title: Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus.
abstract: An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.
authors.affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. y-asami@nagasaki-u.ac.jp.
authors.forename: Asami
authors.initials: A
authors.lastname: Yoshida
authors.affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. megumi-ohta@sea.sannet.ne.jp.
authors.forename: Megumi
authors.initials: M
authors.lastname: Ohta
authors.affiliation: Food Science and Technology Section, Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan. kuwahara012217@pref.nagasaki.lg.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kuwahara
authors.affiliation: College of Food and Biological Engineering, Jimei University, Xiamen 361021, China. mjcao@jmu.edu.cn.
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
authors.affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. hara-k@knwu.ac.jp.|Faculty of Food Nutrition, Kyushu Nutrition Welfare University, Kitakyusyu, Fukuoka 803-8511, Japan. hara-k@knwu.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Hara
authors.affiliation: Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan. osatomi@nagasaki-u.ac.jp.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Osatomi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000737:chemistry / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D010473:Perciformes / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017747:Seafood; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D002401:Cathepsin B
keywords: cathepsin B; endogenous protease; food processing; horse mackerel