Purification and characterization of a thermostable neutrophilic metalloprotease from Pseudomonas sp. DR89

Document Type: Research Paper

Authors

1 Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, P.O. Box 9177948974, Mashhad, I.R. Iran

2 Cellular and Molecular Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, P.O. Box 9177948974, Mashhad, I.R. Iran.

3 Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, P.O. Box 9177948974, Mashhad, I.R. Iran.

Abstract

A novel neutrophilic metalloprotease was isolated from Pseudomonas sp. DR89 isolate which was identified in
a 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 filtration
chromatography. Resuts showed that the enzyme was active at high temperatures and in a wide-range pH of
5-11 with the optimum of 8.0. The zymogram and sodium dodecyl sulfate polyacrylamide gel electrophoresis
analysis revealed the presence of one protease with a molecular weight of 74 kDa. The enzyme activity was
decreased by Zn2+, Mn2+, H2O2 and cetyl trimethylammonium bromide (CTAB), whereas its activity was
increased 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 on
its activity. Casein was a better substrate than bovin serum albumin (BSA) and gelatin for this enzyme. The
kinetic 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.

Keywords