Site-saturation mutagenesis for β-glucosidase 1 from Aspergillus aculeatus to accelerate the saccharification of alkaline-pretreated bagasse.

Aspergillus aculeatus β-glucosidase 1 (AaBGL1) is one of the best cellobiose hydrolytic enzymes without transglycosylation products, among β-glucosidase from various origins, for use in cellulosic biomass conversion with Trichoderma cellulases. However, in our previous report, it was demonstrated that AaBGL1 has lower catalytic efficiency toward cellobiose, which is a major end product from cellulosic biomasses by Trichoderma reesei cellulases, than do gentiobiose and laminaribiose. Thus, we expected that there is room to enhance cellobiose hydrolytic activity of AaBGL1 by increasing catalytic efficiency (k /K ) up to that of gentiobiose or laminaribiose for accelerating the saccharification of cellulosic biomasses, and we performed site-saturation mutagenesis targeting nine amino acids supposed to constitute subsite +1 of AaBGL1. We successfully isolated a mutant AaBGL1 (Q201E) having 2.7 times higher k /K toward cellobiose than the WT enzyme. Q201E showed higher activity toward cellotriose and cellotetraose but lower activity toward gentiobiose and laminaribiose than WT. Kinetic analysis of various Q201 mutants toward cellobiose, gentiobiose, and laminaribiose revealed that only the Q201E mutation resulted in improved k /K toward cellobiose. We demonstrated that side chain length and the nondissociated form of the carboxyl group at E201 in Q201E were required for enhancing the activity toward cellooligosaccharides through supporting nucleophile attack by D280 via changing catalytic environment by pH profile of kinetic parameters and mutation analyses. Moreover, we also demonstrated that Q201E produced more effective synergy with cellulases and xylanases than WT in the saccharification of alkaline-pretreated bagasse.