Design of mutants for enhanced thermostability of β-glycosidase BglY from Thermus thermophilus.

Three design strategies, based on rational and semi-rational approaches, were employed to investigate the functional impact of thermostability-related amino acid substitutions in the β-glycosidase BglY from Thermus thermophilus. Five beneficial mutations were identified, of which 1 mutation was located in the active cavity of the enzyme and contributed to the released substrate inhibition. Combining all 5 beneficial substitutions resulted in the mutant HF5 with a 4.7-fold increase in half-life, with thermal inactivation at 93 °C, and complete lack of substrate inhibition toward the substrate p-nitrophenyl-β-D-glucopyranoside at lower reaction temperatures. The results of this study provide valuable information on amino acid substitutions related to thermostability and substrate inhibition of BglY.