Multi-Method Combined Screening of Agarase-Secreting Fungi from Sea Cucumber and Preliminary Analyses on Their Agarases and Agar-Oligosaccharide Products

Agar can be degraded into agar-oligosaccharides by physical, chemical, and biological methods, but the further industrial application of agar-oligosaccharides has been limited by the environmental pollution of traditional agar-oligosaccharides preparation methods and the lack of novel agarase. In this study, we reported the screening of 12 strains with agar-degrading activity from sea cucumber intestine and mucus using a combination of Gram's iodine staining and 3,5-dinitrosalicylic acid (DNS) method, during which five fungal strains exhibited high agarase activity. Their production of different agarases and agar-oligosaccharides could be visualized by zymogram assay and thin-layer chromatography. A strain ACD-11-B with the highest agarase activity showed 99.79% similarity to <i>Aspergillus sydowii</i> CBS593.65 for ITS rDNA sequence. Strain ACD-11-B produced five possible agarases with predicted molecular weights of 180, 95, 43, 33, and 20 kDa, approximately. The optimal temperature and pH of the crude enzyme production by strain ACD-11-B were 40 °C and 6.0. The crude enzyme was stable at 30 °C, and Ca²⁺, K⁺, and Na⁺ could increase the activity of the crude enzyme. Its agarases demonstrated remarkable salt tolerance and substrate specificity, with neoagarobiose (NA2) identified as the main degradation product. These results indicate that the fungal strain ACD-11-B can secrete agarases with potential in industrial applications, making it a new producer strain for agarase production.