Effects of lignolytic enzymes and the Fenton reaction on lignin depolymerization assayed using lignin films

Depolymerization of lignin by white rot fungi involves oxidative enzymes (laccases (Lac), lignin peroxidases (LiP), and manganese peroxidases (MnP)) as well as small molecules that function as mediators.  Despite identification of the major enzyme components, efficient lignin depolymerization has not been reconstituted in vitro.  Modification of lignin by brown rot fungi involves the Fenton reaction, which generates highly reactive hydroxyl radicals.   An important complicating factor in both cases is that oxidative reactions can polymerize as well as depolymerize lignin, with the relative rates affected by many variables.

The effect of ligninolytic enzymes on lignin is typically assayed by measuring changes in molecular weight distribution of synthetic linear lignin polymers using chromatography. This is most commonly done using C14-labelled dehydrogenation polymers of sinapyl alcohol and coniferyl alcohol. 

We present a new approach to assaying the effects of oxidative enzymes and catalytic reactions on high molecular weight lignin.  The approach utilizes films of lignin where mass loss (or gain) can be detected by ellipsometry and chemical changes can be revealed with vibrational spectroscopy.  In addition, increased polymerization or crosslinking of the lignin particles in the films is revealed by an increased resistance to detergent solution.

Initial results indicate that laccase-activated ABTS results in polymerization or crosslinking of the lignin particles.  ABTS-treated films are highly stable and were treated with LiP/veratryl alcohol, MnP/Mn³⁺, and laccase as well as with the Fenton reagent.  The Fenton reagent was effective in solubilizing a substantial portion of the film after an oxidative pretreatment using H2O2.