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

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.

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。

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、并揭示其生物学功能具有重要意义。本项目首先通过简并引物PCR、巢式PCR和常规分离纯化等方法获得了AgWH50A、AgWH50B、AgWH50C、AgOGA50A、AgOGA50B、AgOGA50C、AgWH16、AgOGA118以及β-琼胶酶a和b等新型琼胶酶，建立了β-琼胶酶资源库。针对产物聚合度较为独特的AgWH50A、AgWH50B、AgWH50C以及β-琼胶酶a和b等新型β-琼胶酶，阐明了其酶学性质和催化机理，揭示了其降解琼胶生成不同聚合度琼胶寡糖的动力学机制和水解模式。研究了AgWH50B结构中的两个碳水化合物结合结构域位点（Carbohydrate-Binding Module, CBM）的功能，根据同源建模的结果，构建了用于研究琼胶酶CBM功能的5个突变体，确定了AgWH50B的两个CBM结构域对于酶与底物的结合具有重要的作用，并且靠近催化结构域上的CBM可能存在着参与催化底物的关键氨基酸。完成了β-琼胶酶AgWH50C的蛋白结晶和结构解析，确定其催化氨基酸分别为Glu-484和Glu-643。通过TK-SA模型、PoPMUSiC和HotMUSiC软件进行计算分析，共筛选了9个突变点进行热稳定性改造，通过理性设计获得了热稳定性提高的2个人工设计酶E38G和K621F。建立了琼胶酶的催化反应体系，制备了新琼二糖、新琼四糖、新琼六糖、新琼八糖和新琼十糖。研究了不同聚合度琼胶寡糖功能活性的差别，发现新琼四糖显著抑制LPS诱导的炎症反应，抑制机制与MAPK和NF-κB调节的iNOS和IL-1β表达有关。建立了抗生素诱导小鼠肠道菌群失调模型，发现具有明显的益生元效应。建立了小鼠力竭运动模型，结果显示新琼四糖能显著缓解力竭运动导致的肠道菌群紊乱，调节肠道菌群结构，增加肠道菌群多样性，并降低肠道致病菌的增殖，有效地缓解力竭运动导致的疲劳应激损伤。本项目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，将对海洋多糖资源的高值化利用产生重要的理论意义和应用价值。