Synergistic mechanism of GH11 xylanases with different action modes from Aspergillus niger An76

Abstract

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.

Functional Specificity of Three α-Arabinofuranosidases from Different Glycoside Hydrolase Families in Aspergillus niger An76

“…Quantitative Real-Time Polymerase Chain Reaction (qPCR) Analysis of Gene Expression. The induction culture conditions of the A. niger An76 strain, RNA extraction, and cDNA synthesis were as described by Zhang et al 19 A 1% (w/v) concentration of different individual carbon sources, including glycerol, xylose, arabinose, XOS, BX, or WAX, was added to the culture medium to induce the production of enzymes. Target genes in the genome of A. niger An76 included five candidate genes encoding Abfs (axhA, g1234.t1; abfA, g10075.t1; abf B, g3885.t1; abf C, g4219.t1; and abf D, g4234.t1).…”

“…XynB from A. niger An76 was produced as previously described. 19 An aliquot (400 μL) of diluted enzyme preparation (XynB, AxhA, AbfB, AbfC) alone or in combination (XynB + AxhA, XynB + AxhA + AbfB, XynB + AxhA + AbfC, XynB + AbfB + AbfC, XynB + AbfB + AbfC, XynB + AxhA + AbfB + AbfC) was mixed with 600 μL of 1% (w/v) WAX, and 100 μL of the reaction mixture was taken out after 10 and 30 min. XynB, AxhA, AbfB, and AbfC were diluted to 0.0125, 0.75 μg/mL, 0.025, and 0.025 mg/mL, respectively.…”

“…The induction culture conditions of the A. niger An76 strain, RNA extraction, and cDNA synthesis were as described by Zhang et al A 1% (w/v) concentration of different individual carbon sources, including glycerol, xylose, arabinose, XOS, BX, or WAX, was added to the culture medium to induce the production of enzymes. Target genes in the genome of A.…”

“…18 Among these enzymes, the biochemical properties and synergistic pattern of key xylanases have also been elucidated. 19 However, Abfs remove arabinose decorations to help xylanase to better convert xylan to xyoligosaccharides in the degradation process of arabinoxylan. 8 Genes encoding several α-Larabinofuranosidases from different GH families have been detected in the genome of A. niger An76.…”

“…In our previous work, Aspergillus niger An76 was shown to possess a comprehensive xylan-degrading enzyme system, in which both xylanases and debranching enzymes were secreted in an orderly manner . Among these enzymes, the biochemical properties and synergistic pattern of key xylanases have also been elucidated . However, Abfs remove arabinose decorations to help xylanase to better convert xylan to xyoligosaccharides in the degradation process of arabinoxylan .…”