Abstract
The enzymatic-oxidative polymerization of phenolic compounds prevents their toxic effects, is a green approach; however, limitations are present, such as limited operational stability, susceptibility to radical deactivation, and difficulties in recovering and reusing free enzymes. Here, an oxidoreductase enzyme, horseradish peroxidase (HRP), immobilized in poly(2-hydroxyethyl methacrylate) (p(HEMA)) cryogels (HRP@p(HEMA)) via the entrapment method during synthesis under cryogenic conditions. The HRP@p(HEMA) cryogels’ activity was determined as 0.056 ± 0.001 unit/mg enzyme, close to free enzyme activity, 0.061 ± 0.003 unit/mg enzyme in phosphate buffer solution (PBS) at pH 6, 25 ℃. The HRP@p(HEMA) cryogels exhibited higher activity% than an equal amount of free enzyme under harsh conditions, e.g., lower/higher pH and temperature environments. After storing at −20, + 4, and + 25 ℃ for 10 days, the HRP@p(HEMA) cryogels preserved almost 95, 65, and 50% of their activity, respectively, higher than the free enzymes stored in the same conditions. The Vmax and Km values, 0.013 ± 0.001 mM/min and 7.69 ± 0.3 mM, respectively, for free HRP enzyme are quite comparable to HRP@p(HEMA) cryogels’ values, 0.011 ± 0.001 mM/min and 7.52 ± 0.2 mM, respectively. Moreover, the HRP@p(HEMA) cryogels used as biocatalyst in enzymatic-oxidative polymerization of 2-naphthol (2-NL) to p(2-NP) resulted in HRP@P(HEMA)-p(2-NL) cryogel composites, which revealed an antioxidant activity of 0.11 ± 0.05 µg/mL Trolox equivalent/g using ABTS.+ scavenging assay and IC50 value of 18.7 ± 3.5 µg/mL via DPPH radical scavenging.