High-efficiency and thermostable β-agarase Aga3 for sustainable production of neoagarooligosaccharides (NAOs) from algal biomass

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 K m and k cat 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.