The role of chitinolytic enzyme in cultured fish

真鲷具有较高的几丁质降解活性。对从真鲷胃中分离纯化的几丁质酶进行了动力学分析。该酶偏爱较长的底物。HPLC分析表明，GlcNAc_n的异头产物为β异头物。异柳胺竞争性抑制酶。这些结果表明，该几丁质酶属于糖基水解酶家族18，为了研究该几丁质酶的一级结构，对其核苷酸序列进行了分析。采用RACE方法克隆了真鲷胃几丁质酶的cDNA。分离了1，428个核苷酸的克隆，发现其含有编码476个氨基酸的蛋白质的开放阅读框。该几丁质酶与酸性哺乳动物几丁质酶、牛血清几丁质酶和蟾蜍胰几丁质酶有很高的相似性。通过北方和RT-PCR分析，几丁质酶基因在真鲷胃、幽门盲囊、肠和肝脏中均有表达，并通过免疫组织化学方法对真鲷生长过程中几丁质酶在各组织中的定位进行了研究。首先，在孵化后3天在肠道中识别出免疫阳性反应。在出壳后20天左右，随着胃的分化，几丁质酶的定位由肠转移到胃。

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.