The effect of temperature on activity was determined by incubating the enzyme in water bath in the range from 30 °C to 90 °C with 10 °C increments for (15 min). The effect of 5 doses of gamma radiation (2, 3, 4, 5 and 6 kGy) on the activity of laccase was studied. Also, the effect of several activators and inhibitors

such as Cu2+, Zn2+ and Mg2+, used as sulphate salts and Ca2+, Cd2+, Co2+ and Ba2+ used as chloride salts and EDTA with the concentration of 1 mM. Laccase activity was monitored under standard assaying conditions. The reaction assay mixture of laccase was incubated with activators or inhibitors, see more optimized buffer and syringaldazine and at respective optimum temperature. The change in absorbance was measured spectrophotometrically to evaluate the influence of these activators and inhibitors on enzyme activity. Results were expressed as percentage of the control (non-treated laccase). 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 MEK activation 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, Phosphoprotein phosphatase 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.