Rational design paving the way for improving glucose tolerance and catalytic properties of a β-glucosidase fromAcetivibrio thermocellus

Cellulases are an ensemble of enzymes that hydrolyse cellulose chains to fermentable glucose, hence, are widely used in bioethanol production. The last enzyme of the cellulose degradation pathway - β-glucosidase, is inhibited by its product – glucose. The product inhibition by glucose hinders cellulose hydrolysis limiting the saccharification during bioethanol production. Therefore, engineered β-glucosidases with improved glucose tolerance along with the catalytic efficiency are the need of the hour. This study focuses on the rational engineering of β-glucosidase from Acetivibrio thermocellus (WT-AtGH1). Recombinant WT-AtGH1 exhibited activity on cellobiose and p-nitrophenyl-β-D-glucosidase as substrates and retained around 80% of its activity over 48 hours at 55°C, pH 5.5. However, WT-AtGH1 showed low glucose tolerance of 380 mM as compared to the required IC 50 value of > 800 mM for industrial use. Therefore, the rational design approach was applied for improving the glucose tolerance of this enzyme. We determined 3 Å resolution crystal structure of WT-AtGH1. The structure-based engineered G168W-AtGH1 and S242W-AtGH1 mutants exhibited improved glucose tolerance of 840 mM and 612 mM, respectively. Surprisingly, S242L-AtGH1 mutant showed ∼ 2.5-fold increase in the catalytic efficiency as compared to WT-AtGH1. A combinatorial effect of improved glucose tolerance, as well as enhanced catalytic efficiency, was observed for the G168W-S242L-AtGH1 mutant. All the mutants with enhanced properties showed considerable stability at industrial operating conditions of 55°C and pH 5.5. Thus, we present the next-generation mutants of WT-AtGH1 with improved glucose tolerance and kinetic properties that have the potential to increase the efficiency of the saccharification process for second generation bioethanol production.