内生枯草芽孢杆菌HD-1β-甘露聚糖酶基因的克隆及结构生物学分析  

Cloning and Structural Biology Analysis of β-mannanase Gene from Endogenous Bacillus subtilis HD-1

作　　者：张建新[1] 刘起丽[2] 胡文波[1] 聂国兴[1] 韩科芳[1] 赵杰[1] 

机构地区：[1]河南师范大学水产学院,河南新乡453007 [2]河南科技学院资源与环境学院,河南新乡453003

出　　处：《河南师范大学学报（自然科学版）》2013年第1期126-129,共4页Journal of Henan Normal University(Natural Science Edition)

基　　金：河南省重点科技攻关项目(112102110118);河南省教育厅科技攻关项目(2010A180014)

摘　　要：采用PCR技术从一株内生枯草芽孢杆菌HD-1基因组DNA中扩增出β-甘露聚糖酶基因核苷酸编码序列.序列分析表明,该基因全长1 089bp,编码362个氨基酸和一个终止密码子,N端前27个氨基酸为其信号肽.该酶氨基酸序列与相同来源的β-甘露糖苷酶同源性最高达98.34%,具有ManB保守结构域,属于糖苷水解酶家族26的一员.通过对该酶分子三维结构的预测分析,该酶的催化域形成TIM桶状结构,Cys92和Cys112形成二硫键,它们之间的部分构成该酶的氧化还原反应的活性中心,Glu100和Glu292分别为该酶的酸碱催化位点和亲核催化位点.三维结构的分析为提高酶的催化活性的和功能方面的定向改造提供了依据.Aβ-mannanase encoding gene was isolated from an endogenous Bacillus subtilis HD-1 by PCR. Sequence analysis showed that the gene was 1 089 bp length encoding 362 amino acids and a termination codon, and the 1-27 amino acid residues of N-terminal was its signal peptide. The amino acid sequence of this enzyme shared the highest up to 98.34% homology with otherβ -mannanases from Bacillus subtilis, and the conserved motifs in otherβ -mannanases of ManB were also recognized in the sequence, so the enzyme should belong to the glycosyl hydrolase family 26. Enzymatic three-dimensional structure analysis showed that the catalytic domain of the enzyme adopted a certain folding way to form a TIM barrel structure. There was a disulfide bond between Cys0.2 and Cys112, and the domain between them formed the redox-active center of the enzyme. Glul00 and Glu292 were the acid-base catalytic site and nucleophilic catalytic site of the enzyme respectively. Enzymatic three-dimensional structure analysis provided a basis for the improvement of the enzymatic catalytic activity and the directed transformation of the enzymatic function.

关 键 词：内生 枯草芽孢杆菌 Β-甘露聚糖酶 三维结构 

分 类 号：Q946[生物学—植物学]