A racemase mutant for racemic p-hydroxyphenylglycine synthesis and application thereof

Abstract

The application provides a racemase mutant for racemization of p-hydroxyphenylglycine and application thereof, and belongs to the technical field of bioengineering.The application uses L-p-hydroxyphenylglycine and PLP as raw materials, and uses a mutant of racemase (LbAAR) with improved activity to perform racemization of L-p-hydroxyphenylglycine, and the overall catalytic process is green, simple and efficient.In the application, no activity of the wild-type to the substrate L-HPG is detected, the activity to the substrate L-HPG is obtained by mutating L307S, and in addition, the enzymatic activity of the optimal mutant can reach 115.2 μmol / min / g, which is 16.5 times higher than that of M1, and the activity of the enzyme is greatly improved.The application provides a new idea for rapid racemization of p-hydroxyphenylglycine.

Description

A racemic enzyme mutant for the synthesis of racemic p-hydroxyphenylglycine and its application Technical Field

[0001] This invention relates to a racemic enzyme mutant for the synthesis of racemic p-hydroxyphenylglycine and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] D-p-hydroxyphenylglycine (D-HPG) is a non-natural amino acid with a chiral center. Its molecular formula is C8H9NO3, and its relative molecular mass is 167.16. It is an enantiomer of L-p-hydroxyphenylglycine. D-HPG is an important intermediate in amino acid biosynthesis and is widely used in the synthesis of peptide hormones and pesticides. In particular, it is widely used as a pharmaceutical intermediate in the preparation of β-lactam antibiotics, such as amoxicillin, ampicillin, and cefoperazone. These antibiotics have good bactericidal effects against Gram-positive bacteria, Gram-negative bacteria, and pathogenic spirochetes, and have advantages such as few side effects and good oral efficacy.

[0003] Currently, there are two main methods for synthesizing D-p-hydroxyphenylglycine: chemical and enzymatic methods. The chemical synthesis method primarily utilizes asymmetric conversion, employing the chiral resolving agent D-3-bromocamphor sulfonate (D-BCS) to induce a configurational change in L-HPG, thereby synthesizing high-purity D-HPG. However, this method suffers from severe pollution problems related to waste. The enzymatic conversion method mainly uses D-p-hydroxyphenylhydantoin (D-HPH) as a substrate, employing D-hydantoinase (Hase) and N-carbamoyl-D-amino acid amide hydrolase (Case) in a two-step hydrolysis process to obtain D-HPG. However, this method is limited by poor substrate solubility, low catalytic efficiency, and high production costs.

[0004] A natural metabolic pathway for the synthesis of L-HPG has been discovered in the biosynthetic pathway of vancomycin antibiotics. Starting with L-tyrosine as a substrate, L-HPG is obtained through the catalysis of L-amino acid dehydrogenase (L-AAD), 4-hydroxymandelate synthase (HmaS), 4-hydroxymandelate oxidase (Hmo), and (S)-3,5-dihydroxyphenylglycine transaminase (HpgT). Based on this pathway, research groups both domestically and internationally have modified it, for example, using D-4-hydroxyphenylglycine transaminase (HpgAT) and meso-diaminopimelic acid dehydrogenase (DAPDH) for D-HPG production. However, the low equilibrium constant of the transaminases, substrate inhibition, and the low catalytic efficiency of meso-diaminopimelic acid dehydrogenase limit the practical application of this pathway. A scheme utilizing racemic enzymes to resolve D-p-hydroxyphenylglycine has not yet been reported. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides a racemic enzyme mutant for the synthesis of racemic p-hydroxyphenylglycine and its application. The aim is to solve the technical problems that limit the production of D-HPG, such as the low reaction equilibrium constant and substrate inhibition of D-4-hydroxyphenylglycine transaminase and the low catalytic efficiency of meso-diaminopimelic acid dehydrogenase.

[0006] The first technical solution provided by this invention is a racemic enzyme (or epimerase) LbAAR and its mutants capable of synthesizing racemic p-hydroxyphenylglycine, the corresponding parental amino acid sequence of which is shown in SEQ ID NO.1 and its nucleotide sequence of which is shown in SEQ ID NO.2. The racemic enzyme LbAAR is derived from Lentilactobacillus buchneri.

[0007] The present invention provides a racemic enzyme LbAAR mutant, which is obtained by mutating one or more of the amino acids at positions 54, 222, 304, and 307 of the racemic enzyme LbAAR parental amino acid sequence as shown in SEQ ID NO. 1.

[0008] In one embodiment of the present invention, the mutant is alanine at position 54 of the racemic enzyme LbAAR parent that is mutated to phenylalanine, aspartic acid at position 222 that is mutated to histidine, leucine at position 307 that is mutated to serine, and / or proline at position 304 that is mutated to glutamic acid, glutamine, aspartic acid, or methionine.

[0009] In one embodiment of the present invention, the mutant is any one of the following (a) to (j):

[0010] (a) The mutant obtained by mutating alanine at position 54 of the racemic enzyme LbAAR parent with the amino acid sequence shown in SEQ ID NO.1 to phenylalanine was named A54F.

[0011] (b) The aspartic acid at position 222 of the racemic enzyme LbAAR parent, whose amino acid sequence is as shown in SEQ ID NO.1, is mutated to histidine, and the mutant is named D222H;

[0012] (c) The mutant obtained by mutating proline at position 304 of the racemic enzyme LbAAR parent with the amino acid sequence shown in SEQ ID NO.1 to glutamate was named P304E.

[0013] (d) The mutant obtained by mutating leucine at position 307 of the racemic enzyme LbAAR parent with the amino acid sequence shown in SEQ ID NO.1 to serine was named L307S.

[0014] (e) The mutant obtained by mutating leucine at position 307 to serine and aspartic acid at position 222 to histidine in the racemic enzyme LbAAR parental amino acid sequence as shown in SEQ ID NO.1 was named L307S / D222H.

[0015] (f) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to glutamic acid in the racemic enzyme parent LbAAR with the amino acid sequence shown in SEQ ID NO.1 is named L307S / D222H / P304E.

[0016] (g) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to glutamine in the racemic enzyme LbAAR parental amino acid sequence as shown in SEQ ID NO.1, is named L307S / D222H / P304Q.

[0017] (h) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to aspartic acid in the racemic enzyme LbAAR parental amino acid sequence as shown in SEQ ID NO.1, is named L307S / D222H / P304D.

[0018] (i) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to methionine in the racemic enzyme parent LbAAR with the amino acid sequence shown in SEQ ID NO.1, is named L307S / D222H / P304M.

[0019] (j) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, proline at position 304 to glutamic acid, and alanine at position 54 to phenylalanine, as shown in SEQ ID NO.1, is named L307S / D222H / P304E / A54F.

[0020] The present invention also provides a second technical solution, which is a gene encoding the mutant described in the first technical solution.

[0021] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0022] In one embodiment of the present invention, the recombinant vector is pET28a as the expression vector.

[0023] The fourth technical solution provided by the present invention is a recombinant cell expressing the mutant described in the first technical solution, or carrying the gene described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0024] In one embodiment of the present invention, the recombinant cells are expressed using bacteria or fungi as expression hosts.

[0025] The fifth technical solution provided by this invention is a method for preparing the racemic enzyme LbAAR mutant described in the first technical solution, the method comprising the following steps:

[0026] (1) Based on the amino acid sequence of racemic enzyme LbAAR, determine the mutation site; design mutation primers for site-directed mutagenesis, and use the vector carrying the racemic enzyme LbAAR gene as a template for site-directed mutagenesis; construct a plasmid vector containing the mutant.

[0027] (2) Transform the mutant plasmid into the host cell;

[0028] (3) Select positive clones for induced fermentation culture and purify the racemic enzyme LbAAR mutant.

[0029] In one embodiment of the present invention, in step (3), the induction is performed at 16°C.

[0030] In one embodiment of the present invention, in step (3), the final concentration of the inducer is 0.5 mM.

[0031] In one embodiment of the present invention, the racemic enzyme is expressed in Escherichia coli BL21(DE3) using pET28a as the expression vector.

[0032] The sixth technical solution provided by this invention is a method for improving the activity of the racemic enzyme LbAAR, wherein the method involves mutating at least one site in the racemic enzyme LbAAR parental amino acid sequence as shown in SEQ ID NO.1, as described in (a) to (d):

[0033] (a) The leucine L at position 307 is mutated to serine S;

[0034] (b) The aspartic acid D at position 222 is mutated to histidine H;

[0035] (c) The proline P at position 304 is mutated to glutamate E, glutamine Q, aspartic acid D, or methionine M.

[0036] (d) The alanine A at position 54 is mutated to phenylalanine F.

[0037] In one embodiment of the present invention, the method comprises mutating the racemic enzyme LbAAR parent with the amino acid sequence as shown in SEQ ID NO.1 at any of the following sites:

[0038] (1) The leucine L at position 307 is mutated to serine S;

[0039] (2) Including the site in (1), and the aspartic acid D at position 222 is mutated to histidine H;

[0040] (3) Including the site in (2), and the proline P at position 304 is mutated to glutamate E, glutamine Q, aspartic acid D or methionine M.

[0041] (4) includes the site in (3), and the alanine A at position 54 is mutated to phenylalanine F.

[0042] The seventh technical solution provided by the present invention is a method for synthesizing racemic p-hydroxyphenylglycine, wherein the method uses L-p-hydroxyphenylglycine or D-p-hydroxyphenylglycine as a substrate and utilizes the mutant described in the first technical solution or the recombinant cells described in the fourth technical solution to produce racemic D / L-p-hydroxyphenylglycine.

[0043] In one embodiment of the present invention, the recombinant cells are expressed in Escherichia coli BL21(DE3) using pET28a as the expression vector, and the racemic enzyme mutant is expressed in the cells.

[0044] In one embodiment of the present invention, the induction temperature is 16–25°C.

[0045] In one embodiment of the present invention, the final concentration of the inducer is 0.25 to 0.50 mM.

[0046] The eighth technical solution provided by the present invention is the application of the mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the method described in the sixth technical solution, or the method described in the seventh technical solution in the preparation of racemic D / L-p-hydroxyphenylglycine or products containing racemic D / L-p-hydroxyphenylglycine.

[0047] Beneficial effects

[0048] This invention provides a racemic enzyme (or epimerase) LbAAR or its mutant for catalytic production of racemic D / L-p-hydroxyphenylglycine. This invention designs a method for the biosynthesis of racemic D / L-p-hydroxyphenylglycine using L-p-hydroxyphenylglycine and PLP as raw materials, utilizing the enhanced activity of the racemic enzyme (or epimerase) LbAAR mutant. The overall catalytic process is simple and efficient. In a 2 mL reaction system, reacting 20 g / L L-p-hydroxyphenylglycine for 1 h yields completely racemic p-hydroxyphenylglycine. Compared to wild-type LbAAR, which cannot catalyze the racemization of p-hydroxyphenylglycine, the LbAAR of this invention... A54F / D222H / P304E / L307S It exhibits high catalytic efficiency, with the catalytic activity of the pure enzyme increased to 165.1 U / mg. This invention provides a new synthetic method for the rapid synthesis of racemic D / L-p-hydroxyphenylglycine, offering a new approach for the industrial production of racemic p-hydroxyphenylglycine. Attached Figure Description

[0049] Figure 1 shows the SDS-PAGE image of recombinant racemic enzyme-induced expression; lane M refers to the low molecular weight protein marker.

[0050] Figure 2 is an HPLC chromatogram of the conversion of L-p-hydroxyphenylglycine to racemic p-hydroxyphenylglycine by enzyme LbAAR using cofactor PLP. A is D / L-p-hydroxyphenylglycine standard, and B is LbAAR reaction solution.

[0051] Figure 3 shows the relationship between the activities of different mutants on L-p-hydroxyphenylglycine. Detailed Implementation

[0052] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and Figures 1 to 3.

[0053] The sources of the genes and vectors involved in the following examples:

[0054] The LbAAR gene involved in this invention is derived from Lentilactobacillus buchneri (Lactobacillus buchneri). The pET28a(+) plasmid was purchased from Novagen (Madison, WI, USA). Restriction endonucleases, T4 DNA ligase, primeSTAR Max, etc., were purchased from TaKaRa (Dalian, China). Standards were purchased from Macklin Reagents (Shanghai, China). LbAAR mutants were all obtained through molecular modification, and all other reagents were purchased commercially.

[0055] The culture media involved in the following examples are as follows:

[0056] Prepare LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and sterilize at 121°C for 20 min.

[0057] Fermentation medium was prepared as follows: tryptone 12 g / L, yeast extract (Angel Yeast Powder 802) 24 g / L, glycerol 4 mL / L, KH2PO4 2.31 g / L and K2HPO4 12.31 g / L.

[0058] The detection methods involved in the following embodiments are as follows:

[0059] Determination of D-HPG content:

[0060] The reaction products were determined by high-performance liquid chromatography (HPLC). An Agilent InfinityLab Poroshell 120 Chiral-T column (4.6 × 150 mm) was used. Mobile phase A consisted of filtered and sonicated degassed methanol solution, and mobile phase B consisted of an aqueous solution containing 20 mM ammonium formate (pH 4.0) at a ratio of 1:9. The injection volume was 5 μL, the column temperature was 25 °C, the UV detector wavelength was 226 nm, the flow rate was 0.5 mL / min, and the sample processing time was 11 min. Under these conditions, the retention time of L-HPG was 3.9 min, and the retention time of D-HPG was 6.7 min.

[0061] Example 1: Screening of enzymes for preparing racemic HPG

[0062] The specific steps are as follows:

[0063] (1) Construction of recombinant strains:

[0064] The racemic enzyme LbAAR, with Uniprot number M1GRN3 and amino acid sequence as shown in SEQ ID NO.1, was synthesized. The corresponding gene fragment was ligated into the empty pET28a vector. The resulting recombinant vectors were introduced into the host cells E.coli BL21(DE3) to obtain recombinant bacteria: E.coli BL21(DE3) / pET28a-LbAAR.

[0065] (2) Detection of the activity of E. coli BL21(DE3) / pET28a-LbAAR against HPG

[0066] Reaction system: After expression and purification of the constructed recombinant bacteria (SDS-PAGE image shown in Figure 1), transformation experiments were performed under the conditions of 10 mL M L-HPG or 10 mM L-phenylglycine (PHG) and 0.5 mM PLP. The reaction temperature was 50℃, pH 7.0, and the reaction time was 30 min. After the reaction was completed, perchloric acid (pH 1.5) was added to terminate the reaction. The sample was filtered through an aqueous membrane and then detected by HPLC. Experimental results (Figure 2): No L-HPG activity was detected in E. coli BL21(DE3) / pET28a-LbAAR, but L-PHG activity was detected.

[0067] Example 2: Construction and screening of single mutants, double mutants, triple mutants, and quadruple mutants

[0068] The specific steps are as follows:

[0069] Design LbAAR L307S LbAAR D222H LbAAR P304E LbAAR A54F The primers for the mutation sites are shown in Table 1. Single mutants were constructed by whole plasmid PCR using the plasmid pET28a-LbAAR, which contains a racemic enzyme (SEQ ID NO.1) from Lentilactobacillus buchneri and was prepared in Example 1, as a template.

[0070] The double mutant in this embodiment is based on the corresponding single mutant LbAAR. L307S Based on the primers in Table 1, double mutants were constructed using whole plasmid PCR. For example, in the LbAAR mutant... L307S Based on this, double mutant LbAAR was detected by whole plasmid PCR using mutant primers D222H-F and D222H-R. L307S / D222H Build.

[0071] The triple mutant in this embodiment is based on the corresponding double mutant LbAAR. L307S / D222H Based on the primers in Table 1, triple mutants were constructed using whole plasmid PCR. For example, in the LbAAR mutant... L307S / D222H Based on this, using the mutation primers P304E-F and P304E-R, the triple mutant LbAAR was detected by whole plasmid PCR. L307S / D222H / P304E Build.

[0072] The quad mutant in this embodiment is based on the corresponding triple mutant LbAAR. L307S / D222H / P304E Based on the primers in Table 1, tetramutants were constructed using whole-plasmid PCR, for example, in the LbAAR mutant. L307S / D222H / P304EBased on this, using the mutant primers A54F-F and A54F-R, the four mutant LbAAR was detected by whole plasmid PCR. L307S / D222H / P304E / A54F Build.

[0073] Table 1. Mutant primer sequences

[0074]

[0075] SEQ ID NO.1:

[0076] MGKLDKASKLIDEENKYYARSARINYYNLVIDHAHGATLVDVDGNKYIDLLASASAINVGHTHEKVVKAIADQAQKLIHYTPAYFHHVPGMELSEKLAKIAPGNSPKMVSFG NSGSDANDAIIKFARAYTGRQYIVSYMGSYHGSTYGSQTLSGSSLNMTRKIGPMLPSVVHVPYPDSYRTYPGETEHDVSLRYFNEFKKPFESFLPADETACVLIEPIQGDGGI IKAPEEYMQLVYKFCHEHGILFAIDEVNQGLGRTGKMWAIQQFKDIEPDLMSVGKSLASGMPLSAVIGKKEVMQSLDAPAHLFTTAGNPVCSAASLATLDVIEYEGLVEKSA TDGAYAKQRFLEMQQRHPMIGDVRMWGLNGGIELVKDPKTKEPDSDAATKVIYYAFAHGVVIITLAGNILRFQPPLVIPREQLDQALQVLDDAFTAVENGEVTIPKDTGKIGW

[0077] Constructing the PCR amplification system: 12.5 μL of 2×PrimeSTAR Max, 0.2 μL each of the two primers for each mutation site, and template (LbAAR). WT 0.5 μL of water and 11.5 μL of water were added. The reaction conditions were: ① 94℃ for 3 min; ② 98℃ for 10 s; ③ 55℃ for 30 s; ④ 72℃ for 1 min and 20 s; ⑤ Repeat steps ② to ④ 30 times; ⑥ 72℃ for 5 min; ⑦ Keep warm at 12℃.

[0078] The above reaction system was incubated at 37°C for 40 min to digest the plasmid template (digestion system: DpnI 0.5 μL, PCR product from the above reaction 8.5 μL, 10×Buffer 1.0 μL). After digestion, the digestion product was introduced into E. coli BL21 competent cells by chemical transformation. The specific steps of the chemical transformation method are as follows:

[0079] (1) 10 μl of homologous recombination product was introduced into 100 μl of BL21 competent cells;

[0080] (2) Ice bath for 15-30 minutes;

[0081] (3) Heat shock in a 42℃ water bath for 90 seconds, then remove and quickly place in ice for 3-5 minutes;

[0082] (4) Add 800 μl of antibiotic-free LB medium and mix well. Incubate at 37°C and 200 rpm for 1 h.

[0083] (5) Centrifuge at 5000 rpm for 2 min to collect bacteria;

[0084] (6) Remove the supernatant, mix the remaining 100-200 μl of culture medium by blowing and aspirating, spread it onto a plate containing 0.05 mg / mL kanamycin resistance, and incubate at 37°C for about 12 h.

[0085] (7) Select a single clone and incubate it in LB containing 0.05 mg / mL kanamycin resistance. After incubation at 200 rpm and 37°C for 12 h, send it to the company for sequencing. The correct sequence is the positive transformant, which means that the genetically engineered bacteria containing the mutant is obtained.

[0086] Example 3: Screening for single mutants, double mutants, triple mutants, and quadruple mutants

[0087] The positive transformants of the mutant recombinant strain prepared in Example 2 were inoculated into LB medium and cultured at 37°C until OD600. 600 When the concentration was 0.6–0.8, 0.2 mM IPTG was added to induce expression. The induction temperature was 16 °C, the induction time was 20 h, and the cells were centrifuged at 8000 rpm for 15 min to obtain wet cells.

[0088] Screening conditions: 100 mM Hepes (pH 7.0) containing 60 mM L-HPG, 0.5 mM PLP, and 20 g / L of wet bacterial cells. The reaction was carried out at 37 °C for 20 min. The contents of L-HPG and D-HPG in the reaction system were measured. The enzyme activity of 1 unit of LbAAR was defined as the amount of wet bacterial cells (U) required to generate 1 μmol of D-HPG product per minute. The results are shown in Figure 3 and Table 2.

[0089] Table 2 Enzyme activities of strains containing LbAAR and some of its mutants

[0090]

[0091]

[0092] Example 4: Determination of kinetic parameters of parental enzyme and mutant

[0093] To evaluate the mutant, this example measured the LbAAR mutant from Example 2. L307S LbAAR L307S / D222H LbAAR L307S / D222H / P304E LbAAR L307S / D222H / P304E / A54F kinetic parameters at 37°C (LbAAR) WT No activity against the substrate L-HPG was detected.

[0094] The mutant LbAAR obtained in Example 2 was used respectively L307S LbAAR L307S / D222H LbAAR L307S / D222H / P304E LbAAR L307S / D222H / P304E / A54F Recombinant strain positive transformants were inoculated into LB medium and cultured at 37°C until OD500. 600 When the concentration is 0.6–0.8, 50 mM IPTG is added for expression at a final concentration. The induction temperature is 16 °C and the induction time is 16–18 h to obtain the fermentation broth.

[0095] Prepare purified enzyme solutions for the corresponding parental enzymes and mutants. First, using a constant flow pump at 4°C, flush the nickel ion affinity chromatography column with ultrapure water (approximately 6–12 column volumes). Then, equilibrate the column environment with 10 mL of binding buffer A. When the pH of the eluent at the bottom of the column matches that of the low-salt buffer pumped into the column (approximately 5 column volumes of buffer are required), add the obtained crude enzyme solution to the column. Wash with binding buffer A to reach baseline equilibration, then elute with elution buffer B (25 mM Tris-HCl, 250 mM NaCl, 500 mM imidazole). Collect and concentrate the eluent with the absorption peak, and determine the enzyme activity.

[0096] The kinetic parameters of the enzyme were determined by measuring the initial rate of the enzymatic reaction over 30 min at different substrate concentrations (0.5–40 mM). Pure enzyme reactions were used for kinetic measurements. The assay system consisted of Na₂HPO₄-KH₂PO₄ solutions (pH 7.5) containing different substrate concentrations (0.5–40 mM), 0.5 mM PLP, and different enzyme concentrations. K was calculated using nonlinear regression based on the Michaelis-Menten equation and Origin software. m value, k cat value and k cat / K m The values ​​are shown in Table 3.

[0097] Table 3 Kinetic parameters of PxAmpC parental enzyme and its mutants

[0098]

[0099] Example 5: Synthesis of racemic p-hydroxyphenylglycine

[0100] LbAAR prepared according to the method in Example 2 L307S / D222H / P304E / A54F The recombinant strain was used to prepare 15 g / L racemic p-hydroxyphenylglycine according to the method in Example 3. 20 g / L LbAAR L307S / D222H / P304E / A54F After reacting the wet bacterial cells for 20 min, the L-HPG:D-HPG ratio was 2.2:1; after reacting for 40 min, the L-HPG:D-HPG ratio was 1.2:1, which is close to racemization; after reacting for 1 h, completely racemic p-hydroxyphenylglycine can be obtained.

[0101] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A racemic enzyme LbAAR mutant, characterized in that, The mutant is any one of the following (1) to (7): (1) obtained by mutating leucine at position 307 of the racemic enzyme LbAAR parent with the amino acid sequence as shown in SEQ ID NO.1 to serine, and the mutant is named L307S; (2) obtained by mutating leucine at position 307 of the racemic enzyme LbAAR parent with the amino acid sequence as shown in SEQ ID NO.1 to serine, and simultaneously mutating aspartic acid at position 222 to histidine, and the mutant is named L307S / D222H; (3) obtained by mutating leucine at position 307 of the racemic enzyme LbAAR parent with the amino acid sequence as shown in SEQ ID NO.1 to serine, mutating aspartic acid at position 222 to histidine, and simultaneously mutating proline at position 304 to glutamic acid, and the mutant is named L307S / D222H / P304E; (4) obtained by mutating leucine at position 307 of the racemic enzyme LbAAR parent with the amino acid sequence as shown in SEQ ID NO.1 to serine, mutating aspartic acid at position 222 to histidine, and simultaneously mutating proline at position 304 to glutamic acid, and the mutant is named L307S / D222H / P304E; The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to glutamine in the racemic enzyme parent shown in SEQ ID NO.1 is named L307S / D222H / P304Q; (5) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to aspartic acid in the racemic enzyme parent shown in SEQ ID NO.1 is named L307S / D222H / P304D; (6) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to aspartic acid in the racemic enzyme parent shown in SEQ ID NO.1 is named L307S / D222H / P304D; The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, and proline at position 304 to methionine in the racemic enzyme parent shown in SEQ ID NO.1 is named L307S / D222H / P304M; (7) The mutant obtained by mutating leucine at position 307 to serine, aspartic acid at position 222 to histidine, proline at position 304 to glutamic acid, and alanine at position 54 to phenylalanine in the racemic enzyme parent shown in SEQ ID NO.1 is named L307S / D222H / P304E / A54F.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant cells expressing the mutant of claim 1, carrying the gene of claim 2, or transformed with the recombinant vector of claim 3.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells use bacteria or fungi as expression hosts.

6. A method for increasing the activity of the racemic enzyme LbAAR, characterized in that, The method involves mutating at least one of the following sites in the racemic enzyme LbAAR parent, whose amino acid sequence is shown in SEQ ID NO. 1: (a) mutating leucine L at position 307 to serine S; (b) mutating leucine L at position 307 to serine S, and aspartic acid D at position 222 to histidine H; (c) mutating leucine L at position 307 to serine S, aspartic acid D at position 222 to histidine H, and proline P at position 304 to glutamate E; (d) mutating leucine L at position 307 to serine S, aspartic acid D at position 222 to histidine H, and proline P at position 304 to glutamate E. (e) Leucine at position 307 is mutated to serine (S), aspartic acid at position 222 is mutated to histidine (H), and proline at position 304 is mutated to aspartic acid (D); (f) Leucine at position 307 is mutated to serine (S), aspartic acid at position 222 is mutated to histidine (H), and proline at position 304 is mutated to methionine (M); (g) Leucine at position 307 is mutated to serine (S), aspartic acid at position 222 is mutated to histidine (H), proline at position 304 is mutated to glutamic acid (E), and alanine at position 54 is mutated to phenylalanine (F).

7. A method for synthesizing racemic p-hydroxyphenylglycine, characterized in that, The method involves using L-p-hydroxyphenylglycine as a substrate and employing the mutant described in claim 1 or the recombinant cells described in any one of claims 4-5 to produce racemic D-p-hydroxyphenylglycine.

8. The method according to claim 7, characterized in that, The recombinant cells were expressed using pET28a as the expression vector in Escherichia coli BL21(DE3) to express the racemic enzyme mutant.

9. The use of the mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant cell of any one of claims 4-5, or the method of any one of claims 6-8 in the preparation of racemic D-p-hydroxyphenylglycine or a product containing racemic D-p-hydroxyphenylglycine.

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