Mutant of cyclodextrin glycosyltransferase

Patent metadata

Field	Value
Publication number	US-11566235-B2
Application number	US-201916388688-A
Country	US
Kind code	B2
Filing date	Apr 18, 2019
Priority date	Dec 7, 2018
Publication date	Jan 31, 2023
Grant date	Jan 31, 2023

Abstract

The present invention discloses a mutant of cyclodextrin glycosyltransferase and belongs to the fields of gene engineering and enzyme engineering. According to the present invention, a mutant having higher disproportionation activity of cyclodextrin glycosyltransferase is obtained by mutating the cyclodextrin glycosyltransferase. The disproportionation activity of enzymes of mutants V6D, S90G, T168A, T171A, T383A, G608A, and V6D/S90G/T168A/T171A/T383A/G608A, is respectively 1.89 times, 1.21 times, 1.21 times, 1.22 times, 1.32 times, 2.03 times, and 3.16 times that of the wild type enzyme in shake flask fermentations.

First claim

What is claimed is:

1. A mutant of cyclodextrin glycosyltransferase, consisting of one of the following mutations:
(a) a mutation of the valine (V) at site 6 to aspartic acid (V6D) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2;
(b) a mutation of the threonine (T) at site 171 to alanine (T171A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2;
(c) a mutation of the threonine (T) at site 383 to alanine (T383A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2;
(d) a mutation of the glycine (G) at site 608 to alanine (G608A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2; or
(e) a mutation of the valine (V) at site 6 to the aspartic acid, a mutation of the serine (S) at site 90 to glycine, a mutation of the threonine (T) at site 168 to alanine, a mutation of the threonine (T) at site 171 to alanine, a mutation of the threonine (T) at site 383 to alanine, and a mutation of the glycine (G) at site 608 to alanine,
wherein the cyclodextrin glycosyltransferase is derived from Bacillus circulans , and
wherein the amino acid sequence of the cyclodextrin glycosyltransferase derived from Bacillus circulans is set forth in SEQ ID NO: 2.

2. The mutant cyclodextrin glycosyltransferase of claim 1 , wherein the mutation is a mutation of the valine (V) at site 6 to aspartic acid (V6D) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2.

3. The mutant cyclodextrin glycosyltransferase of claim 1 , wherein the mutation is a mutation of the threonine (T) at site 171 to alanine (T171A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2.

4. The mutant cyclodextrin glycosyltransferase of claim 1 , wherein the mutation is a mutation of the threonine (T) at site 383 to alanine (T383A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2.

5. The mutant cyclodextrin glycosyltransferase of claim 1 , wherein the mutation is a mutation of the glycine (G) at site 608 to alanine (G608A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO: 2.

6. The mutant cyclodextrin glycosyltransferase of claim 1 , wherein the mutation is a mutation of the valine (V) at site 6 to the aspartic acid, a mutation of the serine (S) at site 90 to glycine, a mutation of the threonine (T) at site 168 to alanine, a mutation of the threonine (T) at site 171 to alanine, a mutation of the threonine (T) at site 383 to alanine, and a mutation of the glycine (G) at site 608 to alanine.

7. A method for constructing the cyclodextrin glycosyltransferase mutant of claim 1 , comprising:
(1) designing a site-directed mutagenesis mutant primer according to the determined mutation site, and performing site-directed mutagenesis using a vector carrying a cyclodextrin glycosyltransferase gene as a template; constructing a plasmid vector comprising a gene encoding the mutant;
(2) transforming the mutant plasmid vector into a host cell;
(3) selecting positive clones for fermentation culture, and centrifuging the supernatant to obtain a crude enzyme solution of the cyclodextrin glycosyltransferase mutant.

8. The method of claim 7 , wherein the plasmid vector is any one of pUC series, pET series, or pGEX.

9. The method of claim 7 , wherein the plasmid vector is cgt/pET20b(+).

10. The method of claim 7 , wherein the host cell is a bacterial or fungal cell.

Assignees

• Univ Jiangnan

Inventors

• Wu Jing
• Su Lingqia
• Du Li

Classifications

• C12Y204/01019 Primary

  Cyclomaltodextrin glucanotransferase (2.4.1.19) · CPC title

• C12N9/1048 Primary

  Glycosyltransferases (2.4) · CPC title

• A23L29/06

  Enzymes · CPC title

• A23L33/13

  Nucleic acids or derivatives thereof (A23L33/145 takes precedence) · CPC title

• A23V2002/00

  Food compositions, function of food ingredients or processes for food or foodstuffs · CPC title