Birchwood xylan

Buffer salts, 1,2,3-trihydroxybenzene (pyrogallol), 3,4,5-trihydroxybenzoic acid (gallic acid), ascorbic acid, TCEP, methoxyhydroquinone, EDTA, 2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene (Zincon), ANS, dextran, CMC, and maltodextrin (dextrose-equivalent 13–17) were purchased from Sigma-Aldrich. Microcrystalline cellulose (20–160 μm) was from Merck, and birchwood xylan (purity >90%) was from Roth. Xyloglucan from Tamarindus indicus (purity ∼95%) and arabinogalactan from larch (purity ∼95%; Ara/Gal = 15:85) were purchased from Megazyme. PASC was prepared by dissolving 4 g of Microcrystalline cellulose in 100 ml of ice-cold 85% (by weight) phosphoric acid. The solubilized cellulose was stirred for 1 h at 4 °C and precipitated by the addition of 1,900 ml of ice-cold HQ-water. The cellulose was washed on a vacuum filtration system with ∼2 liters of ice-cold water, 2 liters of a 2 m sodium bicarbonate solution, and 1 liter of 50 mm sodium phosphate buffer, pH 6. Finally, PASC was homogenized with a high-performance disperser (Ultra Turrax, Ika).

The substrate specificity of AC2aCel5A had previously been determined using Azurine-Crosslinked Polysaccharide (AZCL) substrates, along with activity on insoluble cellulose substrates7 (link). In this study, further characterization of the enzymatic activity was performed using the soluble β-(1,4) linked glucan substrates carboxymethylcelullose (CMC) (Sigma-Aldrich), barley β-glucan (Megazyme), tamarind xyloglucan (Megazyme), lichenan (Sigma-Aldrich), and Birchwood Xylan (Carl Roth). The standard reaction using CMC contained 20 mM BisTris buffer pH 6.5, 25 nM enzyme, 20 mM CaCl₂, and 10 mg/ml substrate in a total volume of 200 μl. Enzyme was added to pre-heated assay mixtures and the reactions were incubated at 40 °C, with 900 rpm vertical shaking. 100 μl sample was taken after 10 minutes, and added to 100 μL DNS reagent40 . The amount of reducing ends released were determined as glucose equivalents using the DNS reducing-end assay and a glucose standard curve. Assays for activity on barley β-glucan, lichenan, xylan and xyloglucan were performed with 0.5% substrate (w/v), and 10, 10, 200 and 200 nM enzyme load, respectively. A Unit of enzyme activity was defined as the amount of enzyme releasing one μmol of glucose equivalents per minute.

The following substrates were used to characterize the substrate specificity of GtLPMO9A-2: phosphoric acid swollen cellulose (PASC), carboxymethyl cellulose (CMC), cellohexaose, cellopentaose, tamarind xyloglucan (partially arabinosylated [XG]), xyloglucan oligosaccharide (XG oligomers), and xyloglucan heptasaccharide (XG7), Konjac glucomannan (GM), ivory nut mannan, wheat arabinoxylan, oat flour mixed-linkage (β-1,3–1,4) glucan, oat spelt xylan, beech wood xylan, and birchwood xylan. PASC was prepared as described earlier (45 ); birchwood xylan was purchased from Carl Roth GmbH (Karlsruhe, Germany). Konjac glucomannan used in the dynamic viscosity experiments was purchased from Wako; Konjac glucomannan used in the coating experiments was purchased from Megazyme (Wicklow, Ireland). Oat spelt xylan was purchased from Serva Electrophoresis GmbH (Heidelberg, Germany), and beech wood xylan was purchased from Sigma-Aldrich (St. Louis, MO). All other substrates were obtained from Megazyme.