Periodic Reporting for period 4 - OXIDISE (Interaction and Kinetics of Oxidative Biomass Degrading Enzymes Resolved by High-Resolution Techniques)
Reporting period: 2021-09-01 to 2022-02-28
The goal of the ERC-CoG project OXIDISE was to develop appropriate methods to characterise lignocellulose degrading oxidoreductases to elucidate their conversion rates and to resolve their distribution and interaction in vicinity of their polymeric substrates. High-resolution techniques were adapted to specifically detect fungal oxidoreductases and hydrolytic enzymes that are involved in the depolymerization of recalcitrant biopolymers and are present in over 90% of fungi. To overcome problems of current detection and assay techniques, such as their low spatial and temporal resolution, OXIDISE developed and applied techniques based on microelectrodes, scanning electron microscopy, surface plasmon resonance and fluorescence microscopy.
The results can be concluded as follows: Enzymes that work together to degrade a certain polymer in the cell wall are co-localised. Their close vicinity supports their interaction. In this way cascadic reactions are performed at a higher speed. The binding sites and the binding affinity of enzymes on different parts of plant cell walls are fine-tuned to specifically bind to the layer of the cell wall where the substrate concentration is highest. But also the desorption of an enzyme from a partially degraded substrate is necessary to allow subsequent enzymes to act. Fungal hyphae degrade wood during their growth and oxidoreductases play an important role during the initial attack and degradation of plant cell walls.
Extracellular lignocellulosic enzymes from two model organisms, the white-rot Phanerochaete chrysosporium and the brown-rot Fomitopsis pinicola, were produced by fermentation in the expression hosts Trichoderma reesei, Pichia pastoris or Escherichia coli. After fermentation and purification the oxidoreductases cellobiose dehydrogenase, laccase, lytic polysaccharide monooxygenase, glyoxal oxidase and pyranose 2-oxidase as well as hydrolases GH45 and GH12 were fluorescence-tagged. Meanwhile, detection methods to asses enzymatic activity and the concentration of various enzyme substrates and products were developed. Four different ultramicroelectrode biosensors for the detection of enzymatic lignocellulolytic reaction products in plant cells were developed: (i) for the detection of the oxidase reaction product hydrogen peroxide, (ii) oxalic acid, (iii) the β-glucosidase reaction product glucose, and (iv) the cellobiohydrolase reaction product and cellobiose dehydrogenase reaction substrate cellobiose.
The results have been disseminated in 23 scientific articles, as well as in numerous conference contributions such as posters and oral presentations. The developed, biosensor-based scanning electrochemical microscope method will be exploited to study biocatalytic reactions on polymeric and/or solid substrates in basic research projects, but also in industrial development projects.
The second major innovation was the development of micro- and nanoelectrodes for SECM to detect of lingocellulolytic breakdown products. In SECM measurements, the ultimate resolution of images or detection sensitivity depends primarily upon the electrodes tip size and shape to enhance mass-transport and reduce the capacitive response for an increased signal-to-noise ratio and temporal resolution. Biosensors with cellobiose dehydrogenase (detecting cellobiose) and glucose oxidase (detecting glucose) were based on novel redox polymers. The redox polymer/biocatalyst mixture was used to modify pyrolised carbon nanoelectrode with diameters down to 500 nm to specifically detect the reaction products of cellulases and glucosidases with the SECM.
The last major innovation was the combination of a video microscope, fluorescence microscope and scanning electrochemical microscope (SECM) to study the action of enzymes on plant cell walls. The main instrument built for the OXIDISE project was a high-resolution SECM that is capable to investigate enzymatic breakdown processes in plant cell walls. This instrument allows the precise positioning of ultramicroelectrodes with a tip diameter as small as 500 nm above plant cell walls. The amount of bound enzymes and their catalytic activity could be determined In combination with fluorescent imaging. In later experiments the secretion of enzymes by growing fungal hyphae was visualized and measured. These experiments go far beyond the state-of-the-art in enzymology and will be continued in future projects to investigate the interaction of enzymes in extracellular reaction pathways on solid materials.