Characterization of extracellular proteases of psychrotrophic pseudomonads

Hamid, Jawed (1983) Characterization of extracellular proteases of psychrotrophic pseudomonads. Masters thesis, Memorial University of Newfoundland.

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Abstract

For this study 8 cultures of P. fluorescens were selected. A protease was isolated from bacterial culture number T20 and was partially purified by a combination of ammonium sulfate precipitation, gel filtration on Sephadex G-200 and affinity column chromatography. The molecular weight of this protease was estimated to be 43,000 by gel filtration on Sephadex G-200. The proteases investigated in this study were active over a wide range of temperatures (5-45°C) with maximum activity at the temperature of 35°C. The pH optimum for T20 protease was 7.2; other proteases were also maximally active at neutral pH. All of the proteases investigated retained considerable activity after a heat exposure of ten minutes at 100°C. The T20 protease was stable over a wide range of pH (5.5-9.0) when stored at 10°C for four days. All of the proteases were found to be metalloproteases requiring divalent cations for activity and were inactivated slightly by sulfhydryl reagents. The synthesis of all proteases by the bacteria was stimulated by addition of milk to the growth medium. The protease activity of all isolates began to increase in early logarithmic phase and continued to increase till late stationary phase, and then began to decline. The proteases from various isolates were found to possess similar antigenic determinants. Each bacterial culture produced only one extracellular protease.

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