PARTIAL PURIFICATION AND CHARACTERIZATION OF PROTEASE IV FROM PSEUDOMONAS AERUGINOSA

Abstract

Clinical strain Pseudomonas aeruginosa No.3 was isolated from humancorneal ulceration. The bacterial cells secreted the extracellular protease inliquid culture. The enzyme was partial purified 191 fold from culture filtrate bysequential steps such as salting out with ammonium sulfate precipitation (80%saturation), ion exchange CM- Cellulose Chromatography, and by SephadexG-75 Gel filtration.Characterization study of the partially purified enzyme revealed that theenzyme had an optimum activity at pH 9.5 and the activity was stable in thealkaline pH range (8- 10 )for 30 min. Enzyme activity toward casein increasedwith temperature raise up to 35°C and maximal activity was attained at 45° C.The enzyme was stable at temperature under 30˚C and approximately 90% ofthe activity was abolished by incubation of the enzyme at 60 ° C for 40 min or at80 ° C for one min. Protease IV activity was partially inhibited byphenylmethylsulfonyl fluoride (20%) and Diisopropyl fluorophosphate (75%).EDTA at 50mM caused a 22% inhibition of protease activity, which suggestedthat the enzyme is a serine protease. The reducing agents dithiothreitol (1.0 mM)and 2- mercaptoethanol (150-mM) also demonstrated complete inhibition of theenzyme, which suggests that the enzyme protein containing disulfide bonds couldbe important in maintaining the molecular conformation required for activity.__________________________________________________________