基于结构生物学与分子模拟的L-阿拉伯糖异构酶催化机制与理性设计

Glycosylation isomerase is an important class of enzyme for food industry. A new member of this family, L-arabinose isomerase (L-AI), is the key enzyme in D-tagatose biosynthesis, thus bearing significant value in terms of industrial application. This project is aimed to analyze the relationship between catalytic activity and its protein structure and improve the industrial applicability of L-AI. Comprehensive utilization of the molecular biology、computational chemistry and molecular biology to carry out structure analysis of thermophilic L-AI enzyme.Creating a multi-variable protein optimization strategy based on the relationship between protein structure and catalytic activity. The ultimate objectives are to havest a number of L-AI mutant proteins with practical value,and a set of universal strategies for biocatalysts rational design. Furthermore, recombinant L-AI producing strain which can be safely used in the food industry is made available, which will contribute to the biosynthesis of D-tagatose. The newly gained bioinformatic knowledge of the glycosylation isomerase has provided a reference for rational modification of relevant enzymes. So it is definite that there are significant academic and practical values in this project.

糖基异构酶是一类重要的食品用酶，该家族中的新成员L-阿拉伯糖异构酶（L-arabinose isomerase, L-AI）是催化合成功能性甜味剂D-塔格糖的关键酶，因此具有重要的工业应用价值。本研究以解析该酶结构与功能对应关系以及提高L-AI酶工业适用性为目标，综合结构生物学，计算化学和分子生物学等多学科力量，开展嗜热L-AI酶结构解析，建立基于酶结构-活性关系的多变量蛋白质优化策略，在此基础上改造获得催化性能改善的L-AI突变酶；构建食品安全的重组L-AI酶生产菌株,推进其在D-塔格糖生产中的应用，为相关酶类的理性改造提供借鉴，具有重要的学术意义和实用价值。

L-阿拉伯糖异构酶（L-arabinose isomerase, L-AI）是催化合成功能性甜味剂D-塔格糖的关键酶，因此具有重要的工业应用价值。对L-阿拉伯糖异构酶（L-AI）的结构进行了精细解析，获得了精准的嗜热型L-AI全酶结构，基于该结构开展了酶分子改造的研究，得到了LFAI突变体D269K/Q270K/Q299K，其最适反应pH由6.5下降为6.0，同时该突变体在pH6.0时的稳定性也提升了48%，催化D-半乳糖生产D-塔格糖的产率最高可达52%，显著提高了该酶的工业适用性。开展了以乳糖为原料，通过构建多顺反子质粒实现发酵乳杆菌来源L-阿拉伯糖异构酶（LFAI）和海栖热袍菌来源β-半乳糖苷酶（β-gal）在E. coli中的高效共表达，共表达菌具有良好的催化稳定性，在70°C连续催化6个批次未见活性显著下降，其中LFAI保留有75%的酶活力而β-gal保留了85%酶活力。首次以食品安全菌株枯草芽孢杆菌为宿主，构建了表面展示固定化L-AI酶系统，该系统提高了L-AI酶的热稳定性，催化生产D-塔格糖的效率显著提高。