A thermophilic GH16 β-agarase from Salegentibacter agarivorans enables efficient and selective production of...

Agarose, a hydrophilic polysaccharide derived from red algae, is widely used in biochemical and agricultural applications, but its intrinsic gelation below ∼40 °C limits enzymatic conversion into high-value neoagarooligosaccharides (NAOS). Here, we report the systematic characterization of a novel thermophilic β-agarase, SaAgaD, identified from Salegentibacter agarivorans. SaAgaD is a 398-residue GH16 enzyme that expresses solubly in Escherichia coli and exhibits a high catalytic efficiency (k_cat/K_m = 1.76 × 10⁴ mM^-1 s^-1) toward agarose. Distinct from many characterized agarases, SaAgaD displays optimal activity at pH 8.0 and 55 °C, retains ∼90% activity at 65 °C, and remains stable under neutral to mildly alkaline conditions, enabling effective hydrolysis above the agarose gelation point. The enzyme preferentially degrades agar and crude Gracilaria agar, with minimal activity on sulfated porphyran. Product profiling by HPAEC-PAD and ESI-MS demonstrates an endolytic mechanism yielding predominantly neoagarotetraose (NA4) and neoagarohexaose (NA6), with a scale-up NAOS yield of ∼75%. Molecular dynamics simulations of SaAgaD and its NA8 complex reveal a stable substrate binding mode and highlight flexible loop regions that coordinate substrate recognition. The combination of high thermal tolerance, catalytic efficiency, and product specificity positions SaAgaD as a promising biocatalyst for industrial NAOS production from marine biomass.