Two Distinct α-l-Arabinofuranosidases in Caldicellulosiruptor Species Drive Degradation of Arabinose-Based Polysaccharides

Abstract

Species in the extremely thermophilic genus Caldicellulosiruptor can degrade unpretreated plant biomass through the action of multimodular glycoside hydrolases. To date, most focus with these bacteria has been on hydrolysis of glucans and xylans, while the biodegradation mechanism for arabinose-based polysaccharides remains unclear. Here, putative α-l-arabinofuranosidases (AbFs) were identified in Caldicellulosiruptor species by homology to less-thermophilic versions of these enzymes. From this screen, an extracellular XynF was determined to be a key factor in hydrolyzing α-1,2-, α-1,3-, and α-1,5-l-arabinofuranosyl residues of arabinose-based polysaccharides. Combined with a GH11 xylanase (XynA), XynF increased arabinoxylan hydrolysis more than 6-fold compared to the level seen with XynA alone, likely the result of XynF removing arabinofuranosyl side chains to generate linear xylans that were readily degraded. A second AbF, the intracellular AbF51, preferentially cleaved the α-1,5-l-arabinofuranosyl glycoside bonds within sugar beet arabinan. β-Xylosidases, such as GH39 Xyl39B, facilitated the hydrolysis of arabinofuranosyl residues at the nonreducing terminus of the arabinose-branched xylo-oligosaccharides by AbF51. These results demonstrate the separate but complementary contributions of extracellular XynF and cytosolic AbF51 in processing the bioconversion of arabinose-containing oligosaccharides to fermentable monosaccharides.IMPORTANCE Degradation of hemicellulose, due to its complex chemical structure, presents a major challenge during bioconversion of lignocellulosic biomass to biobased fuels and chemicals. Degradation of arabinose-containing polysaccharides, in particular, can be a key bottleneck in this process. Among Caldicellulosiruptor species, the multimodular arabinofuranosidase XynF is present in only selected members of this genus. This enzyme exhibited high hydrolysis activity, broad specificity, and strong synergism with other hemicellulases acting on arabino-polysaccharides. An intracellular arabinofuranosidase, AbF51, occurs in all Caldicellulosiruptor species and, in conjunction with xylosidases, processes the bioconversion of arabinose-branched oligosaccharides to fermentable monosaccharides. Taken together, the data suggest that plant biomass degradation in Caldicellulosiruptor species involves extracellular XynF that acts synergistically with other hemicellulases to digest arabino-polysaccharides that are subsequently transported and degraded further by intracellular AbF51 to produce short-chain arabino sugars.

Keywords: Arabinofuranosidase; bioenergy; glycoside hydrolase; hyperthermophiles; synergism.

FIG 1

Activities of endo-xylanases against beechwood xylan and wheat arabinoxylan. (a) Chemical structures of beechwood xylan and wheat arabinoxylan. (b) Specific activities of XynA, XynA-TM, and XynB on beechwood xylan and wheat arabinoxylan. (c) Time course of hydrolysis of beechwood xylan and wheat arabinoxylan by an endoxylanase XynA. (d) Time course of hydrolysis of beechwood xylan and wheat arabinoxylan by XynB. All the reactions were performed at pH 6 and 70°C in citrate buffer.

FIG 2

Schematic structures and genus-wide screening of the putative AbFs in Caldicellulosiruptor species. (a) Schematic diagram of two GH3s, five GH43s, one AbF51, a multimodular XynF enzyme, and two truncated mutants of XynF. (b) SDS-PAGE analysis of two GH3s, five GH43s, and one AbF51 from Caldicellulosiruptor sp. strain F32. (c) Heterologous recombination of a full-length XynF in E. coli by high-resolution mass spectroscopy-guided mutagenesis. (d) Specific activities of AbF51 and XynF on the different substrates.

FIG 3

Biochemical characterizations of XynF and AbF51. (a) Effect of pH and temperature on the activities of XynF. For optimum pH conditions, citrate buffer was used at pH 4 to 6 (black line) and phosphate buffer at pH 6 to 8 (red line). (b) Thermostability of XynF at different temperatures. (c) Effect of pH and temperature on the activities of AbF51. (d) Thermostability of AbF51 at different temperatures. Wheat arabinoxylan and pNP-AraF were used as substrates for XynF and AbF51, respectively. The kinetic parameters of XynF and AbF51 were examined under optimum pH and temperature conditions.

FIG 4

Synergistic effects of AbF51 with three endo-xylanases. A series of enzyme combinations (molar ratio) were used. All the reactions were performed at 70°C against wheat arabinoxylan (0.8%) for three time durations (1 h, 12 h, and 24 h). Reducing sugars were measured by the DNS method. (a) Reducing sugars obtained by AbF51 singly or in combination with XynA. (b) Sugar released by the combined activities of AbF51 and XynA-TM. (c) Sugar released by the combined activities of AbF51 and XynB. (d) Degree of synergy obtained by coupling the action of AbF51 with that of the endoxylanases (separately) for three time durations. Degree of synergy is defined as the ratio of the amounts of saccharides released from the simultaneous activities to the sum of that released by the individual enzyme.

FIG 5

Synergistic effects of XynF with three endo-xylanases (XynA, XynA-TM, and XynB). A series of enzyme combinations (molar ratio) were used. All the reactions were performed as described for Fig. 4. (a) Reducing sugars obtained by XynF singly or in combination with XynA. (b) Sugar released by the combined activities of XynF and XynA-TM. (c) Sugar released by the combined activities of XynF and XynB. (d) Degree of synergy obtained by a coupled action of XynF with the endoxylanases (separately) for three time durations. Degree of synergy is defined as the ratio of the amounts of saccharides released from the simultaneous activities to the sum of that released by the individual enzyme.

FIG 6

Hydrolysis actions of two AbFs on arabinose-based polysaccharides. (a) The natural substrates WAX, SA, and DSA were incubated with AbF51 or XynF for 12 h at 70°C. Hydrolysis products were analyzed by HPAEC-PAD. A, X1, X2, X3, and X4 refer to arabinose, xylose, xylobiose, xylotriose, and xylotetrose, respectively. Std, standard. (b) The chemical structures of SA and DSA. (c) The amounts of arabinose released by AbF51 and XynF on three substrates according to the signal areas of arabinose shown in panel a. AbF51 participant hydrolysis products were diluted 50-fold, and XynF participant products were diluted 200-fold with distilled water. Conc., concentration.

FIG 7

Hydrolysis actions of two AbFs on arabinose-branched XOSs. (a and b) WAX (1%) was hydrolyzed by XynA for 12 h at 70°C. The products were examined by HPAEC-PAD (a) and ¹H-NMR (b). (c and d) WAX (1%) was hydrolyzed by XynA for 12 h followed by AbF51 for 12 h at 70°C. The products were examined by HPAEC-PAD (c) and ¹H-NMR (d). (e and f) WAX (1%) was hydrolyzed by XynA for 12 h followed by XynF for 12 h at 70°C on HPAEC-PAD (e) and ¹H-NMR (f). A, X1, X2, X3, and X4 refer to arabinose, xylose, xylobiose, xylotriose, and xylotetrose, respectively. The signals of 2-O-linked (δ 5.28) and 3-O-linked (δ 5.33) arabinoses were assigned.

FIG 8

Combinational actions of AbF with other hemicellulases on hydrolyzing WAX. WAX (1%) was hydrolyzed by different enzyme combinations, including XynA alone (a), XynA/AbF51 (b), XynA/AbF51/Xyl39B (c), XynA/XynF (d), XynA/XynF/Xyl39B (e), and XynA/XynF/AbF51/Xyl39B (f), for 24 h and then by XynA/XynF for 24 h followed by AbF51/Xyl39B for another 24 h (g). The products were examined by HPAEC-PAD (left panel). The amounts of released sugars were calculated (right panel) according to standard curves of xylo-oligosaccharides. AbF51 participant hydrolysis products were diluted 50-fold, and XynF participant products were diluted 200-fold with distilled water.

FIG 9

A model in Caldicellulosiruptor species for two AbFs involving arabinose-based polysaccharide degradation. (a) A linear route of xylan degradation by xylanases and xylosidases. (b) An interrogative route of arabinoxylan degradation by intracellular AbF51. (c) A route of arabinoxylan degradation by extracellular XynF in Caldicellulosiruptor species. (d) Intracellular route of degradation involving AbF51 on arabinose-containing oligosaccharides.

FIG 10

Phylogenetic tree of XynF-like enzymes in bacteria. An alignment of NCBI sequences was performed using clustalx. A maximum likelihood nearest-neighbor interchange tree was then constructed in MEGA 5.1 and tested with 500 bootstrap replicates. Bootstrap values are listed near each branch. RefSeq numbers are listed in parentheses.