• horseradish peroxidase;
  • Orange II;
  • response surface methodology;
  • electrospray ionization–mass spectrometry

Statistics-based experimental design with response surface methodology (RSM) was applied to optimize the operating conditions in decolorization of Orange II by horseradish peroxidase (HRP) in the presence of H2O2. Four critical factors (i.e., HRP concentration, H2O2 concentration, pH, and reaction time) were used for the optimization of enzymatic decolorization using RSM. The optimized conditions of the enzymatic degradation of Orange II were as follows: HRP concentration, 0.80 U/mL; H2O2 concentration, 0.70 mM; pH, 9.0; reaction time, 40 min. The maximal decolorization efficiency of 95.83% was achieved at the optimized conditions. The analysis of UV–vis spectra and electrospray ionization–time of flight–mass spectrometry indicated that both decomposition and coupling reaction occurred in the enzymatic decolorization. © 2012 American Institute of Chemical Engineers Environ Prog, 32: 294-301, 2013