GB&ELPs双标签重组酶在特殊拓扑结构聚合物双水相浮选体系中传质过程优化调控及机理研究

微生物酶的大规模分离纯化是实现微生物酶催化剂工业化的关键技术问题。本课题拟将目标酶基因表达与下游双水相浮选进行一体化设计，构建温敏单环嵌段聚合物-盐双水相浮选体系，并用于重组酶的选择性分离纯化。主要研究内容如下：① 制备新型温敏单环嵌段聚合物作为成相聚合物，解决聚合物相粘度较大和聚合物分离回收的难题。② 构建高度集成化的温敏单环嵌段聚合物-盐双水相浮选体系分离纯化重组酶，力求缩短整个下游过程的流程和提高单项操作的效率，解决传统多步骤策略所造成的产品收率低、耗时长、生产成本高和酶活损失严重的问题。③ 构建“自捕集重组酶体系”，基于基因工程技术将具有特殊功能的组合标签GB&ELPs引入酶分子，在不添加捕集剂的条件下实现对重组酶的选择性分离纯化，解决无捕集剂浮选体系选择性低和浮选效率不高的问题。④ 解决相关科学问题，从理论上指导应用实践，为工业酶制剂生产开辟全新的思路。

The large-scale separation and purification of microbial enzymes is a key technical problem for realizing the industrialization of microbial enzyme catalyst. This topic is proposed to construct a thermosensitive and monocyclic block polymer-salt aqueous two-phase flotation system (ATPFS) with the integrated design of gene expression and downstream aqueous two-phase flotation process, aiming at separating and purifying recombinase selectively. The main research contents are as follows: ① A series of new type thermosensitive and monocyclic block polymers are to be prepared as phase-forming polymers, solving the problems of high viscosity of polymer-rich phase and recycling of polymer. ② A highly integrated thermosensitive and monocyclic block polymer-salt ATPFS is to be constructed and applied in the selective separation and purification of recombinase, aiming at simplifying the downstream process and improving efficiency of single operation, which can solve the problem of low product yield, time-consuming, high production cost and serious loss of enzyme activity induced by the traditional multi-step strategy. ③ Based on genetic engineering technology, the “self-trapping recombinase system” is to be constructed by introducing GB and ELPs tags with special functions into enzyme molecules, aim at realizing the selective separation and purification of recombinase without the addition of trapping agent, then solving the problem of low selectivity and efficiency for flotation systems free of trapping agent. ④ Some related scientific problems are to be resolved, which can guide application practice in theory and open up a new way of thinking for the production of industrial enzyme.