Stability of immobilized horseradish peroxidase in 1 water-miscible organic solvents

Chitosan beads were prepared with phytic acid to develop a biocompatible matrix for 8 enzyme immobilization. Horseradish peroxidase (HRP) was immobilized on phytic acid modified 9 chitosan beads and its catalytic activity measured using 2-methoxyphenol. The concentration of 10 phytic acid used was varied, with optimal enzyme activity at a concentration of 25 mM phytic 11 acid. The phytic acid/chitosan beads were characterized using FTIR, SEM, and their swelling 12 behavior was investigated at room temperature. The beads exhibited slightly higher levels of water 13 absorption, 20±2 of mass for phytic acid phytic acid/chitosan versus 18.6±1.5 for chitosan beads. 14 The activity of HRP (free and immobilized) was examined in aqueous-nonaqueous mixtures (5:95) 15 of dimethylformamide (DMF), hydroxymethylfurfural (HMF), methanol (MeOH), acetonitrile 16 (ACN), ethanol (EtOH), acetone, and propanol. Immobilized HRP showed good stability in DMF, 17 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 1 support.