Hiromi Kakizaki

Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker

Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)8–fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

Biochemistry of fish stomach chitinase

Fish are reported to exhibit chitinase activity in the stomach. Analyses of fish stomach chitinases have shown that these enzymes have the physiological function of degrading chitinous substances ingested as diets. Osteichthyes, a group that includes most of the fishes, have several chitinases in their stomachs. From a phylogenetic analysis of the chitinases of vertebrates, these particular molecules were classified into a fish-specific group and have different substrate specificities, suggesting that they can degrade ingested chitinous substances efficiently. On the other hand, it has been suggested that coelacanth (Sarcopterygii) and shark (Chondrichthyes) have a single chitinase enzyme in their stomachs, which shows multiple functions. This review focuses on recent research on the biochemistry of fish stomach chitinases.