Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase Pedsa0632

Alginate can be degraded by alginate lyase to produce alginate oligosaccharides (AOs). AOs are widely used in the food, agricultural, and pharmaceutical industries due to their various physiological activities. In this work, alginate lyase Pedsa0632 was successfully characterized, which exists in solution as monomers and oligomers. Pedsa0632 has poor thermal stability, displaying a half-life (t_{1/2, 55 °C}) of merely 6.54 min at its optimum temperature. We attempted to improve the thermal stability of Pedsa0632 by changing the interface and increasing the content of the oligomers. A mutant library was generated through combinatorial engineering of disulfide bonds, intersubunit salt bridges, and PROSS (Protein Repair One-Stop Shop) guided stabilization strategies. Mutant L324 V-D353 V-M363T-T385 V (M3) was finally constructed. The wild-type (WT) enzyme was basically inactivated after 30 min of incubation at 55 °C, whereas M3 still maintained 60% relative activity after 11,000 min of incubation under the same conditions. Further structural comparisons between the WT and M3 revealed that the extraordinary stability of the M3 could be due to the mutation that induced a more stable and compact interface of Pedsa0632, resulting in an increased proportion of oligomer content. The rational design strategy used in this study can effectively improve the enzyme's thermal stability, especially oligomeric enzymes.