Potential applications of laccase-mediator-systems for biorefinery purposes
- Anwendungsmöglichkeiten von Laccase-Mediator-Systemen für Bioraffinerie-Prozesse
Roth, Simon; Spieß, Antje (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen, 2016
Within this project potential applications of fungal laccases in combination with redox mediators (so-called laccase-mediator-systems, LMS) for biorefinery purposes have been investigated. The project was part of the cluster of excellence “Tailor-made Fuels from Biomass” (TMFB) at RWTH Aachen University.Modern biorefinery concepts focus on lignocellulosic biomass as a feedstock for the production of next generation biofuels and platform chemicals. Lignocellulose is a recalcitrant composite consisting of several tightly packed components which are stuck together by the phenolic polymer lignin hampering the access to the carbohydrate compounds of biomass. Therefore, the processing of lignocellulose requires an initial, energy-intensive pretreatment for disintegration. In the context of this work the application of LMS for biomass pretreatment was addressed. It was demonstrated that the enzymatic pretreatment of beech wood with LMS has an enhancing influence on the subsequent enzymatic cellulose hydrolysis. However, this effect was only observed when LMS pretreatment was combined with subsequent freeze-drying and increased hydrolysis yields by a factor of 1.3. It is assumed that freeze-drying loosens up the structure of lignocellulose and thereby increases its surface but simultaneously also exposes more lignin which unproductively binds cellulases. LMS pretreatment most likely reduces unproductive binding of cellulases to the biomass surface by oxidative modifications of the lignin enhancing the hydrolysis performance. Adsorption studies using the fluorescent protein mCherry confirmed that LMS pretreatment significantly reduces unproductive binding of mCherry to the surface of beech wood. With respect to the obtained results LMS pretreatment is considered as promising technique to condition physico-chemically pretreated biomass for the subsequent enzymatic cellulose hydrolysis. For the characterization of biomass regarding the accessibility of the cellulose for cellulases and the impact of different pretreatment methods on this parameter fluorescent protein probes consisting of a cellulose-binding module (CBM) from C. fimi and a fluorescent reporter protein were developed and tested. However, the quantification of cellulase binding sites was not successful since unspecific adsorption of the protein probes to the biomass surface masked the CBM-specific cellulose binding. Furthermore, the application of LMS-catalyzed processes for enzymatic lignin depolymerization in the context of modern biorefineries was validated. First, suitable reaction conditions had to be identified. Therefore, the influence of different organic solvent as well as two redox mediators on the stability of two fungal laccases was investigated. From the obtained results the best possible reaction conditions were derived and applied for further experiments regarding the enzymatic depolymerization of Organosolv lignin with LMS. The influence of LMS on Organosolv lignin was analyzed using gel permeation chromatography (GPC). However, under none of the tested reaction conditions LMS-catalyzed lignin decomposition was observed, rather, incubation of Organosolv lignin with LMS leads to polymerizing reactions increasing the molecular weight of lignin.