Abstract
BackgroundEndo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential.ResultsA HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 °C and 80 °C, with an optimum activity at 60 °C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 °C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides.ConclusionsThe G. thermodenitrificans T12 endo-xylanase, GtXynA1, shows temperature tolerance up to 80 °C and high activity to a variety of xylans. The mode of action of GtXynA1 reveals that arabinose substituents do not hamper substrate degradation by GtXynA1. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates.
Highlights
Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates
We describe the cloning and overproduction in E.coli of GtXynA1, a GH10 family endo-xylanase isolated from Geobacillus thermodenitrificans
The endo-xylanase was purified to apparent homogeneity, as it appeared as a 50 kDa single band on SDS-PAGE after visualization with PageBlue stain
Summary
Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. The most well characterized HUS locus belongs to G. stearothermophilus T-6 and its proposed degradation mechanism is based on a limited number of secreted hydrolytic enzymes and further processing of the generated degradation products intracellularly [3]. One of these secreted hydrolytic enzymes is an endo-xylanase that cleaves the xylan backbone of hemicellulose into shorter oligosaccharides [3, 4]. Substrates from grasses and annual plants are rich in arabinoxylans, whereas hardwoods mostly consist of (4-O-methyl)-α-D-glucuronoxylan This variety of substitutions requires multiple enzymes acting in synergy to fully degrade the xylans. Endo-(1 → 4)-ß-D-xylanases (E.C. 3.2.1.8) hydrolyse the ß-(1 → 4)-xylosidic bonds to release xylose and (substituted) xylo-oligosaccharides of various lengths that can be degraded further by other hemicellulases
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