Determination of Enzyme Specific Activity and Evaluation of D-Lac Variants on D-PL Substrate

The enzyme's specific activity was determined in accordance with established protocols [1]. To evaluate the catalytic efficiency of D-Lac and its various variants towards D-PL, a reaction system was established within 25 mL triangular vials. The reaction mixture comprised 768 mM DL-PL, Tris-HCl buffer (500 mM, pH 7.5), and 200 ﾵL of diluted purified enzyme. After incubating the reaction for 20 minutes at 30ﾰC, the enzyme was inactivated by boiling, and the resulting product was analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC).

The samples were detected in a single layer using a Waters HPLC system equipped with a Chiralcel IG-3 column (4.6 mm ￗ 250 mm, 3 ﾵm, Diacel, Tokyo, Japan). Formic acid/methanol (0.1%, 70:30, v/v) served as the eluent, delivered at a flow rate of 0.3 mL/min at 35ﾰC, with detection at a wavelength of 210 nm. This methodology enabled the precise determination of the specific activity of D-Lac and its variants, providing insights into their catalytic efficiency towards the D-PL substrate.