1 naphtol

Lactococcus. lactis cells were grown as standing cultures at 30°C in peptone-glucose broth (MR-VP broth) for 28 h. This medium was prepared with pancreatic digest of casein 7.0 g, dipotassium phosphate 5.0 g, and dextrose 5.0 g. The components were dissolved in 1 L deionized water, and pH adjusted to 6.9. The medium was autoclaved at 115°C for 15 min.
The Voges-Proskauer reagents were freshly prepared, Barritt’s reagent A: 5% (w/v) 1-naphtol (Sigma-Aldrich, St. Louis, MO, United States) in absolute ethanol. Barritt’s B: 40% (w/v) KOH in deionized water. The VP test was performed as reported previously (Mcdevitt, 2009 ). Briefly, 0.6 mL of Barritt’s reagent A were added to 2.5 mL of the bacterial cultures, then 0.2 mL of Barritt’s reagent B were added. The tubes were shaken for 30 s to expose the culture to atmospheric oxygen, and allowed to stand for 30 min. Within 1 h the tubes were compared. A yellowish color indicates VP-negative and a red color indicates VP-positive.

Mefenamic acid (≥99%), tacrine (≥99%), carvedilol (≥98%), nifedipine (≥98%), ellipticine, α-naphthoflavone (≥97%), ticlopidine (≥99%), 1-naphtol (≥99%), 2-naphtol (≥98%), 4-methoxy-benzaldehyde (≥98%), 2-(p-tolyl)ethylamine (≥97%) were purchased from Sigma-Aldrich (Schnelldorf, Germany); phenacetin was obtained from Brocades-ACF (Maarssen, the Netherlands). 7-Methoxyresorufin was synthesized by the method of Burke and Mayer [28 (link)] and final purity was assessed to be higher than 95%.

Yeast cells were grown to mid-log phase in YPD (1% yeast extract, 2% peptone, 2% glucose) and starved for 4 h in nitrogen starvation medium (SD-N: 0.17% yeast nitrogen base without amino acids, 2% glucose).
In total, 50 OD600 units of yeast culture were harvested by centrifugation. The pellets were washed with dH2O and ice-cold 0.85% NaCl containing 1 mM PMSF and resuspended in 8 μl/OD600 unit lysis buffer [20 mM PIPES pH 6.8, 0.5% Triton X-100, 50 mM KCl, 100 mM potassium acetate, 10 mM MgSO4, 10 μM ZnSO4, 1 mM PMSF, cOmplete protease inhibitor cocktail (Roche)]. Cells were lysed by bead beating, and extracts were cleared by centrifugation. Protein concentration of the supernatant was adjusted to 50 μg in 100 μl lysis buffer. In total, 400 μl reaction buffer (0.4% Triton X-100, 10 mM MgSO4, 10 μM ZnSO4, and 250 mM Tris-HCl pH 8.5) containing 6.25 mM α-naphthylphosphate (Sigma-Aldrich) was added to enzymatic reactions, or only reaction buffer was added to control reactions. Reactions were incubated at 37 °C for 10 min and stopped by adding 500 μl stop buffer (1 M glycine pH 11). A 405 was measured using a plate reader. A standard curve was generated by using a dilution series of the product (1-naphtol, Sigma-Aldrich).
Three independent replicates were performed and activity was calculated the following: activity = [pNPP in nmol]/(t[min]∗[protein in mg]).