D-amino acid oxidase mutant and use thereof

Abstract

The present invention relates to the technical field of biology and in particular to a D-amino acid oxidase mutant and a use thereof in the preparation of L-glufosinate by kinetic resolution of DL-glufosinate and the multi-enzyme catalysis. By means of methods such as protein truncation, multi-site combination mutation, and fusion tag addition, the soluble expression and enzymatic activity of D-amino acid oxidase obtained by means of expression in a host are significantly improved. Further modification of an active pocket of the D-amino acid oxidase provides a variety of amino acid oxidase mutants capable of efficiently catalyzing the synthesis of 2-oxo-4-(hydroxymethylphosphinyl) butyric acid (PPO) from D-glufosinate, such that the enzymatic activity of the D-amino acid oxidase is further improved. A method for preparing an α-keto acid by using the provided D-amino acid oxidase achieves the kinetic resolution of racemic glufosinate and can be widely applied to the production of L-glufosinate.

Description

D-amino acid oxidase mutants and uses thereof

[0001] This application claims priority to the Chinese patent application No. 202410726755.3, filed on June 5, 2024, and entitled "D-amino acid oxidase mutants and uses thereof", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to D-amino acid oxidase mutants and their uses in the kinetic resolution of DL-phosphinothricin and the multi-enzyme catalytic preparation of L-phosphinothricin. BACKGROUND

[0003] Phosphinothricin (also known as Glufosinate), chemically named 2-amino-4- (hydroxymethylphosphinothricin) butyric acid, is a high-efficiency, low-toxicity, broad-spectrum, non-selective contact-kill type organophosphorus herbicide with broad market prospects. Phosphinothricin is the racemic form of D / L-phosphinothricin, among which D-phosphinothricin has herbicidal activity, and L-phosphinothricin is a glutamine inhibitor with more than twice the herbicidal activity of the racemic phosphinothricin, also known as precision phosphinothricin. Because its molecular structure is very similar to that of glutamic acid, it can reversibly bind to the active site of glutamine synthetase, effectively inhibit the synthesis of L-glutamine in plants, cause nitrogen metabolism disorder in plants, excessive accumulation of ammonia, chloroplast disintegration, and thus inhibit photosynthesis, ultimately leading to plant death. Precision phosphinothricin is superior to phosphinothricin in terms of herbicidal efficiency, thoroughness, and stability, and is expected to gradually replace ordinary phosphinothricin in the future. Compared with traditional chemical synthesis, biological methods have the advantages of strict stereoselectivity, mild reaction conditions, high yield, and easy separation and purification of products, and therefore exploring biological synthesis of L-phosphinothricin has important commercial value and significant social benefits.

[0004] D-amino acid oxidase (DAAO) is a typical flavoprotein enzyme with flavin adenine dinucleotide (FAD) as a coenzyme, which belongs to structural selective catalysts and can oxidize the amino group of D-amino acid to produce the corresponding α-keto acid. Therefore, D-amino acid oxidase can be widely used in qualitative and quantitative analysis of D-amino acid, biosensor, production of L-amino acid and α-keto acid. In recent years, it has been found that D-amino acid oxidase can catalyze the oxidation of D-glufosinate in racemic glufosinate substrate to the corresponding α-keto acid, and further convert the keto acid into L-glufosinate by the catalysis of L-amino acid dehydrogenase or transaminase. Therefore, D-amino acid oxidase has great development prospects in the field of L-glufosinate production. However, the natural D-amino acid oxidase still has problems of poor soluble expression, low catalytic activity or no activity, which will lead to problems of increased catalyst consumption and high cost. Therefore, further research is needed to solve the problem of low enzyme activity of wild-type D-amino acid oxidase on substrate D-glufosinate by optimizing protein soluble expression and molecular modification, and to optimize and improve the catalytic activity of D-amino acid oxidase on substrate D / L glufosinate, which will help to realize the industrialized production of glufosinate-p.

[0005] SUMMARY

[0006] Therefore, the present application provides a D-amino acid oxidase mutant and its application in kinetic resolution of DL-glufosinate and multi-enzyme catalytic preparation of L-glufosinate.

[0007] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides the application of any of the following in improving the soluble expression of D-amino acid oxidase, improving the enzyme activity of D-amino acid oxidase, kinetic resolution of DL-glufosinate or preparation of L-glufosinate:

[0009] (I) deletion of 51, 96, 153, 204 bases at the N-terminus and / or deletion of 51 bases at the C-terminus of wild-type D-amino acid oxidase; and / or

[0010] (II) single-point or multiple-site combination mutation of L25P, A79R, T168R, M228P, A254E, G359R of wild-type D-amino acid oxidase; and / or

[0011] (III) single-point or multiple-site combination mutation of L25P, A79R, T168R, M228P, A254E, G359R based on (I).

[0012] In some embodiments of the present application, a T7B9 tag is further added at the N-terminus.

[0013] In some embodiments of the present application, further comprising single point or multiple site combination mutation in R112, V114, R139, Y183, T294, Q312, W318, K336, R382, G409, G410, R411.

[0014] In some embodiments of the present application, further comprising deletion of 39 and / or 78 bases of T7B9 tag at N-terminus.

[0015] In the second aspect, the present application further provides mutants of D-amino acid oxidase, comprising any of:

[0016] (I) mutants obtained by deletion of 51, 96, 153 or 204 bases at N-terminus and / or deletion of 51 bases at C-terminus of wild-type D-amino acid oxidase; and / or

[0017] (II) mutants obtained by single point or multiple site combination mutation in L25P, A79R, T168R, M228P, A254E, G359R of wild-type D-amino acid oxidase; and / or

[0018] (III) mutants obtained by single point or multiple site combination mutation in L25P, A79R, T168R, M228P, A254E, G359R based on the mutants as shown in (I).

[0019] In some embodiments of the present application, further comprising mutants obtained by addition of T7B9 tag at N-terminus.

[0020] In some embodiments of the present application, further comprising mutants obtained by single point or multiple site combination mutation in R112, V114, R139, Y183, T294, Q312, W318, K336, R382, G409, G410, R411.

[0021] In some embodiments of the present application, further comprising mutants obtained by deletion of 39 and / or 78 bases of T7B9 tag at N-terminus.

[0022] In some embodiments of the present application, further comprising mutants obtained by single point or multiple site combination mutation in Q312S, Q312A, V114N, V114Q, G410S, G410A.

[0023] In some embodiments of the present application, further comprising mutants obtained by single point or multiple site combination mutation in Q312S, Q312H, Q312A, W318Y and mutation combination of V114N.

[0024] In some embodiments of the present application, mutants obtained by mutating V114N and / or Q312S are also included.

[0025] In some embodiments of the present application, mutants obtained by deleting 39 and / or 78 bases of the N-terminal T7B9 tag are also included.

[0026] In some embodiments of the present application, the mutants include any one of the following:

[0027] (i) mutants obtained by deleting 51, 96, 153, 204 bases from the N-terminal and / or 51 bases from the C-terminal of wild-type D-amino acid oxidase and adding T7B9 tag at the N-terminal;

[0028] (ii) mutants obtained by mutating L25P, A79R, T168R, M228P, A254E, G359R of wild-type D-amino acid oxidase or mutants as shown in (i) and adding T7B9 tag at the N-terminal;

[0029] (iii) mutants obtained by mutating Q312S, Q312A, V114N, V114Q, G410S, G410A of wild-type D-amino acid oxidase or mutants as shown in (i), (ii);

[0030] (iv) mutants obtained by mutating Q312S, Q312H, Q312A, W318Y of wild-type D-amino acid oxidase or mutants as shown in (i)-(iii) and the combination of V114N;

[0031] (v) mutants obtained by deleting 39 and / or 78 bases of the N-terminal T7B9 tag of wild-type D-amino acid oxidase or mutants as shown in (i)-(iv).

[0032] In some embodiments of the present application, the mutants include:

[0033] (A) mutants obtained by deleting 51, 96, 153, or 204 bases from the N-terminal and / or 51 bases from the C-terminal of wild-type D-amino acid oxidase;

[0034] (B) mutants obtained by mutating L25P, A79R, T168R, M228P, A254E, G359R of mutants as shown in (A) and adding T7B9 tag at the N-terminal;

[0035] (C) mutants obtained by mutating V114N of mutants as shown in (B).

[0036] (D) mutant obtained by single-point mutation of Q312S of the mutant shown in (C);

[0037] (E) mutant obtained by deleting 57 bases at the N terminus of the mutant shown in (D) and deleting or retaining T7B9 tag; and

[0038] (F) mutant obtained by deleting T7B9 tag of 39 and / or 78 bases at the N terminus of the mutant shown in (A).

[0039] In a third aspect, the present application further provides a nucleic acid molecule encoding the mutant.

[0040] In some embodiments of the present application, the nucleic acid molecule has:

[0041] (I) nucleotide sequence shown in any one of SEQ ID NO. 4-26; or

[0042] (II) nucleotide sequence which encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of genetic code; or

[0043] (III) nucleotide sequence obtained by substitution, deletion or addition of one or more nucleotides to the nucleotide sequence shown in (I) or (II), and nucleotide sequence which is functionally identical or similar to the nucleotide sequence shown in (I) or (II); or

[0044] (IV) nucleotide sequence having at least 90% sequence homology with the nucleotide sequence shown in (I), (II) or (III).

[0045] In a fourth aspect, the present application further provides a construct containing a promoter, the nucleic acid molecule and a terminator.

[0046] In a fifth aspect, the present application further provides a plasmid vector comprising any of the following and a backbone vector:

[0047] (I) the nucleic acid molecule; and / or

[0048] (II) the construct.

[0049] In a sixth aspect, the present application further provides a host comprising any of the following:

[0050] (I) the nucleic acid molecule; and / or

[0051] (II) the construct; and / or

[0052] (III) the plasmid vector.

[0053] In some embodiments of the present application, the host comprises E. coli;

[0054] The E. coli comprises BL21 (DE3).

[0055] In a seventh aspect, the present application further provides the use of any of the following in the preparation of D-amino acid oxidase:

[0056] (I) the nucleic acid molecule; and / or

[0057] (II) the construct; and / or

[0058] (III) the plasmid vector; and / or

[0059] (IV) the host.

[0060] In an eighth aspect, the present application further provides a method for preparing D-amino acid oxidase, comprising fermenting the host to obtain a fermentation product.

[0061] In a ninth aspect, the present application further provides the D-amino acid oxidase prepared by the method.

[0062] In a tenth aspect, the present application further provides the use of any of the following in the preparation of α-keto acid, kinetic resolution of DL-glufosinate ammonium or preparation of L-glufosinate ammonium:

[0063] (I) the mutant;

[0064] (II) the nucleic acid molecule;

[0065] (III) the construct;

[0066] (IV) the plasmid vector; and / or

[0067] (V) the host.

[0068] In an eleventh aspect, the present application further provides a method for preparing α-keto acid, comprising using the D-amino acid oxidase to perform an oxidation reaction with D-amino acid or racemic amino acid as a substrate to obtain α-keto acid;

[0069] The substrate concentration in the system of the oxidation reaction is 0.2 mol / L, the reaction temperature is 30°C, the reaction time is 1 h, and the pH value of the reaction solution is 8.0.

[0070] In a twelfth aspect, the present application further provides a method for preparing L-glufosinate ammonium, comprising using D-amino acid or racemic amino acid as a substrate, adding the D-amino acid oxidase, glutamate dehydrogenase, ethanol dehydrogenase, catalase, NADH, and L-glufosinate ammonium to perform an oxidation reaction to obtain L-glufosinate ammonium. +Mixing, stirring, and preparing L-glufosinate.

[0071] In some embodiments of the present application, the concentration of the substrate comprises 0.2 mol / L; the temperature during stirring comprises 40℃; and the pH during stirring comprises 8.0.

[0072] In order to solve the problems of poor soluble expression of the original D-amino acid oxidase (NcDAAO, NCBI Accession No: XP_964990, the amino acid sequence is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2) derived from Neurospora crassa OR74A and low enzyme activity of the substrate D-glufosinate, the present application provides various optimization strategies for optimizing the expression of D-amino acid oxidase in Escherichia coli, improving the catalytic activity, and the application in preparing L-glufosinate. The present application provides various strategies for improving soluble expression and enzyme activity, and provides various D-amino acid oxidase variants. The present application improves the soluble expression and enzyme activity of the D-amino acid oxidase expressed by the host through protein truncation, multi-site combined mutation, and addition of fusion tags, and modifies the active pocket of the D-amino acid oxidase, thereby providing various amino acid oxidase mutants capable of efficiently catalyzing the synthesis of 2-carbonyl-4-(hydroxymethyl phosphine) butyric acid (PPO) from D-glufosinate, and further improving the enzyme activity of the D-amino acid oxidase. The method for preparing alpha-keto acid using the D-amino acid oxidase provided by the present application realizes the kinetic resolution of racemic glufosinate, and can be widely applied to the production and application of L-glufosinate.

[0073] Compared with the prior art, the present application has the following beneficial effects:

[0074] (1) The present application is based on the D-amino acid oxidase (NcDAAO, NCBI Accession No: XP_964990) derived from Neurospora crassa OR74A, and solves the problem of poor soluble expression of NcDAAO through protein truncation, site-directed mutation, and addition of a solubility-promoting tag. Further, the present application solves the problem of low enzyme activity of the substrate D-glufosinate by using a rational design method, and obtains a mutant capable of efficiently catalyzing the preparation of alpha-keto acid and D-glufosinate.

[0075] (2) The rational design method used in the present application can quickly obtain a D-amino acid oxidase mutant with high catalytic activity for the substrate D-glufosinate through screening with a small mutant library.

[0076] (3) The application uses D-glufosinate as a substrate, and uses D-amino acid oxidase to perform oxidation reaction and kinetic resolution to prepare L-glufosinate, and the method has simple process, mild reaction condition, low cost of catalyst, and efficient and green process, and has good catalytic efficiency in the process, the substrate conversion rate is > 99%, the maximum concentration of L-glufosinate can reach 185.06 mM, the ee value is > 99%, and the method has great industrial application prospect, and lays a good foundation for realizing the process. BRIEF DESCRIPTION OF DRAWINGS

[0077] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below.

[0078] Fig. 1 shows the high performance liquid chromatogram of 2-carbonyl-4-(hydroxymethyl phosphine) butyric acid (PPO) standard;

[0079] Fig. 2 shows the high performance liquid chromatogram of D,L-glufosinate standard and L-glufosinate standard;

[0080] Fig. 3 shows the protein electrophoretogram of wild type and D-amino acid oxidase protein truncated genetically engineered bacteria;

[0081] Fig. 4 shows the protein electrophoretogram of NcDAAO2 soluble expression optimized genetically engineered bacteria;

[0082] Fig. 5 shows the protein electrophoretogram of NcDAAO6 iterative saturation mutation screening genetically engineered bacteria;

[0083] Fig. 6 shows the protein electrophoretogram of NcDAAO18 protein truncated genetically engineered bacteria;

[0084] Fig. 7 shows the protein electrophoretogram of NcDAAO20 protein solubilization tag T7B9 truncated genetically engineered bacteria;

[0085] Fig. 8 shows the technical route diagram of preparing L-glufosinate by multi-enzyme cascade reaction;

[0086] Fig. 9 shows the content and ee value changes of D-glufosinate and L-glufosinate in the process of multi-enzyme cascade reaction. DETAILED DESCRIPTION

[0087] The application discloses D-amino acid oxidase mutants and application thereof in kinetic resolution of DL-glufosinate and multi-enzyme catalytic preparation of L-glufosinate, and those skilled in the art can refer to the content of the application and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and are regarded as included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described in the application without departing from the content, spirit and scope of the application, to realize and apply the technical solution of the application.

[0088] The application solves the problems of poor soluble expression of original D-amino acid oxidase (NcDAAO, NCBI accession number: XP_964990, the amino acid sequence is shown as SEQ ID NO: 1, and the nucleotide sequence is shown as SEQ ID NO: 2) derived from Neurospora crassa OR74A and low enzyme activity of the substrate D-glufosinate, provides various optimization strategies for optimizing the expression of D-amino acid oxidase in Escherichia coli, simultaneously improving the catalytic activity, and application in preparation of L-glufosinate. The application provides various strategy schemes for improving soluble expression and enzyme activity, and provides various D-amino acid oxidase variants. The application improves the soluble expression and enzyme activity of D-amino acid oxidase expressed by the host through protein truncation, multi-site combined mutation, addition of fusion tags and other methods, reforms the activity pocket of the D-amino acid oxidase, provides various amino acid oxidase mutants capable of efficiently catalyzing synthesis of 2-carbonyl-4-(hydroxymethyl phosphine) butyric acid (PPO) from D-glufosinate, and further improves the enzyme activity of the D-amino acid oxidase. The method for preparing alpha-keto acid by using the D-amino acid oxidase provided by the application realizes kinetic resolution of racemic glufosinate, and can be widely applied to the production and application of L-glufosinate.

[0089] The specific technical solutions are as follows:

[0090] The application provides any one of the applications in improving the soluble expression and enzyme activity of D-amino acid oxidase:

[0091] (1) a nucleic acid molecule comprising a wild-type D-amino acid oxidase;

[0092] (2) a nucleic acid molecule comprising a D-amino acid oxidase truncated body with 51, 96, 153 or 204 bases deleted from the N terminus and 51 bases deleted from the C terminus of a wild-type D-amino acid oxidase, and a gene element of a tag;

[0093] (3) a nucleic acid molecule encoding a D-amino acid oxidase with any one or more of the following mutations: L25P, A79R, T168R, M228P, A254E, and G359R, and a genetic element of a tag selected from the group consisting of T7B9 and T7B9 mutants;

[0094] (4) a nucleic acid molecule encoding a D-amino acid oxidase mutant with any one or more of the following mutations: L25P, A79R, T168R, M228P, A254E, and G359R, and a N-terminal deletion of 51, 96, 153, or 204 bases, and a genetic element of a tag selected from the group consisting of T7B9 and T7B9 mutants;

[0095] (5) a nucleic acid molecule encoding a D-amino acid oxidase mutant with any one or more of the following mutations: L25P, A79R, T168R, M228P, A254E, and G359R, and a nucleic acid molecule encoding a D-amino acid oxidase mutant with a N-terminal deletion of 51, 96, 153, or 204 bases, and a genetic element of a tag selected from the group consisting of T7B9 and T7B9 mutants; the D-amino acid oxidase variant is a single-point or multiple-point combination mutant of the valine at position 114, the glutamine at position 312, the tryptophan at position 318, and the glycine at position 410 of the amino acid sequence set forth in SEQ ID NO: 1.

[0096] The present application performs multi-strategy superimposed soluble expression optimization on D-amino acid oxidase (NcDAAO) derived from Neurospora crassa OR74A, and performs homology modeling on the optimized protein, molecular docking with the substrate D-glufosinate, and selects 12 key amino acid residues in the side chain group of D-glufosinate within the range, constructs a D-amino acid oxidase mutant library by means of iterative saturation mutation, and screens the optimal combination of D-amino acid oxidase mutants for catalyzing the preparation of PPO.

[0097] Further, the wild-type D-amino acid oxidase has an amino acid sequence as shown in SEQ ID NO: 1.

[0098] The present application provides a D-amino acid oxidase mutant, which includes a wild-type D-amino acid oxidase nucleotide sequence, such as the N-terminal deletion of 51 bases, the N-terminal deletion of 96 bases, and the N-terminal deletion of 153 bases, the N-terminal deletion of 204 bases, and the C-terminal deletion of 51 bases as shown in SEQ ID NO: 2.

[0099] The application provides a D-amino acid oxidase mutant, which comprises any one or more of the following mutation sites: L25P, A79R, T168R, M228P, A254E and G359R.

[0100] The application provides a D-amino acid oxidase mutant, which further comprises one of the following single-point mutations or multi-point combination mutations:

[0101] (1) Q312S, Q312A, V114N, V114Q, G410S, G410A;

[0102] (2) V114N / Q312S, V114N / Q312H, V114N / Q312A, V114N / W318Y

[0103] In the above, " / " represents "and", that is, the two sites before and after " / " are simultaneously mutated; for example: V114N / Q312S represents that the valine at the 114th position is mutated into asparagine, and the amino acid at the 312th position is mutated from glutamine into serine;

[0104] Based on the above research, the D-amino acid oxidase mutant provided by the application further comprises adding a T7B9 tag or a T7B9 mutant at the N terminal.

[0105] The application further provides a coding gene of the D-amino acid oxidase mutant as described in any one of the above.

[0106] The application further provides an expression vector comprising the coding gene as described above. As a preferred, the original expression vector is pET-28a(+).

[0107] The application further provides a genetically engineered bacterium comprising the coding gene as described above. As a preferred, the host cell of the genetically engineered bacterium is E.coli BL21(DE3).

[0108] The application further provides an application of the D-amino acid oxidase mutant as described above in preparing alpha-keto acid and L-phosphinotricine.

[0109] The application further provides an application of the genetically engineered bacterium as described above in preparing alpha-keto acid and L-phosphinotricine.

[0110] The application further provides a detection method of the D-amino acid oxidase, specifically, the substrate concentration is 0.05 mol / L, the oxidation reaction temperature is 30 DEG C, the scanning wavelength is 510 nm, and the scanning time is 200 s.

[0111] The application also provides a preparation method of alpha-keto acid, comprising using D-amino acid or racemic amino acid as a substrate and using the D-amino acid oxidase provided by the application to perform an oxidation reaction.

[0112] Specifically, in the preparation of the alpha-keto acid, the substrate concentration in the oxidation reaction system is 0.2 mol / L, the reaction temperature is 30℃, the reaction time is 1 h, and the pH value of the reaction solution is 8.0.

[0113] The experimental methods in the application are conventional methods unless otherwise specified, and the gene cloning operation can be specifically referred to J. Sambrook et al. Molecular Cloning Guidebook.

[0114] Reagents for upstream genetic engineering: DpnI used in the examples of the application is purchased from TaKaRa, Bao Bioengineering (Dalian) Co., Ltd.; a plasmid extraction kit and a DNA recovery and purification kit are purchased from Axygen Hangzhou Co., Ltd.; E. coli BL21 (DE3) and a plasmid pET-28a (+) are purchased from Novagen Co.; a DNA marker, low molecular weight standard protein and agarose electrophoresis reagent are purchased from Beijing Zhenxi Gold Biotechnology Co., Ltd.; primer synthesis and sequence determination are completed by Chengke Bioengineering Co., Ltd. The reagent usage methods refer to the product instructions. The three-letter or one-letter representation of amino acids used in the application text adopts the IUPAC amino acid code (Eur. J. Biochem., 138: 9-37, 1984).

[0115] The PPO concentration is detected by high performance liquid chromatography (HPLC), and the specific method is as follows: chromatographic conditions: chromatographic column type: QS-C18, 5 μm, 4.6 mm x 250 mm. Mobile phase: 50 mM diammonium phosphate solution, 0.9% 10% tetrabutylammonium hydroxide aqueous solution, 50% phosphoric acid solution (mass fraction) to adjust pH to 3.6, 8% acetonitrile. Detection wavelength: 205 nm. Flow rate: 0.8 mL / min. Column temperature: 40℃.

[0116] The optical purity of glufosinate ammonium is detected by high performance liquid chromatography (HPLC), and the specific method is as follows: pre-column derivatization is used for determination. Derivatization reaction and determination: 100 μL of sample is added to 100 μL of derivatization reagent, mixed and incubated at 25℃ for 5 min. Chromatographic conditions: chromatographic column QS-C18; detection wavelength: 338 nm; column temperature: 30 °C; injection volume: 20 μL; mobile phase: 50 mM sodium acetate aqueous solution: acetonitrile = 9:0.5; flow rate: 1 mL / min.

[0117] 0.2 M boric acid buffer: weigh 7.62 g of sodium tetraborate decahydrate, and dilute with deionized water to 100 mL for standby;

[0118] Derivatization reagent: weigh 0.03 g of o-phthaldehyde and 0.1 g of N-acetyl-L-cysteine, add 400 μL of anhydrous ethanol and 4 mL of boric acid buffer, and ultrasonically dissolve completely, and prepare for use.

[0119] The amino acid sequence of the wild-type NcDAAO derived from Neurospora crassa OR74A is:

[0120] The nucleic acid sequence of the wild-type NcDAAO derived from Neurospora crassa OR74A is:

[0121] The amino acid sequence of the T7B9 tag is:

[0122] The nucleic acid sequence of the NcDAAO1 is:

[0123] The nucleic acid sequence of the NcDAAO2 is:

[0124] The nucleic acid sequence of the NcDAAO3 is:

[0125] The nucleic acid sequence of the NcDAAO4 is:

[0126] The nucleic acid sequence of the NcDAAO5 is:

[0127] The nucleic acid sequence of the NcDAAO6 is:

[0128] The nucleic acid sequence of the NcDAAO7 is:

[0129] The nucleic acid sequence of the NcDAAO8 is:

[0130] The nucleic acid sequence of the NcDAAO9 is:

[0131] The nucleic acid sequence of the NcDAAO10 is:

[0132] The nucleic acid sequence of the NcDAAO11 is:

[0133] The nucleic acid sequence of the NcDAAO12 is:

[0134] The nucleic acid sequence of the NcDAAO13 is:

[0135] The nucleic acid sequence of the NcDAAO14 is:

[0136] The nucleic acid sequence of the NcDAAO15 is:

[0137] The nucleic acid sequence of the NcDAAO16 is:

[0138] The nucleic acid sequence of the NcDAAO17 is:

[0139] The nucleic acid sequence of the NcDAAO18 is:

[0140] The nucleic acid sequence of the NcDAAO19 is:

[0141] The nucleic acid sequence of the NcDAAO20 is:

[0142] The nucleic acid sequence of the NcDAAO21 is:

[0143] The nucleic acid sequence of the NcDAAO22 is:

[0144] The nucleic acid sequence of the NcDAAO23 is:

[0145] The D-amino acid oxidase mutant and the raw materials and reagents used in the application of the D-amino acid oxidase mutant in the kinetic resolution of DL-glufosinate-ammonium and the preparation of L-glufosinate-ammonium by a multi-enzyme catalysis can be purchased from the market.

[0146] The application is further described below in combination with examples:

[0147] Example 1 Construction of a protein-truncated strain of wild-type NcDAAO and determination of activity

[0148] 1. Construction of a wild-type NcDAAO strain

[0149] The gene sequence related information of D-amino acid oxidase NcDAAO derived from Neurospora crassa OR74A (NCBI accession number: XP_964990) was obtained by querying a gene database (https: / / www.ncbi.nlm.nih.gov / genome / ), and a gene synthesis company was commissioned to perform full gene synthesis of the codon-optimized wild-type strain, and the gene was constructed on a plasmid vector pET-28a(+), with NheI and NotI as the enzyme digestion sites; then the constructed plasmid was introduced into an expression host E. coli BL21(DE3) strain, which was a genetically engineered bacterium E. coli BL21(DE3) / pET-28a(+)-NcDAAO.

[0150] 2. Construction of a protein-truncated mutant strain of NcDAAO

[0151] The pET-28a(+)-NcDAAO plasmid was used as a template, and 51, 96, 153 and 204 bases were deleted from the N terminus to construct NcDAAO1-NcDAAO4, respectively; and 51 bases were deleted from the C terminus to construct NcDAAO5. The upstream and downstream primers for protein truncation were designed as follows:

[0152] (1) Whole plasmid PCR:

[0153] Table 1 Primers required for construction of a protein-truncated strain

[0154] PCR amplification system:

[0155] DNA polymerase 25 μL;

[0156] Upstream primer (10 pmol / μL) 1.5 μL;

[0157] Downstream primer (10 pmol / μL) 1.5 μL;

[0158] Template 1.0 μL;

[0159] ddH2O 21 μL.

[0160] PCR amplification conditions:

[0161] 1) Pre-denaturation: 95℃, 5 min;

[0162] 2) Denaturation: 98℃, 10 s; Annealing: 58℃, 15 s; Extension: 72℃, 90 s; Cycle for 30 times;

[0163] 3) Post-extension: 72℃, 10 min;

[0164] 4) Keep at 4℃.

[0165] (2) Template digestion:

[0166] The PCR product was subjected to agarose gel electrophoresis, and after recovery, the plasmid template was digested with Dpn I enzyme. The digestion system was: Dpn I enzyme 1 μL, PCR product 17 μL, Buffer 2 μL. The digestion of the template can be completed at 37℃ for 2 hours.

[0167] (3) Transformation and verification:

[0168] After digestion, the product was verified by nucleic acid agarose gel electrophoresis, and then the 42℃ heat shock method was used to transform the E. coli BL21 (DE3) competent cells. The specific process is as follows:

[0169] 1) The competent cells were placed on ice for 15 min;

[0170] 2) In a sterile environment, 10 μL of DNA was added to 100 μL of competent cells and mixed gently, and placed on ice for 30 min;

[0171] 3) The EP tube was placed in a 42℃ metal bath for heat shock for 90 s, and then placed on ice for cooling for 2 min;

[0172] 4) 800 μL of LB medium was added to the EP tube and mixed with a gun head, and incubated in a 200 rpm shaker at 37℃ for 40-60 min;

[0173] 5) After concentration, an appropriate volume was taken to the corresponding resistant plate, and after 12-16 h of culture in a 37℃ incubator, colonies appeared.

[0174] 3. Induced expression of NcDAAO protein truncated mutant

[0175] The successfully constructed engineering bacteria were inoculated in 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37℃, 220 rpm for 12 h to seed liquid OD 600about 3.0. The bacteria were subcultured into 50 mL LB liquid medium containing 50 μg / mL kanamycin at an inoculum of 2%, and incubated at 37°C until the OD600 reached about 0.6-0.8. IPTG was then added to a final concentration of 0.5 mM / L, and the bacteria were induced to express the protein at 18°C for 16 h. After the incubation, the bacteria were centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The bacteria were resuspended in 100 mM phosphate buffer (pH 8.0), and were broken by ultrasonic treatment at 400 W for 30 times, each time for 3 s with an interval of 7 s. The broken bacteria were centrifuged at 12000 g at 4°C for 10 min to remove the precipitate, and the supernatant was used as the crude enzyme solution.

[0176] 4. Enzyme activity determination of the NcDAAO protein-truncated engineering bacteria

[0177] DL-phosphinothricin was used as the substrate to determine the enzyme activity of the D-amino acid oxidase. The total reaction system was 2 mL, including 1 mL of 200 mM DL-phosphinothricin solution and 1 mL of the crude enzyme solution, which were prepared using 100 mM phosphate buffer (pH 8.0). The reaction was performed at 30°C for 1 h, and was terminated by adding 200 μL of 4 M HCl solution. The reaction mixture was centrifuged at 4000 rpm for 10 min to remove the cells and the enzyme protein. The PPO produced in the reaction system was determined by high performance liquid chromatography. The enzyme activity was defined as the amount of enzyme that could convert 1 μmol of substrate to product per minute at 30°C, and was expressed as U.

[0178] All the results were analyzed by independent T test and the P value was calculated. "n.s" indicates no significant difference compared with the control strain, "*" indicates P < 0.05 compared with the control strain, "**" indicates P < 0.01 compared with the control strain, and "***" indicates P < 0.001 compared with the control strain.

[0179] 5. Soluble expression detection of the NcDAAO protein-truncated engineering bacteria

[0180] The OD of the broken bacteria solution of the engineering bacteria after ultrasonic treatment in step 4 was unified 600After centrifugation at 12000 rpm, 4℃ for 10 min, 15 μL supernatant was mixed with 5 μL 4xSDS-PAGE Loading Buffer; the supernatant was discarded, and the precipitate was resuspended with the same volume of buffer, then 15 μL resuspension was mixed with 5 μL 4xSDS-PAGE Loading Buffer. All protein electrophoresis samples were incubated at 99℃ for 10 min. 10 μL protein sample was taken for protein gel electrophoresis. The results were analyzed by ImageJ software for gray scale analysis of the proportion of soluble D-amino acid oxidase in total cells. Among them, "-" indicates that there are a large number of inclusion bodies, and the protein solubility is less than 10%, "+" indicates that the protein solubility is 10%~50%, "++" indicates that the protein solubility is 50%~90%, "+++" indicates that the protein solubility is 90%~100%. At the same time, "+" indicates the degree of overall protein expression, and "-" indicates low protein expression. As can be seen from Table 2, the wild-type NcDAAO protein expression is high, but the solubility is poor, and it is all insoluble inclusion bodies. The expression of NcDAAO1 protein after N-terminal truncation is almost invisible. NcDAAO2 protein still has a large number of inclusion bodies, but the soluble protein is improved compared with wild-type NcDAAO. The expression of NcDAAO3 protein is high, and all of them are soluble. The expression of NcDAAO4 protein is reduced to almost invisible. The solubility of NcDAAO5 after C-terminal truncation has no significant difference compared with wild-type NcDAAO.

[0181] Table 2 Solubility expression determination of wild-type NcDAAO and protein truncated engineering bacteria

[0182] According to step 3, the wild-type NcDAAO and protein truncated strains were cultured and induced for expression, and the D-amino acid oxidase enzyme activity was determined according to the method of step 4. The results are shown in Table 3. As can be seen from Table 3, with the decrease of N-terminal bases, the enzyme activity increases, wherein the unit OD enzyme activity of NcDAAO2 is improved by 3.9 times compared with wild-type NcDAAO (P<0.001), and the unit OD enzyme activity of NcDAAO3 is improved by 6.43 times compared with wild-type NcDAAO (P<0.001). In summary, combined with the protein expression amount and enzyme activity, it can be seen that the unit mass of NcDAAO2 protein has higher enzyme activity. Therefore, NcDAAO2 with N-terminal truncation of 96 base sequences is preferred as an E. coli gene engineering bacterium for improving soluble expression and enzyme activity.

[0183] Table 3 Enzyme activity determination results of wild-type NcDAAO and protein truncated engineering bacteria

[0184] Example 2 Soluble expression optimization of NcDAAO2

[0185] According to previous work experience, Camsol predicts six-point mutations and the solubility-promoting tag T7B9 can improve the enzyme activity and soluble expression of NcDAAO. Therefore, NcDAAO2 is subjected to six-point mutations L25P, A79R, T168R, M228P, A254E, G359R, and the N-terminal T7B9 tag is added to construct NcDAAO6, and the required primers are shown in Table 4.

[0186] Table 4 Primers required for construction of NcDAAO6

[0187] According to the expression of D-amino acid oxidase and the determination of enzyme activity and protein soluble expression in steps 3-4 of Example 1, the enzyme activity results are shown in Table 5, and the protein soluble expression is shown in Figure 4. As can be seen from Figure 4, the soluble expression of NcDAAO2 after six-point mutation and addition of peptide tag is significantly improved, but the expression amount is reduced. As can be seen from the enzyme activity results in Table 5, the unit OD enzyme activity of NcDAAO6 is increased by 35.54 times compared with wild-type NcDAAO, and is increased by 2.77 times (P<0.001) compared with NcDAAO2. Therefore, the six-point mutations L25P, A79R, T168R, M228P, A254E, G359R and the solubility-promoting tag T7B9 are preferred to improve the soluble expression and enzyme activity of D-amino acid oxidase in Escherichia coli.

[0188] Table 5 Enzyme activity determination results of NcDAAO6 engineering bacteria

[0189] Example 3 Iterative saturation mutation of NcDAAO6 against substrate D- glufosinate

[0190] The D-amino acid oxidase of NcDAAO6 (nucleotide sequence as shown in SEQ ID NO: 9) is subjected to homology modeling, and the substrate D-glufosinate is subjected to molecular docking, and 12 key amino acid residues in the range of D-glufosinate side chain groups R125, V127, R152, Y196, T307, Q325, W331, K349, R395, G422, G423, R424, which correspond to the key amino acid residues of wild-type D-amino acid oxidase (amino acid sequence as shown in SEQ ID NO: 1) R112, V114, R139, Y183, T294, Q312, W318, K336, R382, G409, G410, R411. By means of iterative saturation mutation, a D-amino acid oxidase mutation library is constructed, and the optimal combination of mutations of D-amino acid oxidase mutants for catalyzing the preparation of PPO is screened.

[0191] Step one: first round of single-point saturation mutation screening

[0192] 1. Construction of saturation mutagenesis library

[0193] The pET-28a(+)-NcDAAO6 plasmid was used as a template to design primers (Table 6) covering the upstream and downstream of the mutation point for whole plasmid PCR. The detailed operation is shown in Example 1.

[0194] Table 6 Primers required for construction of single-point saturation mutagenesis library

[0195] 2. Primary screening

[0196] In a sterilized 96-deep well plate, 200 μL of LB medium (containing 50 μg / mL of kanamycin) was added, and a single colony was picked into the 96-deep well plate using a sterilized gun head. Then the deep well plate was placed at 37°C, 220 rpm for 8 h, which was called the primary plate. In another sterilized 96-deep well plate, 400 μL of LB medium (containing 50 μg / mL of kanamycin) was added as the secondary plate, and 50 μL of bacterial solution was taken from the primary plate and added to the secondary plate. The primary plate was added with 20% glycerol and placed in a -80°C refrigerator for long-term preservation. Then the secondary plate was placed at 37°C for 2 h, and then 0.5 mM IPTG was added for induction, and then the secondary plate was placed at 18°C, 220 rpm for 16 h.

[0197] The secondary plate was centrifuged at 4000 rpm, 4°C for 20 min to collect the cells, which were then placed in a -80°C freezer overnight. The secondary plate was taken out from the -80°C freezer, thawed at room temperature for 0.5 h, and then 300 μL of lysis solution (10 mM pH 8.0 phosphate buffer, 750 mg / L lysozyme, 10 mg / L DNase I) was added to each well, the cells were suspended by shaking, and then placed in a 37°C shaker, 220 rpm for 1 h. After incubation, the cells were centrifuged at 4000 rpm, 4°C for 20 min, and the supernatant was taken for enzyme activity determination.

[0198] The enzyme activity determination solution (100 mL) for screening was prepared as follows: 50 mL of color developing agent (0.16 mg / mL 2,4,6-tribromo-3-hydroxybenzoic acid, 1 mg / mL 4-aminoantipyrine, 0.2 mg / mL peroxidase), 50 mL of 50 mmol / L glufosinate solution (pH 8.0), and all the above substances were prepared with 50 mM, pH 8.0 phosphate buffer.

[0199] 100 μL of enzyme activity determination solution was added to each well of a new 96-well plate (reaction plate), and then placed at 30°C for 10 min. 120 μL of enzyme solution was taken and added to the reaction plate to start the reaction, and after 20 min, the absorbance value at 510 nm was determined by an enzyme marker. The higher the absorbance value, the higher the catalytic activity, and the mutant strains with significantly higher absorbance values than the control (wild type) were selected as candidate strains for re-screening.

[0200] 3. Secondary screening

[0201] The mutants showing significant increase in enzyme activity in the primary screening were subjected to secondary screening by chromogenic method. The corresponding mutants on the primary plate were activated by streaking on LB plate, and single colony was inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37 °C for 12 h with shaking until the OD 600 was about 3.0. Then, 2% of the culture was transferred into 50 mL LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37 °C with shaking until the OD 600 was about 0.6-0.8. Then, IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 18 °C for 16 h. After the incubation, the culture was centrifuged at 4000 g at 4 °C for 10 min, and the supernatant was discarded. The collected cells were washed twice with 50 mM phosphate buffer (pH 8.0), and resuspended in the phosphate buffer. The cells were broken by ultrasonication at 400 W for 30 times, with 3 s of ultrasonication and 7 s of interval. The cell lysate was centrifuged at 12000 g at 4 °C for 10 min to remove the precipitate, and the supernatant was used as the crude enzyme solution. 2 mL of enzyme activity assay solution was incubated at 30 °C in a thermostatic metal bath for 10 min, and then transferred into a cuvette. 200 μL of the supernatant was added into the cuvette, mixed well, and then placed into a spectrophotometer. The scanning was started (wavelength 510 nm, scanning time 200 s), and the time was taken as the horizontal coordinate (min), and the absorbance was taken as the vertical coordinate. The slope value was obtained by linear fitting, and the enzyme activity of the crude enzyme solution was calculated.

[0202] Seven positive mutants with significantly increased enzyme activity were obtained in the first round of saturation mutation screening, and the results are shown in Table 7. Among the results of saturation mutation of the 12 target sites, there were 7 positive mutant strains at positions 312, 114 and 410. As shown in Table 7, the enzyme activity of the mutant strain NcDAAO10 was the highest, in which the valine at position 114 was mutated to asparagine, and the enzyme activity per OD was 1.29 times higher than that of the starting strain NcDAAO6 (P < 0.001).

[0203] Table 7. Screening results of positive mutants in the saturation mutation library of NcDAAO6 target sites

[0204] Step two: iterative saturation mutation screening of the remaining 11 sites based on V114N

[0205] The primer for updating position 112 is as follows

[0206] Table 8. Primers required for constructing the single-point saturation mutation library of updated position 112

[0207] According to the method of Example 3 Step 1, the pET-28a(+)-NcDAAO10 plasmid was used as a template to construct the saturated mutation library of the remaining 11 target sites, and high-throughput screening and rescreening were performed. Finally, 6 positive mutant strains with significantly improved enzyme activity were obtained, and the results are shown in Table 9. The results of the saturated mutation of the 11 target sites showed that there were 6 positive mutant strains at positions 312 and 318. After rescreening and detection verification, as shown in Table 9, the NcDAAO18 mutant strain had the highest enzyme activity. The unit OD enzyme activity was significantly improved (P<0.001) after the valine at position 114 was mutated to asparagine and the glutamine at position 312 was mutated to serine. Compared with the starting strain NcDAAO10, the unit OD enzyme activity was improved by 3.5 times, and compared with NcDAAO6, the unit OD enzyme activity was improved by 10.26 times. As shown in Figure 5, the protein expression of the single-point mutant strain NcDAAO10 and the two-point mutant strain NcDAAO18 was still low, and further optimization was needed. Therefore, V114N / Q312S was selected as the optimal combination of mutant strains of NcDAAO.

[0208] Table 9 Positive mutant screening results of iterative saturated mutation library based on V114N

[0209] Example 4 NcDAAO18 protein truncation

[0210] In order to further improve the protein expression and enzyme activity, truncation was continued based on the pET-28a(+)-NcDAAO18 plasmid. The N-terminal 57 base sequences of the NcDAAO18 protein were deleted, and the strain retaining the T7B9 tag was constructed as NcDAAO20, and the strain without the T7B9 tag was constructed as NcDAAO21. The primer sequences are shown in Table 10. The pET-28a(+)-NcDAAO18 was used as a template for whole plasmid PCR, and the detailed operation is shown in Example 1. The strain culture, induction expression, and enzyme activity determination were performed according to the method of Example 3 Step 3, and the soluble expression of D-amino acid oxidase was detected according to Example 1 Step 5. The enzyme activity results are shown in Table 11. The protein soluble expression and expression amount are shown in Table 12. As shown in Tables 11 and 12, the expression amount of NcDAAO20 retaining the T7B9 tag was still low, and the unit OD enzyme activity had no significant difference compared with the starting strain NcDAAO18, and further improvement was needed. The expression amount of the engineered bacteria protein NcDAAO21 with deletion of 57 bases at the N-terminus and deletion of the T7B9 tag was obviously improved, and the enzyme activity was significantly improved. The unit OD enzyme activity was improved by 55.2% (P<0.001) compared with the starting strain NcDAAO18, and the unit OD enzyme activity was improved by 15.14 times compared with the unmodified initial strain NcDAAO6.

[0211] Table 10 Primers related to NcDAAO18 protein truncation construction

[0212] Table 11 Enzyme activity assay of NcDAAO18 and its protein-truncated engineering bacteria

[0213] Table 12 Protein solubility expression assay of NcDAAO18 and its protein-truncated engineering bacteria

[0214] Example 5 Optimization of NcDAAO20 protein expression

[0215] The addition of T7B9 tag can greatly reduce the expression of target protein, therefore, in order to improve the protein expression and further improve the enzyme activity, the T7B9 tag before the N-terminal sequence of NcDAAO20 protein was truncated, and after deleting 39 bases at the N-terminus, NcDAAO22 was constructed, and after deleting 78 bases at the N-terminus, NcDAAO23 was constructed, the primer sequences are shown in Table 13, and the full plasmid PCR was performed with NcDAAO20 plasmid as the template, and the detailed operation is shown in Example 1. The strain culture, induction expression and enzyme activity determination were performed according to the method of step 3 of Example 3, and the soluble expression of D-amino acid oxidase was detected according to step 5 of Example 1. The enzyme activity results are shown in Table 14. The protein soluble expression and expression amount are shown in Table 15.

[0216] As can be seen from the results of Table 14 and Table 15, the protein expression of NcDAAO22 after deleting 39 bases of T7B9 tag has been significantly improved, and the enzyme activity has also been improved, compared with the starting strain NcDAAO20, the unit OD enzyme activity is increased by 60.7% (P < 0.001), and compared with the initial strain NcDAAO6 without modification, the unit OD enzyme activity is increased by 14.85 times. The protein expression of strain NcDAAO23 with 78 bases of T7B9 tag deleted at the N-terminus is significantly decreased, and the unit OD enzyme activity is decreased by 43% compared with the starting strain NcDAAO20. Therefore, the T7B9 mutant with 39 bases of N-terminal deletion is preferred as the tag for improving the protein expression and enzyme activity of NcDAAO.

[0217] Table 13 Primers related to the construction of T7B9 tag truncation in NcDAAO20

[0218] Table 14 Enzyme activity assay of NcDAAO20 T7B9 tag-truncated engineering bacteria

[0219] Table 15 Protein solubility expression assay of NcDAAO20 T7B9 tag-truncated engineering bacteria

[0220] Example 6 Preparation of L-glufosinate by multi-enzyme cascade reaction

[0221] The NcDAAO was modified and the soluble expression was optimized, and we obtained a strain NcDAAO22 with the highest enzyme activity and optimal soluble expression, which was used to build a multi-enzyme cascade system. Starting from DL-glufosinate, D-amino acid oxidase was used to catalyze the oxidation of D-amino acid to produce keto acid, and L-amino acid was completely retained, and then glutamate dehydrogenase was used to reduce the keto acid in situ to L-amino acid, realizing the resolution of D, L-amino acid. At 40°C, pH 8.0 and oxygen conditions, L-glufosinate was prepared by constant temperature magnetic stirrer. The total reaction volume was 100 mL, and the concentrations of the components were as follows: 200 mM DL-glufosinate solution, 300 mM isopropyl alcohol, 0.2 mM NAPD+, 0.1 g / L (dry cell weight) PpGluDH, 0.2 g / L (dry cell weight) alcohol dehydrogenase (BsADH) and 10 g / L (dry cell weight) D-amino acid oxidase (DAAO) of NcDAAO22, and 1000 U / mL catalase (MaCAT). The reaction technical route is shown in Figure 8. The specific operation steps are as follows: 0.2M (NH4)2SO4 aqueous solution is prepared, and 25% ammonium hydroxide is used to adjust the pH to 8.0 to obtain NH3·(NH3)2SO4 buffer solution. DL-glufosinate and isopropyl alcohol are dissolved in the buffer solution, and the pH is adjusted to 8.0 using 25% ammonium hydroxide to obtain a substrate solution (80 mL). The collected PpGluDH, BsADH, DAAO, MaCAT cells are resuspended in the buffer solution, and then NAPD+ is added to obtain an enzyme solution (20 mL). The substrate solution and enzyme solution are mixed and added to a three-necked flask for reaction, and during the reaction, the reaction solution is maintained at pH 8.0 by adding 10% ammonium hydroxide solution.

[0222] The catalytic effect of the multi-enzyme catalytic system was investigated by substrate conversion rate, conversion efficiency and enantiomeric excess value (ee value). The ee value represents the excess of one enantiomer over the other enantiomer, usually expressed in percentage: ee = ([R] - [S] / [R] + [S])*100%.

[0223] The highest enzyme activity of NcDAAO22 D-amino acid oxidase was used to prepare L-glufosinate by multi-enzyme cascade reaction, and the contents of L-, D-glufosinate were determined simultaneously, and the ee value was calculated, as shown in Figure 9. As shown in Figure 9, after 160 min of reaction, D-glufosinate was completely converted to L-glufosinate, and the conversion rate reached 100%. Finally, about 185.06 mM L-glufosinate was generated, and the ee value was greater than 99%.

[0224] Comparative Example 1

[0225] All the mutants of NcDAAO that were modified and optimized for soluble expression were used to build the multi-enzyme cascade system according to the method of Example 6. Starting from DL-phosphinothricin, the D-amino acid oxidase mutant was used to catalyze the oxidation of D-amino acid to produce keto acid, and the L-amino acid was completely retained, and then the glutamate dehydrogenase was used to reduce the keto acid in situ to L-amino acid, realizing the resolution of D, L-amino acid. The catalytic effect of the multi-enzyme catalytic system was investigated by substrate conversion rate, conversion efficiency and enantiomeric excess (ee value). The results are shown in Table 16. The results show that the mutants related to NcDAAO that were modified for soluble expression and enzyme activity can all convert D-phosphinothricin to L-phosphinothricin, and the ee value is greater than 99%.

[0226] Table 16 Multi-enzyme cascade reaction time and ee value determination results of NcDAAO mutants

[0227] The D-amino acid oxidase mutant and its application provided by the present application are described in detail above. The principles and implementation modes of the present application are described by applying specific examples, and the above description of the examples is only used to help understand the method of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. Use of any of the following in improving soluble expression of D-amino acid oxidase, improving enzyme activity of D-amino acid oxidase, kinetic resolution of DL-phosphinothricin or preparing L-phosphinothricin: (I) a mutant of wild type D-amino acid oxidase with 51, 96, 153 or 204 bases deleted from N-terminus and / or 51 bases deleted from C-terminus; and / or (II) a mutant of wild type D-amino acid oxidase with single point or multiple point combination mutation of L25P, A79R, T168R, M228P, A254E, G359R; and / or (III) a mutant of wild type D-amino acid oxidase with single point or multiple point combination mutation of L25P, A79R, T168R, M228P, A254E, G359R on the basis of the mutant of (I).

2. Use according to claim 1, wherein Also included is a mutant with T7B9 tag added to N-terminus.

3. Use according to claim 1 or 2, characterized in that, Also included is a mutant with single point or multiple point combination mutation of R112, V114, R139, Y183, T294, Q312, W318, K336, R382, G409, G410, R411.

4. Use according to claim 2 or 3, wherein the compound is ###0002### Also included is a mutant with 39 and / or 78 bases of T7B9 tag deleted from N-terminus.

5. Mutant of D-amino acid oxidase characterized in that, Also included is any of the following: (I) a mutant of wild type D-amino acid oxidase with 51, 96, 153 or 204 bases deleted from N-terminus and / or 51 bases deleted from C-terminus; and / or (II) a mutant of wild type D-amino acid oxidase with single point or multiple point combination mutation of L25P, A79R, T168R, M228P, A254E, G359R; and / or (III) a mutant of wild type D-amino acid oxidase with single point or multiple point combination mutation of L25P, A79R, T168R, M228P, A254E, G359R on the basis of the mutant of (I).

6. The mutant of claim 5, wherein Also included is a mutant with T7B9 tag added to N-terminus.

7. Mutant according to claim 5 or 6, characterized in that Also included is a mutant with single point or multiple point combination mutation of R112, V114, R139, Y183, T294, Q312, W318, K336, R382, G409, G410, R411.

8. The mutant according to claim 6 or 7, wherein Also included is a mutant with 39 and / or 78 bases of T7B9 tag deleted from N-terminus.

9. Mutant according to any one of claims 5 to 8, characterized in that Also included is a mutant with single point or multiple point combination mutation of Q312S, Q312A, V114N, V114Q, G410S, G410A.

10. Mutant according to any one of claims 5 to 9, characterized in that Also included is a mutant with single point or multiple point combination mutation of Q312S, Q312H, Q312A, W318Y in combination with V114N.

11. Mutant according to any one of claims 5 to 9, characterized in that Also included is a mutant with mutation of V114N and / or Q312S.

12. Mutant according to any one of claims 6 to 11, characterized in that Also included is a mutant with 39 and / or 78 bases of T7B9 tag deleted from N-terminus.

13. A nucleic acid molecule encoding the mutant of any one of claims 5 to 12.

14. The nucleic acid molecule of claim 13, wherein which has: (I) a nucleotide sequence as set forth in any one of SEQ ID NO. 4 to 26; or (I) a nucleotide sequence as set forth in any one of SEQ ID NO. 4 to 26; or (II) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but differing from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (III) a nucleotide sequence obtained by substitution, deletion or addition of one or more nucleotides to the nucleotide sequence shown in (I) or (II), and a nucleotide sequence functionally identical or similar to the nucleotide sequence shown in (I) or (II); or (IV) a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (I), (II) or (III).

15. Construct, characterized in that, comprising a promoter, a nucleic acid molecule according to claim 13 or 14, and a terminator.

16. A plasmid vector, characterized in that, comprising any of the following and a backbone vector: (I) a nucleic acid molecule according to claim 13 or 14; and / or (II) a construct according to claim 15.

17. A host, characterized in that, comprising any of the following: (I) a nucleic acid molecule according to claim 13 or 14; and / or (II) a construct according to claim 15; and / or (III) a plasmid vector according to claim 16.

18. The host of claim 17, wherein, The host comprises E. coli; The E. coli comprises BL21 (DE3).

19. Use of any of the following in the preparation of a D-amino acid oxidase: (I) a nucleic acid molecule according to claim 13 or 14; and / or (II) a construct according to claim 15; and / or (III) a plasmid vector according to claim 16; and / or (IV) a host according to claim 17 or 18.

20. A process for the preparation of a D-amino acid oxidase, characterized in that, comprising fermenting a host according to claim 17 or 18 to obtain a fermentation product.

21. A D-amino acid oxidase prepared according to the method of claim 20.

22. Use of any of the following in the preparation of an α-keto acid, in the kinetic resolution of DL-glufosinate ammonium, or in the preparation of L-glufosinate ammonium: (I) a mutant according to any one of claims 5 to 12; (II) a nucleic acid molecule according to claim 13 or 14; (III) a construct according to claim 15; (IV) a plasmid vector according to claim 16; and / or (V) a host according to claim 17 or 18.

23. A method of producing an α-keto acid, characterized by, comprising an oxidation reaction using a D-amino acid or a racemic amino acid as a substrate and a D-amino acid oxidase according to claim 21 to obtain an α-keto acid; the substrate concentration in the system of the oxidation reaction is 0.2 mol / L, the reaction temperature is 30°C, the reaction time is 1 h, and the pH of the reaction solution is 8.

0.

24. A process for the preparation of L-glufosinate-ammonium, characterized in that, including using D-amino acids or racemic amino acids as substrates, adding a D-amino acid oxidase, glutamate dehydrogenase, alcohol dehydrogenase, catalase, NAD + mixed, stirred, and L-glufosinate was produced.

25. The production method according to claim 24, wherein the substrate concentration comprises 0.2 mol / L; the temperature during stirring comprises 40°C; and the pH during stirring comprises 8.0.

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