Enzyme Activity Determination Using HPLC: A Comprehensive Approach

The enzyme's specific activity was determined in accordance with previously established protocols [1]. A precise quantity of pure enzyme was added to a solution containing 14 mL of 10% (w/v) DL-PL substrate, which had been prepared in a 1 M Tris-HCl buffer with a pH of 7.5. The reaction was terminated by heating the solution to 100°C for a duration of three minutes, after which it was filtered and analyzed using high-performance liquid chromatography (HPLC). The activity of the enzyme was measured in units of D-Lacs, with one unit being defined as the amount of enzyme necessary to release 1 μmol of D-PA per minute. Samples were analyzed using a Waters HPLC system equipped with a Chiralcel IG-3 column (4.6 mm × 250 mm, 3 µm, Diacel, Tokyo, Japan). The eluent used was formic acid/methanol (0.1%, 70:30, v/v), flowing at a rate of 0.3 mL/min, with a temperature of 35°C and a wavelength of 210 nm.