Elastolytic Activity of P. aeruginosa Culture Supernatant

The elastolytic activity of the cell-free culture supernatant of P. aeruginosa was determined using Elastin-Congo red (ECR; Sigma) as the substrate [26 (link)]. 100 μL supernatants of P. aeruginosa incubated in different concentrations of glucose (0, 50, 100, 150 and 200 mg/mL) for 17 h were added to 900 μL of ECR buffer (100 mM Tris, 1 mM CaCl₂, pH 7.5) containing 10 mg of ECR and incubated at 37 °C for 3 h. The reaction was terminated by adding 1 mL of 0.7 M sodium phosphate buffer (pH 6.0) and the tubes were placed in a cold water-bath. The insoluble ECR was removed by centrifugation at 10,000 rpm for 10 min, and the absorbance was measured at OD₄₉₅.