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Chitinolytic activities were measured in two fish species having different feeding habits, chub mackerel (Scomber japonicus) and silver croaker (Pennahia argentata). Chitinase (an endo-type chitinolytic enzyme) activity was measured using pNP-(GlcNAc)n (n = 2, 3) as substrates; its level was significantly high in the stomachs of both species, as well as in the gills, intestine, pyloric appendage, testis, and liver of chub mackerel and in the spleen, kidney, pyloric appendage, ovaries, heart, and liver of silver croaker. β-N-Acetylhexosaminidase (an exo-type chitinolytic enzyme) activity was measured using pNP-(GlcNAc) as a substrate; it was detected at high levels in many parts apart from the digestive tracts of both species. The optimum pH for chitinase activity was 3.0 -5.0 in the stomachs of both species, 4.0 in the liver of chub mackerel, and 4.0 and 8.0 in the kidney of silver croaker. Full-length cDNAs encoding two chitinase isozymes were obtained from the stomachs of the two fish species: SjChi-1 (1604 bp) and SjChi-2 (1512 bp) from chub mackerel and PaChi-1 (1630 bp) and PaChi-2 (1606 bp) from silver croaker. Expression analysis of these genes in the organs of the two species revealed strong expression of SjChi-1 in the stomach of chub mackerel and that of PaChi-1 and PaChi-2 in the stomach of silver croaker. The difference in the expression pattern of these genes is likely attributed to the difference in the feeding habits of the two fish species. Our results suggested the presence of novel chitinases in the two species.

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.

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