Insights into the reaction of laccase/mediator systems with insoluble lignin

Our group has developed a new multiplexed methodology for detecting and quantifying breakdown of high molecular weight polymeric lignin.¹  The assay utilizes films of lignin cast onto silicon wafers. Enzymatic or catalytic activity that results in solubilization of lignin fragments is detected through decrease in film thickness.  A silicone block in the form of a standard 96-well plate is pressed against the silicon wafer to create a highly multiplexed format.  The assay is highly sensitive, quantitative, and compatible with standard 96-well plate robotic fluid handling systems.  This assay enables rapid assessment of the ligninolytic potential of enzymes, catalytic chemistries, and organisms and microbial communities. Here, we report results for breakdown of polymeric lignin films by laccase from T. versicolor along with mediators ABTS and HBT. To complement the film thickness measurements the molecular weight profiles of released lignin oligomers were analyzed by GPC and Maldi-Tof-MS.  The results for the laccase / mediator systems show that laccase is effective at releasing soluble products from lignin only for a limited range of laccase concentration and only when a mediator is present. This suggests that a highly activated laccase system may prohibit the depolymerization of lignin. Further insights into the mediator-assisted mechanisms of laccase in degrading lignin were obtained through studies of depolymerization and/or repolymerization of nonphenolic and phenolic lignin dimers.

1. Kent et al  Green Chemistry 2015, 17, 4830