2 2 azinobis 3 ethylbenzothiazoline 6 sulfonate

The six applied azo dyes (Fig. 1 and Table 1) were supplied by Alvan Sabet Co. (Tehran, Iran). 2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT) were provided by Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany), respectively. The laccase applied in the present study was purified as previously described [25 (link)].Fig. 1

Chemical structures of azo dyes applied in the present study

Table 1

Kinetic and energetic parameters of the laccase on studied synthetic dyes

Dye name	Type	λmax (nm)	Km (mM)	Vmax (mmol/min/mg)	ΔS (Kj/mol/K)	ΔH (kJ/mol)	Ea (kJ/mol)
Acid Orange 67	Monoazo	438	0.49	189	307	93	24
Basic Red 18	Monoazo	489	3.07	64	194	63	28
Basic Yellow 28	Monoazo	450	2.07	100	308	91	29
Disperse Yellow 79	Monoazo	474	2.02	104	189	58	30
Direct Yellow 107	Diazo	368	3.44	47	213	68	26
Direct Black 166	Triazo	602	4.25	33	199	65	31

The Methylobacterium extorquens strain was purchased from JCM Japan. Bacterial strains Escherichia coli DH5α, Escherichia coli BL21 (DE3), and plasmid pET-28a (+) were all kept in our laboratory. Pfu DNA polymerase and protein markers were ordered from TransGen Biotech (Beijing, China). T4 DNA ligase and restriction endonucleases were all purchased from Takara (Dalian, China). All kits, such as the Bacteria DNA Extraction Kit, Gel Extraction and Purification Kit, and Plasmid Mini Kit, were purchased from Bioteke (Wuxi, China). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS); 2,6-dimethylphenol (2,6-DMP), isopropyl-β-d-thiogalactopyranoside (IPTG), and the synthetic dyes Remazol Brilliant Blue R, Crystal Violet, Indigo Carmine, and Congo Red were commercially obtained from Sigma-Aldrich (St. Louis, MO, USA). All the solutions required for the experiment were prepared in deionized water, and reagents and chemicals with the highest purity level were used. Absorption spectra were recorded using SHIMADZU UV-2700 spectrophotometer (Shimadzu, Kyoto, Japan). TECAN infinite F200 Pro (Tecan, Männedorf, Swiss) was used for the protein assay.

All reagents and standards were of analytical grade. Acetone, Folin–Ciocâlteu (FC) reagent, hydrochloric acid, hematoxylin-eosin, paraffin were acquired from Merck (Darmstadt, Germany). Sodium carbonate, sodium chloride, sodium nitrite, acetic acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, bovine serum albumin (BSA), Coomassie Brilliant Blue G (CBB), tris(hydroxymethyl)aminomethane (Tris), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), 6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox), 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA), phosphate-buffered saline (PBS), vanadium (III) chloride, sulphanilic acid, and alpha-naphthylamine were bought from Sigma-Aldrich (Schnelldorf, Germany). The normal saline solution (0.9% sodium chloride) was from B. Braun Melsungen AG (Melsungen, Germany). Neutral buffered formalin was obtained from Chempur (Lodz, Poland), codeine phosphate from Terapia S.A. (Cluj-Napoca, Romania). The water used in our study was ultrapure obtained from a Milli-Q ultrapure water system (Bucharest, Romania).

The Trolox equivalent antioxidant capacity assay was used to compare the antioxidant activity of extracts towards the antioxidant effect of ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, Sigma-Aldrich). A radical cationic solution was used to measure the absorbance differences. Antioxidant extracts would inhibit the absorbance. ABTS^·+ (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate, Sigma Aldrich) was generated by oxidation while using potassium persulfate for at least 12 h. EtOH 96% (Merck KGaA, Darmstadt, Germany) was used to dilute the solution to an absorbance of 734 nm.
Trolox and extracts were diluted to concentrations that were between 0.1 mM and 0.00625 mM with 96% EtoH. For each run, solvent blank and positive controls with ABTS^·+ solution were performed. The absorbance was measured after 6 min. with an Infinite M2000 Pro™ plate reader (Tecan, Crailsheim, Germany). The decreased absorbance that was caused by Trolox was plotted for the different concentrations tested. The resulting plot was used as a calibration curve. The slope of the absorbance inhibition vs. antioxidant concentration plot was divided by the slope of the Trolox plot to calculate the Trolox equivalent antioxidant capacity (TEAC) as Trolox equivalents (TE) in mM/mg plant extract.

Chloroaurate hydrate (99.9%, metal basis) was obtained from Alfa Aesar (USA). Catalase from bovine liver (2000-5000 U/mg), 2,2′-azino-bis(3-ethylbenzothiazoline 6-sulfonate (ABTS), phosphate buffer saline (PBS), dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (USA). NAC and 3,3′,5,5′-tetramethylbenzidine (TMB) were obtained from Aladdin Reagent (China). Hydrogen peroxide (H₂O₂, 30%), hydrochloric acid (36%), and nitric acid were purchased from Guangzhou Chemical Reagent Factory, and used as received. Mito-Tracker Green assay kit was obtained from Beyotime Institute of Biotechnology. The TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) assay kits were bought from ABBkine Scientific Company (USA). All chemicals and solvents were used without further purification. Deionized water, purified by a Milli-Q water purification system (Millipore, USA) to a minimum resistivity of 18.2 MΩ/cm, was used throughout the experiments.

Gallic acid, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), sodium acetate, iron (II) sulphate heptahydrate, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS), potassium peroxodisulfate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, (−)catechin and caffeic acid were purchased from Sigma-Aldrich (Steinheim, Germany). Sulphuric acid, ethanol, hydrochloric acid, iron(III) chloride hexahydrate, acetic acid, sodium hydroxide and methanol were purchased from (Honeywell, Charlotte, NC, USA). Folin–Ciocalteu’s reagent was purchased from Carlo Erba (Chau. du Vexin, France). Citric acid and aluminum chloride were purchased from Merck Group (Darmstadt, Germany). Sodium citrate, sodium nitrite and vanillin were purchased from Semikem (Sarajevo, BiH). Potassium dihydrogen phosphate and di-potassium hydrogen phosphate were purchased from Kemika (Zagreb, Croatia). All standard solutions, electrolytes, and sample solutions were prepared with ultrapure water (0.05 μS cm⁻¹, Millipore Simplicity UV Water Purification System, Millipore, Burlington, MA, USA) using analytical-reagent grade chemicals.