前导肽对BpK9角蛋白酶的效应机制及其协同改造研究

项目名称： 前导肽对BpK9角蛋白酶的效应机制及其协同改造研究

项目编号： No.21676121

项目类型： 面上项目

立项/批准年度： 2017

项目学科： 有机化学

项目作者： 史劲松

作者单位： 江南大学

项目金额： 30万元

中文摘要： 角蛋白酶可特异性降解硬质难溶、结构复杂的角蛋白，在食品加工、生物制革及生态纺织等领域备受关注，被认为是极具发展潜力的新一代蛋白酶。来自短小芽孢杆菌的BpK9是一个新发现的拥有自主知识产权和突出催化性能的S8家族角蛋白酶，具有高表面活性剂耐受性、低胶原活性，但存在底物选择性和稳定性差的问题。近年来发现的S8家族前导肽充当分子伴侣，对蛋白酶构象形成有独特的调控机理，可望成为解决上述问题的一个新的切入点。本课题以BpK9前导肽为研究对象，在序列分析、高级结构预测基础上，通过选择前导序列中特定残基位点和结构区域进行突变，研究突变体对BpK9分子构象和酶学性能的效应，深入理解前导肽与酶体蛋白的相互作用机制；理性设计并构建前导肽与酶体部分的突变体，通过二者协同改造提升突变酶对多种底物的催化性能和稳定性。鉴于S8家族普遍存在前导肽功能的趋同性，本课题的技术理念可发展为角蛋白酶进行分子改造的一种通用策略。

中文关键词： 角蛋白酶；前导肽；S8家族；调控机制；协同改造

英文摘要： The keratinase could specially degrade insoluble and abundant structural keratins, which has attracted substantial attention in the fields of food processing, biological leather processing and eco-friendly textiles. It was considered as a potential candidate of next generation protease for sustainable biotechnology. The keratinase BpK9 from a newly isolated Bacillus pumilus was a member of S8 protease family with independent intellectual property rights and superior catalytic properties. It exhibited high surfactant tolerance and low collagenase activity; however, it suffered from the application problems, namely low substrate selectivity and poor stability. In recent years, the propeptide of S8 protease family was found to be crucial in peculiarly regulating the conformation of protease as the intramolecular chaperone, which might be a new starting point for overcoming the above-mentioned problems. The present project used the propeptide of BpK9 as the research objective. Mutation studies would be performed by selecting specific amino acid residues and structural domains based on sequence analysis and tertiary structure prediction. The effect of BpK9 mutation on molecular conformation and enzymatic properties would be investigated to deeply understand the interaction mechanisms between propeptide and enzyme protein. On this basis, the mutants of propeptide and enzyme protein would be constructed by rational design and cooperative modification to improve the catalytic performance towards several substrates and operational stability. Due to the convergence of ubiquitous propeptide functions of S8 family members, the techniques and concepts of this project are expected to be developed into general strategies for protein engineering of keratinases.

英文关键词： keratinase;propeptide;S8 family;regulation mechanism;cooperative modification