新型β-葡聚糖酶的发掘、性质及应用基础

β-Glucanases are hydrolases which can degrade β-glucans in the endosperm of cereals and cell walls of yeast & medical fungi. They are important biotechnological aids in food, animal feed and energy industries. So far, there are many reports which mainly focus on β-glucanases (β-1,4-glucanases or cellulases) from culturable microbes. However, less research has been done on β-glucanases (especially β-1,3-glucanases, β-1,3-1,4-glucanases) from nonculturable microbes, whose unique properties and action mechanism are not clear. Under support of the previous project from National Natural Science Fundation of China, we have studied two β-1,3-1,4-glucanases from the thermophilic fungi. Besides, One novel glycoside hydrolase (GH) family 81 β-1,3-glucanase has been found and its crystal structure has also been solved. The present project will mine four types of novel β-glucanase genes based on the methods of metagenomes, genomes and breeding microbes. The genes will be overexpressed using the suitable expression systems. The recombinant enzymes will be further purified, characterized and evaluated its potential in the beer malting industry to understand the action mechanism of novel β-glucanases. The results will develop an efficient method for mining enzymes and deepen the research of β-glucanases. Also, the results will be helpful for the action mechanism and industrial application of β-glucanases.

β -葡聚糖酶是一类能降解谷物，酵母、蘑菇等真菌中β-葡聚糖的水解酶总称，可广泛应用于食品、饲料、能源等行业。目前，β-葡聚糖酶特别是β-1,4-葡聚糖酶的研究报道很多，且主要集中于可培养微生物β-葡聚糖酶。不可培养微生物β-葡聚糖酶如β-1,3-1,4-葡聚糖酶、β-1,3葡聚糖酶的研究很少，酶的特性、作用机理等也不清楚。在前一个国家自然科学基金的资助下，课题组完成了两类嗜热真菌β-1,3-1,4-葡聚糖酶的研究工作，并发现一个新的糖苷键水解酶81家族β-1,3葡聚糖酶和解析其晶体结构。本项目拟从宏基因组、基因组和微生物选育等角度发掘4种不同糖苷键水解酶家族的新型β-葡聚糖酶基因，构建基因的高效表达体系，研究重组酶的纯化、性质和啤酒应用，阐明其作用机理。研究成果不仅能开拓食品酶源的发掘方法和深化β-葡聚糖酶的研究，对β-葡聚糖酶的作用机理及工业应用也具有重要的理论意义和生产实践价值。

啤酒酿造过程β-葡聚糖大量溶解在麦芽汁中，使麦芽汁粘度增大，造成麦汁过滤困难，提高了啤酒的生产成本，将β-葡聚糖酶应用于麦芽糖化中可缩短过滤时间，提升啤酒品质。葡寡糖类益生元可通过β-葡聚糖酶水解燕麦麸皮等制备。目前市场上适合于以上应用的β-葡聚糖酶种类少、产量低，因此发掘新型β-葡聚糖酶至关重要。.从土样中筛选到两株高产β-1,3-1,4-葡聚糖酶的真菌，经鉴定分别为泡盛曲霉（Aspergillus awamori）CAU33和嗜热子囊菌（Themoascus aurantiacus）830。嗜热子囊菌经液体发酵产β-1,3-1,4-葡聚糖酶量可达3471 U/mL。泡盛曲霉经液体发酵产酶量的活力达到8447 U/mL，为目前已报道野生型微生物液体发酵产β-1,3-1,4-葡聚糖酶的最高水平。从巴伦葛兹类芽孢杆菌（Paenibacillus barengoltzii）CAU904中克隆到一个GH16 家族β-1,3-1,4-葡聚糖酶基因（PbBglu16A）并在E. coli中成功表达。PbBglu16A对β-1,3-1,4-葡聚糖和β-1,3-葡聚糖均表现出酶活力，且对β-1,3-1,4-葡聚糖水解特性优异，适合制备β-葡寡糖。将米黑根毛霉（RmLic16A）和泡盛曲霉（AaBglu12A）β-1,3-1,4-葡聚糖酶基因成功表达至毕赤酵母GS115中，高密度发酵后胞外酶活力分别可达95,751 U/mL和159,500 U/mL，酶产量为目前报道的最高值。其中嗜热子囊菌和巴伦葛兹类芽孢杆菌β-1,3-1,4-葡聚糖酶在制备葡寡糖方面前景较好。泡盛曲霉和米黑根毛霉β-1,3-1,4-葡聚糖酶在啤酒酿造工业中应用潜力较大。β-1,3-转糖苷酶相对于内切β-葡聚糖酶，工业应用范围较窄。本实验室从米黑根毛霉中克隆表达一个GH17家族β-1,3-转糖苷酶，解析其晶体结构并阐明催化机制，经分子改造，将该酶转变成了内切β-1,3-葡聚糖酶，拓宽了该酶的应用领域。