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Interaction and Kinetics of Oxidative Biomass Degrading Enzymes Resolved by High-Resolution Techniques

Periodic Reporting for period 2 - OXIDISE (Interaction and Kinetics of Oxidative Biomass Degrading Enzymes Resolved by High-Resolution Techniques)

Reporting period: 2018-09-01 to 2020-02-29

The use of biomass is prioritized to support food and feed supply followed by the conversion of biomass into a broad spectrum of goods and products. Biomass as the only renewable carbon source will play a unique role for the production of organic bulk- and fine-chemicals and biorefineries will deconstruct complex biomass constituents into valuable raw-materials. Current processes for lignocellulose breakdown are unspecific and produce some constituents in poor quality. Specific biocatalysts could achieve optimal segregation together with minimal damage to cellulose and lignin and provide high-quality feedstocks for industry. Naturally occurring fungal oxidoreductases perform this task, but their characterisation – and hence their optimisation for industrial application – is difficult because of the experimental challenges. The mission of the ERC-CoG project OXIDISE is to develop appropriate methods to characterise lignocellulose degrading oxidoreductases, i.e. elucidate their conversion rates and to resolve their distribution and interaction in vicinity of their polymeric substrates. High-resolution techniques will be adapted to specifically detect fungal oxidoreductases like lytic polysaccharide monooxygenase, cellobiose dehydrogenase, laccase, lignin peroxidase, or members of the GMC oxidoreductase superfamily. These enzymes are all involved in the oxidative attack of recalcitrant biopolymers and are present in over 90% of fungal genomes. To overcome problems of current assaying techniques such as their low spatial and temporal resolution, OXIDISE will develop and apply techniques based on microelectrodes, scanning electron microscopy, surface plasmon resonance and fluorescence microscopy thereby pursuing three objectives: 1) study the interaction of all major oxidoreductases secreted by fungi in regard to electron transfer, regeneration of redox species and substrate cascading; 2) resolve the distribution of secreted oxidoreductases on cellulosic and lignocellulosic substrates at high resolution; 3) transfer the developed techniques to natural lignocellulose samples with growing fungal hyphae and study the secreted oxidoreductase activities. OXIDISE strives to establish new techniques to elucidate the kinetics and interactions of oxidoreductases – a long neglected enzyme class for lignocellulose depolymerisation.
The ERC-COG project OXIDISE aims to resolve authentic conversion rates of fungal lignocellulose degrading oxidoreductases when bound to their polymeric substrates and to elucidate their distribution and interaction. Three high-resolution techniques – scanning electron microscopy (SECM), surface plasmon resonance (SPR) and fluorescence microscopy – have been adapted to pursue three main objectives: 1) study the interaction of all major oxidoreductases secreted by fungi in regard to electron transfer, regeneration of redox species and substrate cascading, 2) resolve the distribution of secreted oxidoreductases on cellulosic and lignocellulosic substrates at high resolution, 3) transfer the developed techniques to natural lignocellulose samples with growing fungal hyphae and study the secreted oxidoreductase activities. Therefore, the research activities in OXIDISE have been divided in 3 workpackages (WP).

In WP1 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 chromatographic purification the oxidoreductases cellobiose dehydrogenase, laccase, lytic polysaccharide monooxygenase, glyoxal oxidase and pyranose 2-oxidase as well as hydrolases GH45 and GH12 were obtained in quantities >20 mg for subsequent experiments.

In WP2 detection methods to asses enzymatic activity and the concentration of various enzyme substrates and products were developed. Two sensor electrodes for the detection of the fungal enzymes cellobiose dehydrogenase and laccase, three biosensor electrodes for the detection of the cellobiose and cello-oligosaccharides (cellotriose and cellotetraose), one sensor electrode for the detection of the lignin derived syringyl unit syringol (2,6-dimethoxyphenol), one sensor electrode for the electrochemical measurement of pH, and one sensor electrode for the detection of enzymatically released hydrogen peroxide have been build. These biosensors have been further developed and miniaturized to be used in scanning electrochemical microscopy (SECM) in real time and in situ.

In WP3 the integration of enzymes and methods was performed to study the localization, interaction and kinetics of enzymes on biomass. For example, four ultramicroelectrode biosensors for the detection of enzymatic lignocellulolytic reaction products in plant cells are ready: sensors for the detection in solution of the oxidase reaction product hydrogen peroxide, the methylglyoxal reaction product methylglyoxalic acid (pH), the β-glucosidase reaction product glucose, and the cellobiohydrolase reaction product and cellobiose dehydrogenase reaction substrate.
Novel methodologies that have been developed within the project OXIDISE are: 1) a novel way to modify surface resonance plasmon (SPR) spectroscopy probes by a hemicellulose film to study binding affinities of fungal extracellular enzymes, 2) micro-/nanoelectrodes for SECM for the detection of lingocellulolytic breakdown products, 3) the combination of a video microscope, fluorescence microscope and scanning electrochemical microscope (SECM) in combination with a scanning ion conductance microscope (SICM) to study lignocellulose degradation.

1. SPR is a versatile method to determine the binding of proteins to surfaces. In the OXIDISE project, SPR probes are modified by ultrathin cellulose and lignin films to determine the binding affinity and binding capacity of fungal extracellular enzymes to these materials. One of the polymers present in plants is xyloglucan, a hemicellulose that plays an important role in connecting cellulose fibers to other polymers. A new method to deposit ultrathin xyloglucan films on SPR gold targets via cross-linking of xyloglucan to a self-assembled monolayer of bifunctional thioglycerol in a spin-coating process was developed.

2. The development of new, enzyme based micro- and nanoelectrodes for the detection of enzymatic reaction products found in lignocellulose degradation. 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 is used to modify pyrolized carbon nanoelectrode with diameters down to 500 nm to specifically detect the reaction products of cellulases and -glucosidases with the SECM.

3. The main instrument developed for OXIDISE is a high-resolution SECM/SICM that is capable to investigate enzymatic breakdown processes in wood at the cellular level. To establish such an instrument the OXIDISE team has built an extremely heavy vibration dampened table with a resonance frequency <1 Hz and a two-chamber Faraday cage to suppress mainly electric, but also magnetic fields. Into this external structure an inverted fluorescence microscope, a platform for the SECM/SICM and micromanipulators, as well as a video microscope was mounted. First calibration experiments show that micro- and nanoelectrodes can be positioned with the micromanipulators and moved with a resolution of ~10 nm. No drift of the electrode is observed. The video microscope can follow the enzymatic degradation of cell walls and measuring distances/volumes via z-stacking. The fluorescence microscope can follow the localization of fluorescence-tagged enzymes in the conversion process.