D-amino acid oxidase mutant and use thereof

Abstract

The application discloses a D-amino acid oxidase mutant and application thereof, wherein the mutant is obtained by single-point or multi-point mutation of positions 31, 33, 171, 194 and 197 in an amino acid sequence shown in SEQ ID NO. 1. The D-amino acid oxidase mutant and immobilized enzyme thereof are applied to an oxidation reaction of D-glufosinate, and the advantages of the mutant compared with wild-type D-amino acid oxidase are as follows: the catalytic activity is increased by 1200 times, the substrate conversion rate is more than 99.6%, at least 10 batches of immobilized enzyme reaction batches can be used without prolonging the reaction time, and the immobilized enzyme activity is not lost, which can meet the requirements of industrial production application.

Description

Technical fields:

[0001] This invention relates to the field of protein engineering, and specifically to a D-amino acid oxidase mutant and its applications. Background technology:

[0002] Glufosinate ammonium (phosphinothricin, abbreviated as PPT), chemically known as (D,L)-2-amino-4-[hydroxy(methyl)phosphono]butyric acid, has the molecular formula C5H. 12 NO4P, with a relative molecular mass of 181.13, is a broad-spectrum contact herbicide developed by Agerfos (later Bayer) in the 1980s. Its herbicidal mechanism relies on the active ingredient, L-glufosinate, inhibiting glutamine synthase in the plant's nitrogen metabolism pathway. This disrupts normal nitrogen metabolism, leading to ammonium ion accumulation, causing cell membrane dehydration due to changes in ion concentration, chloroplast disintegration, and inhibition of protein and nucleotide biosynthesis. Consequently, photosynthesis is suppressed, preventing the plant from receiving the necessary nutrients for normal physiological metabolism, ultimately resulting in plant death and weed control. Glufosinate is characterized by its broad spectrum of herbicidal activity, low toxicity, high activity, and good environmental compatibility. With the development of transgenic glufosinate-tolerant crops, the ban on paraquat, and the increasing resistance of weeds to glyphosate, glufosinate products have significant market development potential and broad application prospects.

[0003] Glufosinate is a racemic mixture with two enantiomers, L- and D-. However, only the L-form glufosinate is effective in weed control, while the D-form enantiomer has no herbicidal activity. In 2006, Meiji Seika Pharmaceutical Co., Ltd. of Japan successfully developed a mono-isomer, L-form glufosinate (glufosinate-ammonium), whose herbicidal activity is twice that of the racemic DL-form glufosinate mixture. Glufosinate-ammonium has low toxicity, is relatively safe, degrades easily in soil, is safe for crops, has low drift, and is safe for adjacent crops. It has a broad spectrum of weed control (effective against many annual and perennial weeds), can control glyphosate-resistant weeds, has high activity, kills weeds rapidly (between glyphosate and paraquat), and has a long residual effect. At the same time, glufosinate-ammonium has very good water solubility, stable structure, and is easy to process and mix for use.

[0004] If glufosinate products could be used in the L-configuration form of refined glufosinate, the amount of technical grade pesticide used would be greatly reduced. This would play a very important role in improving the economic efficiency of the product, reducing pesticide use, and alleviating environmental pressure. However, currently, glufosinate products sold on the market are generally racemic mixtures. Therefore, developing the production technology of refined glufosinate (L-glufosinate) will have broad market prospects.

[0005] The reported methods for preparing L-glufosinate mainly fall into three categories: chiral resolution, chemical synthesis, and biocatalysis.

[0006] Chiral resolution utilizes racemic D- and L-glufosinate or their derivatives, typically employing chemical chiral resolving reagents to separate the D- and L-isomers, thereby obtaining optically pure L-glufosinate. The main problems with this process are: the need for expensive resolving reagents, making the cost prohibitive; the theoretical yield is only 50%, and the single-step resolution rate is low, while the process is relatively complex.

[0007] Chemical synthesis methods involve synthesizing optically pure L-glufosinate from chiral raw materials. However, the main problems with chemical methods are that they involve many reaction steps, low reaction yields, high production costs, and significant environmental pollution.

[0008] Compared to chemical resolution, biocatalysis has shown its potential to replace traditional chemical synthesis due to its advantages such as strict stereoselectivity, mild reaction conditions, and high yield. A commonly studied route uses D-glufosinate as a raw material, catalyzing D-amino acid oxidase to obtain the precursor of L-glufosinate, 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, which is then catalyzed by transaminase or amino acid dehydrogenase to obtain L-glufosinate.

[0009] In the above-mentioned bioenzymatic catalysis route, the catalytic effect and efficiency of D-amino acid oxidase are particularly crucial. Since D-amino acid oxidase is a typical flavin protease with flavin adenine (FAD) as a cofactor, it can specifically and selectively catalyze the conversion of D-amino acids and their derivatives into keto acids, and it has different catalytic activities for different substrates. Previous studies have reported the application of D-amino acid oxidase mutants in the catalytic synthesis of glufosinate. For example, patent 201811618942.0 discloses a D-amino acid oxidase mutant and its application in glufosinate, achieving enzyme activity and product conversion rates significantly higher than the wild-type mutant, and improving the yield of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO). Patent 201710834958.4 discloses a bioenzymatic deracetamination method for preparing L-glufosinate. In this method, the D-amino acid oxidase is derived from Neurospora crassa, and the intermediate product 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid is catalytically reduced to L-glufosinate by amino acid dehydrogenase, thereby achieving in-situ deracetamination of D and L-glufosinate. The total product yield is ≥99%, and the optical purity can exceed 99%.

[0010] The D-amino acid oxidase of this invention is derived from *Trigonopsis variabilis*, and has a completely different amino acid sequence compared to the D-amino acid oxidases in patents 201811618942.0 and 201710834958.4. Selecting the D-amino acid oxidase from *Trigonopsis variabilis*, modifying and screening it for substrate specificity, improving its catalytic activity for the specific substrate D-glufosinate, shortening the conversion reaction time, and reducing enzyme costs are key technical problems that urgently need to be solved in the enzymatic catalytic production of glufosinate. Summary of the Invention:

[0011] The primary objective of this invention is to provide a D-amino acid oxidase mutant capable of efficiently synthesizing 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO), a precursor of L-glufosinate, and its application. Compared to the wild-type enzyme, this mutant exhibits high catalytic activity, high conversion rate of D-glufosinate, and short reaction time. Furthermore, the immobilized enzyme remains stable after multiple batches of reaction, enabling efficient and low-cost preparation of the intermediate product 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, which can be better applied to the production and preparation of L-glufosinate.

[0012] To achieve this objective, in a basic embodiment, the present invention provides a D-amino acid oxidase mutant obtained by mutating a D-amino acid oxidase derived from the wild-type yeast *Trigonopsis variabilis*. The mutant mutates multiple amino acid sites in the wild-type D-amino acid oxidase of the amino acid sequence shown in SEQ ID NO. 1. These multiple amino acid sites include single-point or multi-point mutations at positions 31, 33, 171, 194, and 197 of the amino acid sequence shown in SEQ ID NO. 1. For example, S194R indicates that the amino acid at position 194 is mutated from S to R.

[0013] The mutants described herein include any one of the following nine mutation types: S194R+V171M; S194R+F197Y; S194R+F33C; S194R+S31D; S194R+F33C+V171M; S194R+F33C+F197Y; S194R+F33C+S31D; S194R+F33C+F197Y+V171M; S194R+F33C+F197Y+S31D.

[0014] Furthermore, the mutation modes include any one of the following seven: S194R+F33C; S194R+S31D; S194R+F33C+V171M; S194R+F33C+F197Y; S194R+F33C+S31D; S194R+F33C+F197Y+V171M; S194R+F33C+F197Y+S31D.

[0015] Furthermore, the mutation modes include any one of the following four: S194R+F33C; S194R+F33C+F197Y; S194R+F33C+F197Y+V171M; S194R+F33C+F197Y+S31D.

[0016] The present invention also provides the nucleotide sequence corresponding to the D-amino acid oxidase mutant.

[0017] A second objective of this invention is to provide applications of D-amino acid oxidase mutants that enable the encoded D-amino acid oxidase mutants to have higher specific activity than the wild-type enzyme and to achieve higher product yields and shorter reaction times in the catalytic preparation of L-glufosinate precursor: 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid.

[0018] To achieve this objective, in a basic implementation scheme, the application of the D-amino acid oxidase mutant of the present invention is to catalyze the reaction of the substrate D-glufosinate with the aforementioned D-amino acid oxidase mutant in a reaction system to prepare 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid.

[0019] In a preferred embodiment, the present invention provides the application of the D-amino acid oxidase mutant, using D-glufosinate as a substrate, to catalyze the synthesis of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid under aerobic conditions.

[0020] The present invention can also carry out the same reaction using a mixture of D-glufosinate and L-glufosinate as a substrate.

[0021] In a preferred embodiment, in every 100 mL of the reaction system of the present invention, the D-amino acid oxidase mutant activity is 100-500 U, the required catalase activity is 100-500 U, and the substrate concentration is 500-750 mM.

[0022] In a preferred embodiment, the reaction temperature of the present invention is 25-30℃, the reaction pH is 8.0-9.0, the reaction stirring speed is 150-200 r / min, and the reaction time is 4-5 h.

[0023] In a preferred embodiment, the D-amino acid oxidase mutant of the present invention is an immobilized D-amino acid oxidase mutant.

[0024] Compared with the prior art, the beneficial effects of the present invention are that the D-amino acid oxidase mutant and its immobilized enzyme of the present invention have higher specific activity than the wild-type D-amino acid oxidase, its catalytic activity is increased by 1200 times, and it can achieve higher product yield and shorter reaction time in the catalytic preparation of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid. Moreover, the immobilized enzyme can react at least 10 batches, which reduces the cost of enzyme production and application and can meet the requirements of industrial production application.

[0025] The amino acid sequence of the wild-type D-amino acid oxidase of this invention, SEQ ID NO.1:

[0026] MAKIVVIGAGVAGLTTALQLLRKGHEVTIVSEFTPGDLSIGYTSPWAGANWLTFYDGGKLADYDAVSYPILRELARSSPEAGIRLINQRSHVLKRDLPKLEGAMSAICQRNPWFKNTVDSFEIIEDRSRIVHDDVAYLVEFASVCIHTGVYLNWLMSQCLSLGATVVKRRVNHIKDAN LLHSSGSRPDVIVNCSGLFARFLGGVEDKKMYPIRGQVVLVRNSLPFMASFSSTPEKENEDEALYIMTRFDGTSIIGGCFQPNNWSSEPDPSLTHRILSRALDRFPELTKDGPLDIVRECVGHRPGREGGPRVELEKIPGVGFVVHNYGAAGAGYQSSYGMADEAISYVERALTRPNL Attached image description:

[0027] Figure 1 This is a schematic diagram of the catalytic reaction of D-amino acid oxidase using a mixture of D-glufosinate and L-glufosinate as substrates;

[0028] Figure 2 This is an SDS-PAGE electrophoresis image of the recombinant D-amino acid oxidase protein derived from Trigonopsis variabilis. Detailed implementation method:

[0029] The following examples are intended to further illustrate the present invention, but not to limit it.

[0030] Example 1: Determination of D-amino acid oxidase activity

[0031] Weigh an appropriate amount of the mixture of substrates D-glufosinate and L-glufosinate (the mixture of D-glufosinate and L-glufosinate in this invention is 50% each), dissolve it thoroughly in 0.1M pH 8.0 phosphate buffer, and bring the volume to 100mL for a final concentration of 100mM. Take 400μl of the substrate solution in a test tube, incubate at 30℃ for 10min, add 50μl of enzyme solution and 0.5μl of catalase, mix well, and start timing. After 10min, add 50μl of 20% trichloroacetic acid to terminate the reaction. Take an appropriate amount of solution, centrifuge at 12000r / min for 5min, and take the supernatant for HPLC liquid chromatography analysis to calculate enzyme activity. Enzyme activity is defined as the amount of enzyme that converts to produce 1 micromolar product per minute under the conditions of 30℃ and pH 8.0, which is defined as 1 unit (1U).

[0032] Example 2: Construction of a recombinant expression strain of D-amino acid oxidase (TvDAAO) derived from Trigonopsis variabilis

[0033] The D-amino acid oxidase sequence of *Trigonopsis variabilis* (accession number: AAR98816.1) was downloaded from the NCBI database. After codon optimization for *E. coli* expression, the sequence was submitted to General Biotechnology (Anhui) Co., Ltd. for synthesis. The gene has an NdeI restriction site at the 5' end, an XhoI restriction site at the 3' end, and a nucleotide sequence with a 6 His tag at the 3' end. The synthesized gene sequence was cloned into the prokaryotic expression plasmid pET30a(+) to construct the recombinant expression plasmid pET30a(+)-TvDAAO. After the recombinant plasmid was verified by sequencing, it was transformed into the *E. coli* expression host *E. coli* BL21(DE3) to obtain a recombinant *E. coli* strain containing the TvDAAO gene.

[0034] Example 3: Expression, purification, and immobilization of TvDAAO

[0035] Using a sterile pipette tip, carefully pick a single colony of the D-amino acid oxidase prokaryotic expression recombinant strain from the above-mentioned LB solid medium plate (containing 50 μg / mL kanamycin), and inoculate it into an Erlenmeyer flask containing 20 mL of LB liquid medium. Incubate overnight at 37°C with shaking at 200 rpm. The next day, inoculate the shake-flask culture at a 1% inoculation rate into an Erlenmeyer flask containing 100 mL of TB liquid medium. Incubate at 37°C with shaking at 220 rpm, and measure the OD value of the culture medium every 1 hour. When the OD value of the culture medium reaches 1.5, add lactose to a final concentration of 1% (m / v), and continue incubating at 25°C with shaking for 4-6 hours, then stop incubation.

[0036] Utilizing the His tag carried in the recombinant protein of D-amino acid oxidase, and employing activated IDA resin (purchased from Anolun (Beijing) Biotechnology Co., Ltd., specific model: His.Bind Resin, Ni-charged), the specific methods and steps are as follows: The fermentation broth was centrifuged at 10000 rpm for 10 min at 4℃, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were washed twice repeatedly with phosphate buffer (pH 8.0, 0.1 mol / L), and after centrifugation, the bacterial cells were concentrated 5 times and resuspended in 20 mL of phosphate buffer (pH 8.0, 0.1 mol / L). The treated bacterial solution was then subjected to ultrasonic disruption in ice water until clear. The ultrasonic disruption conditions were: 2 s operation, 5 s interval, and ultrasonic power of 500 W. The disrupted lysate was centrifuged in a low-temperature high-speed centrifuge (12000 rpm, 4℃, 20 min), and the supernatant was collected to obtain the crude protein. The crude protein was loaded onto the activated resin and eluted with a gradient of imidazole solution (200mM-500mM). The protein was monitored in real time using a protein chromatography system (Bio-Rad), and the stable protein peaks that appeared were collected. These were the purified TvDAAO recombinant protein, which was used for the preparation of immobilized enzymes.

[0037] The purified TvDAAO recombinant protein was used to prepare the immobilized enzyme. The specific method is as follows:

[0038] (1) Activation of immobilized carrier: Accurately measure 30 mL of 60% (m / v) glutaraldehyde and add it together with 4.76 g of dipotassium hydrogen phosphate (K2HPO4·3H2O) to 600 mL of deionized water. After dissolving, bring the volume to 1000 mL with deionized water and adjust the pH to 8.0 with phosphoric acid solution. Add 250 g of epoxy-based carrier ECEP (Resindion S.rl, Italy) to the above solution and activate it by stirring at low speed at 25 °C for 2 h. Filter and collect the carrier, wash it 2-3 times with sterile deionized water, and then vacuum filter it for later use.

[0039] (2) Immobilization of TvDAAO recombinant protein: Take a certain amount of the purified TvDAAO recombinant protein, dilute it with phosphate buffer (pH 8.0, 0.1 mol / L), then add 50 g of the activated carrier, and immobilize it at 25℃ and 120 rpm for 48 h. The immobilized enzyme is washed with deionized water 3-5 times and vacuum filtered to obtain the final immobilized enzyme product.

[0040] Example 4: Construction of a "smart mutant library" of D-amino acid oxidase (TvDAAO) from Trigonopsis variabilis

[0041] D-type glufosinate substrate docking analysis with TvDAAO protein: In order to select key amino acid mutation sites for enzyme-substrate interaction within the 5A range for the construction of a "smart mutant library". The specific steps are as follows: (1) First, the structure of D-amino acid oxidase (TvDAAO) from Trigonopsis variabilis was simulated (the protein crystal structure is not published in the database), and the structure was obtained from the protein database website ( RCSB Protein Data Bank Download the TvDAAO structure prediction PDB file (AlphaFold structure prediction) from the software, process the receptor protein molecule, such as: adding hydrogen, minimizing energy, adding force field, deleting water molecules, removing redundant protein conformations, and supplementing incomplete amino acid residues; (2) Next, draw the molecular structure diagram of the small molecule ligand D-glufosinate in ChemOffice2018 software, convert it into a 3D structure, and save it in mol2 format; (3) Molecular docking: use the molecular simulation software AutoDock Vina to perform molecular docking processing, and analyze the docking results to analyze the key amino acid sites of substrate and enzyme molecule protein binding, and use these as mutation hotspots to construct a smart saturated mutant library of D-amino acid oxidase.

[0042] The construction of the “D-amino acid oxidase smart library” involved selecting key amino acid residues for enzyme-substrate binding, designing saturation mutation primers, and performing saturation mutations. The saturation mutation primers are shown in Table 1, and can also be found in SEQ ID NO: 2-33.

[0043] Table 1: Amino acid mutation sites and saturation mutation primer design in the "Smart Library"

[0044]

[0045]

[0046] The saturated mutant library was constructed using circular PCR. The PCR product was digested with DpnI and then click-transformed into BL21(DE3) competent cells to obtain the TvDAAO saturated mutant library, which was used for subsequent bacterial culture and high-throughput screening experiments.

[0047] Example 5: High-throughput screening of TvDAAO saturated mutant libraries

[0048] Single colonies were selected from the TvDAAO saturated mutant library using sterile toothpicks and cultured in 96-well microplates, each well containing 320 μL of LB medium supplemented with 50 μg / mL kanamycin. The plates were incubated at 37°C for 8 hours on a shaker (240 rpm). Then, 20 μL of the culture was transferred to a new 96-well microplate containing 10% glycerol (v / v) as a daughter plate and stored at 4°C. After preparing the daughter plate, 20 μL of 10% (w / v) lactose was added to the mother plate, and the plate was incubated overnight at 25°C to induce expression of the target protein. Subsequently, 100 μL of the induced bacterial culture was added to a 96-well microtiter plate containing 100 μL of lysis buffer (0.1% (v / v) Triton X-100, 0.5 mg / mL lysozyme, 0.1 M Tris-HCl, pH 8.0). After incubating at 25°C for 1 hour, the cell lysis mixture was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was the crude enzyme solution, which was used for the determination of TvDAAO activity.

[0049] In a 100 μL reaction system, use 20 μL of supernatant and add 80 μL of substrate solution containing a 500 mM mixture of D-glufosinate and L-glufosinate (dissolved in pH 8.0 phosphate buffer). After reacting for 10 min, add 50 μL of reaction solution and react at 25 °C. Observe the color change (reddish-brown). Select clones with darker colors for subsequent rescreening experiments and DNA sequencing analysis. Reaction solution composition: 1000× o-anisidine ammonium stock solution: 53.3 mg / 500 μL, prepared in pH 8.0 phosphate buffer, protected from light, and insoluble. 1000× horseradish peroxidase: 2000 U / 500 μL (13 mg, 160 U / mg), prepared in pH 8.0 phosphate buffer, protected from light, and insoluble. Mix the two in a 1:1 ratio.

[0050] A saturated mutant library was constructed using the saturated mutant library construction method described in Example 4. Then, high-throughput screening of the saturated mutant library was performed using the method described in Example 5. After initial screening of the saturated mutant library at 16 sites, strains showing the deepest color in a 96-well plate were selected for secondary screening and then validated by shake-flask fermentation. The final beneficial mutant strains and their information are shown in Table 2.

[0051] Table 2: Favorable mutant strains obtained from screening Tv-DAAO saturated mutant libraries

[0052]

[0053] Example 6: Construction and Screening of Superimposed Mutant Libraries

[0054] Based on the screening results shown in Example 5, the optimal mutant strain TvDAAO-4 was selected. Using this as a template, superimposed mutants were constructed and screened based on the TvDAAO-4 mutant. The construction and screening methods for superimposed mutants were carried out according to Examples 4 and 5. The beneficial mutant strains obtained through screening are shown in Table 3.

[0055] Table 3: Screening of mutant libraries based on TvDAAO-4

[0056]

[0057] As shown in Table 3, the catalytic activity of the mutant TvDAAO-8 was significantly enhanced through superposition mutation. Furthermore, superposition mutants were constructed based on the TvDAAO-8 mutant strain, and the catalytic performance of these superposition mutants is shown in Table 4.

[0058] Table 4: Screening of mutant libraries based on TvDAAO-8

[0059]

[0060] As shown in Table 4, the catalytic activity of the mutant TvDAAO-11 was most significantly enhanced through superimposed mutations. Furthermore, superimposed mutation libraries were constructed and screened based on the TvDAAO-11 mutant strain.

[0061] Table 5: Screening of mutant libraries based on TvDAAO-11

[0062]

[0063] The results of the above mutation screening show that the mutant TvDAAO-11 has the best effect. Its catalytic performance is the best compared with the wild type and other mutants. It can generate more products per unit time under the same conditions and can be used as a candidate mutant for industrial application.

[0064] Example 7: Verification of the transformation of wild-type enzyme and high-activity mutant enzyme at different substrate concentrations

[0065] To verify the catalytic performance of each mutant enzyme (activity > 1 U / mg), the performance of each mutant strain in catalyzing the conversion of D-glufosinate to keto acid was compared and analyzed under the same D-amino acid oxidase protein dosage. The reaction system was as follows: A mixture of D-glufosinate and L-glufosinate substrates (prepared to substrate concentrations of 500 mM and 750 mM) was dissolved in two equal portions of pH 8.00 and 0.1 M phosphate buffer, respectively. After complete dissolution, 50 mg of catalase and 50 mg of D-amino acid oxidase were added, bringing the total system volume to 100 mL. The reaction conditions were: pH 8.00, temperature 30℃, and oxygenation for 5 h. The product yield was measured, and the conversion rate was calculated. The conversion results are as follows:

[0066] Table 6: Verification of the conversion between mutant and wild-type enzymes at different substrate concentrations

[0067]

[0068]

[0069] As shown in Table 6, under the same enzyme protein dosage, the different mutant strains exhibited different transformation results when transforming a mixture of D-glufosinate and L-glufosinate with the same substrate concentration. Among them, mutant strains TvDAAO11, TvDAAO13, and TvDAAO14 achieved transformation yields of over 90% under both substrate concentrations, demonstrating good performance. In particular, mutant strain TvDAAO11 achieved transformation yields of over 99% under both substrate concentrations, showing potential for industrial application.

[0070] Example 8: Batch Transformation Validation of Immobilized Enzymes

[0071] The mutant TvDAAO-11 was isolated, purified, and immobilized according to the method shown in Example 3. Transformation was performed using the immobilized enzyme. The immobilized enzyme reaction system was as follows: a mixture of D-glufosinate and L-glufosinate substrates was dissolved in 40 mL of pH 8.00, 0.1 M phosphate buffer to prepare a substrate concentration of 750 mM. 50 mg of catalase (total enzyme activity: 100 U) and 50 mg of immobilized D-amino acid oxidase TvDAAO-11 (total enzyme activity: 500 U) were added. The reaction temperature was 30 °C, the stirring speed was 150 r / min, and the reaction was carried out with oxygen for 5 h. After one batch of reaction was completed, the immobilized enzyme was eluted with phosphate buffer, filtered dry, and used for the next batch of reaction. The reaction results are shown in Table 8.

[0072] Table 8: Results of TvDAAO-11 Immobilized Enzyme Transformation Application

[0073]

[0074]

[0075] Analysis of data from the transformation application of immobilized enzymes shows that the mutant TvDAAO-11 exhibits stable reaction yields for the transformation of PPO products, with an average yield of over 99.7%. The reaction time is not prolonged and the activity is not lost, making it suitable for industrial application.

[0076] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention is also intended to include these modifications and variations. The above embodiments or implementations are merely illustrative examples of this invention, and it can also be implemented in other specific ways or forms without departing from its gist or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of this invention should be defined by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of this invention.

Claims

1. A D-amino acid oxidase mutant, characterized in that: it is The amino acid sequence shown in SEQ ID NO.1 was mutated in any of the following nine ways: S194R+V171M; S194R+F197Y; S194R+F33C; S194R+S31D; S194R+F33C+V171M; S194R+F33C+F197Y; S194R+F33C+S31D; S194R+F33C+F197Y+V171M; S194R+F33C+F197Y+S31D.

2. The mutant according to claim 1, characterized in that: The mutation mode is any one of the following 7 types: S194R+F33C; S194R+S31D; S194R+F33C+V171M; S194R+F33C+F197Y; S194R+F33C+S31D; S194R+F33C+F197Y+V171M; S194R+F33C+F197Y+S31D.

3. The mutant according to claim 2, characterized in that: The mutation mode is any one of the following four: S194R+F33C; S194R+F33C+F197Y; S194R+F33C+F197Y+V171M; S194R+F33C+F197Y+S31D.

4. The nucleotide sequence of the D-amino acid oxidase mutant according to any one of claims 1-3.

5. The application of the D-amino acid oxidase mutant according to any one of claims 1-3, characterized in that, 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid was synthesized by catalysis using D-glufosinate as a substrate.

6. The application according to claim 5, characterized in that, Catalytic reaction occurs under aerobic conditions.

7. The application according to claim 6, characterized in that, In every 100 mL of the reaction system, the activity of the D-amino acid oxidase mutant is 100-500 U, the activity of catalase is 100-500 U, and the substrate concentration is 500-750 mM.

8. The application according to claim 6, characterized in that, The reaction temperature is 25-30℃, the reaction pH is 8.0-9.0, the reaction stirring speed is 150-200 r / min, and the reaction time is 4-5 h.

9. The application according to claim 5, characterized in that, The D-amino acid oxidase mutant is an immobilized D-amino acid oxidase mutant.

Images