β-lactamase activity assay

The β-lactamase activity was assayed by using a β-lactamase activity assay kit (Sigma-Aldrich, USA, catalog number MAK221). This assay is based on the hydrolysis of a non-antimicrobial, chromogenic cephalosporin called nitrocefin. Hydrolysis of nitrocefin by β-lactamase enzyme produces a colorimetric product with an absorbance maximum at 490 nm proportional to the enzymatic activity present. The amount of enzyme required to hydrolyze 1.0 μmol of nitrocefin per minute at pH 7.0 at 25°C is equal to one unit of β-lactamase. Assay was done in a 96-well plate using commercially available TEM-1 β-lactamase from E. coli (catalog number PV3575, Thermo Fisher Scientific) and β-lactamase from E. cloacae (catalog number P4524-100 UN, Sigma-Aldrich) in the presence or absence of different concentrations of peptides pep1, pep2 and pep3 (as required). 50 μl of the unknown samples (assay buffer, enzyme, and peptide) were added to wells of a clear flat bottom 96-well plate and supplemented with a reaction mixture containing nitrocefin and assay buffer to a final volume of 100 μl. As needed, 2 μl of 1:100 diluted TEM-1 (10 ng/13.76 nmoles) and 0.0002 units of E. cloacae β-lactamase were used per well for the assay. Immediately after the addition of nitrocefin, absorbance at 490 nm was measured every minute, for 10 minutes at room temperature using a microplate reader (xMark Microplate Reader, BioRad).