Influence of water activity on the competition between β-glycosidase-catalysed transglycosylation and hydrolysis in aqueous hexanol

Five different β-glycosidases (Pyrococcus furiosus β-glucosidase, Sulfolobus solfataricus β-galactosidase, Caldocellum saccharolyticum β-glucosidase, almond β-glucosidase and Escherichia coli β-galactosidase) were evaluated as transglycosylation catalysts in hexanol containing various amounts of water. All enzymes catalysed both hydrolysis and transglycosylation of the glycosidic substrates (pentyl- and p-nitrophenyl-β-glucoside and p-nitrophenyl-β-galactoside). From the concentration ratio (alcohol/water) it was expected that the transglycosylation/hydrolysis ratio would decrease with increasing water activity in the hexanol. However, for all enzymes tested the selectivity for the alcohol increased with increasing water activity. This counteracted the effect of higher water concentration and in most cases the transglycosylation/hydrolysis ratio increased with increasing water activity. On the other hand, in hexanol/water two-phase systems, hydrolysis was by far the dominating reaction even though the total activity increased for all enzymes. The selectivity values were used to predict the maximal reaction yields in the kinetically controlled reactions. However, deviations were found in cases when the reactions became thermodynamically controlled: at high water contents secondary hydrolysis reduced the transglycosylation yields while higher transglycosylation yields than predicted were obtained at low water activity in some cases using enzymes poorly selective for the alcohol.