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 beta-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 degrees C, and complete lack of substrate inhibition toward the substrate p-nitrophenyl-beta-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. (C) 2012 Elsevier Ltd. All rights reserved.