Enzymatic Activity Assessment of Detached Biofilms

Detached biofilms (10 mg) was taken and suspended in 100 ml of isotonic solution. An assessment of enzymatic activities was determined by Iwase et al. (2013 (link)) by plotting a standard curve with the known concentration (or unit of enzyme activity). Standard Catalase was prepared separately in distilled water for each concentration (Catalase from bovine liver 2,950 units/mg solid; 65% protein; 4,540 units/mg protein, the product number C1345, Sigma-Aldrich). Furthermore, bacterial suspension (100 μl) was taken to Pyrex tube (13 mm diameter, 100 mm height, borosilicate glass; Corning, USA). Afterwards, 100 μl of 1% Triton X-100 and 100 μl of undiluted hydrogen peroxide (30%) was taken to the solution and mixed thoroughly and incubated at room temperature. Then the height of the O₂-forming foam was measured using a ruler. We measured the O₂-foam which was constant for 15 min. All these steps performed under ice condition (sample was kept in an ice bucket) to prevent the loss of active enzyme.