Background Xylan is the most abundant hemicellulose polysaccharide in nature, which can be converted into high value-added products. However, its recalcitrance to breakdown requires the synergistic action of multiple enzymes. Aspergillus niger, possessing numerous xylan degrading isozyme-encoding genes, are highly effective xylan degraders in xylan-rich habitats. Therefore, it is necessary to explore gene transcription, the mode of action and cooperation mechanism of different xylanase isozymes to further understand the efficient xylan-degradation by A. niger. Results Aspergillus niger An76 encoded a comprehensive set of xylan-degrading enzymes, including five endo-xylanases (one GH10 and four GH11). Quantitative transcriptional analysis showed that three xylanase genes (xynA, xynB and xynC) were up-regulated by xylan substrates, and the order and amount of enzyme secretion differed. Specifically, GH11 xylanases XynA and XynB were initially secreted successively, followed by GH10 xylanase XynC. Biochemical analyses displayed that three GH11 xylanases (XynA, XynB and XynD) showed differences in catalytic performance and product profiles, possibly because of intricate hydrogen bonding between substrates and functional residues in the active site architectures impacted their binding capacity. Among these, XynB had the best performance in the degradation of xylan and XynE had no catalytic activity. Furthermore, XynA and XynB showed synergistic effects during xylan degradation. Conclusions The sequential secretion and different action modes of GH11 xylanases were essential for the efficient xylan degradation by A. niger An76. The elucidation of the degradation mechanisms of these xylanase isozymes further improved our understanding of GH-encoding genes amplification in filamentous fungi and may guide the design of the optimal enzyme cocktails in industrial applications.

Background: Xylan is the most abundant hemicellulose polysaccharide in nature. Endo-xylanases from GH10 and GH11 families are the most critical xylan degrading enzymes. Filamentous fungi are highly effective xylan degraders and possess numerous xylan degrading isoenzyme-encoding genes, especially Aspergillus niger. Most noteworthy, the amplification of the GH11 xylanase-encoding genes occurs frequently in an organism, but the knowledge of each GH11 xylanases is little known. Results: A. niger An76 encoded a comprehensive set of xylan-degrading enzymes, including five endo-xylanases (one GH10 and four GH11). Quantitative transcriptional analysis showed that three xylanases were up-regulated by xylose substrates, and the order and amount of enzyme secretion differed. Specifically, XynA and XynB were initially secreted successively, followed by XynC. Structural bioinformatics analysis indicated that the different modes of action of the three GH11 xylanases may be due to intricate hydrogen bonding between substrates and functional residues in the active site architectures. Heterologous expression and biochemical characterization of three GH11 xylanases (XynA, XynB and XynD) revealed differences in catalytic performance and product profiles. Furthermore, XynA and XynB displayed obvious synergistic action against beechwood xylan. Conclusions: We investigated subtle differences in the functions of different isoenzymes in the same family using a combination of physiological and biochemical experiments. The transcriptional regulation and catalytic functions of enzymes could be the result of long-term evolutionary adaptation. The finding further expanded our understanding of GH-encoding genes amplification in filamentous fungi, which could guide the design of the optimal enzyme cocktails in industrial applications.