Enzyme mutants and their applications

Abstract

The present invention relates to the field of biocatalysis technology, and more particularly to enzyme mutants and their applications. The present invention provides mutants of D-amino acid oxidase from the yeast Lobelia salmoides, wherein the mutation sites include N54V, C56N, F58H, M216S, R116V, and / or K319T. The mutants have high enzyme activity and high conversion rates, and can efficiently prepare 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid with high product yield.

Description

Technical Field

[0001] The present invention relates to the field of biocatalysis technology, in particular to enzyme mutants and applications thereof. Background Art

[0002] D-amino acid oxidase (DAAO) is a class of flavin adenine dinucleotide-containing oxidoreductases that catalyze the oxidative dehydrogenation of D-amino acids to produce the corresponding α-keto acids, hydrogen peroxide, and ammonia. This class of enzymes is widely distributed in nature, primarily originating from eukaryotes and a few prokaryotes. As a classic biocatalyst, D-amino acid oxidase boasts mild reaction conditions, a broad substrate spectrum, and excellent enantioselectivity, making it valuable in the synthesis of pharmaceuticals, pesticides, and fine chemicals.

[0003] D-amino acid oxidase is currently widely used in the preparation of L-phosphinothricin (L-PPT), the primary active compound in racemic glufosinate that exerts herbicidal activity. Therefore, replacing racemic glufosinate with L-phosphinothricin can save half the amount of glufosinate required, thereby reducing pesticide usage and lowering farmers' weed control costs. Green et al. used RgDAAO and the Escherichia coli transaminase EcgabT to perform a deracemization reaction, converting DL-phosphinothricin to L-phosphinothricin with a conversion efficiency of 85% and an ee value of >99%. Xue Yaping et al. used RgDAAO and Pseudomonas transaminase to perform a deracemization reaction, converting DL-phosphinothricin to L-phosphinothricin. The remaining D-phosphinothricin (D-PPT) was 0.8 mM, with a conversion efficiency of 48.4% (maximum theoretical conversion of 50%) and a resulting concentration of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO) of 20 mM. Tian Zhenhua et al., using site-directed mutagenesis of DAAO from Rhodotorula, improved its enzyme activity, stability, and / or ammonium tolerance. Xie Xinkai et al., using gene mutation and directed enzyme evolution, modified DAAO from the three aforementioned sources, obtaining DAAO with increased stability and / or enhanced activity toward D-phosphinothricin. Yang Lirong et al., using Neurospora crassa NcDAAO and Pseudomonas sp. GluDH, performed a deracemization reaction to convert DL-phosphinothricin to L-phosphinothricin. At a substrate concentration of 20 mM, the conversion rate was 99.4% and the ee value was 99.1%. Cheng Feng et al., using aspartate oxidase CeDAAO and a mutant glutamate dehydrogenase, converted DL-phosphinothricin to L-phosphinothricin, achieving a substrate conversion rate of 98.6% and a product ee value of 99%. Xia Shiwen et al. used D-amino acid oxidase in whole cells of Bacillus xylosinolyticus XX-2 to catalyze the oxidative deamination of D-phosphinothricin to 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid (PPO), while L-phosphinothricin was retained. The overall reaction yield was >70% and the optical purity was >99%.

[0004] The use of D-amino acid oxidase to split D,L-glufosinate to prepare L-glufosinate has become a research hotspot. Therefore, improving the yield of PPO is of great significance. Summary of the Invention

[0005] In view of this, the present invention provides an enzyme mutant and its application, which has the characteristics of high enzyme activity and high conversion rate, can efficiently prepare 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, and has a high product yield.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a mutant of D-amino acid oxidase, comprising:

[0008] The asparagine at position 54 of the D-amino acid oxidase is replaced by valine; and / or

[0009] The cysteine ​​at position 56 of the D-amino acid oxidase is substituted with asparagine; and / or

[0010] The phenylalanine at position 58 of the D-amino acid oxidase is substituted with histidine; and / or

[0011] The methionine at position 216 of the D-amino acid oxidase is substituted by serine.

[0012] In some specific embodiments of the present invention, the mutant further comprises:

[0013] The arginine at position 116 of the D-amino acid oxidase is replaced by valine; and / or

[0014] The 319th lysine of the D-amino acid oxidase is substituted by threonine.

[0015] In some specific embodiments of the present invention, the mutants include:

[0016] (a), the D-amino acid oxidase is derived from Desmodium salmonifolium; and / or

[0017] (b) the D-amino acid oxidase has a specific amino acid sequence; and / or

[0018] (c) the D-amino acid oxidase gene has a specific nucleotide sequence;

[0019] The specific amino acid sequence includes:

[0020] (I), the amino acid sequence shown in SEQ ID NO. 2; or

[0021] (II) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence as shown in (I), and having the same or similar function as (I); or

[0022] (III) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (I) or (II);

[0023] The plurality is 2 to 110;

[0024] The specific nucleotide sequence includes:

[0025] (IV), the nucleotide sequence shown in SEQ ID NO.1; or

[0026] (V) A nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (IV), and having the same or similar function as (IV); or

[0027] (VI) a nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (IV) or (V);

[0028] The plurality is 2 to 330.

[0029] In some specific embodiments of the present invention, the mutant has:

[0030] (1) an amino acid sequence as shown in any one of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10; or

[0031] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0032] (3) an amino acid sequence having at least 70% homology to the amino acid sequence shown in (1) or (2);

[0033] The plurality is 2 to 110.

[0034] The present invention also provides a nucleic acid molecule encoding the above mutant, which has:

[0035] (4) a nucleotide sequence as shown in any one of SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9; or

[0036] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (5), and having the same or similar function as (5); or

[0037] (6) a nucleotide sequence having at least 70% homology to the nucleotide sequence shown in (4) or (5);

[0038] The plurality is 2 to 330.

[0039] The present invention also provides an expression vector comprising the above nucleic acid molecule and acceptable gene elements.

[0040] The present invention also provides a host cell comprising the above nucleic acid molecule or the above expression vector.

[0041] The present invention also provides uses of the mutant, nucleic acid molecule, expression vector and / or host cell in the following aspects:

[0042] (i) increasing the conversion rate of D-phosphinothricin to 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid; and / or

[0043] (ii) Synthesis of L-phosphinothricin.

[0044] The present invention also provides a method for synthesizing 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid, comprising mixing glufosinate-ammonium with any one of the following, and reacting to obtain the 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid:

[0045] (A), the mutants described above; and / or

[0046] (B) the above-mentioned host cells.

[0047] In some specific embodiments of the present invention, in the above-mentioned synthesis method, the reaction temperature is 20-37° C.; and / or

[0048] The reaction time is 2 to 24 hours; and / or

[0049] The reaction is carried out in a phosphate buffer solution with a concentration of 10 to 300 mM; and / or

[0050] The pH of the phosphate buffer is 7.5 to 9.0; and / or

[0051] The concentration of the mutant is 0.1 to 10 g / L; and / or

[0052] The enzyme activity of the mutant is 3.7 to 9.7 U / mL; and / or

[0053] The wet weight of the host cells is 100 g / L.

[0054] The mutant and application of the present invention have the following effects:

[0055] The present invention provides a novel D-amino acid oxidase mutant and its application, targeting the existing process for preparing 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid by catalysis of amino acid oxidase. The mutant has the characteristics of high enzyme activity and high conversion rate, can efficiently prepare 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid, and the yield of the product obtained by catalysis is high. The product can be used as a substrate to directly produce L-phosphinothricin in a one-pot process.

[0056] Among them, high enzyme activity means that the enzyme activity of mutant SS-1 is 3.7U / mL fermentation liquid. By introducing different mutation sites, the enzyme activity of mutant SS-4 is increased to 6.5U / mL fermentation liquid.

[0057] The high conversion rate refers to the use of an enzyme catalytic solution prepared from mutant SS-4 to prepare 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, and the conversion rate reached 99.6% after 7 hours of reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0059] Figure 1 Figure 4 shows the liquid phase diagram of the catalytic test of DAAO-SS-4 mutant; A is the chromatogram; B is the specific data of liquid chromatography;

[0060] Figure 2 Shown is the reaction progress curve of the catalytic test of DAAO-SS-4 mutant. DETAILED DESCRIPTION

[0061] The present invention discloses enzyme mutants and applications thereof. Those skilled in the art can refer to the contents of this article and appropriately improve process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0062] The present invention utilizes a wild-type ssDAAO enzyme derived from the yeast Salmonella (GenBank accession number CEQ39319.1), adds preferred mutation sites, and then constructs an engineered Escherichia coli strain for protein expression. The mutant strain lysate has the ability to catalyze D-phosphinothricin to produce 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid. The inventors then verified the improvement in enzyme activity through a fermentation test in a 10L fermentor. Furthermore, a catalytic test of 50g / L substrate glufosinate was carried out, and the results showed that 0.31mM of D-phosphinothricin remained, with a conversion rate of 99.6%.

[0063] The present invention provides a method for synthesizing 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid, comprising the following steps: using glufosinate-ammonium 2-amino-4-[hydroxy(methyl)phosphonyl]butyric acid as a substrate, utilizing amino acid oxidase or an amino acid oxidase mutant to carry out a catalytic reaction, and the reaction product is 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid.

[0064] In some specific embodiments of the present invention, the amino acid oxidase described in the above synthesis method is derived from Sporidiobolus salmonicolor.

[0065] In some specific embodiments of the present invention, the amino acid sequence of the wild-type amino acid oxidase from Lobelia salmoides described in the above synthesis method is shown in SEQ ID No. 2;

[0066] The amino acid sequence of the wild-type ssDAAO enzyme mutant SS-1 is shown in SEQ ID No. 4;

[0067] The amino acid sequence of the wild-type ssDAAO enzyme mutant SS-2 is shown in SEQ ID No. 6;

[0068] The amino acid sequence of the wild-type ssDAAO enzyme mutant SS-3 is shown in SEQ ID No. 8;

[0069] The amino acid sequence of the wild-type ssDAAO enzyme mutant SS-4 is shown in SEQ ID No. 10;

[0070] In some specific embodiments of the present invention, a tag is connected to the N-terminus and / or C-terminus of the amino acid oxidase described in the above synthesis method.

[0071] In some specific embodiments of the present invention, the mutant of the amino acid oxidase described in the above synthesis method includes: performing the following point mutations on the amino acid sequence shown in SEQ ID No. 2: N54V and / or C56N and / or F58H and / or M216S; performing at least one of the following point mutations on the amino acid sequence shown in SEQ ID No. 4: R116V, K319T.

[0072] In some specific embodiments of the present invention, the amino acid oxidase or the mutant of the amino acid oxidase described in the above synthesis method is added in the form of crude enzyme solution, lyophilized crude enzyme solution powder, pure enzyme or whole cells.

[0073] In some specific embodiments of the present invention, the temperature of the catalytic reaction in the above-mentioned synthesis method is 20 to 37° C., and the time of the catalytic reaction is 2 to 24 hours.

[0074] In some specific embodiments of the present invention, when the alcohol dehydrogenase is added in the form of crude enzyme solution, crude enzyme solution lyophilized powder or pure enzyme in the above-mentioned synthesis method, the concentration of the amino acid oxidase in the reaction system is 0.1 g / L to 10 g / L; when the alcohol dehydrogenase is added in the form of whole cells, the wet weight of the whole cells is 100 g / L.

[0075] In some specific embodiments of the present invention, the catalytic reaction of the above-mentioned synthesis method is carried out in a phosphate buffer solution with a concentration of 10 to 300 mM and a pH of 7.5 to 9.0.

[0076] In some specific embodiments of the present invention, when the amino acid oxidase is added in the form of a crude enzyme solution, a lyophilized crude enzyme solution, or a pure enzyme in the above-mentioned synthesis method, the reaction system of the catalytic reaction needs to be supplied with oxygen or air in addition to the catalase for removing by-products.

[0077] The nucleotide / amino acid sequences involved in the present invention are shown in Table 1:

[0078] Table 1

[0079]

[0080]

[0081]

[0082] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0083] The present invention will be further described below in conjunction with the embodiments:

[0084] Preparation Example

[0085] 1. Preparation process of wild-type DAAO enzyme:

[0086] The nucleotide sequence of the wild-type ssDAAO enzyme from Sporidiobolus salmonicolor with GenBank accession number CEQ39319.1 retrieved by NCBI is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2. The wild-type ssDAAO enzyme gene was fully synthesized, and the gene synthesis was completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd.

[0087] Based on SEQ ID NO. 1, primers were designed to amplify the full-length ssDAAO enzyme gene, introducing an NdeI restriction endonuclease site at the 5' end and an XhoI restriction endonuclease site at the 3' end. The full-length ssDAAO fragment was amplified using a high-fidelity PCR enzyme. The pET28a empty vector plasmid was provided by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The PCR product was purified using a PCR product purification kit purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0088] The purified PCR product was digested with NdeI-XhoI, and simultaneously, the purified pET28a empty vector plasmid was digested with NdeI-XhoI. The size of the digested fragments was verified by 0.7% agarose gel electrophoresis. The fragments were connected using T4 ligase, which was purchased from Takara. The ligation product was used to transform Ecoli.BL21 (DE3) cells, which were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the transformation product was coated on LB solid culture medium containing 50 μg / L kan (kanamycin sulfate). Positive transformants were detected using the colony PCR method, and the positive transformants were used to induce the production of DAAO enzyme.

[0089] 2. Design and screening steps of DAAO enzyme mutants

[0090] Based on the amino acid sequence of the wild-type ssDAAO enzyme, mutation sites were further added, and the mutant sequence was commissioned to Hangzhou Qingke Zixi for sequence synthesis. The amino acid sequence numbering is shown in Table 2, and the strain was constructed according to the above method.

[0091] Table 2

[0092] serial number mutation site Nucleotide number Amino acid number SS-1 N54V-C56N-F58H-M216S SEQ ID NO.3 SEQ ID NO.4 SS-2 N54V-C56N-F58H-R116V-M216S SEQ ID NO.5 SEQ ID NO.6 SS-3 N54V-C56N-F58H-M216S-K319T SEQ ID NO.7 SEQ ID NO.8 SS-4 N54V-C56N-F58H-R116V-M216S-K319T SEQ ID NO.9 SEQ ID NO.10

[0093] Each single colony was inoculated into a 50 mL Erlenmeyer flask containing 20 mL of LB liquid culture medium containing 50 μg / mL Kan, and cultured with shaking at 37°C and 150 rpm for 16 h. Then, 20 mL of LB culture medium containing 50 μg / mL Kan was added at an inoculation ratio of 2%, and cultured with shaking at 37°C and 150 rpm for 4 h. 100 μL of 100 mM IPTG was added, and expression was induced at 28°C and 150 rpm for 6 h. The fermentation supernatant was removed by centrifugation, and the bacterial sludge was treated with BugBuster protein extract (purchased from Novagen) for D-amino acid oxidase activity detection.

[0094] 3. Enzyme activity detection of different DAAO mutants

[0095] Microplate reader assay method: 100 μL of 100 mM substrate (racemic glufosinate ammonium, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) at pH 8.0 was added to 50 μL of a colorimetric solution containing 60 μg / mL TBHBA (3-hydroxy-2,4,6-tribromobenzoic acid) and 1 mg / mL 4-AAP (4-aminoantipyrine), along with 25 μL of horseradish peroxidase (0.1 mg / mL) HRP. Finally, 25 μL of the aforementioned DAAO mutant enzyme solution was added to create a 200 μL reaction system on a microplate. The assay was performed at 30°C and pH 8.0. The absorbance at 510 nm was recorded at 0 and 20 minutes, and the difference was calculated. Positive clones were screened using the wild-type as a reference. The results are shown in Table 3.

[0096] Table 3

[0097]

[0098] 4. Fermentation Scale-up

[0099] The fermentation of Escherichia coli DAAO-SS-1 and Escherichia coli DAAO-SS-4 was carried out in a 10-liter fermenter. The culture medium required for the fermentation was added with the following formula: 10 g / L animal peptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and 0.05% defoamer.

[0100] The fermentation process can be roughly divided into 3 stages:

[0101] (1) Cultivation stage: 1% seed solution inoculation, cultivation at 37°C, initial stirring starting from 200 rpm, adjusted every half hour, and maintained at 500 rpm; ventilation volume from 0.2m 3 / h, and adjust it every half hour synchronously with the stirring to 2m 3 / h to maintain the state, and this stage lasted a total of 4 hours;

[0102] (2) Feeding stage: Glucose (500 g / L) was fed when the dissolved oxygen recovered and the pH increased. The feeding rate was adjusted according to the pH change. Ammonia was used to adjust the acidity and maintain the whole culture state. This stage lasted for 1 h.

[0103] (3) Induction stage: measuring the OD of the fermentation liquid 600 At about 6 to 10 o'clock, the temperature was lowered and induced. The inducer was IPTG at a concentration of 0.1%, the temperature was 28°C, and the rotation speed was 500 rpm. Glucose was continuously added during this period. The induction lasted for 18 hours, and the cells were removed from the tank and the wet cells were collected by centrifugation.

[0104] By measuring the D-amino acid oxidase activity in the fermentation broth at different times, a fermentation progress curve was generated. The results showed that the D-amino acid oxidase activity in the final fermentation broth of the starting strain E. coli DAAO-SS-1 was 5.3 U / mL, while that of the mutant strain E. coli DAAO-SS-4 was 9.7 U / mL, an 83.0% increase over the starting strain, achieving unexpected technical results.

[0105] Example 1: Catalytic reaction test of DAAO mutant (DAAO-SS-1)

[0106] The fermentation broth of the mutant strain Escherichia coli DAAO-SS-1 described in the preparation example was centrifuged to collect wet cells, weighed, and resuspended in 50 mM pH 8.0 phosphate buffer at 10% (wt%). The cells were lysed using an ultrasonic disruptor (power 400 W, 15 s on, 15 s off pulse treatment for 30 min) in an ice bath. The cells were centrifuged at 8000 rpm for 15 min at 4°C, and the precipitate was discarded. The supernatant was the crude enzyme solution of the DAAO mutant and set aside.

[0107] Reaction system: Dissolve 10g of D,L-glufosinate ammonium salt (prepared in-house, purity ≥95%) in a sufficient amount of water, place in a four-necked flask, add 20mL of crude DAAO-SS-1 mutant enzyme solution, add 1mL of catalase (50,000U / mL, purchased externally), add water to 200mL, introduce oxygen (1VVM), start stirring at 200rpm, and begin the reaction. During the process, control the pH between 7.0 and 8.0. Samples are taken every hour until the reaction is complete after 7 hours. The D-glufosinate remaining in the sample at the end of the reaction is 17.48mM, with a conversion rate of 91.3%.

[0108] Example 2: Catalytic reaction test of DAAO mutant (DAAO-SS-2)

[0109] The fermentation broth of the mutant strain Escherichia coli DAAO-SS-2 described in the preparation example was centrifuged to collect wet cells, weighed, and resuspended in 50 mM pH 8.0 phosphate buffer at 10% (wt%). The cells were lysed using an ultrasonic disruptor (power 400 W, 15 s on, 15 s off pulse treatment for 30 min) in an ice bath. The cells were centrifuged at 8000 rpm at 4°C for 15 min, and the precipitate was discarded. The supernatant was the crude enzyme solution of the DAAO mutant and set aside.

[0110] Reaction system: Dissolve 10g of D,L-glufosinate ammonium salt (prepared in-house, purity ≥95%) in a sufficient amount of water, place in a four-necked flask, add 20mL of crude DAAO-SS-2 mutant enzyme solution, add 1mL of catalase (50,000U / mL, purchased externally), add water to 200mL, introduce oxygen (1VVM), start stirring at 200rpm, and begin the reaction. During the process, control the pH between 7.0 and 8.0. Samples are taken every hour until the reaction is complete after 7 hours. The D-glufosinate remaining in the sample at the end of the reaction is 11.92mM, with a conversion rate of 94.1%.

[0111] Example 3: Catalytic reaction test of DAAO mutant (DAAO-SS-3)

[0112] The fermentation broth of the mutant strain Escherichia coli DAAO-SS-3 described in the preparation example was centrifuged to collect wet cells, weighed, and resuspended in 50 mM pH 8.0 phosphate buffer at 10% (wt%). The cells were lysed using an ultrasonic disruptor (power 400 W, 15 s on, 15 s off pulse treatment for 30 min) in an ice bath. The cells were centrifuged at 8000 rpm at 4°C for 15 min, and the precipitate was discarded. The supernatant was the crude enzyme solution of the DAAO mutant and set aside.

[0113] Reaction system: Dissolve 10g of D,L-glufosinate ammonium salt (prepared in-house, purity ≥95%) in a sufficient amount of water, place in a four-necked flask, add 20mL of crude DAAO-SS-3 mutant enzyme solution, add 1mL of catalase (50,000U / mL, purchased externally), add water to 200mL, introduce oxygen (1VVM), start stirring at 200rpm, and begin the reaction. During the process, control the pH between 7.0 and 8.0. Samples are taken every hour until the reaction is complete after 7 hours. The sample at the end of the reaction is tested to have 3.87mM D-glufosinate remaining, with a conversion rate of 98.1%.

[0114] Example 4: Catalytic reaction test of DAAO mutant (DAAO-SS-4)

[0115] The fermentation broth of the mutant strain Escherichia coli DAAO-SS-4 described in the preparation example was centrifuged to collect wet cells, weighed, and resuspended in 50 mM pH 8.0 phosphate buffer at 10% (wt%). The cells were lysed using an ultrasonic disruptor (power 400 W, 15 s on, 15 s off pulse treatment for 30 min) in an ice bath. The cells were centrifuged at 8000 rpm at 4°C for 15 min, and the precipitate was discarded. The supernatant was the crude enzyme solution of the DAAO mutant and set aside.

[0116] Reaction system: Dissolve 10g of D,L-glufosinate ammonium salt (prepared in-house, purity ≥95%) in a sufficient amount of water, place in a four-necked flask, add 20mL of crude DAAO-SS-4 mutant enzyme solution, add 1mL of catalase (50,000U / mL, purchased externally), add water to 200mL, introduce oxygen (1VVM), start stirring at 200rpm, and begin the reaction. During the process, control the pH between 7.0 and 8.0. Samples are taken every hour until the reaction is complete after 7 hours. The sample at the end of the reaction is tested to have 0.31mM D-glufosinate remaining, indicating a conversion rate of 99.6%.

[0117] In the catalytic test of the DAAO-SS-4 mutant, the content of each key component in the samples taken at different times during the reaction process was tracked and detected by high-performance liquid chromatography, and a reaction progress curve was drawn, among which the peak time of PPT was (3.315min) and the peak time of PPO was (6.780min). Figure 1 The liquid phase diagram at 7 hours is shown, and the reaction curve is as follows Figure 2 The data are shown in Table 4.

[0118] Table 4

[0119] time L-PPT (mM) D-PPT (mM) PPO (mM) 0 124.15 124.11 0.0 1.0 125.37 102.25 23.51 2.0 124.58 77.92 46.37 3.0 125.47 41.23 84.72 4.0 125.73 16.23 109.07 5.0 125.33 7.41 117.31 6.0 125.61 0.58 124.83 7.0 125.49 0.31 125.36

[0120] Comparative Example: Wild-type DAAO (DAAO-SS) catalytic reaction test

[0121] The wild-type strain Escherichia coli DAAO-SS fermentation broth was centrifuged to collect wet cells, which were weighed and resuspended in 50 mM pH 8.0 phosphate buffer at 10% (wt%). The cells were lysed using an ultrasonic disruptor (power 400 W, 15 s on, 15 s off pulse treatment for 30 min) in an ice bath. The cells were centrifuged at 8000 rpm for 15 min at 4°C, and the precipitate was discarded. The supernatant was the crude enzyme solution of the DAAO mutant and set aside.

[0122] Reaction system: Dissolve 10g of D,L-glufosinate ammonium salt (prepared in our laboratory, purity ≥95%) in a certain amount of water, place in a four-necked flask, add 20mL of crude enzyme solution of DAAO-SS wild-type strain, add 1mL of catalase (50,000U / mL, purchased externally), add water to 200mL, introduce oxygen (1VVM), start stirring at 200rpm, and start the reaction. During the process, control the pH between 7.0 and 8.0. Samples are taken every hour until the reaction is completed after 7 hours. The D-glufosinate remaining in the sample at the reaction end point is 237.43mM, and the conversion rate is 5.35%.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutant of D-amino acid oxidase, characterized in that The amino acid sequence thereof is shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.

10.

2. A nucleic acid molecule encoding the mutant according to claim 1, characterized in that The nucleotide sequence thereof is shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.

9.

3. An expression vector, characterized in that The method comprises the nucleic acid molecule according to claim 2, and an acceptable genetic element.

4. A host cell, characterized in that comprising the nucleic acid molecule according to claim 2 or comprising the expression vector according to claim 3.

5. Use of the mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression vector according to claim 3 and / or the host cell according to claim 4 in the following aspects: (i) increasing the conversion rate of D-phosphinothricin to 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid; and / or (ii) Synthesis of L-phosphinothricin.

6. A method for synthesizing 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, characterized in that: The method comprises mixing glufosinate-ammonium with any of the following items, reacting the mixture, and obtaining the 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid: (A), the mutant according to claim 1; and / or (B) The host cell according to claim 4.

7. The synthesis method according to claim 6, wherein The reaction temperature is 20-37°C; and / or The reaction time is 2 to 24 hours; and / or The reaction is carried out in a phosphate buffer solution with a concentration of 10 to 300 mM; and / or The pH of the phosphate buffer is 7.5 to 9.0; and / or The concentration of the mutant is 0.1 to 10 g / L; and / or The enzyme activity of the mutant is 3.7 to 9.7 U / mL; and / or The wet weight of the host cells is 100 g / L.

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