A kinetic study of almond-β-glucosidase catalysed synthesis of hexyl-glycosides in low aqueous media: Influence of glycosyl donor and water activity

A variety of alkyl and aryl glycosides were investigated as substrates for almond β-glucosidase catalysed synthesis of hexyl-β--glycosides in low aqueous hexanol media. The rate-limiting step in the organic media was determined to be the glycosylation of the enzyme. The kinetic constants V_max, K_m (glycosyl donor) and V_max/K_m were all influenced by the water activity and they all increased in value with increasing water activity. The increase in V_max/K_m was mainly determined by the increase in V_max and a plot of log(V_max/K_m) versus water activity resulted in a straight line with similar slopes for all glycosides but with different absolute values and thus the most reactive substrate p-nitrophenyl glucoside was the best one in the entire water activity range studied (0.53–0.96). The preference for the two competing acceptors, hexanol and water, was not affected by the aglycon part of the glucoside. Surprisingly, the ratio between trans glycosylation and hydrolysis increased with increasing water activity. A decrease in water activity caused an increase in equilibrium yield of hexyl glycoside, as expected, but was not beneficial for the kinetically controlled yield.