Remazol brilliant blue xylan

The XBP+/XynA+ clones were grown in TB supplemented with 34 µg/mL kanamycin, 100 µg/mL ampicillin for 48 h in 96-well plates (deep well). The supernatants were analyzed for hydrolysis of Remazol Brilliant Blue Xylan (RBB-xylan, Sigma-Aldrich, St. Louis, Missouri, USA), using a modification of a previously described protocol [35 (link)]. Fifty µL of culture supernatant was mixed with 50 µL of a solution containing RBB-xylan (4 mg/mL) in 100 mM acetate buffer (pH 5.5), in the presence or absence of 1 % (m/v) d-xylose (Sigma-Aldrich, St. Louis, Missouri, USA), and incubated at 37 °C for 12 h. After incubation, the reaction was stopped by the addition of 2 volumes (200 µL) of 96 % (v/v) ethanol. The insoluble material was removed by centrifugation (2000g/2 minutes), and the increase in the absorbance of the supernatant was measured at 595 nm. A single clone that showed activity ratio (with xylose)/(without xylose) greater than 1.3 was selected, and nucleotide sequencing indicated that the XynA domain was inserted within the XBP at position 271, and this chimeric enzyme was denominated as XynA–XBP271.

The XynA+ clones were grown in TB supplemented with 34 µg/mL kanamycin, 50 µg/mL streptomycin and 0.5 mM IPTG for 48 h in 96-well plates (deep well). The supernatants were analyzed for hydrolysis of RBB-xylan (Remazol Brilliant Blue Xylan, Sigma), using a modification of the protocol developed by Biely et al. [63 (link)]. In this protocol, 50 µL of supernatants was mixed with 50 µL of a solution containing RBB-xylan (4 mg/mL), in 100 mM acetate buffer (pH 5.5), in the presence or absence of 1% (m/v) d-xylose (Sigma), and incubated at 37°C for 12 h. After incubation, the reaction was stopped by the addition of two volumes (200 µL) of 96% (v/v) ethanol. The insoluble material was removed by centrifugation (2,000g/2 min), and the increase in the absorbance of the supernatant was monitored at 595 nm. The clones that showed the highest activity in the presence of d-xylose compared to the absence of d-xylose were selected.