Polypeptide tag, highly soluble recombinant nitrilase and application thereof in synthesis of pharmaceutical chemicals

Patent metadata

Field	Value
Publication number	US-11807883-B2
Application number	US-202117240715-A
Country	US
Kind code	B2
Filing date	Apr 26, 2021
Priority date	Nov 3, 2020
Publication date	Nov 7, 2023
Grant date	Nov 7, 2023

Abstract

The present invention provides a polypeptide tag and its application in the synthesis of pharmaceutical chemicals, the recombinant nitrilase was obtained by connecting a polypeptide tag to the N-terminus of the amino acid sequence of the nitrilase; wherein amino acids at both ends of the polypeptide tag are uncharged glycine G, and the rest are a random combination of any one or more of glycine G, histidine H, glutamic acid E, aspartic acid D, lysine K and arginine R; The activity of the recombinant nitrilase in the preparation of 1-cyanocyclohexyl acetic acid is up to 3034.7 U/g dcw, the polypeptide tag significantly improves the soluble expression of nitrilase, and the whole cell catalyst hydrolyzes 1M substrate with the same concentration 30 minutes faster than the mother enzyme. The method provided by the present invention can also be used for the biocatalytic reaction of other pharmaceutical intermediates as the substrate catalyzed by the nitrilase, improving the activity of the whole cell catalyst in reaction, and also improving the solubility of other types of nitrilases and the activity of the corresponding whole cell catalysts.

First claim

The invention claimed is:

1. A recombinant nitrilase, comprising:
a polypeptide tag, wherein the polypeptide tag is attached to the recombinant nitrilase on the N terminus, wherein the polypeptide tag is selected from the group consisting of: GKGKG (SEQ ID NO: 3), GKGEG (SEQ ID NO: 4), GKGHG (SEQ ID NO: 5), GRGRG (SEQ ID NO: 6), GRGGG (SEQ ID NO: 7), GHGHG (SEQ ID NO: 8), GKGKGKG (SEQ ID NO: 26), and GKGKGKGKG (SEQ ID NO: 27), wherein the polypeptide tag is universal and enhances soluble expression of the recombinant nitrilase, wherein the recombinant nitrilase has the amino acid sequence set forth in SEQ ID NO: 1, and wherein enzyme activity of the recombinant nitrilase with the polypeptide tag is at least 101.6% of the original strain.

2. The recombinant nitrilase of claim 1 , wherein the polypeptide tag is connected to the recombinant nitrilase by a linker peptide, and wherein the linker peptide is selected from the group consisting of: GKGKG-GS (SEQ ID NO: 31), GKGKG-GGS (SEQ ID NO: 32), GKGKG-GGGS (SEQ ID NO: 33), and GKGKG-GGGGS (SEQ ID NO: 34).

3. The recombinant nitrilase of claim 1 , wherein the polypeptide tag is linked to a linker peptide, and the linker peptide is selected from the group consisting of: GS, GGS, GGGS (SEQ ID NO: 29), and GGGGS (SEQ ID NO: 30).

Assignees

• Univ Zhejiang Technology

Inventors

• Xue Yaping
• Xie Dong
• Xiong Neng
• Zheng Yuguo

Classifications

• C12N9/78 Primary

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title

• C12N9/20 Primary

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12P13/002

  Nitriles (-CN) · CPC title