Granule chitosan was prepared from chitosan,chemically-modified, then used as carrier and covalently linked with polyphenol oxidase(PPO) from tobacco by using glutaraldehyde as cross-linking agent.The desirable granule chitosan could be obtained when the molecular mass of chitosan was about 250000 and its concentration was about 30%.The absorbing ability and the category, distribution and number of active groups on the surface of granule chitosan were all improved by formol titration under weak basic condition after protecting parts of free amino-groups with Cu 2+ .The optimal conditions for immobilization of PPO were as follows: the ratio of PPO to granule chitosan: 0.8∶100(mass);concentration of glutaraldehyde:0.6%; immobilization time: 80 min.The resulting immobilized PPO had specific activity of 73 U·g -1 , and activity retention value of 83.2% was achieved.The K m value of immobilized PPO was 4.67 mmol·L -1 and that of soluble PPO was 1.26 mmol·L -1 .The optimal pH values were 6.0 for soluble PPO and 7.0 for immobilized PPO; the optimal temperature were 40 ℃ for soluble PPO and about 50 ℃ for immobilized PPO.In addition, stability of immobilized PPO to temperature and pH was obviously increased.
