Screening and characterization of a novel ruminal cellulase gene ( Umcel -1) from a metagenomic library of gayal ( Bos frontalis )
Screening and characterization of a novel ruminal cellulase gene ( Umcel -1) from a metagenomic library of gayal ( Bos frontalis )
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DOI：
10.1016/s2095-3119(15)61144-3
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发表时间：
2016-04
影响因子：
4.8
通讯作者：
Bi-feng Li;Zhu Yaxin;Z. Gu;Yuan Chen;J. Leng;X. Gou;Li Feng;Qing Li;D. Xi;H. Mao;Shuli Yang
中科院分区：
文献类型：
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作者：
Bi-feng Li;Zhu Yaxin;Z. Gu;Yuan Chen;J. Leng;X. Gou;Li Feng;Qing Li;D. Xi;H. Mao;Shuli Yang
Gayal是在印度支那发现的稀有半野生牛种。它们可以吃草，包括竹叶，以及芦苇和其他植物物种，并生长到比在类似恶劣环境中饲养的云南黄牛更高的成熟活重。本研究的目的是确定特定纤维素酶在大额牛瘤胃。利用从4头成年山羊瘤胃内容物中提取的基因组DNA构建了宏基因组fosmid文库。该文库含有38400个克隆，平均插入片段大小为35.5kb。Umcel-1基因是从该文库中分离得到的。对24个随机克隆的纤维素酶活性的调查导致鉴定了Umcel-1基因，其表现出最有效的纤维素酶活性。Umcel-1基因的序列分析表明，它包含一个942碱基对的开放阅读框架，编码313个氨基酸的产物。Umcel-1基因产物属于糖基水解酶家族5，与Clostridium lentocellumDSM 5427的纤维素酶（GenBank登录号YP_004310852.1）同源性最高，分别为44%和62%。将Umcel-1基因在大肠杆菌BL 21中异源表达，并纯化重组Umcel-1。对纯化的重组Umcel-1进行了活性测定，结果表明其水解羧甲基纤维素的最适pH为5.5，最适温度为45°C。据我们所知，这项研究提供了第一个证据，纤维素酶的细菌在大额牛瘤胃。
Gayal is a rare semi-wild bovine species found in the Indo-China. They can graze grasses, including bamboo leaves, as well as reeds and other plant species, and grow to higher mature live weights than Yunnan Yellow cattle maintained in similar harsh environments. The aim of this study was to identify specific cellulase in the gayal rumen. A metagenomic fosmid library was constructed using genomic DNA isolated from the ruminal contents of four adult gayals. This library contained 38 400 clones with an average insert size of 35.5 kb. TheUmcel-1gene was isolated from this library. Investigation of the cellulase activity of 24 random clones led to the identification of theUmcel-1 gene, which exhibited the most potent cellulase activity. Sequencing theUmcel-1gene revealed that it contained an open reading frame of 942 base pairs that encoded a product of 313 amino acids. The putative geneUmcel-1product belonged to the glycosyl hydrolase family 5 and showed the highest homology to the cellulase (GenBank accession no. YP_004310852.1) fromClostridium lentocellumDSM 5427, with 44% identity and 62% similarity. TheUmcel-1gene was heterologously expressed inEscherichia coliBL21, and recombinant Umcel-1 was purified. The activity of purified recombinant Umcel-1 was assessed, and the results revealed that it hydrolyzed carboxymethyl cellulose with optimal activity at pH 5.5 and 45°C. To our knowledge, this study provides the first evidence for a cellulase produced by bacteria in gayal rumen.