WP1: In the Quest of Enzyme Candidates for Optimized Synthetic Processes.
WP Leader: Maria L. Mascotti (IHEM - CONICET)
To identify and engineer enzymes capable of catalysing challenging synthetic transformations with high efficiency and selectivity under continuous-flow regimes, a variety of methodologies will be developed. Combining high-throughput screening and enzyme engineering techniques, we aim to identify novel enzyme candidates and enhance the performance of known enzymes. Key strategies will involve optimizing enzymes for specific chemistries, developing multi-component biocatalysts, and addressing enzyme stability and scalability challenges under continuous-flow regimes.
- Task 1.1. Identification of Novel Enzyme Candidates: This task will focus on identifying enzymes that can selectively catalyse desired synthetic reactions. High-throughput screening methods will be implemented to evaluate large enzyme libraries for their production as recombinant proteins and their ability to perform specific transformations with high efficiency and selectivity under continuous-flow conditions.
- Task 1.2. Enzyme Engineering: Benchmark enzymes will be engineered to improve their stability, selectivity, and catalytic efficiency under continuous-flow conditions. Directed evolution campaigns and ancestral sequence reconstruction will be the primary techniques to engineer enzymes for these applications. These will be complemented with computational methods to filter candidates for further experimental production. In addition, to optimize enzyme functional attributes such as catalytic rate, substrate specificity, and resistance to harsh process conditions (e.g., temperature, pH, solvents), sequence diversity will be explored. Ancestral sequence reconstruction will help to identify the specific substitutions that can improve enzyme function for continuous-flow applications.
- Task 1.3. Biocatalyst Production and Scale-Up: To enable industrial applications, the project will address the appropriate-scale production of engineered biocatalysts. Here the participation of non-academic partner such as GECCO Biotech is key. The latest biotechnological methods for enzyme production, purification, and quality control will ensure that the enzymes are produced efficiently and at the required scale.
WP2: Enzyme Immobilization and Process Optimization towards Continuous Synthesis
WP Leader: Fernando López-Gallego (CIC biomaGUNE)
This WP focuses on developing and optimizing enzyme immobilization techniques to enhance enzyme stability, efficiency, and reusability for continuous-flow synthesis processes, addressing Objective 2. Improving enzyme performance will reduce operational costs and makes enzymatic processes more viable for industrial-scale applications.
- Task 2.1. Exploration of Immobilization Methods: A range of enzyme immobilization techniques will be investigated, including covalent bonding, physical adsorption, entrapment, and encapsulation, to enhance enzyme stability and activity under continuous-flow conditions. Enzyme selection will be based on the variants investigated on WP1. Each method will be evaluated based on its ability to preserve enzyme functionality and prevent deactivation over time.
- Task 2.2. Development of Bio-Hybrid Systems: The hybrid biocatalysts, such as heterogeneous biocatalysts composed of either one isolated enzyme and one whole-cell biocatalyst or one biocatalyst (either isolated enzyme or whole-cell) and one chemical catalyst, will facilitate enzyme immobilization. These materials will provide a stable environment for enzymes, ensuring they remain active during continuous-flow reactions and improving overall process efficiency.
- Task 2.3. Long-Term Stability and Reusability of Immobilized Enzymes: The stability and reusability of heterogeneous biocatalysts will be assessed by conducting extended operational cycles. This includes evaluating enzyme deactivation, reactivation potential, and operational lifespan, and it will be performed in close collaboration with WP3.
WP3: Exploring (Bio)Catalysts Reactivity and Process Intensification in Continuous Flow Systems
WP Leader: Gabriela Oksdath-Mansilla (UNC)
The WP3 overall methodology for optimizing catalysed reactions in continuous-flow systems revolves around integrating both biological and (chemo)catalytic processes to enhance reaction efficiency, scalability, and sustainability. The following steps outline the methodology, including the models, assumptions, and key tasks associated with achieving Objective 3:
- Task 3.1. Catalyst Selection, Reactivity, and Integration of (Chemo)enzymatic Processes: This task focuses on optimizing both biocatalytic and (organo)catalytic systems for continuous-flow reactions. Hybrid systems combining the high specificity of enzymes with the reactivity of organocatalysts will be developed and tested under varying conditions (temperature, pressure, flow rate) to evaluate their catalytic activity, stability, and selectivity. The biocatalytic systems designed in WP2 will be implemented to improve the stability of the enzymes and avoid deactivation during continuous flow operations.
- Task 3.2. Development and Optimization of Flow Reactor Designs: Reactor design is critical to maximizing contact between the catalyst and the reactants, ensuring efficient heat and mass transfer. We will implement advanced flow reactor systems, including 3D printed reactors, packed-bed reactors, and spinning reactors, allowing enhanced mixing and surface area contact. These designs aim to improve the overall reaction rates and efficiency.
- Task 3.3. Evaluation of Scalability of the Telescoped Processes: To scale up the catalytic process, we will assess key reactor dynamics such as heat and mass transfer, pressure drops, and flow characteristics. These factors will be evaluated across different reactor sizes to ensure the process can be effectively scaled without losing efficiency or compromising safety.
- Task 3.4. Energy and Environmental Impact Assessment: Energy consumption and environmental impact will be critically evaluated by comparing continuous-flow processes to traditional batch processes. Energy consumption will be monitored by analysing reactor heating/cooling demands and evaluating overall energy efficiency. The environmental impact will be assessed regarding waste generation, solvent use, and the overall sustainability of the process.