Fusion enzyme L-2FL-D for directionally catalyzing conversion of D-chlorophenylglycine into L-chlorophenylglycine as well as preparation method and application of fusion enzyme L-2FL-D

Abstract

The invention provides a fusion enzyme L-2FL-D for directionally catalyzing conversion of D-chlorophenylglycine into L-chlorophenylglycine as well as a preparation method and application of the fusion enzyme L-2FL-D, and belongs to the technical field of enzyme engineering. A seamless cloning technology is used for carrying out homologous recombination on a linearized vector and an insertion fragment to obtain a fusion enzyme L-2FL-D prokaryotic expression vector. And expressing the fusion enzyme L-2FL-D expression vector in a host cell to obtain the fusion enzyme L-2FL-D. According to the method, D-chlorophenylglycine and L-sodium glutamate are taken as substrates, under the action of coenzyme, the directional conversion effect of D-chlorophenylglycine to L-chlorophenylglycine is very remarkable, the substrate conversion rate reaches 98%, and the method has extremely high application value in the aspect of production of L-chlorophenylglycine.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a fusion enzyme L-2FL-D for directionally catalyzing the conversion of D-o-chlorophenylglycine into L-o-chlorophenylglycine, a preparation method thereof, and an application thereof. Background Art

[0002] The phenylglycine derivative o-chlorophenylglycine, with the molecular formula C8H8NO2Cl and a relative molecular mass of 185.61, has two enantiomeric structures, D-type and L-type, in its structure. It is an important derivative of phenylglycine and has a wide range of applications in the synthesis of pharmaceutical intermediates. In particular, L-o-chlorophenylglycine is an important intermediate for the synthesis of many drugs, such as an important intermediate for the anti-thrombotic drug clopidogrel for treating arteriosclerosis. As a non-natural amino acid with biological activity, L-o-chlorophenylglycine is mainly applied to the research and development of anti-thrombotic drugs such as clopidogrel, anti-cancer drugs such as paclitaxel, and various antibiotic drugs. In particular, clopidogrel, as an antiplatelet aggregation drug, is widely used for treating atherosclerosis, ischemic stroke, and preventing thrombosis caused by coronary artery stent implantation. With the development of drug production, the demand for L-o-chlorophenylglycine has increased significantly. Therefore, it is very important to develop L-o-chlorophenylglycine production technology to meet the market demand.

[0003] At present, the preparation of o-chlorophenylglycine mainly includes chemical methods and bioenzymatic methods. The chemical method first synthesizes and then resolves. However, chemical asymmetric synthesis uses expensive chiral sources, chiral auxiliaries, or chiral catalysts, and the chemical resolution process route is complex and causes large environmental pollution. Whether it is chemical asymmetric synthesis or chemical resolution, there are disadvantages such as low yield, low optical purity, and high production cost. The disadvantages of low yield, low optical purity, and high production cost. The biocatalytic method has characteristics such as high stereoselectivity, mild reaction conditions, and environmental friendliness, and is a very useful method for preparing enantiopure non-natural L-amino acids. The biocatalytic method includes enzyme resolution and enzyme-catalyzed asymmetric synthesis. Enzymatic kinetic resolution of racemates is one of the most successful strategies for preparing non-natural L-amino acids, but it involves the derivation of racemic amino acids and the separation of free amino acids and derived amino acids, and the process is complex. The theoretical yield of non-natural L-amino acids is only 50%. Therefore, in order to avoid the waste of D-enantiomers, it is necessary to develop an enzyme-catalyzed or chemoenzymatic synthesis method for non-natural L-amino acids with high yield. Therefore, a detailed study of the directional conversion of D-o-chlorophenylglycine by fusion enzymes is carried out to improve the synthesis efficiency and save production costs. Through the application of synthetic biology technology, the production breadth of non-natural amino acids has been significantly improved, providing important theoretical support for the development of new production technologies and having important significance for the industrial development of the biocatalytic synthesis of L-o-chlorophenylglycine. Summary of the Invention

[0004] In view of this, the present invention provides a fusion enzyme L-2FL-D for directionally catalyzing the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine, its preparation method and application, so as to improve the synthesis efficiency of L-o-chlorophenylglycine.

[0005] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] The present invention provides a preparation method of a fusion enzyme L-2FL-D for directionally catalyzing the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine, which is characterized by including the following steps:

[0007] S1. Using the pET28a-DAAO-6×His plasmid as a template and the primer pair F / R as primers for amplification to obtain an inserted fragment;

[0008] S2. Using the pET28a-LAspAT-6×His plasmid as a template and the vector primers V-F / V-R for amplification to obtain a linearized cloning vector;

[0009] S3. Performing homologous recombination on the linearized vector and the inserted fragment to obtain a prokaryotic expression vector of the fusion enzyme L-2FL-D;

[0010] S4. Expressing the prokaryotic expression vector of the fusion enzyme L-2FL-D in a host cell to obtain the fusion enzyme L-2FL-D for directionally catalyzing the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine;

[0011] The nucleotide sequence of the fusion enzyme L-2FL-D for directionally catalyzing the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine is as shown in SEQ ID NO.10.

[0012] Preferably, the nucleotide sequence of the linearized primer V-F is as shown in SEQ ID NO.5, and the nucleotide sequence of the linearized primer V-R is as shown in SEQ ID NO.6.

[0013] Preferably, the nucleotide sequence of the upstream primer F is as shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer R is as shown in SEQ ID NO.8.

[0014] Preferably, the reaction system for amplifying the linearized vector and the target gene fragment is: Fly Mix(2×) 25 μL, forward primer 1 μL, reverse primer 1 μL, plasmid template 1 μL, and ddH2O is added to make up the volume to 50 μL.

[0015] Preferably, the amplification program for amplifying the linearized vector and the target gene fragment is: 98 °C, 30 s, 65 °C, 20 s, 72 °C, 30 s; extension at 72 °C for 1 min, 4 °C, 60 min.

[0016] Preferably, the molar ratio of the linearized vector to the target gene fragment is 1:2.

[0017] Preferably, the expression conditions are: culturing at 34 - 36 °C and 200 - 240 rpm until the OD 600 reaches 0.8 - 1.2, adding IPTG with a final concentration of 60 - 65 μg / mL, and inducing culture at 24 - 26 °C for 7 - 9 h.

[0018] The present invention also provides the fusion enzyme L-2FL-D obtained by the preparation method described above, and the nucleotide sequence of the fusion enzyme L-2FL-D is as shown in SEQ ID NO.9.

[0019] The present invention also provides the application of the fusion enzyme L-2FL-D in the directional catalysis of the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine.

[0020] By adopting the above technical solutions, the present invention has the following beneficial effects: The present invention conducts in-vivo end-to-end gene fusion of the existing L-aspartate aminotransferase and D-amino acid oxidase, induces under the optimal expression conditions, and finally, under the catalysis of the fusion enzyme, using D-o-chlorophenylglycine and sodium L-glutamate as substrates, and under the action of coenzymes, the directional conversion effect of D-o-chlorophenylglycine to L-o-chlorophenylglycine is very significant, with a substrate conversion rate reaching 98%. The fusion enzyme L-2FL-D has the potential for large-scale production of L-o-chlorophenylglycine and has extremely high application value. Description of the Drawings

[0021] Figure 1 It is a schematic diagram of homologous recombination of the fusion enzyme recombinant vector PET28a-LAspAT-2FL-DAAO-6×His and a gene map of the fusion enzyme recombinant vector PET28a-LAspAT-2FL-DAAO-6×His.

[0022] Figure 2 It is an electrophoresis diagram of the fusion enzyme recombinant vector PET28a-LAspAT-2FL-DAAO-6×His.

[0023] Figure 3 It is a gene map of the fusion enzyme recombinant vector PET28a-LAspAT-2FL-DAAO-6×His.

[0024] Figure 4 It is an SDS-PAGE result diagram of the fusion enzyme L-2FL-D.

[0025] Figure 5 It is a standard curve of L-o-chlorophenylglycine.

[0026] Figure 6It is the liquid phase result diagram of D- and L-o-chlorophenylglycine reference standards.

[0027] Figure 7 It is the chiral distribution diagram of o-chlorophenylglycine after 72 h of fusion enzyme reaction.

[0028] Figure 8 It is the comparison diagram of the catalytic effects of the fusion enzyme and the free enzyme.

[0029] Figure 9 It is the reaction schematic diagram of the free enzyme and the fusion enzyme. Detailed implementation mode

[0030] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0031] The reagents and reagent kits in the embodiments of the present invention are shown in Table 1.

[0032] Table 1 Reagents and reagent kits used in the embodiments

[0033]

[0034]

[0035] Example 1. Construction of the prokaryotic expression vector of the fusion enzyme L-2FL-D

[0036] The amino acid sequences of L-aspartate aminotransferase and D-amino acid oxidase are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. The nucleotide sequences encoding L-aspartate aminotransferase and D-amino acid oxidase are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0037] SEQ ID NO.1:

[0038] MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKTPVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKGSALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSNPSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASVVATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGANRDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVAGMTPDNMAPLCEAIVAVL.

[0039] SEQ ID NO.2:

[0040] MAKIVVIGAGVAGLTTALQLLRKGHEVTIVSEFTPGDLSIGYTSPWAGANWLTFYDGGKLADYDAVSYPILRELARSSPEAGIRLINQRSHVLKRDLPKLEGAMSAICQRNPWFKNTVDSFEIIEDRSRIVHDDEAYLVEFRSVCIHTGVYLNWLMSQCLSLGATVVKRRVNHIKDANLLHSSGSRPDVIVNCSGLFARFLGGVEDKKMYPIRGQVVLVRNSLPFMASFSSTPEKENEDEALYIMTRFDGTSIIGGCFQPNNWSSEPDPSLTHRILSRALDRFPELTKDGPLDIVRECVGHRPGREGGPRVELEKIPGVGFVVHNYGAAGAGYQSSYGMADEAVSYVERALTRPNLLE.

[0041] SEQ ID NO.3:

[0042]

[0043] SEQ ID NO.4:

[0044]

[0045] In the present invention, the pET28a-LAspAT-6×His plasmid carrying the LAspAT gene and the pET28a-DAAO-6×His plasmid carrying the DAAO gene are stored in the laboratory. The DAAO and L-AspAT genes were respectively inserted to obtain the pET28a vector. Use FastPfuFly DNA Polymerase high-fidelity enzyme (TransGen Biotech Co., Ltd., Beijing) for PCR amplification of the linearized vector and the inserted fragment.

[0046] (1) PCR amplification of the linearized vector

[0047] Using the pET28a-LAspAT-6×His plasmid as a template, amplify with the vector forward primer V-F and the vector reverse primer V-R to obtain the linearized vector.

[0048] Vector forward primer V-F: GCTCGAGGGAGGAGGAGGATCCCACCACCACCAC (SEQ ID NO.5);

[0049] Vector reverse primer V-R: CCTCCTCCCAGCACTGCCACAATCGCTT (SEQ ID NO.6).

[0050] The PCR reaction system is: Fly Mix (2×) 25 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, plasmid template 1 μL, add ddH2O to make up the volume to 50 μL.

[0051] Amplification program: 98°C, 1 min for high-temperature denaturation; 30 cycles: 98°C, 30 s, 65°C, 20 s, 72°C, 30 s; 72°C extension for 1 min, 4°C, 60 min.

[0052] (2) PCR amplification of the inserted fragment

[0053] Using the pET28a-DAAO-6×His plasmid carrying the DAAO gene as a template, amplify with the primer pair F / R to obtain the inserted fragment.

[0054] Forward primer F (SEQ ID NO.7) for amplifying the inserted fragment:

[0055] AGTGCTGGGAGGAGGAGGATCCGGAG;

[0056] Reverse primer R (SEQ ID NO.8) for amplifying the inserted fragment:

[0057] CCTCCTCCCTCGAGCAGGTTCGGGCGGGTCAGCGCGCGTT;

[0058] The amplification system and amplification procedure are the same as those in step (1).

[0059] (3) Confirm the size of the target fragment by agarose gel electrophoresis

[0060] After the PCR run, the size of the sample bands is detected by agarose gel electrophoresis. The specific steps are as follows: Weigh 0.8% agarose powder and heat it in 20 mL of TAE buffer until the powder is completely dissolved. After cooling to 55 °C, add 0.1 μL of Gel Red dye (10000×), shake well and pour it into the gel plate. Insert the comb and wait for the gel to solidify before use.

[0061] Mix the PCR product with 10×DNA Loading Buffer and then use a pipette to add it to the agarose gel sample wells. Run at a constant voltage of 120 V for 25 - 30 min, and determine the size of the target band by comparing the image with the Maker bands through a gel imager.

[0062] (5) DNA purification

[0063] After the linearized vector and the target gene are amplified by PCR and identified by agarose gel electrophoresis, use DMT enzyme to digest at 37 °C for 1 h to remove the original template. Then purify the digestion product with reference to the operation instructions of the TransGen purification kit.

[0064] (5) Concentration determination and homologous recombination ligation

[0065] After the linearized vector and the target gene are purified, their concentrations are measured by a Nano Drop spectrophotometer. The linearized vector and the target gene fragment are in a molar ratio of 1:2, and using the homologous recombination kit SeamlessCloning andAssembly Kit, perform homologous recombination of the target gene and the linearized vector according to the instructions to obtain the fusion enzyme recombinant vector PET28a-LAspAT-Linker-DAA0-6xHis. The electrophoresis pattern of the fusion enzyme recombinant vector PET28a-LAspAT-2FL-DAAO-6×His is as Figure 2 shown.

[0066] (6) Chemical transformation

[0067] The recombinant product was transformed into Trans1-1 competent cells by chemical transformation method: ice bath for 30 min, heat shock at 42 °C for 45 s, ice bath for 3 min, add 500 μL of SOC medium, culture at 37 °C and 220 rpm for 1 h. Centrifuge at 5000 rpm / min for 3 min to collect the bacterial cells and resuspend the cells with 50 μL of the supernatant. Use a pipette to take out and evenly spread it onto the kanamycin-resistant (final concentration 50 μg / mL) LB solid medium, and incubate it upside down at 37 °C for 12 h. Pick colonies and inoculate them into 50 mL of kanamycin-resistant (final concentration 50 μg / mL) LB liquid medium, and culture at 37 °C for 12 h. After plasmid miniprep, the bacterial solution was sent to a biological company for nucleotide sequence detection. After comparison, it was determined to be a fusion enzyme strain. The amino acid sequence of the fusion enzyme L-2FL-D is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.7.

[0068] SEQ ID NO.9:

[0069] MFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKTPVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKGSALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSNPSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASVVATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGANRDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVAGMTPDNMAPLCEAIVAVLGGGGSGGGGSMAKIVVIGAGVAGLTTALQLLRKGHEVTIVSEFTPGDLSIGYTSPWAGANWLTFYDGGKLADYDAVSYPILRELARSSPEAGIRLINQRSHVLKRDLPKLEGAMSAICQRNPWFKNTVDSFEIIEDRSRIVHDDEAYLVEFRSVCIHTGVYLNWLMSQCLSLGATVVKRRVNHIKDANLLHSSGSRPDVIVNCSGLFARFLGGVEDKKMYPIRGQVVLVRNSLPFMASFSSTPEKENEDEALYIMTRFDGTSIIGGCFQPNNWSSEPDPSLTHRILSRALDRFPELTKDGPLDIVRECVGHRPGREGGPRVELEKIPGVGFVVHNYGAAGAGYQSSYGMADEAVSYVERALTRPNLLEHHHHHH*.

[0070] SEQ ID NO.10:

[0071]

[0072] (7) Identification and sequencing of positive monoclonal antibodies

[0073] Detection of positive monoclonal antibodies: Pick white independent monoclonal antibodies into a 1.5 mL EP tube, add 500 μL of LB medium containing Amp, and culture at 37 °C for 6 - 8 h. Use it as a template for PCR identification of positive antibodies.

[0074] Plasmid extraction: The positive monoclonal bacterial solution identified by PCR is re - transferred into LB medium containing Amp, and the plasmid is extracted using a plasmid extraction kit. The plasmid is sent to a biological company for sequencing and sequence analysis. The plasmid with correct sequencing is the prokaryotic expression vector of the fusion enzyme L - 2FL - D, and its gene map is as Figure 3 shown.

[0075] Example 2. Chemical transformation of Escherichia coli Rosetta(DE3) competent cells

[0076] The plasmid with correct sequencing is transferred into Rosetta(DE3) competent cells by chemical transformation method. Ice - bath for 30 min, heat - shock at 42 °C for 45 s, ice - bath for 3 min, add 500 μL of SOC medium, and culture at 37 °C and 220 rpm for 1 h. Centrifuge at 5000 rpm / min for 3 min to collect the bacteria, and resuspend the bacteria with 50 μL of the supernatant. Use a pipette to take out and evenly coat it on LB solid medium with kanamycin resistance (final concentration 50 μg / mL), and culture it inverted at 37 °C for 12 h. Pick colonies and inoculate them into 50 mL of LB liquid medium with kanamycin resistance (final concentration 50 μg / mL), culture at 37 °C for 12 h, and pick monoclonal antibodies in the culture dish to activate them into seed liquid for protein fermentation.

[0077] Example 3. Expression of recombinant enzyme and determination of enzyme activity

[0078] The plasmid of L - 2FL - D obtained in Example 2 is inoculated into LB liquid medium with kanamycin resistance at a final concentration of 50 μg / mL by chemical transformation method, and cultured at 35 °C and 220 rpm until the OD 600 reaches 1. Add IPTG at a final concentration of 62.5 μg / mL, and induce culture at 25 °C for 8 h. Centrifuge at 4 °C and 8000 rpm / min for 15 min, discard the supernatant, and collect the precipitate to obtain wet Escherichia coli bacteria containing the recombinant L - aspartate aminotransferase mutant gene.

[0079] The bacterial cells were resuspended using a pH = 8, 100 mM Tris-HCl buffer (1 g corresponding to 5 mL of buffer), washed, and then centrifuged again at 4°C and 8000 rpm / min for 15 min using a low-temperature ultra-high-speed centrifuge. The precipitate was collected, resuspended with the buffer again, and purified. Since a His tag was added during vector construction, affinity chromatography using a Ni 2+ -IDA column was used for protein purification.

[0080] Purification of the recombinant vector protein using a nickel column:

[0081] (1) First, the crude enzyme solution after disruption and centrifugation was filtered using a 0.22 μm pore size filter membrane to filter out larger miscellaneous proteins and cell debris in the crude enzyme solution;

[0082] (2) The filtrate was collected and mixed with the nickel column packing, and incubated at 10°C and 180 rpm for 6 h to allow the target protein to fully bind to the nickel column material;

[0083] (3) The mixture was backfilled into the nickel column and the effluent was collected, and eluted using a pH = 8.0, 100 mM Tris-HCl buffer;

[0084] (4) The protein was eluted and separated using imidazole solutions of different concentrations. The eluents were composed of 20, 40, 80, 120, 160, and 200 mM imidazole dissolved in a pH = 8.0, 100 mM Tris-HCl buffer.

[0085] (5) The effluent was collected as the fused enzyme solution.

[0086] The results of protein purification were analyzed by SDS-PAGE electrophoresis. The results were as Figure 4 shown. The predicted molecular weight of the fused enzyme L-2FL-D was 84.1 kDa, which Figure 4 matched the band in the protein electrophoresis result. Therefore, the purified protein was determined to be the fused enzyme L-2FL-D.

[0087] Determination of the activity of the free enzyme and the fused enzyme: Weigh 20 mM D-phenylglycine and add 1 mL of the L-2FL-D fused enzyme solution and the D-amino acid oxidase solution respectively. React in a shaker at 35°C and 220 rpm / min for 1 h. Pipette 500 μL of the reaction solution and heat it in a boiling water bath for 5 min to terminate the reaction. After the sample was cooled, it was placed in a high-speed centrifuge and centrifuged at 10000 g for 3 min. Use a pipette to aspirate the supernatant and filter it through a 0.22 μm water membrane as the liquid phase detection sample. The concentration of benzoylformic acid was detected by high-performance liquid chromatography.

[0088] Method for detecting 2-chlorophenoxyacetic acid by ultraviolet high performance liquid chromatography: Use an ultraviolet 2998 detector and an organic acid chromatographic column to detect the peak emergence time and peak area of 2-chlorophenoxyacetic acid at a wavelength of 214 nm, and calculate the corresponding concentration according to the standard curve of 2-chlorophenoxyacetic acid. The mobile phase is an aqueous solution of 5 mmol H2SO4 with a pH of 2.0 - 2.2, and the flow rate is 0.6 mL / min.

[0089] Method for detecting D-2-chlorophenylglycine and L-phenylglycine: Use an ultraviolet high performance liquid chromatography 2998 detector, and the chromatographic column is a chiral chromatographic column. Detect the chiral distribution and peak area of 2-chlorophenylglycine at a wavelength of 210 nm, and calculate the corresponding concentration through the standard curve of 2-chlorophenylglycine. The liquid phase conditions are pH = 1.5 perchloric acid buffer (v): acetonitrile (v) = 80:20, and the flow rate is 0.3 mL / min.

[0090] Enzyme activity definition: Under the reaction conditions of 37 °C, the amount of enzyme required to catalyze the reaction of 20 mmol / D-2-chlorophenylglycine to produce 1 μmol of 2-chlorophenoxyacetic acid per 1 h is one enzyme activity unit (U).

[0091] The enzyme activities of free enzyme D-amino acid oxidase and fusion enzyme L-2FL-D are shown in Table 2.

[0092] Table 2 Enzyme activities of D-amino acid oxidase and fusion enzyme L-2FL-D

[0093] Type of enzyme Enzyme activity (U / mL) Free enzyme 8 Fusion enzyme L-2FL-D 40

[0094] It was determined that the enzyme activity of fusion enzyme L-2FL-D is 5 times that of the free enzyme, which is significantly higher than that of the free enzyme, indicating that fusion enzyme L-2FL-D can improve the synthesis efficiency of L-2-chlorophenylglycine.

[0095] Example 45. Preparation of L-2-chlorophenylglycine by catalytic reaction of fusion enzyme L-2FL-D on D-2-chlorophenylglycine

[0096] The recombinant expression vector of fusion enzyme L-2FL-D in Example 2 was induced and expressed according to the methods in Examples 2 and 3 to obtain a crude enzyme solution of recombinant fusion enzyme, which was used as a biocatalyst. Using the racemic mixture of D-2-chlorophenylglycine and L-glutamic acid as substrates, and under the action of coenzyme PLP, L-2-chlorophenylglycine was prepared by biotransformation reaction.

[0097] The catalytic system and catalytic conditions are as follows: In a 5 mL reaction system, 1 mL containing 20 mmol / L D-o-chlorophenylglycine and 40 mmol / L L-glutamic acid is prepared using Tris-Hcl buffer solution with pH = 8. 2.5 mmol coenzyme PLP and 4 mL of the crude enzyme solution prepared in Example 3 are added, and the reaction is carried out in a shaker at 35 °C and 220 rpm. Samples are taken at the 0 h, 24 h, 48 h, and 72 h of the reaction respectively, and the formation of L-o-chlorophenylglycine and the conversion of D-o-chlorophenylglycine are determined by HPLC. The enantiomeric excess percentage during the reaction is shown in Table 3 and Figure 7 , Figure 8 as shown.

[0098] Table 3 Changes in enantiomeric excess percentage during the reaction

[0099] Reaction time (h) ee value of the fusion enzyme catalysis system ee value of the free enzyme catalysis system 0 0％ 0％ 24 61％ 28％ 48 84％ 55％ 72 97％ 72％

[0100] The results show that after 72 h of the catalytic reaction, the conversion rate of D-o-chlorophenylglycine reaches 97%, and the enantiomeric excess percentage reaches 98%, indicating that the fusion enzyme L-2FL-D can catalyze the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine.

[0101] As can be seen from the above examples, the present invention provides a fusion enzyme L-2FL-D for the directional catalysis of the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine, its preparation method and application, which can improve the synthesis efficiency of L-o-chlorophenylglycine.

[0102] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing the fusion enzyme L-2FL-D that directionally catalyzes the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine, characterized in that, Including the following steps: S1. Using the pET28a-DAAO-6×His plasmid as a template, and using the primer pair F / R as primers for amplification to obtain an inserted fragment; S2. Using the pET28a-LAspAT-6×His plasmid as a template, and using the vector primers V-F / V-R for amplification to obtain a linearized cloning vector; S3. Performing homologous recombination on the linearized vector and the inserted fragment to obtain a prokaryotic expression vector of the fusion enzyme L-2FL-D; S4. Expressing the prokaryotic expression vector of the fusion enzyme L-2FL-D in a host cell to obtain the fusion enzyme L-2FL-D that directionally catalyzes the conversion of D-2-chlorophenylglycine to L-2-chlorophenylglycine; The nucleotide sequence of the fusion enzyme L-2FL-D that directionally catalyzes the conversion of D-2-chlorophenylglycine to L-2-chlorophenylglycine is shown in SEQ ID NO.

10.

2. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the linearized primer V-F is shown in SEQ ID NO.5, and the nucleotide sequence of the linearized primer V-R is shown in SEQ ID NO.

6.

3. The preparation method according to claim 1, wherein The nucleotide sequence of the upstream primer F is shown in SEQ IDNO.7, and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO.

8.

4. The preparation method according to claim 3, characterized in that, The reaction system for amplifying the linearized vector and the target gene fragment is: FlyMix(2×) 25 μL, forward primer 1 μL, reverse primer 1 μL, plasmid template 1 μL, and ddH2O is added to make the volume up to 50 μL.

5. The preparation method according to claim 4, characterized in that, The amplification program for amplifying the linearized vector and the target gene fragment is: 98 °C, 30 s, 65 °C, 20 s, 72 °C, 30 s; extension at 72 °C for 1 min, 4 °C, 60 min.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the linearized vector to the target gene fragment is 1:

2.

7. The preparation method according to claim 6, wherein The conditions for the expression are: culturing at 34 - 36°C and 200 - 240 rpm until the OD 600 reaches 0.8 - 1.2, adding IPTG with a final concentration of 60 - 65 μg / mL, and inducing culture at 24 - 26°C for 7 - 9 h.

8. The fusion enzyme L-2FL-D obtained by the preparation method according to any one of claims 1 to 7, characterized in that, The nucleotide sequence of the fusion enzyme L-2FL-D is shown in SEQ ID NO.

9.

9. Use of the fusion enzyme L-2FL-D according to claim 8 in the directional catalysis of the conversion of D-2-chlorophenylglycine to L-2-chlorophenylglycine.

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