Abstract
This paper describes the optimization of the immobilization of the peroxidase from Roystonea regia (RPTP) using the technique of the crosslinking enzyme aggregates. The immobilization was optimized using response surface methodology and after evaluating three different precipitants, ethanol was finally selected. Three variables were analyzed, glutaraldehyde concentration, precipitant concentration and time before collecting the RPTP-CLEAs. The immobilization yield was around 75%. The activity of the RPTP-CLEA was very high, this was even more patent at pH 3, where the free RPTP was fully inactive and the RPTP-CLEA maintained 40% of the maximum activity. In stress inactivations, the RPTP-CLEA maintained the very high thermostability that presented the free enzyme at 90 degrees C and pH 7 (half-live of 50 min). At pH 3, the free enzyme suffered subunit dissociation as a first step of the inactivation, this is not possible using RPTP-CLEAs and provided a very high thermostabilization (that depends on the enzyme concentration). The stability of the enzyme in the presence of hydrogen peroxide is good at low concentrations of this reagent (e.g., 10 mM), but if the concentration is higher (e.g., 300 mM), the enzyme stability drops. The immobilization provides an improved stability in the presence of this oxidant, but the values reached may not be high enough for some applications. The RPTP-CLEAs may be used for the decoloration of methyl orange solutions using 5 mM of hydrogen peroxide for 4 cycles (4 h each cycle) without apparent decrease in activity (but only degrading around 50% of the substrate). Using 225 mM of this oxidative reagent, the activity slowly decreased after each cycle (but enabling the full destruction of the colorant). This immobilized enzyme may be used even at pH 3 and 225 mM hydrogen peroxide, conditions where the free enzyme is completely inactive.