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

ABSTRACTA 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 aKmof 1.64 ± 0.03 mM and akcatvalue 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)6barrel 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.IMPORTANCEA GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme fromPaenibacillus barcinonensisis 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.

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain ofPaenibacillus barcinonensisxylanase 10C containing the CBM22-1–CBM22-2 tandem

A construct containing the CBM22-1–CBM22-2 tandem forming the N-terminal domain ofPaenibacillus barcinonensisxylanase 10C (Xyn10C) has been purified and crystallized. A xylan-binding function and an affinity for mixed β-1,3/β-1,4 glucans have previously been demonstrated for some members of the CBM22 family. The sequence of the tandem is homologous to the N-terminal domains found in several thermophilic enzymes. Crystals of this tandem were grown by the streak-seeding method after a long optimization strategy. The structure has been determined by molecular replacement to a resolution of 2.43 Å and refinement is under way. This study represents the first structure containing two contiguous CBM22 modules, which will contribute to a better understanding of the role that this multiplicity plays in fine-tuning substrate affinity.

Paenibacillus oceanisediminis sp. nov. isolated from marine sediment

A Gram-stain-negative, non-motile, aerobic, endospore forming and rod-shaped bacterium, designated strain L10T, was isolated from marine sediment collected from the South Korean coast. The organism grew optimally under conditions of 30 °C, 1 % (w/v) NaCl and pH 6.0. It was oxidase-negative and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain L10T was associated with the genus Paenibacillus and most closely related to Paenibacillus barcinonensis BP-23T (98.2 % similarity). The major fatty acids of strain L10T were iso-C14 : 0, anteiso-C15 : 0 and iso-C16 : 0. The cell-wall peptidoglycan was the A1γ type, and the predominant isoprenoid quinone was menaquinone-7. Strain L10T contained two unidentified lipids, an unidentified amino-phospholipid, phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The G+C content of the genomic DNA was 44 mol% and the DNA–DNA hybridization values with closely related strains were below 14±2 %. Based on phenotypic, genotypic, and phylogenetic data, strain L10T should be classified as a novel species within the genus Paenibacillus . The name Paenibacillus oceanisediminis sp. nov. is proposed. The type strain is L10T ( = KACC 16203T = JCM 17814T).