Effects of Carbohydrates on the oNPG Converting Activity of ß-Galactosidases

The effects of high concentrations of carbohydrates on the o-nitrophenyl ß-D-galactopyranoside (oNPG) converting activity of ß-galactosidase from Bacillus circulans are studied to get a better understanding of the enzyme behavior in concentrated and complicated systems in which enzymatic synthesis of galacto-oligosaccharides is usually performed. The components that were tested were glucose, galactose, lactose, sucrose, trehalose, raffinose, Vivinal GOS, dextran-6000, dextran-70 000, and sarcosine. Small carbohydrates act as acceptors in the reaction. This speeds up the limiting step, which is binding of the galactose residue with the acceptor and release of the product. Simultaneously, both inert and reacting additives seem to cause some molecular crowding, which results in a higher enzyme affinity for the substrate. The effect of molecular crowding on the enzyme activity is small compared to the effect of carbohydrates acting in the reactions as acceptors. The effects of reactants on ß-galactosidases from B. circulans, A. oryzae, and K. lactis are compared. KEYWORDS: galacto-oligosaccharides, GOS, ß-galactosidase, Bacillus circulans, enzyme activity, carbohydrates, crowding, galactosyl transfer, transgalactosylation, Aspergillus oryzae, Kluyveromyces lacti