基于分子自组装及氧化还原控制的软固定化酶的构建

In this project the molecular self-assembly technique is used to fabricate novel smart cyclodextrin/polyethylene glycol pseudopolyrotaxane hydrogel as the recyclable carrier for enzyme soft-immobilization, which presents the redox-control feature. This technique may provide a novel idea for enzyme immobilization. Three types soft-immobilized enzyme will be fabricated. The type I soft-immobilized enzyme presents the pseudopolyrotaxane of PEGylated enzyme self-assembled with cyclodextrin and capped by another same polypseudorotaxane linked through disulfide bond between each other; the type II has the structure of hydrogel by mixing enzyme and the pseudopolyrotaxane of single-chain polyethylene glycol and cyclodextrin; The type III is the pseudopolyrotaxane hydrogel constructed with enzyme, multi-arms polyethylene glycol and cyclodextrin. The characteristic of them is that it may realize the combination and recycle of both the enzyme and carrier through “thiol/disulfide bond” redox-control. The structural and the comformational change of the two soft-immobilized enzymes will be analyzed by using fluorescent spectrometry, circular dichroism spectrum, zeta potential analysis and X-ray powder diffraction etc. And a further study will be carried out focusing on hydrogel phase transition mechanism and rules, the structural and comformational change of soft-immobilized enzyme, and also the ternary interactions among cyclodextrin, polyethylene glycol and enzyme. The three types of pseudopolyrotaxane hydrogel fabricated in this project is a flexible carrier formed or disassembled in the mild condition, possessing the redox-control feature. Hence they show promising applications as a general carrier for enzyme immobilization.

本项目采用分子自组装技术，构建受氧化/还原控制的新型智能型环糊精/聚乙二醇准聚轮烷水凝胶，并以其为可回收载体，开展酶分子的软固定化研究，为酶的固定化提供了一种崭新的思路。拟构建三类结构不同的软固定化酶，I型是聚乙二醇修饰酶与环糊精自组装并通过二硫键相互连接而封端的产物；II型由环糊精、单链聚乙二醇与酶组装而成；III型为多臂聚乙二醇、环糊精与酶共同组装而成的网格结构水凝胶。其共同特征是通过“巯基/二硫键”氧化/还原控制实现酶与载体的结合与回收。以荧光光谱、圆二色谱、Zeta电位分析、X射线粉末衍射等方法测定软固定化酶结构及酶分子构象的变化，研究水凝胶相变过程的机理和规律，分析软固定化酶分子结构和活性的变化，探讨环糊精、聚乙二醇与酶三者之间的相互作用。本项目构建的准聚轮烷水凝胶是一种柔性载体，通过氧化/还原开关控制，可在温和条件下形成与解体，有望成为一种通用型固定化载体。

本项目将环糊精和聚乙二醇衍生物自组装，通过巯基氧化法，或基于“酶催化自固定化”的巯基-烯点击反应，构建出聚轮烷水凝胶固定化酶。对水凝胶固定化酶进行了FTIR、XRD、SEM等结构表征，测定了其酶学性质，并将水凝胶固定化的漆酶应用于某些染料的脱色研究。. 以胰凝乳蛋白酶、刀豆脲酶、葡萄糖氧化酶和漆酶等四种酶为代表，较为系统地考察了（准）聚轮烷对酶活性和稳定性的影响，分析了环糊精、聚乙二醇与酶分子三者之间的相互作用，并进一步用圆二色谱和同步荧光光谱考察了胰凝乳蛋白酶和脲酶在准聚轮烷影响下的构象变化和表面亲疏水性变化。实验发现准聚轮烷对酶的活性和稳定性基本无明显影响，但聚乙二醇硫醇及其与α-环糊精自组装形成的准聚轮烷却对胰凝乳蛋白酶具有稳定作用，据此提出了胰凝乳蛋白酶与巯基准聚轮烷的可能结合模式，为酶的稳定化提供一种新思路。发现了脲酶表面存在由多个非极性氨基酸残基组成的“疏水穴”。通过荧光探针法，考察了准聚轮烷等添加物对脲酶表面疏水度的影响，对准聚轮烷-脲酶相互作用体系进行了半定量化处理。此法可尝试推广到对多种酶的表面性质研究和酶与其他物质的相互作用研究。. 针对漆酶的催化特性而设计了“酶催化自固定化法”，利用漆酶催化过程中产生的自由基中间体引发聚乙二醇硫醇/聚乙二醇丙烯酸酯端基之间的巯基-烯点击化学反应，同时将漆酶自身包埋于聚轮烷水凝胶中。这种固定化漆酶的方法反应迅速，固定化效率高，可为某些类似酶的固定化提供了一种可借鉴的新方法。聚轮烷水凝胶固定化漆酶对三苯甲烷类染料具有一定的脱色效果，对孔雀石绿的脱色效果尤其突出，在微量Cu2+激活下，孔雀石绿的12 h脱色率可接近90%，显著高于游离漆酶的表现，表明聚轮烷水凝胶载体对漆酶具有较好的保护作用，能够减轻染料对漆酶的抑制，提高漆酶对染料的脱色效果。但水凝胶固定化漆酶的重复使用能力尚待提高。