Amino acid dehydrogenase mutant and application in synthesis of L-glufosinate-ammonium thereof
11408016 · 2022-08-09
Assignee
Inventors
- Yaping Xue (Zhejiang, CN)
- Feng Cheng (Zhejiang, CN)
- Heng Li (Zhejiang, CN)
- Yuguo Zheng (Zhejiang, CN)
- Jianmiao Xu (Zhejiang, CN)
Cpc classification
International classification
Abstract
The present invention discloses an amino acid dehydrogenase mutant and application thereof in synthesizing L-glufosinate-ammonium, the amino acid dehydrogenase mutant is obtained by a single mutation or a multi-site mutation of the amino acid at position 95, 108, 172, 303 of the amino acid sequence shown in SEQ ID No. 2. The amino acid dehydrogenase mutant DyGDH-F95I-A108T-R172P-R303H prepared by the present invention has a specific enzyme activity that is 33 times higher than that of the original Aldo-keto reductase, and the concentration of the largest substrate, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid reaches 500 mM, the amino acid dehydrogenase mutant has more industrial application prospects. Using the amino acid dehydrogenase mutant to produce L-glufosinate-ammonium, the reaction time is significantly shortened, the general process takes 20 hours, and the reaction time of the present invention only requires 120 minutes, which shows that the amino acid dehydrogenase mutant has a good industrial application prospect.
Claims
1. An amino acid dehydrogenase mutant, wherein the amino acid dehydrogenase mutant is obtained by a single mutation or multi-site mutations of the amino acid at position 95, 108, 172, 303 of the amino acid sequence shown in SEQ ID NO: 2, wherein the single mutation or the multi-site mutations is/are selected from the group consisting of: (1) phenylalanine at position 95 of the amino acid sequence shown in SEQ ID NO: 2 into leucine or isoleucine; (2) phenylalanine at position 95 of the amino acid sequence shown in SEQ ID NO: 2 into leucine, and alanine at position 108 into threonine, serine or tyrosine; (3) phenylalanine at position 95 of the amino acid sequence shown in SEQ ID NO: 2 into isoleucine, and arginine at position 172 into proline or valine; (4) phenylalanine at position 95 of the amino acid sequence shown in SEQ ID NO: 2 into isoleucine, alanine at position 108 into threonine, and arginine at position 172 into proline or valine; (5) phenylalanine at position 95 of the amino acid sequence shown in SEQ ID NO: 2 into isoleucine, alanine at position 108 into tyrosine, and arginine at position 172 into proline; and (6) phenylalanine at position 95 of the amino acid sequence shown in SEQ ID NO: 2 into isoleucine, alanine at position 108 into threonine, arginine at position 172 into proline and arginine at position 303 into histidine.
2. The amino acid dehydrogenase mutant as claimed in claim 1, wherein the amino acid dehydrogenase mutant is obtained by mutating phenylalanine at position 95 of the amino acid sequence shown in SEQ ID NO: 2 into isoleucine, alanine at position 108 into threonine, arginine at position 172 into proline and arginine at position 303 into histidine.
3. A method of performing asymmetric reductive amination of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid to L-glufosinate-ammonium, the method comprising contacting the amino acid dehydrogenase mutant as claimed in claim 1 with 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid.
4. The method as claimed in claim 3, wherein the method is carried out as follows: wet cells obtained by induction of a recombinant genetically engineered strain containing the gene of the amino acid dehydrogenase mutant and wet cells obtained by induction of an engineered strain containing the gene of the glucose dehydrogenase are mixed and resuspended in a pH7.4, 100 mM phosphate buffer, the resulting mixture is subjected to ultrasonication and centrifugation, then the resulting supernatant is used as catalyst, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid is used as substrate and glucose is used as auxiliary substrate, a reaction is carried out under the conditions of 35° C. and 400-600 rpm, after the reaction is completed, the reaction solution is separated and purified to obtain L-glufosinate-ammonium.
5. The method as claimed in claim 4, wherein in the reaction, the final concentration of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid is 300-500 mM, the final concentration of glucose is 450-750 mM, the amount of the catalyst is 50-100 g/L calculated by the total amount of the wet cells before ultrasonication, the wet cells obtained by induction of the recombinant genetically engineered strain containing the gene of the amino acid dehydrogenase mutant and the wet cells obtained by induction of engineered strain containing the gene of the glucose dehydrogenase are mixed at the mass ratio of 3:1.
6. The method as claimed in claim 4, wherein the wet cells containing the amino acid dehydrogenase mutant are prepared by a method as follows: the recombinant genetically engineered strain containing the gene of the amino acid dehydrogenase mutant is inoculated into LB liquid medium containing 50 g/mL (final concentration) ampicillin, cultured at 37° C. for 8 hours, the resulting inoculum is inoculated with 2% incubating volume to fresh LB liquid medium containing 50 g/mL (final concentration) ampicillin, cultured at 37° C. and 180 rpm for 1.5 h, then added with IPTG at a final concentration of 0.1 mM, cultured at 18° C. for 20 h, and centrifuged at 4° C. and 8000 rpm for 10 min, thereby obtaining the wet cells containing the amino acid dehydrogenase mutant; the wet cells containing the glucose dehydrogenase are prepared by the following method: the engineered strain containing the gene of the glucose dehydrogenase is inoculated into LB liquid medium containing 50 g/mL (final concentration) kanamycin, cultured at 37° C. for 9 hours, the resulting inoculum is inoculated with 2% incubating volume to fresh LB liquid medium containing 50 g/mL (final concentration) kanamycin, cultured at 37° C. and 180 rpm for 1.5 h, then added with IPTG at a final concentration of 0.1 mM, cultured at 28° C. for 10 h, and centrifuged at 4° C. and 8000 rpm for 10 min, thereby obtaining the wet cells containing the glucose dehydrogenase.
7. The method as claimed in claim 4, wherein the ultrasonication is carried out as follows: the wet cells obtained by induction of the recombinant genetically engineered strain containing the gene of the amino acid dehydrogenase mutant and the wet cells obtained by induction of engineered strain containing the gene of the glucose dehydrogenase are mixed and resuspended in the pH7.4, 100 mM phosphate buffer, the resulting mixture is subjected to ultrasonication for 15 min, and the conditions of the ultrasonication are 400 W, 1 second on, and 5 seconds off.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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SPECIFIC EMBODIMENTS
(5) The present invention is further illustrated below with specific examples.
Example 1: Construction and Screening of the Amino Acid Dehydrogenase Mutant Library
(6) The gene of the amino acid dehydrogenase cloned from Pseudomonas monteilii WP_060477601.1 (the nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2) was used to construct the expression vector pETDEut-dygdh, then the obtained vector was transformed into E. coli to obtain the original strain E. coli BL21(DE3)/pETDEut-dygdh.
(7) The amino acid dehydrogenase mutant library was prepared by 4 rounds of site-directed saturation mutagenesis. The primer design was shown in Table 1. In the first round, with the vector pETDEut-dygdh as a template and F95F and F95R in Table 1 as primers, saturation mutation PCR was performed to mutate phenylalanine at position 95 of the amino acid sequence of the amino acid dehydrogenase shown in SEQ ID No. 2 into other 19 amino acids, the PCR product was transformed and spread, and the superior strain was then selected, thereby obtaining the amino acid dehydrogenase mutant pDyGDH-F95L. In the second round, with mutant pDyGDH-F95L as a template and A108F and A108R as primers, saturation mutation PCR was carried out, the PCR product was subjected to transformation and spreading, and the superior strain was selected, thereby obtaining the amino acid dehydrogenase mutant pDyGDH-F95L-A108T. In the third round, with mutant pDyGDH-F95L-A108T as a template and R172F and R172R as primers, saturation mutation PCR was carried out, the PCR product was subjected to transformation and spreading, and the superior strain was selected, thereby obtaining the amino acid dehydrogenase mutant pDyGDH-F95L-A108T-R172P. In the fourth round, with mutant pDyGDH-F95L-A108T-R172P as a template and R303F and R303R as primers, saturation mutation PCR was carried out, the PCR product was subjected to transformation and spreading, and the superior strain was selected, thereby obtaining the amino acid dehydrogenase mutant pDyGDH-F95L-A108T-R172P-R303H. And in the following experiments, all the other single mutants pDyGDH-A108T, pDyGDH-R172P and pDYGDH-R303H were constructed by the same method.
(8) The mutation PCR system was as follows: 2×Phanta Max buffer 25 μL, dNTPs 1 μL, forward and reverse primers 1 μL respectively, template 1 μL, Phanta Super-Fidelity DNA polymerase 0.5 μL, and ddH.sub.2O to a final volume of 50 μL. The conditions of the PCR procedure were as follows: pre-denaturation at 95° C. for 5 min; 30 cycles of 90° C. for 30 s, 62° C. for 30 s and 72° C. for 7 min, and finally extension at 72° C. for 5 min. The PCR result was checked by DNA agarose gel electrophoresis. The PCR product was digested with restriction endonuclease DpnI at 37° C. and 220 rpm for 1 h, the DpnI was heat inactivated at 65° C. for 1 min, the digested PCR product was transformed by heat shock treatment, competent E. coli BL21(DE3) was activated, cultured at 37° C. and 220 rpm for 1 h, then was spread onto an LB plate with 50 μg/mL ampicillin, and invertedly incubated overnight at 37° C., superior mutants were selected from the obtained mutants, sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing confirmation and stored.
(9) TABLE-US-00001 TABLE 1 Primers designing for the site-directed saturation mutagenesis of the amino acid dehydrogenase Primer Primer sequence (5′-3′) F95 F:GGTTTGCGTNNKCACCCTTCCGTGAATCT R:GGAAGGGTGMNNACGCAAACCTCCCTTAT A108 F:AAATTCTTGNNKTTTGAACAAGTTTTTAAA R:TTGTTCAAAMNNCAAGAATTTCAGTACAC R172 F:GTAGGAGCCNNKGAGATTGGGTTTATGTT R:CCCAATCTCMNNGGCTCCTACTCCAATATC R303 F:CTGGAATTCNNKAAGGGCCAGA R:CTGGCCCTTMNNGAATTCCAGACC
Example 2: Induced Expression of the Original Amino Acid Dehydrogenase, its Mutant and the Glucose Dehydrogenase
(10) The gene of the glucose dehydrogenase esgdh (the nucleotide sequence was shown in SEQ ID No. 3, and the amino acid sequence was shown in SEQ ID No. 4) was cloned from Exiguobacterium sibiricum and connected to vector pET-28b (+) by double digestion, the recombinant plasmid was transformed into E. coli BL21(DE3), thereby obtaining a recombinant glucose dehydrogenase strain E. coli BL21(DE3)/pET28b-esgdh.
(11) The original strain E. coli BL21(DE3)/pETDEut-dygdh and the strain containing the amino acid dehydrogenase mutant from Example 1 were respectively inoculated into LB liquid medium containing 50 μg/mL (final concentration) ampicillin, cultured at 37° C. for 8 hours, the resulting inoculum was inoculated with 2% incubating volume to fresh LB liquid medium containing 50 μg/mL (final concentration) ampicillin, cultured at 37° C. and 180 rpm for 1.5 h, then added with IPTG at a final concentration of 0.1 mM, cultured at 18° C. for 20 h, and centrifuged at 4° C. and 8000 rpm for 10 min, thereby obtaining respective wet cells.
(12) The strain E. coli BL21(DE3)/pET28b-esgdh containing the recombinant glucose dehydrogenase was inoculated into LB liquid medium containing 50 μg/mL (final concentration) kanamycin, cultured at 37° C. for 8 hours, the resulting inoculum was inoculated with 2% incubating volume to fresh LB liquid medium containing 50 μg/mL (final concentration) kanamycin, cultured at 37° C. and 180 rpm for 1.5 h, then added with IPTG at a final concentration of 0.1 mM, cultured at 28° C. for 10 h, and centrifuged at 4° C. and 8000 rpm for 10 min, thereby obtaining wet cells containing glucose dehydrogenase.
(13) The above cells with corresponding proteins can be used for preparation of pure protein enzyme liquid and used as crude enzyme for application in asymmetric reductive amination of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid to give L-glufosinate-ammonium.
Example 3: Screening of the Mutant Library
(14) The wet cells of the mutant strain and that of the glucose dehydrogenase obtained by induced expression from Example 2 were mixed at the mass ratio of 3:1, and resuspended in a pH 7.4, 100 mM phosphate buffer at a ratio of 50 g of the total cell amount per liter, the resulting mixture was subjected to ultrasonication on an ice-water mixture for 15 minutes, the conditions of the ultrasonication were 400 W, 1 second on, and 5 seconds off, thereby obtaining crude enzyme liquid. Under the same conditions, the wet cells of the original strain E. coli BL21(DE3)/pET2Deut-dygdh was used to replace that of the mutant strain to prepare crude enzyme liquid of the original strain.
(15) The crude enzyme liquid of the mutant strain or the original strain was used as a catalyst, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid was used as a substrate, glucose was used as an auxiliary substrate, and the endogenous NADPH in the cells rather than exogenous NADPH or NADP+ were used to establish a coenzyme circulatory system. The reaction system was selected as 10 mL, the amount of catalyst was 50 g of the wet cells before ultrasonication per liter, the final concentration of substrate was 300 mM, and the final concentration of glucose was 450 mM, after the reaction was carried out at 30° C. and 600 rpm for 10 min, 100 μL of the reacting solution was taken and added with 5 μL of hydrochloric acid to end the reaction, and then added with ultrapure water to a final volume of 1 mL, that was, the reaction solution was diluted 10 times, the diluted reaction solution was first subjected to derivatization treatment: 200 μl of the diluted reacting solution+400 μL derivatization reagent (a pH 9.8 borate buffer containing 15 mM o-phthalaldehyde and 15 mM N-acetyl-L-cysteine) were subjected to derivatization for 5 min at 30° C., 400 μL of ultrapure water was added to a final volume of 1 mL, the resulting mixture was centrifuged for 1 min at 12000 rpm, the supernatant was passed through a 0.22 μm membrane filter, the filtrate was taken as a liquid sample, and HPLC was used to detect 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid, L-glufosinate-ammonium, D-glufosinate-ammonium and de.sub.p values. Using the product L-glufosinate-ammonium and e.e. as indicators, superior mutants were screened, and the experimental results were shown in Table 2.
(16) HPLC conditions of detecting 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid were as follows: the chromatographic column was Unitary® C18 (4.6×250 mm, Acchrom, China), the mobile phase was acetonitrile: 50 mM ammonium dihydrogen phosphate (pH3.8, containing 10% tetrabutylammonium hydroxide) at a volume ratio of 12:88. The flow rate was 1.0 mL/min, the test wavelength was 232 nm, the injection volume was 10 μL, the column temperature was 30° C., and the retention time of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid was 9.7 min.
(17) HPLC conditions of detecting glufosinate-ammonium: the chromatographic column was Unitary® C18 (4.6×250 mm, Acchrom, China), the mobile phase was methanol: 0.05M ammonium acetate (pH5.7) at a volume ratio of 10:90. The flow rate was 1.0 mL/min, the test wavelength Ex=232 nm, the injection volume was 10 μL, the column temperature was 30° C., and the retention time of L-glufosinate-ammonium and D-glufosinate-ammonium was 10.6 min and 12.6 min respectively.
(18) TABLE-US-00002 TABLE 2 Catalytic performance and stereoselectivity of pDyGDH and its mutants L-glufosinate-ammonium e.e. Strains (mM) .sup.a (%) pDyGDH 39.4 ± 0.6 99.5 pDyGDH-F95L 145.2 ± 0.6 99.5 pDyGDH-F95I 247.2 ± 0.6 99.5 pDyGDH-F95A 38.3 ± 2.2 99.5 pDyGDH-F95W 30.2 ± 0.7 99.5 pDyGDH- F95I-A108T 255.6 ± 1.2 99.5 pDyGDH- F95I-A108F 20.8 ± 0.8 99.5 pDyGDH- F95I-A108H 30.0 ± 0.1 99.5 pDyGDH- F95I-A108D 15.2 ± 3.1 99.5 pDyGDH- F95I-A108C 10.2 ± 0.1 99.5 pDyGDH- F95I-A108S 252.6 ± 2.0 99.5 pDyGDH- F95I-A108Y 51.8 ± 2.0 99.5 pDyGDH- F95I-R172P 252.9 ± 0.9 99.5 pDyGDH- F95I-R172V 250.5 ± 1.8 99.5 pDyGDH- F95I-R172A 38.4 ± 1.6 99.5 pDyGDH- F95I-R172N 35.5 ± 0.7 99.5 pDyGDH- F95I- R172K 39.0 ± 0.3 99.5 pDyGDH- F95I-R172D 19.8 ± 1.7 99.5 pDyGDH- F95I-R172H 31.0 ± 0.6 99.5 pDyGDH- F95I-R172S 45.1 ± 1.1 99.5 pDyGDH- F95I-A108T-R172P 272.1 ± 1.2 99.5 pDyGDH- F95I-A108T-R172V 255.4 ± 2.3 99.5 pDyGDH- F95I-A108S-R172P 263.5 ± 0.7 99.5 pDyGDH-F95I-108T-R172P-R303H 285.3 ± 2.4 99.5
Example 4: Purification of the Original Amino Acid Dehydrogenase and its Mutants
(19) The superior mutants obtained in Example 3(pDyGDH-F95I, pDyGDH-A108T, pDyGDH-R172P, pDyGDH-R303H, pDyGDH-F95I-A108T, pDyGDH-F95I-A108T-R172P, pDyGDH-F95I-A108T-R172P-R303H) were used to obtain wet cells of the amino acid dehydrogenase mutants according to the method in Example 2, the wet cells were respectively suspended in buffer A (pH 8.0, 50 mM sodium phosphate buffer containing 0.3 M NaCl and 30 mM imidazole), subjected to sonication for 20 min (ice bath, 400 W, 1 second on and 5 seconds off), and centrifuged for 20 min at 4° C. and 12000 rpm, then the supernatant was collected. The mutant proteins were purified by Ni-NTA column (1.6×10 cm, Bio-Rad, the USA), and the specific operation was carried out as follows: {circle around (1)} a Ni column was equilibrated with 5 column volumes of binding buffer (pH 8.0, 50 mM sodium phosphate buffer containing 0.3 M NaCl) until the baseline was steady; {circle around (2)} the sample was loaded with a flow rate of 1 mL/min, the amount of the loading sample was 25-40 mg/mL calculated by the volume of the column, thereby attaching the target protein to the Ni column; {circle around (3)} the column was washed with 6 column volumes of buffer A (pH 8.0, 50 mM sodium phosphate buffer containing 0.3 M NaCl and 30 mM imidazole) at a flow rate of 1 mL/min until the baseline was steady; {circle around (4)} the column was washed with buffer B (pH 8.0, 50 mM sodium phosphate buffer containing 0.3 M NaCl and 500 mM imidazole) at a flow rate of 1 mL/min, the target protein was collected and dialyzed overnight in a pH 7.5, 20 mM phosphate buffer, thereby obtaining the pure enzyme; and {circle around (5)} the Ni column was washed with 5 column volumes of binding buffer (pH 8.0, 50 mM sodium phosphate buffer containing 0.3 M NaCl) until the baseline was steady, and the Ni column was stored in 5 column volumes of ultrapure water containing 20% ethanol.
(20) The pure amino acid dehydrogenase of the original strain E. coli BL21(DE3)/pETDEut-pdygdh was collected by the same conditions.
Example 5: Specific Enzyme Activity Determination of the Original Amino Acid Dehydrogenase and its Mutants
(21) The enzyme activity unit (U) is defined as follows: under the conditions of 35° C. and pH 7.4, the amount of enzyme required for producing 1 μmol L-glufosinate-ammonium in one minute is one enzyme activity unit, U. Specific enzyme activity is defined as activity units of one milligram of enzyme protein, U/mg.
(22) Standard conditions of enzyme activity detection: 100 mM 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid, 10 mM NADPH, 0.02 ug/uL enzyme liquid, 30° C., pH 7.4 and 600 rpm for 10 min, the resulting sample was treated and then analyzed by HPLC.
(23) The protein concentration was determined by BCA protein determination kit (NanJing Key Gen Biotech Co., Ltd, NanJing).
(24) Specific enzyme activity of the original amino acid hydrogenase and its mutants was shown in Table 3.
(25) TABLE-US-00003 TABLE 3 Relative activity e.e. Enzyme (%) (%) pDyyGDH 100.sup.a 99.5 pDyGDH-F95I 411.1 ± 4.2 99.5 pDyGDH-A108T 398.1 ± 2.1 99.5 pDyGDH-R172P 417.3 ± 8.0 99.5 pDyGDH-R303H 418.0 ± 7.3 99.5 pDyGDH- F95I-A108T 531.0 ± 10.0 99.5 pDyGDH-F95I-A108T-R172P 1171.6 ± 1.1 99.5 pDyGDH- F95I-A108T-R172P-R303H 3320 ± 3.2 99.5 .sup.aunder standard conditions, the primary enzyme actively of pDyGDH is defined as 100%
Example 6: Kinetic Parameter Determination of the Original Amino Acid Dehydrogenase and its Mutants
(26) Kinetic parameters of the amino acid hydrogenase and its mutants were examined, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid was taken as substrate, the concentration was set as 2-10 mM (2, 4, 6, 8, 10 mM), the concentration of exogenous coenzyme NADPH was set as 1-5 mM (1, 2, 3, 4, 5 mM), 100 uL of pure enzyme solution was added (according to Example 4).
(27) The reaction system was 500 μL, the pure enzyme solution collected in Example 4 was diluted 10 times with pH 7.4, 100 mM phosphate buffer, then 100 μL of the resulting solution was sampled, added with the substrate and exogenous coenzyme NADPH, pH 7.4, 100 mM phosphate buffer was taken as a reaction medium, after reacting at 35° C. and 600 rpm for 10 min, the concentration of L-glufosinate-ammonium in the reacting solution was determined by HPLC (according to Example 4).
(28) According to the sequential mechanism of amino acid dehydrogenase's catalytic reaction, v.sub.max, K.sub.m.sup.A, K.sub.m.sup.B can be calculated by dual-reciprocal graph, the results were shown in Table 4. By comparing k.sub.cat and K.sub.m, it can be found that the Km values of pDyGDH on 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid and NADPH were 3.45 mM and 0.11 mM respectively. Except for the mutant pDyGDH-A08T, the rest of the mutants had a certain decrease which meant an increase of the affinity with 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid and NADPH. The catalytic efficiency k.sub.cat/K.sub.m.sup.B of the mutant pDyGDH-F95I-A108T-R172P-R303H on 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid reached 363.1 s.sup.−1.Math.mM.sup.−1, which was 29.3 times higher than that of the original enzyme (k.sub.cat/K.sub.m.sup.B=12.41 s.sup.−1.Math.mM.sup.−1), and its catalytic efficiency on coenzyme NADPH reached 14382.3 s.sup.−1.Math.mM.sup.−1, which was 37.3 times higher than that of the original enzyme (k.sub.cat/K.sub.m.sup.B=385.67 s.sup.−1.Math.mM.sup.−1).
(29) TABLE-US-00004 TABLE 4 Comparison of kinetic parameter of the original pDyGDH and its mutants k.sub.cat K.sub.m.sup.A K.sub.m.sup.B k.sub.cat/K.sub.m.sup.A k.sub.cat/K.sub.m.sup.B Enzyme (s.sup.−1).sup.a (mM).sup.b (mM).sup.b (s.sup.−1 .Math. mM.sup.−1) (s.sup.−1 .Math. mM.sup.−1) original pDyGDH 42.81 ± 3.10 0.11 3.45 385.67 12.41 pDyGDH-F95I 45.01 ± 2.91 0.092 2.67 489.23 16.85 pDyGDH-A108T 33.80 ± 2.21 0.16 4.91 209.93 6.88 pDyGDH-R172P 50.98 ± 1.04 0.071 2.12 718.03 24.05 pDyGDH-R303H 48.21 ± 0.98 0.090 3.30 535.67 14.61 pDyGDH-F95I-A108T 53.45 ± 4.01 0.097 3.01 551.03 17.75 pDyGDH-F95I-A108T-R172P 651.9 ± 3.12 0.046 2.19 14171.7 297.7 pDyGDH-F95I-A108T- 733.5 ± 3.83 0.051 2.02 14382.3 363.1 R172P-R303H
Example 7: Asymmetric Reductive Amination of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric Acid Using the Amino Acid Dehydrogenase Mutant pDyGDH-F95I-A108T-R172P-R303H
(30) According to Example 2, 3 g of the cells of the amino acid dehydrogenase mutant pDyGDH-F95I-A108T-R172P-R303H and 1 g of the cells of the glucose dehydrogenase EsGDH cells obtained by fermentation were mixed and resuspended in 40 mL of the pH 7.4, 100 mM phosphate buffer, the resulting mixture was subjected to ultrasonication on ice (the conditions of the ultrasonication are 400 W, 1 second on, and 5 seconds off), all the broken mixture (crude enzyme liquid) was added with 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid at a final concentration of 500 mM and glucose at a final concentration of 750 mM, thereby constructing the reaction system with a volume of 50 ml, the reaction was carried out at 35° C. and 300 rpm, ammonia water was flow-added to maintain the pH of the reaction solution at 7.4. The liquid phase method shown in Example 3 was used to detect the synthesis of the product L-glufosinate-ammonium and the change of e.e. value during the reaction, and the reaction processing curve was shown in