Preparation and kinetic studies of surfactant–horseradish peroxidase ion paired complex in organic media

Abstract

Horseradish peroxidase was solubilized in organic medium using the technique of ion pairing with the anionic surfactant Aerosol OT (AOT). System parameters like surfactant concentration, pH, ionic strength of the aqueous phase, cosolvent concentration etc. were found to influence the solubilization of HRP. Kinetic behaviour of the ion paired complex towards oxidation of o-phenylenediamine (OPD) in three different solvent systems—aqueous, aqueous-organic monophasic and aqueous-organic biphasic system was investigated. Steady state kinetics of ion paired HRP revealed that ion pairing of the enzyme with the surfactant resulted in an increase in the apparent K m and a decrease in V max values of the enzyme in aqueous medium. Enzyme–substrate affinity ( K m) showed an improvement for the ion paired enzyme in organic solvents and was found to decrease with increase in hydrophobicity of the solvent with the ion paired enzyme exhibiting the least K m value in the most hydrophobic solvent toluene. There was an eightfold decrease in K m value in toluene compared to that in aqueous medium. Among the two types of non-aqueous systems used for the study, V max was found to be higher in the microaqueous system (toluene). Ion paired HRP exhibited very high catalytic efficiency ( K cat/ K m) in toluene compared to that of native HRP.