Arabinofuranosidases are important accessory enzymes involved in the degradation of arabinose-containing poly- and oligosaccharides. Two arabinofuranosidases from the recently described novel anaerobic cellulolytic bacterium Acetivibrio mesophilus, designated AmAraf51 and AmAraf43, were heterologously expressed in Escherichia coli and biochemically characterized. AmAraf51 not only removed arabinose moieties at O-3, O-2 and terminal O-5 positions of arabinose-containing oligosaccharides, but also exhibited exo-β-xylosidase side activity. In comparison, AmAraf43 preferably cleaved 1,3-linkages from arabinosyl disubstitutions. AmAraf51 and AmAraf43 demonstrated maximum activity at 70 °C and 57 °C, respectively. Judging from the genetic context and substrate specificity, AmAraf51 may decompose internalized arabino/xylo-oligosaccharides. The embedding of the AmAraf43 gene between genes for several putative xylanolytic enzymes, along with its enzymatic properties suggests that AmAraf43 cleaves arabinose decorations from heteroxylans extracellularly. The enzymes revealed completely converse activity profiles towards arabinan/arabinoxylan: AmAraf51 displayed strong activity on arabinan, while AmAraf43 prefers arabinoxylan. AmAraf51 dramatically stimulated the saccharification level of wheat arabinoxylan (WAX-RS) and sugar beet arabinan when administered along with xylanase M_Xyn10 or arabinanase PpAbn43, respectively. For WAX-RS degradation, the yield of arabinose and xylose was boosted 13.77-fold and 4.96-fold, respectively. The bifunctional activity, thermostability and high catalytic efficiency make AmAraf51 an interesting candidate for industrial applications.

Background Plant cell walls represent the most plentiful renewable organic resource on earth, but due to their heterogeneity, complex structure and partial recalcitrance, their use as biotechnological feedstock is still limited. Results In order to identify efficient enzymes for polysaccharide breakdown, we have carried out functional screening of metagenomic fosmid libraries from biogas fermenter microbial communities grown on sugar beet pulp, an arabinan-rich agricultural residue, or other sources containing microbes that efficiently depolymerize polysaccharides, using CPH (chromogenic polysaccharide hydrogel) or ICB (insoluble chromogenic biomass) labeled polysaccharide substrates. Seventy-one depolymerase-encoding genes were identified from 55 active fosmid clones by using Illumina and Sanger sequencing and dbCAN CAZyme (carbohydrate-active enzyme) annotation. An around 56 kb assembled DNA fragment putatively originating from Xylanivirga thermophila strain or a close relative was analyzed in detail. It contained 48 ORFs (open reading frames), of which 31 were assigned to sugar metabolism. Interestingly, a large number of genes for enzymes putatively involved in degradation and utilization of arabinose-containing carbohydrates were found. Seven putative arabinosyl hydrolases from this DNA fragment belonging to glycoside hydrolase (GH) families GH51 and GH43 were biochemically characterized, revealing two with endo-arabinanase activity and four with exo-α-l-arabinofuranosidase activity but with complementary cleavage properties. These enzymes were found to act synergistically and can completely hydrolyze SBA (sugar beet arabinan) and DA (debranched arabinan). Conclusions We screened 32,776 fosmid clones from several metagenomic libraries with chromogenic lignocellulosic substrates for functional enzymes to advance the understanding about the saccharification of recalcitrant lignocellulose. Seven putative X. thermophila arabinosyl hydrolases were characterized for pectic substrate degradation. The arabinosyl hydrolases displayed maximum activity and significant long-term stability around 50 °C. The enzyme cocktails composed in this study fully degraded the arabinan substrates and thus could serve for arabinose production in food and biofuel industries.