| Title | Purification and Characterization of Extracellular Temperature-Stable Serine Protease from Aeromonas hydrophila |
|---|---|
| Author | Soo-Jin Cho 1 , Jong-Ho Park 1 , Seong Joo Park 1 , Jong-Soon Lim 2 , Eung Ho Kim 3 , Yeon-Jae Cho 4 , and Kwang-Soo Shin 1 , * |
| Address | 1 Department of Microbiology, College of Sciences, Daejeon University, Daejeon300-716, Korea ; 2 College of Oriental Medicine, Daejeon University, Daejeon 300-716, Korea ; 3 Major in Civil Engineering, School of Urban & Civil Engineering, College of Engineering, Hong-Ik University, Seoul 121-791, Korea ; 4 Seil Technology Co., LTD., Seoul 150-800, Korea (Received April 18, 2003 / Accepted May 28, 2003) |
| Bibliography | Journal of Microbiology, 41(3),207-211, 2003, |
| DOI | |
| Key Words | Aeromonas hydrophila, characterization, extracellular protease, purification |
| Abstract | Extracellular protease, from Aeromonas hydrophila Ni 39, was purified 16.7-fold to electrophoretic homogeneity with an overall yield of 19.9%, through a purification procedure of acetone precipitation, and Q Sepharose and Sephacryl S-200 chromatographies. The isoelectric point of the enzyme was 6.0 and the molecular mass, as determined by Sephacryl S-200 HR chromatography, was found to be about 102 kDa. SDS/PAGE revealed that the enzyme consisted of two subunits, with molecular masses of 65.9 kDa. Under standard assay conditions, the apparent K_m value of the enzyme toward casein was 0.32 mg/ml. About 90% of the proteolytic activity remained after heating at 60 ℃ for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60℃, and the optimum pH of the enzyme was 9.0. The enzyme was inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), by about 87.9%, but not by E64, EDTA, pepstatin or 1,10-phenanthroline. The enzyme activity was inhibited slightly by Ca_2^+, Mg_2^+ and Zn_2^+ ions. |
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