厌氧菌株Clostridium sp.WGC702的筛选及其β-葡萄糖苷酶基因的克隆

摘要

An anaerobic bacteria strains WGC702 for product β-gluoosidase was isolated from biogas digester and was identified as a species of Clostridium sp. ( named Clostridium sp. WGC702) based on its 16S rDNA sequence. After digestion of the choromasomal DNA with BamHI, the 2～10 kb DNA fragment was gel-purified and ligated to pGEM-3zf( + )vector to prepare the genomic library. Approximatdy 120 dones were screened by media with Esculin, and one of the positive done was analyzed by sequencing. The results showed that an ORF of 1347 bp was included, encoding a protein of 448 amino acids. This β-glucosidase gene was analyzed by NCBI gene data base in amino acid level, it indicated that had 48 ％ identity with the sequence of the beta-galactosidase from Clostridium cellulovorans 743B (EES30417.1). The predicted pI and Mw were 4.94 and 53978.6Da respectivdy. It might be a new β-glucosidase gene.%从沼气池中筛选一株产β-葡萄糖苷酶的厌氧菌菌株WGCT02,基于16S rDNA序列及生理特性将其确定为梭菌属,命名为Clostridium sp.WGC702.用Bam HI不完全酶切WGC702的总DNA,回收2～10 kb范围内的DNA片段,连接到pGEM-3zf(+)载体上,电转化至大肠杆菌E.coli DH5α中构建基因文库,利用七叶苷平板筛选到120个能产黑色水解圈的阳性克隆.将其中一个含3.5 kb左右大小的外源片段的阳性克隆子进行测序,结果发现其中含有一个1 347 bp大小的完整ORF框,进一步分析表明该片段编码448个氨基酸组成的蛋白质,氨基酸序列与来自Clostridium cellulovorans 743B(登录号为EES30417.1)的β-葡萄糖苷酶具有48%的同源性,其预测的等电点和分子量分别为4.94和53 978.6 Da,可能为一个新的β-葡萄糖苷酶基因.