Enzymatic green removal of anisidines from wastewater using partially purified Phaseolus vulgaris peroxidase

Crude green bean (Phaseolus vulgaris L.) peroxidase (GBP) was extracted from fresh pods and partially purified.Following ammonium sulfate fractionation, dialysis, ion-exchange chromatography (DEAE-cellulose), and finally gel filtration, the specific activity obtained was 40.8 U mg⁻¹ corresponding to a purification fold of 32.9 and a yield of 49.35%.An enzyme activity assay was optimized, and maximal activity was reached in the dark, at pH 7.5 and 25 °C; kinetic studies based on substrate variation showed a Km value of 0.064 mM, with a Vmax of 4.91 U mL⁻¹.Anisidine removal by GBP was studied in batch reactor experiments.At H₂O₂ of 0.2 mM, and at enzyme activity of 3.1 U mL⁻¹, > 95% removal was achieved within 3 h for both p-anisidine (at pH = 4.5) and o-anisidine (at pH = 5.5).Pseudo-first-order kinetics demonstrated speedy degradation within the initial 30 minutes, exhibiting rate constants of 3.93 × 10⁻² min⁻¹ for p-anisidine and 3.54 × 10⁻² min⁻¹ for o-anisidine, with corresponding half-lives of 17.6 and 19.6 minutes.Subsequent to 180 minutes, the rates diminished, and the half-lives extended to 26.3 and 36.3 minutes.A 95 Eco-Scale score indicates a highly sustainable procedure utilizing plant-based peroxidase under moderate conditions, a solvent-free process for anisidine removal.