点青霉葡萄糖氧化酶热稳定性关键氨基酸研究

项目名称： 点青霉葡萄糖氧化酶热稳定性关键氨基酸研究

项目编号： No.31201313

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 食品科学、农学基础与作物学

项目作者： 张宇宏

作者单位： 中国农业科学院生物技术研究所

项目金额： 23万元

中文摘要： 葡萄糖氧化酶（Glucose oxidase，GOD，EC 1.1.3.4）可催化葡萄糖生成葡萄糖酸和过氧化氢，在食品工业如面包烘焙、生产葡萄糖酸以及生物传感器等领域应用广泛。来源于点青霉Penicillium notatum F4的葡萄糖氧化酶GODp与同类酶相比具有更强的催化活性，但其热稳定性较差，并直接影响了其应用范围和使用效果。本研究以稳定酶蛋白整体结构为出发点，从增加蛋白质区域构象稳定性，提高其解折叠自由能，优化蛋白表面电荷分布和增强辅酶FAD结合强度四个层面入手，通过计算机辅助分子模拟分析，选取可能与酶蛋白热稳定性相关的氨基酸，再利用定点突变等理性设计策略进行突变研究，揭示影响GODp热稳定性的关键氨基酸和结构特性，进而获得高效且稳定性好的葡萄糖氧化酶，为通过理性设计改善蛋白质热稳定性的研究提供新思路。

中文关键词： 葡萄糖氧化酶；热稳定性；青霉；理性设计；

英文摘要： Glucose oxidase (GOD, EC 1.1.3.4) are a class of enzymes that catalyze the oxidation of D-glucose to gluconic acid and hydrogen peroxide. Glucose oxidase has emerged as an important biocatalyst in food industry, such as beverage, gluconic acid production and biosensors. A glucose oxidase GODp from Penicillium notatum F4 exhibits preferable catalytic activity than other glucose oxidases. The poor thermostability, however, limit its application effectiveness. We sought to enhance the structural stability of the GODp using protein engineering, including increase conformational stability and unfolding free energy of the GODp, optimize surface charge distribution, and enhance combination stability between coenzyme FAD and the protein. Firstly, computer-assisted rational design is used to select appropriate amino acid associated with protein thermostability. Then, the mutants are obtained by strategies of site-directed mutagenesis. The purpose is to explore critical sites relate with thermostability of the GODp and how to improve the thermostability of this protein. Furthermore, mutants which exhibited better thermostability than the widetype enzyme are desire to obtain. This research can provide a valid method to improve protein thermostability by way of computer-assisted rational design.

英文关键词： Glucose oxidase；thermostability；Penicillium；rational design；