Insights into thermostability enhancement of a PL-5 alginate lyase through rational design.
Efficient utilization of natural alginate is restricted by its high viscosity. Alginate lyase degrades alginate via β-elimination to reduce viscosity, supporting its development and application. However, natural alginate lyases exhibit poor thermostability, limiting their application in high-temperature environments. Enhancing the thermostability of natural alginate lyase would better meet the demands of industrial production. We cloned and characterized RwAly5A, a PL-5 family alginate lyase from Ralstonia wenshanensis 56D2. Recombinant RwAly5A exhibited maximal catalytic activity at pH 8.0, with robust tolerance over the pH range of 4.6-10.6. Its maximum activity is observed at 50 °C, and although the protein remains fairly stable at 0-40 °C, its thermostability deteriorates rapidly at temperatures above 50 °C. Additionally, RwAly5A exhibited optimal activity at 100 mM NaCl. Its activity was promoted by a broad spectrum of cations, including monovalent (K+, NH4+, and Li+) and divalent (Ca2+ and Mg2+) ions. In contrast, Mn2+, Ba2+, Fe3+, and EDTA suppressed the enzyme, and SDS almost completely abolished its function. Based on molecular dynamics (MD) simulations, residues predicted to be located in the flexible-loop region were chosen as targets for proline substitution, and the resulting variants were subjected to thermostability mechanism studies. Proline substitutions were introduced in flexible loops, generating D20P, D227P, and A229P mutants, which exhibited half-lives at 50 °C that increased to 1.14‑, 4.69‑, and 2.28‑fold, respectively, while retaining most of the catalytic activity. These findings provide new strategies for improving alginate lyases for commercial applications.