P. Fusi

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

Abstract The adsorption, desorption, catalytic activity, and susceptibility to microbial degradation of the enzyme horseradish peroxidase (HRP E.C. 1.11.1.7), on Wyoming montmorillonite (M) homoionic to Na + or Ca 2+ were investigated. Adsorption at equilibrium was reached after 1xa0h of contact between the clay and HRP. The adsorption isotherms were of the L type and fitted the Freundlich equation on M-Na and the Langmuir equation on M-Ca. Adsorption was greater on M-Na than on M-Ca and was maximal at pH 3.0, i.e., below the isoelectric point ( pI =pH 9) of the protein. Only 10–25% of HRP was desorbed from 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 M-Ca, but complete intercalation was observed only at pH 3. The enzymatic activity of HRP measured immediately after the preparation of the complexes at all concentrations was greatly reduced when bound on M-Na (about 90%), regardless of the loading of HRP. The reduction in activity of M-Ca-HRP was related to the amount of bound protein (no reduction for the highest and 60% for the lowest amount bound). After 24xa0h, pure HRP in dilute solution lost about 10% of its catalytic activity daily, whereas when bound on M-Ca, a greater reduction was 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 the 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. Consequently, 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 for its substrate. The availability of HRP bound on M-Na as a source of carbon and/or nitrogen for soil microorganisms was reduced by 90% in comparison with the free enzyme.

Interaction of catalase with montmorillonite homoionic to cations with different hydrophobicity: effect on enzymatic activity and microbial utilization

Abstract The exchange sites of montmorillonite (M) were made homoionic to calcium (Ca), hexadecyltrimethylammonium (HDTMA) or pyridinium (PY) cations. The clays were used as adsorbents for the enzyme, catalase (CA). Equilibrium adsorption and binding isotherms (i.e., after washing of the clay–CA complexes after adsorption at equilibrium until no CA was desorbed) were of the L 3 -type and fitted the Langmuir equation in the initial, but not in the later, portions of the isotherms. The amounts adsorbed and bound at the plateau, as well as the affinity, were higher for the hydrophobic clays (M–HDTMA and M–PY) as indicated by the Langmuir parameter, B max and K eq . In all three systems, there was additional adsorption after the initial plateau at higher concentrations of CA. In the case of M–Ca–CA this probably resulted from some penetration of CA into the interlayer spaces of the clay, as shown by X-ray diffraction analysis. No penetration of the interlayers was observed in the M–HDTMA–CA and M–PY–CA systems. The additional adsorption that occurred after the initial plateau in these systems may have resulted from the formation of multilayers of CA or from a change in the orientation of CA on these clay-organic surfaces, which may also have occurred in the M–Ca–CA system in addition to intercalation. Most of the CA adsorbed at equilibrium was bound on the clays (85–90%). Fourier-transform infrared difference spectra showed a shift in the Amide I and Amide II frequencies for only M–Ca–CA and M–PY–CA, which was consistent with the hypothesis of a conformational modification of the structure of CA on M–Ca and M–PY. The enzymatic activity of CA adsorbed at equilibrium on the three clays was lower than that of free CA and decreased in the order of M–Ca–CA>M–PY–CA>M–HDTMA–CA. As shown by the values of the overall first-order rate constant, K 1 , there was a further reduction in activity when CA was bound on the clays, especially on M–Ca. The pH optimum for the activity of CA remained essentially unchanged when adsorbed or bound on all clays. CA bound on the clay systems, except on M–PY–CA, was poorly utilized in comparison with the free enzyme as a sole source of carbon or nitrogen.

Adsorption of isoproturon on homoionic clays

The adsorption of isoproturon (3-(4-isopropylphenyl)- 1,1 dimethylurea)] on a montmorillonite and on a kaolinite was studied by batch experiments and Fourier transform infrared spectroscopy (FT-IR). The clays were saturated with cations such as Al3+, Ca2+, Cu2+, Na+ and hexadecyl-trimethylammonium (HDTMA). The studied clays did not show detectable catalytic activity on abiotic degradation of isoproturon, as evidenced by HPLC analysis. Adsorption was found related to clay specific external surface for HDTMA-saturated clays, while it seems independent from clay kind and inorganic cation. For clays saturated with inorganic cations, no penetration into the mont-morillonite interlayer was found and FT-IR spectroscopy showed the involvement of Cue5fbO group of isoproturon in adsorption.

Humic-like compounds formed from l-tryptophan and d-glucose in the presence of CU (II)

Abstract This study concerns the humic-like polymeric compounds formed by D-glucose plus L-tryptophan and by L-tryptophan alone, both on Cu-saturated nontronite, montmorillonite and kaolinite and on quartz in the presence of Cu(II) ions. These mineral-organic systems were kept in thermostat at 70°C for 30 days and submitted to alternate wetting-drying cycles. Higher amounts of humic-like compounds were extracted from quartz-organic than from clay-organic systems, showing the remarkable action of Cu(II), which even overcame the high catalytic power of clays.

Interactions of two acetanilide herbicides with clay surfaces modified with Fe(III) oxyhydroxides and hexadecyltrimethyl ammonium

Abstract The adsorption at 22° and 5°C of two acetanilide herbicides (Alachlor and Metolachlor) on montmorillonite saturated with HDTMA+ cation or coated with Fe(III) oxyhydroxides (modified clays) was studied. The isotherms were of L, S and C type depending on the type of the adsorbent. The calculated values of isosteric adsorption heat (ΔH) and desorption experiments suggest that weak bonds take place. FT-IR analyses showed that both molecules are adsorbed on clays by hydrogen bond between the C-O group and OH groups of Fe(III) oxyhydroxy cation coating the clay. Also on HDTMA-Montmorillonite the C-O group is involved in the adsorption but with lower strenght. Alachlor is only partially decomposed to 2-chloro-2′,6′-diethylacetanilide when the dirty clays complexes are heated at 70°C for 18 h. X-ray analyses showed that both herbicides were unable to penetrate into the inter-layers of the clays.