title: Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi).
abstract: Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21kDa and 21.5kDa, respectively, as estimated by SDS-PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35°C and 40°C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45°C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca(2+) and Mg(2+) and inactivated by Fe(2+), Zn(2+), Mn(2+), Cu(2+), Al(3+), Ba(2+) and Co(2+) to different degrees. Apparent Km values of trypsin A and B were 2.18μM and 1.88μM, and Kcat values were 81.6S(-1) and 111.3S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Jimei, Xiamen 361021, China.
authors.forename: Bao-Ju
authors.initials: BJ
authors.lastname: Lu
authors.affiliation: Institute of Food Processing, Zhejiang Academy of Agriculture Sciences, Hangzhou 310021, China.
authors.forename: Li-Gen
authors.initials: LG
authors.lastname: Zhou
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Jimei, Xiamen 361021, China.
authors.forename: Qiu-Feng
authors.initials: QF
authors.lastname: Cai
authors.affiliation: Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Hara
authors.affiliation: Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Maeda
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Jimei, Xiamen 361021, China.
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Jimei, Xiamen 361021, China. Electronic address: mjcao@jmu.edu.cn.
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
keywords: Homology; Immunoblotting; Purification; Siniperca chuatsi; Trypsins