Engineering T. naphthophila β-glucosidase for enhanced synthesis of galactooligosaccharides by site-directed mutagenesis.

Hydrolysis is a significant problem in enzymatic production of galactooligosaccharide (GOS) via β-glucosidase catalyzed transgalactosylation. 7 Mutant β-glucosidases engineered from hyperthermophilic Thermotoga naphthophila RKU-10 (TN0602) are created through site(F414)-directed mutagenesis scenario with modeling-assisted design, for the purpose of obtaining new enzyme species for improved production of GOS. The enhancement in GOS production is achieved by continuous accumulation of GOS with less degradation as the hydrolysis activity of the mutants is strongly suppressed. The mutant F414S is found to be the most promising engineered glucosidase. Compared to the wild-type (WT), the hydrolytic activity of the mutant F414S deducts by up to 350-fold and the molecular catalytic efficiency ( k cat /K m ) for hydrolysis deducts by 22-fold, whereas the production of GOS increases from 138.58 (WT) to 207.63 mM (F414S) by 50%. Most of the mutants result in alteration of catalytic specificity, yielding both galactooligotetrasaccharide (GOS4) and galactooligotrisaccharide (GOS3) compared to the sole product GOS3 by the WT. The molecular docking study presents a plausible explanation for reaction specificity alteration and hydrolysis inhibition for most of the mutants. [ABSTRACT FROM AUTHOR]