【作者】 封毅； 段承杰； 庞浩； 许跃强； 张鹏； 莫新春； 吴春凤； 唐纪良； 冯家勋；

【Author】 Feng Yi Duan Chengjie Pang Hao Xu Yueqiang Zhang Peng Mo Xinchun Wu Chunfeng Tang Jiliang Feng Jiaxun College of Life Science and Technology,Guangxi University at the Eastern Campus,Nanning,Guangxi 530005

【机构】 广西亚热带生物资源保护与利用重点实验室； 广西大学生命科学与技术学院；

【摘要】 草食动物依靠消化道中的微生物来分解富含纤维素的食物。盲肠是兔子等非反刍草食动物最重要的消化器官,盲肠中许多共生微生物能分泌纤维素酶、半纤维素酶、果胶酶等降解植物的细胞壁。在本研究中,我们构建了一个兔子盲肠未培养微生物宏基因组的柯斯质粒文库,文库含3.25×10~4个克隆,平均插入片段为35.1 Kb,文库总容量为1.14×109 bp。对文库进行活性筛选,获得四个具有内切葡聚糖酶的独立克隆和七个具有β-葡萄糖苷酶的独立克隆。对活力较强的两个内切葡聚糖酶活性克隆和两个β-葡萄糖苷酶活性的克隆分别进行了亚克隆,并对它们所含的纤维素酶基因进行了测序分析。两个内切葡聚糖酶基因(umcel5G 和umcel5H)的表达产物(Umcel5G 和Umcel5H)均属糖苷水解酶家族5的成员。Umcel5G 与源自Bacillus sp.N-4的内切葡聚糖酶B 的氨基酸同源性最高,具有46%的一致性和64%的相似性。Umcel5H 与Bacillus subtilis DLG 的内切β-1,4-葡聚糖酶同源性最高(46%的一致性和66%的相似性)。与两个β-葡萄糖苷酶基因编码产物同源性最高的是来自Butyrivibrio fibrisolvens H17c 的β-葡萄糖苷酶A,相似性为50%,一致性低于40%。所获得的四个基因与已知的纤维素酶基因的同源性较低,由此可见,兔子盲肠是研究和发现新的纤维素酶基因的很好材料。本文进行了内切葡聚糖酶基因umcel5G 在大肠杆菌中的异源表达,表达产物对羧甲基纤维素(CMC)的最佳作用温度为55℃,最佳作用pH 为6.5;对CMC 底物的Km 值为16.07 mg/ml,最大反应速度Vmax 为417.5 U/mg。更多还原

【Abstract】 Metagenomic DNA of uncultured microorganisms was isolated from the contents of rabbit cecumand cloned into a cosmid library with an average size of insert fragment of 35.1 Kb and a total capacity of 1.14×109 bp.Four clones expressing endoglucanase activity and seven clones expressing β-glucosidase activity wereisolated.Four genes,umce15G,umce15H,umbg13B and umbg13C were sequenced and analyzed.The putativeproduct ofumce15G shared highest homology to endoglucanase B,a glycosyl hydrolase family 5(GHF 5)enzymefrom Bacillus sp.N-4,with 46% identity and 64% positive.The putative product of umce15H also belonged toGHF 5 and was most closely related to endo-β-1,4-glucanase from Bacillus subtilis DLG with 46% identity and66% positive.Both umbg13B and umbg13C encoded GHF 3 β-glucosidase showing highest similarity to β-glu-cosidase A of Butyrivibrio fibrisolvens H17c with 50% positive and less than 40% identity.umce15G was heterol-ogously expressed in Escherichia coli and the recombinant protein was purified and characterized.The optimalpH and temperature of the recombinant Umce15G on carboxymethyl cellulose(CMC)were 6.5 and 55℃,respec-tively.The Km and Vmax toward CMC were 16.07 mg/ml and 417.5 U/mg protein,respectively.The high fre-quency of positive clones isolated from the metagenomic library and the relatively low similarities of the fourgenes’products to known cellulases suggested that rabbit cecum is a rich reservoir of novel cellulases.更多还原