Studies on primary structures and genes of cellulases from marine invertebrates

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Distribution of cellulases in marine invertebrates was investigated by measuring the cellulase activity (CMCase activity) of digestive fluids from various invertebrates. Accordingly, herbivorous invertebrates such as abalone, sea urchin, and scallop were found to possess cellulases, whereas carnivorous invertebrates like starfish and Neptune snail were considered to possess no cellulases. Subsequently, cellulases (endo-1,4-β-glucanases) were isolated from abalone, sea urchin, and scallop by conventional column chromatographies and their cDNA and structural genes were cloned to deduce their primary structure. According to the deduced structures, the abalone, sea urchin, and scallop enzymes were classified to glycoside hydrolase family 9 (GHF9) as in case of termite and crayfish cellulases previously reported. Phylogenetic analysis of cellulase genes indicated that abalone, sea urchin, and scallop cellulases were derived from a common ancestral gene along with arthropod enzymes. In the abalone digestive fluid, an isozyme possessing a family-II carbohydrate-binding module in the N-terminus was found. This enzyme is the first animal cellulase possessing the family-II CBM in the N-terminus of the GHF9-type catalytic domain. Degrading abilities of the invertebrate cellulases toward cello-oligosaccharides were investigated. The abalone cellulase could degrade trisaccharide to disaccharide and glucose ; however, the sea urchin and scallop enzymes could not. Thus smallest substrate was trisaccharide for the abalone enzyme but tetrasaccharide for sea urchin and scallop enzymes. Further, when tetrasaccharide was allowed to react, the abalone enzyme split it into two moles of disaccharide, while the sea urchin and scallop enzymes split into trisaccharide and glucose as well as 2 moles of disaccharide. Therefore, the catalytic mechanism of invertebrate cellulases was considered to vary depending on the animal species although the enzymes all belong to GHF9.