基于易错PCR技术的中性内切葡聚糖酶基因的定向进化  

Directed Evolution of Neutral Endoglucanase Gene by Error-prone PCR

作　　者：姚友旭[1] 李雨霏[1] 侯晟琦[1] 李春梅[1] 陈惠[1] 廖燕[1] 

机构地区：[1]四川农业大学生命科学与理学院,雅安625014

出　　处：《农业生物技术学报》2011年第6期1136-1143,共8页Journal of Agricultural Biotechnology

基　　金：四川省科技支撑计划(No.2008GZ0150)资助

摘　　要：纤维素酶的工业应用一直受酶活性低,成本高的限制,为了提高中性内切葡聚糖酶的活性,本研究利用易错PCR技术对来自枯草芽胞杆菌(Bacillus subtilis)C-36的内切葡聚糖酶基因进行定向进化研究,在酶分子水平上改造内切葡聚糖酶分子。实验获得了最佳突变株b-15和b-28,其内切葡聚糖酶活力比亲本酶分别提高了2.1和3.6倍。序列分析表明:突变体b-15有6个碱基发生了突变,分别是A360G、T402A、A419T、T648A、A1208G和A1397T,导致4个氨基酸突变,分别是N134K、Q140L、N403S和Q466L;b-28只有1个碱基发生了突变,导致了398位Lys突变为Arg。通过SWISS-MODEL服务器模拟酶的三维结构,分析表明,b-15改变的4个氨基酸分别位于催化结构域的第4和第5个α螺旋之间的转角和结合结构域的第5个β折叠及第9和第10个β折叠之间的转角;b-28的突变位于结合结构域的第4个β折叠。同时,对获得的两株突变株b-15和b-28用正交试验优化产酶条件,优化后的酶活分别达到4.542和5.136 U/mL,是优化前的2.2和1.5倍。实验获得了酶活性提高的内切葡聚糖酶菌株,为进一步在分子水平研究内切葡聚糖酶的功能和其应用打下了基础,也为高酶活酶分子在其他高表达系统的表达提供了基础材料。Low enzymatic activity and high cost are the two main problems that limit the industrial applications of cellulose.In order to enhance the enzymatic activity of neutral endoglucanase activity,error-prone PCR was conducted to engineer the Bacillus subtilis C-36 endoglucanase gene.Two optimum mutants,b-15 and b-28 were obtained,with an endoglucanase activity 2.1 folds and 3.6 folds increased,respectively.The sequence of b-15 endoglucanase gene showed six nucleotide substitutions leading to four mutated amino acids;and b-28 endoglucanase gene showed one nucleotide substitution leading to one mutated amino acid.According to the 3D structure of endoglucanase mimicked by SWISS-MODEL,the four mutated amino acids of b-15 were either located at the corner between the fourth and fifth α-helix in the catalytic domain or at the fifth β-fold or the corner between the ninth and tenth β-fold in the carbohydrate-binding domain.And the mutation of b-28 was located at the fourth β-fold in the carbohydrate-binding domain.Following the orthogonal experiment,the mutant b-15 and b-28 could reach to an endoglucanase activity of 4.542 U/mL and 5.136 U/mL through fermentation,respectively,both of which were much higher than the wild-type control.These results have provided a base for further research of endoglucanase.

关 键 词：枯草芽胞杆菌 内切葡聚糖酶 定向进化 产酶条件 

分 类 号：S565.4[农业科学—作物学]