Polyphenol oxidase was purified from leaves of Nicotiana tobaccum and the needle shape crystals were formed. The enzyme was immobilized on nylon membrane with glutaraldehyde as the cross linking agent. The appropriate conditions for immobilization were as follows: (1) The concentration of enzyme, 13 5 U/ml (concentration of protein: 0 03 mg/ml); (2) The optimum time for cross linking by glutaraldehyde was 20 minutes; (3)The optimum time for immobilization was 7 hours; (4) The optimum concentration of glutaraldehyde was 0 25%; (5) The optimum temperature was 4℃. The activity of immobilized enzyme reached 35—40 U/g matrix, and the activity recovery was 76 2% and the coupling efficiency was 76 3%. The stability of immobilized enzyme under acid, basic and high temperature conditions were enhanced and shifted toward basic pH range. It will be beneficial for industrial utilization.
