Preparation of LRP
Lignin-Rich Precipitate (LRP) from Norwegian birchwood was prepared as previously reported [28]. 15 g raw birchwood was mixed with 135 mL 50 v/v-% ethanol in a batch reactor (300 mL HC EZE-Seal, Parker Autoclave Engineers, Pennsylvania USA). The reactor was heated to 180 °C and kept at 180 °C for 1 h with stirring at 600 rpm. After extraction, the reactor was cooled and the material retrieved. The pre-treated liquor was separated from the solid biomass by filtration using Ashless 40 filter paper, 8 µm (Whatman). The solids (named Cellulose-Rich Precipitate) were washed with 50 v/v-% EtOH and freeze dried. The pre-treated liquor (280 mL in total including washings) was diluted with three volumes of water (840 mL) resulting in the precipitation of a Lignin-Rich Precipitate (LRP). pH in the pre-treated liquor was measured to 3.8. The suspension of lignin-rich precipitate and the pre-treated liquor was separated by centrifugation. The separated liquor phase consisted of a hemicellulose-rich liquid (HRL). The lignin-rich precipitate was washed in water and freeze dried prior to use for hydrolysis experiments. A schematic overview of the extraction process can be found in Additional file 6. All fractions were subjected to acid hydrolysis for determination of monomeric components (see Additional file 2). A small portion of the LRP was subjected to NaOH treatment in order to release all esterified carbohydrates from the lignin matrix. 0.5 M NaOH was added to approx. 11.25 mg of LRP in suspension in water to reach pH > 11 and kept at room temperature overnight. Hereafter, absolute ethanol was added to a final concentration of 90 v/v-% and kept overnight at 4 °C. The precipitate was retrieved by centrifugation, re-dissolved in water, and analyzed by HPAEC-PAD as LRP alkali and represented the majority of the carbohydrate moiety of the lignin-rich fraction. It is expected that the precipitation of short, linear, neutral xylo-oligosaccharides under these conditions is incomplete. The resulting concentration in the LRP alkali fraction analyzed by HPAEC-PAD originated from 37.5 mg/mL. As described below, the enzyme hydrolysis samples were performed with 5 mg/mL substrate and hence the final concentration of analytes in LRP alkali was approx. 7.5 times higher than in the enzyme hydrolysis samples.
Enzymatic hydrolysis
Lignin-rich precipitate was suspended in 25 mM sodium acetate buffer pH 6 to a concentration of 5 mg/ml. CuGE (for expression and purification protocol see Additional file 2) and GH10 endo-β1,4-xylanase Shearzyme® 500 L (batch CDN00486) (donated by Novozymes A/S) were added either individually or together to the reactions to a final concentration of 30 mg enzyme protein/g dry matter and 10 mg enzyme protein/g dry matter, respectively, and incubated for 24 h at 50 °C. After reaction, the solid substrate was removed by centrifugation and the supernatant used for analysis.
HPAEC-PAD
Enzyme reaction products and substrate (LRP alkali) were analyzed by HPAEC-PAD using a CarboPac PA100 (4.6 × 250 mm) and guard (4.6 × 50 mm) on a Dionex ICS3000 system (Thermo Fischer Scientific, Sunnyvale, CA, USA). The column was operated at 1 mL/min with eluent A (water), eluent B (500 mM NaOH), and eluent C (500 mM sodium acetate) according to the following gradient: 0–2 min isocratic 40% B and 1% C, 2–35 min linear gradient to 40% B and 45% C, hereafter immediately shifted to 5% B and 90% C and these conditions were kept for 4 min and ended by shifting back to starting conditions and reconditioning for 6 min.
LC–MS analysis
Identification and quantification of enzyme reaction products were performed by LC–MS analysis on a UHPLC Dionex UltiMate 3000 system (Thermo Fischer Scientific, Sunnyvale CA, USA) connected to an ESI-iontrap (model AmaZon SL from Bruker Daltonics, Bremen, Germany) operated in MRM mode (multiple reaction monitoring) or fullscan mode. The UHPLC was equipped with a porous graphitized carbon column (Hypercarb PGC, 150 × 2.1 mm; 3 µm, Thermo Fischer Scientific, Waltham, MA, USA) including a guard column of same brand (10 × 2.1 mm). The column was operated at 0.4 mL/min at 70 °C with eluent A (0.1% formic acid) and B (acetonitrile) according to the following gradient: 0–1 min 0% B, from 1 to 15 min linear gradient to 50% B, from 15 to 20 min linear gradient to 80% B, 20–30 isocratic 80% B, hereafter immediately back to starting conditions followed by reconditioning for 10 min.
The ESI was operated in positive mode with spray nebulizer at 3 bar nitrogen, a dry gas flow, and temperature of 12 L/min and 280 °C, respectively. The capillary cap voltage was set to 4.5 kV and end-plate offset of 0.5 kV. Target mass was set to 500 and for MRM conditions, a pre-determined list of ions representing the sodium adducts of exact masses of neutral and charged species was applied: Xyl2 (m/z 305), Xyl2Ac (m/z 347), Xyl2Ac2 (m/z 389), Xyl3 (m/z 437), Xyl3Ac (m/z 479), Xyl3Ac2 (m/z 521), Xyl3Ac3 (m/z 563), XylMeGlcA (m/z 231), Xyl2MeGlcA (m/z 495), Xyl2MeGlcAAc (m/z 537), Xyl3MeGlcA (m/z 627), Xyl3MeGlcAAc (m/z 669), Xyl3MeGlcAAc2 (m/z 711), Xyl4MeGlcA (m/z 759), Xyl4MeGlcAAc (m/z 801), and Xyl4MeGlcAAc2 (m/z 843). CID fragmentation was performed using SmartFrag enhanced amplitude ramping of 100% and helium as the colliding gas. Data analysis and quantification were performed by Compass DataAnalysis 4.2 and Compass QuantAnalysis 2.2 from Bruker Daltonics.
Quantification of enzyme reaction products was done against external calibration curves of authenticated standards of xylobiose, xylotriose, xylotetraose, and reduced aldotetrauronic acid all of which were purchased from Megazyme, Ireland. Samples for quantification purposes were treated with NaOH prior to analysis (and after enzyme reaction) in order to raise pH > 11 and hereby remove all acetylations. This procedure resulted in a much simplified mixture of reaction products mainly consisting of xylose, xylobiose, xylotriose, xylotetraose, aldotriuronic acid, aldotetrauronic acid, and aldopentauronic acid (see Additional file 14). The aldouronic acids were quantified relative to the signal response for reduced aldotetrauronic acid. Elaborate description of the quantification procedure is provided in the Additional files 2, 15.