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Immobilized transaminase and application of immobilized transaminase in synthesis of sitagliptin intermediate

A technique for immobilizing transaminase and transaminase, which is applied in the asymmetric synthesis of sitagliptin intermediates. In the field of immobilized transaminase, it can solve the problems of difficult mass transfer, large loss of enzyme activity, and difficult separation of transaminase, and achieve simple separation. , less loss of enzyme activity, environmentally friendly effect

Active Publication Date: 2016-01-06
ABIOCHEM BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The present invention aims at the problems such as the difficult separation of the transaminase derived from Mycobacterium vanbaalenii PYR-1 in the enzyme catalysis process, the large loss of enzyme activity or the difficulty of mass transfer in the process of enzyme immobilization, and the commercialized epoxy resins are sequentially Derivatization with iminodiacetic acid (IDA), cobalt chloride to obtain an enzyme-immobilized carrier, immobilized with histidine-tagged Mycobacterium vanbaalenii PYR-1-derived transaminase, and treated with H2O2 to enhance the binding capacity of the enzyme

Method used

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  • Immobilized transaminase and application of immobilized transaminase in synthesis of sitagliptin intermediate
  • Immobilized transaminase and application of immobilized transaminase in synthesis of sitagliptin intermediate
  • Immobilized transaminase and application of immobilized transaminase in synthesis of sitagliptin intermediate

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Embodiment 1

[0050] The preparation of embodiment 1 immobilized carrier A

[0051] Under stirring, slowly add 100 grams of amino resin of the type D301 of Shanghai Huazhen Company to 200 mL of 5% (v / v) ethylene glycol diglyceryl ether in toluene solution, stir and react at 60°C for 1 hour, and use Wash with toluene and water, then place in 350mL of 100mM iminodiacetic acid (IDA), 10mM triethanolamine, pH 9.0 aqueous solution, react at 60°C for 2 hours, filter and wash with water, and reconstitute with 200mL of 10mM cobalt chloride solution Suspended, stirred at room temperature for 30 min, filtered and washed with water to obtain immobilized carrier A.

Embodiment 2

[0052] Embodiment 2 Preparation of immobilized carrier B

[0053] Add 100 grams of ion chelating resin model HZD401 from Shanghai Huazhen Company under stirring to 200 mL of 10 mM cobalt chloride solution for resuspension, stir at room temperature for 30 min, filter and wash with water to obtain immobilized carrier B.

Embodiment 3

[0054] Embodiment 3 Preparation of recombinant transaminase expression transformant

[0055] According to Chinese patent application number 201410169882.4, the recombinant expression vector pET21a-MvAT containing SEQNo.1 was constructed, digested with restriction endonucleases NdeI and EcoRI at 37°C for 8h, purified by agarose gel electrophoresis, and used agarose gel DNA The recovery kit recovers the target fragment. Under the action of T4 DNA ligase, the target fragment was ligated with the plasmid pET28a digested by NdeI and EcoRI at 16°C overnight to obtain the recombinant expression plasmid pET28a-MvAT. The recombinant expression plasmid was transformed into Escherichia coli (E.coli) DH5α competent cells, the transformation conditions were 45°C, heat shock for 90 seconds, and the positive recombinants were screened on the resistance plate containing kanamycin, Pick a single clone and verify the positive clone by colony PCR (see figure 1 A). Cultivate the recombinant ba...

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Abstract

The invention provides immobilized transaminase. The immobilized transaminase is obtained by fixing transaminase, with histidine tag, derived from Mycobacterium vanbaalenii PYR-1 onto an enzyme immobilization carrier; the enzyme immobilization carrier is obtained by derivatization of epoxy resin through IDA (imino diethyl acetic acid) and cobalt chloride or derivatization of ionic chelate resin through cobalt chloride. The immobilized transaminase has the advantages of firmness in combination, low enzyme activity loss, simplicity in separation and high reusability; low cost of an asymmetric conversion process, mild reaction conditions, environment friendliness, simplicity and convenience in operation, easiness in industrial expanding and promising industrial application prospect are realized.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an immobilized transaminase, a preparation method of the immobilized transaminase, and an application of the immobilized transaminase as a catalyst in the asymmetric synthesis of sitagliptin intermediates. Background technique [0002] Enzyme is a class of biomacromolecular substances produced by organisms with catalytic functions. As a biocatalyst, enzymes can catalyze various organic chemical reactions under mild conditions of normal temperature and pressure. Enzyme catalysis has high efficiency, which is 10 times higher than that of general catalysts 7 ~10 13 times; enzyme catalysis has high specificity, can reduce or avoid side reactions, and obtain products with high purity; enzyme-catalyzed reactions also have high stereoselectivity and regioselectivity, without protection and deprotection. These advantages of enzymes have greatly promoted the research o...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N11/08C12P13/00C12P17/18
CPCY02P20/50
Inventor 罗煜丁时澄瞿旭东
Owner ABIOCHEM BIOTECH CO LTD
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