Enzyme immobilization on thermosensitive hydrogel microspheres

Precipitation polymerization of N-isopropylacrylamide, acrylamide, and methylenebisacrylamide in water at 70°C resulted in thermosensitive hydrogel microspheres. Carboxyl groups on the microspheres were introduced by hydrolysis, and amino groups by the Hofmann reaction of amide units on the microspheres. Trypsin was immobilized on the carboxylated microspheres using carbodiimide. Phase transitions were detected using a hydrophobic fluorescence probe. The temperatures at which a phase transition occurred were increased by immobilizing enzymes. The enzymatic activity of the immobilized enzymes decreased above the transition temperature. This was attributed to (i) a decrease in the diffusion of substrate; and (ii) entrapment of enzyme in the surface layer of the microspheres. In an attempt to overcome the entrapment, enzymes were immobilized via a hydrophilic spacer (α-(carboxymethyl)-ω-(carboxymethoxy)-poly(oxy-1,2-ethanediyl), PEO acid) to the microspheres. These enzyme-carrying hydrogel microspheres were found to show an enzymatic activity independent of temperature, even though these conjugates show a phase transition at the lower critical solution temperature.