Improving high-degree-polymerization GOS synthesis of a marine cold-active β-galactosidase via semi-rational design

Cold-active β-galactosidase has significant application value in the enzymatic synthesis of galacto-oligosaccharides (GOS); however, its low transglycosylation efficiency and poor selectivity toward high-degree polymerization (DP) products remain major challenges. In this study, a cold-active β-galactosidase (K3-β-gal) from marine Bacillus sp. K-3 was cloned and expressed. Four mutants (R252M, F261W, Y281R, and F355K) were constructed via semi-rational design, among which R252M exhibited markedly enhanced transglycosylation activity. Compared with the wild-type enzyme, R252M showed no change in optimum temperature (20 °C) or optimum pH (6.0), and retained good stability under low-temperature and weakly acidic conditions. The total GOS yield increased from 21.68% to 38.12% (a 75.83% improvement), and the DP4/DP3 ratio increased from 0.95 to 2.6, while the hydrolytic activity (k_cat/k_m) decreased by approximately 44%, indicating a shift in the catalytic equilibrium from hydrolysis toward transglycosylation. Molecular dynamics simulations revealed that R252M remodels the hydrogen-bond network in the active pocket, enhances protein rigidity, and reduces dynamic perturbations around the active site, which may contribute to substrate binding and transglycosylation efficiency.", This mutant holds promising potential for the green synthesis of high-DP GOS.