Trypsin (EC 3.4.21.4) has been used in various industrial processes, such as treatment of milk, limited proteolysis, peptide synthesis and affinity purification of trypsin inhibitors. The use of immobilized enzymes has been widely applied to overcome the limited large-scale applications of them. In this work, we presented a new composite support: PVA-Glutaraldehyde-Polyaniline-Glutaraldehyde (PVAG-PANIG) and its use for trypsin immobilization. The support was characterized by FT-IR, photomicrography and the immobilized derivative was submitted to tests of optimization concerning enzyme retention capacity, pH and time for complete enzyme immobilization. Some of their proprieties were investigated (kinetic parameters, optimal pH and temperature and reaction time) and compared to the native trypsin. Storage conditions, thermal stability and reuse capacity were also optimized. Analysis of FT-IR spectra showed bands of PVA (3300 cm^-1, 2930 cm^-1 and 1440 cm^-1) and PANI (1594 cm^-1 and 1100 cm^-1) in composite discs and appearance of the green emeraldine color of PANI. The best immobilization conditions were achieved at trypsin concentration of 0.01 mg mL^-1, pH 7.6 and 60 min of immobilization, resulting in 21.1 units immobilized enzyme per 150 mg disc (10% of enzyme retention). The PVAG-PANIG-trypsin showed optimal pH and temperature similar to those reported for the native enzyme. Apparently, the Vmax_app decreased considerably. However, PVAG-PANIG provided to trypsin reusability, storage and thermal stability and the possibility to remove immediately enzyme from the bulk reaction and to stop catalysis with high precision.
Financial support: CNPq and PrP/UEG