In order to investigate the characterizations of chitosan nano-particles prepared by ionization gelation method, neutral proteinase was selected as a model enzyme to assess its potential applicability for the enzyme immobilization. The characterizations of immobilized enzyme were studied. The highest enzyme activity of immobilized enzyme was allowed at pH 7.5, identical to that of free enzyme. but the range of the optimum pH was broadened. The optimal temperature of immobilized enzyme was 55 degrees C, and the range of the optimum temperature was also broadened. The thermal, operational and storage stabilities of immobilized enzyme were improved after they were immobilized on chitosan nano-particles. Chitosan nano-particles were applied to protect the activity of neutral proteinase. They could improve 13.17% of neutral proteinase activity than that of free neutral proteinase at the same conditions. The Michaelis constant K for immobilized enzyme was disclosed to 0.6629 mg/ml by Line weaver-Burk plot at 40 degrees C. (c) 2005 Published by Elsevier Ltd.