The improvement of stability, activity, and substrate promiscuity of glycerol dehydrogenase substituted by divalent metal ions

The substitution of the catalytic zinc ion of glycerol dehydrogenase (GDH) from Klebsiella pneumonia sp. by divalent metal ions, Mn²⁺ and Mg²⁺, enabled improvements of activity, substrate promiscuity and stability. The activity of Mn-GDH and Mg-GDH improved several folds in comparison to the native GDH. The activity of substituted GDH towards non-natural substrates, 4-chloroacetoacetate, 3-chloroacetylpyridine, p-chloroacetophenone, and acetophenone was 30 folds higher than native GDH. Manganese substitution increased the half-life of GDH by 6 folds at 60 and 70°C. The two-fraction first order inactivation models fitted the nonlinear thermal inactivation curves well. Combined with the kinetic and thermodynamic analysis, further mechanistic insights to the metal ion roles in thermostability enhancements were studied. The thermodynamic parameters of inactivation, enthalpy, entropy and the Gibbs free energy indicated that Mn-GDH was stabilized entropically and elucidated the mechanisms of enzyme inactivation.