菌株Chitinophaga sp.CH-1阿拉伯糖苷酶Ara1805基因克隆与表达

Cloning and Expression of Arabinosidase Ara1805 Gene from Chitinophaga sp.CH-1

阿拉伯糖苷酶协同木聚糖酶在低聚木糖的生产、废物的生物处理、食品或饲料养分的提高以及半纤维素的生物转化等方面起着非常重要的作用。噬几丁质细菌是可以滑动并形成粘孢子的革兰氏阴性细菌,因具有较强的几丁质酶活性而得名。采用PCR方法从噬几丁质菌株Chitinophagasp.CH-1基因组DNA中克隆出编码阿拉伯糖苷酶基因ara1805,以pET29a为表达质粒,与其C末端6个组氨酸标签序列融合,在大肠杆菌中得到高效可溶性表达。SDS-PAGE测得酶的分子量为38 ku,与理论推算值相吻合。表达菌株粗酶经检测表现出α-L-阿拉伯糖苷水解酶活性。蛋白质序列分析和其他来源的阿拉伯糖苷水解酶表明Ara1805中可能存在的活性中心为Asp67,Glu186和Asp236,说明该蛋白确实可能具有阿拉伯糖苷酶功能。研究为今后理性设计阿拉伯糖苷水解酶以期提高其水解活性奠定了一定的理论基础。

Arabinosidase synergistic xylanase plays a major role in the degradation of hemicellulose,low polyxylose production and food or feed nutrient enhancement.Chitinophagais a motile,gram-negative bacterium that is characterised by its ability to utilise the eponymous insect and fungal polysaccharide chitin.The gene of arabinosidase was obtained from a genome DNA of Chitinophaga sp.CH-1.Two primer were designed on the basis of nucleotide sequence of ara1805 gene.The ara1805 gene fragments were successfully amplified by PCR reaction under the suitable condictions.E.coli BL21（DE3） competent cells were transformed with the ligated pET29 a.ara1805 with Nde I/Xho I site.The recombinant protein presented on SDS-PAGE with a molecular weight of 38 ku.Among all the tested substrates,the enzyme exhibited the specific activity towards pnitrophenylα-L-arabinofuranoside（pNPAF）.Sequence alignment analysis of arabinosidases revealed that the Ara1805 has a Asp_（67）-Glu_（186）-Asp_（236） catalytic triad,which indicated that the protein may indeed have arabinose hydrolytic activity.This study lays a theoretical foundation for the future rational design of arabinose hydrolyzyme in order to improve its hydrolysis activity.