Neutral protease was immobilized on glutaraldehyde-pretreated N-succinyl chitosan hydrogel beads and the biocatalyst obtained was used for the preparation of low molecular weight chitosan and chito-oligomers with molecular weight of 1.9-23.5 kDa from commercial chitosan. Factors affecting the chitinolytic hydrolysis were described. The degradation was monitored by gel permeation chromatography. The structure of degraded chitosan was characterized by Fourier transform infrared, X-ray diffraction and liquid chromatography-mass spectrometry. Immobilized neutral protease showed optimal depolymerization at pH 5.7 and 50 degrees C. The degree of deacetylation of the hydrolysates did not change compared to that of the initial chitosan. The decrease of molecular weight led to transformation of crystal structure but the chemical structures of residues were not modified. The degree of polymerization of chito-oligomers was mainly from 3 to 8. The method allows cyclic procedures of immobilized enzyme and N-succinyl chitosan support utilization, and is suitable for a large-scale production of the low molecular weight chitosan and chito-oligomers free of protein admixtures. (c) 2006 Wiley Periodicals, Inc.