Immobilization of alcohol dehydrogenase from Saccharomyces cerevisiae onto carboxymethyl dextran-coated magnetic nanoparticles: a novel route for biocatalyst improvement via epoxy activation
Katja Vasić, Željko Knez, Maja Leitgeb
Abstract
Abstract A novel method is described for the immobilization of alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae onto carboxymethyl dextran (CMD) coated magnetic nanoparticles (CMD-MNPs) activated with epoxy groups, using epichlorohydrin (EClH). EClH was used as an activating agent to bind ADH molecules on the surface of CMD-MNPs. Optimal immobilization conditions (activating agent concentration, temperature, rotation speed, medium pH, immobilization time and enzyme concentration) were set to obtain the highest expressed activity of the immobilized enzyme. ADH that was immobilized onto epoxy-activated CMD-MNPs (ADH-CMD-MNPs) maintained 90% of the expressed activity. Thermal stability of ADH-CMD-MNPS after 24 h at 20 °C and 40 °C yielded 79% and 80% of initial activity, respectively, while soluble enzyme activity was only 19% at 20 °C and the enzyme was non-active at 40 °C. Expressed activity of ADH-CMD-MNPs after 21 days of storage at 4 °C was 75%. Kinetic parameters ( K M , v max ) of soluble and immobilized ADH were determined, resulting in 125 mM and 1.2 µmol/min for soluble ADH, and in 73 mM and 4.7 µmol/min for immobilized ADH.