Partial purification and characterization of protease IV from Pseudomonas aeruginosa

Abstract EN

Clinical strain Pseudomonas aeruginosa No.

3 was isolated from human corneal ulceration.

The bacterial cells secreted the extracellular protease in liquid culture.

The enzyme was partial purified 191 fold from culture filtrate by sequential steps such as salting out with ammonium sulfate precipitation (80 % saturation), ion exchange CM- Cellulose Chromatography, and by Sephadex G-75 Gel filtration.

Characterization study of the partially purified enzyme revealed that the enzyme had an optimum activity at pH 9.

5 and the activity was stable in the alkaline pH range (8-10) for 30 min.

Enzyme activity toward casein increased with 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 % of the activity was abolished by incubation of the enzyme at 60 ° C for 40 min or at 80 ˚C for one min.

Protease IV activity was partially inhibited by phenylmethylsulfonyl fluoride (20 %) and Diisopropyl fluorophosphate (75 %).

EDTA at 50mM caused a 22 % inhibition of protease activity, which suggested that the enzyme is a serine protease.

The reducing agents dithiothreitol (1.

0 mM) and 2- mercaptoethanol (150-mM) also demonstrated complete inhibition of the enzyme, which suggests that the enzyme protein containing disulfide bonds could be important in maintaining the molecular conformation required for activity.