Colloidal coomassie brilliant blue r 250

The G6PD::6PGL protein was expressed and purified as previously reported by Morales-Luna et al. [19 (link)]. The E. coli BL21(DE3)Δzwf::kan^r cells containing the pET-3a/g6pd::6pgl vector were induced with 1 mM of isopropyl-β-d-thiogalactoside (IPTG) for 18 h at 25 °C in Luria Bertani (LB) culture medium. The cells were pelleted by centrifugation, suspended in lysis buffer, and disrupted by sonication. Then, by centrifugation (10,000× g for 15 min at 4 °C), the crude extract was obtained and used for protein purification employing a 2′,5′-ADP Sepharose 4B affinity column (GE Healthcare, Chicago, IL, USA) and a Sephacryl 200 (16/60) gel filtration column (GFC) (GE Healthcare, Chicago, IL, USA) [19 (link)]. Purified G6PD::6PGL protein was analyzed in 12% SDS–PAGE gel [20 (link)] and stained with colloidal Coomassie Brilliant Blue (R-250) (Sigma-Aldrich, San Luis, MO, USA). The protein concentration was quantified according to Lowry et al. [21 (link)] using bovine serum albumin (BSA) as the standard.

The EECE were used to determine the extracellular laccase activity by spectrophotometry, monitoring the change in absorbance due to the oxidation of 2,6-dimethoxyphenol (2,6-DMP) using the JENWAY 6715 UV/Vis spectrophotometer (Cole Parmer, Vernon Hills, IL, USA). The standard reaction mixture (1 mL) contained 2,6-DMP (2 mM) in K₂HPO₄ (0.1 M, pH 6.5) and 40 µL of EECE. The reaction was monitored at 468 nm for 4 min at 39 °C [40 (link),41 ]. The enzymatic activity was expressed in International Units (IU), where 1 IU was defined as the amount of enzyme that catalyzes the transformation of 1 µmol of 2,6-DMP into product per minute. The total protein concentration in the EECE was determined following Lowry et al. [42 (link)]. Furthermore, the EECE was used to determine the laccase activity by zymography in semi-denaturing SDS–PAGE in 11% acrylamide. The electrophoresis conditions were 150 V for 1–1.25 h. The zymogram was revealed using 2,6-DMP (2 mM) as substrate in K₂HPO₄ (0.1 M, pH 6.5). The semi-denaturing and denaturing SDS–PAGE gels were stained with colloidal Coomassie brilliant blue (R-250) (Sigma–Aldrich, Waltham, MA, USA).