CN-119776301-B - Alcohol dehydrogenase, recombinant genetically engineered bacterium and application thereof in asymmetric reduction of potential chiral carbonyl compounds

Abstract

The invention discloses an alcohol dehydrogenase, recombinant genetic engineering bacteria and application thereof in asymmetric reduction of a potential chiral carbonyl compound, wherein the alcohol dehydrogenase has good biological performance, heterologous expression in the engineering bacteria of escherichia coli is realized, intracellular coenzyme can be directly utilized when whole cells are used as catalysts, zero addition of the coenzyme is realized, and the enzyme has the advantages of high activity, high stereoselectivity, wider substrate spectrum and the like, can catalyze the transformation of the potential chiral ketone compound into the corresponding chiral alcohol in a green and efficient way, and solves the problems of low transformation rate, low yield, low e.e. value, complex reaction process, multiple byproducts and the like in the traditional chemical method. The alcohol dehydrogenase has good tolerance to isopropanol, and can realize the recycling of coenzyme by taking the isopropanol as a hydrogen source. And the by-product of the enzyme catalytic reaction, namely acetone and the residual isopropanol of the reaction can be separated and recovered by a technical means, so that the production cost is greatly reduced.

Inventors

• YU XINYAN
• WANG JIARUI
• Hu Wenye
• WU YOUCAI

Assignees

• 杭州文德阶生物科技有限公司

Dates

Publication Date

20260505

Application Date

20241210

Claims (3)

1. 1. The application of the alcohol dehydrogenase in preparing chiral alcohol by catalyzing asymmetric reduction of a latent chiral carbonyl compound is characterized in that wet thalli obtained by fermenting and culturing recombinant genetic engineering bacteria containing an alcohol dehydrogenase encoding gene are used as a catalyst, a latent chiral ketone compound is used as a substrate, isopropanol is used as a reaction medium to form a reaction system, the reaction is carried out at 200-700rpm and 37 ℃, a reaction liquid containing chiral alcohol is obtained after the reaction is finished, the reaction liquid is separated and purified to obtain the required chiral alcohol, the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO.2, and the latent chiral ketone compound is 3-methyl-2-butanone, 4-methyl-2-pentanone, 2-hexanone, 2' -chloroacetophenone, 3' -chloroacetophenone, 2' -fluoroacetophenone, o-nitroacetophenone, p-bromoacetophenone, 4-iodoacetophenone, 4-acetylpyridine, 2-naphthyridine, 1-naphthyridine ketone, 2-acetylthiazole and 1- (3-bromopyridine-2-yl) ethanone.
2. 2. The use according to claim 1, wherein the catalyst is used in an amount of 50 to 300g/L based on the weight of wet cells and the initial substrate addition concentration is 0.1 to 1M in the reaction system.
3. 3. The method according to claim 1, wherein the wet cell is prepared by inoculating recombinant engineering bacteria containing genes encoding alcohol dehydrogenase into LB culture solution containing kanamycin at a final concentration of 50mg/L, culturing for 8 hours at 37 ℃ to obtain seed solution, inoculating the seed solution into sterile LB liquid culture medium containing kanamycin at a final concentration of 50mg/L at a volume concentration of 2%, culturing for 1.5-2.5 hours at 37 ℃ to obtain a cell concentration OD600 of 0.4-0.8, adding isopropylthio-beta-D-galactoside at a final concentration of 0.1-1.0mM into the culture solution, and centrifuging for 10-20 minutes at 4 ℃ and 4000rpm after induction expression for 12 hours at 26 ℃, and collecting the wet cell.

Description

Alcohol dehydrogenase, recombinant genetically engineered bacterium and application thereof in asymmetric reduction of potential chiral carbonyl compounds Field of the art The invention belongs to the technical field of bioengineering, and particularly relates to an alcohol dehydrogenase, a coding gene, recombinant engineering bacteria and application thereof in preparing chiral alcohol with high optical activity by asymmetrically reducing latent chiral carbonyl compounds. (II) background art Chiral alcohols are important intermediates for many off-the-shelf drugs and chiral chemicals. Wherein chiral alpha-phenethyl alcohol and derivatives thereof are important chiral building blocks widely applied to the manufacture of high added value products such as medicines, fine chemicals, agrochemicals and the like, for example, (1S) -2-chloro-1- (3, 4-difluorophenyl) ethanol is a novel anti-platelet aggregation medicine intermediate, (R) -3, 5-bis (trifluoromethyl) -phenethyl alcohol is a traditional Chinese medicine intermediate of a chemotherapeutical antiemetic agent aprepitant, atorvastatin calcium is a third-generation total synthetic statin medicine, the blood lipid reducing effect is outstanding, the application in the prevention and treatment field of cardiovascular and cerebrovascular diseases is wide, the only prescription medicine which accumulates and sells more than 1500 hundred million dollars at present, (S) -4-chloro-3-hydroxybutyrate ethyl ester is the first chiral hydroxyl for constructing atorvastatin calcium, carbapenem and carbapenem medicines have wide antibacterial spectrum, outstanding antibacterial activity and stability to most beta-lactamase, are considered as a first-line medicine for treating serious hospital infection, 4-acetoxyazetidine [ (2R, 3R) -3- (t-butyloxy) ethyl-4-butanone-4-oxetane is a key intermediate in the production of 2-acetyl-azetidine acetate, methyl (2S, 3R) -2- [ (benzoylamino) methyl ] -3-hydroxybutyrate is a hotspot for the synthesis of 4-acetoxyazetidinone. The novel phosphine ligand has the advantages of high reactivity, enantioselectivity and the like, can be used for various metal catalytic reactions, and is widely applied to chiral side chain frameworks in the latest synthetic route of the phosphine ligand, such as chiral symmetrical diols of (2R, 3R) -2, 3-butanediol and the like. Alcohol dehydrogenase (Alcohol dehydrogenase, ADH), which is a key enzyme for metabolism of major short-chain alcohols in organisms, is found in large numbers in human and animal livers, plants and microbial cells, plays an important role in many physiological processes, has a wide substrate specificity, and is a key enzyme for catalyzing asymmetric reduction of prochiral ketones to chiral alcohols. The traditional chiral alcohol synthesis requires an expensive chiral noble metal catalyst, severe reaction conditions and a flammable and explosive borohydride reducing agent, and has the defects of low optical purity of products, complex separation and purification process, difficult three-waste treatment, high production cost and the like. ADH catalyzed carbonyl asymmetric reduction reaction has the outstanding advantages of wide enzyme source, outstanding activity and stereoselectivity, mild reaction condition, high optical purity of products, low production cost, environmental friendliness and the like, and has wide application prospect in chiral alcohol synthesis. Therefore, finding an economically readily available ADH capable of catalyzing high stereoselective asymmetric reduction at high substrate concentrations, with no or very little coenzyme added, is certainly of great significance. (III) summary of the invention The invention aims to provide an alcohol dehydrogenase, a coding gene, a recombinant genetic engineering bacterium and application thereof in preparing chiral alcohol with high optical activity by asymmetrically reducing a latent chiral carbonyl compound, wherein the enzyme has the advantages of high activity, strong stereospecificity, mild reaction conditions, simplicity and convenience in operation and the like, can economically and environmentally produce various chiral alcohols with high optical selectivity, has good application prospects, and solves the problems of low optical purity, complex separation and purification process, difficult three-waste treatment, high production cost and the like of products in the traditional chiral alcohol production method. The technical scheme adopted by the invention is as follows: the invention provides a high-activity alcohol dehydrogenase derived from candida utilis (Candida orthopsilosis Co), and the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO. 2. The invention also provides a coding gene of the alcohol dehydrogenase, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. The invention also relates to a recombinant vector containing the coding gene of the alcohol dehydrogenas