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

Abstract

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-β-D-glucosaminidase activity (8-18 × 10³ U·ml^-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-β-1.4-glucosaminidase is 41 kDa according to SDS-PAGE. The purified enzyme has optima pH and temperature 4.0 and 50-55 °C, respectively, pI 4.9; it is stable under pH 3.0-8.0 and 55 °C. Activity of the enzyme is strongly inhibited by 1 mM Hg²⁺ and Ag⁺, in less degree-10 mM Cu²⁺, Zn²⁺, Ni⁺ and Fe²⁺, slightly activated-with 1 mM Mg²⁺, 10 mM Ca²⁺, tween-80 (10 mM) and Triton X-100 (1 mM). 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 µM × min^-1 × mg^-1, K_M ~ 0.83 mg/ml). Purified exo-chitosanase also degraded laminarin, β-glucan, colloidal chitin and showed significant chitobiohydrolase activity (V ~ 50 µM × ml^-1 × min^-1 for pNP-GlcNAc₂) but no hydrolyzed CMC, cellulose, xylan and galactomannan. It is found that crude and partially purified exo-β-1.4-glucosaminidase inhibits in vitro the growth of some phytopathogenic fungi that is first report for antifungal activity of exo-chitosanase.

Keywords: Antifungal activity; Chitosanase; Exo-β-1.4-glucosaminidase; N-acetyl-β-D-glucosaminidase; Penicillium sp. IB-37-2A.