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 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.

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纤维二糖差向异构酶（CsCE）同时具有差向异构化和结构异构化活性，能够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。本项目通过共结晶法首次获得了CsCE与结构异构产物葡萄糖基-β1,4-D-果糖(Glc-Fru)的晶体结构。同时构建了低催化活性的CsCE突变体H247F，通过筛选和解析H247F的复合晶体结构，捕捉CsCE催化的中间产物，明确了CsCE催化的第一步和最后一步为开环和闭环反应，此外，突变实验推测His247可能参与底物的固定和催化过程中底物的正确伸展和引导。随后结合LC-MS和NMR监测，验证了CsCE的差向异构和结构异构催化机制均为cis-enediol机制。随后通过分子动力学模拟和QM/MM计算提出了由His377和His247/Glu250催化的差向异构催化机制及His377和His188共同催化的吡喃糖基和果糖基相互转化的结构异构催化机制。在此基础上，通过理性设计、计算机辅助设计等分子生物学方法对CsCE进行分子改造，构建了基于CsCE的单位点和多位点突变体。获得了结构异构活性显著提高的突变体：CsCE/V177D（1.5倍）、CsCE/I178D（1.7倍）、RmC（2.2倍）、CsCE/Q371E（3.3倍）。其中突变体CsCE/Q371E的底物结合力得到了显著改善，且反应平衡被拉动，乳果糖：依匹乳糖：乳糖的最终比例由60:10:30变为72:8:20。