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

Abstract

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.