L-glutamate dehydrogenase mutant and application thereof
11634693 · 2023-04-25
Assignee
Inventors
- Zhenhua Tian (Shanghai, CN)
- Zhanbing Cheng (Shanghai, CN)
- Shaonan Ding (Shanghai, CN)
- Wenxuan Xu (Shanghai, CN)
- Ruru Wang (Shanghai, CN)
- Qi JIAO (Shanghai, CN)
- Yao Huang (Shanghai, CN)
Cpc classification
C12Y104/01004
CHEMISTRY; METALLURGY
International classification
Abstract
Provided are an L-glutamate dehydrogenase mutant and an application thereof, the mutant mutating the amino acid residue A at position 166 and/or the amino acid residue V at position 376 shown in SEQ ID NO. 1 into a hydrophilic or small sterically hindered amino acid residue, the application performing an amination reaction of 2-oxo-4-(hydroxymethylphosphinyl)butyrate in the presence of an L-amino acid dehydrogenase mutant, an inorganic amino donor, and a reduced coenzyme NADPH, and performing an acidification reaction on the obtained L-glufosinate salt to obtain L-glufosinate. Compared to wild L-glutamate dehydrogenase, the present L-glutamate dehydrogenase mutant has a higher concentration of substrates that can be catalysed when preparing L-glufosinate, thereby increasing the efficiency of the action of the enzyme and reducing reaction costs.
Claims
1. An L-glutamate dehydrogenase mutant, wherein the amino acid sequence of the L-glutamate dehydrogenase mutant is SEQ ID NO: 34.
2. A method for producing an L-glufosinate salt, wherein the method comprises the following step: combining the L-glutamate dehydrogenase mutant of claim 1, 2-oxo-4-(hydroxymethylphosphinyl) butyrate, an inorganic amino donor, and NADPH in a reaction solvent to produce the L-glufosinate salt.
3. The method of claim 2, wherein the method further comprises the following step: combining a D-glufosinate salt with a D-amino acid oxidase to produce the 2-oxo-4-(hydroxymethylphosphinyl) butyrate.
4. The method of claim 2, wherein the method comprises one or more of the following conditions: the L-glutamate dehydrogenase mutant has a concentration of 0.09-3 U/ml; the inorganic amino donor has a concentration of 100-2000 mM; the 2-oxo-4-(hydroxymethylphosphinyl) butyrate has a concentration of 100-600 mM; the molar ratio of NADPH to the 2-oxo-4-(hydroxymethylphosphinyl) butyrate is 1:30000-1:1000; the inorganic amino donor is one or more of ammonia, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, ammonium acetate, ammonium formate, and ammonium bicarbonate; the reaction solvent is water; a pH of 7-9; and a temperature of 20-50° C.
5. The method of claim 2, wherein the method further comprises the following step: the NADPH is oxidized to NADP.sup.− and the NADP.sup.− is reduced to NADPH by a dehydrogenase and a hydrogen donor.
6. The method of claim 5, wherein the method comprises one or more of the following conditions: the dehydrogenase that reduces NADP.sup.− to NADPH has a concentration of 0.6-6 U/mL; the NADP.sup.− has a concentration of 0.02-0.1 mM; the hydrogen donor has a concentration of 100-1000 mM; a pH of 7-9; and a temperature of 20-50° C.
7. An isolated nucleic acid comprising a nucleotide sequence encoding the L-glutamate dehydrogenase mutant of claim 1.
8. The method of claim 3, wherein the method comprises one or more of the following conditions: the D-glufosinate salt is purified or is mixed with an L-glufosinate salt; the D-amino acid oxidase has a concentration of 0.6-6 U/mL; the combining is performed in the presence of oxygen; the combining is performed in the presence of catalase; the D-glufosinate salt has a concentration of 100-600 mM; a pH of 7-9; and a temperature of 20-50° C.
9. The method of claim 5, wherein the dehydrogenase that reduces NADP.sup.− to NADPH is a glucose dehydrogenase, an alcohol dehydrogenase or a formate dehydrogenase; and/or, the hydrogen donor is glucose, isopropanol or formate.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
(7) 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.
(8) 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.
(9) 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.
(10) TABLE-US-00001 TABLE 1 Name of Amino Acid Three-Letter Code Single Letter Code alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutanine Gln Q glutamic acid Glu E glycine Gly G histidine His H isoleucine Ile I leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr Y valine Val V
(11) 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.
(12) TABLE-US-00002 TABLE 2 First Second Nucleotide Third Nucleotide T C A G Nucleotide 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
(13) pET28a, pET21a and bugbuster protein extraction reagent were purchased from Novagen; Dpnl enzyme was purchased from Invitrogen Shanghai Trading Co., Ltd.; 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.
(14) 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:
(15) (1) Chromatographic conditions: Agilent ZORBAX Eclipse plus C18, 3.5 μm, 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.
(16) (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.
(17) (3) Derivatization reaction: The reaction solution was diluted 100 times and added with equal volume of Marfey's reagent for derivatization. 10 μl of mixture was injected for analysis.
Conversion rate=(reactant-remaining reactant)/reactant×100%
(18) 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 μm, 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.
Example 1 Construction of L-Glutamate Dehydrogenase Mutant Library
(19) Table 3 shows the sequence of primers designed for the construction of a mutant library with mutations at positions 166, 376, and 196 of SEQ ID NO. 1 in the sequence list.
(20) TABLE-US-00003 TABLE 3 Mutation Site and No. Primer Name Primer Sequence SEQ ID NO. 1 A166-forward primer GAATATCGCGATGTTCCGNNKGGTGATATTGGTG 2 TGGG 2 A166-reverse primer CCCACACCAATATCACCMNNCGGAACATCGCGA 3 TATTC 3 V376-forward primer GCAAATGCCGGCGGTNNKGCGACCAGTGCACTG 4 4 V376-reverse primer CAGTGCACTGGTCGCMNNACCGCCGGCATTTGC 5 5 T196-forward primer GAATCAGGTGTGCTGNNKGGTAAAGGCCTGACC 6 6 T196-reverse primer GGTCAGGCCTTTACCMNNCAGCACACCTGATTC 7
(21) Wherein, N represents any one of nucleotide A, G, C, T, and M represents A or C, and K represents G or T; the corresponding nucleotide was selected according to the nucleotides encoding the desired amino acid to be mutated at the specific position. For example, NNK in A166-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine) or AGT (serine), etc. The nucleotides corresponding to specific amino acids can be found in Table 2.
(22) Gene cgGLUDH (Corynebacterium glutamicum) was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. (Building C3, Bionano Technology Park, Xinghu Street 218, Suzhou Industrial Park) according to the sequence of SEQ ID NO. 1 in the sequence list, and the PDB number of cgGLUDH is 5IJZ. Then NdeI and HindIII restriction sites were introduced to the plasmid pET21a to construct plasmid pET21a-cgGLUDH. Using the plasmid pET21a-cgGLUDH as a template, the target band was amplified by PCR.
(23) The PCR amplification system is as follows:
(24) TABLE-US-00004 Reagents Dosage (μL) 2 × PCR buffer (contains high fidelity enzyme) 25 Primer F 1 Primer R 1 Template 1 Deionized Water 22
(25) The PCR amplification procedure is as follows:
(26) TABLE-US-00005 95° C. 5 min 95° C. 40 s 50° C. 40 s {close oversize bracket} 30 cycles 72° C. 6 min 72° C. 10 min 12° C. heat preservation
(27) The PCR product was digested with Dpnl at 37° C. for 2 hr. Then the product was transformed into E. coli BL21 (DE3) competent cells after the reaction was completed, which were spreaded on LB medium containing 100 μg/mL ampicillin and cultured at 37° C. overnight. The bacteria was harvested, and transformants containing the mutant library were obtained.
Example 2 High-Throughput Screening of Mutant Libraries
(28) Screening was performed according to the following experimental steps:
(29) The transformants were inoculated and cultured in 96-wells plate and induced with IPTG at 30° C. overnight. The bacteria was harvested, and lysed by bugbuster protein extraction reagent, thus obtaining the enzyme solution by centrifugation.
(30) A reaction solution with final concentrations of 20 mM PPO, 200 mM NH.sub.4Cl, and 0.37 mM NAD was prepared. 180 μL of the reaction solution was pipetted to the microplate and then 20 μL of enzyme solution was added to obtain a total system of 200 μL. OD.sub.340 value was measured by the microplate reader. Taking the wild type as reference system, positive clones were selected, sequenced and the enzyme activity of which was detected. Sequence was conducted by Shenggong Bioengineering (Shanghai) Co., Ltd., No. 698, Xiangmin Road, Songjiang District, Shanghai.
(31) Selected positive clones were cultivated as follows:
(32) 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, sterilized at 121° C. for 20 min for later use.
(33) A single clone was selected and inoculated into 5 ml LB liquid medium containing 100 μg/ml ampicillin, and cultured with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 50 ml fresh LB liquid medium containing 100 μg/ml ampicillin, shook at 37° C. until 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.
(34) After culturing, the collected bacteria 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 L-glutamate dehydrogenase mutant.
(35) The detection method of enzyme activity is as follows: 25 g/L wet bacteria (lysed by homogenizer), 10 mM PPO, 20 mM coenzyme (NADPH), 750 mM NH.sub.4Cl, with a total system of 400 μL and a pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer as reaction medium. The total system was reacted in a metal bath shaking reactor at 30° C. for 6 h, and the reaction was terminated by adding 2 times of acetonitrile. After the sample was diluted by a certain folds, the concentration of L-glufosinate was detected by pre-column derivatization high performance liquid phase, and the enzyme activity was calculated. The results are shown in Table 4.
(36) The unit of enzyme activity is defined by the amount of enzyme required to produce 1 μmol of L-glufosinate per minute under specific reaction conditions (30° C.).
(37) TABLE-US-00006 TABLE 4 Amino Nucleo- Mutant Enzyme Acid SEQ tide SEQ No. Mutation Site Acitivity ID NO. ID NO. WT * 1 1 V376A ** 8 9 2 V376G ** 10 11 3 A166G, V376P ** 12 13 4 A166G, V376A ** 14 15 5 A166G, V376S, T196V ** 16 17 6 A166E, V376G ** 18 19 7 A166C, V376A ** 20 21 8 A166G *** 22 23 9 A166G, V376G *** 24 25 10 A166G, V376E *** 26 27 11 A166G, V376Q *** 28 29 12 A166G, V376S, T196S *** 30 31 13 A166T *** 32 33 14 A166G, V376S **** 34 35 15 A166G, V376S, T196C **** 36 37 16 A166H, V376S **** 38 39 Wherein, * means that the enzyme activity is less than 1 U/ml, ** means that the enzyme activity is between 3-5 U/ml; *** means that the enzyme activity is between 5-10 U/ml; **** means that the enzyme activity is 10 U/ml or more.
(38) 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
(39) The whole gene of DAAO was synthesized according to the gene sequence of AC302 DAAO described in U.S. Pat. No. 9,834,802B2. 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
(40) 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.
(41) 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.
(42) After activating the engineered strain containing DAAO gene by streaking on a plate, a single colony was selected and inoculated into 5 ml of LB liquid medium containing 100 μg/ml ampicillin, and cultured with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 50 ml of fresh LB liquid medium containing 100 μg/ml ampicillin, 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
(43) After culturing, 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.
(44) The detection method of enzyme activity is as follows: 100 μL 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 μL of indicator (60 μg/mL of 2,4,6-tribromo-3-hydroxybenzoic acid and 1 mg/mL of 4-aminoantipyrine), 50 μL 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.
(45) The unit of enzyme activity is defined by the amount of enzyme required to produce 1 μmol of PPO per minute under specific reaction conditions (30° C.).
(46) 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 Glucose Dehydrogenase Gene
(47) The whole gene of glucose dehydrogenase was synthesized according to the glucose dehydrogenase gene sequence from Bacillus subtilis 168 (NCBI accession number: NP 388275.1).
(48) 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.
(49) Glucose 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 glucose dehydrogenase gene. After activating the engineered strains containing glucose dehydrogenase gene by streaking them on a plate, a single colony was selected and inoculated into 5 ml of LB liquid medium containing 100 μg/ml ampicillin, and cultured with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 50 ml of fresh LB liquid medium containing 100 μg/ml ampicillin, 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 Glucose Dehydrogenase Solution and Detection of Enzyme Activity
(50) After culturing, the collected bacteria in Example 6 was washed twice with 50 mM pH 8.0 phosphate buffer solution, resuspended in pH 8.0 phosphate buffer solution, 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 glucose dehydrogenase.
(51) The detection method of enzyme activity is as follows: in 1 mL reaction system and under the condition of 25° C., first, 980 μL of 50 mM of sodium hydrogen phosphate-sodium dihydrogen phosphate buffer (containing 400 mM glucose) with pH 7.0 was added, then 10 μL of NADP.sup.+ (25 mM) was added, and finally 10 μL of appropriate enzyme solution was added, thus measuring OD value at 340 nm by an ultraviolet spectrophotometer.
(52) The unit of enzyme activity is defined by the amount of enzyme required to produce 1 μmol of NADPH per minute under specific reaction conditions (30° C.).
(53) Methods for preparing the crude enzyme solution of glucose dehydrogenase used in the following examples are all as described above.
Example 8 Preparation of Crude Alcohol Dehydrogenase Solution and Enzyme Activity Test
(54) The whole gene of alcohol dehydrogenase was synthesized according to the Cyclopentanol dehydrogenase gene sequence from Lactobacillus brevis KB290 (Genbank accession number: BAN05992.1).
(55) 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 glucose dehydrogenase gene. After activating the engineered strains containing glucose dehydrogenase gene by streaking them on a plate, a single colony was selected and inoculated into 5 ml of LB liquid medium containing 100m/m1 ampicillin, and cultured with shaking at 37° C. for 12 h. 2% of inoculum was transferred to 50 ml of fresh LB liquid medium containing 100m/m1 ampicillin, 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.
(56) 50 ml of 100 mM pH7.5 ammonium phosphate buffer was added to 10 g of bacteria mud, stirred well, and lysed homogenously at 500 bar to obtain a crude enzyme solution. 10% of flocculant was added dropwise under stirring conditions (in a final concentration of 2-2.5%0). After stirring for 5 minutes, the solution was centrifuged at 4,000 rpm for 10 minutes to obtain a clear enzyme solution. The supernatant was took to detect enzyme activity.
(57) 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 μL of 400 mM isopropanol (prepared with 100 mM phosphate buffer) with pH 8.0 was added first, then 50 μL of NADP.sup.+ (25 mM) was added. After adjusting the UV spectrophotometry meter to zero, then 100 μL of enzyme solution diluted 100 folds was added, and OD value at 340 nm was measured by an ultraviolet spectrophotometer.
(58) The unit of enzyme activity is defined as follows: the amount of enzyme required to produce 1 μmol of NADPH per minute under specific reaction conditions (25° C., pH 8.0) is defined as 1 U.
(59) Methods for preparing the crude enzyme solution of alcohol dehydrogenase used in the following examples are all as described above.
Example 9 Preparation of L-Glufosinate Catalysed by L-Glutamate Dehydrogenase Mutant
(60) PPO, NADP.sup.+, NH.sub.4Cl and glucose were weighed and added to the reaction flask, and dissolved completely with 50 mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH 8.0. The pH was adjusted to 8.0 with 25% concentrated ammonia water. 15 mL of the crude enzyme solution of L-glutamate dehydrogenase mutants 1(1 U/mL), 2 (1.3 U/mL), 8 (2.1 U/mL), 14 (3 U/mL), 15 (2.8 U)/mL) and 16 (2.5 U/mL) prepared according to the method in Example 2, and 1 mL of the crude enzyme solution of glucose dehydrogenase (100 U/mL) prepared according to the method in Example 7 were added, 50 mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH 8.0 was used to make the volume constant to 50 mL, so that the final concentration of PPO, ammonium chloride, glucose and NADP.sup.+ were 300 mM 600 mM, 360 mM and 0.03 mM, respectively. During the reaction process, the pH was controlled at 8.0 with ammonia water, and the residual concentration of PPO was measured by ion-pair HPLC after reacting in water bath using magnetic stirring for 10 h at 37° C. Meanwhile the production and ee value of L-glufosinate were determined by pre-column derivatization high performance liquid chromatography.
(61) The data at the end of the reaction are shown in Table 5. In the best embodiment of CN106978453A, the substrate that can be catalysed by 10 mL of L-glutamate dehydrogenase is in the concentration of 10-100 mM, while the substrate that can be catalysed by 15 mL of L-glutamate dehydrogenase mutant has reached a concentration of 300 mM in the present example.
(62) The HPLC analysis results of D-glufosinate and L-glufosinate in the products are shown in
(63) The ion-pair HPLC analysis results of the prepared PPO are shown in
(64) Although L-glutamate dehydrogenase mutant 14 is taken as an example in the above results, experiments on all other mutations were conducted by inventors to verify that the substrate can by catalyzed by these mutations of the present invention when participating in above reaction, thereby producing correct products.
(65) TABLE-US-00007 TABLE 5 Enzyme No. of Mutant PPO conversion rate ee value 1 10% 90% 2 15% 92% 8 45% 95% 14 99% >99% 15 98% >99% 16 97% >99%
Example 10 Preparation of L-Glufosinate Catalyzed by DAAO and L-glutamate Dehydrogenase Mutants
(66) D, L-glufosinate, NADP.sup.+, NH.sub.4Cl and glucose were weighed and added to the reaction flask, and dissolved completely with 50 mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0. The pH was adjusted to 8.0 with 25% concentrated ammonia water. 15 mL of crude enzyme solution of DAAO enzyme (12 U/mL) prepared according to the method in Example 5, 0.2 g of 200,000 U/g catalase, 15 mL of crude enzyme solution of L-glutamate dehydrogenase mutant 1 (1 U/mL) prepared according to Example 2 or L-glutamate dehydrogenase mutant 14 (3 U/mL), and 1 mL of crude enzyme solution of glucose dehydrogenase (100 U/mL) prepared according to the method in Example 7 were added, and 50 mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0, was used to make the volume constant to 50 mL, so that the concentration of glufosinate, ammonium chloride, glucose and NADP.sup.+ were 600 mM, 600 mM, 360 mM and 0.03 mM, respectively. During the reaction process, the pH was controlled at 8.0 with ammonia water, and magnetic stirring was carried out in a water bath at 37° C. Air was ventilated at 1 VVM (ventilate 1 times the reaction volume of air per minute), 200 μL of defoamer was added to prevent foaming, and the residual concentration of PPO was determined by ion-pair HPLC after reaction for 24 h, thereby determining the production mass and ee value of L-glufosinate by pre-column derivatization high performance liquid chromatography simultaneously. The data at the end of the reaction is shown in Table 6.
(67) TABLE-US-00008 TABLE 6 Enzyme No. of Mutant PPO conversion rate ee value 1 90% 90% 14 99% >99%
Example 11 Preparation of L-Glufosinate Catalysed Stepwise by DAAO and L-Glutamate Dehydrogenase Mutants
(68) 80 g of D, L-glufosinate was weighed, and dissolved completely with 50 mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0. 5 g of 200,000 U/g catalase was added, 150 mL of crude enzyme solution of DAAO enzyme (12 U/mL) prepared according to the method in Example 5 was added, and the pH was adjusted to 8.0 with 25% concentrated ammonia water. 50 mM of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 8.0, was used to make the volume constant to 1 L. The reaction was performed in a water bath at 20° C. and mechanically stirred, and oxygen was ventilated at 0.5 VVM (ventilate 0.5 times the reaction volume of oxygen per minute). 1 mL of defoamer was added to prevent foaming, the production concentration of PPO was determined by ion-pair HPLC, and the production mass and ee value of L-glufosinate were determined with pre-column derivatization high performance liquid chromatography simultaneously. The reaction was terminated when the ee value was greater than 99%.
(69) 2 aliquots of 50 mL of the above reaction solutions 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 according to the method in Example 8 and 1 mL of crude enzyme solution of L-glutamate dehydrogenase mutant were added, respectively. The pH was adjust to 8.5 with ammonia water, and the reaction temperature was controlled by performing reaction in water bath and magnetically stirred at 37° C. The residual concentration of PPO was determined by ion-pair HPLC, and the production mass and ee value of L-glufosinate were determined by pre-column derivatization high performance liquid chromatography simultaneously. The data at the end of the reaction is shown in Table 7.
(70) TABLE-US-00009 TABLE 7 Enzyme No. of Mutant PPO conversion rate ee value 1 92% 90% 14 99% >99%
(71) Although specific embodiments of the present invention have been described above, it shall be understood by those skilled in the art that the foregoing description of embodiments is intended to be purely illustrative of the invention, and various changes or modifications can be made without departing from the principle and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.