Beads of cross-linking chitosan-coated silica gel (CTS-SiO2) were prepared. The surface layer of chitosan had a porous structure and could provide sufficient amount of amino groups, which could be easily activated with the functional group of epoxy, diazo and aldehyde, respectively. Trypsin (EC 3.4.21.4) could be efficiently immobilized on the three different activated matrices. The stability of trypsin immobilized via different methods in long term of storage, the thermal stability and pH dependence in comparison with the free enzyme were investigated. Results showed that trypsin immobilized via all employed methods could tolerate relatively tough environmental conditions such as high temperature and wide pH range. The immobilized enzyme was obviously stable in long storage time and was reusable. The best results were obtained when trypsin was immobilized via direct epoxy activation, which might be ascribed to the multi-point attachment (MPA) between enzyme and the support. The results were explained and discussed by analyzing the protein structure such as surface exposure rate and the number of amino acid residues related to enzyme coupling. The distance between these. amino acids and the catalytic site of trypsin was also predicted by protein structure prediction and display software. (c) 2005 Elsevier Ltd. All rights reserved.