高温纤维素降解菌的筛选及内切酶基因的克隆表达  

Isolation of Thermophilic Cellulose-Decomposing Bacteria and Cloning and Expression of an Endoglucanase Gene

作　　者：田伟[1] 王磊[1] 李刚[1] 汪贞[1] 李妍[1] 张纪兵[1] 

机构地区：[1]环境保护部南京环境科学研究所,南京210042

出　　处：《农业环境科学学报》2014年第4期788-793,共6页Journal of Agro-Environment Science

基　　金：环保公益性行业科研专项(201209036);环保公益性行业科研专项(201309036);中央级公益性科研院所基本科研业务专项"猪粪有机肥在有机蔬菜生产过程中的安全评价研究"

摘　　要：以滤纸条作为唯一碳源从以牛粪和砻糠为原料的堆肥样品中分离到了8株具有纤维素降解特性的微生物,其中菌株T1和T2在刚果红培养基上培养48 h后的水解圈明显大于其他菌株。对菌株T1和T2进行了生理生化和16S rRNA序列分析,克隆了菌株T2的内切酶基因并对该基因进行了结构域分析,同时构建了表达载体p28-egB并将其转化至E.coli BL21(DE3)感受态细胞中进行表达。研究结果表明:(1)菌株T1和T2可分别鉴定为Bacillus cereus和Bacillus subtilis;(2)菌株T2的内切酶基因片段大小为1500 bp,编码499个氨基酸,结构域分析显示该酶由两个不连续的结构域组成,其一为N端催化结构域,由糖基水解酶家族5组成,其二为C端底物结合结构域,由碳水化合物绑定结构域家族3组成;(3)在E.coli BL21中成功表达了菌株T2的内切酶基因,SDS-PAGE电泳图谱显示该蛋白大小约为50 KD,浓度约为93.14 mg·L-1。Thermophilic cellulose-decomposing bacteria could shorten digesting time of composts.Here we isolated eight strains of thermopilic cellulose-decomposing bacteria from composts produced from dairy manure and rice chaff using filter paper as a sole carbon source.After 48 hour cultivation on CMC-congo red medium,strains T1 and T2 had bigger clear zones than the other strains.Based on the morphological,physiological and biochemical properties as well as 16S rRNA homology analysis,the strains T1 and T2 were identified as Bacillus cereus and Bacillus subtilis respectively.Endoglucanase gene of T2 was cloned and domains analysis showed that the endoglucanase gene contained an open reading frame of 1500 bp,corresponding to 499 amino acids.The endoglucanase was comprised of two discontinuous domains：one was N terminal catalytic domain belonging to glycosyl hydrolase family 5,and the other was C terminal substrate binding domain belonging to carbohydrate binding domain family 3.Using PET28a as the expression plasmid,the endoglucanase gene of strain T2 was successfully expressed in E.coli BL21（DE3）.SDS-PAGE analysis showed that molecular weight of the expressed protein was about 50 KD and the concentration was about 93.14 mg·L^-1.

关 键 词：纤维素降解菌 内切酶基因 克隆 表达 结构域 

分 类 号：X172[环境科学与工程—环境科学]