木聚糖高温水解关键酶分子改造及其热稳定和耐糖机理

Enzymatic hydrolysis of cellulose and hemicellulose at high temperature has become a research focus in the field of biomass energy and chemicals. In view of existing key problem in the pyrohydrolysis of xylan, this research project is aimed to study the molecular modification and mechanism of thermostbility and xylose tolerance of the key enzymes (xylanase and beta-xylosidase) for the hydrolysis of xylan at high temperature. Based on the sequence alignment of proteins, 3D structure of xylanase A and beta-xylosidase from extremely thermophilic bacteria-Thermotoga thermarum will be constructed by using homology modeling, CD spectra and protein crystal X-ray diffraction. Thereafter, according to the biological properties of the key enzymes the site-directed mutagenesis of their functional amino acids is conducted using rational design of molecules to obtain very efficient xylanase and xylosidase with high temperature resistance. Furthermore, the pyrohydrolysis of xylan in aspen wood and cornstalk catalyzed by the enzymes is evaluated, and the mechanism of their thermostability and xylose tolerance is also investigated, to provide theoretical and practical basis for the development and application of efficient xylan hydrolase with high thermostability. This research has great significance in science and application.

研究开发耐高温酶催化纤维素和半纤维素水解，是近年来生物质能源与化学品领域的热点研究。针对目前木聚糖（半纤维素的主要组成）高温降解中存在的关键问题，本项目拟对木聚糖高温水解关键酶木聚糖酶和木糖苷酶的分子改造及其热稳定和耐糖机理进行研究。对来源于一株极耐热菌Thermotoga thermarum的耐高温木聚糖酶A和beta-木糖苷酶，在蛋白序列比对的基础上利用同源建模、圆二色光谱和蛋白单晶X-射线衍射等方法构建其3D结构模型,并基于分子理性设计对两种关键酶的相关功能氨基酸进行定点突变，以获得高效极耐热木聚糖酶和木糖苷酶，研究其对杨木和玉米秸秆纤维木聚糖的高温催化降解性能，阐明其热稳定和耐木糖机理，为耐高温高效木聚糖水解酶的开发利用提供理论和实践依据。项目研究具有重要的科学理论意义和应用价值。

1、项目背景.高温酶催化因其具有诸多优点已成为现代生物催化技术的重要发展方向之一，尤其是在纤维生物质原料水解转化制备生物质能源与化学品领域，耐高温半纤维素酶和纤维素酶的开发利用已成为研究热点。但迄今为止，对于耐高温半纤维素酶的结构特征及其耐热稳定性和机制研究，国内外鲜有报道。本项目研究目的是构建极耐热木聚糖酶Xyn10A的3D结构，阐明其Ca2+促进热稳定性及机制，研究获得高效木聚糖类半纤维素水解关键酶。..2、主要研究内容.1）极端嗜热菌Thermotoga thermarum来源的耐高温木聚糖酶的基因克隆、表达及酶学性质；.2）极耐热木聚糖酶C端和N端碳水化合物结合域CBM的功能作用；.3）Ca2+-木聚糖酶的空间结构特征及Ca2+促进热稳定性；.4）极耐热木聚糖酶Xyn10A的分子定向改造；.5）极耐热木聚糖酶、β-木糖苷酶催化效果。..3、重要结果及关键数据.1）通过同源建模构建了极耐热木聚糖酶Xyn10A的3D结构，采用圆二色谱方法对其高温结构特性进行了分析表征，研究确定了其Ca2+结合区域并成功对其进行了分子定向改造；.2）研究获得了可在80-95oC下催化水解木聚糖且热稳定性好的耐高温Ca2+-木聚糖酶催化体系；.3）项目研究成果已发表论文11篇，其中SCI收录论文9篇，获授权发明专利1件、申请发明专利1项。