| Abstract [eng] |
Immobilized enzymes are a common tool in modern biotechnology and diagnostics. Such enzymes often have a wider pH and temperature limits of operation and enhanced thermostability. They are easy to use and can be adapted for multiple use, for that reason their market is rapidly growing. Another widely used tool in biotechnology - magnetic particles. Their synthesis is easy, the produced particles are homogeneous and correct size. Magnetic particles used in the experiments had differently modified hydrophobic and hydrophilic surfaces. Direct coupling - enzyme immobilization onto the magnetic particles through the tosyl or epoxy groups, which results in a covalent bond between the magnetic bead and the primary amine of the enzyme (https://tools.thermofisher.com/content/sfs/brochures/Surface_Activated_Dynabeads.PDF). Indirect coupling – immobilization of biotinylated enzyme on magnetic particles coated with streptavidin, which comprises a non-covalent complex (https://tools.thermofisher.com/content/sfs/brochures/Dynabeads_Streptavidin_Products_and_Applications.pdf). Polymerase and nuclease were selected for the experiments. Specific and functional activities, multi-use of coupled enzymes were studied during the experiments. It was found that bound enzymes show significantly lower specific activity. Nuclease is more likely directly couple to magnetic beads with hydrophobic surface, and polymerase – indirectly to magnetic beads with hydrophilic surface. Nuclease retained specific activity after re-use, while polymerase lost it. |
