Re-Engineered β-Agarase for Efficient Agarose Oligosaccharide Preparation with Enhanced Thermostability

Agarases are enzymes that serve as valuable catalysts in producing bioactive agar oligosaccharides. The thermostability of agarase is crucial for its industrial application since the sol–gel transition of the agar substrate is temperature-dependent. An exo -β-agarase, Aga50D, from Saccharophagus degradans 2–40 was re-engineered based on a single mutation near the interface. The A86D mutant exhibited a melting temperature increase of 23.3 °C with more than a 7-fold improvement in catalytic performance for agarose hydrolysis at 50 °C for 1 h. Small-angle X-ray scattering (SAXS), differential fluorimetry scanning (DFS), and size exclusion chromatography (SEC) analyses provided convincing evidence that the mutation significantly promoted the dimerization. Molecular dynamics simulations of the enzyme further supported our findings, demonstrating that the single mutation near the interface induced the rearrangement of the intrachain salt bridges between the loops and strengthened the interchain salt-bridge interaction at the interface, which were identified as critical mechanisms for improving its thermostability. Meanwhile, the stable interface interaction may play an important role in the dimerization of A86D. These results highlight the design of intersubunit interactions to obtain high-stability enzymes via targeted mutations near the subunit interface. The robust A86D mutant shows great potential in industrial agar oligosaccharide production.