V 560 uv vis

Laccase activity measurement was performed spectrophotometrically (JASCO V/560 UV/Vis, Japan) at wavelength of 525 nm in a reaction medium containing 1 mM syringaldazine (ϵ = 65 mM⁻¹ cm⁻¹), 50 mM phosphate buffer pH 5 and culture filtrate. Oxidation of syringaldazine was monitored by measuring the increase in absorbance for 4 min. Enzyme activity was expressed in units (U); one unit was defined as 1 μmol of syringaldazine oxidized per min [15] .

Five dyes namely methyl orange, trypan blue, ramazol brilliant red, ramazol brilliant blue and ramazol brilliant yellow (Dye Star company, Germany) were chosen to test the enzyme’s ability to remove their color. A volume of 0.1 ml of the stock solution (20 ppm) was added to 2 ml distilled water and 2 ml of the partially purified enzyme extract with activity 417 U/ml respectively, the percentage reduction of color was monitored for 3 h and was determined spectrophotometrically (JASCO V/560 UV/Vis, Japan) by monitoring the absorbance at the characteristic wavelength of each dye. The decolorization efficiency (R%) was calculated as follows:Dye decolorization percentage = [(Initial absorbance − final absorbance)/(initial absorbance)] × 100
Initial absorbance indicated absorbance of the untreated dye at the characteristic peak and the final absorbance indicated absorbance of dye after treatment with laccase at the same peak after 3 hours.

GNPs were prepared as previously described [19] , briefly, to 3 ml of laccase enzyme, containing 417 IU/mg, 0.1 ml of tetrachloroauric acid with concentration of (10 mg/1 ml) was added, (49% purity). The reaction mixture was stirred properly using magnetic stirrer, within 90 min the yellow colored solution started changing to pink then violet, detected visually and by UV/Visible spectrophotometer indicating the formation of GNPs. Average particle size and size distribution were determined by (PSS-NICOMP 380-ZLS) particle sizing system (St. Barbara, California, USA). UV/Visible Spectra of GNPs were recorded using a spectrophotometer (JASCO V-560UV/Vis, Japan) operated at a resolution of 1 nm from range of 200–700 nm and observed absorption peak at 550 nm due to excitation of surface plasmon vibration in GNPs solution or the SPR band. FT-IR measurements were carried out using a spectrophotometer (JASCO FT/IR-6300 infra-red) by employing KBr pellet technique. The size and morphology of synthesized GNPs were recorded using transmission electron microscope, TEM (JEM-100CX.TEM JEOL, Japan). TEM studies were carried by drop coating GNPs onto carbon-coated TEM grids. The film on the TEM grids were allowed to dry, the extra solution was removed using blotting paper.