Chemical modification and characterisation of horseradish peroxidase and its derivatives for use in environmental applications

The biochemistry and molecular properties o f the widely-used horseradish peroxidase (HRP) are reviewed. The applications of HRP in diagnostics, sensors and waste treatment are also surveyed. The research aims were to stabilise HRP by chemical modification to ascertain the characteristics of stabilised derivatives and to explore the capabilities of these derivatives in waste treatment applications. Assay methods for HRP activity were compared and thermoinactivation experiments were undertaken using a variety of conditions in order to devise workable, reproducible protocols for routine use. Systematic chemical modification of the available reactive HRP amino residues (Arg, Tyr and Lys) were performed. Arginine and tyrosine modified derivatives yielded little gain in stability Modification of lysine residues with cyclic anhydrides of aromatic carboxylic acids was undertaken. Of the compounds used, only phthalic anhydride yielded a thermally stabilised HRP (PA HRP). The overall stability characteristics of this derivative were investigated fully together with previously reported crosslinked HRP modifications. The tolerances of these two derivatives towards heat, organic solvents, hydrogen peroxide, pH and dénaturants was enhanced. Both were immobilised on adipic acid dihydrazide-agarose (AADH-A). The stabilities of the immobilised, modified HRPs were compared with the modified-only counterparts. Unfolding of native and both modified HRPs were investigated using urea, guanidine chloride (GnCl), EDTA and heat. Unfolding over time was monitored using fluorescence and binding of the hydrophobic probe Nile Red. Results of the two methods agreed closely. Attempts were made to fragment the HRP polypeptide so as to demonstrate the presence of crosslinks and, possibly, to identify the modified Lys residues. Unfortunately these experiments proved inconclusive. It was shown that recombinant HRP which, lacks the carbohydrate moiety, could withstand a chemical modification and be stabilised in a similar manner to the plant HRP. A kinetic investigation of the HRP forms took place. Binding of the inhibitor BHA was found to be slightly less in the case of PA HRP. The pre-steady state and steady state kinetics of the HRPs using a variety of substrates were found to be in close agreement with each other. The significance of these results is discussed. The use of both modified HRPs in waste treatment was explored. Modified HRP was as effective as native in the precipitation of phenols at 37 °C. Modified HRPs were more effective than the native at higher temperature where the native is inactivated. In the bleaching of dyes, modified HRPs were as effective as the native. Thus, a new stabilised chemically modified form of HRP (PA HRP) has been described and the capabilities of this and the EG HRP in two distinct types of wastewater treatment have been characterised