# US 11,078,465 B2 · Alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohols

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- Number: US 11,078,465 B2
- Document ID: US11078465B2
- Status: Granted
- Kind: B2
- Application number: 16/521,067
- Filed: 2019-07-24
- Published: 2019-11-14
- Granted: 2021-08-03
- Earliest claimed: 2018-02-12
- Assignees: Jiangnan University
- Applicants: Jiangnan University
- Inventors: Ye Ni; Yue Wang; Wei Dai; Guochao Xu; Jieyu Zhou
- Web page: https://patentgrep.com/patent/US11078465B2

## CPC

- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12P 17/12: containing a six-membered hetero ring
- C12P 41/002: {by oxidation/reduction reactions}
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

The present disclosure discloses an alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohols, and belongs to the technical field of bioengineering. The alcohol dehydrogenase mutant of the present disclosure has excellent catalytic activity and stereoselectivity, and may efficiently catalyze the preparation of a series of chiral diaryl alcohols in R- and S-configurations. By coupling alcohol dehydrogenase of the present disclosure to glucose dehydrogenase or formate dehydrogenase, the synthesis of chiral diaryl alcohol intermediates of various antihistamines may be achieved. Compared with the prior art, a method for preparing diaryl chiral alcohols through asymmetric catalytic reduction using the alcohol dehydrogenase of the present disclosure has the advantages of simple and convenient operation, high substrate concentration, complete reaction and high product purity, and has great industrial application prospects.

## Claims

### Claim 1

An alcohol dehydrogenase mutant, wherein the alcohol dehydrogenase mutant comprises an amino acid sequence having all of SEQ ID NO:2 except for:
a substitution of valine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2;
a substitution of tyrosine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2;
a substitution of isoleucine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2;
a substitution of glutamine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2;
a substitution of serine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2;
a substitution of asparagine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2;
a substitution of arginine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2;
a substitution of valine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of alanine for serine at position 237;
a substitution of tyrosine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of alanine for serine at position 237;
a substitution of isoleucine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of alanine for serine at position 237;
a substitution of glycine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of cysteine for serine at position 237;
a substitution of glutamine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of cysteine for serine at position 237;
a substitution of serine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of cysteine for serine at position 237;
a substitution of asparagine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of cysteine for serine at position 237; and
a substitution of arginine for glutamate at position 214 of the amino acid sequence SEQ ID NO: 2, and the substitution of cysteine for serine at position 237, and
wherein the alcohol dehydrogenase mutant has alcohol dehydrogenase activity.

### Claim 2 (depends on 1)

A method for producing chiral (4-chlorophenyl)-(pyridin-2-yl)-methanol (CPMA), which comprises:
combining the alcohol dehydrogenase mutant of claim 1 at a concentration of 1 to 10 kU/L with prochiral (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK) at a concentration of 10 to 500 mM, and NADP+at a concentration of 0.1 to 1.0 mM;
adding a coenzyme circulation system comprising glucose dehydrogenase at a concentration of 1 to 10 kU/L, D-glucose at a concentration of 20 to 1000 mM, and a phosphate buffer;
incubating the coenzyme circulation system with the alcohol dehydrogenase mutant, CPMK, and NADP+at 30 to 35° C. and a pH of 6 to 8 for 1 to 24 hours to produce CPMA; and
extracting the CPMA by adding an organic solvent after an asymmetric reduction reaction;
wherein the coenzyme circulation system further comprises: (i) phosphite and phosphite dehydrogenase (FTDH), (ii) formic acid and formate dehydrogenase (FDH), (iii) lactic acid and lactate dehydrogenase (LDH), or (iv) glycerol and glycerol dehydrogenase.