High Galacto-Oligosaccharide Production and a Structural Model for Transgalactosylation of β‑Galactosidase II from Bacillus circulans

The transgalactosylase activity of β-galactosidase produces galacto-oligosaccharides (GOSs) with prebiotic effects similar to those of major oligosaccharides in human milk. β-Galactosidases from Bacillus circulans ATCC 31382 are important enzymes in industrial-scale GOS production. Here, we show the high GOS yield of β-galactosidase II from B. circulans (β-Gal-II, Lactazyme-B), compared to other commercial enzymes. We also determine the crystal structure of the five conserved domains of β-Gal-II in an apo-form and complexed with galactose and an acceptor sugar, showing the heterogeneous mode of transgalactosylation by the enzyme. Truncation studies of the five conserved domains reveal that all five domains are essential for enzyme catalysis, while some truncated constructs were still expressed as soluble proteins. Structural comparison of β-Gal-II with other β-galactosidase homologues suggests that the GOS linkage preference of the enzyme might be quite different from other enzymes. The structural information on β-Gal-II might provide molecular insights into the transgalactosylation process of the β-galactosidases in GOS production.