Enhancing the Thermostability of α-Agarase CmAga through Rational Design in Flexible Regions

α-Agarases hydrolyze agarose into bioactive agaro-oligosaccharides but often suffer from poor thermostability, limiting industrial application above the agarose gelling temperature. Here, we enhanced the thermostability of a multidomain α-agarase from Catenovulum maritimum STB14 (CmAga) through rational design targeting its flexible regions. Molecular dynamics simulations identified six highly flexible segments. Key residues were subjected to site-directed mutagenesis, yielding stabilizing single mutants (D156N and D381R) and a double mutant with enhanced thermal resistance. Disulfide bond engineering further provided a stabilized variant (D579C–W597C). The combination mutant of the two strategies exhibited a 3.15 °C increase in melting temperature and a 16.05-fold longer half-life at 50 °C. Structural analyses indicated that stabilization arises from reduced overall conformational fluctuations and an optimized hydrogen-bond network, demonstrating that the four-site mutations act cooperatively across the entire molecular structure. This work provides thermostable α-agarase variants for industrial use and a rational design framework for engineering flexible multidomain glycosidases.