Tyramine modified alginates via periodate oxidation for peroxidase induced hydrogel formation and immobilization

Phenol and amino groups were introduced into alginate to different degrees via oxidation with 2.5, 5, 10, 15 and 20 mol% of periodate and reductive amination by tyramine. Modification of alginate with tyramine was confirmed by FTIR spectroscopy and UV-VIS spectroscopy, while concentration of phenol and ionizable groups was determined using absorbance at 275 nm and acid-base titration. All tyramine-alginates were able to form hydrogels after cross-linking with horse radish peroxidase (HRP) and hydrogen peroxide. Tyramine-alginates oxidized with up to 10 mol% of periodate were also capable of forming hydrogels with calcium ions. Tyramine-alginates were tested for HRP immobilization within micro-beads obtained by peroxidase catalyzed droplet polymerization using internal delivery of hydrogen peroxide via glucose oxidase and glucose. Highest activity of immobilized peroxidase was obtained with 20% (w/v) tyramine-alginate obtained via 20 mol% periodate oxidation. Immobilized enzyme was not leaking from the micro-beads and was further kinetically characterized for pyrogallol oxidation. Km for pyrogallol was increased after immobilization from 1.93 mM for soluble HRP to 7.34 mM for immobilized HRP. The optimum pH was also increased from pH 7.0 to 8.0. Temperature and organic solvent stability improved significantly after immobilization, so that half-life at 70 °C increased around four times, while half-life in 80% (v/v) dioxane increased 22 times. After repeated use of 6 times in batch reactor for pyrogallol oxidation immobilized HRP retained 45% of original activity.