A GH42 β-Galactosidase from the Human Isolate <i>Bifidobacterium breve</i> DSM 20213: Biochemical and Transgalactosylation Properties Reveal the Potential for Galacto-oligosaccharides Synthesis

A β-galactosidase (<i>Bbre</i>βgal-III) derived from the human isolate <i>Bifidobacterium breve</i> DSM 20213, which belongs to the glycoside hydrolase (GH) family 42, was successfully overproduced in <i>Escherichia coli</i>, purified to apparent homogeneity, and studied with respect to its biochemical and molecular properties as well as its transgalactosylation activity for the synthesis of galacto-oligosaccharides (GOS) in comparison to the previously reported two GH2 β-galactosidases, <i>Bbre</i>βgal-I and <i>Bbre</i>βgal-II, originating from this strain. <i>Bbre</i>βgal-III is a homotrimer with a molecular mass of approximately 240 kDa and shows distinct characteristics in terms of catalytic efficiency, pH dependence of activity and stability over a broad pH range, substrate specificities, and preference for the formation of specific GOS structures when compared with the other two GH2 β-galactosidases. These results suggest that multiple β-galactosidases in <i>B. breve</i> play divergent roles in substrate utilization under various conditions, especially in the degradation of various human milk oligosaccharide structures containing one or more β-linked galactose moieties as selective substrates for the growth and well-being of breast-fed infants. <i>Bbre</i>βgal-III displays transgalactosylation activity with a total GOS yield of 17% of total sugars with the preference to form 6'-galactosyllactose, which is also a predominant product during transgalactosylation of lactose. This finding paves the way for further work on this enzyme using engineering strategies to enhance transgalactosylation activity and hence the yield of the transgalactosylation products or for the biosynthesis of the GOS mixtures with specific structures, which could result in functionally enhanced prebiotic GOS.