Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi) |
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Authors: | Bao-Ju Lu Li-Gen Zhou Qiu-Feng Cai Kenji Hara Asami Maeda Wen-Jin Su Min-Jie Cao |
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Affiliation: | 1. College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Jimei, Xiamen 361021, China;2. Institute of Food Processing, Zhejiang Academy of Agriculture Sciences, Hangzhou 310021, China;3. Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan |
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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 21 kDa and 21.5 kDa, 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 Ca2+ and Mg2+ and inactivated by Fe2+, Zn2+, Mn2+, Cu2+, Al3+, Ba2+ and Co2+ to different degrees. Apparent Km values of trypsin A and B were 2.18 μM and 1.88 μM, and Kcat values were 81.6 S−1 and 111.3 S−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. |
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Keywords: | Trypsins Siniperca chuatsi Purification Immunoblotting Homology |
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