Biochemical characterization of Aspergillus niger CfcI, a glycoside hydrolase family 18 chitinase that releases monomers during substrate hydrolysis

Abstract

The genome of the industrially important fungus Aspergillus niger encodes a large number of glycoside hydrolase family 18 members annotated as chitinases. We identified one of these putative chitinases, CfcI, as a representative of a distinct phylogenetic clade of homologous enzymes conserved in all sequenced Aspergillus species. Where the catalytic domain of more distantly related chitinases consists of a triosephosphate isomerase barrel in which a small additional (a+b) domain is inserted, CfcI-like proteins were found to have, in addition, a carbohydrate-binding module (CBM18) that is inserted in the (a+b) domain next to the substratebinding cleft. This unusual domain structure and sequence dissimilarity to previously characterized chitinases suggest that CfcI has a novel activity or function different from chitinases investigated so far. Following its heterologous expression and purification, its biochemical characterization showed that CfcI displays optimal activity at pH 4 and 55-65 6C and degrades chitin oligosaccharides by releasing N-acetylglucosamine from the reducing end, possibly via a processive mechanism. This is the first fungal family 18 exochitinase described, to our knowledge, that exclusively releases monomers. The cfcI expression profile suggests that its physiological function is important in processes that take place during the late stages of the aspergillus life cycle, such as autolysis or sporulation. INTRODUCTIONAspergillus niger is a filamentous ascomycete of significant industrial importance. This fungus is the main producer of citric acid and is used as an expression host for both heterologously and homologously (over)expressed extracellular enzymes (Schuster et al., 2002). The genome sequences of two A. niger strains each revealed the presence of 16 genes encoding putative chitin-degrading enzymes (Andersen et al., 2011;Pel et al., 2007). Chitinolytic enzymes are of major physiological importance in their fungal hosts. They are involved in the degradation of chitin substrates (Lopez-Mondéjar et al., 2009) and may become expressed during mycoparasitism as discussed by Seidl (2008). In addition, chitinases are thought to modify chitin present as a structural component in the fungal cell wall and thereby play a role during morphological changes such as autolysis, hyphal branching or germination of conidia (Shin et al., 2009;Yamazaki et al., 2008).The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology distinguishes two chitinolytic activities (IUBMB, 1992). The b-N-acetylhexosaminidases (EC 3.2.1.52) release N-acetyl-D-hexosamine residues from the non-reducing terminus of N-acetyl-b-D-hexosaminides, such as chitin oligosaccharides. All fungal enzymes with this activity belong to family 20 of the sequence-and fold-based glycoside hydrolase (GH) families, as described in the CAZy database (Cantarel et al., 2009). Chitinases (EC 3.2.1.14) randomly hydrolyse b(1,4) linkages between N-acetyl-b-D-glucosamines in chitin. Fungal chitinases belong to GH18. Me...

In Vitro Analyses of Mild Heat Stress in Combination with Antifungal Agents against Aspergillus fumigatus Biofilm

“…The release of eDNA (which is genomic DNA coming from autolysis) of ECM in A. fumigatus biofilms was lower with higher-temperature treatment but phase dependent (release at 8 h Ͻ at 12 h Ͻ at 24 h Ͻ at 48 h) in another study (47). The reason for this situation may be that long-term mild heat stress will force chitinase, a regulator of autolysis (51), to lose its activity (52). The evidence shown that only 75% Ϯ 4%, 34% Ϯ 1%, and 0% chitinase activities were retained, respectively, after 30 min at 40, 50, and 60°C, although the activity can reach its maximum at 55 to 65°C (52).…”

“…The reason for this situation may be that long-term mild heat stress will force chitinase, a regulator of autolysis (51), to lose its activity (52). The evidence shown that only 75% Ϯ 4%, 34% Ϯ 1%, and 0% chitinase activities were retained, respectively, after 30 min at 40, 50, and 60°C, although the activity can reach its maximum at 55 to 65°C (52). Thus, we presume that increasing the temperature would decrease the activity of chitinase and the release of eDNA, which may increase susceptibility of A. fumigatus biofilms to antifungal drugs.…”

Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides

“…U mg -1 and 2.1 mg protein per liter culture reported previously for CfcI-MBP 17 . Kinetic parameters of CfcI were determined with three substrates using the validated HPAEC-PAD method.…”

“…One of these chitinases, CfcI, is a representative of a phylogenetic cluster conserved within Aspergilli. We recently showed that CfcI differs from previously characterized fungal family GH18 members in displaying a novel reaction specificity: CfcI releases GlcNAc monomers from the reducing end of COS 17 .…”

Genetic and Genomic Manipulations in Aspergillus niger

“…Among the 14 genes encoding GH18 proteins in the A. niger genome, the product of cfc1 was singled out for characterization because it and its close orthologues in other Aspergillus species occupy a distinct clade in the phylogenetic tree of GH18 chitinases. Biochemical analysis of Cfc1 revealed that it is an exochitinase that releases monomers of N -acetylglucosamine from the reducing end of chitin oligosaccharides (van Munster et al 2012 ). The vmaD gene was identifi ed by sequence similarity to the subunit of the eukaryotic vacuolar-H(+)-ATPase.…”