Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, <i>Aplysia kurodai</i>: Unique properties of two α‐amylases and two α‐glucosidases

Tsuji, Akihiko; Nishiyama, Nami; Ohshima, Miki; Maniwa, Saori; Kuwamura, Shuji; Shiraishi, Manabu; Yuasa, Keizo

doi:10.1016/j.fob.2014.06.002

Cited by 12 publications

(11 citation statements)

References 44 publications

(91 reference statements)

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“…Our findings on the optimal pH for the lobster α-amylase (pH 5.5) agree with values obtained before from crude extracts [ 11 ] and other invertebrate purified α-amylases [ 19 , 69 ], with the pH of gastric juice in different spiny lobsters (e.g., 6.4 to 5.8 [ 8 , 11 , 73 ]), and with a pH allowing of high enzyme stability. The strong reduction of α-amylase activity we observed below pH 4 was also found in P .…”

Section: Discussionsupporting

confidence: 90%

“…argus crude extracts [ 11 ] and in α-amylases from distant groups (e.g., human salivary α-amylase [ 74 ]), in agreement with high sequence and structure conservation. Also, the relationship between stability of the lobster α-amylase and temperature is consistent with those observed for other α-amylases [ 69 ]. In addition, using CNP-G3 as the substrate we demonstrated that the lobster α-amylase has lower K m (0.36 mM) than pancreatic and salivary human α-amylases (1.15 mM) [ 75 ].…”

Section: Discussionsupporting

confidence: 83%

“…It is well known that ions such as sodium, chloride and calcium, are important for the activity and stability of α-amylases. Lobster α-amylase activity increased with NaCl up to the optimum, and then only slightly declined at higher concentrations as observed for other α-amylases [ 19 , 68 , 69 ]. Optimum NaCl concentration for the lobster enzyme was 0.3 mM and activity remained high even at salt concentrations higher than those found in sea water (~0.5–0.6 M).…”

Section: Discussionsupporting

confidence: 55%

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Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

et al. 2016

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Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 83%

Section: Discussionsupporting

confidence: 55%

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Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

et al. 2016

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“…The East-Asian marine gastropod, sea hare ( A . kurodai ) consumes seaweed as staple food [ 18 ] and, although we have clarified the cellulose [ 18 ] and starch digestion systems [ 19 ], its defense mechanisms against seaweed secondary metabolites remain unclear. A .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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“…kurodai digestive fluid which was incubated with cellulose fiber (CF-11) or semi-crystalline starch used as control. SDS-PAGE analysis revealed 21-kDa and 80-kDa proteins bound to cellulose and starch, respectively ( Fig 1A ), which were identified as ß-1,4 endoglucanase (AkEG21) and α-amylase (ApAmy80) based on exact similarity of N-terminal amino acid sequences EQKCQPNSHGVRMYQ [ 22 ] and ASAYHDP [ 33 ], respectively. Although AkEG21 does not possess a CBD [ 23 ], the enzyme showed strong binding to CF-11, filter paper, Avicel and ß-1,3-glucan curdlan, but not to starch and chitin ( Fig 1B ), indicating that AkEG21 has specific affinity to ß-1, 4 and ß-1,3 glucans.…”

Section: Resultsmentioning

confidence: 99%

Cellulose-binding activity of a 21-kDa endo-ß-1,4-glucanase lacking cellulose-binding domain and its synergy with other cellulases in the digestive fluid of Aplysia kurodai

2018

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Endo-ß-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo ß-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another ß-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.

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Comprehensive enzymatic analysis of the amylolytic system in the digestive fluid of the sea hare, Aplysia kurodai: Unique properties of two α‐amylases and two α‐glucosidases

Cited by 12 publications

References 44 publications

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

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

Cellulose-binding activity of a 21-kDa endo-ß-1,4-glucanase lacking cellulose-binding domain and its synergy with other cellulases in the digestive fluid of Aplysia kurodai

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