Led five mm TCI gradient probe with inverse geometry. The lignosulfonate samples (40 mg initial weight, ahead of therapies) have been dissolved in 0.75 mL of deuterated DMSO-d6. The central solvent peak was utilized because the internal reference (at CH 39.52.49 ppm), and also the other signals were normalized for the very same intensity of the DMSO signals (because the very same DMSO volume and initial amount of sample was utilized in each of the situations). The HSQC experiment utilised Bruker’s “hsqcetgpsisp.2” adiabatic pulse plan 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 within the 13C dimension. The 1JCH applied was 145 Hz. Processing utilised common matched Gaussian Danofloxacin Epigenetics apodization in the 1 H dimension and squared cosine-bell apodization inside the 13C dimension. Before Fourier transformation, the information matrices were zero-filled to 1024 points in the 13C dimension. Signals had been assigned by literature comparison [32, 51, 58, 692]. Inside the aromatic region in the spectrum, the C2 2, C5 five and C6 six correlation signals have been integrated to estimate the quantity of lignins as well as the SG ratio. Within the aliphatic oxygenated area, the signals of methoxyls, and C (or C ) correlations inside the side chains of sulfonated and non-sulfonated -O-4, phenylcoumaran and resinol substructures were integrated. The intensity corrections introduced by the adiabatic pulse plan 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 inside the HSQC 2D-NMR spectra (at 20.52.23 ppm) towards the total quantity of lignin aromatic units (G + S + S). To overcome variations in coupling constants of aliphatic and aromatic 13 1 C- H couples, the latter was estimated from the intensity of the methoxyl signal, taking into account the SG ratio of the sample, and also the variety of methoxyls of G and S units [73].S zJim ez et al. Biotechnol Biofuels (2016) 9:Web page 11 ofAdditional fileAdditional file 1. More figures which includes VP cycle, and added kinetic, PyGCMS, SEC and NMR final 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 hard wood lignosulfonates: CTPI-2 Purity native VP vs W164S variant. Fig. S3. Lignosulfonate permethylation: PyGCMS of softwood lignosulfonate before and following 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 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 devoid of 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, plus the corresponding controls without enzyme. Fig. S9. Difference spectra of peroxidasetreated softwood lignosulfonates minus their controls. Fig. S10. Distinction spectra of peroxidasetreated hardwood lignosulfonates minus their controls.Received: 16 August 2016 Accepted: 9 Septem.