前导肽对BpK9角蛋白酶的效应机制及其协同改造研究

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.

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。

微生物角蛋白酶能够特异性的降解结构稳定且抗性较强的角蛋白质为可被吸收和利用的多肽和氨基酸，在食品、饲料、医药等多个领域具有重要的应用价值和研究意义。目前研究主要集中于野生菌的挖掘与性质研究，克隆表达研究较浅，表达水平普遍较低，严重影响了角蛋白酶的工业化生产应用。基于前导肽序列对成熟酶的正确折叠起着至关重要的指导作用，本项目以课题组前期筛选获得的角蛋白酶基因为研究对象，对其前导肽序列进行了一系列的改造及机制分析工作，主要开展了以下方面的研究工作：.1.通过对角蛋白酶前导肽序列的缺失表达及不同长度的截短突变，验证了前导肽序列对该角蛋白酶表达酶活具有至关重要的作用，并确定了其功能区域为前导肽全长。.2.通过对前导肽切割位点进行氨基酸替换，确定了苯丙氨酸更有利于对前导肽的切割，并通过替换成功提高了前导肽的切割效率，从而提高成熟酶的释放量，将酶活从179 U/mL提升至440 U/mL。.3.通过与数据库中已报道的多条高酶活角蛋白酶序列进行一级结构的氨基酸序列比对分析，选取了18个位点进行定点突变，确定了6个位点对酶活具有显著促进作用。.4.对以上六个位点进行了组合饱和突变，构建突变体库并从中筛选获得酶活显著提升突变株，将酶活进一步提升至1140 U/mL。.5.对最优突变株进行了15 L发酵罐产酶研究，在发酵32小时后酶活达到最高值3040 U/mL，为目前国际报道领先水平。.