Molecular Characterization and Mechanistic Insights of a Thermostable Neoagarobiose Hydrolase Aga2457 from Alteromonas sp.

The enzymatic valorization of agarose, a major polysaccharide in red algae, is critical for its application in the food, pharmaceutical, and biotechnology industries. In this study, a gene encoding a thermostable α-neoagarobiose hydrolase, aga2457, was cloned from an epiphytic bacterium associated with Indonesian macroalgae. Unlike typical mesophilic GH117 enzymes, recombinant Aga2457 displayed a higher optimal temperature at 50 °C and retained 55% activity after 12 days of incubation at 50 °C. The enzyme specifically hydrolyzes neoagarobiose into D-galactose and 3,6-anhydro-L-galactose, thereby facilitating the complete depolymerization of agarose. Combined molecular dynamics (MD) simulations and site-directed mutagenesis revealed that residues P253, N256, and Q285 are pivotal for substrate recognition and active site stability. These findings highlight Aga2457 as a robust biocatalyst for industrial agar processing and provide structural insights for the rational design of thermostable agarolytic enzymes.