海洋Bacillus sphaericus 蛋白酶有机溶剂耐受性的分子机制研究

Short peptides have wide industrial applications in food stuff and medication. As compared with the chemical method, enzymatic peptide synthesis in organic solvent media has the advantages of the chemoselectivity of enzyme allowing the use of minimally protected substrates, the reaction being tereospecificity without racemization, and less environmental pollution. While enzymes are often inactivated and give low rate of reactions in organic solvent media. In this study, directed evolution techniques will be used to change the organic solvent stability of the protease from marine Bacillus sphaericus (OSP), and the high throughput screening will be used to obtain the mutated enzymes (OSPM). The OSP and OSPM will be grown crystals, and X-ray diffraction techniques will be used to resolve their structure and function. The three-dimensional structures of OSPM and OSP will be compared and analyzed. The molecular mechanisms related to organic solvent stability of the OSP will be investigated from the results of three aspects by employing a combination of crystallographic techniques, biophysical techniques, and molecular dynamics simulation described below. The difference of three-dimensional structures between OSP and OSPM will be compared. The microenvironment and conformation changes of OSP and OSPM in different organic solvents will be not only investigated with intrinsic fluorescence, UV measurements, and circular dichroism analysis, but also investigated by molecular dynamics simulation with Gromacs software and Packmol software. Based on the molecular mechanisms related to organic solvent stability of the OSP, the key amino acids for the organic solvent stability of the OSP will be selected for site-directed mutagenesis. The molecular mechanisms will be confirmed through comparing the organic solvent stability of the mutants and the results of theoretical analysis. The results would provide a theoretical basis and experimental evidence for the directed evolution of protease for improving organic solvent stability with rational design.

蛋白酶在有机相催化用于食品和医药工业的短肽合成，较化学合成方法，具有减少保护基团、避免氨基酸消旋作用，减少污染等优点，但存在酶失活或活性迅速降低的问题。本项目以海洋球状芽孢杆菌耐有机溶剂蛋白酶（OSP）为研究对象，定向进化后获得耐有机溶剂变化的突变酶（OSPM），制备并用X-射线衍射技术解析野生酶和突变酶的晶体结构。比较OSP和OSPM的晶体结构差异；通过荧光光谱法、紫外光谱法和圆二色谱法测定OSP和OSPM在不同有机溶剂中微环境和二级结构的变化；利用Gromacs软件和Packmol软件对OSP和OSPM在不同有机溶剂中的构象变化规律进行分子动力学模拟分析；综合分析上述三方面获得的结果，阐明OSP有机溶剂耐受性的分子机制。本项目获得的耐有机溶剂蛋白酶晶体结构和蛋白酶有机溶剂耐受性的分子机制为蛋白酶通过理性设计提高有机溶剂耐受性提供理论和实验依据。

较化学合成法，利用蛋白酶在有机相催化合成短肽，具有减少保护基团、避免氨基酸消旋作用，减少污染等优点，但存在酶失活或活性迅速降低的应用瓶颈。本项目定向进化了球状芽孢杆菌耐有机溶剂蛋白酶，获得了有机溶剂耐受性变化的突变酶，基于野生型酶晶体结构，模拟分析了其与突变酶的三维结构差异，通过荧光光谱法、紫外光谱法和圆二色谱法测定野生型酶和突变酶在不同有机溶剂中微环境和二级结构的变化，分子动力学模拟分析了有机溶剂中的酶构象变化规律，阐明了球状芽孢杆菌耐有机溶剂蛋白酶有机溶剂耐受性的分子机制，并通过定点突变对稳定性机制进行验证。主要结果如下：.重要结果一：通过Error prone-PCR和DNA shuffling 技术获得了在25%乙醇、30℃、180 rpm孵育1 d后，较野生型高34.8%和56.3%，以及降低5.4%的突变酶。重要结果二：随着甲醇浓度的增加，越来越多的甲醇分子进入到蛋白酶内部，导致更多的疏水基团暴露，疏水作用减弱，二级结构被破坏，酶的结构变松散，稳定性降低，另外，甲醇分子进入酶分子内部后，酶分子的必须水被逐渐替代，导致酶活性逐渐降低。重要结果三：Bacillus sphaericus蛋白酶有机溶剂耐受性的机制：1. Loop环柔性显著影响B. sphaericus蛋白酶有机溶剂耐受性。降低Loop环的柔性，可以提高酶对有机溶剂的耐受性；2. 有机溶剂耐受性提高的突变酶的溶剂可及表面积值低于野生型和有机溶剂耐受性降低的酶的溶剂可及表面积，表明有机溶剂耐受性提高突变酶蛋白质内部的一些疏水基团不易暴露在高浓度的甲醇中，导致蛋白酶的结构被破坏的程度较野生型酶低。以上研究成果为通过理性设计提高蛋白酶有机溶剂耐受性提供理论和实验依据。