环糊精葡萄糖基转移酶催化合成长链糖基化染料木素的pH调控机制研究

Cyclodextrin glycosyltransferase (CGTase) is a multifunctional enzyme, which is able to catalyze four reactions such as cyclization, coupling, disproportionation and hydrolysis. CGTase has been widely applied in food, cosmetics and medicine fields. In the study on genistein glycosylation by CGTase for water solubility improvement, the results show that long glycosylated genistein products with great water solubility can be specifically produced in low pH condition, whereas the glycosylation efficiency is too low. In order to understand the pH-specific regulation mechanism of CGTase for glycosylation products, acid-tolerant mechanism of CGTase, and efficient synthesis mechanism of long glycosylated products, in this project we will reveal the pH-specific regulation mechanism for glycosylation products by studying the “synthesis” and “hydrolysis” mechanism of long glycosylated products, elucidate CGTase acid-tolerant mechanism by rational design of active sites “pKa values”, and study the efficient synthesis mechanism of long glycosylated products by semi-rational or rational design for substrate binding subsites. The results and methods will provide referential value for other flavone glycosylation and for pH tolerance mechanism study of other enzyme with same type of CGTase.

环糊精葡萄糖基转移酶(CGTase)是一种可催化环化、偶合、歧化、水解反应的多功能酶，被广泛应用在食品、化妆品和医药领域。在CGTase催化染料木素糖基化提高其水溶性研究中，发现低pH条件下可特异性合成水溶性更好的长链糖基化产物，但酸性条件下CGTase糖基化反应效率较低。为了解析pH对CGTase糖基化产物特异性调控机制，以及CGTase耐酸机制和长链糖基化产物高效合成机制。本项目拟通过研究酸性条件下长链糖基化产物“开源节流机制”，解析pH对CGTase糖基化产物特异性调控机制；基于活性位点“pKa值”的理性设计解析CGTase耐酸机制，并通过CGTase底物结合亚位点的理性-半理性改造，进一步研究长链糖基化产物高效合成机制。本项目的研究结果及研究方法对其他黄酮类物质糖基化以及与CGTase同类型酶的pH调控及耐受机制研究具有重要的借鉴意义。

染料木素具有多种药理作用，广泛应用于制药和食品等领域。但是，染料木素难溶于水的特性极大限值了其应用范围。通过对染料木素的糖基化修饰，其在水中溶解度可以随着糖链长度的延长而增加。所以，本文通过对软化类芽孢杆菌来源的环糊精葡萄糖基转移酶(PmCGTase)分子改造，提高其催化合成长链糖基化染料木素的产物特异性。主要研究结果如下：1）研究发现低pH条件有利于提高长链糖基化染料木素特异性。通过不同pH条件下WT以及突变酶的酶活和产物特异性分析，揭示了pH通过调控PmCGTase的四种酶活来调节糖基化染料木素的合成和水解，进而解析pH对染料木素糖基化产物特异性调控机制。2）通过对丙氨酸 156、丙氨酸 166、甘氨酸 173 和亮氨酸 174 的饱和诱变，大大提高了长链糖基化产物 (LCGS) 的特异性。动力学模拟结果表明，变体LCGS特异性的提高可能归因于对长链底物亲和力的增强，这可能是由“-5”、“-6”和“-7”亚位点处氢键相互作用的变化引起的。3）通过对S77、Y195位点的定点饱和突变，提高长链糖基化槐角苷LCGS产物特异性。双点突变酶S77N/Y195I 的转化率分别比WT提高了20.6%，糖基化产物LCGS的比例分别提高了119%。分子对接和模拟结果表明，突变酶S77N/Y195I糖基化效率提高的原因可能与突变后底物作用力的增加和底物结合位点氨基酸更加灵活有关。4）通过对D182位点的定点饱和突变，提高对短链糖基化槐角苷 SCGS 产物产量。 结果表明突变酶D182C较WT转化率提高了13.42%，主要糖基化产物（槐角苷单糖苷、二糖苷、三糖苷）分别提高了39.35%、56.05%和64.81%。同源建模及分子对接结果表明，突变酶 D182C 糖基化效率提高的原因可能与底物作用力增加有关。