Purification and characterization of exo-β-1,4-glucosaminidase produced by chitosan-degrading fungus, Penicillium sp. IB-37-2A

Chitosan-degrading fungal strain, Penicillium sp. IB-37-2A, produced mainly extracellular chitosanolytic enzymes under submerged agitating cultivation in presence of soluble chitosan or colloidal chitin as main carbon source. Significant N-acetyl--d-glucosaminidase activity (8-18x10(3) Uml(-1)) was also detected in culture filtrate of the fungal strain. Alone major exo-chitosanase from culture filtrate of Penicillium sp. IB-37-2A was purified in 46-fold using ultrafiltration, affinity sorption on colloidal chitosan and hydrophobic chromatography on Phenyl-Sepharose CL 4B and characterized. Molecular weight of the exo--1.4-glucosaminidase is 41kDa according to SDS-PAGE. The purified enzyme has optima pH and temperature 4.0 and 50-55 degrees C, respectively, pI 4.9; it is stable under pH 3.0-8.0 and 55 degrees C. Activity of the enzyme is strongly inhibited by 1mM Hg2+ and Ag+, in less degree10mM Cu2+, Zn2+, Ni+ and Fe2+, slightly activatedwith 1mM Mg2+, 10mM Ca2+, tween-80 (10mM) and Triton X-100 (1mM). Viscosimetric assay confirmed reported earlier exo-splitting manner of the enzyme activity. Soluble chitosan (deacetylation degree (DD) 80-85%) is most rapidly hydrolyzed by the enzyme (V-max=7.635 mu Mxmin(-1)xmg(-1), K-M similar to 0.83mg/ml). Purified exo-chitosanase also degraded laminarin, -glucan, colloidal chitin and showed significant chitobiohydrolase activity (V similar to 50 mu Mxml(-1)xmin(-1) for pNP-GlcNAc(2)) but no hydrolyzed CMC, cellulose, xylan and galactomannan. It is found that crude and partially purified exo--1.4-glucosaminidase inhibits in vitro the growth of some phytopathogenic fungi that is first report for antifungal activity of exo-chitosanase.