Abts 2 2 azino bis 3 ethylbenzthiazoline 6 sulfonate

Laccases from P. sanguineus CS43 were obtained from a tomato medium as described in our previous study (Ramírez-Cavazos et al. 2014a (link)). Mycelia were removed from the culture supernatant by filtration using two tangential flow filters in series, with pore sizes 0.5 and 0.2 μm. After that, the 0.2-μm filtered culture supernatant (laccase cocktail) was ultra-filtered by using a membrane cut-off of 10 kDa. The ultrafiltration process avoids the presence of lower molecular weight solutes present in the culture that can represent an environmental risk. Standards of BPA, NP, EE2 and TCS (high purity grade), 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonate (ABTS), dibasic sodium phosphate and citric acid salt were obtained from Sigma Aldrich, USA. Methanol, acetonitrile and ethanol (trace analysis quality) were supplied from Fisher Scientific, Tedia and Fermont, respectively.

Copper (II) sulfate pentahydrate, CuSO₄∙5H₂O (>98.0%); ammonium sulfate, (NH₄)₂SO₄ (99%); calcium chloride, CaCl₂ (99.9%); magnesium sulfate heptahydrate, MgSO₄∙7H₂O (99%); potassium chloride, KCl (99%); potassium phosphate monobasic, K₂HPO₄ (99%); thiamine hydrochloride (99%) sodium citrate dihydrate HOC(COONa)(CH₂COONa)₂∙2H₂O (>99%), and sodium hydroxide anhydrous pellets, NaOH (>98%) were purchased from Merck (Darmstadt, Germany). The citric acid (99.5%), malt extract agar, and yeast extract were supplied by Scharlau (Barcelona, Spain). 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonate), ABTS (>98.0%), 3,5-dinitrosalicylic acid, and DNS (>98.0%) were manufactured from Sigma–Aldrich, St. Louis, MO, USA. D(+)-Glucose anhydrous PA-ACS and sodium acetate anhydrous (99.9%) were obtained from PanReac AppliChem (Barcelona, Spain). Acrylamide and bis-acrylamide solution (30%, 29:1), 0.5 M Tris-HCl, pH 6.8, 1.5 M Tris-HCl, pH 8.8 solutions were provided for Bio-Rad, Alcobendas, Spain.