Mutant of D-carbamoylase and application thereof

Abstract

The invention discloses a D-carbamoylase mutant, a preparation method of the D-carbamoylase mutant and application of the D-carbamoylase mutant in catalytic synthesis of D-phenylglycine. The mutant disclosed by the invention comprises five kinds of D-carbamoyl hydrolase mutants, namely, A273T-T2S, A273T-Q291K, A273T-H58L, A273T-T262S and A273T-E303Q, and the D-carbamoyl hydrolase mutants disclosed by the invention comprise five kinds of D-carbamoyl hydrolase mutants disclosed by the invention. N-carbamyl-D-phenylglycine is used as a substrate to prepare D-phenylglycine, N-carbamyl-D-phenylglycine can be selectively and excellently catalyzed, high enzyme activity is shown, and the conversion rate is obviously higher than that of wild type D-carbamyl hydrolase. Therefore, the D-carbamoylase mutant disclosed by the invention has a wide application prospect in the industrial production of D-phenylglycine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and enzyme molecular modification technology, and particularly relates to a mutant of D-carbamoylase and application thereof. BACKGROUND

[0002] D-phenylglycine (D-PG) as an important chiral amino acid derivative has wide application value in the fields of medicine, pesticide and fine chemical industry. In the field of medicine, D-phenylglycine is a key chiral intermediate for the synthesis of β-lactam antibiotics, especially an important side chain structural unit for the semi-synthesis of penicillin and cephalosporin antibiotics; in the synthesis of chiral drugs, it can be used as a chiral auxiliary agent or a synthon for constructing complex drug molecules; in the field of pesticide, its derivatives can be used for developing chiral pesticide active ingredients with high efficiency and low toxicity; in addition, some derivatives can also be used as food additives or feed supplements to improve the efficiency of nutrient absorption.

[0003] L-phenylglycine is relatively common in nature, while D-phenylglycine with important application value needs to be synthesized by chemical or biocatalytic methods. Traditional chemical synthesis method has the disadvantages of severe reaction conditions, serious environmental pollution, low yield and high cost, and has been gradually eliminated. In contrast, enzyme synthesis has the significant advantages of mild reaction conditions (no need for high temperature and high pressure), low cost, environmental friendliness, high substrate conversion rate and optical purity. At present, two-step enzyme method is mainly used in industry: first, D-hydantoinase (D-hydantoinase) is used to catalyze the asymmetric ring opening of phenylhydantoin under the alkaline conditions of in situ racemization to generate N-carbamoyl-D-phenylglycine, and then N-carbamoyl-D-phenylglycine hydrolase (i.e. D-carbamoylase, DCase) is used to catalyze the hydrolysis to obtain the final product D-phenylglycine.

[0004] However, in the industrial production process, it is found that due to the low catalytic efficiency and poor stability of DCase, the intermediate product N-carbamoyl-D-phenylglycine accumulates in large amounts, which seriously affects the yield of the final product, and DCase becomes the rate-limiting enzyme of the entire enzyme reaction system. With the development of genetic engineering technology, especially the continuous improvement of E. coli expression system, it is possible to realize the efficient expression of DCase through genetic engineering means. It is worth noting that although natural enzyme molecules have undergone a long natural evolution process, due to the significant difference between the natural environment and the actual application environment, these enzyme molecules still have great potential for modification. SUMMARY

[0005] In view of this, one of the purposes of the present application is to provide a D-carbamoyl hydrolase mutant, which is obtained by mutating the amino acid sequence of D-carbamoyl hydrolase shown in SEQ ID No. 1 (D-carbamoyl hydrolase derived from Agrobacterium sp. (strain KNK712)) as follows:

[0006] 1) the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 2 is mutated from threonine to serine; the amino acid sequence of this mutant is shown in SEQ ID NO. 3;

[0007] 2) the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 291 is mutated from glutamine to lysine; the amino acid sequence of this mutant is shown in SEQ ID NO. 4;

[0008] 3) the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 58 is mutated from histidine to leucine; the amino acid sequence of this mutant is shown in SEQ ID NO. 5;

[0009] 4) the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 262 is mutated from threonine to serine; the amino acid sequence of this mutant is shown in SEQ ID NO. 6;

[0010] 5) the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 303 is mutated from glutamic acid to glutamine; the amino acid sequence of this mutant is shown in SEQ ID NO. 7.

[0011] The second purpose of the present application is to provide a nucleic acid molecule encoding the above-mentioned D-carbamoyl hydrolase mutant.

[0012] The third purpose of the present application is to provide a biological material containing the above-mentioned nucleic acid molecule, which is a recombinant DNA, an expression cassette, a transposon, a recombinant plasmid, a viral vector or an engineered bacterium. For example, a D-carbamoyl hydrolase mutant DNA fragment is linked to a vector pET21a to obtain a recombinant plasmid; the recombinant plasmid is transformed into E. coli BL21 (DE3) for culture to obtain an engineered bacterium capable of expressing D-carbamoyl hydrolase mutant.

[0013] The fourth object of the present application is to provide a preparation method of the D-carbamoylase mutant, comprising the following steps: connecting a DNA fragment of the D-carbamoylase mutant with a vector pET21a to obtain a recombinant plasmid; transforming the recombinant plasmid into E. coli BL21 (DE3) to obtain an engineering strain; inducing expression of the engineering strain; and obtaining the D-carbamoylase mutant recombinant protein by using multi-channel ultrasonic crushing and affinity chromatography purification. As for the expression vector, pET21a is taken as an example, but other vectors can also be used to construct corresponding expression vectors, such as commonly used expression vectors like pET system, pSU system, pTrc system, pMW system, pKK system, RSF1010, etc. Similarly, the constructed corresponding expression vector can also be transformed into other microorganisms to obtain corresponding transformed microorganisms, such as Pseudomonas, Flavobacterium, Bacillus, Serratia, Agrobacterium, Corynebacterium or Brevibacterium, etc.

[0014] The fifth object of the present application is to provide an application of the D-carbamoylase mutant in catalyzing synthesis of D-phenylglycine.

[0015] The sixth object of the present application is to provide a method for synthesizing D-phenylglycine, comprising the following steps: using the D-carbamoylase mutant as a catalyst to catalyze N-carbamoyl-D-phenylglycine to obtain D-phenylglycine.

[0016] Preferably, the catalyzed reaction condition is 25-42 ℃, 180-250 rpm, and pH 7.5-9.0.

[0017] Preferably, the N-carbamoyl-D-phenylglycine is obtained by comprising the following steps:

[0018] 1) connecting a DNA fragment of the D-hydantoinase mutant with a vector pET21a to obtain a recombinant plasmid;

[0019] 2) transforming the recombinant plasmid into E. coli BL21 (DE3) to obtain an engineering strain;

[0020] 3) inducing expression of the engineering strain;

[0021] 4) obtaining the D-hydantoinase mutant recombinant protein by using multi-channel ultrasonic crushing and affinity chromatography purification;

[0022] 5) the D-hydantoinase mutant recombinant protein as catalyst, catalyzing the phenylhydantoin to obtain N-carbamoyl-D-phenylglycine;

[0023] The DNA segment of the D-hydantoinase mutant is one of the following A) -E) sequences:

[0024] A) R36S-I114A-T403S: that is, based on the sequence of SEQ ID NO. 9, the arginine codon CGT at position 36 is mutated to the serine codon AGC, the isoleucine codon ATT at position 114 is mutated to the alanine codon GCA, and the threonine codon ACC at position 403 is mutated to the serine codon AGT;

[0025] B) E30A-Q113K-N384A: that is, based on the sequence of SEQ ID NO. 9, the glutamic acid codon GAA at position 30 is mutated to the alanine codon GCC, the glutamine codon CAG at position 113 is mutated to the lysine codon AAG, and the asparagine codon AAC at position 384 is mutated to the alanine codon GCT;

[0026] C) E44T-C91V-Q189A: that is, based on the sequence of SEQ ID NO. 9, the glutamic acid codon GAG at position 44 is mutated to the threonine codon ACG, the cysteine codon TGC at position 91 is mutated to the valine codon GTC, and the glutamine codon CAG at position 189 is mutated to the alanine codon GCG;

[0027] D) V141E-K408H-A457S: that is, based on the sequence of SEQ ID NO. 9, the valine codon GTT at position 141 is mutated to the glutamic acid codon GAG, the lysine codon AAG at position 408 is mutated to the histidine codon CAC, and the alanine codon GCA at position 457 is mutated to the serine codon TCT;

[0028] E) E95A-S166A-M196L: that is, based on the sequence of SEQ ID NO. 9, the glutamic acid codon GAA at position 95 is mutated to the alanine codon GCA, the serine codon AGC at position 166 is mutated to the alanine codon GCT, and the methionine codon ATG at position 196 is mutated to the leucine codon CTG.

[0029] The five D-carbamoyl hydrolase mutants A273T-T2S, A273T-Q291K, A273T-H58L, A273T-T262S and A273T-E303Q provided by the application can selectively catalyze N-carbamoyl-D-phenylglycine well, have good stability and higher product conversion rate. The obtained recombinant proteins are used to prepare D-phenylglycine with N-carbamoyl-D-phenylglycine as the substrate, and are compared with the D-carbamoyl hydrolase with the sequence of SEQ ID NO. 1. The results show that the D-carbamoyl hydrolase mutants provided by the application are different from the currently known D-carbamoyl hydrolases, and have higher enzyme activity when N-carbamoyl-D-phenylglycine is used as the substrate, and the conversion rate is obviously higher than that of the D-carbamoyl hydrolase with the sequence of SEQ ID NO. 1. Therefore, the D-carbamoyl hydrolase mutants provided by the application have broad application prospects in the industrial production of D-phenylglycine. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The peak with a retention time of 9.656 is the L configuration, and the peak with a retention time of 10.290 is the D configuration, which are the spectra of DL-phenylglycine detected by HPLC;

[0031] Figure 2 The SDS-PAGE electrophoretogram of the D-carbamoyl hydrolase mutants expressed in E. coli (wherein, 1 represents A273T-T2S, 2 represents A273T-Q291K, 3 represents A273T-H58L, 4 represents A273T-T262S, and 5 represents A273T-E303Q);

[0032] Figure 3 The conversion rates of different D-carbamoyl hydrolase mutants on N-carbamoyl-D-phenylglycine (wherein, 1 represents A273T-T2S, 2 represents A273T-Q291K, 3 represents A273T-H58L, 4 represents A273T-T262S, and 5 represents A273T-E303Q);

[0033] Figure 4 The spectra of D-phenylglycine generated by different D-carbamoyl hydrolase mutants. DETAILED DESCRIPTION

[0034] The application will be described in detail below with reference to the examples, which are only illustrative and do not limit the application. The application is not limited to the following embodiments or examples, and any modification and transformation made without departing from the spirit of the application shall be included in the scope of the application. The experimental materials or reagents used in the following examples are commercially available unless otherwise specified. In the quantitative experiments in the following examples, three repeated experiments are set, and the results are taken from the average values.

[0035] In the following examples, the mutant starting gene, DCase gene, is derived from D-aminoacylase of Agrobacterium sp. (strain KNK712), the DNA sequence and amino acid sequence of which are shown in SEQ ID NO. 2 and SEQ ID NO. 1, respectively. The genes of the obtained mutants (DCase-M) differ from the DCase gene as follows:

[0036] (1) A273T-T2S, in which the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 2 is mutated from threonine to serine; the amino acid sequence of this mutant is shown in SEQ ID NO. 3;

[0037] (2) A273T-Q291K, in which the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 291 is mutated from glutamine to lysine; the amino acid sequence of this mutant is shown in SEQ ID NO. 4;

[0038] (3) A273T-H58L, in which the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 58 is mutated from histidine to leucine; the amino acid sequence of this mutant is shown in SEQ ID NO. 5;

[0039] (4) A273T-T262S, in which the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 262 is mutated from threonine to serine; the amino acid sequence of this mutant is shown in SEQ ID NO. 6;

[0040] (5) A273T-E303Q, in which the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 303 is mutated from glutamic acid to glutamine; the amino acid sequence of this mutant is shown in SEQ ID NO. 7.

[0041] (6) A273T, in which the amino acid at position 273 is mutated from alanine to threonine; the amino acid sequence of this mutant is shown in SEQ ID NO. 8.

[0042] Example 1. Construction of expression vector containing target gene and engineering bacteria

[0043] The target gene sequence was directly synthesized by Kingsriver Biotech Co., Ltd. to obtain a pET21a expression vector plasmid containing the target gene (the target gene is directly connected to a 6x histidine tag sequence), which was then transformed into E. coli BL21 (DE3) to obtain recombinant E. coli. The target gene sequence is as follows:

[0044] Sequence 1: The starting nucleotide sequence, as shown in SEQ ID NO. 2 (the encoded amino acid sequence is shown in SEQ ID NO. 1);

[0045] Sequence 2: A273T-T2S: Based on Sequence 1, the amino acid at position 273 is mutated from alanine to threonine (codon: GCG→ACC), and the amino acid at position 2 is mutated from threonine to serine (codon: ACA→AGC);

[0046] Sequence 3: A273T-Q291K: Based on Sequence 1, the amino acid at position 273 is mutated from alanine to threonine (codon: GCG→ACC), and the amino acid at position 291 is mutated from glutamine to lysine (codon: CAG→AAG);

[0047] Sequence 4: A273T-H58L: Based on Sequence 1, the amino acid at position 273 is mutated from alanine to threonine (codon: GCG→ACC), and the amino acid at position 58 is mutated from histidine to leucine (codon: CAT→CUG);

[0048] Sequence 5: A273T-T262S: Based on Sequence 1, the amino acid at position 273 is mutated from alanine to threonine (codon: GCG→ACC), and the amino acid at position 262 is mutated from threonine to serine (codon: ACC→UCG);

[0049] Sequence 6: A273T-E303Q: Based on Sequence 1, the amino acid at position 273 is mutated from alanine to threonine (codon: GCG→ACC), and the amino acid at position 303 is mutated from glutamic acid to glutamine (codon: GAG→CAG).

[0050] Sequence 7: A273T: Based on Sequence 1, the amino acid at position 273 is mutated from alanine to threonine (codon: GCG→ACC)

[0051] Example 2, Expression of the target protein of the recombinant strain

[0052] The plate on which the E. coli expression strain was constructed was placed, and a single colony was picked into 5 mL of LB liquid medium containing 50 μg / mL of ampicillin, and cultured at 37 °C, 220 rpm, overnight. The overnight cultured strain was inoculated into LB liquid medium containing 50 μg / mL of ampicillin at an inoculation amount of 1%, and cultured at 37 °C, 220 rpm, until the OD 600When the OD600 is about 0.6, IPTG is added to a final concentration of 0.5 mM, and the culture is induced at 16 ℃ for 12-16 hours. The culture medium is centrifuged at 8000 rpm for 10 min at 4 ℃, and the supernatant is discarded. The cell pellet is used for enzyme purification.

[0053] Example 3, purification of the target protein

[0054] All purification steps are performed at 4 ℃. The cell pellet is resuspended in buffer containing 20 mM Tris-HCl pH 7.4, 150 mM sodium chloride at 75-100 g / L. An ultrasonic disrupter is used to sonicate at 4 ℃ for 10 min at 60% power, 3 s on and 3 s off. The lysate is collected and centrifuged at 12000 rpm for 30 min at 4 ℃. The supernatant is collected to obtain the crude enzyme solution. According to the histidine tag of the recombinant protein, the crude enzyme solution is purified using an affinity chromatography column-nickel column (purchased from Tiandihuan Biotechnology Co., Ltd.). Gradient elution is set, and 20, 50, 30 mM imidazole is used for elution. After elution, the protein is mixed with 25% of 50% glycerol to make the final glycerol concentration 10%. The concentration is determined using a nanodrop1 from Thermo, and the calculated value is greater than 2 mg. The sample is stored in a -80 ℃ refrigerator for subsequent use. The collected target protein is identified by protein electrophoresis SDS-PAGE, and the results are shown in FIG. 2. A single band is shown to obtain the pure enzyme. Figure 2

[0055] Example 4, preparation of D-phenylglycine by pure enzyme

[0056] The enzyme in Example 3 is used in the hydrolysis reaction. The reaction system is 2 mL, and the specific conditions are as follows: the amount of enzyme used in the reaction is 4 μg (the volume is calculated according to the concentration and amount of the enzyme), the amount of N-carbamoyl-D-phenylglycine intermediate (purchased from Aladdin) added is 2 mg, and 50 mM Tris-HCl (pH 8.5) is added to the reaction system to 2 mL. Then the reaction is carried out in a constant temperature shaker at 30 ℃, 220 rpm for 4 h, and the pH of the reaction is 8.5. The sample of the reaction solution after conversion is added with 200 μL of 1 M hydrochloric acid to stop the reaction. Centrifugation is performed at 12000 rpm for 10 min, the supernatant is diluted with ultrapure water, and then filtered through a 0.22 μm water membrane. High performance liquid chromatography (HPLC) analysis is performed. The HPLC (Shimadzu model DGU-20A) detection method is as follows: detector LC-20AD, sample size 20 μL, using a chromatographic column (CHIRALPAK ZWIX(+) 3 μm, 3x150 mm, purchased from Shimadzu Corporation), mobile phase is methanol: acetonitrile: H2O = 49:49:2 (V / V / V) (containing 50 mM formic acid and 25 mM diethylamine), flow rate 0.5 mL / min. The results are shown in FIG. 3.​Figure 4 As shown in the figure, data 1-8 are HPLC chromatograms of DL-phenylglycine control, 5 double-site mutants, A273T mutant and the starting protein (wild type) in turn; the DL-phenylglycine control is purchased from Baoeinxintebio Technology (Suzhou) Co., Ltd., and its HPLC chromatogram can be seen in Figure 1 , which is incorporated herein as background; the peak with a retention time of 10.29 in data 2-8 is in turn A273T-Q291K mutant, A273T-T2S mutant, A273T-E303Q mutant, A273T-T262S mutant, A273T-H58L mutant, A273T mutant and wild type protein from high to low. It can be seen that the mutant and the wild type protein of the present application can both selectively convert the N-carbamoyl-D-phenylglycine intermediate into D-phenylglycine. Figure 4

[0057] According to the HPLC chromatograms, the conversion rate of each reaction is calculated by dividing the peak area of the product by the sum of the peak area of the substrate and the peak area of the product, as shown in Figure 4 Figure 3 As shown in the figure, the conversion rates of the 5 double-site mutants are significantly higher than those of the wild type and the A273T mutant. The conversion rates of the mutants are directly compared with the conversion rate of the original sequence, and the conversion rates are increased by 5.5 times, 5.6 times, 5.3 times, 5.3 times and 5.4 times, respectively; at the same time, compared with the mutant A273T, the 5 double-site mutants are increased by 4.2 times, 4.2 times, 3.9 times, 4.0 times and 4.0 times, respectively. Therefore, the double-site mutant of the present application has higher catalytic activity, significantly improves the conversion rate of D-phenylglycine, has significant progress, and has a broad application prospect in the industrial production of D-phenylglycine by using double-enzyme method.

[0058] Example 5, preparation of N-carbamoyl-D-phenylglycine intermediate

[0059] The substrate N-carbamoyl-D-phenylglycine intermediate used in the hydrolysis reaction in Example 4 can also be obtained by the following method:

[0060] (1) Preparation of expression vector and engineering strain of mutant expressing D-hydantoinase

[0061] The gene sequence of the mutant of D-hydantoinase is directly synthesized into a pET21a expression vector plasmid containing the target gene (the gene sequence of the mutant is directly connected with a 6x histidine tag sequence) by Kingsri Biotechnology Co., Ltd., and the recombinant Escherichia coli is obtained by transforming the mutant gene sequence into Escherichia coli BL21 (DE3). The D-hydantoinase mutant gene sequence is any one of the following A) to E) sequences:

[0062] ​​A) R36S-I114A-T403S: i.e. based on the sequence of SEQ ID NO. 9, the arginine at position 36 of the amino acid sequence encoded thereby is mutated to serine (codon: CGT→AGC), the isoleucine at position 114 is mutated to alanine (codon: ATT→GCA), and the threonine at position 403 is mutated to serine (codon: ACC→AGT);

[0063] B) E30A-Q113K-N384A: i.e. based on the sequence of SEQ ID NO. 9, the glutamic acid at position 30 of the amino acid sequence encoded thereby is mutated to alanine (codon: GAA→GCC), the glutamine at position 113 is mutated to lysine (codon: CAG→AAG), and the asparagine at position 384 is mutated to alanine (codon: AAC→GCT);

[0064] C) E44T-C91V-Q189A: i.e. based on the sequence of SEQ ID NO. 9, the glutamic acid at position 44 of the amino acid sequence encoded thereby is mutated to threonine (codon: GAG→ACG), the cysteine at position 91 is mutated to valine (codon: TGC→GTC), and the glutamine at position 189 is mutated to alanine (codon: CAG→GCG);

[0065] D) V141E-K408H-A457S: i.e. based on the sequence of SEQ ID NO. 9, the valine at position 141 of the amino acid sequence encoded thereby is mutated to glutamic acid (codon: GTT→GAG), the lysine at position 408 is mutated to histidine (codon: AAG→CAC), and the alanine at position 457 is mutated to serine (codon: GCA→TCT);

[0066] E) E95A-S166A-M196L: i.e. based on the sequence of SEQ ID NO. 9, the glutamic acid at position 95 of the amino acid sequence encoded thereby is mutated to alanine (codon: GAA→GCA), the serine at position 166 is mutated to alanine (codon: AGC→GCT), and the methionine at position 196 is mutated to leucine (codon: ATG→CTG).

[0067] (2) Expression and purification of D-hydantoinase mutants

[0068] The obtained recombinant E. coli expression strain was activated, and a single colony was picked into 5 mL LB liquid medium containing 50 μg / mL ampicillin and cultured at 37 °C, 220 rpm, overnight. The overnight cultured strain was inoculated into LB liquid medium containing 50 μg / mL ampicillin at an inoculation amount of 1%, and cultured at 37 °C, 220 rpm, until the OD 600 ≈0.6, 0.5 mM IPTG was added, and the culture was induced at 16 °C for 12-16 hours. The medium liquid was centrifuged at 4 °C, 8000 rpm, for 10 min, and the supernatant was discarded. The cell pellet was purified at 4 °C using an affinity chromatography column-nickel column.

[0069] (3) Catalytic synthesis of N-carbamoyl-D-phenylglycine intermediate

[0070] The purified D-hydantoinase mutant was used in the catalytic reaction, and the reaction system was 2 mL. Specifically, the enzyme was used in an amount of 2 μg (the mass of the enzyme was calculated according to the concentration and volume of the enzyme), and hydantoin (purchased from Aldrich) was added in an amount of 2 mg. The reaction system was adjusted to 2 mL by adding 50 mM Tris-HCl (pH 8.5). Then the reaction was carried out at 30 °C, 220 rpm, for 4 h, and the pH was 8.5. The reaction sample was terminated by adding 200 μL of 1 M hydrochloric acid after centrifugation at 12000 rpm for 10 min. The supernatant was diluted with ultrapure water, filtered through a 0.22 μm water membrane, and then analyzed and separated by high-performance liquid chromatography (HPLC). The HPLC (Shimadzu model DGU-20A) detection method was as follows: detector LC-20AD, using a chromatographic column (CHIRALPAK ZWIX(+) 3 μm, 3x150 mm, purchased from Shimadzu Corporation), mobile phase was methanol:acetonitrile:H2O=49:49:2 (V / V / V) (containing 50 mM formic acid and 25 mM diethylamine), flow rate 0.5 mL / min. The product N-carbamoyl-D-phenylglycine was collected from the peak with a retention time of 6.265.

[0071] The conventional techniques and schemes not described in detail in the above examples are well known in the art, and therefore will not be described in detail here. The above examples and / or experimental examples describe the preferred embodiments of the present application in detail, however, the present application is not limited to the specific details in the above embodiments, and within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A D-carbamoyl hydrolase mutant, characterized in that, The mutant was obtained by mutagenesis based on the amino acid sequence of D-carbamoyl hydrolase as shown in SEQ ID No. 1, with the mutation site being one of the following 1)-5): 1) In A273T-T2S, the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 2 is mutated from threonine to serine. 2) In A273T-H58L, the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 58 is mutated from histidine to leucine. 3) In A273T-T262S, the amino acid at position 273 is mutated from alanine to threonine, and at the same time, the amino acid at position 262 is mutated from threonine to serine. 4) In A273T-Q291K, the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 291 is mutated from glutamine to lysine. 5) In A273T-E303Q, the amino acid at position 273 is mutated from alanine to threonine, and the amino acid at position 303 is mutated from glutamic acid to glutamine.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the D-carbamoyl hydrolase mutant of claim 1.

3. A biomaterial containing the nucleic acid molecule of claim 2, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, recombinant plasmid, viral vector, or engineered bacteria.

4. The method for preparing the D-carbamoyl hydrolase mutant according to claim 1, characterized in that, The procedure includes the following steps: ligating a DNA fragment of a D-carbamoyl hydrolase mutant to the vector pET21a to obtain a recombinant plasmid; transforming the recombinant plasmid into E. coli BL21(DE3) to obtain an engineered strain; inducing expression in the engineered strain; and using multichannel sonication and affinity chromatography to purify the D-carbamoyl hydrolase mutant recombinant protein.

5. The application of the D-carbamoyl hydrolase mutant according to claim 1 in the catalytic synthesis of D-phenylglycine.

6. A method for synthesizing D-phenylglycine, characterized in that, The method includes the following steps: using the D-carbamoyl hydrolase mutant of claim 1 as a catalyst to catalyze N-carbamoyl-D-phenylglycine to obtain D-phenylglycine.

7. The method as described in claim 6, characterized in that, The catalytic reaction conditions were 25–42 °C, 180–250 rpm, and pH 7.5–9.

0.

8. The method as described in claim 7, characterized in that, The N-carbamoyl-D-phenylglycine is obtained by comprising the following steps: 1) The DNA fragment of the D-hynanase mutant was ligated into the vector pET21a to obtain a recombinant plasmid; 2) The recombinant plasmid was transformed into E. coli BL21(DE3) to obtain the engineered strain; 3) The engineered strain was induced to express its contents; 4) The D-hydantoin mutant recombinant protein was obtained by multi-channel ultrasonic disruption and affinity chromatography purification. 5) Using the D-hydantoin mutant recombinant protein as a catalyst, phenylhydantoin is catalyzed to obtain N-carbamoyl-D-phenylglycine; The DNA fragment of the D-hynanase mutant is one of the following sequences A)-E): A) R36S-I114A-T403S: Based on SEQ ID NO.9, the arginine codon CGT at position 36 is mutated to the serine codon AGC, the isoleucine codon ATT at position 114 is mutated to the alanine codon GCA, and the threonine codon ACC at position 403 is mutated to the serine codon AGT. B) E30A-Q113K-N384A: Based on SEQ ID NO.9, the glutamate codon GAA at position 30 is mutated to the alanine codon GCC, the glutamine codon CAG at position 113 is mutated to the lysine codon AAG, and the asparagine codon AAC at position 384 is mutated to the alanine codon GCT. C) E44T-C91V-Q189A: Based on SEQ ID NO.9, the glutamate codon GAG at position 44 is mutated to the threonine codon ACG, the cysteine ​​codon TGC at position 91 is mutated to the valine codon GTC, and the glutamine codon CAG at position 189 is mutated to the alanine codon GCG. D) V141E-K408H-A457S: Based on SEQ ID NO.9, the valine codon GTT at position 141 is mutated to the glutamate codon GAG, the lysine codon AAG at position 408 is mutated to the histidine codon CAC, and the alanine codon GCA at position 457 is mutated to the serine codon TCT. E) E95A-S166A-M196L: Based on SEQ ID NO.9, the glutamate codon GAA at position 95 is mutated to the alanine codon GCA, the serine codon AGC at position 166 is mutated to the alanine codon GCT, and the methionine codon ATG at position 196 is mutated to the leucine codon CTG.

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