Determination of D-Lac Enzyme Activity and Variant Analysis Using Reversed-Phase HPLC

The enzyme's specific activity was defined and determined in accordance with established protocols [1]. To evaluate the specific activity of D-Lac and its various variants towards D-PL, a reaction system was meticulously prepared in 25 mL triangular vials. This system comprised 768 mM DL-PL, Tris-HCl buffer (500 mM, pH 7.5), and 200 uL of diluted purified enzyme. The reaction was allowed to proceed for a duration of 20 minutes at a controlled temperature of 30°C. Subsequent to the reaction, the enzyme was inactivated through a heat treatment process involving boiling. The resulting product was then subjected to analysis using the sophisticated technique of reversed-phase high-performance liquid chromatography (HPLC). Samples were detected in a single layer using a state-of-the-art Waters HPLC system equipped with a Chiralcel IG-3 column (4.6 mm × 250 mm, 3 µm, Diacel, Tokyo, Japan).