耐热β-糖苷酶的基因克隆、表达和耐热性研究

Cloning and Expression of a Thermostable β-glycosidase Gene from Thermus nonproteolyticus HG102

从非解朊栖热菌HG10 2 (Thermusnonproteolyticus)染色体文库中 ,筛选得到耐热 β 糖苷酶 (Tn gly)基因。该基因位于重组质粒 2 6kbHindⅢ插入片段上 ;并在E .coli中表达 ,酶比活力提高 17倍。经序列测定 ,该基因1311bp ,编码 436个氨基酸 ,前端与部分糖透性酶基因紧密相连 ,G +G含量为 71% ,有明显的RBS序列 ,启动子序列不明显。经序列同源性比较 ,其氨基酸序列属糖苷水解酶家族 1,有 N E P 和 T E N 保守序列 ,Glu16 4和Glu338推测是催化活性中心。其氨基酸组成中 ,疏水氨基酸含量较高 (Ala 12 .8%、Leu 10 .9% ) ,Arg(9 6 % )、Glu(9 44 % )和Pro(8.0 % )含量显著较高。理论预测二级结构中 ,α 螺旋占 41 4% ,β 折叠占 16 2 % ,β 转角占 14 4% ,并有大量的Pro位于 β 转角的第二位。疏水作用、盐键、α 螺旋和Pro对Tn gly的突出热稳定性有重要贡献。经系统进化树分析发现 ,β

The gene coding for β glycosidase (EC3.2.1.21)from \%Thermus nonproteolyticus\% HG102 has been cloned and expressed in E.coli.The gene open reading frame was 1311bp and it codes for 436 amino acids.The deduced amino acid sequence of the enzyme showed identity with members of glycosyl hydrolase family I.The enzyme had high content of hydrophobic amino acid (Ala 12.8%\,Leu 10.9%),Arg(9.6%),Glu(9.4%)and Pro(8.0%),but low content Cys(0.45%) and Met (0.9%).From the alignment of enzyme amino acid sequence with other glycosyl hydrolase family I members,Glu164 and Glu338 were predicated as the proton donor and nucleophile group.The DNASTAR program was used to predict the secondary structure.According to the Chou-Fasman model,the enzyme has 41.4% of α-helics,16.2%,β strands,14.4%,β turns.14 of the 35 Pro were located at the second sites of β turns.Hydrophobic interaction,ion bond,α helics and Pro had important contribution to Tn-gly thermostability.