能降解天然纤维素的地衣芽孢杆菌GXN151的分离鉴定及其一个纤维素酶基因(cel5A)的克隆和测序分析  

Isolation and identification of a natural cellulose degrading Bacillus licheniformis strain GXN151 and cloning and sequencing of a cellulase gene cel5A from the bacterium

作　　者：刘永生[1] 冯家勋[2] 段承杰[2] 莫新春[2] 柏学亮[2] 张成刚[1] 唐纪良[2] 马庆生[2] 

机构地区：[1]中国科学院沈阳应用生态研究所,辽宁沈阳110015 [2]广西大学分子遗传学研究所

出　　处：《广西农业生物科学》2003年第2期132-138,共7页Journal of Guangxi Agricultural and Biological Science

基　　金：国家863计划"(2001AA214151);"国际科技合作重点项目计划"(2002AA217121)

摘　　要：从广西大学农场陈旧稻草堆中分离出1株具有降解天然纤维素稻草粉、甘蔗渣粉活性的细菌GXN151,经形态观察、16SrDNA序列分析和丙酸盐利用试验将其鉴定为地衣芽孢杆菌(Bacilluslicheniformis)。通过连接经BamHI酶切的pBluescriptKS(+)和回收得到的经Sau3AI部分酶切的GXN151的3～10kbDNA在大肠杆菌JM109中构建了该菌的含7099个克隆的基因文库,文库克隆中插入片段最大的为11.0kb,最小的为3.1kb,平均大小为4.6kb,含GXN151基因组中任一基因的概率P为99.6%。筛选该文库获得8个表达羧甲基纤维素酶活性的克隆,DNA测序和PCR分析表明,该8个克隆可归为3类不重叠的独立克隆,其中一个克隆pGXNL2的测序分析表明其上的cel5A基因为1626bp,可编码含542个氨基酸的羧甲基纤维素酶,Cel5A的N-末端的42个氨基酸具有信号肽特征,它的第66～321氨基酸为家族5糖基水解酶(glycosylhydrolase)功能域、第391～472氨基酸为家族3碳水化合物结合组件(carbohydrate-bind-ingmodule,CBM)。本工作首次报道地衣芽孢杆菌能降解结晶纤维素及从该种细菌中克隆到纤维素酶基因。A bacterial strain GXN151 which can efficiently degrade natural cellulose such as rice straw and bagasse, was isolated from rice straw haystack at the farmland of Guangxi University The strain was identified as Bacillus licheniformis by morphology, 16S rRNA gene sequence analysis and testing for utilization of propionate A genomic library of GXN151 was constructed in Escherichia coli JM109 by ligating BamHI-digested pBluescript KS(+) vector with recovered 3~10 kb Sau3AI-partially digested GXN151 DNA The library contains 7 099 clones with biggest insert of 110 kb, smallest insert of 31 kb, and the average insert size of 46 kb The library has 996% chances (P=996%)to cover any given gene in the genome of GXN151 Eight clones expressing carboxymethylcellulase(CMCase) activity were isolated by screening this library DNA sequencing and PCR-based screening of the eight clones revealed that these clones fell into 3 types of non-overlapping independent clones Sequencing analysis revealed that cel5A gene on pGXNL2 was 1 626 bp, encoding an enzyme with 542 amino acids The N-terminal 42 amino acids of Cel5A was characteristic of signal peptide Amino acid 66～321 of Cel5A formed family 5 glycosyl hydrolase catalytic domain, and amino acid 391～472 of the enzyme formed family 3 carbohydrate-binding module(CBM) This is the first report that Bacillus licheniformis can degrade natural cellulose and a cellulase gene was cloned from this bacterium

关 键 词：天然纤维素 地衣芽孢杆菌 分离 鉴定 降解 家族5糖基水解酶 碳水化合物结合组件 

分 类 号：Q785[生物学—分子生物学]