Purification and characterization of extracellular temperature-stable serine protease from Aeromonas hydrophila

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 60degreesC for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60degreesC, 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 Ca2+, Mg2+ and Zn2+ ions.