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Esterase mutant and application thereof

A technology of mutants and esterases, which is applied in the field of preparation of optically active 2-aryl propionic acid, can solve the problems of poor stereoselectivity of ketoprofen, and achieve reduced production costs, high enzyme activity and/or stereoselectivity Sexuality, the effect of improving production efficiency

Active Publication Date: 2019-03-01
HUANGSHI SHIXING PHARMA +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The document "Cloning and Characterization of Thermostable Esterase from Archaeoglobus fulgidus" records that Seung-Bum Kim et al. isolated a thermostable esterase Est-AF from the extremophile Archaeoglobus fulgidus DSM 4304. Est-AF has excellent thermal stability and can withstand Subject to 70~90 ℃ high temperature, but it is relatively poor in the stereoselectivity of ketoprofen (dexketoprofen ee%=30.2%)

Method used

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  • Esterase mutant and application thereof
  • Esterase mutant and application thereof
  • Esterase mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Codon optimization of Est-AF

[0031] According to the codon usage frequency distribution table of E. coli, all amino acids in the esterase Est-AF sequence SEQ ID NO: 1 were translated using the optimal codons in the codon usage frequency distribution table. In addition, it is necessary to avoid repetitive sequences and avoid NcoI and HindIII restriction endonuclease sites generated during codon optimization. Finally, the optimized sequence SEQ ID NO:2 was obtained.

[0032] The optimized sequence GC content distribution is as follows figure 1 shown. Finally, NcoI and HindIII restriction sites were added to the two ends of the sequence, which was synthesized by General Biosystems (Anhui) Co., Ltd., and the synthesized sequence was named Est1.

Embodiment 2

[0033] Example 2 Construction of Est1 expression strain

[0034] Using the synthesized Est1 sequence as a template, the following primers were used to amplify the Est1 gene fragment with a length of about 740 bp.

[0035] 5'-CC CCATGG AACGTATTACCCTG-3' (SEQ ID NO: 3)

[0036] 5'-CC AAGCTT TTACAGTTTTTCAATAAA-3' (SEQ ID NO: 4)

[0037] PCR system: 5×PrimeSTAR PCR HS Buffer 10μL, upstream primer and downstream primer 1μL (10pM), DNA template 1μL (0.1μg), dNTPs (2.5mM) 4μL, PrimeSTAR PCR HS Polymerase 0.5μL and ddH 2 0 to 50 μL.

[0038] PCR amplification steps are: (1) Pre-denaturation at 98°C for 20s; (2) Denaturation at 98°C for 10s; (3) Annealing at 60°C for 10s; (4) Extension at 72°C for 60s; (2)~(4) repeat 30s (5) Continue to extend for 10 min at 72°C.

[0039] The PCR product was purified by agarose gel electrophoresis, and the target band of about 1100 bp was recovered using an agarose gel DNA recovery kit.

[0040] The PCR product was double-digested with restric...

Embodiment 3

[0042] Expression of embodiment 3 recombinant esterases

[0043]The recombinant Escherichia coli E. coli BL21(DE3) / pET28a-Est1 obtained in Example 2 was inoculated into LB medium containing kanamycin (50 mg / L), and cultured overnight at 37° C. with shaking. According to the inoculum amount of 2‰ (v / v), insert into the 250ml Erlenmeyer flask that 50ml LB culture medium is housed, put 37 ℃, 200rpm shaker shaking culture. When the OD600 of the culture medium reached 0.8, IPTG with a final concentration of 0.5 mmol / L was added for induction. After induction at 30°C for 8 hours, the culture medium was centrifuged to collect the cells to obtain about 0.3 g of wet cells.

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Abstract

The invention relates to an esterase mutant and application thereof to preparation of optical active 2-aryl propionic acid (APA). The esterase mutant is obtained by carrying out amino acid mutation onone or more sites on the basis of SEQ ID NO:1; a mutation site is a 138th site and / or a 200th site. Compared with wild type esterase, the mutant has higher stereoselectivity on the basis of keeping thermal stability; compared with a chemical synthesis method, the application of the esterase mutant to the preparation of the optical active 2-APA has the advantages that the production cost can be reduced and the production efficiency is improved; the esterase mutant is adaptive to the requirements of industrial production.

Description

technical field [0001] The invention relates to an esterase mutant and its application in preparing optically active 2-aryl propionic acid. Background technique [0002] Among the enantiomers of 2-arylpropionic acid (2-APA) NSAIDs, only the right-handed configuration shows high pharmacological activity, and the left-handed configuration has low or no effect. In view of the chiral center generated by general chemical reactions, an equal amount of a pair of enantiomeric mixtures is always obtained, which is called a racemate. To obtain optically pure dextrorotatory 2-APA, splitting the racemate is one of the main methods. [0003] Ketoprofen is a 2-aryl propionic acid non-steroidal anti-inflammatory drug (NSAIDs), its anti-inflammatory and analgesic effect is 150 times that of aspirin, and its antipyretic effect is 100 times that of aspirin. This medicine has been reported since the 1970s to synthesize a lot of methods, but they are all synthetic racemates. Therefore, prepar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12P7/40
CPCC12N9/16C12P7/40
Inventor 张向阳王小龙张鸿鲍素敏林洁李文辉韦念钻张琼光王松何志祥刘元胜丁国柱余晓骁程冲黄杰张希袁蕤
Owner HUANGSHI SHIXING PHARMA
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