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CN-121975753-A - Ethanol dehydrogenase mutant, engineering bacterium and application thereof in synthesis of cinnolamine ester intermediate

CN121975753ACN 121975753 ACN121975753 ACN 121975753ACN-121975753-A

Abstract

The invention discloses an alcohol dehydrogenase mutant, engineering bacteria and application thereof in synthesizing a cinnolamine ester intermediate, wherein the alcohol dehydrogenase mutant is used for catalyzing prochiral ketone (B) to synthesize chiral alcohol (A), the method has high substrate concentration, simple and convenient post-treatment steps, is more suitable for industrial scale-up production, and solves the problems of low substrate concentration, complex post-treatment and the like in the prior art.

Inventors

  • YU XINYAN
  • WANG JIARUI

Assignees

  • 杭州文德阶生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260204

Claims (7)

  1. 1. A high-activity and stereoselective alcohol dehydrogenase mutant is characterized in that the alcohol dehydrogenase mutant is obtained by single mutation or multiple mutation of amino acid 187 and/or 211 of an amino acid sequence shown in SEQ ID NO. 2.
  2. 2. The alcohol dehydrogenase mutant according to claim 1, wherein the alcohol dehydrogenase mutant is characterized in that the amino acid sequence shown in SEQ ID NO.2 is mutated into one of (1) threonine at position 187 into glycine, (2) leucine at position 211 into alanine, (3) threonine at position 187 into glycine, and leucine at position 211 into alanine.
  3. 3. A recombinant genetically engineered bacterium comprising a gene encoding the alcohol dehydrogenase mutant of claim 1.
  4. 4. An application of the alcohol dehydrogenase mutant in synthesizing the intermediate of cinnolamine ester, which is characterized in that wet thalli obtained by fermenting recombinant genetically engineered bacteria expressing the alcohol dehydrogenase mutant are used as catalysts, 1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethane-1-ketone is used as a substrate, organic alcohol is used as a reaction medium to form a reaction system, the reaction is carried out at 200rpm and 30-37 ℃, after the reaction is finished, a reaction solution containing (R) -1- (2-chlorophenyl) -2- (2H tetrazol-2-yl) ethanol is obtained, and the reaction solution is separated and purified to obtain (R) -1- (2-chlorophenyl) -2- (2H tetrazol-2-yl) ethanol.
  5. 5. The use according to claim 4, wherein the organic alcohol comprises isopropanol.
  6. 6. The process according to claim 4, wherein the catalyst is used in an amount of 10 to 200g/L based on the weight of wet cells and the substrate is added in a concentration of 300 to 500g/L.
  7. 7. The method according to claim 4, wherein the wet cells are prepared by inoculating recombinant engineering bacteria expressing an alcohol dehydrogenase mutant into LB liquid medium containing kanamycin at a final concentration of 50mg/L, culturing for 8 hours at 37 ℃ to obtain seed liquid, inoculating the seed liquid into sterile LB liquid medium containing kanamycin at a final concentration of 50mg/L at an inoculum size of 1-2% by volume, culturing for 1.5-2.5 hours at 37 ℃ and 180rpm to obtain a cell concentration OD 600 of 0.4-0.8, adding isopropylthio-beta-D-galactoside at a final concentration of 0.1-1.0mM into the culture liquid, inducing expression for 12-14 hours at 26 ℃, centrifuging for 30 minutes at 4 ℃ and 4000rpm, and collecting the wet cells.

Description

Ethanol dehydrogenase mutant, engineering bacterium and application thereof in synthesis of cinnolamine ester intermediate Field of the art The invention belongs to the field of biochemical engineering, and in particular relates to an ethanol dehydrogenase mutant, recombinant genetic engineering bacteria and application thereof in catalyzing and synthesizing a cinnolamine ester intermediate. (II) background art Cinnolamine ester (cenobamate), chemical name [ (1R) -1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethyl ] carbamate. Developed by SK biopharmaceutical (SK Biopharmaceuticals) in korea, approved by FDA at 11 months in 2019, under the trade name Xcopri. As a sodium channel blocker [ gamma-aminobutyric acid (gamma-aminobutyric acid, GABAA) positive allosteric modulator of ion channels ], can inhibit voltage-gated sodium current, reduce repetitive neuron discharge, is mainly used for treating partial epileptic seizures of adult patients clinically, provides a new choice for treating adult patients with focal epileptic seizures, and has wide market prospect. Industrial synthesis of cinnolamine esters there are a number of synthetic routes, the most critical of which is how to efficiently prepare the intermediate (R) -1- (2-chlorophenyl) -2- (2H tetrazol-2-yl) ethanol (a) in high purity. Improvements in the technical route in recent years have focused mainly on optimizing the steps for the synthesis of 1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethane-1-one (B) in the preparation of higher purity 1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethane-1-one (B) as in patent CN102803233A and patent CN 102574821A. However, in the step of converting the ketone (B) into the chiral alcohol (A), the existing technical scheme has the problems of low substrate concentration, complex post-treatment and the like, and limits the production efficiency. (III) summary of the invention The invention aims to provide an alcohol dehydrogenase mutant, engineering bacteria and application thereof in synthesizing a cinnolamine ester intermediate, wherein the alcohol dehydrogenase mutant is used for catalyzing prochiral ketone (B) to synthesize chiral alcohol (A), the method has high substrate concentration, simple and convenient post-treatment steps, is more suitable for industrial scale-up production, and solves the problems of low substrate concentration, complex post-treatment and the like in the prior art. The technical scheme adopted by the invention is as follows: The invention provides a high-activity and stereoselective alcohol dehydrogenase mutant, which is obtained by single mutation or multiple mutation of amino acid 187 and/or 211 of an alcohol dehydrogenase amino acid sequence which is shown in SEQ ID NO.2 and is derived from candida utilis (Candida orthopsilosis Co). Further, the alcohol dehydrogenase mutant is one of (1) mutation of threonine at position 187 into glycine (T187G), (2) mutation of leucine at position 211 into alanine (L211A), (3) mutation of threonine at position 187 into glycine, and mutation of leucine at position 211 into alanine (T187G/L211A) by mutating the amino acid sequence shown in SEQ ID NO. 2. The recombinant vector of the invention is not limited as long as the recombinant vector can keep the replication or autonomous replication in various host cells of procaryotic and eucaryotic cells, and the vector can be various vectors conventional in the art, such as various plasmids, phage or viral vectors, and the like, preferably pET-28a (+) plasmid is used as an expression vector, and Escherichia coli is used as an expression host (Escherichia coli BL21 cells or Escherichia coli DH5 alpha). The invention provides an application of an alcohol dehydrogenase mutant in the synthesis of a cinnolate intermediate, namely, catalyzing 1- (2-chlorophenyl) -2- (2H-tetrazole-2-yl) ethane-1-ketone to synthesize (R) -1- (2-chlorophenyl) -2- (2H-tetrazole-2-yl) alcohol, wherein the application method comprises the following steps: the method comprises the steps of taking wet thalli obtained by fermenting and culturing recombinant genetically engineered bacteria expressing an alcohol dehydrogenase mutant as a catalyst, taking 1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethane-1-ketone as a substrate, taking organic alcohol as a reaction medium to form a reaction system, reacting at 200rpm and 30-37 ℃, obtaining a reaction solution containing (R) -1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethanol after the reaction is finished, and separating and purifying the reaction solution to obtain (R) -1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethanol. Further, the organic alcohol includes isopropyl alcohol. Further, in the reaction system, the catalyst is used in an amount of 10 to 200g/L (preferably 50 g/L) based on the weight of the wet cell, and the substrate is added in a concentration of 300 to 500g/L (preferably 400 g/L). Further, the wet cell is prepared by inoculating recombinant engineering bacteria expressing an