瑞氏木霉内切葡聚糖酶Ⅰ及其CBD的噬菌体表面展示系统的构建

Phage display of endoglucanase Ⅰ from Trichoderma reesei and its cellulose-binding domain

为研究纤维素酶的酶活性和吸附特性之间的关系,将瑞氏木霉内切葡聚糖酶Ⅰ(endoglucanaseⅠ,EGⅠ)和其吸附结构域(cellulose-binding domains,CBD)的基因经PCR扩增后,连接到载体pCANTAB5E上,构建成噬菌粒展示载体pCANTAB5E-egⅠ和pCANTAB5E-egⅠ-cbd。将这些噬菌体展示载体转化到大肠杆菌TGⅠ中,在辅助噬菌体M13KO7的帮助下得到重组噬菌体。用ELISA和Somogyi方法分别测定了EGⅠ及其CBD的表面展示噬菌体对纤维素的亲和性和EGⅠ的CMC活性,结果显示EGⅠ及其CBD已功能展示于噬菌体表面。该系统的成功构建为今后利用噬菌体展示的方法进行纤维素酶的酶分子改造提供了基础。

To study the relationship between the binding ability and the catalytic activity of cellulase, endoglucanase Ⅰ （EG Ⅰ ） of Trichoderma reesei and its cellulose-binding domain （CBD） were constructed into phagemids PCANTABSE respectively as pCANTABSE-eg Ⅰ and pCANTABSE-eg Ⅰ-cbd, which were then trans- formed into E. coli TG Ⅰ I, expressing EG Ⅰ and its CBD on the phage tip by the co-infected with M13K07 helper phage. The ability of binding cellulose of these phage displayed enzymes was proved by ELISA, while the endoglucanase activity was estimated by Somogyi method. All these results showed that EG Ⅰ and its CBD are successfully displayed on the M13 phage, and it would be greatly helpful for directed evolution of cellulase using phage display as a tool.