固氮类芽孢杆菌YUPP-5中环糊精糖基转移酶基因的克隆与功能分析

从魔芋根际分离的固氮类芽孢杆菌Paenibacillus azotofixans YUPP-5对多种β-1,4糖苷键连接的多糖具有水解作用。通过构建该菌的fosmid文库,克隆到2 157 bp的基因片段,编码环糊精糖基转移酶。大肠杆菌中表达此酶,能降解葡甘聚糖、羧甲基纤维素钠盐、几丁质、木聚糖等多种β-1,4糖苷键连接的多糖,同时该酶还能以葡甘聚糖为底物生成β-1,4糖苷键连接的环糊精,而文献报道这种酶仅能利用α-1,4糖苷键连接的淀粉为底物生成环糊精;并展示了环糊精糖基转移酶的一些新功能。

Cloning and Functional Analysis of CGTase Gene from Paenibacillus azotofixans YUPP-5

Paenibacillus azotofixans YUPP-5,which was isolated from the rhizosphere of Amorphophallus konjac,is able to hydrolyze polysaccharide with β-1,4 linkage,including glucomannan,galactomannan,xylan,carboxymethyl cellulose,and chitin.To clone the relative encoding genes,we constructed the fosmid library of strain YUPP-5 to pick out the transformant with activity of degrading glucomannan.By screening fosmid library,the encoding gene had an open reading frame of 2 157 bp,which deduced cyclodextrin glycosyltransferase(CGTase),including 718 amino acids with a signal peptide in the N-terminal region.The gene was expressed in Escherichia coli BL21.The purified CGTase exhibited strong activity in degrading polysaccharides with β-1,4 linkage,and forming cyclodextrin using glucomannan as substrate.The CGTase with some new function was different from others reported in previous literature.