Periodic Reporting for period 1 - ReversE (Modifying Enzyme with Solid-Binding Peptide for Site-specific and Reversible Enzyme Immobilization)
Berichtszeitraum: 2023-08-01 bis 2025-07-31
ReversE (Modifying enzyme with solid-binding peptide for site-specific and reversible enzyme immobilization) aims to address a key question, with a special focus on the longevity of the labile enzyme-based systems, which remains a bottleneck in enzyme-electrode biotechnological applications. Redox enzymes catalyze numerous biological processes, such as hydrogen evolution and uptake, carbon dioxide reduction, and nitrogen reduction, which are also relevant for a variety of applications, including enzymatic biofuel cells and the electrosynthesis of complex molecules. However, enzyme’s limited stability diminishes their potential to be exploited in biotechnological applications. The project’s overall goal is to develop a reversible immobilization approach that can attach enzymes on an electrode surface in a defined orientation and enable its controlled release on demand upon stimulation. This paves the way for enzyme regeneration on the electrode surface, which will be groundbreaking in the context of sustainable deployment of enzyme‒electrode systems. To accomplish this goal, the project is structured around three core objectives.
Objective 1 – To genetically engineer redox metalloenzymes for site-specific immobilization
Objective 2 – To establish methods for releasing bound enzymes from electrodes under mild conditions, preserving the properties of the electrode surface, and allowing new cycles of enzyme immobilization
Objective 3 – To demonstrate the practical use of the reversible binding for device reusability and prolonged operation in energy-conversion applications
Pathway to impact
Scientific: the findings are crucial for fundamental understanding of intricate mechanisms behind the association and dissociation of solid-binding peptide-fused enzyme on the solid electrode surface.
Technological: develop a blueprint for a reusable enzyme-electrode suitable for practical implementation in future technologies.
Industrial & economic: enables the regeneration of devices, making future enzymatic biofuel cells and biosensor technologies more sustainable, which would otherwise be hampered by costly and elaborate surface modifications.
Societal & environmental: the reuse of gold or other solid electrodes supports the objectives of the European Green Deal by minimizing environmental impact and decreasing reliance on critical raw materials.
Objective 1 – To genetically engineer redox metalloenzymes for site-specific immobilization
Objective 2 – To establish methods for releasing bound enzymes from electrodes under mild conditions, preserving the properties of the electrode surface, and allowing new cycles of enzyme immobilization
Objective 3 – To demonstrate the practical use of the reversible binding for device reusability and prolonged operation in energy-conversion applications
Pathway to impact
Scientific: the findings are crucial for fundamental understanding of intricate mechanisms behind the association and dissociation of solid-binding peptide-fused enzyme on the solid electrode surface.
Technological: develop a blueprint for a reusable enzyme-electrode suitable for practical implementation in future technologies.
Industrial & economic: enables the regeneration of devices, making future enzymatic biofuel cells and biosensor technologies more sustainable, which would otherwise be hampered by costly and elaborate surface modifications.
Societal & environmental: the reuse of gold or other solid electrodes supports the objectives of the European Green Deal by minimizing environmental impact and decreasing reliance on critical raw materials.
The main work performed within the project is summarized as follows;
- Model enzymes including Fe-Fe hydrogenase, tungsten-selenocysteine formate dehydrogenase, and eukaryotic nitrate reductase that are involved in critical reactions; a) H2 evolution and uptake, b) CO2 reduction, c) nitrate reduction, were identified and their functional recombinant production in Escherichia coli was successfully established.
- Gold-binding peptides with various amino acid compositions were identified and genetically fused to model enzymes to enhance their affinity for gold and promote electronic coupling on the gold electrode surface.
- The expression, production, and activity of the mutants and wild type in in vitro solution assays were investigated. Genetic incorporation of gold-binding peptides at either the N- or C-terminus of enzymes does not compromise their bioactivity
- The mutants were screened using microarrays with gold as the working electrode, providing a potential platform for assessing direct electron transfer at the enzyme–electrode interface.
- The bioelectrocatalytic properties of enzymes on electrodes were quantified through both direct and mediated electron transfer.
Contingency study: While successful conjugation of enzymes to the electrode surface was achieved, a significant direct-electron transfer signal for the Fe-Fe hydrogenase fused with gold-binding peptides has not yet been realized. Contingency study of truncation of model enzymes, [FeFe]-hydrogenase, tungsten-selenocysteine formate dehydrogenase, and nitrate reductase is still ongoing and is promising for promoting direct electron transfer at the enzyme-electrode interface. Additionally, various redox polymers are available in the host group, which will be explored for the immobilization of enzymes from Work Package 1 onto electrode for hydrogen evolution and oxidation, carbon dioxide uptake, and nitrate biosensing.
- Model enzymes including Fe-Fe hydrogenase, tungsten-selenocysteine formate dehydrogenase, and eukaryotic nitrate reductase that are involved in critical reactions; a) H2 evolution and uptake, b) CO2 reduction, c) nitrate reduction, were identified and their functional recombinant production in Escherichia coli was successfully established.
- Gold-binding peptides with various amino acid compositions were identified and genetically fused to model enzymes to enhance their affinity for gold and promote electronic coupling on the gold electrode surface.
- The expression, production, and activity of the mutants and wild type in in vitro solution assays were investigated. Genetic incorporation of gold-binding peptides at either the N- or C-terminus of enzymes does not compromise their bioactivity
- The mutants were screened using microarrays with gold as the working electrode, providing a potential platform for assessing direct electron transfer at the enzyme–electrode interface.
- The bioelectrocatalytic properties of enzymes on electrodes were quantified through both direct and mediated electron transfer.
Contingency study: While successful conjugation of enzymes to the electrode surface was achieved, a significant direct-electron transfer signal for the Fe-Fe hydrogenase fused with gold-binding peptides has not yet been realized. Contingency study of truncation of model enzymes, [FeFe]-hydrogenase, tungsten-selenocysteine formate dehydrogenase, and nitrate reductase is still ongoing and is promising for promoting direct electron transfer at the enzyme-electrode interface. Additionally, various redox polymers are available in the host group, which will be explored for the immobilization of enzymes from Work Package 1 onto electrode for hydrogen evolution and oxidation, carbon dioxide uptake, and nitrate biosensing.
Within this MSCA postdoctoral fellowship project, the gold-binding peptide enzymes mutants with gold-binding affinity that retain most of enzyme activity were identified, produced, and investigated. These findings lay the groundwork for further investigation into potential enzymes and materials relevant to desired biotechnological applications. Next, the researcher has successfully identified and recombinantly produced two redox metalloenzymes as model enzymes, a tungsten-selenocysteine formate dehydrogenase and a eukaryotic nitrate reductase, which are involved in two critical reactions, CO2 to formate and nitrate reduction, respectively. Specifically, a CO2 reductase is a highly valuable and important enzyme because of its ability to directly convert CO2 to formate. This result is of interest for potential scientific collaborations with the scientific community working on CO2 reduction applications, including biocatalysis, enzymatic cascades, enzymatic electrosynthesis, and light-driven CO2 reduction. In fact, as the production of tungsten-selenocysteine formate dehydrogenase in higher yield capable of CO2 reduction activity is being optimized at the time of writing, this will directly impact contemporary societal challenges (climate change and global warming) by facilitating an important step towards achieving the goal of upcycling CO2 into energy-rich long-chain compounds, representing a substantial opportunity to tackle environmental issues and achieve a circular economy. This aligns with the core ambition of the European Green Deal. In addition, the researcher has successfully established the production of yeast nitrate reductase in E. coli and subsequently worked on its engineering to have an enzyme with increased KM for use in nitrate quantification in plants, and therefore would be of scientific as well as economic impact for the agricultural sector.