N-Acetyl-lactosamine (LacNAc, β-D-Gal-(1→4)-β-D-GlcNAc), forming the core structure of certain human milk oligosaccharides, can be synthesized via transgalactosylation catalyzed by β-galactosidases, using lactose as the galactosyl donor and N-acetyl-glucosamine (GlcNAc) as the galactosyl acceptor. To improve LacNAc yield, a GH2 β-galactosidase (Bbreβgal-II) from Bifidobacterium breve DSM 20213, was subjected to alanine-scanning mutagenesis targeting the galactose-binding amino acid residues at its active site. Disruption of the hydrogen bonds at non-conserved residues severely reduced enzymatic activity. Loosening the active site reduced the substrate binding capacity but enhanced regioselectivity toward β-(1→4) linkage formation in transgalactosylated products. All active mutants increased LacNAc yield compared to wild-type Bbreβgal-II. The Bbreβgal-II-H558A variant showed a threefold increase in LacNAc yield, while the formation of its regioisomeric by-product, N-acetyl-allolactosamine (alloLacNAc, β-D-Gal-(1→6)-β-D-GlcNAc), remained comparable to the wild type. This straightforward approach of alanine scanning mutagenesis may be applied for different glycosidases that are of interest for the enzymatic synthesis of other desired glycans.