The Glycoside Hydrolase Family 8 Reducing-End Xylose-Releasing Exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 Is Active on Branched Xylooligosaccharides.

A GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylose-releasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a Km of 1.64 ± 0.03 mM and a kcat value of 118.8 s(-1) Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (α/α)6 barrel fold where the loops connecting the α-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a -2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts. A GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report provides original and useful information on the properties of a new example of the rarely studied Rex enzymes. Depolymerization of highly substituted xylans is crucial for biomass valorization as a platform for generation of biofuels, chemicals, and solvents.

Valenzuela SV ，Lopez S ，Biely P ，Sanz-Aparicio J ，Pastor FI ... -  《-》

Structural analysis of the reducing-end xylose-releasing exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 deciphers its molecular specificity.

Reducing-end xylose-releasing exo-oligoxylanases (Rex) are GH8 enzymes that depolymerize xylooligosaccharides complementing xylan degradation by endoxylanases in an exo manner. We have studied Paenibacillus barcinonensis Rex8A and showed the release of xylose from xylooligomers decorated with methylglucuronic acid (UXOS) or with arabinose (AXOS). This gives the enzyme a distinctive trait among known Rex, which show activity only on linear xylooligosaccharides. The structure of the enzyme has been solved by X-ray crystallography showing a (α/α)6 folding common to GH8 enzymes. Analysis of inactived Rex8A-E70A complexed with xylotetraose revealed the existence of at least four binding subsites in Rex8A, with the oligosaccharide occupying subsites -3 to +1. The enzyme shows an extended Leu320-His321-Pro322 loop, common to other Rex, which blocks the binding of longer substrates to positive subsites further than +1 and seems responsible for the lack or diminished activity of Rex enzymes on xylan. Mutants with smaller residues in this loop failed to increase Rex8A activity on the polymer. Analysis of the complexes with AXOS showed the accommodation of arabinose at subsite -2, which cannot be allocated at subsite -1. Arabinose substitutions at the xylose O2 or O3 are accommodated by hydrophobic interaction and seem tolerated rather than recognized by Rex8A. A strained binding of the branch is facilitated by the lack of direct polar interactions of the xylose occupying this subsite, its water-mediated links allowing some conformational flexibility of the sugar. The plasticity of Rex8A is a notable property of the enzyme for its application in xylan deconstruction and upgrading. DATABASE: Structural data are available in PDB database under the accession numbers 6SRD (native form), 6TPP (E70A mutant in complex with EDO), 6TOW (E70A in complex with Xyl4), 6SUD (L320A mutant in complex with xylose), 6SHY (L320A/H321S double mutant in complex with EDO), 6TO0 (E70A in complex with AX3), and 6TRH (E70A in complex with AX4).

Jiménez-Ortega E ，Valenzuela S ，Ramírez-Escudero M ，Pastor FJ ，Sanz-Aparicio J ... -  《-》

A novel reducing-end xylose-releasing exo-oligoxylanase (PphRex8A) from Paenibacillus physcomitrellae XB.

In order to better understand the function of a putative GH8 xylanase gene in the xylan-degrading bacterium Paenibacillus physcomitrellae XB, a novel reducing-end xylose-releasing exo-oligoxylanase (Rex) PphRex8A of GH8 family was characterized. Phylogenetic analysis showed that it was clustered tightly with other published GH8 Rexs and exhibited the highest amino acid sequence identity (77.4 %) with the Rex of PbRex8 from P. barengoltzii G22. The three-dimensional (3D) structure of PphRex8A was also built based on the template of PbRex8 (5YXT) and three conserved catalytic active sites (Glu71, Asp263, and Asp129) were predicted and further confirmed by the enzymatic inactivity of their mutants (E71A, D129A, and D263A). The hydrolysis assay of PphRex8A showed that it could hydrolyze xylo-oligosaccharides (XOSs) with a degree of polymerization (DP) ≥ 3, such as xylotriose (X3) through xylohexaose (X6), and some natural XOSs, such as corncob xylan (CCX) and oat spelt xylan (OSX) to release xylose. However, it could not hydrolyze p-nitrophenyl-β-D-xylopyranoside (pNPX), which suggested that it mainly released xylose from the reducing-end of XOSs and belonged to a Rex. In addition, PphRex8A also could deconstruct xylans with high DP, such as wheat arabinoxylan (AX) and beech wood xylan (BWX) to produce XOSs with DP3-6. Moreover, PphRex8A had synergistic effects with other xylanolytic enzymes of P. physcomitrellae XB, such as with PphXyn10 or PphXyn11 at a ratio of 1:3, or with PphXyl43B as a ratio of 3:1, significantly increasing the amounts of reducing sugars toward different xylan substrates. Thus, PphRex8A could be an exo-xylanase toward XOSs and could improve the deconstruction capability of high DP xylans, thereby complementing other xylanolytic enzymes to contribute to xylan degradation and improve the efficiency of converting hemicellulose biomass into energy by P. physcomitrellae XB.

Wang L ，Zhang XJ ，Li YH 《-》

The effect of an oligosaccharide reducing-end xylanase, BhRex8A, on the synergistic degradation of xylan backbones by an optimised xylanolytic enzyme cocktail.

Xylan, the most abundant hemicellulose in lignocellulosic biomass, requires a consortium of xylanolytic enzymes to achieve its complete de-polymerisation. As global interest in using xylan-containing lignocellulosic feedstocks for biofuel production increases, an accompanying knowledge on how to efficiently depolymerise these feedstocks into fermentable sugars is required. Since it has been observed that the same enzyme [i.e. an enzyme with the same EC (Enzyme Commission) classification] from different GH families can display different substrate specificities and properties, we evaluated GH10 (XT6) and 11 (Xyn2A) xylanase performance alone, and in combination, during xylan depolymerisation. Synergistic enhancement with respect to reducing sugar release was observed when Xyn2A at 75% loading was supplemented with 25% loading of XT6 for both beechwood glucuronoxylan (1.14-fold improvement) and wheat arabinoxylan (1.1-fold improvement) degradation. Following this, the optimised xylanase mixture was dosed with an oligosaccharide reducing-end xylanase (Rex8A) from either Bifidobacterium adolescentis or Bacillus halodurans for further synergistic enhancement. Dosing 75% of the xylanase mixture (Xyn2A:XT6 at 75:25%) with 25% loading of Rex8A led to an enhancement of reducing sugar (up to an 1.1-fold improvement) and xylose release (up to an 1.5-fold improvement); however, this effect was both xylan and Rex8A specific. Using thin layer chromatography, synergism appeared to be a result of the GH10 and 11 xylanases liberating xylo-oligomers that are preferred substrates of the processive Rex8As. Rex8As then hydrolysed xylo-oligomers to xylose - and xylobiose which was the preferred substrate for xylosidase, SXA. This likely explains why there was a significant improvement in xylose release in the presence of Rex8As. Here, it was shown that Rex8As are key enzymes in the efficient saccharification of hetero-xylan into xylose, a major component of lignocellulosic substrates.

Malgas S ，Pletschke BI 《-》

Characterization of two β-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides.

Lagaert S ，Pollet A ，Delcour JA ，Lavigne R ，Courtin CM ，Volckaert G ... -  《-》