Elucidation of the structure of cellulolytic enzyme lignin

Abstract

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.

Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins (and an indicator for cinnamoyl CoA reductase deficiency)

“…The control and CCR-downregulated poplar samples were those used in a companion paper (Leplé et al, 2007). Briefly, lignins were prepared by ball-milling solvent-extracted cell walls, digesting away most of the polysaccharides with crude cellulases, and dissolving and acetylating the so-called 'cellulolytic enzyme lignin' (Hu et al, 2006) using our cell-wall dissolution method . Acetylated lignins (approximately 60 mg) were dissolved in CDCl 3 for NMR.…”

HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state 10th EWLP, Stockholm, Sweden, August 25–28, 2008

“…Other approaches are: 1) subjecting the milled wood to enzymatic treatment with cellulolytic enzymes to remove most of the carbohydrate components (Chang et al 1975;Wu and Argyropoulos 2003;Holtman et al 2004;Hu et al 2006); 2) completely dissolving ball-milled wood in a solvent system (dimethylsulfoxide, DMSO, and N-methylimidazol, NMI) followed by precipitation in dioxane/water in the course of which lignin and carbohydrate fractions are separated (Fasching et al 2008); or 3) isolating most of the lignin as lignin-carbohydrate complexes after endoglucanase treatment (Henriksson et al 2007). However, it is well recognized that these lignin preparations, in particular the MWL due to its low yield, represent only a part of the native lignin in the wood cell wall and may not be representative of the whole lignin present.…”

“…Indeed, it has also been demonstrated that MWL can undergo some structural modifications during isolation, especially during the milling process (Fujimoto et al 2005;Guerra et al 2006;Hu et al 2006;Balakshin et al 2008). Because lignin is intimately interpenetrating the other major components (cellulose and hemicelluloses), it is obvious that its truly native form can only be studied by analytical methods applicable directly on the whole plant material.…”

Determination of Arylglycerol−β-Aryl Ether Linkages in Enzymatic Mild Acidolysis Lignins (EMAL): Comparison of DFRC/31P NMR with Thioacidolysis

“…13,33 The high relative contents of lignin from ML contaminate lignin preparations obtained at low yields, explaining the structural differences observed in MWL released during the early stages of ball milling, when low yields are obtained. 13 However, the effects of such contamination diminish as the yield of lignin from the S 2 increases. Since the EMAL yields were always higher than 20%, regardless of the ball-milling time evaluated, the contamination by ML lignin is minimized and the EMALs isolated at yields ranging from 20% to 62% have similar structures ( Figure 4).…”

“…[7][8][9]13 Intensive milling protocols offered by vibratory-and orbital-milling devices provide higher lignin yields within relatively short milling intervals, although at the expense of the integrity of the lignin macromolecule and associated condensation and oxidation reactions. 8,9,13,14 Low-intensity milling minimizes structural changes during wood pulverization, but moderate yields are usually achieved under such conditions. As such, the representative nature of the resulting lignin may be questionable.…”