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Studies on primary structures and genes of cellulases from marine invertebrates

海洋无脊椎动物纤维素酶的一级结构和基因研究

基本信息

批准号：
15380138
负责人：
OJIMA Takao
金额：
$ 3.9万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

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.

通过测量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，鲍鱼、海胆和扇贝等草食性无脊椎动物被发现含有纤维素酶，而海星和海王星蜗牛等肉食性无脊椎动物被认为不含有纤维素酶。随后，通过常规柱色谱从鲍鱼、海胆和扇贝中分离出纤维素酶（内切-1,4-β-葡聚糖酶），并克隆其cDNA和结构基因以推断其一级结构。根据推导的结构，鲍鱼、海胆和扇贝酶被归类为糖苷水解酶家族 9 (GHF9)，就像之前报道的白蚁和小龙虾纤维素酶一样。纤维素酶基因的系统发育分析表明，鲍鱼、海胆和扇贝纤维素酶与节肢动物酶源自共同的祖先基因。在鲍鱼消化液中，发现了一种在 N 末端具有 II 族碳水化合物结合模块的同工酶。该酶是第一个在 GHF9 型催化结构域 N 末端具有家族 II CBM 的动物纤维素酶。研究了无脊椎动物纤维素酶对纤维寡糖的降解能力。鲍鱼纤维素酶可将三糖降解为二糖和葡萄糖；然而，海胆和扇贝酶却不能。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，当四糖发生反应时，鲍鱼酶将其分解为两摩尔二糖，而海胆和扇贝酶则分解为三糖和葡萄糖以及2摩尔二糖。因此，无脊椎动物纤维素酶的催化机制被认为根据动物种类而变化，尽管这些酶都属于GHF9。