Ment with native VP (Fig. 4b, e) the intensity on the signals in the different aromatic ligninunits (G, S, S and S) and side-chain interunit linkages (A, A, B and C) decreased simultaneously, preserving related linkage percentages. However, the methoxyl numbers per unit improved as much as twofold. In the hardwood lignosulfonate, this was accompanied by larger abundance of C-oxidized syringyl units (S) with respect to total syringyl units, even though the SG ratio also PZ-128 web enhanced (from 2.0 within the handle to 3.5 inside the 24-h treated sample). Concerning side-chain signals, only these from the key sulfonated -O-4 substructures (A, A along with a) remained inside the softwood lignosulfonate, though these 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 be observed inside the hardwood lignosulfonate, albeit with low intensities. Much more interestingly, inside the lignosulfonates treated for 24 h with the W164S variant (Fig. 4c, f ) only minor changes in the aliphaticaromatic HSQC signals had been observed (spectra with equivalent intensities of most signals, and only slight increases of methoxyl content and SG ratio compared with the control).S zJim ez et al. Biotechnol Biofuels (2016) 9:Page five ofaPhenolic-AcAlcoholic-AcaA280 ( )010 11 12 13 14 15 16 17b2.two.2.1.HA280 ( )b0 5 six 7 8 9 10 11 12 13 14 15 16 17cA280 ( )two.2 2.1 two.0 1.H10 11 12 13 14 15 16 17 18 Volume (mL)Fig. two Lignosulfonate permethylation: 1HNMR evaluation just after second ary acetylation confirming the earlier comprehensive methylation of softwood lignosulfonate (b) compared together with the untreated sample (a). Regions of phenolic and alcoholic acetates are indicatedSteadystate treatment of nonphenolic vs native lignosulfonatesWith the goal of additional investigating lignosulfonate modification by VP, including the observed compact alterations by the W164S variant, derivatized (nonphenolic) lignosulfonates have been treated in new steady-state experiments. The native VP was able to modify the nonphenolic lignosulfonates however the modifications within the molecular-mass distribution (Additional file 1: Bendazac web Figure S4, green continuous line) and molecular structure of lignins (More file 1: Figure S5b, e) were modest, compared with those observed for the native (partially phenolic) lignosulfonates (Fig. 3a, b, green continuous line, and Fig. 4b, e, respectively). These modifications involve lower-intensity signals in the NMR spectra of nonphenolic hardwood lignosulfonate (the S signal being the exception) and displacement of your Mp inFig. three SEC profiles of softwood (a) and hardwood (b) lignosulfonates treated for 24 h with native VP and its W164S variant and handle devoid of enzyme, and sulfonated polystyrene standards (c). Lignosul fonate samples (12 g L-1) right after a 24h therapy with 1.2 native VP (green line) and its W164S variant (dashes) in presence of 9.five mM H2O2, and the corresponding softwood (red) and hardwood (blue) ligno sulfonate controls with no enzyme, have been analyzed in a Superdex75 column applying 0.15 M NaOH as eluent (0.five mL in-1) and detection at 280 nm. Sulfonated polystyrenes (Mp 78,400, 29,500, ten,200 and 4210 Da, from left to correct) were utilised as molecular mass standards in c (arrow shows the excluded blue dextran elution volume)the SEC profile, although reduced modifications have been observed for the nonphenolic softwood lignosulfonate. In contrast, the SEC profiles of the W164S-treated (green dashed lines) and c.
