Objective: The aim of this study was to investigate the laccase production of Trametes versicolor under submerged fermentation condition. Then, dye decolorization by laccase was optimized using Box-Behnken methodology. Methods: The optimal culture conditions for producing high amount of laccase were determined using Taguchi methodology. The experiments were designed with five factors (glucose, yeast extract, CuSO 4 , inoculum size and pH) at three levels with orthogonal array layout of L27 (3 5 ). Then, the optimum conditions for high decolorization activity of Reactive Blue 49 by obtained crude laccase were also investigated using Box-Behnken methodology. Results: The optimum culture conditions for production of high amounts of laccase were detected as 2 g L -1 of glucose, 5 g L -1 of yeast extract, 2mM of CuSO 4 , 4% of inoculum amount and pH 5.5. Yeast extract was the most effective factor, followed by CuSO 4 , inoculum, glucose and pH. Under these conditions, predicted values were in a good agreement with the actual experimental one. The predicted results showed that the maximum of Reactive Blue 49 decolorization as 98% could be obtained under the optimum conditions of pH 2.95, initial dye concentration 55.6 mg L -1 , enzyme amount 0.76 mL and reaction time 46.91 min. The validity and practicability of this statistical optimization strategy was confirmed with the relation between predicted and experimental values.
Conclusion:The results suggested that Taguchi method can be used in the optimization of laccase production process. Production of laccase by Trametes versicolor 2008001 can be effectively used for enzymatic decolorization according to the results of decolorization experiments in optimal levels. Key Words: Laccase, Taguchi Method, Box-Behnken Methodology, dye decolorization.
Conflict of Interest:The authors declare no conflict of interest.
Materials and Methods
Microorganism, culture conditions and dyeTrametes versicolor ATCC (200801) originally isolated and cultured by Dr. O Yesilada was used in this study. This fungus was subcultured on Malt Extract Agar (MA) plates of 30 °C and stored at 4 °C.Laccase production of T. versicolor was tested under agitated culture conditions. To this end, firstly this fungus was cultured at 30 ˚C on slant MA for one week. Then, mycelial suspension was prepared and this suspension was inoculated into 250 mL Erlenmeyer flask containing 100 mL Potato Dextrose Broth (PDB). This culture was incubated at 30 °C and 150 rev min -1 for 4 days. After that, the culture was homogenized and determined culture of homogenized culture was transferred in 250 mL Erlenmeyer flasks with 100 mL Stock Basal Medium (SBM) that contain K 2 PO 4 : 0.2 g L