基于多功能融合纤维素酶分离纯化的温敏类弹性蛋白双水相浮选体系的构筑及传质过程调控研究

With the increasing application of cellulase industrialization, the large-scale separation and purification of cellulase is the key technical problem in cellulase industrialization. This topic is proposed to construct a thermosensitive elastin-like protein aqueous two-phase flotation system (ATPFS) with the integrated design of “upstream” gene expression strategy and “downstream” separation and purification method of target cellulase, aiming at selectively separating and purifying fusion cellulase with GB and RLPs tags. The main research contents are as follows: ① The thermosensitive elastin-like proteins which are constructed by recombinant gene technique are to be prepared as phase-forming polymers, radically solving the environmental and resource issues in chemical synthesis. ② The thermosensitive elastin-like protein ATPFS is to be constructed and applied in selective separation and purification of fusion cellulase, which can solve the complicated process of separation and purification, high production cost and instability of enzyme activity. ③ Based on genetic engineering technology, the multifunctional fusion cellulase which has the abilities of adsorption at hydrophobic interface and thermosensitive phase transition at UCST is to be constructed by introducing GB and RLPs tags, then solving the problem of low selectivity and efficiency for flotation systems, difficult separation of target from flotation solvent. ④ Some related scientific problems are to be resolved, the new method is expected to provide a systematic solution that is comprehensive and scale for the separation and purification of bioactive molecules.

随着纤维素酶工业应用的日益广泛，纤维素酶的大规模分离纯化是纤维素酶工程中的关键技术问题。本课题拟将目标纤维素酶的“上游”表达策略与“下游”分离纯化方法进行一体化设计，构建温敏类弹性蛋白双水相浮选体系并用于“GB&RLPs双标签融合纤维素酶”的选择性分离纯化。主要研究内容如下：①通过基因重组技术制备温敏类弹性蛋白作为成相聚合物，从根本上解决化学合成法所面临的环境和资源问题；②构建温敏类弹性蛋白双水相浮选并用于融合纤维素酶的选择性分离纯化，解决目前纤维素酶分离纯化过程复杂、生产成本高和酶活稳定性差等固有问题。③在基因水平融合具有疏水界面吸附能力和UCST温敏相变能力的组合标签GB&RLPs，构建多功能融合纤维素酶，解决无捕集剂体系浮选率和选择性不高以及目标物从浮选上相中难分离的问题。④探究解决相关科学问题，使新方法有望为生物活性分子的下游分离纯化提供具有普适性及规模化生产前景的系统解决方案。

随着纤维素酶工业应用的日益广泛，纤维素酶的大规模分离纯化是纤维素酶工程中的关键技术问题。本课题拟将目标纤维素酶的“上游”表达策略与“下游”分离纯化方法进行一体化设计，构建亲和双水相浮选体系并用于“GB&ELPs双标签融合纤维素酶”的选择性分离纯化。主要研究内容如下：①筛选温敏线性聚合物作为成相聚合物，规模化应用的成相聚合物可有效解决聚合物回收的难题；②构建亲和双水相浮选并用于融合纤维素酶的选择性分离纯化，解决目前纤维素酶分离纯化过程复杂、生产成本高和酶活稳定性差等固有问题。③在基因水平融合具有疏水界面吸附能力和LCST温敏相变能力的组合标签GB&ELPs，构建多功能融合纤维素酶，解决无捕集剂体系浮选率和选择性不高以及目标物从浮选上相中难分离的问题。④探究解决相关科学问题，使新方法有望为生物活性分子的下游分离纯化提供具有普适性及规模化生产前景的系统解决方案。