β-Glucosidase from Thermotoga naphthophila RKU-10 for exclusive synthesis of galactotrisaccharides: Kinetics and thermodynamics insight into reaction mechanism

- This work presents a new enzymatic system to valorize milk processing waste lactose.
- A highly selective β-glucosidase for exclusive synthesis of GOS3 is identified.
- TN0602 β-glucosidase affords 23.28 g L−1 h−1 yield of GOS3 with 100% selectivity.
- Deep pocket with a narrow entrance to catalytic site dedicates high selectivity.
- Catalytic specificity is elucidated by thermodynamic analysis & molecular docking.

This work reports a novel thermophilic β-glucosidase (TN0602) from Thermotoga naphthophila RKU-10, demonstrating exceptionally high catalytic selectivity (100%) for the exclusive synthesis of prebiotic galactotrisaccharides (GOS3) in a high volumetric production yield of 23.28 g L−1 h−1 (higher than the highest value ever reported) at pH 6.5 and 75 °C, with milk processing waste lactose as both the galactosyl donor and acceptor. A comparative study with commercial β-galactosidase from Aspergillus oryzae (AO) with respect to reaction kinetics, enzyme-substrate thermodynamic binding (substrate induced fluorescence quenching) and molecular docking simulation studies showed that β-glucosidase TN0602 has a deep catalytic “pocket” with a narrow entrance that prevents simultaneous access of lactose and GOS3 to the catalytic site, explaining its distinct catalytic specificity and reaction kinetics. The findings revealed in this work offer an improved understanding of how enzyme protein structure determines catalytic specificity, which serves as new knowledge to engineer β-glucosidase for the biosynthesis of designer GOS.