Research Article: 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

Date Published: January 27, 2017

Publisher: Public Library of Science

Author(s): Akihiko Tsuji, Shuji Kuwamura, Akihiro Shirai, Keizo Yuasa, Erik Sotka.


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.

Partial Text

Global warming and the depletion of fossil fuels are of great concern, leading to the urgent search for alternative sustainable, renewable, efficient and cost-effective energy sources with reduced greenhouse gas emissions. Biomass seems to be as an excellent alternative source to meet present and future fuel demands. Two of the most common biofuels currently produced are ethanol derived from corn or sugarcane and biodiesel produced from a variety of oil crops, such as soybean and oil perm [1–3]. However the appropriation of significantly higher amounts of corn, sugarcane, or soybean to produce biofuels could have devastating effects on global food availability. To avoid this situation, lignocellulosic feedstocks are being developed as a second-generation of bioethanol sources [4, 5]. However, bioethanol produced from lignocellulosic feedstocks has higher costs, due to the pretreatment required (e.g. steam explosion) to remove lignin and make cellulose accessible for efficient enzymatic digestion [6], and to the larger amount of enzymes needed to produce glucose from cellulosic-based feedstocks than required for starch enzymatic saccharification. Thus, novel technologies for reducing the cost of cellulose saccharification are urgently required [7].

The most striking finding in the present investigation was the identification of a novel 25 kDa EHEP, which binds to polyphenols in brown seaweed. To survive against predation by marine animals such as sea urchin and gastropods, seaweeds produce feeding deterrents [16, 17], such as the phlorotanins of E. bicyclis that have been shown to have a deterrent effect against the feeding behavior of the herbivorous gastropod Turbo cornuts [26]. In the present study, we showed that E. bicyclis extract contained a strong inhibitor of A. kurodai β-glucosidases, particularly the 110 kDa BGL, which was highly sensitive to E. bicyclis and A. nodosum extracts. Although purified laminaran was efficiently digested to glucose by the 110 and 210 kDa BGLs, saccharification of E. bicyclis laminaran by these BGLs was almost completely blocked by E. bicyclis compounds; however the inhibitory activity of the E. bicyclis extract was completely neutralized with EHEP. E. bicyclis contains several kinds of phlorotannins, eckol (phloroglucinol trimer), phlorofucoeckol A (pentamer), dieckol and 8,8’-bieckol (hexamers) [22, 23, 38]. Although we did not identify individual phlorotannin-inhibiting β-glucosidases, several results suggested phlorotannins as the most plausible candidates for BGL inhibitors: 1) BGLs were inhibited in brown seaweed extracts containing a high content of phlorotannins, as is the case of E. bicyclis and A. nodosum, and extracts of the green seaweed, U. pertusa had no effect on β-glucosidase activity; 2) the phlorotannin fraction isolated from the 80% ethanol extract of E. bicyclis (phlorotannin A) strongly inhibited BGLs, but was neutralized when incubated with EHEP, and EHEP was precipitated by phlorotannins; 3) phloroglucinol, a constituent of phlorotannins, also inhibited BGL, and EHEP was precipitated by phloroglucinol, although a higher concentration of phloroglucinol was required for BGL inhibition. In addition, the affinities of EHEP toward phlorotannins and phloroglucinol were higher than that of BSA, EHEP and BSA had similar affinities to tannic acid, and EHEP was more specific toward phlorotannins comprising phloroglucinol than for tannin comprising gallic acid.




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