With growing concerns about the circular economy and carbon neutrality, attention has increasingly focused on the discovery and industrial application of powerful renewable resources. Agarose, a major structural component of red algae and classified as a third-generation biomass, has emerged as a promising renewable substrate for the production of high-value chemicals and bioactive compounds. In this study, we identified GH16 β-agarase, designated as Aga3, from the marine agarolytic bacterium, Cellulophaga omnivescoria MSK1 and conducted comprehensive biochemical characterization of the enzyme. Aga3 exhibited excellent agarose-degrading activity, with Km and kcat values of 0.93 mg/mL and 1300 s−1, respectively. The enzyme also showed remarkable thermal stability above 50°C, and its activity increased more than six-fold in the presence of 5 mM cobalt ions. Under optimized conditions, Aga3 efficiently produced neoagarobiose (NA2) (6.61 ± 0.55 g/L) and neoagarotetraose (NA4) (10.4 ± 1.30 g/L) with maximum conversion rate of 88.8%. These results demonstrate that Aga3 is a robust and highly efficient biocatalyst with substantial potential for sustainable bioprocesses.
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