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Alcohol dehydrogenase, gene and recombinase thereof, and application of alcohol dehydrogenase in synthesis of chiral diaryl secondary alcohol

An alcohol dehydrogenase and gene-encoding technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of low enzyme yield, low overall catalytic activity, and narrow asymmetric reduction substrates, and achieve atom economy. High, environmentally friendly, and simple preparation methods

Inactive Publication Date: 2016-09-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The technical problem to be solved in the present invention is aimed at using Kluyveromyces sp. CCTCCM2011385 as the CPMK asymmetric reduction catalyst when the enzyme yield is low so that the overall catalytic activity is not high and the asymmetric reduction substrate spectrum is narrow. Provides an alcohol dehydrogenase with excellent asymmetric catalytic activity, wide substrate applicability, environment-friendly and its encoding gene, as well as a recombinant expression vector containing the gene, a recombinant expression transformant, and a method for preparing the recombinant enzyme , and its application in the preparation of chiral bisaryl alcohols

Method used

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  • Alcohol dehydrogenase, gene and recombinase thereof, and application of alcohol dehydrogenase in synthesis of chiral diaryl secondary alcohol
  • Alcohol dehydrogenase, gene and recombinase thereof, and application of alcohol dehydrogenase in synthesis of chiral diaryl secondary alcohol
  • Alcohol dehydrogenase, gene and recombinase thereof, and application of alcohol dehydrogenase in synthesis of chiral diaryl secondary alcohol

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Experimental program
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Embodiment 1

[0044] Cloning of embodiment 1 alcohol dehydrogenase gene

[0045] The above Kluyveromyces alcohol dehydrogenase gene was cloned by shotgun method.

[0046] (1) First, the above-mentioned Kluyveromyces sp. CCTCCM2011385 was cultured overnight at 37° C. using LB medium. After centrifugation to obtain the bacterial cells, use a conventional yeast genome extraction kit to obtain total genomic DNA.

[0047] (2) Digest the total DNA with restriction endonuclease Sau3AI to form GATC cohesive ends. By controlling the enzyme dosage and reaction time, the total DNase is cut into 2-6kb fragments and recovered. These fragments were ligated with the pET28a plasmid digested by BamH I (recognition sequence GGATCC, cohesive end GATC), and the ligated product was transformed into competent cells E.coliBL21 ((DE3) After culturing at 37°C for 12-16 hours on a plain LB solid plate, pick a single colony for the next step of activity screening.

[0048] (3) Pick a single colony and inoculate i...

Embodiment 2

[0054] Example 2 Construction and cultivation of recombinant Escherichia coli BL21(DE3) / pET28a-kpadh

[0055] The gene kpadh recovered in Example 1 and the plasmid pET28a were double digested with restriction enzymes NdeI and BamH I in a water bath at 37°C overnight, purified by agarose gel electrophoresis the next day, and the target was recovered using an agarose recovery kit fragment. 4°C, use T 4 DNA ligase overnight ligase-cut gene kpadh and plasmid pET28a to obtain the recombinant expression vector pET28a-kpadh ( figure 2 ). The constructed recombinant expression vector pET28a-kpadh was thermally transferred into Escherichia coli BL21 (DE3) competent, coated with Kan-resistant LB solid plate, and colony PCR verification was carried out after overnight culture. The positive clone was the recombinant Escherichia coli BL21 ( DE3) / pET28a-kpadh. Pick positive clones and culture them overnight in LB medium, then transfer them into fresh LB culture at 2% transfer amount th...

Embodiment 3

[0056] The separation and purification of embodiment 3 alcohol dehydrogenase

[0057] Suspension-cultured recombinant cells were placed in liquid A (20mmol·L -1 Sodium phosphate, 500mmol·L -1 NaCl, 20mmol·L -1 imidazole, pH 7.4), the crude enzyme solution was obtained after sonication and centrifugation. The column used for purification is an affinity column HisTrap FFcrude (nickel column), which is accomplished by using the histidine tag on the recombinant protein for affinity binding. First, use solution A to equilibrate the nickel column, load the crude enzyme solution, continue to use solution A to elute the breakthrough peak, and after equilibrium, use solution B (20mmol L -1 Sodium phosphate, 500mmol·L -1 NaCl, 1000mmol·L -1 imidazole, pH 7.4) for gradient elution to elute the recombinant protein bound to the nickel column to obtain recombinant alcohol dehydrogenase. Enzyme activity assay (CPMK as substrate, NADPH as coenzyme) and SDS-PAGE analysis were carried out...

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Abstract

The invention discloses an alcohol dehydrogenase, a gene thereof, a recombinant expression vector and a recombinant expression transformant respectively containing the gene, a recombinase of the alcohol dehydrogenase, and an application of the alcohol dehydrogenase in asymmetric reduction synthesis of chiral diaryl secondary alcohol as a catalyst, and belongs to the technical field of bioengineering. The alcohol dehydrogenase is from Kluyveromyces sp. CCTCCM2011385, has a carbonyl group reduction function, and also has a hydroxy group oxidation function. Extra addition of glucose dehydrogenase and other enzymes used for cofactor circulation is not needed when the alcohol dehydrogenase is used in the reduction of diaryl ketone into the chiral diaryl secondary alcohol as a biocatalyst, and the alcohol dehydrogenase has the advantages of high catalysis efficiency, mild reaction conditions, easy product recovery and low cost, so the alcohol dehydrogenase has very good application and exploitation prospect in the production of antihistamine medicines.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an alcohol dehydrogenase derived from Kluyveromyces and its gene, a recombinant expression vector containing the gene, a recombinant expression transformant, the recombinant enzyme and the preparation of the recombinant enzyme The method, and the application of the recombinant enzyme as a catalyst in the preparation of optical bisaryl secondary alcohols by asymmetric reduction. Background technique [0002] Chiral bisaryl secondary alcohols are an important class of chiral compounds. Many drugs have a bisaryl secondary alcohol building block structure, among which chiral (4-chlorophenyl)-(pyridine-2 -Base)-methanol can be used to synthesize the antiallergic drug bepotahistine. The process of synthesizing chiral bisaryl alcohols from latent chiral bisaryl ketones through asymmetric reduction has the advantage of high atom economy. [0003] The chemical asymmetr...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12P17/12
CPCC12N9/0004C12P17/12
Inventor 倪晔许国超唐铭烩董晋军边雅倩
Owner JIANGNAN UNIV
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