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 to Chinese patent application No. 2022101146927, filed on January 30, 2022. This application cites the above Chinese patent application in full.

[0002] The present invention is in the field of biotechnology, specifically relating to D-amino acid oxidase and its use in the production of L-glufosinate or its intermediates. [Background technology] Glufosinate, glyphosate, and paraquat are the three major herbicides in the world, all of which are non-selective. Among them, paraquat is highly toxic, so its use has been severely restricted or banned in many countries recently, while glyphosate is widely used, but China has excluded it from the approved A-grade green food list. Glufosinate has the characteristics of high activity, wide range of herbicidal spectrum, and excellent environmental compatibility, and exerts herbicidal activity at a faster rate than glyphosate and can be used in areas where glyphosate-resistant weeds exist, so glufosinate has great potential for market development and broad application prospects.

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

[0004] Commercially available glufosinate is a racemic mixture with two isomers, D and L. However, only the L form has activity, is easily decomposed in soil, and has low toxicity. There are mainly two methods for the production of L-glufosinate: chemical and biological. The chemical production of glufosinate mainly includes the Strecker process and the pyrolysis-ACA process. Among them, the Srecker process is complicated, flammable and explosive raw materials are used, and the product yield is low. The pyrolysis-ACA process is complicated, and methyldichlorophosphine (MDP) is an important intermediate in the previous step of this process, which is a technical barrier for the process. The biological method has mild reaction conditions, simple production steps, easy product separation, low yield, high cost, and has not yet been industrialized.

[0005] In CN105603015A, 2-oxo-4-(hydroxy(methyl)phosphono)butyric acid (PPO) and its salts are used as substrates, alanine is used as amino donor, and in vitro transaminase or cells expressing transaminase in vitro are used to catalyze the substrate, and alanine and a transaminase reaction are carried out to obtain L-glufosinate. In CN106978453B, a method for producing L-glufosinate using amino acid dehydrogenase is provided, in which L-glufosinate is obtained by carrying out a transamination reaction using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid or its salts as a substrate. This method has a high raw material conversion rate, but the substrate synthesis process is complicated and costly.

[0006] [ka]

[0007] In US9834802B2, it was invented 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, and PPO is catalyzed by amino acid aminotransferase to produce L-glufosinate. The composition obtained by the reaction according to this method contains L-glufosinate, D-glufosinate, and PPO, in which the content of L-glufosinate reaches 80% or more, the content of D-glufosinate is less than 10%, and the content of PPO is less than 20%. However, the stability of its enzyme activity still needs to be improved.

[0008] [ka]

[0009] Therefore, there is an urgent need to discover D-amino acid oxidase with high stability of enzyme activity, high D-glufosinate conversion rate, and high ee value of L-glufosinate. [Summary of the Invention] The technical problem that the present invention aims to solve is to overcome the shortcomings of the prior art, such as low enzyme activity, poor stability of enzyme activity, and low yield of L-glufosinate. In order to solve the above technical problems, the present invention provides a D-amino acid oxidase and its use in the production of L-glufosinate or its intermediate. The use of a D-amino acid oxidase with higher thermostability not only improves the enzyme activity, but also expands the temperature range of the enzyme, and when used at low temperatures, the enzyme's life can be extended, and when used at high temperatures, the catalytic efficiency of the enzyme can be improved. Enzymes with high thermostability have the advantages of increasing the chemical reaction rate, improving product quality, stable activity, and long storage life.

[0010] In a first aspect of the present invention, the amino acid sequence comprises 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; In addition, the present invention provides a D-amino acid oxidase that has activity and / or thermostability that is not lower than that of the D-amino acid oxidase represented by the amino acid sequence of SEQ ID NO:1.

[0011] In the present invention, a " / " between amino acid residues means that the position corresponding to the amino acid residue has a choice of different amino acid residue differences. 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 invention, the amino acid sequence of the D-amino acid oxidase compared to SEQ ID NO:1 comprises two or more amino acid residue differences selected from V42Y, E195Y, V326W, C234L.

[0013] Preferably, the D-amino acid oxidase of the present invention comprises an amino acid residue difference of C234L compared to SEQ ID NO:1, and further comprises 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 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 invention provides an isolated nucleic acid encoding a D-amino acid oxidase according to the first aspect of the invention. A third aspect of the invention provides a recombinant expression vector comprising the nucleic acid according to the second aspect of the invention.

[0019] In a fourth aspect, 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 cell for the transformant according to the invention may be any conventional in the art, 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, which may be present alone or together with L-glufosinate or a salt thereof, e.g., 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 a D-amino acid oxidase cell, 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 preferably 0.17 mol / L. The pH of the reaction system in 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 of the oxidation reaction preferably further comprises a buffer, which may be any buffer commonly used in the art, preferably a phosphate buffer, such as ammonium dihydrogen phosphate and 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. The seventh aspect of the present invention is Step 1: obtaining 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof by the method according to the sixth aspect of the present invention; Step 2, in the presence of glutamate dehydrogenase, an inorganic amino donor and a reduced auxiliary enzyme, 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 auxiliary enzyme is preferably NADPH or NADH. In the method for producing L-glufosinate or a salt thereof described in 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 form of 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 in 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 of the ammonification reaction is preferably 28 to 35°C, and 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 subjecting an oxidized coenzyme to a reduction reaction 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, and then an ammonification reaction may be carried out to produce L-glufosinate or a salt thereof, and further, a "one-pot method" may be carried out, for example, by mixing all the 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 producing 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 D-amino acid oxidase substrate in the present invention is calculated using D-glufosinate or a salt thereof.

[0038] As long as it does not violate the 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 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, it is possible not only to improve the enzyme activity, but also to expand the operating temperature range of the enzyme, extend the enzyme's life span when used at low temperatures, and improve the catalytic efficiency when used at high temperatures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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, the experimental methods for which no specific conditions are described are selected according to the usual 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 shown below. (1) Chromatographic conditions: Agilent ZORBAX Eclipse plus C18, 3.5 μm, 150 × 4.6 mm. Mobile phase A: 0.1% TFA + H 2O, mobile phase B: 0.1% TFA+CAN. 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 ultrasonic treatment was performed for 5 minutes, followed by adding purified water to the mark to dilute and mix uniformly. 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, covered, and heated in a 50 °C oven in the dark for 1 hour. After the reaction was completed, 0.1 mL of hydrochloric acid solution was added and mixed uniformly.

[0043] 1 mL of the above mixed solution was weighed out, 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 analysis method is as shown below.

[0044] Chromatographic conditions: μLtimate 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 H 2 O solution. 10 μL was injected and analyzed. 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 of 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 as shown in Table 2.

[0049] [Table 2]

[0050] pET28a plasmid and bugbuster protein extraction reagent were purchased from Novagen, NdeI enzyme, 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 peptone 10 g / L, yeast powder 5 g / L, and NaCl 10 g / L, which was dissolved in deionized water, adjusted to volume, and sterilized for use at 121° C. for 20 minutes. The solid medium was LB medium supplemented with 2% agar.

[0053] After streaking the DAAO enzyme-processing bacteria on a plate, 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 150 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 about 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 completion of the cultivation, the collected cells were washed twice with 50 mM pH 8.0 phosphate buffer, then resuspended in 50 mL pH 8.0 phosphate buffer, homogenized and disrupted, and the disrupted solution was centrifuged to remove the precipitate to obtain a crude enzyme solution containing the recombinant DAAO enzyme.

[0055] Example 2 Construction of D-amino acid oxidase (DAAO) mutants 1. Activation of process bacteria and extraction of plasmids The activated DAAO enzyme process bacteria described in Example 1 was placed in a test tube containing 5 mL of LB medium and cultured at 37 ° C and 200 rpm for 8 to 12 hours. After obtaining the cultured bacteria, the plasmid was extracted according to the instruction manual of the Sangong plasmid extraction kit. The obtained plasmid can 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) Gene mutations were obtained using whole-plasmid PCR. Using the plasmid extracted in the above step as a template, PCR primer sequences for a mutation library were constructed and designed to mutate the K29, V42, E195, C234, and V326 positions of the mutated D-amino acid oxidase sequence, and the target mutation genes were obtained. The primer sequences are shown in Table 3.

[0057] [Table 3]

[0058] Here, N represents any one of the nucleotides A, G, C, or 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] DpnI enzyme was added to digest the PCR products for 2 hours at 37° C. After the reaction was completed, the products were transformed into E. coli BL21 competent cells, inoculated onto LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37° C. The bacteria were then harvested to obtain transformants containing the mutation library.

[0063] 3. Combinatorial Mutations The beneficial mutations screened were subjected to overlap PCR to achieve various mutation combinations 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 induced overnight at 30° C. The bacteria were then harvested, dissolved in bugbuster protein extraction reagent, and centrifuged to remove the supernatant, yielding a DAAO mutant enzyme solution.

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

[0066] Enzyme activity detection analysis: 100 μL of 100 mM substrate (D,L-glufosinate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) with pH 8.0 was taken, and 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) were added, and finally 25 μL of the above DAAO mutant enzyme solution was added to obtain a 200 μL enzyme plate reaction system, which was analyzed under the conditions of 30 ° C and pH 8.0. The absorbance at 510 nm was recorded at 0 minutes and 20 minutes, respectively, and the difference was confirmed, and then the wild type was used as the reference system to screen for positive clones. The positive clones with enzyme activity equal to or higher than Enz.01 and improved thermostability were screened and shown in Table 6.

[0067] [Table 6] TIFF2025504067000009.tif41170

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

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

[0070] [Table 7] TIFF2025504067000011.tif47170

[0071] As shown in the table above, most of the single point mutant enzymes and combination mutant 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 peptone 10 g / L, yeast powder 5 g / L, and NaCl 10 g / L, 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 of NdeI and HindIII, and 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 then 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 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 cryogenic refrigerator at -20°C for use.

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

[0077] 4600g of water was added to a 10L jacketed reaction kettle, 0.28g of ammonium dihydrogen phosphate and 5.57g of diammonium hydrogen phosphate were added and stirred to dissolve transparently, then 400g of D,L-glufosinate (ammonium salt) was added to the reaction kettle and stirred to dissolve, the pH of the solution was adjusted to 7.9-8.1 with ammonia water, 5g of catalase (enzyme activity 800,000μ / g) was added to the reaction kettle, 1000mL (200g of bacteria) of mutant DAAO enzyme solution (Enz.27, Enz.29 or Enz.32) was added, and the reaction was carried out for 20 hours by bubbling with air, controlling the ventilation volume to 1 reaction volume per minute, and controlling the temperature to 25℃.

[0078] The temperature was controlled at 30-33°C, and 0.4 g of NADP + , 78g of isopropanol, 50g of ammonium chloride were added, the pH was adjusted to 8.4-8.6 with ammonia water, and 0.35g of alcohol dehydrogenase (ADH) bacteria was added. When the temperature and pH became normal, 160g of glutamate dehydrogenase bacteria (mutant 1-4 of our CN201910434350.1) was added to the reaction kettle to start the reaction. After 6 hours of reaction, the PPO remaining amount 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, indicating that they were significantly superior to Enz.1.

Claims

1. the amino acid sequence compared to SEQ ID NO:1 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, and the activity and / or thermostability is not lower than that of the D-amino acid oxidase represented by the amino acid sequence of SEQ ID NO: 1; D-amino acid oxidase.

2. The D-amino acid oxidase according to claim 1, characterized in that, compared with SEQ ID NO: 1, the amino acid sequence of the D-amino acid oxidase contains two or more amino acid residue differences selected from V42Y, E195Y, V326W, and C234L.

3. The D-amino acid oxidase comprises an amino acid residue difference of C234L compared to SEQ ID NO:1, and further comprises one or two amino acid residue differences of V42Y, E195Y, and V326W; Preferably, the D-amino acid oxidase has the following sequence as compared to SEQ ID NO: 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; 3. The D-amino acid oxidase according to claim 2, characterized in that it has a difference in an amino acid residue selected from any one of the groups:

4. the D-amino acid oxidase comprises an amino acid difference of E195Y compared to SEQ ID NO:1 and further comprises an amino acid residue difference of V42Y or V326W; Preferably, the D-amino acid oxidase has the following sequence as compared to SEQ ID NO: E195Y and V42Y; E195Y and V326W; E195Y, K29Q and V42Y; E195Y, K29W and V42Y; E195Y, K29Y and V42Y; 3. The D-amino acid oxidase according to claim 2, characterized in that it has a difference in an amino acid residue selected from any one of the groups:

5. The amino acid sequence of the D-amino acid oxidase compared to SEQ ID NO:1 is: K29G; K29H; K29I; K29N; K29Q; K29W; K29Y; V42C; V42D; V42E; V42H; V42P; V42Y; E195H; E195N; E195Y; E195Q; C234L; 2. The D-amino acid oxidase according to claim 1, characterized in that it has a difference in an amino acid residue selected from any one of the groups:

6. An isolated nucleic acid encoding the D-amino acid oxidase according to any one of claims 1 to 5.

7. A recombinant expression vector comprising the nucleic acid of claim 6.

8. 8. A transformant comprising a nucleic acid according to claim 6 or a recombinant expression vector according to claim 7, preferably wherein the host cell is Escherichia coli, such as E. coli BL21(DE3).

9. A method for producing the D-amino acid oxidase according to any one of claims 1 to 5, comprising culturing the transformant according to claim 8 under conditions suitable for expressing the D-amino acid oxidase.

10. The method includes a step of oxidizing a substrate in the presence of the D-amino acid oxidase according to any one of claims 1 to 5 to obtain 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof, Preferably, The substrate is 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, and / or the oxidation reaction is carried out under ventilation conditions, the ventilation being preferably by injecting air or oxygen, the ventilation rate being preferably between 0.5 VVM and 1.5 VVM, for example 1 VVM; 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, preferably 0.17 mol / L; and / or the pH of the reaction system of the oxidation reaction is 7 to 9, preferably 8; And / or, the temperature of the reaction system of the oxidation reaction is 20 to 50°C, preferably 25°C; More preferably, the mass ratio of the D-amino acid oxidase cells to the substrate is 1:(0.5 to 3), for example, 1:1, and / or the mass ratio of the catalase to the D-amino acid oxidase cells is 1:(20 to 60), for example, 1:

40.

11. The method according to claim 10, characterized in that the reaction system of the oxidation reaction contains a buffer solution, and the buffer solution is preferably a phosphate buffer solution, for example, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

12. Step 1: obtaining 2-oxo-4-(hydroxymethyloxyphosphino)butyric acid or a salt thereof by the method according to claim 10 or 11; 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 auxiliary enzyme 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 hydrogen carbonate, the form of ammonia used being preferably aqueous ammonia, and / or the pH of the reaction system of the ammonification reaction is 7 to 10, preferably 8.4 to 8.6; And / or the method according to claim 12, characterized in that the reaction temperature of the ammonification reaction is 28 to 35°C, preferably 30 to 33°C.

14. Use of the D-amino acid oxidase according to any one of claims 1 to 5 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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