Cellotetraose

To study the production of xylanolytic enzymes, F. oxysporum was first grown in medium containing 0.2 M sodium phosphate buffer, 0.3 g/L MgSO₄·7H₂O, 1 g/L KH₂PO₄, 10 g/L (NH₄)₂HPO₄, and 1% w/v sucrose. The medium was inoculated with 6% v/v of pre-culture and incubated for 2 days in a shaking incubator. Cells were harvested through a sterile vacuum filter (0.45 µm, Sarstedt, Nümbrecht, Germany), washed with sterile milliQ water, and transferred to the same medium as above, but without sucrose. Cells were incubated for 24 h to ensure that the mycelium was free of any residual sugars. Then, individual compounds with potential inducer activity were added at the following final concentrations: 0.1% w/v sophorose (Megazyme, Wicklow, Ireland); 0.2 and 0.3% w/v cellobiose; 0.2 and 0.3% w/v lactose; 0.1% w/v xylobiose, xylotetraose, and xylohexaose (Megazyme); and 0.1% w/v cellotetraose and cellohexaose (Megazyme). The fungal culture was incubated at 29 °C and 190 rpm. To minimize errors during sampling, each sample point consisted of a separate flask, whose entire content was harvested and filtered. The supernatant and biomass fractions were used to determine extracellular and cell-bound xylanase activity, respectively, as described below.

Cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose were from Megazyme (Bray, Ireland). Sodium cacodylate, manganese (II) chloride tetrahydrate, uridine diphosphate galactose (UDP-Gal), fluorescein isothiocyanate (isomer I) and galactosyl transferase from bovine milk were from Sigma. AlexaFluor488 C5-aminooxyacetamide, and bis(triethylammonium) salt were from Invitrogen (Nærum, Denmark). 2-(aminooxy)-1-ethanaminium dichloride was from ABCR GmbH (Karlsruhe, Germany). 2,5-Dihydroxy-benzoic acid was from Bruker Daltonics (Bremen, Germany). The LPMO used in this study, from Neurospora crassa (NcLPMO9A), and Cellobiose dehydrogenase from Myrococcum thermophilum (MtCDH) were produced and purified according to Petrovic et al. 2019^{26 (link)} and Flitsch et al. 2019^{28 (link)}, respectively. The endocellulase Cel5A from Hypocrea jecorina was produced according to Saloheimo et al. 1988^{41 (link)} and the exocelluase Cel6B from Thermobifida fusca was produced according to Vuong and Wilson 2009^{42 (link)}.

The following substrates were used: phosphoric acid swollen cellulose (PASC, prepared from Avicel PH-101 (Sigma, Deisenhofen, Germany), as described earlier [43 ]), cellotriose, cellotetraose, cellopentaose, and cellohexaose (Megazyme, Wicklow, Ireland), and the hemicelluloses sugar beet arabinan, konjac glucomannan, barley β-glucan, tamarind xyloglucan (XG) (Megazyme) and beechwood xylan (Sigma, Deisenhofen, Germany).

XG from tamarind (Tamarindus indica, TXG) seed, TXG oligosaccharide standards (xyloglucan hepta + octa + nona saccharides) and XEG (GH5) from Paenibacillus sp. were purchased from Megazyme (Bray, Ireland). XG from black currants (Ribes nigrum L., BCXG) was available in our laboratory (fraction CASS) extracted by Hilz and coworkers [17 (link)]. Glucose was purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and Asc was purchased from VWR International (Radnor, PA, USA). Cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose were used as standards and purchased from Megazyme. Water used in all experiments was generated by a Milli-Q system (Millipore, Molsheim, France), unless mentioned otherwise.

Syringol, pyrogallol, and
ammonium acetate were purchased from Sigma-Aldrich (St. Louis, Missouri).
Cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose
were purchased from Megazyme (Bray, Ireland). Regenerated amorphous
cellulose (RAC) was prepared from Avicel PH-101 (Sigma-Aldrich) as
described previously.^{29 (link)} Ascorbic acid (Asc)
was purchased from VWR International (Radnor, Pennsylvania). Other
aromatic compounds used were purchased from Sigma-Aldrich or VWR International.
Other carbohydrate substrates used were purchased from Sigma-Aldrich
or Megazyme. Glucose oxidase from Aspergillus niger (AnGOX, 10 000 U g^–1 powder)
was purchased from Sigma-Aldrich. All water used was produced by a
Milli-Q system (Merck Millipore, Molsheim, France), unless stated
otherwise.

All buffering chemicals and substrates including glucose, xylose, mannose, galactose, cellobiose, lactose, mannobiose, β-(1 → 4)-_D-galactobiose, isomaltose and maltose were purchased from Sigma-Aldrich (Germany). The other substrates including cellotriose, cellotetraose, xylobiose, xylotriose, xylotetraose, 3²-α-l-arabinofuranosyl-xylobiose (A³X), 2³-α-l-arabinofuranosyl-xylotriose (A²XX), 2²-(4-O-methyl-α-d-glucuronyl)-xylobiose (U^4m2X), 2³-(4-O-methyl-α-d-glucuronyl)-xylotriose (U^4m2XX), chitosanbiose, 3²-β-d-glucosyl-cellobiose, β-(1 → 4)-_D-glucosyl-_D-mannose, gentiobiose, kojibiose, sophorose, laminaribiose, and nigerose were purchased from Megazyme (Ireland). The laccase from T. versicolor (38429, Sigma-Aldrich, Germany) was used in oxidation reactions to recycle the electron acceptor, 1,4-benzoquinone (BQ, PHR1028, Sigma-Aldrich, Germany). Methylated cellobiose and lactose was produced in house (details in Additional File 2).