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

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, Sa AgaD, identified from Salegentibacter agarivorans . Sa AgaD is a 398-residue GH16 enzyme that expresses solubly in Escherichia coli and exhibits a high catalytic efficiency ( k cat / K m  = 1.76 × 10 4  mM −1  s −1 ) toward agarose. Distinct from many characterized agarases, Sa AgaD 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 Sa AgaD 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 Sa AgaD as a promising biocatalyst for industrial NAOS production from marine biomass.