Preparation and application of cyclodextrin glucosyltransferase mutant

Patent metadata

Field	Value
Publication number	US-10876099-B2
Application number	US-201815991322-A
Country	US
Kind code	B2
Filing date	May 29, 2018
Priority date	Jan 15, 2018
Publication date	Dec 29, 2020
Grant date	Dec 29, 2020

Abstract

The present invention relates to preparation and application of a cyclodextrin glucosyltransferase mutant, belonging to the fields of gene engineering and enzyme engineering. By mutating amino acids of cyclodextrin glucosyltransferase, the enzyme activity of the obtained mutant can reach 2.5 times that of wild enzyme. In addition, the cyclodextrin glucosyltransferase mutant obtained in the present invention is simple in purification and suitable for industrial production.

First claim

What is claimed is:

1. A cyclodextrin glucosyltransferase mutant, wherein the cyclodextrin glucosyltransferase mutant has the amino acid sequence SEQ ID NO:1
except for one or more mutations selected from the group consisting of
K124E, G450S, I465F, I641T, K647E, I631T, double mutant I641T and K647E, and double mutant I641T and 1631T.

2. A method of constructing a recombinant bacterium expressing the mutant of claim 1 , comprising the following steps:
designing mutagenic primers of site-directed mutagenesis,
performing site-directed mutagenesis by using a vector comprising a gene encoding a parent cyclodextrin glucosyltransferase of SEQ ID NO:1 as a template to generate a gene encoding the mutant of claim 1 ;
constructing a plasmid vector containing the gene encoding the mutant of claim 1 ;
transforming the vector into a host cell;
selecting a positive clone,
culturing the positive clone, and
centrifuging the culture to generate a supernatant, wherein the supernatant is a crude enzyme solution of the cyclodextrin glucosyltransferase mutant of claim 1 .

3. The method of claim 2 , wherein the vector is a pUC, pET, or pGEX vector.

4. The method of claim 2 , wherein the host cell is a bacterial or fungal cell.

Assignees

• Univ Jiangnan

Inventors

• Wu Jing
• Su Lingqia
• Tao Xiumei
• Wang Yusheng
• Dong Junchen

Classifications

• C12N9/1074 Primary

  Cyclomaltodextrin glucanotransferase (2.4.1.19) · CPC title

• C12P19/60

  having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin {(C12P19/605)} · CPC title

• C12Y204/01019

  Cyclomaltodextrin glucanotransferase (2.4.1.19) · CPC title

• C12N15/70

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