Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades
Idania L López1, Mercedes Sánchez-Costa1, Alejandro H Orrego1
1Heterogeneous Biocatalysis laboratory Center for Cooperative Research in Biomaterials (CIC biomaGUNE) - Basque, Research and Technology Alliance (BRTA), Paseo de Miramón, 182., 20014, Donostia-San Sebastián, Spain.
Angewandte Chemie (International Ed. in English)
|July 22, 2024
Summary
Developing efficient immobilized biocatalysts for multi-enzyme systems is challenging. This study introduces a high-throughput screening method and software to rapidly identify optimal immobilization protocols, accelerating enzyme pathway development for chemical manufacturing.
Area of Science:
- Applied biocatalysis and enzyme immobilization.
- Chemical engineering and process development.
- High-throughput screening methodologies.
Background:
- Enzyme immobilization is crucial for biocatalyst separation, recovery, and reuse in applied biocatalysis.
- Developing a unified immobilization protocol for multi-enzyme systems is complex, often requiring extensive trial-and-error.
- Efficient heterogeneous biocatalysts are essential for sustainable chemical manufacturing.
Purpose of the Study:
- To develop a rapid and systematic method for screening enzyme immobilization protocols.
- To identify optimal immobilization chemistries for diverse enzymes within a multi-enzyme system.
- To accelerate the prototyping of immobilized multi-enzyme pathways for industrial applications.
Main Methods:
- Utilized a 96-well microtiter plate format to screen 17 carriers and 21 immobilization protocols for up to 18 enzymes.
- Incorporated activity and stability assays to evaluate biocatalyst performance.
- Developed a Python-based application (CapiPy) for rationalizing screening data.
- Scored screening results to determine a consensus immobilization protocol.
Main Results:
- Successfully screened multiple immobilization conditions for a large number of enzymes simultaneously.
- Identified optimal immobilization strategies leading to highly active and stable heterogeneous biocatalysts.
- Assembled an immobilized four-enzyme system for the conversion of vinyl acetate to (S)-3-hydroxybutyric acid using the consensus protocol.
- Demonstrated the effectiveness of the CapiPy application in data analysis and protocol selection.
Conclusions:
- The developed high-throughput screening methodology significantly accelerates the identification of optimal immobilization protocols for multi-enzyme systems.
- This approach enables faster prototyping and development of immobilized enzyme pathways for chemical manufacturing.
- The CapiPy software provides a valuable tool for rationalizing and optimizing enzyme immobilization strategies.
