Fusion enzyme l-2fl-d for stereoselectively catalyzing conversion of d-o-chlorophenylglycine into l-o-chlorophenylglycine, and preparation method and application thereof

Abstract

The application provides a fusion enzyme L-2FL-D for directionally catalyzing conversion of D-ortho-chlorophenylglycine into L-ortho-chlorophenylglycine, and a preparation method and application thereof, and belongs to the technical field of enzyme engineering. The application uses seamless cloning technology to perform homologous recombination on a linearized vector and an insertion fragment, so as to obtain a prokaryotic expression vector of the fusion enzyme L-2FL-D. The fusion enzyme L-2FL-D expression vector is expressed in a host cell, so as to obtain the fusion enzyme L-2FL-D. The application takes D-ortho-chlorophenylglycine and L-glutamic acid sodium salt as substrates, and under the action of coenzyme, the directionally catalyzing conversion effect of D-ortho-chlorophenylglycine into L-ortho-chlorophenylglycine is very remarkable, the substrate conversion rate reaches 98 %, and the application has extremely high application value in production of L-ortho-chlorophenylglycine.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to the fusion enzyme L-2FL-D, which catalyzes the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine, its preparation method, and its applications. Background Technology

[0002] L-chlorophenylglycine, a phenylglycine derivative, has the molecular formula C8H8NO2Cl and a relative molecular mass of 185.61. Structurally, it has both D- and L-enantiomers. It is an important derivative of phenylglycine and has wide applications in the synthesis of pharmaceutical intermediates. In particular, L-chlorophenylglycine is an important intermediate in the synthesis of many drugs, such as clopidogrel, an antithrombotic drug used to treat arteriosclerosis. As a biologically active non-natural amino acid, L-chlorophenylglycine is mainly used in the research and development of antithrombotic drugs such as clopidogrel, anticancer drugs such as paclitaxel, and various antibiotics. Clopidogrel, in particular, as an antiplatelet aggregation drug, is widely used to treat arteriosclerosis, ischemic stroke, and to prevent thrombosis after coronary artery stenting. With the development of pharmaceutical production, the demand for L-chlorophenylglycine has increased significantly; therefore, developing L-chlorophenylglycine production technologies to meet market demand is crucial.

[0003] Currently, the preparation of o-chlorophenylglycine mainly involves chemical and enzymatic methods. Chemical methods involve synthesis followed by resolution, but chemical asymmetric synthesis requires expensive chiral sources, chiral auxiliaries, or chiral catalysts, while chemical resolution is complex and environmentally polluting. Both chemical asymmetric synthesis and chemical resolution suffer from low yields, low optical purity, and high production costs. Biocatalytic methods, characterized by high stereoselectivity, mild reaction conditions, and environmental friendliness, are extremely useful for preparing enantiomeric non-natural L-amino acids. Biocatalytic methods include enzymatic resolution and enzymatically catalytic asymmetric synthesis. Enzymatic kinetic resolution of racemic amino acids is one of the most successful strategies for preparing non-natural L-amino acids, but it involves the derivatization of racemic amino acids and the separation of free and derived amino acids, making the process complex, and the theoretical yield of non-natural L-amino acids is only 50%. Therefore, to avoid wasting D-enantiomers, it is necessary to develop high-yield enzymatic or chemoenzymatic synthetic methods for non-natural L-amino acids. Therefore, a detailed study was conducted on the directed conversion of D-o-chlorophenylglycine using fusion enzymes to improve synthesis efficiency and reduce production costs. The application of synthetic biology techniques has significantly broadened the production scope of non-natural amino acids, providing important theoretical support for the development of novel production technologies and holding great significance for the industrialization of L-o-chlorophenylglycine synthesis via enzymatic methods. Summary of the Invention

[0004] In view of this, the present invention provides a fusion enzyme L-2FL-D for the directed catalytic 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] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing the fusion enzyme L-2FL-D, which catalyzes the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine, characterized by comprising the following steps:

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

[0008] S2. Using pET28a-LAspAT-6×His plasmid as a template, amplification was performed using vector primers VF / VR to obtain a linearized cloning vector;

[0009] S3. Homologous recombination of the linearized vector and the inserted fragment was performed to obtain the prokaryotic expression vector of the fusion enzyme L-2FL-D;

[0010] S4. Express the prokaryotic expression vector of fusion enzyme L-2FL-D in host cells to obtain the fusion enzyme L-2FL-D that directionally catalyzes the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine;

[0011] The nucleotide sequence of the fusion enzyme L-2FL-D, which catalyzes the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine, is shown in SEQ ID NO.10.

[0012] Preferably, the nucleotide sequence of the linearized primer VF is shown in SEQ ID NO.5, and the nucleotide sequence of the linearized primer VR is shown in SEQ ID NO.6.

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

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

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

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

[0017] Preferably, the expression conditions are: incubation at 34–36°C and 200–240 rpm until OD. 600 The concentration of the solution was 0.8–1.2, and IPTG was added to a final concentration of 60–65 μg / mL. The mixture was then induced and cultured at 24–26 °C for 7–9 h.

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

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

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

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

[0022] Figure 2 Electrophoresis image of the recombinant vector PET28a-LAspAT-2FL-DAAO-6×His, which is a fusion enzyme.

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

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

[0025] Figure 5 This is the standard curve for L-o-chlorophenylglycine.

[0026] Figure 6The liquid chromatography results are for D- and L-type o-chlorophenylglycine standards.

[0027] Figure 7 The chiral distribution of o-chlorophenylglycine after 72 hours of fusion enzyme reaction.

[0028] Figure 8 This is a comparison of the catalytic effects of fusion enzymes and free enzymes.

[0029] Figure 9 This is a schematic diagram of the reaction between free enzyme and fusion enzyme. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

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

[0032] Table 1. Reagents and kits used in the examples.

[0033]

[0034]

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

[0036] The amino acid sequences of L-aspartate transaminase and D-amino acid oxidase are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The nucleotide sequences encoding L-aspartate transaminase 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 this invention, the pET28a-LAspAT-6×His plasmid carrying the LAspAT gene and the pET28a-DAAO-6×His plasmid carrying the DAAO gene were preserved in the laboratory. The DAAO and L-AspAT genes were inserted into the pET28a vector, respectively. FastPfuFly DNA Polymerase (Beijing TransGen Biotechnology Co., Ltd.) is used for PCR amplification of linearized vectors and inserts.

[0046] (1) PCR amplification of linearized vector

[0047] Using pET28a-LAspAT-6×His plasmid as a template, the vector was amplified using the forward primer VF and the reverse primer VR to obtain a linearized vector.

[0048] Forward vector VF: GCTCGAGGGAGGAGGAGGAGTCCCACCACCACCAC (SEQ ID NO.5);

[0049] Reverse vector VR: CCTCCTCCCAGCACTGCCACAATCGCTT (SEQ ID NO.6).

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

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

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

[0053] Using the pET28a-DAAO-6×His plasmid carrying the DAAO gene as a template, the insert fragment was amplified using primer pair F / R.

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

[0055] AGTGCTGGGAGGAGGAGGATCCGGAG;

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

[0057] CCTCCTCCCTCGAGCAGGTTCGGGCGGGTCAGCGCGCGTT;

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

[0059] (3) Agarose gel electrophoresis to confirm the size of the target fragment

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

[0061] After mixing the PCR product with 10×DNA Loading Buffer, add it to the sample wells of the agarose gel using a pipette. Run the gel at a constant voltage of 120V for 25-30 minutes. The size of the target band is determined by imaging the gel and comparing it with the Mark band.

[0062] (5) DNA purification

[0063] The linearized vector and target gene were amplified by PCR and identified by agarose gel electrophoresis. The original template was removed by digestion with DMT enzyme at 37°C for 1 hour. The digestion product was then purified according to the instructions of the total gold purification kit.

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

[0065] After purification, the linearized vector and target gene were concentrated using a Nano Drop spectrophotometer. The linearized vector and target gene fragment were then mixed at a molar ratio of 1:2 using a homologous recombination kit. The Seamless Cloning and Assembly Kit was used to perform homologous recombination of the target gene and the linearized vector according to the instructions, yielding the fusion enzyme recombinant vector PET28a-LAspAT-Linker-DAA0-6xHis and the electrophoresis images of the fusion enzyme recombinant vector PET28a-LAspAT-2FL-DAAO-6×His are shown below. Figure 2 As shown.

[0066] (6) Chemical transformation

[0067] The recombinant product was transformed into Trans1-1 competent cells via chemical transformation: the cells were incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, incubated on ice for 3 min, and then 500 μL of SOC medium was added. The cells were incubated at 37℃ and 220 rpm for 1 h. The cells were collected by centrifugation at 5000 rpm / min for 3 min and resuspended in 50 μL of supernatant. The cells were then pipetted and evenly spread onto kanamycin-resistant (final concentration 50 μg / m) LB solid medium and incubated upside down at 37℃ for 12 h. Colonies were then picked and inoculated into 50 mL of kanamycin-resistant (final concentration 50 μg / m) LB liquid medium and incubated at 37℃ for 12 h. The bacterial culture was then subjected to plasmid mini-extraction and sent to a biotechnology company for nucleotide sequence analysis. After alignment, the strain was identified as 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] Positive monoclonal detection: Select white independent monoclonal clones into 1.5 mL EP tubes, add 500 μL of LB medium containing Amp, incubate at 37℃ for 6-8 h, and use as templates for PCR identification of positive results.

[0074] Plasmid extraction: Positive monoclonal bacterial cultures identified by PCR were re-transferred into LB medium containing Amp. Plasmids were extracted using a plasmid extraction kit and sent to a biotechnology company for sequencing and sequence analysis. Plasmids with correct sequencing were identified as the L-2FL-D fusion enzyme prokaryotic expression vector, and their gene map is shown below. Figure 3 As shown.

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

[0076] The correctly sequenced plasmid was transformed into Rosetta (DE3) competent cells via chemical transformation. The cells were incubated on ice for 30 min, heat-shocked at 42°C for 45 s, and then incubated on ice for 3 min. 500 μL of SOC medium was added, and the cells were incubated at 37°C and 220 rpm for 1 h. The cells were collected by centrifugation at 5000 rpm / min for 3 min and resuspended in 50 μL of supernatant. The cells were then pipetted and evenly spread onto kanamycin-resistant (final concentration 50 μg / m) LB solid medium and incubated upside down at 37°C for 12 h. Colonies were picked and inoculated into 50 mL of kanamycin-resistant (final concentration 50 μg / m) LB liquid medium and incubated at 37°C for 12 h. Single colonies were then picked from the culture dishes and activated to create a seed culture for protein fermentation.

[0077] Example 3. Expression and enzyme activity assay of recombinase

[0078] The L-2FL-D plasmid obtained in Example 2 was inoculated into LB liquid medium containing kanamycin resistance at a final concentration of 50 μg / mL via chemical transformation and cultured at 35°C and 220 rpm until OD200. 600 Once the concentration reaches 1, add IPTG to a final concentration of 62.5 μg / mL, induce culture at 25℃ for 8 h, centrifuge at 4℃ and 8000 rpm / min for 15 min, discard the supernatant, collect the precipitate, and obtain wet Escherichia coli cells containing the gene expressing the recombinant L-aspartate transaminase mutant.

[0079] The bacterial cells were resuspended in 100 mM Tris-HCl buffer (pH 8, 1 g = 5 mL buffer) and washed. The cells were then centrifuged again at 4°C, 8000 rpm / min for 15 min using a low-temperature ultracentrifuge. The precipitate was collected and resuspended in buffer for purification. Because a His tag was added during vector construction, Ni... 2+ Protein purification is performed using an IDA column.

[0080] The recombinant vector protein was purified using a nickel column:

[0081] (1) First, the crude enzyme solution after crushing and centrifugation is filtered. Larger impurities and cell debris in the crude enzyme solution are filtered using a 0.22μm pore size filter membrane.

[0082] (2) Collect the filtrate and mix it with the nickel column packing material. Incubate at 10℃ and 180rpm for 6h to allow the target protein to fully bind with the nickel column material.

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

[0084] (4) Proteins were eluted and separated using imidazole solutions of different concentrations. The eluent consisted of 20, 40, 80, 120, 160, and 200 mM imidazole dissolved in 100 mM Tris-HCl buffer at pH 8.0.

[0085] (5) Collect the effluent, which is the fusion enzyme solution.

[0086] Protein purification results were analyzed by SDS-PAGE electrophoresis, and the results are as follows: Figure 4 As shown, the predicted molecular weight of the fusion enzyme L-2FL-D is 84.1 kDa, which is consistent with... Figure 4 The bands in the protein electrophoresis results matched, therefore the purified protein was identified as the fusion enzyme L-2FL-D.

[0087] Determination of free enzyme and fusion enzyme activities: Weigh 20 mM D-phenylglycine and add 1 mL of L-2FL-D fusion enzyme solution and D-amino acid oxidase solution, respectively. React in a shaker at 35℃ and 220 rpm / min for 1 h. Terminate the reaction by heating 500 μL of the reaction solution in a boiling water bath for 5 min. After cooling, centrifuge at 10000 g for 3 min. Pipette the supernatant and filter through a 0.22 μm water membrane as the liquid chromatography sample. Detect the concentration of benzoylformic acid by high performance liquid chromatography.

[0088] A method for detecting o-chlorophenoxyacetic acid by ultraviolet-high performance liquid chromatography (UV-HPLC): An UV-2998 detector and an organic acid column were used. The peak time and peak area of ​​o-chlorophenoxyacetic acid were detected at a wavelength of 214 nm. The corresponding concentration was calculated based on the standard curve of o-chlorophenoxyacetic acid. The mobile phase was an aqueous solution of 5 mmol H₂SO₄ with a pH of 2.0-2.2, and the flow rate was 0.6 mL / min.

[0089] Methods for detecting D-o-chlorophenylglycine and L-phenylglycine: Ultraviolet-high performance liquid chromatography (UV-HPLC) with a 2998 detector and a chiral column was used. The chiral distribution and peak area of ​​o-chlorophenylglycine were detected at a wavelength of 210 nm. The corresponding concentrations were calculated using a standard curve of o-chlorophenylglycine. The HPLC conditions were: pH = 1.5, perchlorate buffer (v): acetonitrile (v) = 80:20, and a flow rate of 0.3 mL / min.

[0090] Enzyme activity is defined as the amount of enzyme required to catalyze the reaction of 20 mmol / L D-o-chlorophenylglycine to produce 1 μmol of o-chlorophenoxyacetic acid per hour under reaction conditions of 37°C.

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

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

[0095] Example 45. Preparation of L-chlorophenylglycine from D-o-chlorophenylglycine catalyzed by the fusion enzyme L-2FL-D.

[0096] The recombinant expression vector of the fusion enzyme L-2FL-D in Example 2 was induced to express the crude recombinant fusion enzyme according to the methods in Examples 2 and 3. The crude enzyme solution was then used as a biocatalyst to prepare L-o-chlorophenylglycine by biotransformation reaction with D-o-chlorophenylglycine mixture and L-glutamate as substrates and under the action of coenzyme PLP.

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

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

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

[0100] The results showed that the conversion rate of D-o-chlorophenylglycine reached 97% and the enantiomeric excess percentage reached 98% after 72 h of catalytic reaction, 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 embodiments, the present invention provides a fusion enzyme L-2FL-D for the directed catalytic 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 description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for the preparation of the fusion enzyme L-2FL-D, which catalyzes the directed conversion of D-o-chlorophenylglycine into L-o-chlorophenylglycine, characterized in that, Comprising the following steps: S1. Using pET28a-DAAO-6xHis plasmid as a template, primer pair F / R as primers to amplify the insertion fragment; S2. Using pET28a-LAspAT-6xHis plasmid as a template, vector primers V-F / V-R to amplify the linearized cloning vector; S3. Homologous recombination of the linearized vector and the insertion fragment to obtain the fusion enzyme L-2FL-D prokaryotic expression vector; S4. Expressing the fusion enzyme L-2FL-D prokaryotic expression vector in host cells to obtain the fusion enzyme L-2FL-D that catalyzes the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine in a targeted manner; The nucleotide sequence of the fusion enzyme L-2FL-D that catalyzes the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine in a targeted manner is shown as SEQ ID NO. 10; The nucleotide sequence of the primer V-F is shown as SEQ ID NO. 5, and the nucleotide sequence of the primer V-R is shown as SEQ ID NO. 6; The nucleotide sequence of the upstream primer F is shown as SEQ ID NO. 7, and the nucleotide sequence of the downstream primer R is shown as SEQ ID NO.

8.

2. The production method according to claim 1, characterized by, The reaction system for amplifying the linearized vector and the target gene fragment is: FlyMix (2x) 25 μL, forward primer 1 μL, reverse primer 1 μL, plasmid template 1 μL, ddH2O to 50 μL.

3. The preparation method according to claim 2, characterized in that, The amplification program for amplifying the linearized vector and the target gene fragment is: 98℃, 30s, 65℃, 20s, 72℃, 30s; 72℃ extension for 1 min, 4℃, 60 min.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the linearized vector to the target gene fragment is 1:

2.

5. The preparation method according to claim 4, characterized in that, The expression conditions are: 34-36℃, 200-240rpm, culture to OD 600 0.8-1.2, IPTG is added to a final concentration of 60-65μg / mL, and the culture is induced at 24-26℃ for 7-9h.

6. The fusion enzyme L-2FL-D obtained by the production method according to any one of claims 1 to 5, characterized in that, The amino acid sequence of the fusion enzyme L-2FL-D is shown as SEQ ID NO.

9.

7. The use of the fusion enzyme L-2FL-D of claim 6 in catalyzing the conversion of D-o-chlorophenylglycine to L-o-chlorophenylglycine in a targeted manner.

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