表面氨基酸残基对脂肪酶SMG1耐受有机溶剂的影响及分子保护机制研究

The use of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages compared to traditional aqueous reaction systems. Despite the advantages, native enzymes almost universally exhibit low activities and/or stabilities in the presence of organic solvents. This inactivation of the enzymes by organic solvents results in significant limitation of the enzymatic reaction process. Research indicated that animo acides on the surface of enzyme have potential to protect the stability of enzyme in the organic solvent media. However, the detail mechanism still needs further research..Present study choose lipase SMG1, which has a better research background and tolerance to organic solvents, to investigate the protective mechanism of surface animo acids on the lipase to organic solvents. First, by using molecular dynamic simulation and computational calculation, the predict the potential residues target are determined, combined with the site-directed mutagenesis method to construct and express the muations. After that, the enzymatic stability characteristics of various lipase are validated from the direction of activity, thermodynamics and catalysis dynamic information. Meanwhile, the biochemical changes of lipase on molecular conformation, micro-water environment and enzymatic characters are investigated by using various spectrums and CD methods. Finally, informations on the animo acid residue sites that have close correlation with the stability of SMG1 to organic solvent medium are determined. Combining with molecular simulation results, the mechanisms on surface amino acids mediate protective effect to lipase stability in organic solvents are clarified. Present studies have great significance to industry catalysis process under organic solvents system.

脂肪酶在有机相中的催化反应可有效解决水相催化所面临的诸多困难，使许多重要化合物合成变为现实。然而酶在有机介质中稳定性普遍降低的现实成为制约其大规模应用的瓶颈问题。已有研究表明酶表面某些氨基酸残基对保护酶在有机介质中的稳定性起重要作用，但具体残基信息及其影响机制有待进一步研究。项目选取课题组有较好研究基础的有机溶剂耐受性脂肪酶SMG1作为酶代表，首先采用分子动力学模拟计算方法分析预测潜在的残基靶标及突变信息；结合定点突变基因工程技术构建表达突变体；继而采用酶工程技术从酶活性、热力学、催化动力学等角度对突变体在有机介质中的稳定性进行分析验证；利用光谱、CD等方法研究突变体在有机介质中的分子构象、微水环境等性质的变化。最终确定与在有机介质中稳定性相关的氨基酸残基位点信息。结合分子动力学模拟，探讨其与有机溶剂的相互作用及其分子保护机制。本研究对推动有机溶剂体系下酶法工业催化发展有重要科学价值。

研究表明酶表面某些氨基酸残基对保护酶在有机介质中的稳定性起重要作用，但具体残基信息及其影响机制未知。本项目以脂肪酶SMG1为代表，开展影响酶蛋白有机溶剂耐受性的关键氨基酸研究。研究内容和结果为：首先，采用分子动力学模拟计算，预测盖子结构上的Leu103，Phe104以及催化口袋周围的Trp116，Trp229， Asn277和Phe278位点与酶蛋白的有机溶剂耐受性存在相关性；其次，结合定点突变技术构建针对上述位点的系列突变体(L103G, F104G, W116A, W116F, W116H, W229A, W229F, W229H, W116F-W229F, N277D, N277F, N277L, N277V, F278A, L103G-F278A, F104G-F278A, Delete(103-104))；继而采用酶工程技术从酶活性、热力学、催化动力学等不同角度对突变体的各项酶学性质进行分析验证；结果表明，上述位点突变会导致酶蛋白在不同有机溶剂中耐受性改变，通过实验进一步证实上述位点与酶在有机介质中稳定性相关。除此之外，研究还同时发现上述位点还与酶蛋白热稳定性（Asn277，Trp229）、底物选择性(Leu103，Phe104,Phe278)等酶学性质密切相关；最后，采用结构分析手段，探讨不同位点分子保护机制。从结构上分析发现：Leu103 和 Phe104 是盖子区 N 端的两个疏水性残基，且侧链伸向蛋白质表面而不是插入到催化口袋中，两个位点通过与口袋中的疏水残基相互作用来稳定盖子的构象； W116、A113、H111之间的CH-π键及氢键相互作用对SMG1脂肪酶盖子构象的变化具有重要的作用，破坏之间的相互作用不利于盖子的构象变化，不利于酶催化反应的进行；残基N277位于催化位点的入口，亲水性的残基277和E275, D279, D280，R277一起形成一个高度亲水的区域，有利于酶的稳定性，提高其有机溶剂耐受性；Phe278 位于催化口袋边缘，His281 的上方， Phe278 与 底物（PNPO）的苯环之间存在一个π-π作用力，这个作用力有利于稳定 PNPO 在催化口袋中的构象，从而保持酶活力；Trp229与Phe278之存在的π-π相互作用对该区域的稳定性具有重要的作用。本研究结果对推动有机溶剂体系下酶法工业催化发展有重要科学价值。