Elucidation of the structure of cellulolytic enzyme lignin

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 beta-O-4 structures in lignin. However, even after extensive ball milling, less than 25% of the beta-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.