Characterization of Two α-l-Arabinofuranosidases from Acetivibrio mesophilus and Their Synergistic Effect in Degradation of Arabinose-Containing Substrates

Abstract

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.

Arabinan saccharification by biogas reactor metagenome-derived arabinosyl hydrolases

“…The activities of both enzymes against internally di-arabinosylated XA 2,3 XX were negligible. In comparison, with terminally double-substituted side chains in A 2,3 XX and internally di-arabinosyl side chains in AA 2,3 A, the enzymes displayed activity, which is similar to the GH51 isolated from A. mesophilus [ 52 ]. All these enzymes, including MC57GH51 and MC60GH51, did not show any activity with WAX, despite the presence of the same arabinosyl side-chain substitution in this substrate polymer.…”

“…The PCR amplicons were then cloned in NdeI / XhoI linearized pET24c vector by Gibson Assembly (New England Biolabs, Massachusetts, USA). Transformation and induction, purification and quantification of the recombinant proteins were performed following the same procedures as described in our earlier study [ 52 ], except a lower concentration of IPTG of 0.05 mM and a shorter incubation time of 6 h at 30 °C was applied to protein MC60GH51 and MC68GH43-2 induction to avoid inclusion body formation.…”

“…The enzyme activity was measured with p -nitrophenyl-α- l -arabinofuranoside ( p NP-AF, Megazyme), various polysaccharide substrates (Megazyme, Wicklow, Ireland), including insoluble wheat arabinoxylan, arabinoxylan for reducing sugar assay (both from wheat flour), sugar beet arabinan (SBA; monomer composition: arabinose: galactose: rhamnose: galacturonic acid: other sugar = 69: 18.7: 1.4: 10.2: 0.7), and debranched arabinan (DA; monomer composition: arabinose: galactose: rhamnose = 71: 26: 3), and oligosaccharides (Megazyme, Wicklow, Ireland), including O-ATR (Arabinotriose), O-A4B (3 2 -α- l -arabinofuranosyl-(1,5)-α- l -arabinotriose), O-A5B MIX (2 2 ,3 2 -di-α- l -arabinofuranosyl-(1,5)-α- l -arabinotriose plus 3 2 -α- l -arabinofuranosyl-(1,5)-α- l -arabinotetraose, O-AX3 (2 3 -α- l -arabinofuranosyl-xylotriose), O-XAXX MIX (3 3 -α- l - plus 2 3 -α- l -arabinofuranosyl-xylotetraose (XA 3 XX/XA 2 XX) mixture), O-XA3XX (3 3 -α- l -arabinofuranosyl-xylotetraose), O-A2X3 (2 2 , 3 2 -di-α- l -arabinofuranosyl-xylotriose), O-XA23XX (2 2 , 3 2 -di-α- l -arabinofuranosyl-xylotetraose) (the nomenclature and the structure of oligosaccharides are guided by Fauré et al . and Liu et al) [ 52 , 62 ]. All insoluble substrates were washed with Milli-Q water to remove the soluble reducing sugars before starting the enzyme reaction.…”

“…To determine which products appeared after cleavage of SBA and DA, reactions were carried out by incubating the different enzymes with either SBA or DA or both substrates in 25 mM citrate–phosphate buffer at optimal temperature and pH for different time spans (0 h, 0.5 h, 1.5 h, 4 h, 8 h, 20 h). Thin-layer chromatography (TLC) was used for the determination of hydrolysis products as described in our previously study [ 52 ].…”

Biochemical and Molecular Dynamics Study of a Novel GH 43 α-l-Arabinofuranosidase/β-Xylosidase From Caldicellulosiruptor saccharolyticus DSM8903

“…The first chain of this heteropolysaccharide is created from β-1,4-linked D-xylopyranosyl sugar with L-arabinose scattered randomly. Furthermore, the backbone of arabinan and arabinogalactan consists of a linear 1,5-linked L-arabinofuranosyl polymer and 1,6-glycosidically connected D-galactopyranose residues, respectively (Liu et al, 2021).…”