Molecular dynamics and NMR data for: Active-site engineering of a GH2 bifidobacterial β-galactosidase for improved formation of N-acetyl-lactosamine - a core structure of human milk oligosaccharides

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.N-乙酰乳糖胺（N-Acetyl-lactosamine, LacNAc, β-D-Gal-(1→4)-β-D-GlcNAc）是部分人乳寡糖的核心结构，可通过β-半乳糖苷酶催化的转糖基反应合成，以乳糖作为半乳糖基供体、N-乙酰葡糖胺（GlcNAc）作为半乳糖基受体。为提升LacNAc产率，研究以短双歧杆菌DSM 20213来源的GH2家族β-半乳糖苷酶（Bbreβgal-II）为对象，针对其活性位点的半乳糖结合氨基酸残基开展丙氨酸扫描诱变。破坏非保守残基处的氢键会显著降低酶活性；松动活性位点虽会降低底物结合能力，却能提升转糖基产物中β-(1→4)糖苷键形成的区域选择性。所有活性突变体的LacNAc产率均高于野生型Bbreβgal-II，其中Bbreβgal-II-H558A突变体的LacNAc产率提升了三倍，但其区域异构体副产物N-乙酰别乳糖胺（alloLacNAc, β-D-Gal-(1→6)-β-D-GlcNAc）的生成量与野生型相当。这种简便的丙氨酸扫描诱变策略，可推广应用于其他可用于酶法合成目标聚糖的糖苷酶类研究。