枯草芽孢杆菌解聚酶YwtD调节γ-聚谷氨酸链长的结构基础研究

Poly-γ-glutamate (γ-PGA) is an excellent biocompatible and biodegradable polymer with potential and extensive applications in different areas. γ-PGA is currently under active investigation and has become the hotspots in biopolymer research field. However, the metabolic control mechanism of γ-PGA, especially the mechanism of the regulation of γ-PGA chain length is still unclear . To solve this scientific problem, which has become a bottleneck for the improvement of γ-PGA production, we will focus our study on the structural and functional characteristics of γ-PGA depolymerization enzyme YwtD in Bacillus subtilis. First, we will perform cloning, expression, purification and crystallization of YwtD, and next determine the structure of YwtD by X-ray diffraction. Second, we will combine site-directed mutagenesis and biochemical experiments based on the structure information to identify the key residues essential for the depolymerization activity and specific substrate recognition of YwtD. At last, we will integrate the structural information and functional experimental results to analysis the regulatory function of YwtD in γ-PGA chain length and the molecular mechanism of specific substrate recognition. This study will help us understand the metabolic control mechanism of γ-PGA in-depth at the molecular level, and provide theoretical basis for the molecular modification of depolymerase and the construction of a controllable pathway of γ-PGA synthesis. This study is of great value for scientific research and industrial application.

γ-聚谷氨酸（γ-PGA）作为一种优良的“绿色”高分子材料，具有较大的应用前景和商业价值，近年来成为生物多聚物研究领域中的热点。但是，γ-PGA在微生物细胞内的代谢机制，尤其是其链长的调节机制目前仍不清楚，成为制约γ-PGA产量进一步提高的瓶颈和亟需解决的难题。针对这一科学问题，本课题拟以枯草芽孢杆菌中调节γ-PGA链长的解聚酶YwtD作为研究对象。首先，对YwtD进行外源表达、纯化和结晶，并通过X-射线衍射法测定YwtD的晶体结构。然后，根据结构信息对YwtD进行定向突变，通过生化实验确定影响γ-PGA解聚酶活性及底物特异性的关键氨基酸残基。最终，通过综合结构信息与功能实验结果阐明YwtD调节γ-PGA链长及特异性识别底物的结构学基础和分子机制。本课题的研究结果将为基于结构对该酶进行理性分子改造提供理论基础，并为建立γ-PGA分子量可控的合成途径提供借鉴，具有重大的科学意义和应用价值。

γ-聚谷氨酸（γ-PGA）作为一种优良的“绿色”高分子材料，具有较大的应用前景和商业价值，近年来成为生物多聚物研究领域中的热点。YwtD是一类γ-PGA的解聚酶，通过YwtD酶解来实现γ-PGA分子量可控的具有潜在应用前景。然而目前，YwtD的结构一直未知。在本项目研究中，通过小角散射实验技术我们分析了YwtD在溶液中的整体构象，结合其他实验方法如动态光散射，定点突变等， 进一步验证了YwtD的整体结构模型以及存在的底物传递通道。由于 γ-PGA 在各个领域具有广泛的应用， 本项目的研究结果可为应用酶解方法来实现γ-PGA分子量的可控提供参考。