Bacillus circulans β-galactosidase catalyses the synthesis of N-acetyl-lactosamine in a hydro-organic medium via a steady-state ordered Bi Bi reaction mechanism

Starting from a galactosyl donor, the β-galactosidase from Bacillus circulans (Lactoles® L3; BcβGal) catalyses the transgalactosylation of N-acetyl-d-glucosamine (GlcNAc) in hydro-organic media, leading to the synthesis of N-acetyl-lactosamine (LacNAc). The aim of this study was to determine the kinetic properties of LacNAc synthesis and sought to highlight the effect of the organic solvent on the enzyme kinetic parameters. Analyses of enzyme kinetics were performed in aqueous solutions and water/tert-butanol mixtures. A steady-state ordered Bi Bi mechanism was characterised for the transgalactosylation of GlcNAc catalysed by BcβGal in water/tert-butanol (70:30, v/v) mixtures, starting from o-nitrophenyl-β-d-galactopyranoside (oNPG) as a galactosyl donor. Moreover, tert-butanol was shown to have various effects on the kinetic parameters of the total transformation of oNPG and lactose catalysed by BcβGal, which led us to liken this solvent to a noncompetitive inhibitor towards the former and a competitive inhibitor towards the latter.