“Fishes-in-net” 酵母孢子微胶囊式近平滑假丝酵母SCRII酶有机相高效手性合成机制研究

Candida parapsilosis CCTCC M203011 is an excellent strain for chiral alcohol production with independent intellectual property rights. The stereoselective oxidoreductase SCRII catalyzes asymmetric reaction towards low-concentrated substrate and exhibits strong substrate specificity (Bioresour Technol, 2011, 102:483-89; PLoS ONE, 2013, 8:e83586; Microb Cell Fact, 2012, 11:167-75). The project focuses on construction of spore-encapsulation in Saccharomyces cerevisiae with osw1 gene delection and enhanced permeability. The spore-encapsulation follows the “Fishes-in-net” model: the substrates and cofactors can smoothly get into and out of the spores, while enzyme is immobilized in spores. The physiological, biochemical and dynamic characteristics, substrate and product stereroselectivity, and interaction model between encapsulated enzyme and cofactor will be studied. The cofactor-regeneration metabolic pathways will be strengthened in the yeast-spore encapsulation system. The project will provide theoretical basis of novel and stable carbonyl reductases with high stereoselectivity for efficient chiral synthesis in organic phase.

近平滑假丝酵母CCTCC M203011是一株高效转化手性苯乙二醇，具有自主知识产权的优良菌株。其立体选择性氧化还原酶SCRII催化不对称反应底物浓度低且专一性强（Bioresour Technol2011,102:483-89；PLoS ONE,2013, 8:e83586；Microb Cell Fact,2012,11:167-75）。本研究拟构建孢子微胶囊酶，该酶采用“Fishes-in-net”催化模式，底物和辅酶自由进出孢子，酶固定于孢子内。研究微胶囊酶催化立体异构反应的生理生化与动力学特性、立体选择性、及酶与辅酶协同和稳定催化的作用模式，为获得稳定的高选择性新立体选择性还原酶有机相高效手性合成提供理论基础。

近平滑假丝酵母CCTCC M203011是一株具有自主知识产权的优良菌株。其立体选择性氧化还原酶SCRII催化不对称反应底物浓度低且专一性强。本研究理性设计SCRII的定点突变E228S，改变了酶的底物特异性，与葡萄糖脱氢酶相偶联。以敲除osw1基因通透性增强的酿酒酵母孢子为载体，将偶联酶构建在孢子中，形成微胶囊酶，该酶采用“Fishes-in-net”催化模式，底物和辅酶自由进出孢子，酶固定于孢子内。解析了微胶囊酶催化立体异构反应的生理生化与动力学特性、立体选择性、及酶与辅酶协同和稳定催化的作用模式，和酶有机相催化高浓度底物的分子机制。另外，我们设计了特定的一种固定在多孔ZnO/C纳米颗粒上具有双功能的“kiwifruit-assembly”的氧化还原酶：SCRII和GDH显著提高温度、pH和有机溶剂的稳定性。当底物浓度为75 mM时，反应仅需1 h，产物的产率高达100%。纳米酶重复使用10个批次，转化产率高于72%。纳米酶的生产力比游离酶提高了4.5–8.0倍，反应时间缩短了2.0–8.0倍。纳米酶通过激发质子偶联电子传递，不仅高效手性催化，且具有长期和优秀的再生稳定性。酵母孢子微胶囊酶和纳米酶中强化辅酶代谢，为获得稳定的高选择性新立体选择性还原酶有机相高效手性合成奠定了较坚实的研究基础。