Studies on primary structures and genes of cellulases from marine invertebrates

通过测定各种无脊椎动物消化液的纤维素酶活性（CMCase活性）来研究纤维素酶在海洋无脊椎动物中的分布。因此，草食性无脊椎动物如鲍鱼、海胆和扇贝被发现具有纤维素酶，而肉食性无脊椎动物如海星和海王星蜗牛被认为不具有纤维素酶。采用柱层析法从鲍鱼、海胆和扇贝中分离得到纤维素酶（endo-1，4-β-glucanases），并克隆其cDNA和结构基因，推测其一级结构。根据推导的结构，鲍鱼，海胆和扇贝的酶被归类为糖苷水解酶家族9（GHF 9）的情况下，白蚁和小龙虾纤维素酶先前报道。系统发育分析表明，鲍鱼、海胆和扇贝纤维素酶与节肢动物纤维素酶沿着来自一个共同的祖先基因。在鲍鱼消化液中发现了一种N-末端含有Ⅱ族碳水化合物结合模块的同工酶，该酶是第一个在GHF 9型催化结构域N-末端含有Ⅱ族CBM的动物纤维素酶。研究了无脊椎动物纤维素酶对纤维低聚糖的降解能力。鲍鱼纤维素酶能将三糖降解为二糖和葡萄糖，而海胆和扇贝纤维素酶不能。因此，鲍鱼酶的最小底物是三糖，而海胆和扇贝酶的最小底物是四糖。此外，当允许四糖反应时，鲍鱼酶将其分解成2摩尔二糖，而海胆和扇贝酶分解成三糖和葡萄糖以及2摩尔二糖。因此，尽管无脊椎动物纤维素酶都属于GHF 9，但它们的催化机制因动物种类而异。

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.