Transaminase mutant and screening method thereof

Abstract

The invention relates to a transaminase mutant and a screening method thereof, and belongs to the technical field of transaminase preparation. The invention provides a transaminase mutant and a screening method thereof, the transaminase mutant is an omega-transaminase mutant, the Genbank registration number of the omega-transaminase mutant is 3WWJA, and the amino acid sequence of the omega-transaminase mutant is shown as SEQ ID NO.1; the omega-transaminase mutant is obtained by performing single-site or multi-site mutation on one or more of 67th, 69th, 155th or 283th sites of an omega-transaminase amino acid sequence shown in SEQ ID NO: 1; the method has the advantages of high conversion efficiency and good catalytic benefit on the whole.

Description

Technical Field

[0001] The invention relates to a transaminase mutant and a screening method thereof, belonging to the technical field of transaminase preparation. Background Art

[0002] Transaminase is a key enzyme in the asymmetric synthesis of optically pure chiral amines. It is a coenzyme pyridoxal phosphate (PLP)-dependent enzyme. During the reaction, PLP and 5'-pyridoxamine phosphate (PMP) will convert into each other and catalyze the reversible transfer of the amino group from a suitable donor to a carbonyl acceptor. According to the amino group being transferred to the amino acceptor at different positions, transaminase can be divided into α-transaminase and ω-transaminase. α-transaminase requires a carbonyl acceptor at the α position to exert its catalytic function, while ω-transaminase can catalyze keto acids, ketones, aldehydes and amines of any structure and has excellent stereoselectivity. Therefore, it has higher application value and can be more widely used in the synthesis of pharmaceutical intermediates.

[0003] R-3-aminopiperidine is an important pharmaceutical intermediate, mainly used to synthesize dipeptidyl peptidase-IV (DPP-IV) inhibitors, such as alogliptin, linagliptin and other hypoglycemic drugs. The synthesis of (R)-3-aminopiperidine by chemical synthesis involves many reagents, and the operation is complicated, the process is cumbersome, the cost is high, and the target product with high optical purity cannot be obtained. The method of synthesizing (R)-3-aminopiperidine by transaminase has high conversion rate, good selectivity, simple process flow, green and environmental protection, and is suitable for large-scale promotion and application.

[0004] In the industrial production of (R)-3-aminopiperidine, further improving the catalytic activity of ω-transaminase, thereby maximizing the catalytic benefit and reducing the production cost, is the focus and difficulty of the current research of technicians, among which the screening of transaminase mutants is another difficult problem. Summary of the invention

[0005] In view of the defects in the above prior art, the present invention provides a transaminase mutant and a screening method thereof, so as to solve the technical problem of preparing a transaminase with high conversion efficiency and good catalytic benefit.

[0006] The present invention can be achieved by the following technical scheme: a wild-type transaminase, wherein the wild-type transaminase mutant from Arthrobacter is an ω-transaminase, the Genbank accession number of the ω-transaminase is 3WWJ_A, and the amino acid sequence thereof is shown in SEQ ID NO.1; The ω-aminase mutant is obtained by performing single-site or multi-site mutation at one or more of positions 67, 69, 155 or 283 of the ω-aminase amino acid sequence shown in SEQ ID NO:1.

[0007] Furthermore, the ω-aminase mutant is obtained by mutation of the ω-aminase amino acid shown in the sequence SEQ ID NO.1 at one or more of the following sites: the tyrosine at position 67 of the ω-aminase is mutated to arginine, lysine or isoleucine; the threonine at position 69 of the ω-aminase is mutated to lysine, methionine or leucine; the glutamine at position 155 of the ω-aminase is mutated to cysteine, serine or arginine; the threonine at position 283 of the ω-aminase is mutated to phenylalanine, lysine or methionine.

[0008] Furthermore, the recombinant expression vector of the ω-aminotransferase mutant is constructed by connecting the nucleic acid sequence of the recombinant aminotransferase gene to various vectors.

[0009] Furthermore, the vector is selected from one of pET-30a, pET-22b, pREST A, etc.; Preferably, the plasmid vector is Pet-30a.

[0010] Furthermore, the host cell of the ω-aminotransferase mutant is an Escherichia coli cell and its derivative strains.

[0011] Furthermore, the host cell is selected from one of Escherichia coli BL21 (DE3), Rosseta (DE3), Escherichia coli W3110, etc.; Preferably, the host cell is Escherichia coli BL21 (DE3).

[0012] The aminotransferase mutant is obtained by subjecting one or more of the 67th, 69th, 155th or 283rd positions of the amino acid sequence shown in SEQ ID NO:1 to single-site or multi-site mutation, wherein the tyrosine at the 67th position is mutated to arginine, lysine or isoleucine; the threonine at the 69th position is mutated to lysine, methionine or leucine; the glutamine at the 155th position is mutated to cysteine, serine or arginine; the threonine at the 283rd position is mutated to phenylalanine, lysine or methionine, and finally a ω-aminotransferase mutant with higher enzyme activity is obtained, and its catalytic activity is 3 times that of the wild-type aminotransferase, and it can efficiently catalyze the synthesis of R-3-aminopiperidine.

[0013] A high-throughput screening method for ω-aminotransferase mutants is disclosed. In the high-throughput screening method for mutants, piperidone is used as a substrate, pyridoxal phosphate is used as a coenzyme, D-alanine is used as an amino donor, glucose, glucose dehydrogenase, lactate dehydrogenase and NAD are used to construct a coenzyme coupling system, and phenol red is used as a pH indicator; an ω-aminotransferase mutant enzyme solution is added to a phosphate buffer, and the absorbance is detected by a spectrophotometer to screen positive mutants.

[0014] Furthermore, the catalytic force of the ω-aminase mutant that has been screened is screened. During the catalytic force screening of the ω-aminase mutant, the reaction solution is used with piperidone as a substrate, pyridoxal phosphate as a coenzyme, and isopropylamine as an ammonia donor; the ω-aminase mutant enzyme solution is added to the Tris-HCl buffer, and after the reaction is completed, the catalytic force of the ω-aminase mutant is determined by HPLC detection using dichloromethane extraction.

[0015] In summary, compared with the prior art, the present invention has the following advantages: In the present invention, some sites that affect the aminotransferase activity are obtained by homology modeling and molecular docking, and site-directed mutagenesis is performed on them, and finally a ω-aminotransferase mutant with higher enzyme activity is obtained, and its catalytic activity is 3 times that of the wild-type aminotransferase, that is, in the same time, for 20 g / L of substrate, the conversion rate is increased from 32% to 96%, which can efficiently catalyze the synthesis of R-3-aminopiperidine. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further specifically described below through specific embodiments, but the present invention is not limited to these embodiments.

[0017] Enzyme source and gene synthesis: According to the gene sequence of Arthrobacter sp KNK168 aminotransferase included in Genbank (NCBI accession number: 3WWJ_A), the gene fragment was artificially synthesized, and NdeⅠ and XhoⅠ endonuclease sites were added on both sides to insert the gene into the pET-30a plasmid to obtain the recombinant plasmid TA-pET-30a. The recombinant plasmid was transferred into Escherichia coli BL21 (DE3) to establish the aminotransferase gene engineering bacteria.

[0018] Vector construction of mutants: Using the recombinant plasmid TA-Pet30a as a template, primers were designed to mutate the amino acids at positions 67, 69, 155, and 283, wherein the mutation primers are: 67F:GATGCCACCNNKACCACCTTTTCATGTTTGG 67R:CATGAAAGGTGGTMNNGGTGGCATCACTGG 69F:GATGCCACCTATAACCNNNKTTTCATGTTTGG 69R:CCAAACATGAAAMNNGGTATAGGTGGCATC 155F:CCTGCCCGTATNNKTGGATTGTTCCGTTTG 155R:GGAACAATCCAMNNATACGGGGCAGGCACTC 283F:GTTCTGGGTTGTAGCNNKGGTGGTGGCGTTTG 283R:CAAACGCCACCACCMNNGCTACAACCCAGAAC The PCR reaction system was: 10 μL of 5x buffer, 4 μL of dNTP, 1 μL of forward primer, 1 μL of reverse primer, 0.5 μL of DNA polymerase, 1 μL of DNA template, and finally fixed to 50 μL with ddH2O.

[0019] PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 55 min, for a total of 30 cycles, and final extension at 72°C for 10 min.

[0020] Take 5 μL of the PCR product and transfer it into the competent cells of Escherichia coli BL21 (DE3). The transformation conditions are 42°C and heat shock for 90 seconds. Rapidly place it on ice for 2 minutes. Add 600 μL of LB liquid culture medium to the tube. Culture at 37°C, 200 r / min for 45 minutes. Centrifuge at 4000 rpm for 3 minutes. Discard 600 μL of supernatant. Mix the remaining 100 μL of bacterial solution thoroughly and apply it to the kanamycin LB resistance plate. After the bacterial solution is completely absorbed by the culture medium, invert and culture at 37°C for 12 hours.

[0021] High-throughput screening of mutants: In a 96-well culture plate, 300 μL of LB culture medium containing a final concentration of 50 μg / mL kanamycin was added to each well, and different transformed colonies were inoculated. The culture was cultured at 37°C and 150 r / min for 12 h. 50 μL of the bacterial solution was transferred to a new 96-well culture plate containing 600 μL of culture medium in each well, and cultured at 37°C and 150 r / min for 2 h. IPTG with a final concentration of 0.4 mM was then added to the culture medium. After inducing expression at 28°C for 10 h, the culture was centrifuged at 4°C and 8000 r / min for 10 min, and the supernatant was discarded.

[0022] The collected bacteria were placed in a -80°C refrigerator and frozen for 12 h. 200 μL of lysozyme solution was added. The mixture was shaken at 37°C and 150 r / min for 1 h and centrifuged at 4°C and 8000 r / min for 10 min.

[0023] Add 100 μL of enzyme solution to each well of a 96-well plate containing 400 μL of color reaction mixture, react at 35°C for 2 hours, detect the absorbance at OD560, screen out mutants that perform better than the control, re-plate the plate to preserve the bacteria, and send to a sequencing company for sequencing.

[0024] The color reaction mixture consists of 40 mM / L piperidone, 500 mM / L D-alanine, 20 mM / L pyridoxal phosphate, 50 mM / L glucose, 1 g / L glucose dehydrogenase, 1 g / L lactate dehydrogenase, 1 g / L NAD, and 2 g / L ω-transaminase. The reaction is carried out in phosphate buffer, the color is observed, and the absorbance is detected at OD560 using a spectrophotometer.

[0025] Preparation of mutant crude enzyme solution: The mutant re-preserved in the above Example 3 and the TA-Pet-30a strain were inoculated into 100 mL of culture medium containing kanamycin resistance, and cultured at 37°C and 200 rpm for 3.5 h. When the OD600 value of the bacterial solution was between 0.6 and 0.8, IPTG with a final concentration of 0.4 mMol / L was added and induced at 28°C for more than 12 h; the cells were collected by centrifugation at 4°C and 8000 r / min for 10 min, and an appropriate amount of phosphate buffer was added for ultrasonic disruption in an ice-water bath to obtain crude enzyme solutions of the mutant and the control group, which were frozen and stored at -20°C.

[0026] Screening of mutant catalytic activity: Reaction system: 100mMol / L piperidone, 400mMol / isopropylamine hydrochloride, 0.5mMol / pyridoxal phosphate, add 100mMol / L Tris-HCl buffer to 20mL, adjust the pH to 8.5 with isopropylamine solution, add 200μL crude enzyme solution, shake at 35℃ for 3h, extract with dichloromethane, take the extract for HPLC detection, compare the catalytic effect between the mutant and TA-Pet-30a, and between the mutants. The specific results are shown in Table 1.

[0027] The implementation of the present invention is not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and detail, and these are considered to fall within the protection scope of the present invention.

Claims

1. A transaminase mutant, characterized in that: The transaminase mutant is an ω-transaminase mutant, the Genbank accession number of the ω-transaminase is 3WWJ_A, and the amino acid sequence thereof is shown in SEQ ID NO.1; The ω-aminase mutant is obtained by performing single-site or multi-site mutation at one or more of positions 67, 69, 155 or 283 of the ω-aminase amino acid sequence shown in SEQ ID NO:

1.

2. A transaminase mutant according to claim 1, characterized in that: The ω-aminase mutant is obtained by mutation of the ω-aminase amino acid shown in the sequence SEQ ID NO.1 at one or more of the following sites: the tyrosine at position 67 of the ω-aminase is mutated to arginine, lysine or isoleucine; the threonine at position 69 of the ω-aminase is mutated to lysine, methionine or leucine; the glutamine at position 155 of the ω-aminase is mutated to cysteine, serine or arginine; the threonine at position 283 of the ω-aminase is mutated to phenylalanine, lysine or methionine.

3. A transaminase mutant according to claim 2, characterized in that: The recombinant expression vector of the ω-aminotransferase mutant is constructed by connecting the nucleic acid sequence of the recombinant aminotransferase gene to various vectors.

4. A transaminase mutant according to claim 3, characterized in that: The vector is selected from one of pET-30a, pET-22b, pREST A and the like.

5. A transaminase mutant according to claim 4, characterized in that: The host cell of the ω-aminotransferase mutant is an Escherichia coli cell and its derivative strains.

6. A transaminase mutant according to claim 5, characterized in that: The host cell is selected from one of Escherichia coli BL21 (DE3), Rosseta (DE3), Escherichia coli W3110 and the like.

7. A method for screening the ω-aminotransferase mutant according to any one of claims 1 to 6, characterized in that: The screening method includes high-throughput screening and catalytic screening; In the high-throughput screening, the reaction solution is used with piperidone as a substrate, pyridoxal phosphate as a coenzyme, D-alanine as an amino donor, glucose, glucose dehydrogenase, lactate dehydrogenase, and NAD to construct a coenzyme coupling system, and phenol red is used as a pH indicator; an ω-transaminase mutant enzyme solution is added to a phosphate buffer, and the absorbance is detected by a spectrophotometer to screen positive mutants.

8. The method for screening transaminase mutants according to claim 7, characterized in that: The catalytic force of the ω-aminase mutants that have been screened is screened. During the catalytic force screening of the ω-aminase mutants, the reaction solution is used with piperidone as a substrate, pyridoxal phosphate as a coenzyme, and isopropylamine as an ammonia donor; the ω-aminase mutant enzyme solution is added to the Tris-HCl buffer, and after the reaction is completed, the catalytic force of the ω-aminase mutant is determined by HPLC extraction and detection with dichloromethane.

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