低聚龙胆糖高效酶法制备的分子基础

Gentiooligosaccharides are a kind of high value-added functional oligosaccharides with broad application prospects for its capacity of preventing starch aging, excellent intestinal benefits and unique bitter flavor. Two major limiting factor in the present industrial preparation of gentiooligosaccharides were the lack of β-glucosidase with high transglycosylation activity and the low product conversion rate of the existing reaction system using high concentration of glucose as substrate. This project was designed to research the structure foundation and catalytic mechanism of β-glucosidase transglycosylation, and obtain β-glucosidases with high transglycosylation activity by performing molecular modification and function optimization based on the relationship between enzyme structure and function; to investigate the optimal mix proportion, reaction parameters and synergetic pattern of β-glucosidase and cellulases, and then establish a multi-enzyme synergistic reaction system which could prepare gentiooligosaccharides from cellulose efficiently. This project would not only deepen the theoretical understanding of transglycosylation of β-glucosidase, and also provide a theoretical basis for the large-scale industrial production of gentiooligosaccharides.

低聚龙胆糖具有抗淀粉老化和益肠道等特点，且具备独特的提神苦味，是一种极具应用前景的高附加值功能性低聚糖。目前工业化生产低聚龙胆糖的主要限制因素在于缺少高转糖苷活性β-葡萄糖苷酶，以及现有的以高浓度葡萄糖为底物的反应体系转化率不高。本项目拟研究β-葡萄糖苷酶转糖苷活性结构基础和内在机理，基于酶结构与功能的关系开展β-葡萄糖苷酶的分子改造和功能优化，获得高性能β-葡萄糖苷酶；摸索β-葡萄糖苷酶与纤维素酶的最优复配比例和反应参数，明确β-葡萄糖苷酶与纤维素酶的协同反应机制，建立以纤维素直接制备低聚龙胆糖的多酶协同反应体系。本项目的开展既能加深对β-葡萄糖苷酶转糖苷活性的理论认知，又能为低聚龙胆糖大规模工业化生产提供理论基础。

针对低聚龙胆糖酶法制备应用瓶颈和技术难题，本项目比较了不同来源的β-葡萄糖苷酶转糖苷活性，根据已报道的高逆水解或转苷活性的GH3 β-葡萄糖苷酶序列特征和进化关系分析，挖掘适用于低聚龙胆糖制备的高转糖苷活性β-葡萄糖苷酶，获得了具有较高转糖基活性/逆水解活性的热袍菌Thermotoga sp. KOL6 β-葡萄糖苷酶TsBgl1A和T. piceae β-葡萄糖苷酶TpBgl3A。进一步探究了β-葡萄糖苷酶转糖苷活性的结构基础和催化机理，开展高转糖苷活性β-葡萄糖苷酶的分子改造和功能优化，改造获得高转糖苷活性突变体7个，其中Tsbgl-G224A制备低聚龙胆糖产率为17.99%，较野生型提高72.8%；TpBgl3A-Q34G/Y200F产率为19.1%，较野生型提高75.2%。此外，建立了并优化以纤维素高效制备低聚龙胆糖的β-葡萄糖苷酶与纤维素酶协同反应体系，以及β-葡萄糖苷酶和β-1,6-葡聚糖酶双酶协同反应体系，其中TcBgn16与TcBgl3A双酶复配反应体系制备低聚龙胆糖产率最高可达23.89%，较目前报道最高水平提高50%以上。相关研究成果发表论文6篇(SCI 5篇)，已接收中文核心期刊2篇，申请发明专利10件（国际专利4件），其中1件中国发明专利已获授权。本项目丰富了低聚龙胆糖高效酶法制备的理论知识及反应体系，可为低聚龙胆糖工业生产提供关键技术及理论依据。