Pseudomonas aeruginosa Elastase Activity Analysis

The elastase activity of all P. aeruginosa strains was determined using Elastin-Congo red (ECR; Sigma-Aldrich). Briefly, for each sample, 100 μL concentrated filter-sterilized bacterial exoprotein was added to 900 μL of ECR buffer (100 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 20 mg ECR) and incubated for 18 h at 37°C with agitation at 200 rpm. Then insoluble ECR was removed by centrifugation at 16,000 g for 15 min at room temperature, and the absorbance was measured at 495 nm. Fresh LB medium was used as the blank control, and LB with ECR as the negative control. Secreted elastase activity was expressed as the ratio of the OD495 and OD600 absorbance value.