Immobilization of horseradish peroxidase onto clay minerals using soil organic matter for phenol removal

Horseradish peroxidase (HRP) was immobilized on inorganic natural materials for phenol removal. Clays and soil organic matter (SOM) were screened as the enzyme carrier (support) and binding agent (spacer), respectively. Montmorillonite activated with fulvic acid was found to be the best surface-activated carrier because the 2:1 dioctahedral structure clay that has inner pores comprised of planar layers at nanoscale distance demonstrated the highest sorptive capacity for SOM, and the fulvic acid provided various stable chemical bonds with enzymes owing to its high content of various functional groups. The enzyme immobilization yield was 66%, and the high level of phenol removal activity was preserved during this process (91% of enzyme activity was retained). The Michaelis–Menten model parameters for the immobilized HRP were somewhat different from those for free HRP (νmax = 7.83 mM/min and KM = 8.41 mM for free HRP and νmax = 4.29 mM/min and KM = 12.96 mM for immobilized HRP). However, in general, the immobilized enzyme was almost as effective as free enzyme. While free HRP was sensitive to environmental factors such as pH, temperature, ionic strength, fairly stable operating enzyme activities were observed for immobilized HRP, even after long-term storage and repetitive operations. The results of this study are expected to provide a useful enzyme immobilization technique for the environmental applications.