Antioxidant activity of xylooligosaccharides (XOS) released from beechwood and birchwood glucuronoxylans by two different xylanases, one from family GH10 (Xyn10A) and another from family GH30 (Xyn30D) was examined. The ABTS (2, 2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) method was used, since it resulted more accurate for the antioxidant activity determination of XOS. Thin layer chromatography and MALDI-TOF MS analysis showed that Xyn10A produced a mixture of neutral and acidic XOS whereas the XOS produced by Xyn30D were all acidic, containing a methylglucuronic acid (MeGlcA) ramification. These acidic XOS, MeGlcA substituted, showed a strongly higher antioxidant activity than the XOS produced by Xyn10A (80% vs. 10% respectively, at 200 μg mL-1). Moreover, the antioxidant activity increased with the degree of polymerization of XOS, and depended on the xylan substrate used. The antioxidant capacity of eucalyptus autohydrolysates after xylanase treatment was also analysed, showing a decrease of their antioxidant activity simultaneous with the decrease in XOS length.

A Gram-positive, endospore-forming, xylanase-producing bacterium isolated from a rice field was studied taxonomically. The strain grows at 10–40 °C and in the presence of lysozyme or 5 % (w/v) NaCl. Chemotaxonomic analysis revealed that MK-7 was the predominant menaquinone of the isolated strain, while the major fatty acid was anteiso-C_15 : 0. Comparison of 16S rRNA gene sequences showed that strain BP-23^T fell within the radiation of the cluster comprising Paenibacillus species. The highest 16S rRNA gene sequence similarities were found with Paenibacillus illinoisensis (97·4 %), Paenibacillus pabuli (97·1 %) and Paenibacillus amylolyticus (96·9 %). The DNA–DNA relatedness of strain BP-23^T with respect to these three species was very low (32·7, 31·6 and 23·0 %, respectively). On the basis of phenotypic and genotypic data, strain BP-23^T should be placed in the genus Paenibacillus and designated a novel species, for which the name Paenibacillus barcinonensis sp. nov. is proposed. The type strain is BP-23^T (=CECT 7022^T=DSM 15478^T).

Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K _m of 14.72 mg/ml and a k _cat value of 1,510 min ⁻¹ . The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α) ₈ barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.