Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAcGlcN and GlcN-GlcN in its substrate, suggesting that it is a subclass I chitosanase. In addition, an extensive site-directed mutagenesis study on 10 conserved carboxylic amino acids of glycosyl hydrolase family 75 was performed. This showed that among these various mutants, D160N and E169Q lost nearly all activity. Further investigation using circular dichroism measurements of D160N, E169Q, wild-type CSN, and other active mutants showed similar spectra, indicating that the loss of enzymatic activity in D160N and E169Q was not because of changes in protein structure but was caused by loss of the catalytic essential residue. We conclude that Asp 160 and Glu 169 are the essential residues for the action of A. fumigatus endo-chitosanase.Chitosanase (EC 3.2.1.132) is a hydrolytic enzyme acting on the ␤-1,4-glycosidic linkage of chitosan, a linear biopolymer of ␤-1,4-linked GlcN, to release chito-oligosaccharides. The oligomers GlcNAc and GlcN have interesting biological activities (1), including anti-tumor effects (2, 3), hypo-cholesterolemic effects (4), anti-microbial activities (5, 6), disease-resistance responses, and as phytoalexin elicitors in higher plants (7,8). Hence chitosanase, chito-oligosaccharides and their derivatives have attracted interest from the food and pharmaceutical industries because they can be used as edible additives, agricultural immunity controls and promise many other prospective applications as prophylactic agents for liver diseases (4), atherosclerosis, and hypertension.