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

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.