For growth tests, a synthetic minimal medium (MM) (2 g L−1 NaNO3, 1 g L−1 K2HPO4, 0.5 g L−1 KCl, 0.5 g L−1 MgSO4·7H2O, 0.01 g L−1 FeSO4·7H2O) was prepared and supplemented with 1% (w/v) carbon source (glucose, sucrose, carboxymethyl cellulose (CMC), galacturonic acid (GA), polygalacturonic acid (PGA), xylose, xylan from beechwood (Carlroth, Karlsruhe, Germany), tween 80, olive oil, triolein, sodium acetate (NaAc)) and 1.5% agar (Oxoid, LP0011, Swedesboro, NJ, USA). pH was adjusted and buffered at 5 or 7 with a McIlvaine buffer. For inoculation, 7-mm diameter plugs with actively growing mycelium were used. The experiment was repeated independently three times (biological replicates), including three technical replicates (three plates) and the mean was calculated with 9 measures. The Petri dishes were incubated at 21 °C in the dark, and the growth diameter of the wild type, mutant, and complemented strains was measured to 4 dpi.
Xylan from beechwood
Manufactured by Carl Roth
Sourced in Germany, Ireland, France
Xylan from beechwood is a natural polysaccharide extracted from the hemicellulose fraction of beechwood. It is a linear polymer of β-1,4-linked xylose units.
Automatically generated - may contain errors
Lab products found in correlation
Lp0011, by Thermo Fisher Scientific (1 mentions)
Xylose, by Carl Roth (1 mentions)
Copycontrol fosmid library production kit, by Illumina (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
T4 dna ligase, by Thermo Fisher Scientific (1 mentions)
Fosmidmax dna purification kit, by Illumina (1 mentions)
Congo red, by HiMedia (1 mentions)
E coli nzy5α, by NZYTech (1 mentions)
Lipase from candida rugosa, by Merck Group (1 mentions)
Xylanase from thermomyces lanuginosus, by Merck Group (1 mentions)
Nucleospin plasmid quickpure kit, by Macherey-Nagel (1 mentions)
Nzycolour protein marker 2, by NZYTech (1 mentions)
3 protocols using xylan from beechwood
1
Evaluating Carbon Source Utilization in Fungi
2
Fosmid Library Production and Xylanase Purification
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The CopyControl™ Fosmid Library Production kit, including the autoinduction solution (500 ×), and the FosmidMAX™ DNA Purification kit were provided by Epicentre Biotechnologies (Madison, Wisconsin, USA). The NucleoSpin® Gel and PCR Clean-up kit and the NucleoSpin® Plasmid QuickPure™ kit were obtained from Macherey-Nagel (Düren, Germany). Azurin-crosslinked xylan (AZCL-xylan) was supplied by Megazyme (Wicklow, Ireland) and xylan from beechwood was obtained from Carl Roth (Karlsruhe, Germany). Congo red was acquired from HIMEDIA Laboratories Pvt. Ltd. (Maharashtra, India). NdeI and BamHI restriction enzymes and T4 DNA ligase were provided by Thermo Fisher Scientific (Waltham, Massachusetts, USA). pETDuet-1 expression vector was supplied by Novogene (Cambridge, UK). E. coli NZY5α, E. coli BL21 (DE3), bovine serum albumin (BSA), and NZYColour Protein Marker II were delivered by NzyTech (Lisboa, Portugal). Xylanase from Thermomyces lanuginosus, lipase from Candida rugosa, and other assay reagents used in this work (analytical grade) were purchased from Sigma-Aldrich (St. Louis, IL, USA).
3
Thermobacillus xylanilyticus Cultivation
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Thermobacillus xylanilyticus XE9/11/91, isolated from a farm soil under a manure heap in northern France, was used in this study. The bacterium was cultivated on basal medium composed by three different solutions, a macromineral solution, a vitamin solution, and a metallic trace solution complemented with NH 4 Cl (1 g/L), yeast extract (2 g/L) and KHCO 3 (5 g/L). The medium was supplemented with 10% (v/v) CO 2 as previously described by [7] . Cultivation volumes are 10 mL of media in sealed contents (100 mL bottles). Various carbon sources were used: glucose 5 g/L (Sigma Aldrich), xylan from beechwood 5 g/L (Roth), destarched wheat bran 10 g/L, and wheat straw 15 g/L, (ground to 1-2 mm) (ARD Pomacle-Bazancourt, France) (Table 1).
The complementation of xylan medium with glucose was done by reducing the xylan concentration from 5 to 4 g/L (ratio 80/20) or 2.5 g/L (ratio 50/50) before completing with glucose. The final glucose concentration was 1 g/L or 2.5 g/L for the ratios 80/20 and 50/50, respectively, to reach the total carbon source concentration of 5 g/L. The addition of glucose was realized after sterilization of the medium.
The complementation of xylan medium with glucose was done by reducing the xylan concentration from 5 to 4 g/L (ratio 80/20) or 2.5 g/L (ratio 50/50) before completing with glucose. The final glucose concentration was 1 g/L or 2.5 g/L for the ratios 80/20 and 50/50, respectively, to reach the total carbon source concentration of 5 g/L. The addition of glucose was realized after sterilization of the medium.
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