| Budget Amount *help |
¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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.
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