Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers |
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Authors: | Olivera Prodanović Miloš Prokopijević Dragica Spasojević Željko Stojanović Ksenija Radotić Zorica D. Knežević-Jugović Radivoje Prodanović |
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Affiliation: | 1. Institute for Multidisciplinary Research, University of Belgrade, Kneza Vi?eslava 1, 11030, Belgrade, Serbia 2. Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njego?eva 12, 11001, Belgrade, Serbia 3. Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11120, Belgrade, Serbia 4. Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000, Belgrade, Serbia
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Abstract: | A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g?1 dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K m) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity. |
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