Isolation of a multi-functional endogenous cellulase gene from mollusc, Ampullaria crossean.
ABSTRACT The cellulase genes of some animals, most coding for endo-beta-1,4-glucanases, were found and cloned. There has been no reports about genes encoding exo-beta-1,4-glucanase or endo- -1,4-xylanase from animal. Here we cloned the cDNA of a cellulase designated as EGX from mollusc, Ampullaria crossean, and expressed it in Pichia pastoris for the first time. The cellulase EGX is a multi-functional beta cellulase with the activities of exo-beta-1,4-glucanase, endo-beta-1,4-glucanase and endo-beta-1,4-xylanase. The opening reading frame of EGX cDNA is 1185 bp and encodes 395 amino acids. The EGX gene can also be amplificated from the genomic DNA by PCR, which verified the endogenous origin of this gene. This EGX gene was the first multi-functional cellulase gene that was directly isolated from animals.
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ABSTRACT: The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95, 66, 45, and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40°C, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55°C for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic β-1,4-glucanase (EC 220.127.116.11). A cDNA of 1013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.Frontiers in Chemistry 08/2014; 2:60.
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ABSTRACT: We isolated a cellulase from the digestive organs of the short-spined sea urchin Strogylocentrotus intermedius using a combination of ion-exchange chromatography and gel filtration together with an assay for carboxymethylcellulase activity. The isolated cellulase was stained as a single band by Congo red. The molecular weight of the isolated cellulase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, was 59 kDa. The isolated cellulase exhibited hydrolytic activity toward carboxymethyl cellulose, with an optimum temperature and pH of 30 °C and pH 8.0, respectively. The thermal stability of the enzyme was characterized by determining the temperature at which activity decreased by 50 % with treatment for 30 min at pH 7.0 and found to be 32 °C. Cellulase activity remained at a high level at 5–20 °C, which is the growth temperature of the short-spined sea urchin. These results confirm that the short-spined sea urchin should preferably be reared at a water temperature of <20 °C.Fisheries Science 78(5). · 0.86 Impact Factor
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ABSTRACT: a b s t r a c t We examined the feeding niche of four species of molluscs by analysis of their stable isotope signatures and cellulase activities to determine if they could utilize terrestrial organic matter. The molluscs and potential food sources were collected from the upper, middle and lower estuary of the Yura River from spring 2007 to winter 2008. All species showed positive cellulase activity which highlighted their potential to digest terrestrial organic matter. Consumption and assimilation of terrestrial organic matter by estuarine molluscs however varied spatially and temporally, reflecting species-specific differences in feeding niche and in response to variations in food availability in the estuary. Thus, terrestrial primary production in the catchment area supports secondary production of molluscs in the Yura River estuary.