Improving thermostability of α-L-fucosidase from Pedobacter sp. via consensus-guided engineering and directed...

The α-L-fucosidase from Pedobacter sp. (PbFuc), a glycoside hydrolase capable of catalyzing the synthesis of 2'-fucosyllactose (2'-FL) from 4-nitrophenyl α-L-fucopyranoside (pNP-Fuc) and D-lactose, exhibits limited industrial applicability due to inherent thermostability constraints. This study implemented a combinatorial strategy integrating consensus-guided engineering and directed evolution to engineer the thermal stability of PbFuc, resulting in the identification of six critical mutants (G53C, M54I, N59S, T71H, R125C, S199P) and the subsequent construction of the combinatorial mutant M6. Thermostability assays revealed complete inactivation of the wild-type enzyme after 30-min incubation at 45 °C, whereas M6 retained approximately 40 % residual activity under equivalent conditions at 60 °C, accompanied by an increase in the optimal reaction temperature from 35 °C to 40 °C. Structural mechanism analysis demonstrated that the enhanced thermostability of M6 originated from synergistic multilevel structural optimization and reorganization of molecular interaction networks: Conformational stabilization manifested through prolonged maintenance of stable secondary structural conformations during thermal stress and reduced amplitude of tertiary structural fluctuations; Global structural compaction decreased solvent-accessible surface area, thereby minimizing thermal energy transfer; Local structural reinforcement occurred via the formation of novel hydrogen bonds, enhanced rigidity through π-π stacking, and neutralization of electrostatic repulsion via charge compensation.