多酶-氧化硅杂化三维有序大孔生物催化剂的制备及性能

With the development of the biocatalysis that carried out from using single enzyme to multienzyme, rational construction of highly reactive and easily recyclable multienzyme systems and realizing effective cascade reactions are the hot and difficult issues in this field. In this project, we will construct three dimensionally ordered macroporous (3DOM) multienzyme-silica hybrid biocatalysts and investigate their catalytic properties. Firstly, enzyme-based 3DOM biocatalyst will be prepared through the combination of colloidal crystal templating (CCTs) method and cross-linked enzyme aggregates method. Secondly, to improve the mechanical stability and operational stability of the enzyme-based 3DOM biocatalyst, biomimetic silicification will be introduced in the preparation process, and then 3DOM enzyme-silica hybrid biocatalysts will be obtained. In order to achieve feasible and optimal implementation of this preparation process, the formation and regulation mechanisms will be investigated. Thirdly, based on the above investigation, 3DOM multienzyme-silica hybrid biocatalysts comprising nitrile hydratase and penicillin G acylase will be constructed. The relationship among the structure, enzyme microenvironment and catalytic activity will be clarified. Finally, this multienzyme system will be employed to catalyze the synthesis of cephalexin, and the process intensification will be studied. The success of this project will provide novel theoretical basis and technical support for the rational design of 3DOM co-immobilization of multienzymes and the applications of the multienzyme system. Additionally, the results of this project will open up a new avenue for industrial production of cephalexin.

随着工业酶催化由单酶催化体系向多酶级联催化体系的方向发展，构建高活性、易回收的多酶共固定化系统，实现高效多酶协同催化成为该领域的研究热点和难点。为此，本项目提出构建多酶-氧化硅杂化三维有序大孔（3DOM）生物催化剂的研究思路。首先基于温敏性的胶体晶体模板，通过交联酶聚集体技术制备酶分子为骨架的3DOM生物催化剂；为进一步提高其机械强度和操作稳定性，合理引入仿生硅化过程，考察其制备过程及机理，实现在温和可控条件下酶-氧化硅杂化3DOM生物催化剂的制备；以腈水合酶和青霉素酰化酶为模型酶，构建多酶-氧化硅杂化3DOM生物催化剂，并研究多酶-氧化硅杂化3DOM生物催化剂级联催化制备头孢立新的过程机制，实现头孢立新多酶催化的高效制备。本项目的成功实施，不仅为新型3DOM多酶共固定化体系的理性构建、微环境调控及应用提供理论基础和技术支持，还可促进多酶法制备头孢立新的工业化进程。

本项目提出了构建多酶-氧化硅杂化三维有序大孔（3DOM）生物催化剂的研究思路，由单酶催化体系向多酶级联催化体系方向发展，构建了高活性、易回收的多酶共固定化系统，实现了高效的多酶协同催化。主要研究内容：(1)基于温敏性的胶体晶体模板，通过交联酶聚集体技术，制备得到了以酶分子为骨架的3DOM生物催化剂，研究了受限空间内酶分子沉积过程及酶与模板间的相互作用、交联剂浓度、交联时间等因素对单酶3DOM形貌、孔径、活性等的影响；相对于游离酶，单酶3DOM具有更好的热稳定性、酸碱耐受性和重复使用性。(2)在3DOM生物催化剂中合理引入仿生硅化过程，制备了酶-氧化硅杂化三维有序大孔生物催化剂(3DOM-Si-enzyme)，考察了制备过程及机理，实现了在温和可控条件下具有三维有序大孔结构的生物催化剂的制备；考察了3DOM-Si-enzyme的稳定性及动力学参数，3DOM-Si-enzyme较游离酶具有更好的热稳定性和酸碱耐受性；以青霉素酰化酶(PGA)为模型酶，3DOM-Si-PGA重复使用9次之后仍能保持初始活性的71%。(3)以聚N-异丙基丙烯酰胺(PNIPAM)胶体晶体为模板，正硅酸甲酯(TMOS)为硅前驱体，实现了多酶-氧化硅杂化3DOM生物催化剂的制备，并以腈水合酶和青霉素酰化酶为模型酶通过级联反应合成了头孢立新；通过考察反应温度、反应时间、底物配比等，实现了头孢立新的高效制备。本项目不仅为新型3DOM生物催化剂的可控构建提供理论和技术支持，促进酶催化的工业化应用，还为新型酶固定化技术的开发奠定了基础。本项目具有较强的理论研究意义和实用价值。