title: Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker.
abstract: 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.
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. kawashima.tp@gmail.com.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kawashima
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. mg7759@gmail.com.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ikehata
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. mackerel@mrj.biglobe.ne.jp.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Tada
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. ogino19900829@yahoo.co.jp.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Ogino
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. himibambi@hotmail.co.jp.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Kakizaki
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. perfect-love@road.ocn.ne.jp.
authors.forename: Mana
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. fukushima.hideto@nihon-u.ac.jp.
authors.forename: Hideto
authors.initials: H
authors.lastname: Fukushima
authors.affiliation: Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan. matsumiya@brs.nihon-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Matsumiya
mesh_terms: D000818:Animals; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D005399:Fishes; D005753:Gastric Mucosa / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007564:Japan; D010802:Phylogeny; D012623:Seawater
chemical_list: D007527:Isoenzymes; D002688:Chitinases
keywords: Japanese sardine Sardinops melanostictus; cDNA cloning; fish stomach chitinase isozymes; fish-specific acidic fish chitinase; long linker