Purification and Characterization of a Thermostable Neutrophilic Metalloprotease from Pseudomonas sp. DR89

A novel neutrophilic metalloprotease was isolated from Pseudomonas sp. DR89 isolate which was identified ina mineral spring in Iran. The enzyme was purified from the isolate to 21-folds in a three-step procedure involving ammonium sulfate precipitation, Q-Sepharose ionic exchange and Sephadex G-100 gel filtrationchromatography. Resuts showed that the enzyme was active at high temperatures and in a wide-range pH of5-11 with the optimum of 8.0. The zymogram and sodium dodecyl sulfate polyacrylamide gel electrophoresisanalysis revealed the presence of one protease with a molecular weight of 74 kDa. The enzyme activity wasdecreased by Zn2+, Mn2+, H2O2 and cetyl trimethylammonium bromide (CTAB), whereas its activity wasincreased by Ca2+, Mg2+, Cu2+ and dimethyl sulfoxide (DMSO). Na+, phenylmethyl sulfonylfluoride (PMSF),β-mercaptoethanol, sodium dodecyl sulfate (SDS), and Triton X-100 did not show a considerable effect onits activity. Casein was a better substrate than bovin serum albumin (BSA) and gelatin for this enzyme. Thekinetic parameters (Km and Vmax) of the purified protease towards caseinolytic activity were also determined.These properties of the enzyme make it suitable for use in food industries.