Ment with native VP (Fig. 4b, e) the intensity with the signals in the distinct aromatic ligninunits (G, S, S and S) and side-chain interunit linkages (A, A, B and C) decreased simultaneously, maintaining related linkage percentages. Nonetheless, the methoxyl numbers per unit enhanced up to twofold. In the hardwood lignosulfonate, this was accompanied by larger abundance of C-oxidized syringyl units (S) with respect to total syringyl units, whilst the SG ratio also increased (from 2.0 within the manage to 3.5 within the 24-h treated sample). Regarding side-chain signals, only these in the key sulfonated -O-4 substructures (A, A in addition to a) remained inside the softwood lignosulfonate, while those of phenylcoumaran (B), resinol (C) and -O-4 (A) non-sulfonated side chains disappeared. In contrast, signals of sulfonated (A) and non-sulfonated -O-4 (A) and resinol (C) side chains could possibly be observed inside the hardwood lignosulfonate, albeit with low intensities. Extra interestingly, in the lignosulfonates treated for 24 h using the W164S variant (Fig. 4c, f ) only minor changes inside the aliphaticaromatic HSQC signals were observed (spectra with comparable intensities of most signals, and only slight increases of methoxyl content material and SG ratio compared with the control).S zJim ez et al. Biotechnol Biofuels (2016) 9:Web page 5 ofaPhenolic-AcAlcoholic-AcaA280 ( )010 11 12 13 14 15 16 17b2.two.two.1.HA280 ( )b0 five six 7 8 9 10 11 12 13 14 15 16 17cA280 ( )2.two 2.1 2.0 1.H10 11 12 13 14 15 16 17 18 Volume (mL)Fig. two Lignosulfonate permethylation: 1HNMR evaluation soon after second ary acetylation confirming the prior comprehensive methylation of softwood lignosulfonate (b) compared using the untreated sample (a). Regions of phenolic and alcoholic acetates are indicatedSteadystate treatment of nonphenolic vs native lignosulfonatesWith the goal of further investigating lignosulfonate modification by VP, which A11466 5 cathepsin Inhibitors Reagents includes the observed compact modifications by the W164S variant, derivatized (nonphenolic) lignosulfonates had been treated in new steady-state experiments. The native VP was capable to modify the nonphenolic lignosulfonates but the modifications inside the molecular-mass distribution (Further file 1: Figure S4, green continuous line) and molecular structure of lignins (Additional file 1: Figure S5b, e) have been modest, compared with those observed for the native (partially phenolic) lignosulfonates (Fig. 3a, b, green continuous line, and Fig. 4b, e, respectively). These modifications include lower-intensity signals within the NMR spectra of nonphenolic hardwood lignosulfonate (the S signal being the exception) and displacement on the Mp inFig. 3 SEC profiles of softwood (a) and hardwood (b) lignosulfonates treated for 24 h with native VP and its W164S variant and manage without the need of enzyme, and sulfonated polystyrene standards (c). Lignosul fonate samples (12 g L-1) soon after a 24h therapy with 1.two native VP (green line) and its W164S variant (dashes) in presence of 9.five mM H2O2, and also the corresponding softwood (red) and hardwood (blue) ligno sulfonate controls devoid of enzyme, were analyzed within a Superdex75 column applying 0.15 M NaOH as eluent (0.5 mL in-1) and detection at 280 nm. Sulfonated polystyrenes (Mp 78,400, 29,500, 10,200 and 4210 Da, from left to suitable) were made use of as molecular mass standards in c (arrow shows the excluded blue dextran elution volume)the SEC profile, even though reduce changes were observed for the nonphenolic softwood lignosulfonate. In contrast, the SEC profiles with the W164S-treated (green dashed lines) and c.
