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

Abstract

Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAcGlcN 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 Asp 160 and Glu 169 are the essential residues for the action of A. fumigatus endo-chitosanase.Chitosanase (EC 3.2.1.132) is a hydrolytic enzyme acting on the ␤-1,4-glycosidic linkage of chitosan, a linear biopolymer of ␤-1,4-linked GlcN, to release chito-oligosaccharides. The oligomers GlcNAc and GlcN have interesting biological activities (1), including anti-tumor effects (2, 3), hypo-cholesterolemic effects (4), anti-microbial activities (5, 6), disease-resistance responses, and as phytoalexin elicitors in higher plants (7,8). Hence chitosanase, chito-oligosaccharides and their derivatives have attracted interest from the food and pharmaceutical industries because they can be used as edible additives, agricultural immunity controls and promise many other prospective applications as prophylactic agents for liver diseases (4), atherosclerosis, and hypertension.

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

“…In order to determine the number of putative chitosanase genes in the M. oryzae , the A. fumigatus Csn protein sequence (Genbank AAO41660.1; Cheng, Chang, Wu, & Li, 2006) was used as a Basic Local Alignment Search Tool (BLAST) query against the M. oryzae protein database (http://www.broadinstitute.org/annotation/genome/magnaporthe comparative/MultiHome.html). This identified just a single putative chitosanase (MGG07656).…”

“…were an exception, with four, as discovered previously (Cheng et al . , 2006). Thus, the distribution and number of chitosanases do not mirror that of the CDAs.…”

Cloning and Characterization of a Gene Coding for a Major Extracellular Chitosanase from the Koji MoldAspergillus oryzae

“…1). 9,10) CsnC had a unique domain at the Cterminus, which was joined to the catalytic domain by a linker rich in Thr and Pro residues (Fig. 1).…”

Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

“…Based on similarity of amino acid sequences, GH has been classified into 115 families [http://www.cazy.org/], among which GH-5 [Tanabe et al, 2003], -8 [Mitsutomi et al, 1998;Kimoto et al, 2002;Yatsunami et al, 2002;Choi et al, 2004;Kim et al, 2004;Ogura et al, 2006], -46 [Masson et al, 1994[Masson et al, , 1995Akiyama et al, 1999;Shimosaka et al, 2000;Yoon et al, 2000;Lee et al, 2006], -75 [Zhang et al, 2001;Shimosaka et al, 2005;Cheng et al, 2006] and -80 [Park et al, 1999;Yun et al, 2005] are known to have chitosanase activity. Founded on substrate specificity, these chitosanases have been further divided into 3 subclasses [Izume et al, 1992;Fukamizo et al, 1994Fukamizo et al, , 1995.…”