Carbonyl reductase oligomers and their application in synthesis of chiral alcohols

Patent metadata

Field	Value
Publication number	US-10337033-B2
Application number	US-201715650852-A
Country	US
Kind code	B2
Filing date	Jul 15, 2017
Priority date	Dec 30, 2016
Publication date	Jul 2, 2019
Grant date	Jul 2, 2019

Abstract

Disclosed are methods for the synthesis of chiral alcohols by Sortase A-mediated oxidoreductase oligomers, which relates to the field of biocatalysis. In the present disclosure, oxidoreductase oligomers were used as biocatalysts for chiral alcohol preparation. Compared to wild-type enzymes, the oxidoreductase oligomers significantly improved catalytic activity and thermal stability. The sortase A-mediated oxidoreductase oligomers had 6-8 folds improvement in specific activity over that of the wild-type enzymes. The oligomers displayed a Tm value 6-12° C. higher than that of the wild-type, suggesting the sortase A-mediated oxidoreductase oligomers significantly improved thermostability of the enzymes. The oxidoreductase oligomers catalyzed asymmetric transformation to produce (S)-1-phenyl-1,2-ethanediol or (R)-1-phenethyl alcohol within 3-6 hr, with an optical purity of 98%-100% and a yield of 98%-99%.

First claim

What is claimed is:

1. A carbonyl reductase oligomer, wherein said carbonyl reductase oligomer comprises two of the same carbonyl reductase polypeptide, wherein the C-terminus of a first carbonyl reductase polypeptide is connected to the N-terminus of a second carbonyl reductase polypeptide by a sortase A (Srt A) recognition sequence via a Srt A mediated ligation mechanism, wherein said carbonyl reductase is selected from a group consisting of SCRII of SEQ ID NO: 1, SCR1 of SEQ ID NO:18, SCR3 of SEQ ID NO:19, CR2 of SEQ ID NO:20, and CR4 of SEQ ID NO:21; and wherein said carbonyl reductase oligomer has higher specific activity than that of the wild-type counterpart enzyme.

2. The carbonyl reductase of claim 1 , wherein said Srt A recognition sequence is LPXTG, wherein X is any amino acid moiety.

3. The carbonyl reductase oligomer of claim 1 , wherein said oxidoreductase oligomer is prepared as follows:
(1) obtaining the first carbonyl reductase polypeptide with said SrtA recognition sequence added to its C-terminus;
(2) using SrtA to cleave said SrtA recognition sequence to obtain a thioester intermediate; and
(3) using said thioester intermediate of the first carbonyl reductase polypeptide to react with a Gly at the N-terminus of the second carbonyl reductase to form said carbonyl reductase oligomer.

4. The carbonyl reductase oligomers of claim 3 , wherein step (3) is carried out in the presence of Ca 2+ .

5. The carbonyl reductase oligomer of claim 1 , wherein said SrtA is from Staphylococcus aureus.

6. A method for preparing chiral alcohols, comprising contacting a carbonyl reductase substrate with the carbonyl reductase oligomer of claim 2 to produce said chiral alcohol.

7. The method of claim 6 , wherein the chiral alcohols are (S)-1-phenyl-1,2-ethanediol or (R)-1-phenethyl alcohol.

Assignees

• Zhang Rongzhen
• Xu Yan
• Li Kunpeng
• Univ Jiangnan

Inventors

• Zhang Rongzhen
• Xu Yan
• Li Kunpeng

Classifications

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12N9/0006

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

• C07B2200/07

  Optical isomers · CPC title

• C12P7/22

  aromatic · CPC title