聚糖醛酸裂解酶多结构域功能解析及嗜盐嗜温机制研究

Polyuronide lyase (PL) is a group of enzymes used to breakdown long chain natural gels such as pectin and alginate into bioactive oligosaccharides. However, some PLs originated form non-terrestrial bacteria usually have low thermal stability and high halotolerance. Excessive waste water may be generated during its reaction process so it is not suitable for food industry application. Studies showed thermal stability of PLs was determined by carbohydrate binding modules (CBM), while halotolerance of halophilic enzymes was related to the amino acids on the surface of active site. The purpose of this study was to improve the thermal stability and decrease halophilicity of PLs. Alginate lyase (Aly01) would be used as a representative PLs. The structure activity relationship of different domains and halophilic mechanism will be elucidated by protein structure determination, chemical modification, site-specific mutagenesis and fusion expression. And finally, to obtain enzymes that can be readily used in food industry. This project innovatively focus on the halophilic mechanism of alginate lyase. It would provide detailed structure of CBM in alginate lyase. Eventually, it would provide some important implication for the development and application of similar PLs.

聚糖醛酸裂解酶（PLs）是一类用于裂解天然胶类物质如果胶和褐藻胶生产功能性低聚糖的酶。但非陆生来源的PLs常具有热稳定性差、对氯化钠依赖性强等特点，反应过程产生大量废水，不适于实际生产。研究表明多结构域PLs中的碳水化合物结合结构域（CBM）对酶的热稳定性有重要作用，而嗜盐酶的嗜盐特性则由催化活性域表面的氨基酸决定。为提高非陆生来源的PLs的热稳定性，同时降低酶反应对氯化钠的依赖性，本项目以氯化钠严格依赖型褐藻胶裂解酶为研究对象，以解析该酶多结构域结构的构效关系及各结构域间的相互作用机制为核心，利用蛋白结构解析、化学修饰、定向改造、融合表达等方法，阐明其底物结合、多结构域相互作用及嗜盐机制，并获得去盐依赖性和热稳定性高的产业化用酶。本项目首次聚焦该类酶的嗜盐机制，将填补PLs CBM结构研究空白，为类似PLs的开发和应用提供全新理论基础，具有重要的科学价值。

聚糖醛酸裂解酶（PLs）是一类用于裂解天然胶类物质如果胶和褐藻胶生产功能性低聚糖的酶。但非陆生来源的PLs常具有热稳定性差、对氯化钠依赖性强等特点，反应过程产生大量废水，不适于实际生产。研究表明多结构域PLs中的碳水化合物结合结构域（CBM）对酶的热稳定性有重要作用，而嗜盐酶的嗜盐特性则由催化活性域表面的氨基酸决定。为提高非陆生来源的PLs的热稳定性，同时降低酶反应对氯化钠的依赖性，本项目以氯化钠严格依赖型褐藻胶裂解酶为研究对象，以解析该酶多结构域结构的构效关系及各结构域间的相互作用机制为核心，利用蛋白结构解析、化学修饰、定向改造、融合表达等方法，阐明其底物结合、多结构域相互作用及嗜盐机制，并获得去盐依赖性和热稳定性高的产业化用酶。本项目首次聚焦该类酶的嗜盐机制，将填补PLs CBM结构研究空白，为类似PLs的开发和应用提供全新理论基础，具有重要的科学价值。