Ment with native VP (Fig. 4b, e) the intensity in the signals with the different aromatic ligninunits (G, S, S and S) and side-chain interunit linkages (A, A, B and C) decreased simultaneously, sustaining comparable linkage percentages. Nonetheless, the methoxyl numbers per unit improved up to twofold. In the hardwood lignosulfonate, this was accompanied by greater abundance of C-oxidized syringyl units (S) with respect to total syringyl units, even though the SG ratio also elevated (from 2.0 inside the handle to three.5 in the 24-h treated sample). Concerning side-chain signals, only those from the main sulfonated -O-4 substructures (A, A and a) remained in the softwood lignosulfonate, even though 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 be Antipain (dihydrochloride) Inhibitor observed within the hardwood lignosulfonate, albeit with low intensities. Much more interestingly, in the lignosulfonates treated for 24 h with the W164S variant (Fig. 4c, f ) only minor adjustments in the aliphaticaromatic HSQC signals have been observed (spectra with equivalent intensities of most signals, and only slight increases of methoxyl content and SG ratio compared with the manage).S zJim ez et al. Biotechnol Biofuels (2016) 9:Page 5 ofaPhenolic-AcAlcoholic-AcaA280 ( )010 11 12 13 14 15 16 17b2.2.two.1.HA280 ( )b0 5 6 7 8 9 10 11 12 13 14 15 16 17cA280 ( )two.two two.1 two.0 1.H10 11 12 13 14 15 16 17 18 Volume (mL)Fig. two Lignosulfonate permethylation: 1HNMR analysis just after second ary acetylation confirming the prior full 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 purpose of further investigating lignosulfonate modification by VP, such as the observed compact adjustments by the W164S variant, derivatized (nonphenolic) lignosulfonates have been treated in new steady-state experiments. The native VP was capable to modify the nonphenolic lignosulfonates however the changes within the molecular-mass distribution (Extra file 1: Figure S4, green continuous line) and molecular structure of lignins (Added file 1: Figure S5b, e) have been modest, compared with these observed for the native (partially phenolic) lignosulfonates (Fig. 3a, b, green continuous line, and Fig. 4b, e, respectively). These modifications contain lower-intensity signals in the NMR spectra of nonphenolic hardwood lignosulfonate (the S signal becoming 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 without enzyme, and sulfonated polystyrene standards (c). Lignosul fonate samples (12 g L-1) after a 24h therapy with 1.two native VP (green line) and its W164S variant (dashes) in presence of 9.five mM H2O2, as well as the corresponding softwood (red) and hardwood (blue) ligno sulfonate controls with no enzyme, were analyzed in a Superdex75 column utilizing 0.15 M NaOH as eluent (0.five mL in-1) and detection at 280 nm. Sulfonated polystyrenes (Mp 78,400, 29,500, 10,200 and 4210 Da, from left to right) had been utilised as molecular mass requirements in c (arrow shows the excluded blue dextran elution volume)the SEC profile, although decrease modifications were observed for the nonphenolic softwood lignosulfonate. In contrast, the SEC profiles with the W164S-treated (green dashed lines) and c.