Characterization of a β-galactosidase from Bacillus subtilis with transgalactosylation activity

Microbial β-galactosidases (EC 3.1.2.23) have applications in the production of galacto-oligosaccharides, which are established prebiotic food ingredients. The β-galactosidase from Bacillus subtilis (YesZ) was expressed as a heterologous protein in Escherichia coli, and presented an optimum activity at pH 6.5 and 40 °C. The catalytic constants K_m and V_max of the enzyme were 8.26 mM and 1.42 μmol·min^-1·mg^-1 against pNP-β-d-galactopyranoside, respectively. Structural characterization revealed that YesZ is a homotrimer in solution, and homology modeling suggested that the YesZ conserves a Cys cluster zinc binding site. Flame photometry experiments confirmed the presence of bound zinc in the recombinant enzyme, and YesZ activity was inhibited by 1 mM zinc, copper and silver ions. Transgalactosylation activity of YesZ was observed with the synthetic substrate p-NP-βGal in the presence of a d-xylose acceptor, producing a β-d-galactopyranosyl-(1 → 4)-d-xylopyranose disaccharide. Analysis of this disaccharide by MALDI-ToF-MS/MS suggested a β-1,4 glycosidic linkage between a non-reducing galactose residue and the xylose. The β-galactosidase YesZ from B. subtilis is a candidate for enzymatic synthesis showing favorable thermostability (with residual activity of 50% after incubation at 30 °C for 25 h) and transgalactosylation activity.