Led 5 mm TCI gradient probe with inverse geometry. The lignosulfonate samples (40 mg initial weight, ahead of remedies) had been dissolved in 0.75 mL of deuterated DMSO-d6. The central solvent peak was used because the internal reference (at CH 39.52.49 ppm), and also the other signals were normalized for the same intensity from the DMSO signals (because the similar DMSO volume and initial level of sample was applied in all the instances). The HSQC experiment utilised Bruker’s “hsqcetgpsisp.2” adiabatic pulse program with spectral widths from 0 to ten ppm (5000 Hz) and from 0 to 165 ppm (20,625 Hz) for the 1H and 13C dimensions. The number of transients was 64, and 256 time increments had been normally recorded in the 13C dimension. The 1JCH made use of was 145 Hz. Processing made use of standard matched Gaussian apodization in the 1 H dimension and squared cosine-bell apodization in the 13C dimension. Before Fourier transformation, the data matrices had been zero-filled to 1024 points within the 13C dimension. Signals had been assigned by literature comparison [32, 51, 58, 692]. Inside the aromatic region of the spectrum, the C2 2, C5 five and C6 six correlation signals were integrated to estimate the volume of lignins and also the SG ratio. Within the aliphatic oxygenated region, the signals of methoxyls, and C (or C ) correlations within the side chains of sulfonated and non-sulfonated -O-4, phenylcoumaran and resinol substructures have been integrated. The intensity corrections introduced by the adiabatic pulse system permits to refer the latter integrals for the previously obtained number of lignin units. The percentage of phenolic structures was calculated by referring the phenolic acetate signal in the HSQC 2D-NMR Ai ling tan parp Inhibitors products spectra (at 20.52.23 ppm) to the total number of lignin aromatic units (G + S + S). To overcome differences in coupling constants of aliphatic and aromatic 13 1 C- H couples, the latter was estimated from the intensity of your methoxyl signal, taking into account the SG ratio of the sample, along with the number of methoxyls of G and S units [73].S zJim ez et al. Biotechnol Biofuels (2016) 9:Web page 11 ofAdditional fileAdditional file 1. Additional figures including VP cycle, and extra kinetic, PyGCMS, SEC and NMR results. Fig. S1. VP catalytic cycle and CI, CII and resting state electronic absorption spectra. Fig. S2. Kinetics of CI reduction by native, acetylated and permethylated softwood and tough wood lignosulfonates: Native VP vs W164S variant. Fig. S3. Lignosulfonate permethylation: PyGCMS of softwood lignosulfonate before and immediately after 1 h methylation with methyl iodide. Fig. S4. SEC profiles of softwood and hardwood nonphenolic lignosulfonates treated for 24 h with native VP and its W164S variant and controls without having enzyme. Fig. S5. HSQC NMR spectra of acetylated softwood and hardwood lignosulfonates treated for 24 h with native VP and its W164S variant, and manage without enzyme. Fig. S6. Kinetics of reduction of LiP CII by native and permethylated softwood and hardwood lignosulfonates. Fig. S7. SEC profiles of soft wood and hardwood lignosulfonates treated for 24 h with native LiP and controls with no enzyme. Fig. S8. HSQC NMR spectra of native softwood and hardwood lignosulfonates treated for 3 and 24 h with LiPH8, and the corresponding controls without having enzyme. Fig. S9. Distinction spectra of (E)-2-Methyl-2-pentenoic acid site peroxidasetreated softwood lignosulfonates minus their controls. Fig. S10. Difference spectra of peroxidasetreated hardwood lignosulfonates minus their controls.Received: 16 August 2016 Accepted: 9 Septem.