农业生物技术学报2011，Vol.19Issue(6)：1136-1143,8.DOI:10.3969/j.issn.1674-7968.2011.06.021
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
摘要
Abstract
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, 6-75 and b-28 were obtained, with an endoglucanase activity 2.1 folds and 3.6 folds increased, respectively. The sequence of 6-75 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 6-75 were either located at the corner between the fourth and fifth a-helix in the catalytic domain or at the fifth p-fold or the corner between the ninth and tenth P-fold in the carbohydrate-binding domain. And the mutation of b-28 was located at the fourth P-fold in the carbohydrate-binding domain. Following the orthogonal experiment, the mutant 6-75 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Key words
Bacillus subtilis/ Endoglucanase/ Directed evolution/ Fermentation conditions引用本文复制引用
姚友旭,李雨霏,侯晟琦,李春梅,陈惠,廖燕..基于易错PCR技术的中性内切葡聚糖酶基因的定向进化[J].农业生物技术学报,2011,19(6):1136-1143,8.基金项目
本研究由四川省科技支撑计划(No.2008GZ0150)资助 （No.2008GZ0150）
