Fig 5.

Cascade degradation of MLX by the xylanolytic enzymes of Polaribacter sp. Q13. (A) Cleavage sites of xylanases Xyn26A (27), Xyn10A, Xyn10B, and Xyn10C in MLX. The cleavage sites were determined by analyzing the hydrolytic products of xylanases separately. Reaction mixtures containing 90 µL MLX (5 mg/mL) and 10 µL each of xylanases Xyn10A (0.5 mg/mL), Xyn10B (2.4 mg/mL), and Xyn10C (0.6 mg/mL) were incubated at 30°C for 24 h. The products were analyzed by HPAEC-PAD. Xylose, β1,4-xylooligosaccharides (β1,4XOs; β1,4X2-X6), β1,3-xylooligosaccharides (β1,3X2-X3), X3X4X, and X4X3X were used as standards to identify the peaks in chromatograms, and the standard chromatogram of a mixture of xylose and β1,4X2-X6 is shown. The cleavage sites are indicated by arrows, and the indeterminate ones are indicated by dotted arrows. (B) Xylooligosaccharides in the culture supernatant of strain Q13 grown on MLX. Xylose, β1,4-xylooligosaccharides (β1,4XOs; β1,4X2-X6), β1,3-xylooligosaccharides (β1,3X2-X3), X3X4X, and X4X3X were used as standards to identify the peaks in chromatograms, and the standard chromatogram of a mixture of xylose and β1,4X2-X6 is shown. (C) Degradation of xylooligosaccharides in the culture supernatant by periplasmic xylosidases Xyl43B and Xyl3A. A zoom-in view of a part of the chromatogram is shown below. (D) Thin-layer chromatography (TLC) analysis of Rex10A activity on β1,4-xylotriose (β1,4X3) and the reduced β1,4X3 by NaBH₄. The + and − indicate the presence and absence of the components, respectively. (E) Schematics of the degradation of xylooligosaccharides by periplasmic (Xyl43B and Xyl3A) and cytoplasmic (Rex10A and Xyl43A) enzymes. Cleavage sites are indicated by arrows with different colors. Experiments were performed in triplicate, and representative data are shown. MLXOs, mixed-linkage xylooligosaccharides; unidentified MLXOs, mixed-linkage xylooligosaccharides whose structures were not identified using our available standard xylooligosaccharides.