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A kind of transaminase and its application in the synthesis of sitagliptin intermediate

A transaminase and transamination technology, applied in the field of bioengineering, can solve the problems of poor stereoselectivity, expensive catalyst, low yield and the like

Active Publication Date: 2018-06-05
弈柯莱(台州)药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Aiming at the problems of low yield, poor stereoselectivity, expensive catalyst and difficult solvent recovery in the reported asymmetric synthesis of sitagliptin and its intermediates, the invention provides a high catalytic activity, enantiomeric Enzymatic synthesis of R-3-amino-4-(2,4,5-trifluorophenyl)-butyric acid methyl ester by transaminase with strong selectivity, good substrate tolerance and solvent tolerance, and further synthesis Enzyme-Chemical Synthesis of Sitagliptin

Method used

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  • A kind of transaminase and its application in the synthesis of sitagliptin intermediate
  • A kind of transaminase and its application in the synthesis of sitagliptin intermediate
  • A kind of transaminase and its application in the synthesis of sitagliptin intermediate

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Embodiment 1

[0075] Cloning of embodiment 1 mycobacterium PYR-1 transaminase gene

[0076] According to the gene sequence predicted to be mycobacterium PYR-1 transaminase included in Genbank (NCBI accession number: YP_955297.1), PCR primers were designed as the upstream and downstream primers for gene cloning in Table 1. Wherein, the underlined part of the upstream primer is the NdeI restriction site, and the underlined part of the downstream primer is the EcoRI restriction site.

[0077] PCR amplification was performed using the genomic DNA of mycobacterium PYR-1 as a template. PCR system: 10×KOD-Plus PCR buffer 2μL, 25mM MgSO 4 1.2 μL, 2 mM dNTP 2 μL, KOD-Plus PCR high-fidelity enzyme 0.3 μL, DNA template 0.5 μL (including 0.1 μg DNA template), ddH 2 013 μL, each 0.5 μL (10 mmol / L) of the gene cloning upstream primers and the gene cloning downstream primers (SEQ ID No: 11 and 12) in Table 1 were used for PCR amplification. PCR amplification steps are: (1) 95°C, pre-denaturation for 3m...

Embodiment 2

[0080] The construction of embodiment 2 recombinant expression vector

[0081] The transaminase gene DNA fragment obtained in Example 1 was double-digested with restriction endonucleases NdeI and EcoRI at 37°C for 8 h, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit. Under the action of T4 DNA ligase, the target fragment was ligated with the plasmid pET21a digested with NdeI and EcoRI at 16°C overnight to obtain the recombinant expression plasmid pET21a-MvAT.

Embodiment 3

[0082] Embodiment 3 Preparation of recombinant expression transformant

[0083] Transform the recombinant expression plasmid into Escherichia coli (E.coli) DH5α competent cells, the transformation conditions are 45°C, heat shock for 90 seconds, and positive recombinants are screened on the resistance plate containing ampicillin, and picked Monoclonal, positive clones verified by colony PCR (see figure 2 Lane A in ). Cultivate the recombinant bacteria, extract the plasmid after the plasmid is amplified, retransform into E.coli BL21(DE3) competent cells, spread the transformation solution on the LB plate containing ampicillin, and culture it upside down at 37°C overnight to obtain positive recombinant Transformant E.coli BL21(DE3) / pET21a–MvAT, positive clones verified by colony PCR (see figure 2 Lane B in ).

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Abstract

The invention provides a new aminotransferase, a gene of the new aminotransferase, a recombinant expression vector containing the gene, a recombinant expression transformant, a recombinase, a preparation method of the recombinase, and application of the aminotransferase to preparation of active chiral amine by performing asymmetrical transamination on a carbonyl compound. The aminotransferase is derived from mycobacterium (mycobacterium vanbaalenii) PYR-1, and is applied to preparation of (R)-3-amino-4-(2, 4, 5-trifluorophenyl)-methyl butyrate. Compared with other preparation methods, the preparation methods provided by the present invention have the advantages that catalyzed and prepared products are high in concentration, enantioselectivity is high, reaction conditions are mild, operations are simple and convenient, enlargement is easy and the like, so that the industrial application prospect in production of sitagliptin phosphate is excellent.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a transaminase, a recombinant expression vector containing the gene encoding the enzyme and a recombinant expression transformant, the expressed recombinant enzyme and the preparation method of the recombinant enzyme, and the transaminase used as a catalyst in asymmetric synthesis The application of sitagliptin intermediate. Background technique [0002] Diabetes mellitus is a metabolic disease that occurs due to changes in insulin secretion, leading to insulin deficiency and weakened action, or decreased insulin activity, or under the combined influence of the two. It is characterized by high blood sugar and accompanied by protein, sugar and fat metabolism. disorder. Diabetes and its complications are the third most harmful to human health after cardiovascular diseases and tumors, becoming an important disease that endangers human health. The International Di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/00C12P17/18
Inventor 罗煜丁时澄瞿旭东
Owner 弈柯莱(台州)药业有限公司
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