27家族α-半乳糖苷酶的Loop区影响其底物特异性的分子机理

Galacto-oligosaccharides (i.e. stachyose and raffinose) commonly exist in feed and are indigestible by monogastric animals. α-Galactosidase has hydrolysis ability to reduce these anti-nutritional factors and has great application value in feed industry. In our previous study, two acidic and high-temperature-active α-galactosidases of glycosyl hydrolase family 27, Gal27A and Gal27B, were identified in the thermophilic fungus Neosartorya fischeri P1. The enzymes had similar tertiary structures but varied in loop regions and substrate specificity. Gal27A showed higher activity towards raffinose, which was 4.9- and 3.8-fold for that of melibiose and stachyose. Whereas stachyose was a preferred substrate for Gal27B. Its activity to stachyose was 9.6- and 4.4-fold of melibiose and raffinose, respectively. The objective of this project is to undermine the effects of the loop structures on enzyme functions. Hybrid recombination enzymes will be constructed by loop replacement of Gal27A and Gal27B. Molecular docking and site-directed mutagenesis will be performed to study the enzyme-substrate complex and key regions involved in substrate binding. Site-directed mutagenesis will be further performed to determine the loop regions and key amino acid sites related to enzyme-substate interaction. This study will reveal the molecular mechanisms of substrate specificity of GH27 α-galactosidases and provide valuable theoretical basis for enzyme improvement.

α-半乳糖苷酶能有效降解饲料中普遍存在的抗营养因子棉籽糖和水苏糖等低聚半乳糖，是一种广泛应用的饲料用酶。从嗜热真菌费氏新萨托菌中克隆到27家族酸性高温α-半乳糖苷酶Gal27A和Gal27B，它们除Loop结构显著差异外其三级结构高度相似，但它们的底物特异性不同，前者降解三糖(棉籽糖)的活性较降解二糖(蜜二糖)和四糖(水苏糖)分别高4.9倍和3.8倍，而后者具有更高的四糖降解活性，较降解二糖和三糖分别高9.6倍和4.4倍。本项目聚焦酶的Loop结构，以上述两个酶为核心材料，构建差异Loop区替换的杂合重组酶，并运用分子对接方法构建底物结合模型，探索Loop区对酶底物特异性的影响，进一步采用定点突变技术，确定影响底物结合功能的Loop区上结构模块和关键氨基酸位点，研究其在影响酶与不同底物互作中的功能，阐明27家族α-半乳糖苷酶的底物特异性机理，为酶分子改良提供重要理论基础。

α-半乳糖苷酶作为饲料用酶能有效降解饲料中普遍存在的抗营养因子棉籽糖和水苏糖等低聚半乳糖，具有广阔应用前景，研究其Loop结构在影响酶与不同底物互作中的功能，阐明27家族α-半乳糖苷酶的底物特异性机理，为酶分子改良提供重要理论基础。本项目的主要内容包括：1）确定影响嗜热真菌费氏新萨托菌来源27家族酸性高温α-半乳糖苷酶Gal27A和Gal27B底物结合功能的Loop区上结构模块和关键氨基酸位点及其在影响酶与不同底物互作中的功能；2）分析影响这两个酶底物结合功能的Loop结构上关键氨基酸位点对27家族其它α-半乳糖苷酶底物特异性的影响，阐明27家族α-半乳糖苷酶底物特异性机理，获得具有应用潜力的α-半乳糖苷酶。本项目构建了上述两种酶差异Loop区替换的杂合重组酶，探索了Loop区对酶底物特异性的影响，进一步采用定点突变技术，确定了影响底物结合功能的Loop区上结构模块和关键氨基酸位点，已取得了较好的研究进展和学术成绩：1）L3、L4和L6与Gal27A和Gal27B的底物结合功能相关；2）Gal27A L4的L177-202区段提高三糖（棉子糖）的降解能力；Gal27B L6的L231-237区段提高四糖（水苏糖）的降解能力；Gal27B L3的L401-447区段提高四糖及大分子多糖瓜尔胶和角豆胶的降解能力。3）不同区域底物结合位点对不同底物结合起到不同功能，Gal27B L4的F162和Gal27B L6的Y234促进四糖的特异结合，Gal27A L4的W195促进三糖的特异结合，Gal27B L6的G233有利于底物进入，Gal27B L3的Y433促进四糖的结合。4）上述底物结合功能Loop区上关键位点在影响嗜热蓝状菌来源27家族α-半乳糖苷酶Aga27A与不同底物互作中具有相似功能。5）27家族α-半乳糖苷酶的Loop结构影响底物结口袋大小，导致容纳不同底物，不同位点残基的侧链大小和极性影响底物的识别与结合。部分研究结果已发表在Journal of Bioscience and Bioengineering（2016, 121(1):7-12. SCI IF=1.884）杂志上，并申请中国发明专利1项（专利名称：α-半乳糖苷酶突变体Gal7B-A16及其编码基因和应用，申请号201810033238.2），已完成了任务指标。