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

Patent metadata

Field	Value
Publication number	US-2019345457-A1
Application number	US-201916521656-A
Country	US
Kind code	A1
Filing date	Jul 25, 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 of one or two amino acid sites comprises mutation of amino acid phenylalanine at position 161 and mutation of amino acid serine at position 196 in the 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 serine at position 196 of the amino acid sequence in SEQ ID No. 2; a substitution of tryptophan for serine at position 196 of the amino acid sequence in SEQ ID No. 2; a substitution of proline for serine at position 196 of the amino acid sequence in SEQ ID No. 2; a substitution of glycine for serine at position 196 of the amino acid sequence in SEQ ID No. 2; and a substitution of glycine for serine at position 196 of the amino acid sequence in SEQ ID No. 2, and a substitution of valine for phenylalanine at position 161.

4 . The mutant according to claim 2 , wherein the mutation comprises substitution in any one of the following:
a substitution of valine for serine at position 196 of the amino acid sequence in SEQ ID No. 2; a substitution of tryptophan for serine at position 196 of the amino acid sequence in SEQ ID No. 2; a substitution of proline for serine at position 196 of the amino acid sequence in SEQ ID No. 2; a substitution of glycine for serine at position 196 of the amino acid sequence in SEQ ID No. 2; and a substitution of glycine for serine at position 196 of the amino acid sequence in SEQ ID No. 2, and a substitution of valine for phenylalanine at position 161.

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 claim 2 .

10 . A recombinant strain expressing the mutant according to 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 resulting recombinant vector into a host to obtain a recombinant transformant, and culturing the resulting recombinant expression 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 mutant for efficient expression of a recombinant alcohol dehydrogenase.

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 medium solution reaches 0.5-1.0, and inducing for 5-10 h to obtain the alcohol dehydrogenase mutant.

18 . A method for producing chiral CPMA using the alcohol dehydrogenase of claim 1 , wherein the method comprises the following steps: constructing a reaction system, wherein CPMK concentration is 10-500 mM, an amount of the alcohol dehydrogenase mutant is 1-10 kU/L, and an amount of NADP + is 0.1-1.0 mM; adding a coenzyme circulation system, wherein the coenzyme circulation system contains glucose dehydrogenase GDH and D-glucose, an amount of glucose dehydrogenase GDH is 1-10 kU/L, an amount of D-glucose dosage is 20-1000 mM, and a concentration of a phosphate buffer is 0.1-0.2 M; performing reaction at 30-35° C. and pH 6-8 for 1-24 h; and extracting the chiral CPMA from a reaction solution according to an organic solvent extraction method after asymmetric reduction reaction; where the coenzyme circulation system comprises phosphite/phosphite dehydrogenase (FTDH), formic acid/formate dehydrogenase (FDH), lactic acid/lactate dehydrogenase (LDH) or glycerol/glycerol dehydrogenase.

19 . A method for producing chiral CPMA using the alcohol dehydrogenase of claim 2 , wherein the method comprises the following steps: constructing a reaction system, wherein CPMK concentration is 10-500 mM, an amount of the alcohol dehydrogenase mutant is 1-10 kU/L, and an amount of NADP + is 0.1-1.0 mM; adding a coenzyme circulation system, wherein the coenzyme circulation system contains glucose dehydrogenase GDH and D-glucose, an amount of glucose dehydrogenase GDH is 1-10 kU/L, an amount of D-glucose dosage is 20-1000 mM, and a concentration of a phosphate buffer is 0.1-0.2 M; performing reaction at 30-35° C. and pH 6-8 for 1-24 h; and extracting the chiral CPMA from a reaction solution according to an organic solvent extraction method after asymmetric reduction reaction; wherein the coenzyme circulation system comprises phosphite/phosphite dehydrogenase (FTDH), formic acid/formate dehydrogenase (FDH), lactic acid/lactate dehydrogenase (LDH) or glycerol/glycerol dehydrogenase.

20 . A method for producing chiral CPMA using the alcohol dehydrogenase of claim 3 , wherein the method comprises the following steps: constructing a reaction system, wherein CPMK concentration is 10-500 mM, an amount of the alcohol dehydrogenase mutant is 1-10 kU/L, and an amount of NADP + is 0.1-1.0 mM; adding a coenzyme circulation system, wherein the coenzyme circulation system contains glucose dehydrogenase GDH and D-glucose, an amount of glucose dehydrogenase GDH is 1-10 kU/L, an amount of D-glucose dosage is 20-1000 mM, and a concentration of a phosphate buffer is 0.1-0.2 M; performing reaction at 30-35° C. and pH 6-8 for 1-24 h; and extracting the chiral CPMA from a reaction solution according to an organic solvent extraction method after asymmetric reduction reaction; wherein the coenzyme circulation system comprises phosphite/phosphite dehydrogenase (FTDH), formic acid/format

Assignees

• Univ Jiangnan

Inventors

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

Classifications

• C12Y101/01001

  Alcohol dehydrogenase (1.1.1.1) · CPC title

• C12N9/0006 Primary

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

• C12P17/12

  containing a six-membered hetero ring · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title