EcXyl43 β-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction

Abstract

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 β-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of β-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented β-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction

“…Reducing sugars released from the reactions were measured by dinitrosalicylic acid (DNS) method (Miller 1959) using glucose or xylose standard curves. β-Glucosidase, cellobiohydrolase, β-xylosidase and α-larabinofuranosidase activities were assayed, using 5 mM pnitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), p-nitrophenyl-β-D-xylopyranoside (pNPX) and p-nitrophenyl-α-L-arabinofuranoside (pNPA) (Sigma-Aldrich, STL, USA) as substrates, according to previously established protocols (Ontañon et al 2018). In brief, reactions were performed by combining 100 μL of LEE (10×) or LIE fraction with 100 μL of 2.5 mM substrate in citrate buffer (pH 6, 100 mM), and incubating at 40°C for 20 min.…”

β-Xylosidases: Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides

“…The X19 domain, which is only found in a subset of GH43 subfamilies [61], appeared to be crucial for catalytic activity of the type III GH43 β-xylosidases, since removing this domain abolished the activity of the GH43_11 β-xylosidases from Thermobifida fusca YX [71] and Enterobacter sp. [72]. In fact, a loop from the X19 domain contributes a Phe residue to the active site of the type III β-xylosidases [66,67,68,70], which is spatially conserved among all GH43 β-xylosidase structures.…”

“…Finally, the β-xylosidase from Enterobacter sp. is competitively inhibited by l -arabinose, but with a quite high K i value of 102 mM [72], indicating that the enzyme has only low affinity for l -arabinose.…”

Research progress of fusion glycoside hydrolase in biomass conversion

“…利用糖苷水解酶(glycoside hydrolases, [1,7~10] . 例如, 微生物来源的一些β-木糖苷酶有2个结 构域: 一个是起催化作用的主要结构域, 另一个是具有 底物结合或其他糖苷水解酶活性的辅助结构域, 它们 的多功能特性能有效地实现异质木聚糖的降解 [11,12] .…”