The role of chitinolytic enzyme in cultured fish

罗非鱼具有较高的几丁质分解活性。对从罗非鱼胃中提纯的几丁质酶进行了动力学分析。该酶倾向于较长的底物。经高效液相色谱分析，产物为β异构体。阿司他丁对该酶有竞争性抑制作用。为了研究几丁质酶的一级结构，对其核苷酸序列进行了分析。采用RACE方法，克隆了团头鱼胃几丁质酶基因。1,428个核苷酸的克隆被分离出来，发现它含有一个编码476个氨基酸的蛋白质的开放阅读框。该酶与酸性哺乳动物几丁质酶、牛血清几丁质酶和蟾蜍胰腺几丁质酶具有较高的相似性。经Northern和RT-PCR分析，几丁质酶在胃、幽门盲囊、肠道和肝脏中均有表达。采用免疫组织化学方法检测了生长过程中几丁质酶在各组织中的定位。首先，在孵化后3天，在肠道中发现了免疫阳性反应。随着孵化后20d左右出现胃的分化，几丁质酶的定位从肠道向胃转移。

Red sea bream has high chitinolytic activity. Kinetic analysis was done on the chitinase purified from the stomach of red sea bream. The enzyme preferred the longer substrate. The anomeric forms of the products from GlcNAc_n were analyzed to be β anomer by HPLC. Allosamidin inhibited competitively the enzyme. These results suggest that the red sea bream chitinase belong to family 18 of glycosyl hydrolase.In order to investigate on the primary structure of the chitinase, its nucleotide sequence was analyzed. Using RACE method, cDNA encoding red sea bream stomach chitinase was cloned. The clone of 1,428 nucleotides was isolated, and found to contain an open reading frame encoding a protein of 476 amino acids. This chitinase has high similarity to acidic mammalian chitinase, bovine serum chitinase and toad pancreatic chitinase. By northern and RT-PCR analysis of chitinase-encoding mRNA, it was expressed in the stomach, pyloric caeca, intestine and liver.Using immunohistochemistry, the localization of chitinase in the tissues of red sea bream during the growth was examined. First, the immunopositive reaction was recognized in the intestine at 3 days after hatching. As the differntation of the stomach was seen at about 20 days after hatching, the localization of chitinase moved to the stomach from the intestine.