Topspin 3.2 mac software

Cell walls were characterised without fractionation using two-dimensional (2D) solution-state NMR (Kim and Ralph, 2010 (link); Mansfield et al., 2012 (link)). Straw (2-mm pieces) was pre-ground using a Mixer Mill MM400 (Retsch; 30/s vibrational frequency for 90–120 s). Samples were extracted three times with water, three times with 80% ethanol and once with acetone, then allowed to dry. The pre-ground extracted samples were ball-milled using a Fritsch Planetary micro mill Pulverisette 7 vibrating at 600 rpm with zirconium dioxide (ZrO₂) vessels containing ZrO₂ ball-bearings (10 mm × 10) with 5-min milling and 5-min cooling per milling cycle (cycle number depended on the amount of sample). The ball-milled samples were subjected to digestion (72 h × 2) to obtain ‘enzyme lignin’ (EL) by Cellulysin^® cellulase from Trichoderma viridae (Calbiochem), at 35°C in acetate buffer (pH 5.0). The ELs were dissolved into DMSO-d₆/pyridine-d₅ (4:1 v/v) and subjected to NMR on a Bruker Biospin AVANCE-III 700 MHz spectrometer equipped with a 5-mm QCI ¹H/³¹P/¹³C/¹⁵N cryoprobe with inverse geometry (proton coil closest to the sample). 2D-¹H–¹³C HSQC spectra were acquired using Bruker’s pulse program (hsqcetgpsip2.2). Bruker’s Topspin 3.2 (Mac) software was used to process spectra. The central DMSO peak was used as internal references (δ_C: 39.51, δ_H: 2.49 ppm).

Cell walls were characterised without fractionation using two‐dimensional (2D) solution‐state NMR (Kim and Ralph, 2010; Mansfield et al., 2012). Straw (2‐mm pieces) was pre‐ground using a Mixer Mill MM400 (Retsch; 30/s vibrational frequency for 90–120 s). Samples were extracted three times with water, three times with 80% ethanol and once with acetone, then allowed to dry. The pre‐ground extracted samples were ball‐milled using a Fritsch Planetary micro mill Pulverisette 7 vibrating at 600 rpm with zirconium dioxide (ZrO₂) vessels containing ZrO₂ ball bearings (10 mm × 10) with 5‐min milling and a 5‐min cooling per milling cycle (cycle number depended on the amount of sample). The ball‐milled samples were subjected to digestion (72 h × 2) to obtain ‘enzyme lignin’ (EL) by Cellulysin^® Cellulase, Trichoderma viridae (Calbiochem), at 35 °C in acetate buffer (pH 5.0). The EL were dissolved into DMSO‐d₆/pyridine‐d₅ (4 : 1) and subjected to NMR on a Bruker Biospin AVANCE‐III 700 MHz spectrometer equipped with a 5‐mm QCI ¹H/³¹P/¹³C/¹⁵N cryoprobe with inverse geometry (proton coil closest to the sample). 2D‐¹H‐¹³C HSQC spectra were acquired using Bruker's pulse program (hsqcetgpsip2.2). Bruker's Topspin 3.2 (Mac) software was used to process spectra. The central DMSO peak was used as internal references (δ_C: 39.51, δ_H: 2.49 ppm).