Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju, 500-757, Republic of Korea.
J Microbiol. 2011 Feb;49(1):141-5. doi: 10.1007/s12275-011-0205-3. Epub 2011 Mar 3.
A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.
使用从鲍鱼肠道中分离的基因组 DNA 构建了宏基因组 fosmid 文库。对 3840 个克隆的文库进行筛选，发现了一个纤维素酶阳性克隆（pAMHElO）的 36 kb 插入片段。使用阳性克隆（pAMHElO）构建了一个 shotgun 克隆文库，并使用约 5 kb 插入大小的刚果红覆盖物进一步筛选了 3840 个 shotgun 克隆，仅发现一个纤维素酶阳性克隆（pAMHL9）。pAMHL9 由 5293bp 的 DNA 序列和三个开放阅读框（ORF）组成。在这三个 ORF 中，只有 ORF3 编码的重组蛋白（CelAMll）显示出纤维素酶活性，该蛋白与来自 Algino 溶藻弧菌 12G01 的外膜蛋白 A 具有 100%的同一性，但与已知的纤维素酶没有显著的序列同源性。表达的蛋白（CelAMll）分子量约为 37 kDa，在 pH 7.0 和 37°C 时观察到最高的 CMC 水解活性。羧甲基纤维素酶活性通过同工酶活性染色测定，当使用纤维四糖和纤维五糖作为底物时，获得了 CelAMll 的不同降解产物谱，而当使用纤维二糖和纤维三糖等低聚糖作为底物时，没有观察到底物水解。
