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Papain (from papaya latex; EC 3.4.22.2) and Pronase (from Streptomyces griseus; EC 3.4.24.31) caused optimum depolymerization of chitosan at pH 3.5 and 37• C, resulting in LMMC (low molecular mass chitosan) and chito-oligomeric-monomeric mixture. The yield of the latter was 14-16 % and 14-19 % respectively for papain-and Pronase-catalysed reactions, depending on the reaction time (1-5 h). HPLC revealed the presence of monomer(s) and oligomers of DP (degree of polymerization) 2-6, which was also confirmed by matrix-assisted laser-desorption ionization-time-of-flight MS. Along with the chito-oligomers, the appearance of only GlcNAc (N-acetylglucosamine) in Pronasecatalysed chitosanolysis was indicative of its different action pattern compared with papain. Fourier-transform infrared, liquidstate 13 C-NMR spectra and CD analyses of chito-oligomericmonomeric mixture indicated the release of GlcNAc/GlcNAc-rich oligomers. The monomeric sequence at the non-reducing ends of chito-oligomers was elucidated using N-acetylglucosaminidase. The chito-oligomeric-monomeric mixture showed better growth inhibitory activity towards Bacillus cereus and Escherichia coli compared with native chitosan. Optimum growth inhibition was observed with chito-oligomers of higher DP having low degree of acetylation. The latter caused pore formation and permeabilization of the cell wall of B. cereus, whereas blockage of nutrient flow due to the aggregation of chito-oligomers-monomers was responsible for the growth inhibition and lysis of E. coli, which were evidenced by scanning electron microscopy analysis. The spillage of cytoplasmic enzymes and native PAGE of the cellfree supernatant of B. cereus treated with chito-oligomeric-monomeric mixture further confirmed bactericidal activity of the latter. Use of papain and Pronase, which are inexpensive and easily available, for chitosanolysis, is of commercial importance, as the products released are of considerable biomedical value.

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