环糊精糖基转移酶定点突变及其产物特异性分析

doi:10.7506/spkx1002-6630-201617022

摘要/Abstract

摘要：

环糊精葡萄糖基转移酶（cyclodextrin glycosyltransferase，CGTase）是一种多功能酶，能够催化淀粉生成环糊精（cyclodextrin，CD），本研究考察了Bacillus cereus的CGTase活性区域43位氨基酸与CGTase酶活力及其催化玉米淀粉形成γ-CD能力的联系。实验中共构建17 株突变株，并在大肠杆菌BL21中实现异源表达。结果表明：相比于原始酶，H43E、H43F、H43W、H43Y突变酶的活力均有所提高；产物特异性方面，除H43W突变酶外，其余突变酶的催化产物中γ-CD比例均有所升高，将43位组氨酸突变成脯氨酸、异亮氨酸和苏氨酸后得到的突变酶作用于玉米淀粉，酶的催化产物中β-CD含量均下降，γ-CD含量由20%分别升高到30.2%、28.4%、29.2%，实际产量由4.76 g/L升高到7.64、6.05、6.29 g/L，由此可见Bacillus cereus的CGTase活性区域中43位氨基酸对于CGTase的活力和产物特异性具有重要影响。

关键词: 环糊精糖基转移酶, 定点突变, 产物特异性

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

中图分类号:

Q814

凌凯，张洪斌，刘鹏飞，胡雪芹. 环糊精糖基转移酶定点突变及其产物特异性分析[J]. 食品科学, doi: 10.7506/spkx1002-6630-201617022.

LING Kai, ZHANG Hongbin, LIU Pengfei, HU Xueqin. Site-Directed Mutation of Cyclodextrin Glycosyltransferase and Analysis of Its Product Specificity[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201617022.

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环糊精糖基转移酶定点突变及其产物特异性分析

Site-Directed Mutation of Cyclodextrin Glycosyltransferase and Analysis of Its Product Specificity

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