Interaction of horseradish peroxidase with montmorillonite homoionic to Na+ and Ca2+: effects on enzymatic activity and microbial degradation

The adsorption, desorption, catalytic activity, and susceptibility to micro bial degradation of the enzyme horseradish peroxidase (HRP E.C. 1.11.1.7), on Wyoming montmorillonite (M) homoionic to Na+ or Ca2+ were investigated. Adsorption at equilibrium was reached after 1 h of contact between the clay and HRP. The adsorption isotherms were of the L type and fitted the Freund lich equation on M-Na and the Langmuir equation on M-Ca. Adsorption was gre ater on M-Na than on M-Ca and was maximal at pH 3.0, i.e., below the isoele ctric point (pI = pH 9) of the protein. Only 10-25% of HRP was desorbed fro m the equilibrium M-Na-HRP complexes, whereas 20-30% was desorbed from the equilibrium M-Ca-HRP complexes with 4-7 washes with double distilled water. HRP partially penetrated the interlayers of M-Na and hi-Ca, but complete i ntercalation was observed only at pH 3. The enzymatic activity of HRP measu red immediately after the preparation of the complexes at all concentration s was greatly reduced when bound on M-Na (about 90%), regardless of the loa ding of HRP. The reduction in activity of M-Ca-HRP was related to the amoun t of bound protein (no reduction for the highest and 60% for the lowest amo unt bound). After 24 h, pure HRP in dilute solution lost about 10% of its c atalytic activity daily, whereas when bound on M-Cal a greater reduction wa s observed (about 30% for the highest and 60% for the lowest amount bound). FT-IR analyses indicated only small changes in the secondary structure of HRP as a result of binding on the clays. Electronic absorption spectra in t he UV region of bound HRP did not show the typical 'red-shift' of the Soret band that usually results from binding of HRP with its substrate. Conseque ntly, the reduction in the activity of bound HRP was probably the result of the inaccessibility and/or of modifications of the active center of HRP fo r its substrate. The availability of HRP bound on M-Na as a source of carbo n and/or nitrogen for soil microorganisms was reduced by 90% in comparison with the free enzyme. (C) 2001 Elsevier Science Ltd. All rights reserved.