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Purification and Characterization of an Extracellular Protease from Pseudomonas aeruginosa Isolated from East Calcutta Wetland

Author(s): Jaysankar Sing Yadav | Sanhita Chowdhury | Shaon Ray Chaudhuri

Journal: Journal of Biological Sciences
ISSN 1727-3048

Volume: 10;
Issue: 5;
Start page: 424;
Date: 2010;
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Keywords: Bheri | endoprotease | aprA gene | metalloprotease | hydrophobic interaction chromatography

In an earlier study, we had isolated and identified a protease producing bacterium as Pseudomonas aeruginosa from the Charakdanga Bheri waters of East Calcutta Wetland. The enzyme was concentrated by lyophilization and finally purified by hydrophobic interaction chromatography using Phenyl Sepharose CL-4B column resulting in 1.2 fold increase in specific activity and 28% recovery. The crude extracellular protein was run on a 12% SDS-PAGE; two bands were found, one between 43 and 66 kDa and another between 29 and 43 kDa. The molecular weight of the purified protein was 36.18 kDa as determined from ESI-Mass Spectroscopy; corresponding to the lower band of the crude sample. The zymogram showed a sharp zone of clearance corresponding to the lower band confirming protease activity. The optimum temperature for the protease activity was 40°C. The protease activity showed a wide range of pH tolerance with almost similar activity ranging from pH 5 to 8. The activity of the enzyme was totally lost in the presence of chelating agent EGTA (10 μM), Phosphoramidone (500 μg mL-1) and 1,10 Phenanthroline (2 μM), suggesting the purified enzyme to be a metalloprotease, while other protease inhibitors had no effect on the enzyme activity. Moreover, since phosphoramidone is a metallo-endoprotease inhibitor, this finding was further confirmed. The endoprotease nature was confirmed through BSA digested banding pattern generation. The kinetics of protease activity revealed that BSA was completely digested within 8 to 9 min and a minimum of 5 μL (6 mg mL-1) of crude protease soup was required for digestion of 50 μg BSA solution.
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