β-Galactosidase expression on Lactococcus sp. KTH0-1S as a whole-cell biocatalyst for efficient lactosucrose...

Lactosucrose is a functional trisaccharide with prebiotic properties and growing potential in food and agricultural applications; however, its industrial production is limited by low conversion efficiency and high downstream processing costs. In this study, a whole-cell biocatalyst was developed by displaying β-galactosidase on Lactococcus sp. KTH0-1S using a nisin-inducible expression system, the USP45 signal peptide, and a LysM anchoring domain. The engineered strain exhibited a maximum whole-cell β-galactosidase activity of 146.0 Miller units after 4 h of induction. The whole-cell enzyme efficiently catalyzed lactosucrose synthesis from lactose and sucrose, with optimal activity observed at pH 6.0 and 50 °C. Substrate molar ratio significantly influenced product formation, and the highest lactosucrose concentration (15,940 mg/L) was achieved at a lactose:sucrose ratio of 1:3. Reusability studies revealed high initial catalytic activity but limited stability upon repeated reaction cycles, likely due to the non-covalent nature of LysM-mediated surface anchoring. Molecular docking analysis supported the experimental observations by identifying a high-affinity substrate-binding pocket capable of accommodating both donor and acceptor substrates, providing a structural rationale for enhanced transgalactosylation under sucrose-enriched conditions. Overall, this study demonstrates the feasibility of using a whole-cell biocatalyst based on Lactococcus sp. for efficient lactosucrose production and highlights its potential as a cost-effective and food-compatible platform for the synthesis of functional oligosaccharides.