Molecular cloning of a putative gastric chitinase in the toad Bufo japonicus

On the basis of our preliminary observation that a crude extract of the stomach of the toad Bufo japonicus exhibited a chitinase activity with its optimum pH around 3.0, we undertook molecular cloning of a cDNA encoding this putative gastric chitinase. By use of 2 degenerate oligonucleotide primers derived from the 2 conserved regions of the vertebrate chitinases, a reverse transcription-PCR product was obtained. This product was used as a probe to screen a cDNA library constructed from the toad stomach. The longest positive clone was revealed to contain an open reading frame for a putative chitinase protein of 484 amino acids, which protein exhibited sequence similarity to the known vertebrate chitinases. Our data also revealed this putative gastric chitinase to be distinct from the chitinase that we had previously isolated from the pancreas of the same species. In this putative gastric chitinase, both the N-terminal catalytic domain and the C-terminal chitin-binding domain were perfectly conserved, suggesting this protein to function as chitinase in the toad stomach.