Xylo oligosaccharides

EpiQuik DNA Methyltransferase 1 kit and a 3B Activity/Inhibitor Screening Assay kit (Epigentek, Brooklyn, NY) were used to access inhibition of DNMT1 and DNMT3B, respectively. Ellagic acid, urolithin A, urolithin B, cyanidin-glucoside, cyanidin-rutinoside, protocatechuic acid, fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, butyric acid, acetic acid, propionic acid, and valenic acid were purchased from SigmaAldrich (St. Louis, MO). Each compound was dissolved in dimethyl sulfoxide to prepare stock solution, then diluted to 1 nM, 100 nM, and 10 μM and tested in DNMT1 and DNMT3B inhibition assays. Each compound was tested in both assays in three independent runs.

Reaction systems (1 mL) containing 0.5 U of Xyn10A, Xyn11A or Xyl43A and 1% (w/v) substrate (beechwood xylan, birchwood xylan or soluble wheat arabinoxylan) in McIlvaine buffer (pH 6.5) were incubated at room temperature for 12 h and treated as baselines. The simultaneous reactions of Xyn10A, Xyn11A and Xyl43A contained 0.5 U of each enzyme. For sequential reactions, first reactions containing either Xyn10A or Xyn11A (0.5 U of each) were incubated at room temperature for 12 h, boiled in water bath for 10 min to inactivate the enzyme(s), followed by secondary and tertiary incubation(s) with the other xylanase and xylosidase for another 12 h. The released reducing sugars in the supernatants were measured as xylose equivalents by using the DNS method, and the appropriately diluted hydrolysates (50–200 times) were analyzed by the HPAEC (model 2500, Dionex) equipped with a pulsed amperometric detector (PAD) and xylooligosaccharides (Sigma-Aldrich) as standards. The XCR was defined as the percentage of xylose amount against the total amount of reducing sugars (xylose equivalents) released. The degree of synergy was defined as the ratio of xylose released when enzymes were incubated simultaneously or sequentially to the sum of the xylose released by each enzyme alone^{93 (link)}.