Purification and characterization of chitosanase and exo-beta-D-glucosaminidase from a Koji Mold, Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillu s oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kD a in molecular masses, were purified from the culture fluid of A. oryzae IA M2660. Viscosimetric assay and an analysis of reaction products by thin-lay er chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, design ated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers la rger than pentamer, glycol chitosan, and chitosan with a low degree of acet ylation (0-30%). The 135-kDa enzyme, named exo-beta-D-glucosaminidase, rele ased a single GlcN residue from the GlcN oligomers and chitosan, but did no t release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.