水解壳聚糖活性的双功能酶结构与功能研究

项目名称： 水解壳聚糖活性的双功能酶结构与功能研究

项目编号： No.20876068

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 夏文水

作者单位： 江南大学

项目金额： 35万元

中文摘要： 本项目在前期研究基础上，对绿色木霉非专一性产具有壳聚糖活性的两种双功能酶CCBE和Csn、米曲霉所产一种双功能酶CNBE和两种壳聚糖酶CSN、CSX进行了鉴定，明确了以上五种酶的糖苷水解水解酶家族归属、其水解壳聚糖的作用模式以及CCBE、CSN、CSX的基因序列，确证了丝状真菌绿色木霉所产壳聚糖酶与纤维素酶具有同源性；同时综合采用化学修饰、FT-IR、圆二色谱分析、删减突变、定点突变以及生物信息学分析等手段，进一步对绿色木霉产双功能酶CCBE和米曲霉产双功能酶CNBE、内切壳聚糖酶CSN作用壳聚糖的结构与功能关系进行了深入探讨，发现绿色木霉产双功能酶CCBE对壳聚糖的结合结构域与CMC并不相同：位于N-端附近，与C-CBD区域不相关；催化结构域位于序列中上游部位，与作用CMC的部分重叠；羧酸氨基酸Asp214和Glu217分别在双功能酶CCBE催化壳聚糖过程中起到亲核试剂和质子供体的作用。而米曲霉所产内切壳聚糖酶CSN作用壳聚糖结构域与已报告GH-75族壳聚糖酶基本一致。本研究为下一步研究丝状真菌发酵产壳聚糖酶的调控机制奠定了良好的理论基础。

中文关键词： 壳聚糖酶;分子克隆;重组表达；定点突变;酶结构与功能

英文摘要： Based on the previous research, two bifunctional chitosanase-cellulases(CCBE and Csn) from Trichoderma viride ,one bifuntional chitosanase-chitinase(CNBE) and two specific chitosanases(CSN and CSX) from Aspergillus oryzae are identified in glycoside hydrolase belongings and action-modes.The genes of CCBE and two specific chitosanases(CSN and CSX) have also been cloned,which confirmed that the chitosanases and cellulases secreted by Trichoderma viride shared homology. Moreover, the correlations between structure and function of the above CCBE and CNBE,CSN have also been discussed with the methods of chemical modification,FT-IR and circular dichroism spectrum,deletion and site-directed mutagenesis as well as the bio-informatics analysis,as a result, the dual activity of CCBE from Trichodema viride shared two distinct catalytic domains: the catalytic and the substrate-binding domains of CCBE for chitosan hydrolysis located in the upper-middle reaches of N-terminal, while those for CMC analysis located in the lower reaches of C-terminal. C-CBD is not essential for its chitosan catalysis. Asp214 and Glu217 may act as proton donor and nucleophile in its chitosan hydrolysis reaction,respectively. Whereas,the active domains of CSN from Aspergillus oryzae showed similar to those of the reported GH-75 family endo-chitosanases.The above results provided the theoretical guidance for studing the regulation mechanism of filamentous fungi secreting chitosanases.

英文关键词： chitosanase;molecular clone; recombination and expression;site-directed mutagenesis；enzyme structure and function