Alcohol Dehydrogenase Mutant and Application thereof in Synthesis of Diaryl Chiral Alcohols

Patent metadata

Field	Value
Publication number	US-2019345455-A1
Application number	US-201916521067-A
Country	US
Kind code	A1
Filing date	Jul 24, 2019
Priority date	Feb 12, 2018
Publication date	Nov 14, 2019
Grant date	—

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.

First claim

What is claimed is:

1 . An alcohol dehydrogenase mutant, wherein an amino acid sequence of the mutant comprises mutation of one or more amino acid sites in an amino acid sequence in SEQ ID NO. 2.

2 . The mutant according to claim 1 , wherein the mutation comprises mutation of amino acid glutamate at position 214 and amino acid serine at position 237 in an amino acid sequence in SEQ ID No. 2.

3 . The mutant according to claim 1 , wherein the mutation comprises substitution in any one of the following:
a substitution of valine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of tyrosine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of isoleucine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of glycine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of glutamine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of serine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of asparagine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of arginine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of valine for glutamate at position 214 of the amino acid sequence in 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 in 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 in 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 in 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 in 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 in 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 in 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 in SEQ ID No. 2, and the substitution of cysteine for serine at position 237.

4 . The mutant according to claim 2 , wherein the mutation comprises substitution in any one of the following:
a substitution of valine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of tyrosine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of isoleucine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of glycine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of glutamine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of serine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of asparagine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of arginine for glutamate at position 214 of the amino acid sequence in SEQ ID No. 2; a substitution of valine for glutamate at position 214 of the amino acid sequence in 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 in 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 in 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 in 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 in 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 in 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 in 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 in SEQ ID No. 2, and the substitution of cysteine for serine at position 237.

5 . A nucleotide sequence encoding the mutant according to claim 1 .

6 . A nucleotide sequence encoding the mutant according to claim 2 .

7 . A nucleotide sequence encoding the mutant according to claim 3 .

8 . A recombinant strain expressing the mutant according to claim 1 .

9 . A recombinant strain expressing the mutant according to any one of claim 2 .

10 . A recombinant strain expressing the mutant according to any one of claim 3 .

11 . A method for constructing the recombinant strain according to claim 8 , wherein the method comprises the following steps: cloning a nucleotide sequence encoding the mutant into a recombinant vector, transforming the resulting recombinant vector into a host to obtain a recombinant transformant, and culturing the recombinant transformant and conducting isolation and purification to obtain the mutant.

12 . The method according to claim 11 , wherein the host of the recombinant strain is Escherichia coli , and plasmid is pET28a (+).

13 . The method according to claim 11 , wherein the host of the recombinant strain is E. coli BL21 (DE3).

14 . The method according to claim 12 , wherein the host of the recombinant strain is E. coli BL21 (DE3).

15 . A method for producing an alcohol dehydrogenase mutant by using the recombinant strain according to claim 8 , wherein the method comprises: inoculating the recombinant strain into an LB medium containing 40-60 μg/mL kanamycin sulfate for shake cultivation at 30-40° C. and 100-200 rpm, adding 0.05-1.0 mM isopropyl-β-D-thiogalactofuranoside (IPTG) for induction at an inducing temperature of 16-30° C. when the absorbance OD 600 of a medium solution reaches 0.5-1.0, and inducing for 5-10 h to obtain the alcohol dehydrogenase mutant.

16 . A method for producing an alcohol dehydrogenase mutant by using the recombinant strain according to claim 9 , wherein the method comprises: inoculating the recombinant strain into an LB medium containing 40-60 μg/mL kanamycin sulfate for shake cultivation at 30-40° C. and 100-200 rpm, adding 0.05-1.0 mM isopropyl-β-D-thiogalactofuranoside (IPTG) for induction at an inducing temperature of 16-30° C. when the absorbance OD 600 of a medium solution reaches 0.5-1.0, and inducing for 5-10 h to obtain the alcohol dehydrogenase mutant.

17 . A method for producing an alcohol dehydrogenase mutant by using the recombinant strain according to claim 10 , wherein the method comprises: inoculating the recombinant strain into an LB medium containing 40-60 μg/mL kanamycin sulfate for shake cultivation at 30-40° C. and 100-200 rpm, adding 0.05-1.0 mM isopropyl-β-D-thiogalactofuranoside (IPTG) for induction at an inducing temperature of 16-30° C. when the absorbance OD 600 of a medi

Assignees

• Univ Jiangnan

Inventors

• Ni Ye
• Wang Yue
• Dai Wei
• Xu Guochao
• Zhou Jieyu

Classifications

• C12Y101/01001

  Alcohol dehydrogenase (1.1.1.1) · CPC title

• C12P17/12 Primary

  containing a six-membered hetero ring · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title