Notes
Doctoral thesis consists of two parts, in the first part synthesis of magnetic carrier and modification with organic polymer carboxymethyl dextran (CMD) was performed. Three different CMD concentrations were applied into the synthesis process (0,25 g/mL, 0,40 g/mL in 0,50 g/mL CMD), which resulted in three different modified magnetic carriers (CMD1-MNPs, CMD2-MNPs and CMD3-MNPs). All CMD-MNPs were characterized with various analytical methods: Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDX), transmission electron microscopy (TEM), dynamic light scattering (DLS). Magnetic properties were analyzed with electron paramagnetic resonance (EPR) and magnetization measurements with vibrating sample magnetization (VSM). CMD-MNPs were successfully synthesized with average diameters from 27-30 nm and size distribution revealed most consistent sizes of CMD3-MNPs. Hydroxyl and carboxyl groups were confirmed on the CMD-MNPs surface, which confirms the presence of CMD layer on the surface of CMD-MNPs. EPR and VSM measurements confirmed magnetic properties of all CMD-MNPs and a ferromagnetic system. Inhibition properties were determined of all CMD-MNPs on two different bacterial cultures, where CMD3-MNPs displayed inhibition zone, confirming antimicrobial properties, whereas with other CMD-MNPs no inhibition was detected. Toxicity was also determined on MNPs coated with chitosan (HIT-MNPs) and aminosilane (AMS-MNPs) on five different bacterial cultures, where no inhibition was detected. CMD3-MNPs was chosen for further investigation of enzyme immobilization in the second part of doctoral thesis, because of its most favourable properties.%In second part of doctoral thesis nano-carrier CMD3-MNPs was surface functionalized with epoxy cross-linking, using epoxy cross-linker epichlorohydrin (EClH) to covalently bind enzyme alcohol dehydrogenase (ADH). With optimization of process parameters EClH with 4 % (v/v) was used, which resulted in the highest residual activity of immobilized ADH. Epoxy-functionalized CMD3-MNPs were used in immobilization protocol, where effect of process parameters was investigated on the residual activity and immobilization efficiency of ADH. After successful optimization ADH immobilized onto CMD3-MNPs managed to obtain 89,6 % of residual activity and 99,5 % of immobilization efficiency. Further on, co-immobilization of enzyme ADH and cofactor %-nicotinamide adenine dinucleotide (%-NAD) was performed. Again, effect of process parameters on residual activity and immobilization efficiency of co-immobilization were investigated. After successful optimization, ADH co-immobilized with %-NAD onto CMD3-MNPs managed to obtain 73,3 % of residual activity and 93,8 % of immobilization efficiency.%Thermal stability of immobilized ADH at different temperatures was investigated. ADH immobilized onto CMD3-MNPs obtained almost 60 % of its initial activity after 24 hours at 20 °C and 40 °C, ADH co-immobilized with %-NAD onto CMD3-MNPs managed to obtain 75,4 % of its initial activity at 30 °C and 66,5 % of its initial activity at 50 °C after 5 hours. Storage stability of ADH immobilized onto CMD3-MNPs and ADH co-immobilized with %-NAD onto CMD3-MNPs were investigated at 4 °C, where both obtained almost 60 % of its initial activity after three weeks.%Magnetic carriers HIT-MNPs and AMS-MNPs were functionalized with cross-linker glutaraldehyde (GA) and amino-donor pentaethylenehexamine (PEHA). Functionalized HIT-MNPs and AMS-MNPs were immobilized with enzyme %-galactosidase (%-GAL). When using combination of 0,5 % (v/v) GA and 30 % (v/v) PEHA 128,9 % of residual activity was achieved, which resulted in hyper-activation of enzyme due to its conformational changes. Hyper-activation was achieved also with immobilizing %-GAL onto AMS-MNPs and the highest residual activity was obtained with with 20 % (v/v) of PEHA (154,4 %).