耐热β-半乳糖苷酶在枯草杆菌芽孢表面展示过程中酶活影响因素与构象变化分析

Due to the reasonable safety and high stability features, the new recently developed Bacillus subtilis spore-surface display that enables the expression of large and complex enzymes on the spore surface shows great potential for the development of vaccines and the production of industrial enzymes. However, the low display efficiency is a main barrier that blocks the development of this display technology. Note that the spore has a complex and highly ordered structure named the coat. It appears that the processes of spore formation and surface-expression might disturb the target enzyme’s structure and harm enzyme’s activity, but little is known about the mechanism involved. In this project, a thermostable β-galactosidase (β-Gal) was used as a model enzyme. The effects of scaffoldin composition and architecture as well as expression ratio for bgaB encoding β-Gal on the expression level and characteristics of β-Gal will be evaluated. Deletion of several abundant and non-fully cooperative coat proteins will then be performed to investigate the the effect of the altered spore coat on the localization of β-Gal and its enzymatic features. Finally, dynamic processes and interactions between β-Gal-fused anchoring protein and some key coat proteins in vitro will be studied by atomic force microscopy based single molecule force spectroscopy. Based on the data from conformational changes of β-Gal and its measured catalytic activity, the results of the present project will offer an insight into the critical factors in determining B. subtilis spore display performance of β-Gal but also the mechanism about how the assembly of coat protein affects the structure and activity of β-Gal.

枯草芽孢杆菌芽孢表面展示技术具有公认的食用安全性、良好的稳定性、可展示高分子量多聚体蛋白等优点，在疫苗制备、工业用酶生产等领域展现出巨大应用潜力。但也存在外源酶蛋白展示效率不高的问题，主要原因是对酶蛋白在复杂而高度规则的芽孢结构组装与展示表达过程中对其构效的影响因素及机理尚不清楚。本项目在前期研究基础上，以耐热β-半乳糖苷酶（β-Gal）为模型，研究其在不同脚手架骨架组成和空间结构及β-Gal表达比例下的活力与酶学性质的变化，通过敲除部分高丰度、非必须型衣壳蛋白分析芽孢结构的改变对β-Gal在芽孢表面的空间分布和酶学特性的影响，并运用基于原子力显微镜的单分子力谱技术解析自组装过程中与β-Gal融合的锚定蛋白与一些关键衣壳蛋白间的相互作用及动力学过程，以及结合β-Gal在自组装过程中的构象变化，阐明影响展示过程中β-Gal展示效率的关键因素及可能的影响机制。

枯草杆菌芽孢表面展示系统是近年来发展最为迅速的微生物表达系统之一，在疫苗开发、工业用酶生产等领域展现出巨大应用潜力。然而，如何提高枯草杆菌芽孢表面展示系统的展示效率是其进一步发展应用的关键。针对上述问题，本项目开展了以下研究工作：.1.利用基于cohesin-dockerin作用原理构建了5种β-Gal芽孢表达展示体系，发现β-Gal-DocⅠ-3/CotG-CohⅠ-3展示系统的效率高于锚定蛋白直接融合表达展示体系和基于Pcry1Aa表达的无锚定蛋白芽孢表面展示体系，并且β-Gal-DocⅠ-3/CotG-CohⅠ-3具有良好的热稳定性和有机溶剂耐受性，底物亲和力较强。.2.通过敲除部分非必须型衣壳蛋白改变芽孢衣壳“骨架”构成，分析芽孢衣壳改构前后β-Gal的展示效率以及酶学性质的变化，结果显示：cotV、cotX和ytdA三个衣壳蛋白基因敲除能提高β-Gal展示活力和赋予β-Gal优良的酶学特性，发现这是通过修饰芽孢衣壳结构即破坏crust层来实现；敲除cotC基因会促使芽孢衣壳内层与外层分离、部分crust层破坏，疏水性升高、抗热性下降，其表面展示β-Gal对有机溶剂的耐受性变差、Km值高达15.58mM；敲除ytdA后芽孢衣壳crust层呈散开网状分布，疏水性显著升高，其表面展示β-Gal的Km（2.87mM）低于游离β-Gal（2.97mM）。低温培养（25℃）不能提高所有不同基因敲除型芽孢表面展示β-Gal的表达水平。.3.探索了3种不同类型大肠杆菌表达系统对cotG、cotG-bgaB融合基因进行表达，均以失败告终。利用圆二色谱解析了β-Gal、CotH和CotZ的二级结构，由无锚定蛋白型β-Gal展示体系中分离得到的β-Gal的特征吸收峰发生了蓝移（从280nm偏移到260nm）。