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

Abstract

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.

Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing

“…Native EHEP (25 kDa; GenBank BAV38197.1; Table 1) was purified from the natural digestive fluid of A. kurodai as described previously (Tsuji et al, 2017). This protein was then further purified using size-exclusion chromatography (SEC) on a Superdex 200 10/300 GL column (GE Healthcare) which was equilibrated with 20 mM sodium acetate buffer containing 100 mM sodium chloride pH 6.0.…”

“…A previous study predicted that native EHEP possesses a signal peptide in its N-terminus (Tsuji et al, 2017). To confirm the molecular weight of mature native EHEP, MALDI-TOF mass-spectrometric analysis of purified EHEP was carried out using an Autoflex III Smartbeam mass spectrometer (Bruker).…”

“…The sea hare Aplysia kurodai consumes brown algae as a staple food, using -glucosidases (akuBGLs) as catalysts to hydrolyze the glycosidic bonds to produce glucose (Tsuji et al, 2013) and making it an excellent model for investigating the biofuel production process. However, phlorotannin, which is also abundant in brown algae (La Barre et al, 2010), inhibits the hydrolytic reaction of akuBGLs (Tsuji et al, 2017). Moreover, previous studies have reported that phlorotannin also inhibits the activity of several other enzymes, such as hyaluronidase (Shibata et al, 2002), -glucosidase (Kellogg et al, 2014) and -amylase (Kellogg et al, 2014).…”

“…Moreover, previous studies have reported that phlorotannin also inhibits the activity of several other enzymes, such as hyaluronidase (Shibata et al, 2002), -glucosidase (Kellogg et al, 2014) and -amylase (Kellogg et al, 2014). Interestingly, Eisenia hydrolysis-enhancing protein (EHEP), which was recently identified in the digestive fluid of A. kurodai, protects akuBGLs from this inhibition by binding and then precipitating with phlorotannin, despite not possessing a catalytic (Tsuji et al, 2017). Furthermore, it has been shown that EHEP is a novel and unique protein, with no proteins with homologous amino-acid sequences occurring in any other organism, and it also binds to analogs of phlorotannin such as gallic acid and tannic acid (Tsuji et al, 2017).…”

“…Interestingly, Eisenia hydrolysis-enhancing protein (EHEP), which was recently identified in the digestive fluid of A. kurodai, protects akuBGLs from this inhibition by binding and then precipitating with phlorotannin, despite not possessing a catalytic (Tsuji et al, 2017). Furthermore, it has been shown that EHEP is a novel and unique protein, with no proteins with homologous amino-acid sequences occurring in any other organism, and it also binds to analogs of phlorotannin such as gallic acid and tannic acid (Tsuji et al, 2017). Consequently, EHEP has a high potential for application in the biofuel industry.…”