Abstract:

Cyclodextrin glycosyltransferase (CGTase) is a multifunctional enzyme that converts starch into cyclodextrins
(CD). In this research, we applied site-directed mutagenesis to histidine residue 43 in the active site region of Bacillus
cereus cyclodextrin glycosyltransferase with the aim to study mutation effect on the enzyme activity and product specificity
of CGTase. A total of 17 mutant CGTases were obtained and heterologously expressed in E. coli BL21. Compared to the
wild-type CGTase, most of the mutations showed decreased enzyme activity expect H43F, H43W, H43Y and H43E; for
mutant H43P, H43I and H43T, the percentage of β-CD in hydrolysis products was decreased and γ-CD was increased
from 20% to 30.2%, 28.4% and 29.2%, respectively. The actual yield of γ-CD was increased from 4.76 g/L to 7.64, 6.05
and 6.29 g/L, respectively. It can be seen that the active-site residue 43 has important impact on CGTase activity from
Bacillus cereus as well as its product specificity.

Key words: cyclodextrin glycosyltransferase, site-directed mutagenesis, product specificity