The Preparation and Properties of Immobilized Trypsin on Polyacrylonitrile Resin

Trypsin was immobilized on partially imidoesterized polyacrylonitrile. The activity and the amounts of immobilized enzyme on the carrier resin were depended on the conditions of the immobilization reaction (pH and temperature). The optimum pH in the immobilization had a broad peak in the range from pH 4.5 to 5.5. The optimum temperature for the activity was observed at 20°C, but the amounts of the immobilized enzyme was gradually increased with the elevation of the temperature. The relative activity of the immobilized trypsin was found to be in the range from 80% to 90% of the equivalent amount of the native enzyme. The optimum pH of the immobilized trypsin were slightly shifted to the alkaline side (pH 8.1→pH 8.6) and the optimum temperature moved to the lower temperature side (49°C→47°C) comparing with those of native enzyme. The pH stability of the immobilized trypsin were almost identical to those of the native enzyme. The value of the apparent Michaelis constant (Km) was estimated to be about 0.065wt% for the immobilized trypsin and 0.047wt% of the native enzyme respectively. The endurance test of the immobilized trypsin column was pursued by the continuous operation for two weeks with a flow rate of 0.5ml/min at room temperature and 40°C. The presence of ionic bond was suggested besides the covalent one of amidine bonding in mode of chemical attachment of enzyme to the carrier by the repeated hydrolysis of casein in batch process.