D-amino acid oxidase and its use in the production of L-glufosinate or its intermediates

Abstract

The present invention provides a D-amino acid oxidase and its use in the production of L-glufosinate or an intermediate thereof. The D-amino acid oxidase has an amino acid sequence that, compared with SEQ ID NO: 1, differs by one or more amino acid residues selected from K29G / H / I / N / Q / W / Y / C / L; V42C / D / E / H / Y; E195N / Y / Q; C234L; V326W, and has activity and / or thermostability not lower than that of the D-amino acid oxidase represented by the amino acid sequence of SEQ ID NO: 1. The present invention provides a D-amino acid oxidase with high thermostability, which not only improves the enzyme activity but also expands the temperature range in which the enzyme can be used, and further extends the life of the enzyme when used at low temperatures and improves the catalytic efficiency of the enzyme when used at high temperatures.

Description

Detailed Description of the Invention

[0001] [Technical field] This application claims priority from Chinese Patent Application No. 2022101146927, filed on January 30, 2022. This application cites the above Chinese patent application in its entirety.

[0002] The present invention is in the field of biotechnology, and specifically relates to D-amino acid oxidase and its use in the production of L-glufosinate or its intermediates. [Background technology] Glufosinate, glyphosate, and paraquat are all non-selective herbicides in the world. Due to its highly toxic nature, paraquat's use has recently been severely restricted or banned in many countries. While glyphosate is widely used, China has excluded it from its list of approved Class A green foods. Glufosinate boasts high activity, a broad herbicidal spectrum, and excellent environmental compatibility. It exerts herbicidal activity more quickly than glyphosate and can be used in areas where glyphosate-resistant weeds exist. Therefore, glufosinate has great market development potential and broad application prospects.

[0003] The global market for glufosinate reached $1.05 billion in 2020, becoming the fastest-growing non-selective herbicide with a global market growth rate of 6.3% over the past six years. Therefore, developing or optimizing L-glufosinate manufacturing processes is of great significance for increasing economic benefits, lowering application costs, and reducing environmental pressure.

[0004] Commercially available glufosinate is a racemic mixture of two isomers, D and L. However, only the L-isomer is active, readily decomposed in soil, and less toxic. There are two main methods for producing L-glufosinate: chemical and biological. Chemical production of glufosinate primarily involves the Strecker process and the pyrolysis-ACA process. The Srecker process is complex, uses flammable and explosive raw materials, and has low product yields. The pyrolysis-ACA process is complex, and methyldichlorophosphine (MDP), a key intermediate in the early stages of this process, poses a technical barrier to the process. The biological method has mild reaction conditions, a simple production process, and easy product separation, but it has low yields and high costs, and has not yet been industrialized.

[0005] CN105603015A provides a method for producing L-glufosinate using amino acid dehydrogenase, which involves a transamination reaction using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) or its salts as a substrate and alanine as an amino donor, using an in vitro transaminase or cells expressing the transaminase in vitro to catalyze the substrate and alanine. CN106978453B provides a method for producing L-glufosinate using amino acid dehydrogenase, which involves a transamination reaction using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid or its salts as a substrate to obtain L-glufosinate. This method has a high raw material conversion rate, but the substrate synthesis process is complicated and costly.

[0006] [ka]

[0007] US9834802B2 discloses that D,L-glufosinate is used as a raw material, and the intermediate product PPO is produced by the catalytic action of a D-amino acid oxidase mutant, which is then catalyzed by an amino acid aminotransferase to produce L-glufosinate. The composition obtained by this reaction contains L-glufosinate, D-glufosinate, and PPO, with an L-glufosinate content of 80% or more, a D-glufosinate content of less than 10%, and a PPO content of less than 20%. However, the stability of the enzyme activity still needs to be improved.

[0008] [ka]

[0009] Therefore, there is an urgent need to discover a D-amino acid oxidase with high stability of enzyme activity, a high D-glufosinate conversion rate, and a high ee value of L-glufosinate. [Summary of the Invention] The technical problem addressed by the present invention is to overcome the drawbacks of prior art D-amino acid oxidases, such as low enzymatic activity, poor enzymatic activity stability, and low L-glufosinate yield. To address these technical problems, the present invention provides a D-amino acid oxidase and its use in producing L-glufosinate or its intermediates. The use of a D-amino acid oxidase with higher thermostability not only improves enzymatic activity but also broadens the operating temperature range of the enzyme, extending its lifespan when used at low temperatures and improving its catalytic efficiency when used at high temperatures. Highly thermostable enzymes have the advantages of increasing chemical reaction rates, improving product quality, stable activity, and a long shelf life.

[0010] In a first aspect of the present invention, the amino acid sequence contains one or more amino acid residue differences selected from K29G / H / I / N / Q / W / Y / C / L; V42C / D / E / H / Y; E195N / Y / Q; C234L; V326W compared to SEQ ID NO: 1, Furthermore, the present invention provides a D-amino acid oxidase that is not less active and / or thermostable than the D-amino acid oxidase represented by the amino acid sequence of SEQ ID NO:1.

[0011] In the present invention, " / " between amino acid residues means that there are different amino acid residue differences at the position corresponding to the amino acid residue. For example, E195N / Y / Q means that the difference at position 195 can be N, Y, or Q.

[0012] In some preferred embodiments of the present invention, the amino acid sequence of the D-amino acid oxidase compared to SEQ ID NO: 1 contains two or more amino acid residue differences selected from V42Y, E195Y, V326W, and C234L.

[0013] Preferably, the D-amino acid oxidase of the present invention contains an amino acid residue difference of C234L compared to SEQ ID NO: 1, and further contains one or two amino acid residue differences of V42Y, E195Y, and V326W.

[0014] More preferably, the D-amino acid oxidase has an amino acid residue difference selected from any one of the following groups compared to SEQ ID NO:1: C234L and V42Y; C234L and E195Y; C234L and V326W; C234L, K29C and V42Y; C234L, K29G and V42Y; C234L, K29L and V42Y; C234L, V326W and V42Y; C234L, V42Y and E195Y; C234L, E195Y and V326W.

[0015] In some other preferred embodiments of the invention, the D-amino acid oxidase comprises an E195Y amino acid difference compared to SEQ ID NO: 1 and further comprises a V42Y or V326W amino acid residue difference; Preferably, the D-amino acid oxidase has an amino acid residue difference selected from any one of the following groups compared to SEQ ID NO:1:

[0016] E195Y and V42Y; E195Y and V326W; E195Y, K29Q and V42Y; E195Y, K29W and V42Y; E195Y, K29Y and V42Y.

[0017] In some other preferred embodiments of the present invention, the D-amino acid oxidase has an amino acid residue difference selected from any one of the following groups compared to SEQ ID NO:1: K29G;K29H;K29I;K29N;K29Q;K29W;K29Y;V42C;V42D;V42E;V42H;V42P;V42Y;E195H;E195N;E195Y;E195Q;C234L;V326W.

[0018] A second aspect of the present invention provides an isolated nucleic acid encoding a D-amino acid oxidase according to the first aspect of the present invention. A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid according to the second aspect of the present invention.

[0019] A fourth aspect of the present invention provides a transformant comprising the nucleic acid according to the second aspect of the present invention or the recombinant expression vector according to the third aspect of the present invention. The host cells for the transformants according to the present invention may be any conventional cell in the art, and are preferably Escherichia coli, such as E. coli BL21(DE3).

[0020] A fifth aspect of the present invention provides a method for producing the D-amino acid oxidase described in the present invention, which comprises culturing the transformant described in the fourth aspect of the present invention under conditions suitable for expressing the D-amino acid oxidase.

[0021] A sixth aspect of the present invention provides a method for producing 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof, comprising the step of oxidizing a substrate in the presence of the D-amino acid oxidase described in the first aspect of the present invention to obtain 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof.

[0022] The substrate is preferably D-glufosinate or a salt thereof, and the D-glufosinate or a salt thereof may be present alone or together with L-glufosinate or a salt thereof; for example, the substrate is present in the form of racemic glufosinate or a salt thereof.

[0023] The oxidation reaction is preferably carried out under ventilation conditions, the ventilation preferably being air or oxygen injection, and the ventilation rate is preferably 0.5 VVM to 1.5 VVM, for example 1 VVM.

[0024] The oxidation reaction is preferably carried out in the presence of catalase. The D-amino acid oxidase is preferably present in the form of D-amino acid oxidase cells, a crude enzyme, a pure enzyme, or an immobilized enzyme.

[0025] The concentration of the substrate is preferably 0.1 to 0.5 mol / L, and more preferably 0.17 mol / L. The pH of the reaction system of the oxidation reaction is preferably 7 to 9, for example 8.

[0026] The temperature of the reaction system for the oxidation reaction is preferably 20 to 50°C, for example 25°C. In some preferred embodiments, the mass ratio of the D-amino acid oxidase cells to the substrate is 1:(0.5 to 3), for example, 1:1.

[0027] In some more preferred embodiments, the mass ratio of the catalase to the D-amino acid oxidase in bacterial cells is 1:(20 to 60), for example, 1:40. The reaction system for the oxidation reaction preferably further comprises a buffer solution, which may be a buffer solution commonly used in the art, and is preferably a phosphate buffer solution, such as ammonium dihydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0028] The buffer is used to adjust the pH of the reaction system, and preferably the pH of the buffer is 8.0. A seventh aspect of the present invention is Step 1: obtaining 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof by the production method described in the sixth aspect of the present invention; Step 2, in the presence of glutamate dehydrogenase, an inorganic amino donor, and a reduced coenzyme, ammonifies the 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof obtained in Step 1 to obtain the L-glufosinate or a salt thereof; The present invention provides a method for producing L-glufosinate or a salt thereof, comprising:

[0029] In the method for producing L-glufosinate or a salt thereof according to the present invention, the reduced coenzyme is preferably NADPH or NADH. In the method for producing L-glufosinate or a salt thereof according to the present invention, the inorganic amino donor is preferably one or more of ammonia, ammonium sulfate, ammonium chloride, diammonium hydrogen phosphate, ammonium acetate, ammonium formate, and ammonium hydrogen carbonate, and the ammonia used is preferably aqueous ammonia.

[0030] In the method for producing L-glufosinate or a salt thereof according to the present invention, the pH of the reaction system for the ammonification reaction is preferably 7 to 10, and more preferably 8.4 to 8.6. In the method for producing L-glufosinate or a salt thereof according to the present invention, the reaction temperature for the ammonification reaction is preferably 28 to 35°C, more preferably 30 to 33°C.

[0031] In the method for producing L-glufosinate or a salt thereof according to the present invention, the glutamate dehydrogenase is preferably any one of mutants 1 to 4 of CN201910434350.1. In the method for producing L-glufosinate or a salt thereof according to the present invention, the mass ratio of the glutamate dehydrogenase to the substrate D-glufosinate is preferably 1:(0.5 to 3), for example, 1:1.25.

[0032] In the method for producing L-glufosinate or a salt thereof according to the present invention, the molar ratio of the substrate D-glufosinate to the inorganic amino donor is preferably 1:(1 to 1.5), for example, 1:1.

[0033] The method for producing L-glufosinate or a salt thereof according to the present invention preferably further comprises a step of reducing an oxidized coenzyme in the presence of a dehydrogenase and a hydrogen donor to obtain the reduced coenzyme. The dehydrogenase is preferably glucose dehydrogenase, alcohol dehydrogenase or formate dehydrogenase.

[0034] The hydrogen donor is preferably glucose, isopropanol or formate. In some preferred embodiments of the invention, when the dehydrogenase is an alcohol dehydrogenase, the hydrogen donor is isopropanol; when the dehydrogenase is a glucose dehydrogenase, the hydrogen donor is glucose; and when the dehydrogenase is a formate dehydrogenase, the hydrogen donor is formate.

[0035] The method for producing L-glufosinate or a salt thereof according to the present invention may be carried out in separate steps. For example, an oxidation reaction may be carried out first to produce 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof, followed by an ammonification reaction to produce L-glufosinate or a salt thereof, and the process may be further carried out in a "one-pot" manner, for example, by mixing all raw materials to produce L-glufosinate or a salt thereof.

[0036] An eighth aspect of the present invention provides use of the D-amino acid oxidase described in the first aspect of the present invention in the production of L-glufosinate or a salt thereof, or 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof.

[0037] The DAAO enzyme and D-amino acid oxidase referred to in this invention can be used interchangeably. The amount of the D-amino acid oxidase substrate in the present invention is calculated using D-glufosinate or a salt thereof.

[0038] Unless contrary to common knowledge in the art, the above-mentioned preferable conditions can be arbitrarily combined to obtain each preferable embodiment of the present invention. The reagents and raw materials used in the present invention are commercially available.

[0039] The positive effects of the present invention are as follows: By providing a highly thermostable D-amino acid oxidase, not only can the enzyme activity be improved, but also the operating temperature range of the enzyme can be expanded, thereby extending the enzyme's lifespan when used at low temperatures and improving the catalytic efficiency when used at high temperatures. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be further described below in the form of examples, but the present invention is not limited to the scope of the examples. In the following examples, experimental methods for which specific conditions are not described are selected according to conventional methods and conditions or product instructions.

[0041] Chiral analysis and concentration analysis of the product L-glufosinate were carried out by pre-column derivatization high performance liquid chromatography, and the specific analytical method is as follows. (1) Chromatography conditions: Agilent ZORBAX Eclipse plus C18, 3.5 μm, 150 × 4.6 mm. Mobile phase A: 0.1% TFA + H2O, Mobile phase B: 0.1% TFA + CHCl3. Detection wavelength: 340 nm, Flow rate: 1.0 mL / min, Column temperature: 30 °C.

[0042] (2) Derivatization reagent: Marfey's reagent (3) Derivatization reaction: 50 mg of sample was weighed and placed in a 25 mL volumetric flask, 15 mL of dilution solution (pure water:acetonitrile = 50:50) was added, and the mixture was sonicated for 5 minutes. Purified water was added up to the mark to dilute the mixture and mix evenly. 1 mL of the above solution was transferred to a 5 mL volumetric flask, and 1 mL of Marfey's reagent solution and 0.1 mL of sodium bicarbonate (1 M) solution were added. The flask was then covered with a cover and heated in a 50°C oven for 1 hour in the dark. After the reaction was complete, 0.1 mL of hydrochloric acid solution was added and mixed evenly.

[0043] 1 mL of the above mixed solution was weighed, and 4 mL of the diluted solution was added, mixed uniformly, and poured into an injection bottle. 10 μL was injected and analyzed. PPO is analyzed by ion pair chromatography, and the specific analytical method is as follows.

[0044] Chromatographic conditions: MLtimate AQ-C18, 5 μm, 4.6 × 250 mm; mobile phase: 0.05 mol / L diammonium hydrogen phosphate (PH = 3.6): 10% aqueous tetrabutylammonium hydroxide solution: acetonitrile = 91:1:8; detection wavelength: 205 nm; flow rate: 1.0 mL / min; column temperature: 25 °C.

[0045] Sample: 5 mg / mL solution in H2O. 10 μL was injected for analysis. The experimental methods of the present invention are conventional methods unless otherwise specified, and the gene cloning procedure can be specifically referred to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0046] Unless otherwise specified, all abbreviations for amino acids in the present invention are conventional symbols in the art, and the amino acids corresponding to specific abbreviations are as shown in Table 1.

[0047] [Table 1]

[0048] The codons corresponding to the above amino acids are also common in the art, and the correspondence between specific amino acids and codons is shown in Table 2.

[0049] [Table 2]

[0050] The pET28a plasmid and bugbuster protein extraction reagent were purchased from Novagen, NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher, E. coli BL21 competent cells were purchased from Beijing Ding Guo Prosperous Biotechnology Co., Ltd., and catalase was purchased from Shandong Fengtai Biotechnology Co., Ltd.

[0051] Example 1: Preparation of D-amino acid oxidase (DAAO) The DAAO enzyme processing strain is derived from the processing strain containing seq. 79 disclosed in Patent CN111019916B, the amino acid sequence of which is shown in SEQ ID NO: 1, and the nucleotide sequence of which is shown in SEQ ID NO: 12.

[0052] The LB liquid medium composition was 10 g / L peptone, 5 g / L yeast powder, and 10 g / L NaCl, dissolved in deionized water, adjusted to volume, and sterilized at 121°C for 20 minutes before use. The solid medium was LB medium supplemented with 2% agar.

[0053] After streaking the DAAO enzyme-process bacteria onto a plate, a single colony was selected and inoculated into 5 mL of LB liquid medium containing 50 μg / mL of kanamycin. The resulting mixture was cultured at 37°C for 12 hours with shaking. 2% of the inoculum was then transferred to 150 mL of fresh LB liquid medium containing 50 μg / mL of kanamycin. The OD 600 The mixture was shaken at 37°C until the pH reached approximately 0.8, then the temperature was lowered to 30°C, IPTG was added to a final concentration of 0.5 mM, and induction culture was carried out for 16 hours. After the culture was completed, the medium was centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the cells were collected and stored in a refrigerator at -20°C for use.

[0054] After the cultivation was completed, the collected cells were washed twice with 50 mM pH 8.0 phosphate buffer, then resuspended in 50 mL of pH 8.0 phosphate buffer, homogenized, and disrupted. The disrupted solution was then centrifuged to remove the precipitate, yielding a crude enzyme solution containing the recombinant DAAO enzyme.

[0055] Example 2 Construction of D-amino acid oxidase (DAAO) mutants 1. Process: Activation of bacteria and extraction of plasmids The activated DAAO enzyme-processing bacteria described in Example 1 were placed in a test tube containing 5 mL of LB medium and cultured at 37°C and 200 rpm for 8-12 hours. After the cultured bacteria were obtained, the plasmid was extracted according to the instructions of the Sangong Plasmid Extraction Kit. The resulting plasmid could be used directly for point mutation or stored in a refrigerator at -80°C for long-term storage.

[0056] 2. Construction of mutation libraries (K29, V42, E195, C234, and V326 positions) The gene mutation was obtained using whole-plasmid PCR. Using the plasmid extracted in the above step as a template, PCR primer sequences were constructed and designed for a mutation library that mutated K29, V42, E195, C234, and V326 of the mutated D-amino acid oxidase sequence to obtain the target mutant gene. The primer sequences are shown in Table 3.

[0057] [Table 3]

[0058] Here, N represents any one of the nucleotides A, G, C, and T, M represents A or C, and K represents G or T, which are selected based on the nucleotide encoding the amino acid to be mutated at the corresponding site. For example, NNK in the K29-forward primer can represent AAG (lysine), AAT (aspartic acid), AGG (arginine), or AGT (serine), etc. The PCR amplification system is as follows:

[0059] [Table 4]

[0060] The PCR amplification procedure is as follows:

[0061] [Table 5]

[0062] The PCR product was digested with DpnI enzyme for 2 hours at 37°C. After the reaction was completed, the product was transformed into competent E. coli BL21 cells, which were then inoculated onto LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. The cells were then harvested to obtain transformants containing the mutation library.

[0063] 3. Combinatorial Mutation The screened beneficial mutations were then combined with various mutations via overlap PCR to form new mutant transformants.

[0064] Example 3 Preliminary screening of high-throughput mutation libraries The transformants obtained in Example 2 were inoculated into a 96-well plate, IPTG was added to a final concentration of 0.5 mM, and then induction was carried out overnight at 30°C. The cells were then harvested and lysed with Bugbuster protein extraction reagent. The cells were then centrifuged and the supernatant was collected to obtain a DAAO mutant enzyme solution.

[0065] Thermostability screening method: The supernatant was placed in a water bath at 60°C for 20 minutes and heat-treated. Then, positive clones were screened using the "enzyme activity detection assay."

[0066] Enzyme activity detection assay: 100 μL of 100 mM substrate (D,L-glufosinate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) at pH 8.0 was added to 50 μL of color-developing solution (60 mg / mL TBHBA (3-hydroxy-2,4,6-tribromobenzoic acid) and 100 mg / mL 4-AAP (containing 4-aminoantipyrine)) and 25 μL of HRP (horseradish peroxidase, 0.1 mg / mL). Finally, 25 μL of the above DAAO mutant enzyme solution was added to obtain a 200 μL enzyme plate reaction system, which was analyzed at 30°C and pH 8.0. The absorbance at 510 nm was recorded at 0 and 20 minutes, and the difference was confirmed. After confirming the difference, positive clones were screened using the wild-type as a reference system. Positive clones with enzyme activity equal to or greater than that of Enz.01 and improved thermostability were screened and are listed in Table 6.

[0067] [Table 6] TIFF0007825310000009.tif41170

[0068] Example 4 Rescreening for mutants with improved thermostability Enzyme activity detection method for rescreening mutants: To a 5 mL reaction mixture, 1 mL of 500 mM D,L-glufosinate (ammonium salt), 0.25 mL of heat-treated (60°C, 20 min) DAAO mutant crude enzyme solution, 1.25 mL of horseradish peroxidase (HRP), and 2.5 mL of color-developing dye solution (containing 60 mg / mL TBHBA and 100 mg / mL 4-AAP) were added. The reaction medium was disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 8.0). The reaction was carried out with shaking on a shaker at 30°C. The absorbance of the reaction solution at 510 nm was scanned every 2 minutes to generate an enzyme reaction kinetic curve of absorbance versus time (min). The enzyme activity was calculated based on the slope of the curve.

[0069] Definition of unit enzyme activity: The amount of enzyme required to produce 1 μmol H2O2 per minute under specific reaction conditions (30°C), and the unit of enzyme activity is U. The enzyme activity before and after heat treatment was detected according to the enzyme activity measurement method described above, and the fold increase in thermostability relative to Enz.1 was calculated. The results are shown in Table 7 below. * indicates that thermostability was improved by 1 to 1.2 times (excluding 1.2), ** indicates that thermostability was improved by 1.2 to 2 times (excluding 2), and *** indicates that thermostability was improved by more than 2 times. The calculation method for the fold increase in thermostability: Ratio of the enzyme activity after heat treatment of the mutant to the enzyme activity before treatment / Ratio of the enzyme activity after heat treatment of Enz.01 to the enzyme activity before treatment.

[0070] [Table 7] TIFF0007825310000011.tif47170

[0071] As shown in the table above, most of the single point mutation enzymes and combination mutation enzymes have significant effects on improving thermostability, with Enz.27, Enz.29, and Enz.32 showing better effects.

[0072] Experimental Example 5: Production of L-glufosinate The reaction pathway according to the present invention is as follows:

[0073] [ka]

[0074] The alcohol dehydrogenase gene was completely synthesized based on the cyclopentanol dehydrogenase gene sequence from Lactobacillus brevis KB290 (GenBank accession number: BAN05992.1).

[0075] The LB liquid medium composition was 10 g / L peptone, 5 g / L yeast powder, and 10 g / L NaCl, which was dissolved in deionized water, allowed to stand, and sterilized at 121°C for 20 minutes before use. The alcohol dehydrogenase gene was ligated into pET28a, with enzyme cleavage sites NdeI and HindIII. The recombinant vector was transformed into host E. coli BL21 (DE3) competent cells to obtain a process strain containing the alcohol dehydrogenase gene. The process strain containing the alcohol dehydrogenase gene was streaked onto a plate, and a single colony was selected and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for 12 hours with shaking. 2% of the inoculum was transferred to 50 mL of fresh LB liquid medium containing 50 μg / mL kanamycin, and the OD 600 The mixture was shaken at 37°C until the pH reached approximately 0.8, IPTG was added to a final concentration of 0.5 mM, and induction culture was carried out at 18°C ​​for 16 hours. After completion of the culture, the medium was centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the cells were collected and stored in a deep-freezer at -20°C for use.

[0076] Preparation of enzyme solution: The bacterial cells with high enzyme activity screened in Example 4 (enzyme numbers Enz. 27, Enz. 29, Enz. 32) were homogenized in a 50 mmol / L ammonium phosphate buffer solution at pH 7.0, with the ratio of bacterial cells to buffer solution being 1:5 (g:mL). After homogenization, a flocculant was added to flocculate the cells, and the mixture was centrifuged to obtain the supernatant.

[0077] A 10-liter jacketed reactor was charged with 4,600 g of water, and 0.28 g of ammonium dihydrogen phosphate and 5.57 g of diammonium hydrogen phosphate were added and stirred to dissolve until clear. Then, 400 g of D,L-glufosinate (ammonium salt) was added to the reactor and stirred to dissolve. The pH of the solution was adjusted to 7.9-8.1 with aqueous ammonia. 5 g of catalase (enzyme activity 800,000 μg / g) and 1,000 mL (200 g of bacterial cells) of mutant DAAO enzyme solution (Enz. 27, Enz. 29, or Enz. 32) were added to the reactor. The reaction was carried out for 20 hours while bubbling with air, controlling the ventilation rate at 1 reaction volume per minute, and controlling the temperature at 25°C.

[0078] The temperature was controlled at 30-33°C, and 0.4 g of NADP was added. + 78g of isopropanol and 50g of ammonium chloride were added, and the pH was adjusted to 8.4-8.6 with aqueous ammonia. 0.35g of alcohol dehydrogenase (ADH) cells were added. Once the temperature and pH were normalized, 160g of glutamate dehydrogenase cells (mutant 1-4 of our CN201910434350.1) were added to the reaction kettle to initiate the reaction. After 6 hours of reaction, the remaining PPO was detected to be ≦2%.

[0079] [Table 8]

[0080] The reaction results are shown in Table 8. After 24 hours of reaction, the ee values ​​of Enz.27, Enz.29 and Enz.32 were all 98% or higher, demonstrating that they were significantly superior to Enz.1.

Claims

1. A D-amino acid oxidase comprising an amino acid sequence that differs from SEQ ID NO: 1 by a difference in a residue selected from the following: C234L and V42Y; C234L and E195Y; C234L and V326W; C234L, K29C and V42Y; C234L, K29G and V42Y; C234L, K29L and V42Y; C234L, V326W and V42Y; C234L, V42Y and E195Y; C234L, E195Y and V326W; E195Y and V42Y; E195Y, K29Q and V42Y; E195Y, K29W and V42Y; E195Y, K29Y and V42Y; K29H; K29I; K29Q; V42E; V42H; And V42Y.

2. The D-amino acid oxidase of claim 1, wherein the amino acid sequence differs from SEQ ID NO:1 by a residue difference selected from: C234L and V42Y; C234L, E195Y and V326W; and E195Y and V42Y.

3. An isolated nucleic acid encoding the D-amino acid oxidase 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 producing the D-amino acid oxidase of claim 1, comprising culturing the transformant of claim 5 under conditions suitable for expressing the D-amino acid oxidase.

7. A method for producing 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof, comprising the step of oxidizing a substrate in the presence of the D-amino acid oxidase according to claim 1 to obtain 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof.

8. The substrate is D-glufosinate or a salt thereof, and the D-glufosinate or a salt thereof may exist alone or together with L-glufosinate or a salt thereof; and / or the oxidation reaction is carried out under ventilated conditions; and / or the oxidation reaction is carried out in the presence of catalase; and / or the D-amino acid oxidase is present in the form of a D-amino acid oxidase cell, a crude enzyme, a pure enzyme, or an immobilized enzyme; and / or the concentration of the substrate is 0.1 to 0.5 mol / L; and / or the pH of the reaction system of the oxidation reaction is 7 to 9; And / or the method according to claim 7, characterized in that the temperature of the reaction system of the oxidation reaction is 20 to 50°C.

9. The substrate is present in the form of racemic glufosinate or a salt thereof, The ventilation is injecting air or oxygen, and the ventilation rate is 0.5 VVM to 1.5 VVM; The concentration of the substrate is 0.17 mol / L; The pH of the reaction system of the oxidation reaction is 8, The method according to claim 8, wherein the temperature of the reaction system for the oxidation reaction is 25°C.

10. The method described in claim 8, characterized in that the mass ratio of the D-amino acid oxidase cells to the substrate is 1: (0.5 to 3), and / or the mass ratio of the catalase to the D-amino acid oxidase cells is 1: (20 to 60).

11. The method according to any one of claims 7 to 10, wherein the reaction system for the oxidation reaction contains a buffer solution.

12. Step 1: obtaining 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof by the production method according to any one of claims 7 to 10; Step 2: ammonifying the 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof obtained in Step 1 in the presence of glutamate dehydrogenase, an inorganic amino donor, and a reduced coenzyme to obtain the L-glufosinate or a salt thereof. A method for producing L-glufosinate or a salt thereof, comprising:

13. the reduced coenzyme is NADPH or NADH, 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 pH of the reaction system of the ammonification reaction is 7 to 10; And / or the reaction temperature of the ammonification reaction is 28 to 35°C.

14. Use of the D-amino acid oxidase according to claim 1 or 2 in the production of L-glufosinate or a salt thereof, or 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof.

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