4 methyl umbelliferone mu

Different strains of Xcc harboring the chromosomal transcriptional fusions with GUS reporter (S2 Table; see Supporting Materials and Methods) were grown in rich PS medium with appropriate antibiotics at 28°C and 200 rpm for overnight. 0.2% primary inoculum was transferred to the PS, low-iron (PS + 100 μM 2, 2′-dipyridyl) and FeSO₄ supplemented low-iron (PS + 100 μM 2, 2′-dipyridyl + 100 μM FeSO₄) media and grown at 28°C and 200 rpm in shaking incubator (New Brunswick Scientific, Innova 43, Edison, NJ, USA). At regular time intervals, absorbance was measured at 600 nm, cells were harvested from 1ml culture, washed with sterile miliQ water, resuspended in 250 μl of 1 mM MUG (4-methylumbelliferyl β-d-glucuronide) extraction buffer (50mM sodium dihydrogen phosphate [pH 7.0], 10 mM EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100, and 10 mM β-mercaptoethanol) (Jefferson et al., 1987) and incubated at 37°C. Subsequently, reactions were terminated after addition of 675 μl of 0.2 M Na₂CO₃ into 75μl of reaction mixture and fluorescence was measured with 4-methyl-umbelliferone (MU; Sigma) as the standard at excitation 365nm and emission 455 nm of wavelength. β-Glucuronidase activity for GUS assays was expressed as nanomoles of MU produced/minute.

For the characterization of CPC fractions, commercial standards of glucose, cellobiose, and 5-hydroxymethylfurfural (HMF) were purchased from Alfa-Aesar (Haverhill, MA); xylose, galactose, arabinose, mannose, and furfural were purchased from Sigma-Aldrich (C2730, St. Louis, MO). Standards for acetic acid and formic acid were purchased from Amresco (Solon, OH). Folin-Ciocalteu's (F-C) phenol reagent and sulfuric acid (98%) were obtained from Thermo Fisher Scientific (Fair Lawn, NJ).
Recombinant enzymes were purified from transgenic corn grain. A commercial T. reesei cellulase cocktail was purchased from Sigma-Aldrich (C2730, St. Louis, MO). Four-methylumbelliferyl-β-D-cellobioside (MUC) (Gold Biotechnology) and 4-methylumbelliferone (MU) (Sigma-Aldrich, M1381) were used in the MUC assay as the substrate and for standard curve construction. Sigmacell cellulose (Sigma-Aldrich, S3504), 3,5-dinitrosalicylic acid (DNS) (Sigma-Aldrich, 128848) and D-glucose (Fisher Scientific) were used in the cellulose-DNS assay as the substrate and for standard curve construction.