Identification and characterization of GH11 xylanase and GH43 xylosidase from the chytridiomycetous fungus, Rhizophlyctis rosea

Abstract

The early-lineage, aerobic, zoosporic fungi from the Chytridiomycota constitute less than 1% of the described fungi and can use diverse sources of nutrition from plant or animal products. One of the ancestral sources of fungal nutrition could be products following enzymatic degradation of plant material. However, carbohydrate-active enzymes from these ancient fungi have been less studied. A GH11 xylanase (RrXyn11A) (EC 3.2.1.8) and a GH43 xylosidase (RrXyl43A) (EC 3.2.1.37) were identified from an early-lineage aerobic zoosporic fungus, Rhizophlyctis rosea NBRC 105426. Both genes were heterologously expressed in Pichia pastoris and the recombinant enzymes were purified and characterized. The optimal pH for recombinant RrXyn11A and RrXyl43A was pH 7. RrXyn11A had high stability over a wide range of pH (4-8) and temperature (25-70 °C). RrXyn11A also showed high substrate specificity on both azurine-cross-linked (AZCL) arabinoxylan and AZCL xylan. RrXyl43A had β-xylosidase and minor α-L-arabinofuranosidase activity. This enzyme showed low product inhibition and retained 51% activity in the presence of 100 mM xylose. A combination of RrXyn11A and RrXyl43A exhibited significantly higher hydrolytic and polymer degradation capability and xylose release on wheat bran and beechwood xylan compared to treatment with commercial enzymes. This study was the first to heterologously express and characterize the GH11 xylanase (RrXyn11A) and GH43 xylosidase (RrXyl43A) from the ancient fungus, R. rosea. Meanwhile, this study also demonstrated that the enzymes from the ancient fungus R. rosea can be easily handled and heterologously expressed in Pichia, which presents a promising path to a new source of enzymes for biomass degradation.

Keywords: Chytridiomycota; GH11 xylanase; GH43 xylosidase; HotPep; Rhizophlyctis rosea.

Fig. 1

3D model structure analyses of xylanase and xylosidase from Rhizophlyctis rosea. 3D model structure of RrXyn11A (a) and a xylohexaose superimposed structure of RrXyn11A (b); 3D model structure of RrXyl43A (c), a xylobiose superimposed structure (d), and a Ara-(α1-3)Xyl superimposed structure of RrXyl43A (e). The amino acids of the active site are highlighted as yellow sticks. The peptides with highest frequency from HotPep analysis (RrXyn11A: DGGTYD, and RrXyl43A: GWTTHH) are highlighted in pink. The peptides with second highest frequency from HotPep analysis (RrXyn11A: QYWSVR, and RrXyl43A: WAPDAA) are highlighted in green. Ca²⁺ and Na⁺ ions are shown as green and purple spheres, respectively. Modeled or superimposed ligands are shown as cyan sticks. Residues involved in substrate binding are highlighted as sticks with the following colors: aromatic residues in magenta, protein backbone in purple, and residue sidechains in orange. Polar interactions between substrate and protein are indicated as yellow dotted lines

Fig. 2

Phylogenic analyses of GH11 xylanases and GH43 xylosidases. a Radial tree of GH11 xylanases including RrXyn11A from Rhizophlyctis rosea; b circular tree of GH43 xylosidases including RrXyl43A from Rhizophlyctis rosea. The outer ring is the taxonomy of the origins of each sequence (taxonomy color legend (left)); the inner ring is the CAZy subfamilies (subfamily color legend (right)) and EC number and CAZy subfamily number (b) of each sequence. The accession numbers of the sequences which were selected for the phylogenetic tree can be found in Supplemental Fig. S1 and S2

Fig. 3

SDS-PAGE and western blot analysis of purified recombinant RrXyn11A and RrXyl43A. a SDS-PAGE, lane 1: Precision Plus Protein™ Unstained Protein Standards; lane 2: RrXyn11A L (larger molecular weight); lane 3: RrXyn11A S (smaller molecular weight); and lane 4: RrXyl43A. b Western blot, lane 1: RrXyn11A L; lane 2: Endo H treated RrXyn11A L; lane 3: RrXyn11A S; lane 4: Endo H treated RrXyn11A S; lane 5: RrXyl43A; lane 6: Endo H treated RrXyl43A; lane 7: Precision Plus Protein™ Dual Color Standards

Fig. 4

Effects of temperature and pH on recombinant RrXyn11A and RrXyl43A activity and stability. a pH profile of RrXyn11A L, RrXyn11A S, and RrXyl43A; b pH stability of RrXyn11A L, RrXyn11A S, and RrXyl43A; c temperature profile of RrXyn11A L, RrXyn11A S, and RrXyl43A; d thermostability of RrXyn11A L, RrXyn11A S, and RrXyl43A

Fig. 5

Reducing sugar after enzyme hydrolysis of beechwood xylan, wheat bran, and corn bran. commercial_pul, commercial Pulmozyme HC. commercial_xyl, commercial 1.4-β-D-xylosidase. Statistical significance is indicated by capital letters

Fig. 6

Xylo-oligosaccharide concentration after enzyme hydrolysis of different substrates. Xylo-oligosaccharide concentration after monocomponent enzyme and combination of enzymes hydrolysis of wheat bran (a), beechwood xylan (b), and corn bran (c). commercial_pul, commercial Pulmozyme HC (Novozyme, Bagsvaerd, DK). commercial_xyl, commercial 1,4-β-d-xylosidase (Megazyme, Bray, IE). Statistical significance is indicated by capital letters