Cellohexaose

Phosphoric acid swollen cellulose (PASC) was prepared as follows. 5 g of Avicel^® PH-101 was moistened with water and treated with 150 mL ice cold 85% phosphoric acid, stirred on an ice bath for 1 h. Then 500 mL cold acetone was added while stirring. The swollen cellulose was filtered on a glass-filter funnel and washed 3 times with 100 mL ice cold acetone and subsequently twice with 500 mL water. PASC was then suspended in 500 mL water and blended to homogeneity.
High-purity pachyman (β-d-1,3-glucan), barley β-glucan (β-d-1,3-1,4-glucan), lichenan (from Icelandic moss, β-d-1,3-1,4-glucan), mannan (borohydride reduced), konjac glucomannan (β-d-1,4), carob galactomannan, larch arabinogalactan, wheat arabinoxylan, cellotriose, cellotetraose, cellopentaose, cellohexaose, mannobiose, and xylobiose were purchased from Megazyme. Locust bean gum, carboxymethyl cellulose (CMC), beechwood xylan, and cellobiose were purchased from Sigma. 5-bromo-4-chloro-3-indolyl-β-d-cellobioside was purchased from Santa Cruz Biotechnology.

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)}.