A quantitative approach to characterize lignin-carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.

Keywords Milled wood lignin Cellulolytic enzyme lignin Enzyme lignin Liquidambar styraciflua Picea abies Sweetgum Spruce Schlüsselwörter (Sachgebiete) B j örkman-Lignin Cellulolytisch.es Enzym-Lignin Enzym-Lignin Liquidambar styraciflua Picea abies Amberbaum Fichte Comparative Studies on Cellulolytic Enzyme Lignin and Milled Wood Lignin of Sweetgum and Spruce SummaryMilled wood lignin (MWL) and Cellulolytic enzyme lignin (CEL) were isolated from the same batch of ball milled sapwood of sweetgum and Norway spruce. Ball milling decreased the molecular weight and increased the -carbonyl and the phenolic-hydroxyl content of lignin. Thus, the content of these functional groups in the original lignin in wood is less than that of isolated MWL and CEL. Compared to MWL, CEL of each species was very similar in elemental composition and both UV and IR spectra. A slightly greater extent of condensation and, in the case of sweetgum, a slightly smaller ratio of syringyl-to guaiacyl-propane units were found in MWL than in CEL. This indicates probable structural differences in lignin in various regions of wood cell walls. Despite these small differences, however, we conclude that MWL is adequately representative of the total lignin in wood but CEL is preferable for future studies of lignin structure because it is more representative of the total lignin in wood and can be obtained in good yield with less degradation due to ball milling. Vergleichende Untersuchungen an Cellulase-Enzym-Lignin und Björkman-Lignin von Sweetgum und Fichtenholz ZusammenfassungSplintholz von Sweetgum (Liquidambar styraciflua L.) und Fichte (Picea abies L.) wurde in einer Kugelschwingmühle gemahlen. Aus jeweils denselben Ansätzen wurde Björkman-Lignin ("Milled Wood Lignin", MWL) und durch Ein Wirkung von Cellulase freigelegtes Lignin (Cellulolytisches Enzym-Lignin, CEL) isoliert. Der Mahlvorgang führte zur Verminderung des Molekulargewichts und zur Erhöhung des Gehaltes an a-Carbonylgruppen und phenolischem Hydroxyl der untersuchten Lignine. Der Vergleich von MWL mit CEL beider Arten zeigte die große Ähnlichkeit dieser Ligninpräparate in bezug auf Elementarzusammensetzung und spektrale Eigenschaften (IR, UV). Die MWL zeigten einen etwas höheren Kondensationsgrad und das MWL von Sweetgum ein etwas niedrigeres Verhältnis von Syringylpropan-zu Guajacylpropaneinheiten als die CEL. Dies dürfte auf die unterschiedliche Zusammensetzung der Lignine in verschiedenen Teilen der Zellwand zurückzuführen sein. Trotz dieser geringen Unterschiede ergibt sich, daß MWL zureichend repräsentativ für das gesamte Lignin des Holzes ist. Trotzdem ist CEL dem MWL vorzuziehen, da es in noch höherem Grade dem Lignin in situ entspricht und außerdem in guten Ausbeuten auch bei geringerer Mahldauer auf schonende Weise erhalten werden kann.

The structures of milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and residual lignin (REL) from a loblolly pine were analyzed using a modified derivatization followed by reductive cleavage (DFRC) method developed to allow the quantitative determination of three different structural monomeric products originating in lignin: phenolic beta-O-4, alpha-O-4, and etherified beta-O-4 structures. Results show that MWL and CEL are structurally identical, with an increased phenolic beta-O-4 content compared to that of the original Wiley milled wood. These results indicate that the portion of lignin linked to carbohydrates and that not linked to carbohydrates are structurally the same. Modified DFRC analysis of the effect of ball milling on the structure of lignin in wood, MWL, CEL, and REL indicate that vibratory ball milling does not change the lignin structure provided certain precautions are taken. Specifically, dry vibratory ball milling under a nitrogen atmosphere causes substantial structural changes including condensation, whereas vibratory ball milling in toluene had little effect on the lignin structure. This indicates that the structural differences observed in MWL and CEL arise because of the extraction procedure, which preferentially extracts phenolic lignin structures. MWL and CEL are representative of the total lignin in wood; however, due primarily to the solvent extraction process, higher phenolic hydroxyl contents are observed. Nitrobenzene oxidation showed structural results similar to those from the modified DFRC method.

The impact of lignin-derived inhibition on enzymatic hydrolysis is investigated by using lignins isolated from untreated woods and pretreated wood pulps. A new method, biomass reconstruction, for which isolated lignins are precipitated onto bleached pulps to mimic lignocellulosic biomass, is introduced, for the first time, to decouple the lignin distribution issue from lignin chemistry. Isolated lignins are physically mixed and reconstructed with bleached pulps. Lignins obtained from pretreated woods adsorb two to six times more cellulase than lignins obtained from untreated woods. The higher adsorption of enzymes on lignin correlates with decreased carbohydrate conversion in enzymatic hydrolysis. In addition, the reconstructed softwood substrate has a lower carbohydrate conversion than the reconstructed hardwood substrate. The degree of condensation of lignin increases significantly after pretreatment, especially with softwood lignins. In this study, the degree of condensation of lignin (0.02 to 0.64) and total OH groups in lignin (1.7 to 1.1) have a critical impact on cellulase adsorption (9 to 70%) and enzymatic hydrolysis (83.2 to 58.2%); this may provide insights into the more recalcitrant nature of softwood substrates.

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Cellulolytic enzyme lignin (CEL) and milled wood lignin (MWL) were prepared by three different ball-milling methods. The structure of CEL at various yields was elucidated and compared with MWL using wet chemical analysis, FTIR and solution-state NMR techniques. Results show that ball milling of wood degrades β-O-4 structures in lignin. However, even after extensive ball milling, less than 25% of the β-O-4 structures were degraded. The extent of degradation was less for softwood than for hardwood lignin. Extractable lignin yield, either MWL or CEL, was the best way to assess the extent and effect of ball milling. CEL is preferred over MWL, as it can be isolated in higher yield with less degradation. CEL was isolated at yields ranging from 20% to 86%. Over this range the CEL had similar structures, suggesting that lignin in the secondary wall is uniform in structure. The residual enzyme lignin (REL) was structurally different from CEL and may originate mainly from the middle lamella. In this paper we propose a new procedure for the isolation of lignin for use in structural studies, whereby wood is sufficiently milled and successively extracted to produce three lignin fractions representing the total lignin in wood.

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