsGluDH-A175G-V386A 9 10

Example 2 Test of Specific Activity of Enzyme on Mutant Enzymes

[0099] The substrate solution is prepared as: 355 .Math.L of 2.25 M PPO (in a final concentration of 20 mM) (prepared by inventors, and the method for preparation was referred to US8017797B, FIG. 6 is the corresponding mass spectrum) and 0.4 g of NH.sub.4Cl (in a final concentration of 200 mM) were added. The pH was adjusted by ammonia to 8.5, and the volume was made constant to 40 mL by 50 mM Tris-HCl buffer solution of pH 8.5.

[0100] The method for testing enzyme activity is as follows:

[0101] The total reaction system is 1 mL, and absorbance value was measured at OD340 nm. 940 .Math.L of the substrate solution was added to a cuvette of 1 mL, and the value was adjusted to zero; then 10 .Math.L of 25 mM NADPH was added; finally 50 .Math.L of the crude enzyme solution was added in order. The value changes in 0-10 min were recorded, then one value was picked every 30 s, and a plot was made with the reaction time as abscissa and the absorbance value at 340 nm of wavelength as Y-axis. The slope was used to calculate the reducing rate of NADPH, and the enzyme activity was calculated.

[0102] The unit of enzyme activity is defined as the amount of enzyme required to reduce 1 .Math.mol of NADPH per minute under specific reaction conditions (30° C.).

[0103] The specific activity of enzyme is the activity units contained in per mg of enzyme protein, and the calculation formula is: enzyme acitivity/protein content, the unit of which is U/mg or U/g. The results are shown in Table 4.

[0104] It is known in CN108588045A that the enzyme activity of wildtype LsGluDH-WT (WP_012293812.1) is much lower than that of single-position mutant, and a person skilled in the art can figure out that the specific activity of enzyme of wildtype LsGluDH-WT (WP_012293812.1) is much lower than that of mutants. Therefore, the specific activity of enzyme of wildtype LsGluDH-WT (WP_012293812.1) was not tested in the present invention.

TABLE-US-00004 Mutant No. Mutation Site Specific activity of enzyme (U/g) SEQ ID NO. of amino acid sequences SEQ ID NO. of nucleotide sequences 1-1 LsGluDH-A175G 38.19 3 4 1-2 LsGluDH-V386A 26.64 5 6 1-3 LsGluDH-A175G-V386S 49.44 7 8 1-4 LsGluDH-A175G-V386A 58.73 9 10

[0105] Methods for preparing the crude enzyme solution of L-glutamate dehydrogenase used in the following examples are all as described above.

Example 3 Acquisition of D Amino Acid Oxidase (DAAO) Gene

[0106] The whole gene of DAAO was synthesized according to the gene sequence of AC302 DAAO described in Patent US9834802B2. Synthesis was conducted by Suzhou Genewiz Biological Technology Co., Ltd., No. 211 Pubin Road, R & D Park, Jiangbei New District, Nanjing, Jiangsu Province.

Example 4 Expression of D Amino Acid Oxidase (DAAO) Gene

[0107] The composition of LB liquid medium is as follows: peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, after dissolving them in deionized water and calibrated to a constant volume, LB liquid medium was sterilized at 121° C. for 20 min for later use.

[0108] DAAO gene synthesized in Example 3 was ligated to pET28a, with restriction sites NdeI & HindIII, and the ligated vector was transformed into host E. coli BL21 (DE3) competent cells to obtain engineered strains containing DAAO.

[0109] After activating the engineered strain containing DAAO gene by plate-streaking, a single colony was selected and inoculated into 5 mL of LB liquid medium containing 50 .Math.g/mL kanamycin, and cultured with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 50 mL of fresh LB liquid medium containing 50 .Math.g/mL kanamycin, shook at 37° C. until the OD.sub.600 value reached about 0.8. IPTG was added to a final concentration of 0.5 mM for induced culturing at 18° C. for 16 h. After cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacteria was collected and stored in an ultra-low temperature refrigerator at -20° C. for later use.

Example 5 Preparation of D Amino Acid Oxidase (DAAO) Crude Enzyme Solution and Enzyme Activity Detection

[0110] After cultivation, the collected bacteria in Example 4 was washed twice with 50 mM phosphate buffer solution, pH 8.0, resuspended in phosphate buffer solution with pH 8.0, and lysed homogeneously at low temperature and high pressure. The lysis liquid was centrifuged to remove cell pellets, thus obtaining supernatant as a crude enzyme solution containing recombinant DAAO.

[0111] The detection method of enzyme activity is as follows: 100 .Math.L of pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (containing 50 mmol/L of D-glufosinate and 0.1 mg/mL of peroxidase), 50 .Math.L of indicator (60 .Math.g /mL of 2,4,6-tribromo-3-hydroxybenzoic acid and 1 mg/mL of 4-aminoantipyrine), 50 .Math.L of DAAO enzyme were added, the concentration of H.sub.2O.sub.2 was determined by detecting UV absorption at 510 nm, the concentration of PPO was calculated and the enzyme activity was obtained.

[0112] The unit of enzyme activity is defined as the amount of enzyme required to produce 1 .Math.mol of PPO per minute under specific reaction conditions (30° C.).

[0113] Methods for preparing the crude enzyme solution of DAAO enzyme used in the following examples are all as described above.

Example 6 Acquisition and Expression of Alcohol Dehydrogenase Gene

[0114] The whole gene of alcohol dehydrogenase was synthesized according to the gene sequence of Cyclopentanol dehydrogenase from Lactobacillus brevis KB290 (Genbank Accession No.: BAN05992.1).

[0115] The composition of LB liquid medium consists of: peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, after dissolving them in deionized water, make the volume constant, and sterilized at 121° C. for 20 min for later use.

[0116] The alcohol dehydrogenase gene was ligated to pET28a, with restriction sites NdeI & HindIII, and the ligated vector was transformed into host E. coli BL21 (DE3) competent cells to obtain engineered strains containing the alcohol dehydrogenase gene. After activating the engineered strains containing the alcohol dehydrogenase gene by plate-streaking, a single colony was selected and inoculated into 5 mL of LB liquid medium containing 50 .Math.g/mL kanamycin, and cultured with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 50 mL of fresh LB liquid medium containing 50 .Math.g/mL kanamycin the same, shook at 37° C. until the OD.sub.600 value reached about 0.8. IPTG was added to a final concentration of 0.5 mM for induced culturing at 18° C. for 16 h. After cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacteria was collected and stored in an ultra-low temperature refrigerator at -20° C. for later use.

Example 7 Preparation of Crude Alcohol Dehydrogenase Solution And Measurement of Enzyme Activity

[0117] 10 g of bacterial sludge was taken from the collected bacteria in Example 6 and added with 50 mL of 100 mM ammonium phosphate buffer solution with pH 7.5. The mixture was stirred well, and lysed homogeneously at 500 bar as crude enzyme solution. 10% flocculant was added dropwise under the condition of stirring (in a final concentration of 2-2.5%.sub.0), centrifuged at 4,000 rpm for 10 min after stirred for 5 minutes to obtain clear enzyme solution. The enzyme activity of the supernatant was measured.

[0118] The method for detecting enzyme activity is as follows: in a 3 mL of reaction system and under the condition of 25° C., first, a 2850 .Math.L of 400 mM isopropanol (prepared with 100 mM phosphate buffer) with pH 8.0 was added first, then 50 .Math.L of NADP.sup.+ (25 mM) was added. After adjusting the UV spectrophotometry meter to zero, then 100 .Math.L of enzyme solution diluted 100 folds was added, and OD value at 340 nm was measured by an ultraviolet spectrophotometer.

[0119] The unit of enzyme activity is defined as follows: the amount of enzyme required to produce 1 .Math.mol of NADPH per minute under specific reaction conditions (25° C., pH 7.0) is defined as 1 U.

[0120] Methods for preparing the crude enzyme solution of glucose dehydrogenase used in the following examples are all as described above.

Example 8 Preparation of L-glufosinate Catalysed by DAAO Enzyme and L-glutamate Dehydrogenase Mutant

[0121] 200 g of bacteria of L-glutamate dehydrogenase mutant (prepared according to Example 1) was resuspended by 50 mM phosphate buffer solution with pH 8.0, and the volume was made constant to 1 L. The solution was lysed homogeneously at low temperature and high pressure. The lysis liquid was centrifuged to remove precipitation, thus obtaining the crude enzyme solution containing L-glutamate dehydrogenase mutant.

[0122] 80 g of D, L-glufosinate was weighed and dissolved completely with 50 mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH 8.0. 2.5 g of 0.4 million U/g catalase was added, and 150 mL of the crude enzyme solution of DAAO enzyme (12 U/mL) prepared by Example 5 was added. The pH was adjusted to 8.0 by ammonia, and the volume was made constant to 1 L by 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. The reaction was conducted in water bath using mechanical stirring at 20° C., and under the condition of ventilation by introducing air at 1 VVM (introduce one-fold of reaction volume of air per minute). 1 mL of defoamer was added to avoid foaming, and the production concentration of PPO was determined by ion-pair HPLC. Meanwhile the content and ee value of remaining L-glufosinate were determined by pre-column derivatization high performance liquid chromatography. When the ee value was greater than 99%, the reaction was stopped. At this time, more than 99% of the D-glufosinate was reacted to producing PPO, wherein the residual amount of the D-glufosinate accounts for less than 1% of the initial amount of the D-glufosinate, accounts for less than 0.5% of the initial amount of the D, L-glufosinate. In the reaction solution, PPO is more than 49.5% (in mole) of the total amount of PPO, L-glufosinate and D-glufosinate; D-glufosinate is less than 0.5% (in mole) of the total amount of PPO, L-glufosinate and D-glufosinate; L-glufosinate is 50% (in mole) of the total amount of PPO, L-glufosinate and D-glufosinate, which is equal to the initial proportion of L-glufosinate in D, L-glufosinate.

[0123] Four aliquots of 50 mL of the reaction solution above were added with 0.54 g of ammonium chloride, 0.4 mg of NADP.sup.+ and 0.73 g of isopropanol respectively. 1 mL of alcohol dehydrogenase (300 U/mL) prepared in Example 7 was added, and 1 mL of the crude enzyme solution of L-glutamate dehydrogenase mutant was added respectively. The pH was adjusted by ammonia to 8.5, and the reaction temperature was controlled at 37° C. by performing reaction in water bath and magnetically stirred. The residual concentration of PPO was determined by ion-pair HPLC, and meanwhile the content and ee value of L-glufosinate were determined by pre-column derivatization high performance liquid chromatography. The date at the end of the reaction is shown in Table 5.

[0124] The HPLC analysis results of D-glufosinate and L-glufosinate in the products after reaction completion (18 h) are shown in FIG. 2 (in the drawings, L-glutamate dehydrogenase mutant 1-4 (LsGluDH-A166G-V376A) is used as an example for illustration ), wherein L-glufosinate has the retention time of 13.820 min, and D-glufosinate with the retention time of 12.469 min is almost undetectable; the HPLC chromatogram of Marfey’s reagent pre-column derivatization of the racemic glufosinate standard (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) is shown in FIG. 1 (the retention time of L-glufosinate and D-glufosinate are 13.683 min and 12.016 min, respectively). The peak time of the composition of the product prepared in this example is generally the same with that of the L-glufosinate in the standard product. In addition, it is indicated that L-glufosinate was prepared in present example by conducting acidification treatment, concentration, purification by column, recrystallization on products obtained, to obtain pure L-glufosinate, which has [a]D.sup.25=+28.2° (C=1,1N HCl) determined by optical rotation (the optical rotation of L-glufosinate has been reported in prior art, such as US4389488).

[0125] The ion-pair HPLC analysis results of the reaction solution after reaction completion (18 h) are shown in FIG. 5, wherein no peak at the peak position of PPO, and 3.828 min is the peak position of glufosinate. The ion-pair HPLC spectrum of the PPO standard product (the standard product was prepared by inventors self, and the method for preparing was referred to US8017797B, FIG. 6 is the corresponding mass spectrum) is shown in FIG. 4, wherein the retention time of PPO standard product is 9.520 min. The ion-pair HPLC spectrum of the racemic glufosinate standard (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) is shown in FIG. 3, wherein the retention time of racemic glufosinate standard is 3.829 min. It can be seen that PPO was completely reacted to conversion finally, and the peak time of glufosinate product is generally consistent with the peak time of the standard product.

[0126] Although L-glutamate dehydrogenase mutant 1-4 (LsGluDH-A166G-V376A) is taken as example in the above figure results, experiments on all other mutants were conducted by inventors to verify that the substrate can by catalyzed by these mutants in the present invention when participating in above reaction, thereby producing correct products.

TABLE-US-00005 Mutant No. Mutation Site Conversion rate at 2h Conversion rate at 18 h ee value at 18 h 1-1 LsGluDH-A175G 59.8% >99% >99% 1-2 LsGluDH-V386A 62.9% >99% >99% 1-3 LsGluDH-A175G-V386S 70.1% >99% >99% 1-4 LsGluDH-A175G-V386A 76.1% >99% >99%

Compare Example

[0127] A mutant enzyme of glutamate dehydrogenase derived from Pseudomonas putida (Genbank Accession No.: NP_742836.1) disclosed in CN108588045A (referred to as PpGluDH hereinafter) was obtained by performing the same method with that in Example 1, and the specific activity of enzyme was tested by the same method described in Example 2, and the results are shown in Table 6:

TABLE-US-00006 Mutant No. Mutation Site Specific activity of enzyme (U/g) SEQ ID NO. of amino acid sequence SEQ ID NO. of nucleotide sequence 1′ PpGluDH-WT (NP_742836.1) N.D. 11 12 1′-1 PpGluDH-A167G 15.25 13 14 1′-2 PpGluDH-V378A 27.73 15 16 1′-4 PpGluDH-A167G-V378A 16.84 17 18

[0128] It can be seen in Table 6 that, the specific activity of enzyme mutants mutating at homologous sites of glutamate dehydrogenase derived from pseudomonas putida is significantly lower than the mutants obtained in the present invention, and therefore, not all mutants with mutation at two sites have better effects than mutant with mutation at single site.