Mutant of Cyclodextrin Glycosyltransferase

Patent metadata

Field	Value
Publication number	US-2020181585-A1
Application number	US-201916388688-A
Country	US
Kind code	A1
Filing date	Apr 18, 2019
Priority date	Dec 7, 2018
Publication date	Jun 11, 2020
Grant date	—

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 enzyme in shake flask fermentations. The present invention has certain significance for the industrial production of cyclodextrin glycosyltransferase, and improves the application potential of the enzyme in food, medicine and chemical industries.

First claim

What is claimed is:

1 . A mutant of cyclodextrin glycosyltransferase, comprising one or more mutations of valine (Val) at site 6, serine (Ser) at site 90, threonine (Thr) at site 168, threonine (Thr) at site 171, threonine (Thr) at site 383 and glycine (Gly) at site 608 in cyclodextrin glycosyltransferase derived from Bacillus circulans.

2 . The mutant of claim 1 , wherein the amino acid sequence of the cyclodextrin glycosyltransferase derived from Bacillus circulans is set forth in SEQ ID NO. 2.

3 . The mutant of claim 1 , comprising:
(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; or (b) a mutation of the serine (S) at site 90 to glycine (S90G) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (c) a mutation of the threonine (T) at site 168 (T168A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (d) a mutation of the threonine (T) at site 171 (T171A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (e) a mutation of the threonine (T) at site 383 (T383A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (f) a mutation of the glycine (G) at site 608 (G608A) to alanine in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2; or (g) 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 (V6D/S90G/T168A/T171A/T383A/G608A) in the cyclodextrin glycosyltransferase with the amino acid sequence set forth in SEQ ID NO. 2.

4 . A method for constructing the 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 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.

5 . A method for constructing the mutant of claim 2 , 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 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.

6 . A method for constructing the mutant of claim 3 , 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 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.

7 . The method of claim 4 , wherein the plasmid vector is any one of pUC series, pET series, or pGEX.

8 . The method of claim 4 , wherein the plasmid vector is any one of pUC series, pET series, or pGEX.

9 . The method of claim 4 , wherein the plasmid vector is any one of pUC series, pET series, or pGEX.

10 . The method of claim 4 , wherein the plasmid is cgt/pET20b(+).

11 . The method of claim 6 , wherein the plasmid is cgt/pET20b(+).

12 . The method of claim 4 , wherein the host cell is a bacterial or fungal cell.

13 . The method of claim 6 , wherein the host cell is a bacterial or fungal cell.

14 . A method to use the mutant of claim 1 in the fields of food, medicine and chemical industry, comprising adding the mutant as an enzyme to a preparation of a food, a medicine or a chemical.

Assignees

• Univ Jiangnan

Inventors

• Wu Jing
• Su Lingqia
• Du Li

Classifications

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• A23L29/06

  Enzymes · CPC title

• C12Y204/01019 Primary

  Cyclomaltodextrin glucanotransferase (2.4.1.19) · CPC title

• C12N9/1048 Primary

  Glycosyltransferases (2.4) · CPC title

• C12N2800/101

  for bacteria · CPC title