Soybean Peroxidase-CATALYZED OLIGOMERIZATION OF aRYLAMINES in water: OPTIMIZATION, KINETICS, Products AND COST

Abstract Water contamination by refractory and persistent organic aromatic compounds such as poly-substituted mono-cyclic, 4-chloro-o-toluidine and bis-anilino compound, 4,4'-methylenebis-(2-chlororaniline), is an emerging environmental concern and the compounds are considered priority pollutants by U.S. Environmental Protection Agency. Thus, there is a search for new, efficient and eco-friendly treatment methods for converting and eliminating these pollutants at the point of release. The crude form of soybean seedcoat peroxidase, was able to catalyze the oxidation of these pollutants by generating reactive radicals which couple to form insoluble products. The reaction parameters, pH, hydrogen-peroxide-to- substrate molar ratio and minimum effective enzyme concentration were optimized to achieve ≥95% removal of these substrates in a 3-h reaction time. The pH optima for 4-chloro-o-toluidine and 4,4'-methylenebis (2-chlororaniline) were 4.4 and 4.2, respectively. Consumption of H2O2 of both substrates was near the theoretical stoichiometric value (i.e. H2O2/substrate ≤ 1.0). For 95% removal of 1.0 mM 4-COT and 0.1 mM MOCA, 0.009 and 0.10 U/mL of enzyme were required, respectively. The enzyme kinetic mechanism based on the Michaelis-Menten model was determined for a group of aromatic amines, p-cresidine, 4,4′-oxydianiline, 4-chloro-o-toluidine and 4,4'-methylenebis-(2-chlororaniline). The lowest KM was for 4,4'-methylenebis (2-chlororaniline), 1.70 ± 0.14 µM, indicating highest affinity for the enzyme compared to other amines studied. In addition, product analysis by mass spectrometry revealed presence of “oxidative oligomers” and “oxidized oxidative oligomers” (azo compounds) after the enzymatic treatment. A pro-forma cost estimation emphasized the feasibility of commercialization of the enzymatic treatment, showing it to be 5 to 8 times lower than conventional methods.