Directed Evolution of Clostridium phytofermentans Glycoside Hydrolase Family 9 Endoglucanase for Enhanced Specific Activity on Solid Cellulosic Substrate

被引:9
作者
Ahmad, Sajjad [1 ,2 ]
Ma, Hui [3 ]
Akhtar, Muhammad Waheed [2 ]
Zhang, Yi-Heng Percival [1 ,3 ,4 ]
Zhang, Xiao-Zhou [1 ,3 ]
机构
[1] Virginia Polytech Inst & State Univ, Dept Biol Syst Engn, Blacksburg, VA 24061 USA
[2] Univ Punjab, Sch Biol Sci, Lahore 54590, Pakistan
[3] Gate Fuels Inc, Blacksburg, VA 24060 USA
[4] Virginia Polytech Inst & State Univ, Inst Crit Technol & Appl Sci, Blacksburg, VA 24061 USA
基金
美国国家科学基金会;
关键词
Bacillus subtilis; Clostridium phytofermentans; Directed evolution; Endoglucanase; Glycoside hydrolase family 9; Cellulose; BACILLUS-SUBTILIS; BIOFUELS; LIGNOCELLULOSE; CELLULASES; EXPRESSION; SELECTION; PROTEIN; MICROBE; SYSTEM;
D O I
10.1007/s12155-013-9382-8
中图分类号
TE [石油、天然气工业]; TK [能源与动力工程];
学科分类号
080707 [能源环境工程]; 082001 [油气井工程];
摘要
Increasing specific activity of cellulase on solid cellulosic materials would be among the top priorities for second-generation biorefineries. However, the complicated relationship among the heterogeneity of solid cellulosic materials and different action mode cellulase components results in great challenges in cellulase engineering. We applied directed evolution to a Clostridium phytofermentans ISDg glycoside hydrolase family 9 processive endoglucanase (CpCel9) for enhanced hydrolytic performance by using Bacillus subtilis as a host for cloning and expression. Several CpCel9 mutants with both increased expression level and enhanced specific activity on the solid cellulosic material were obtained. The most active mutant, which also exhibits an increased expression level, had more than threefold specific activity than that of wild type on regenerated amorphous cellulose. Most mutation sites were located in the family 3 cellulose-binding module near to its catalytic module, which might guide the entrance of glucan into the catalytic module. This study suggested that directed evolution by combining B. subtilis secretory protein expression host and solid cellulosic substrates would be a powerful tool to evolve more active cellulase mutants for cost-effective biosaccharification process.
引用
收藏
页码:381 / 388
页数:8
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