Heterologous expression of lignin-degrading enzymes for biological depolymerization of lignin

Dissertation, RWTH Aachen University, 2018; Aachen 1 Online-Ressource (IV, 138 Seiten) : Illustrationen, Diagramme (2018). = Dissertation, RWTH Aachen University, 2018
The decrease in the amount of the fossil fuels and significant environmental issues arisen from their consumption drives our currently fossil fuel-based society towards more sustainable sources. Lignocellulose is the most abundant sustainable biomass on earth. It has a very promising potential for the production of renewable fuels and chemicals. Two of the main components of lignocellulosic material, cellulose and hemicellulose, are evaluated for the use in industrial processes since they can be degraded easily by identified enzymes and chemical hydrolysis. However, at present very little is known about lignin, the third component of the lignocellulosic material. Insufficient knowledge and technology in lignin degradation, in part due to its recalcitrant nature, prevents industry from fully exploiting the potential of this polymer. The effort shown in the last decades about enzymatic lignin degradation has provided many bacterial and fungal organisms, eventually specific enzymes, which take part in efficient lignin degradation in the nature. Promising enzymes from bacteria and fungi have been identified; however, many drawbacks and difficulties stand between these enzymes and efficient exploitation of them in the industrial processes. The main objective of this dissertation was to establish a versatile set of knowledge-based lignin-degrading enzymes from oxidoreductases to β-etherases and to achieve the heterologous expression of those enzymes to develop efficient methods for enzymatic lignin degradation. For this purpose, individual lignin-degrading enzymes were selected and heterologously expressed initially in E. coli. Expression yields of certain enzymes were improved by changing the host to P. fluorescens. The recombinant enzymes were purified via immobilized metal affinity chromatography and characterized using simple lignin-model substrates. Following basic characterization assays, which helped to understand the best conditions for the activity assays, the recombinant enzymes were tested with more complex lignin-model compounds and polymeric Kraft lignin. Two different immobilization methods were applied successfully to a laccase as a model for the recycling of these enzymes to reduce the production costs. Eventually a directed evolution library for a DyP-type peroxidase was created and an ultra-high-throughput method based on FACS was developed to screen the library for the variants with higher H2O2 stability. This dissertation presents efficient heterologous expression of bacterial lignin-degrading enzymes and offers ways to obtain a decent amount of recombinant enzymes. Most of the expression yields reported here represent the highest yields achieved so far in the literature, which gives the flexibility of performing a wide range of activity assays with simple and complex lignin-model compounds and polymeric lignin samples from different sources. Immobilization and protein engineering were applied to the selected enzymes to develop advanced methods for improving the activity and stability of lignin-degrading enzymes economically.
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