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 (Vmax = 7.635 µM × min−1 × mg−1, KM ~ 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.

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Acknowledgements

The authors with appreciation acknowledge the partial financial support provided by the Russian Science Foundation (Grant No. 16-14-00046).

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Correspondence to Gleb E. Aktuganov.

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Aktuganov, G.E., Galimzianova, N.F., Gilvanova, E.A. et al. Purification and characterization of exo-β-1,4-glucosaminidase produced by chitosan-degrading fungus, Penicillium sp. IB-37-2A. World J Microbiol Biotechnol 35, 18 (2019). https://doi.org/10.1007/s11274-019-2590-4

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Keywords

  • Exo-β-1.4-glucosaminidase
  • Chitosanase
  • N-acetyl-β-d-glucosaminidase
  • Penicillium sp. IB-37-2A
  • Antifungal activity