Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa

Abstract

Exocellular protease production was examined in 2 separate strains of P. aeruginosa, 1 a clinical isolate and the other a laboratory strain. Both strains produced 2 separate proteases (proteases 1 and 2) which were indistinguishable from 1 strain to the other. The 2 proteases were purified by a 2-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were distinct serologically and unrelated by physicochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was estimated to have a MW of 34,850 and contained 10% glucosamine by weight. Protease 2, in contrast, had an estimated MW of 52,750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity and a limited collagenase activity. Specificity of the 2 proteases against synthetic peptides was quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophobic R group.