D glucose god pod

Microbial dry biomass was estimated gravimetrically in duplicate. Each sample of 50 mL was filtered through quantitative filter paper (VWR International, Leuven, The Netherlands). The fungal pellets were dried at 60 °C for 48 h. The samples were placed in a desiccator for 60 min and weighted afterwards. The concentration of biomass was calculated as grams of dry biomass per liter of medium. To determine glucose consumption in fungal cultures, the glucose concentration was measured using the commercial kit D-Glucose GOD-POD (NzyTech, Lisbon, Portugal).
APAP concentrations were quantified in the fungal culture supernatants by using a high-performance liquid chromatography system L-7100 (LaChrom HPLC System, Merck, Darmstadt, Germany) consisting of a quaternary gradient pump, and L-7400 UV detector. The whole system was controlled using the Merck HPLC System Manager software. The separation of the analytes was performed at ambient temperature in a Waters Spherisorb ODS2 column (5.0 µm, 4.6 × 250 mm) (Waters, Milford, MA, USA), using an isocratic condition with a mobile phase composed of water (pH 3.5, adjusted with orthophosphoric acid) and acetonitrile, at a 90:10 (v/v) and a flow rate of 1.0 mL/min. Detection was performed at 254 nm. Under these conditions, APAP, HQ, CAT, and APA could be separated within 12 min.

Kinetics of lactose hydrolysis were measured by the glucose produced by the enzyme at different lactose concentrations. Purified samples were diluted in Z buffer. The initial velocity was measured in triplicate with 5.5 μg mL⁻¹ enzyme and lactose from 0 to160 mM. The reaction times were 6–20 min at 30 °C. The reaction was stopped by heating to 96 °C for 5 min.
β-galactosidase activity is expressed in enzymatic units (U), defined as the amount of enzyme capable of liberating 1 μmol of product (D-glucose) per min under the experimental conditions (i.e. μmol min⁻¹ mL⁻¹).
Glucose concentration was measured using the commercial kit D-Glucose GOD-POD (Nzytech). Non-linear fitting by the least squares method was used to infer the apparent enzymatic kinetic parameters from Michaelis-Menten plots (via Prism 6).