Temperature- and pH-sensitive hydrogels, based on N-isopropylacrylamide (NiPAAm) and itaconic acid (IA), were synthesized by free radical crosslinking copolymerization in the presence of lipase from Candida rugosa. The samples were characterized for their sensitivity to the changes of external conditions and the ability to control the release of a hydrophilic model protein, lipase. These hydrogels were highly responsive to temperature and pH, at constant ionic strength. Parameters, such as the crosslinking degree and non-ionic/ionic (NiPAAm/IA) ratio, were found to impact the hydrogel structure, mechanical properties, morphology and swelling kinetics at different pH and temperatures. The hydrogels demonstrated protein loading efficiency as high as 95 wt%. Release studies of a hydrophilic model protein at a physiological temperature of 37 degrees C were performed at different pH values. High dependence of lipase release kinetics on hydrogel structure and the environmental pH was found, showing generally low release rates, lower in acidic media (pH 2.20) and higher at higher pHs (6.80). Lipase activity was retained even after treatment conditions that would provoke denaturation of the enzyme if it was not protected in the gel. The obtained hydrogels were found suitable for releasing therapeutic proteins in a controlled manner at specific sites in gastrointestinal tract.