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The digestive fluid of the sea hare  Aplysia kurodai  can liberate approximately 2.5 mg of glucose from 10 mg of dried  Eisenia bicyclis  powder. Although laminaran, a major storage polysaccharide in  E .  bicyclis , is easily digested to glucose by the synergistic action of the 110 and 210 kDa  A .  kurodai  β-glucosidases (BGLs), glucose is not liberated from  E .  bicyclis  by direct incubation with these BGLs. To clarify this discrepancy, we searched for an  Eisenia  hydrolysis enhancing protein (EHEP) in the digestive fluid of  A .  kurodai . A novel 25 kDa protein that enhances  E .  bicyclis  saccharification by β-glucosidases was purified to a homogeneous state from the digestive fluid of  A .  kurodai , and its cDNA was cloned from total cDNAs reverse-transcribed from hepatopancreas total RNA. The  E .  bicyclis  extract strongly inhibited BGLs, suggesting some compound within this brown alga functioned as a feeding deterrent. However, when  E .  bicyclis  was incubated with BGLs in the presence of EHEP, glucose production was markedly increased. As  E .  bicyclis  is rich in phlorotannin, which are only found in brown algae, our study suggested that these compounds are the main BGL inhibitors in  E .  bicyclis  extract. EHEP protects BGLs from phlorotannin inhibition by binding to phlorotannins and forming an insoluble complex with phloroglucinol and phlorotannins. These findings indicated that EHEP plays a key role in the saccharification of brown seaweeds containing phlorotannins in the digestive fluid of  A .  kurodai . This is the first report of EHEP as a phlorotannin-binding protein that protects BGLs from inhibition.

plos one

Identification and Characterization of a 25 kDa Protein That Is Indispensable for the Efficient Saccharification of Eisenia bicyclis in the Digestive Fluid of Aplysia kurodai