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Appl Environ Microbiol, March 1998, p. 890-895, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Purification and Characterization of Exo-beta -D-Glucosaminidase from a Cellulolytic Fungus, Trichoderma reesei PC-3-7

Masahiro Nogawa, Hiroya Takahashi, Aya Kashiwagi, Kenji Ohshima, Hirofumi Okada, and Yasushi Morikawa*

Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan

Received 17 July 1997/Accepted 23 December 1997

Chitosan-degrading activities induced by glucosamine (GlcN) or N-acetylglucosamine (GlcNAc) were found in a culture filtrate of Trichoderma reesei PC-3-7. One of the chitosan-degrading enzymes was purified to homogeneity by precipitation with ammonium sulfate followed by anion-exchange and hydrophobic-interaction chromatographies. The enzyme was monomeric, and its molecular mass was 93 kDa. The optimum pH and temperature of the enzyme were 4.0 and 50°C, respectively. The activity was stable in the pH range 6.0 to 9.0 and at a temperature below 50°C. Reaction product analysis from the viscosimetric assay and thin-layer chromatography and 1H nuclear magnetic resonance spectroscopy clearly indicated that the enzyme was an exo-type chitosanase, exo-beta -D-glucosaminidase, that releases GlcN from the nonreducing end of the chitosan chain. 1H nuclear magnetic resonance spectroscopy also showed that the exo-beta -D-glucosaminidase produced a beta -form of GlcN, demonstrating that the enzyme is a retaining glycanase. Time-dependent liberation of the reducing sugar from partially acetylated chitosan with exo-beta -D-glucosaminidase and the partially purified exo-beta -D-N-acetylglucosaminidase from T. reesei PC-3-7 suggested that the exo-beta -D-glucosaminidase cleaves the glycosidic link of either GlcN-beta (1right-arrow4)-GlcN or GlcN-beta (1right-arrow4)-GlcNAc.

* Corresponding author. Mailing address: Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan. Phone: 81-258-479407. Fax: 81-258-479400. E-mail: yasushi{at}nagaokaut.ac.jp.




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