Construction and optimization of a monophasic organic-water system for enzymatic synthesis of p-nitrobenzyl β-d-glucopyranosides by reverse hydrolysis

A monophasic organic-water system for efficient enzymatic synthesis of β-d-glucopyranoside by reverse hydrolysis was constructed and optimized. p-Nitrobenzyl alcohol (pNBA), selected as a model substrate alcohol, was readily glucosylated with d-glucose through reverse hydrolysis using almond β-d-glucosidase in a monophasic aqueous-organic medium, producing a new glucoside, p-nitrobenzyl β-d-glucopyranoside (pNBG). The effects of different buffers, organic solvents and water contents were investigated. Buffer type and pH affected the initial reaction rate but had little effect on the final yields. The ratio of organic solvent to water plays a crucial role in shifting the reaction equilibrium toward synthesis, but a minimum amount of water is necessary to maintain the enzyme activity. Dioxane, which was previously known as an unsuitable solvent for β-d-glucosidase-catalyzed reactions, was found to be the most appropriate solvent for this synthetic procedure. The reaction equilibrium and enzyme stability in the reaction medium were also investigated. Under the optimal reaction conditions, i.e. 90% dioxane (v/v) + 10% buffer (Na2HPO4-KH2PO4, 70 mM, pH 6.0) with alcohol-to-glucose molar ratio of 9:1, p-nitrobenzyl β-d-glucopyranoside was produced with a maximum yield (13.3%).