Stability of Immobilized Horseradish Peroxidase in Water-Miscible Organic Solvents

Abstract

Chitosan beads were prepared with phytic acid to develop a biocompatible matrix for enzyme immobilization. Horseradish peroxidase (HRP) was immobilized on phytic acid-modified chitosan beads, and its catalytic activity was measured using 2-methoxyphenol. The concentration of phytic acid used was varied, with optimal enzyme activity at a concentration of 25 mM phytic acid. The phytic acid/chitosan beads were characterized using FTIR, SEM, and their swelling behavior was investigated at room temperature. The beads exhibited slightly higher levels of water absorption, 20 ± 2 of mass for phytic acid/chitosan versus 18.6 ± 1.5 for chitosan beads. The activity of HRP (free and immobilized) was examined in aqueous-nonaqueous mixtures (5:95) of dimethylformamide (DMF), hydroxymethylfurfural (HMF), methanol (MeOH), acetonitrile (ACN), ethanol (EtOH), acetone, and propanol. Immobilized HRP showed good stability in DMF, MeOH, ACN, EtOH, and acetone, maintaining over 40% of its initial activity after 6 h incubation in the solvent mixtures, demonstrating the successful use of phytic acid-chitosan as an enzyme support.