The immobilization of trypsin onto polyaniline for protein digestion

Trypsin (E.C. 3.4.21.4) was successfully immobilised by covalent binding to carbonyl groups present on the surface of polyaniline that had been pre-treated with 2.5% (v/v) glutaraldehyde (PANIG). Under optimised conditions (0.5 mg mL− 1 enzyme in 0.1 mol L− 1 sodium phosphate buffer, pH 7.6, 1 h), 5.0 mg of PANIG retained 11 U of trypsin (2200 g− 1). Immobilised trypsin was more stable than the free and showed higher activities at elevated temperatures (45–55 °C) and in the basic pH region (7–10). PANIG-trypsin retained 58% of its initial activity after 49 days of storage at 4 °C in glycine buffer (0.1 mol L− 1, pH 3.6) containing CaCl2 (0.6 mmol L− 1). High enzymatic activities were recovered from stored samples of lyophilised PANIG-trypsin. The hydrolysis products from casein, BSA and skimmed milk formed in the presence of free and immobilised trypsin were analysed by SDS-PAGE and the results confirmed the absence of autolysis in PANIG-trypsin.