【作者】 付亚丽；

【导师】 姚冬生； 刘大岭；

【作者基本信息】 暨南大学 ， 遗传学， 2008， 硕士

【摘要】 β-1,4-D-甘露聚糖酶(β-1,4-D-mannan mannohydrolase;Ec.3.2.1.78),简称β—甘露聚糖酶(β-mannanase),是一类能够水解含有β-1,4-D-甘露糖苷键的甘露寡糖、甘露多糖的内切水解酶,它属于半纤维素酶类,广泛存在于动物、植物和微生物中。迄今,所报道的β—甘露聚糖酶均归属糖苷水解酶家族5和26。该酶已在饲料、食品、造纸、石油开采及生物质能等多方面有了广泛的应用。本研究通过诱导培养本实验室保藏菌种A.tabescens EJLY2098,且对其分泌的β—甘露聚糖酶Man78蛋白进行纯化,获得了电泳纯的β—甘露聚糖酶,利用MS/MS技术分析了其肽段氨基酸序列,并以此获得的肽段氨基酸序列设计简并引物,以基因组DNA为模板进行简并PCR,再通过SMART-RACE技术,获得β—甘露聚糖酶的末端cDNA序列,最后进行成熟肽PCR,获得全长cDNA序列。对获得的全长cDNA序列进行ORF分析,获得蛋白序列。然后对所获得的蛋白序列进行同源性、一级结构、二级结构、三级结构的生物信息学分析。经过分析发现,所获得的A.tabescensβ—甘露聚糖酶Man78与现已报道的属糖苷水解酶家族5和26的β—甘露聚糖酶没有同源性,存在氧化还原酶结构域,推测该酶属于一个新的糖苷水解酶家族。本研究的完成标志A.tabescens所产的两个组分的β—甘露聚糖酶的分离和基因的全长cDNA克隆全部完成。A.tabecscensβ—甘露聚糖酶Man78可能属一个新的糖苷水解酶家族,深入开展β—甘露聚糖酶Man78的研究对认识β—甘露聚糖酶有十分重要的意义。更多还原

【Abstract】 β-mannanase is a short name forβ-1,4-D-mannan mannanohydrolase.It is a kind of endohydro-enzyme that can hydrolyze t he substance wit hβ-1,4-D-indican-bond.It universally exits in the nature,including the inferior animals and legume plants,whereas microorganism is the main source forβ-mannanase.All mannanases belong to GHF 5 and GHF26.So,it is a new age for the research and usage of beta—mannanase.By inducing A.tabescens EJLY2098,and purifing the protein that A.tabescens EJLY2098 secreted,a single band on the SDS-PAGE was obtained,β-mannanase. Basing on this,the purifiedβ-mannanase was enzymolysis in gel,then analyzed the partial peptides in the protein.Three random peptides was obtained.Acording these sequences,we designed degenerate primers to amplify.A 1457bp DNA fragment was amplidied from A.tabescens EJLY2098 with the primers by degenerate PCR with the genome DNA as template.Gene specific primers derived from this partial sequence were used for the amplication of the 3’and 5’ends of this cDNA by RACE method,then splicing to a full-length cDNA sequence.Then the analysis including the ORF,homologous, primary structure,secondary structure,tertiary structure was made with the sequence.This work means that it has been finished that the seperation and the genes cloning of beta-mannanase Man45 and Man78 which produced by A.tabescens EJLY2098.The beta -mannanase Man 78 maybe a member of another new GHF but of GHF5 and GHF26.The penetrating research of the beta-mannanase Man78 is very important for the knowledge of beta-mannanases.更多还原