Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside.

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived beta-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (k(cat)/K-m of 561.72 mM(-1) s(-1)) toward rho-nitrophenyl-beta-d-glucopyranoside (rho NPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 degrees C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to beta-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel beta-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.