融合基因umcel5N-CBM的构建、表达及融合酶性质分析

Construction and Expression of umcel5N-CBM Encoding Fused Endoglucanase Catalytic Domain and Carbohydrate-Binding Module and Characterization of the Fusion Enzyme

碳水化合物结合组件(carbohydrate-binding module,CBM)是一些糖基水解酶分子上的结构域,它在纤维素酶降解不可溶纤维素中起着重要的作用。本研究的目的是检测一个新的内切葡聚糖酶Umcel5N(GenBank登录号为ACH67609)加上一个碳水化合物结合组件后得到的融合酶是否获得降解结晶纤维素的能力。本文将编码内切葡聚糖酶Umcel5N的催化结构域(catalytic domain,CD)的序列与编码Umcel6A的CBM序列通过接头序列进行基因融合,得到融合基因umcel5N-CBM,并实现了融合基因在大肠杆菌BL21(DE3)pLysS中的表达。研究结果表明,融合酶Umcel5N-CBM与结晶纤维素(avicel)以及滤纸粉末的结合能力比原始酶Umcel5N提高了约一倍,但未显示出降解结晶纤维素的新活性,说明在结晶纤维素的降解过程中,纤维素酶的催化功能域起到关键作用。

Carbohydrate-binding module （CBM） is an important domain of some cellulases, which plays a key role in hydrolysis of insoluble cellulose. The purpose of this study is to detect crystalline cellulose degrading ability of fusion enzyme in which a new endoglucanase Umcel5N （GenBank accession No. ACH67609） was fused with a carbohydrate binding module. In this study, DNA fragment coding for the catalytic domain （CD） of Umcel5N was combined with sequence that encodes the CBM of Umcel6A to construct a fusion gene, umcel5N-CBM. The umcel5N-CBM was expressed in E. coli. The results showed that the binding of the fusion enzyme Umcel5N-CBM with crystalline cellulose （Avicel）, as well as with filter paper powder was double compared to that of wild-type enzyme Umcel5N. However, no new hydrolysis activity of the fusion enzyme on crystalline cellulose was observed. These data may indicate that the catalytic domain ofcellulase plays essential role in the hydrolysis of crystalline cellulose.