Rational Engineering of Heparinase III from Pedobacter schmidteae to Improve the Thermostability via Distal...

Heparinase III (Hep-III) plays an important role in the degradation of heparan sulfate and heparin, but its instability limits its applications. Herein, a distal mutagenesis strategy based on sequence conservation analysis and molecular dynamics (MD) simulations was employed to enhance the thermostability of Hep-III from Pedobacter schmidteae (PsHep-III). The V183A/D378G mutant (M2a) exhibited a 31.21-fold increase in half-life (t_1/2) at 35 °C relative to the wild type (WT). Furthermore, the melting temperature (T_m) and the temperature at which 50% residual activity was retained after 1 min (T₅₀¹) of M2a were increased by 23.66 and 8.02 °C, respectively. The specific activity of M2a was 104.16% relative to that of the WT. Analysis of intramolecular interactions, protein surface charge and MD simulations indicated that enhanced local rigidity and increased surface potential were the main contributors to the overall stability improvement of M2a. This study provides an effective strategy for improving the thermostability of Hep-III.