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J. Biol. Chem., Vol. 281, Issue 6, 3137-3144, February 10, 2006

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Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus^*

From the Center for Interdisciplinary Molecular Science and Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan

A powerful endo-chitosanase (CSN) previously described for a large scale preparation of chito-oligosaccharides (Cheng, C.-Y., and Li, Y.-K. (2000) Biotechnol. Appl. Biochem. 32, 197–203) was cloned from Aspergillus fumigatus and further identified as a member of glycosyl hydrolase family 75. We report here a study of gene expression, functional characterization, and mutation analysis of this enzyme. Gene cloning was accomplished by reverse transcription-PCR and inverse PCR. Within the 1382-bp Aspergillus gene (GenBank^TM accession number AY190324 [GenBank] ), two introns (67 and 82 bp) and an open reading frame encoding a 238-residue protein containing a 17-residue signal peptide were characterized. The recombinant mature protein was overexpressed as an inclusion body in Escherichia coli, rescued by treatment with 5 M urea, and subsequently purified by cation exchange chromatography. A time course ¹H NMR study on the enzymatic formation of chito-oligosaccharides confirmed that this A. fumigatus CSN is an inverting enzyme. Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAc-GlcN and GlcN-GlcN in its substrate, suggesting that it is a subclass I chitosanase. In addition, an extensive site-directed mutagenesis study on 10 conserved carboxylic amino acids of glycosyl hydrolase family 75 was performed. This showed that among these various mutants, D160N and E169Q lost nearly all activity. Further investigation using circular dichroism measurements of D160N, E169Q, wild-type CSN, and other active mutants showed similar spectra, indicating that the loss of enzymatic activity in D160N and E169Q was not because of changes in protein structure but was caused by loss of the catalytic essential residue. We conclude that Asp¹⁶⁰ and Glu¹⁶⁹ are the essential residues for the action of A. fumigatus endo-chitosanase.

Received for publication, November 22, 2005

^* This work was supported by the National Science Council of Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hseh Rd., Hsinchu 30030, Taiwan. Tel.: 886-3-5731985; Fax: 886-3-5723764; E-mail: ykl{at}cc.nctu.edu.tw.

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