新型β-琼胶酶分子改造及其对琼胶寡糖聚合度的影响机制研究

海洋寡糖，因其在结构和功能方面显示出的巨大研究价值和开发潜力，成为本世纪的研究热点。琼胶酶降解琼胶生成的琼胶寡糖在食品、医药行业有着广泛应用，而不同聚合度的琼胶寡糖功能活性和应用范围相差很大，因此酶法获得特定聚合度的琼胶寡糖具有重要意义。本项目组积累了多个在稳定性和产物聚合度方面具有显著特性的β-琼胶酶，并进行了重组表达；结合已报道的β-琼胶酶结构，对其进行了生物信息学和同源建模分析。在此基础上，拟采用同源建模和理性设计，结合基于酶序列-活性关系(ProSAR)的多变量蛋白质优化策略，进行β-琼胶酶的催化机理和分子改造研究，从酶分子与基因水平上阐明琼胶酶降解琼胶生成不同聚合度琼胶寡糖的理性调控机制，获得高活力、高稳定性和高专一性的新型β-琼胶酶，并降解琼胶生成不同聚合度的琼胶寡糖。为我国海洋寡糖工业生产技术的升级、改善产物聚合度分布以及提高产品质量与产率方面提供重要的理论依据和技术平台。

Agars are important gelling agents for biochemical use and the food industry, owing to their high viscosity, gelling ability and stabilizing properties. β-agarases, catalyzing the hydrolysis of agar, could cleave β-1,4 linkage to produce neoagarooligosaccharides, which have potential applications in food, pharmaceutical, and cosmetic industries owing to their physiological and biological activities. Neoagarooligosaccharides in different degree of polymerization (DP) have been found show various physiological activity and applications, so intensive study has been focused on the on the modification of agarase to search for a β-agarases with good substrate specificity.Our research team have found many β-agarases from Marine Agarivorans genus recently, and the β-agarases shows excellent characteristics in substrate specificity and stability. The cloning, expression, production, and purification of the recombinant β-agarases have been described. .In this research, according to the reported agarase structure, homology modeling and crystallization analysis of the above β-agarases would been carried out to understand the variability of substrate specificity. These three-dimensional structures help to find the amino acid residues involved in the recognition and cleavage of agarose and to understand the determinants of substrate specificity by comparison with the known structures of the reported agarase. In order to validate the relationship between the enzyme sequence, structure and the function of the above β-agarases, site-directed mutagenesis experiment would be carried out based on the above analysis. On these basis obtained, rational design, directed evolution and the protein sequence-activity relationship algorithm would be combined to research the molecular modification of the β-agarases, and to get the novel β-agarase with high activity, high stability and high specificity. With the novel β-agarases obtained in the former research, neoagarooligosaccharides in different degree of polymerization would be prepared, and their structure-activity relationship would also be studied. .Our research would elucidate the mechanism of the degradation pattern for agar hydrolyzing and the rational regulation for the different degree of polymerization of neoagarooligosaccharides. What's more, this research would promote the understanding for the relationship between the structure and function of agarases and other glycoside hydrolases.