An immobilized monoamine oxidase and its application in the synthesis of chiral azabicyclic compounds

A monoamine oxidase and compound technology, applied in the field of bioengineering, can solve the problems of large loss of enzyme activity, difficult separation of monoamine oxidase, difficult mass transfer, etc., and achieve the effects of less loss of enzyme activity, good industrial application prospects, and simple separation

Active Publication Date: 2020-03-17
弈柯莱生物科技(集团)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0037] The present invention aims at the problems such as difficult separation of monoamine oxidase in the enzymatic catalysis process, large loss of enzymatic activity or difficult mass transfer in the process of enzymatic immobilization, and the commercialized epoxy resin is sequentially treated with iminodiacetic acid (IDA), sulfuric acid Derivatization with nickel to obtain an enzyme-immobilized support, followed by immobilization with monoamine oxidase

Method used

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  • An immobilized monoamine oxidase and its application in the synthesis of chiral azabicyclic compounds
  • An immobilized monoamine oxidase and its application in the synthesis of chiral azabicyclic compounds
  • An immobilized monoamine oxidase and its application in the synthesis of chiral azabicyclic compounds

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Embodiment 1

[0069] The preparation of embodiment 1 immobilized carrier

[0070] Slowly add 100 grams of 335 amino resin from Shanghai Huazhen Company under stirring to 200 mL of 5% (v / v) toluene solution of ethylene glycol diglyceryl ether, stir and react at 60°C for 1 hour, and then use Wash with toluene and water, then place in 350mL of 20mM sodium phosphate buffer (pH 7.5) containing 100mM iminodiacetic acid (IDA), react at 60°C for 2 hours, filter and wash with water, and then use 200mL of 40mM nickel sulfate solution Resuspend, stir at room temperature for 30 min, filter and wash with water to obtain the immobilized carrier.

Embodiment 2

[0071] Example 2 Preparation of recombinant monoamine oxidase expression transformant

[0072] According to Chinese patent application number 201410441595.4, a recombinant expression vector pET28a-BYK-MAON containing monoamine oxidase BYK-MAON (the ORF nucleotide sequence and its encoded amino acid sequence are respectively shown in SEQ ID No.1 and SEQ ID No.2) was constructed. The recombinant expression plasmid was transformed into Escherichia coli (E.coli) DH5α competent cells, the transformation conditions were 45°C, heat shock for 45 seconds, and the positive recombinants were screened on the resistance plate containing kanamycin, Single clones were picked, and positive clones were verified by colony PCR. 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 kanamycin, and culture it upside down at 37°C overnight to obtain Posi...

Embodiment 3

[0073] Embodiment 3 Expression of recombinant monoamine oxidase

[0074] The recombinant E.coli BL21(DE3) / pET28a-BYK-MAON obtained in Example 3 was inoculated into LB medium containing kanamycin (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0 ), shake culture overnight at 37°C, insert 1% (v / v) inoculum into a 500mL Erlenmeyer flask containing 200mL LB medium, and culture on a shaking table at 37°C at 200rpm, when the OD of the culture solution 600 When it reached 0.8, IPTG with a final concentration of 0.1 mmol / L was added as an inducer, and after induction at 26°C for 12 hours, the culture medium was centrifuged to collect cells and washed twice with saline to obtain resting cells. The resulting resting cells were suspended in a buffer solution with pH 7.5, ultrasonically disrupted in an ice bath, and the supernatant was collected by centrifugation, which was the crude enzyme solution of the recombinant monoamine oxidase. Protein concentration was determined by the Br...

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Abstract

The invention provides an immobilized monoamine oxidase with firm combination and less enzyme activity loss, and an application thereof in enzyme catalysis synthesis of a (1S,2S,5R)-aza-dicyclic compound. Compared with other immobilized enzymes prepared through other methods, the immobilized enzyme prepared in the invention has the advantages of firm combination, less enzyme activity loss, simple separation, high reuse frequency, low cost of the oxidation-addition process, mild reaction conditions, environmental protection, simple operation, and easy industrial amplification, so the immobilized enzyme has very good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an immobilized monoamine oxidase, a preparation method of the immobilized monoamine oxidase, and an application of the immobilized monoamine oxidase as a catalyst in the asymmetric synthesis of chiral azabicyclic compounds. 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 promo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/70C12N11/089C12P17/10
Inventor 罗煜丁时澄瞿旭东钱龙
Owner 弈柯莱生物科技(集团)股份有限公司
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