Caldicellulosiruptor saccharolyticus纤维二糖差向异构酶CsCE催化乳糖异构化的机制研究及高乳糖异构化活性酶的构建

Caldicellulosiruptor saccharolyticus纤维二糖差向异构酶CsCE不仅具有差向异构化活力，还能催化单一底物乳糖直接异构化生成乳果糖，是目前最为高效、最有可能实现工业化的乳果糖制备用酶。目前CsCE差向异构化和结构异构化的催化机理尚不清楚，且存在底物特异性较差、产物不单一、热稳定性有待提高等不足，本项目拟首先通过获取糖-酶晶体并解析结构功能关系，明晰CsCE催化乳糖异构化制备乳果糖的作用机制；在此基础上，结合定点突变解析底物特异性机理；通过理性设计、计算机辅助设计等分子生物学方法对CsCE进行分子定向改造，获取底物特异性专一、异构化活力高且热稳定性强的适合工业化应用的正向突变体酶，并构建具有高乳糖异构化活性的食品级表达载体，推动CsCE酶法制备乳果糖技术的发展。

Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) can not only catalyze the epimerization, it can additionally isomerize the single substrate lactose into lactulose and is the most efficient enzyme ever found for the enzymatic synthesis of lactulose. However, the utilization of CsCE for industrial lactulose bioconversion which exhibits a tremendous advantage has not yet been fully studied. The detailed catalytic mechanism of CsCE is unclear due to the lack of structural data in complex with ligands. Besides, low specificity and weak thermostability as well as the formation of by-product obviously restricted its further industrial amplification. This project plans to investigate and propose the catalytic mechanism by detecting the crystal structures of the CsCE with different substrates, analogs of substrates and intermediate analogs. Based on the determined crystal structures of CsCE-ligands, rational design and computational design were applied to enhance the thermostability、improve the isomerization activity and increase the substrate specificity of CsCE. Food grade expression system with high isomerization activity was constructed in order to facilitate the enzymatic bioconversion of lactulose on an industrial scale by CsCE.