Enzyme-encapsulated protein frameworks for optimized cascade biocatalysis - Project Summary While incorporating multiple enzymes into a solid matrix holds significant potential for enhancing catalytic efficiency in complex cascade reactions, progress in this direction is hampered by two critical challenges: (1) a lack of understanding of how structural factors such as substrate accessibility and spatial arrangement of multi- enzyme assemblies affect overall catalytic efficiency, and (2) enzyme instability, which limits the long-term reuse of biocatalysts. To address these challenges, we will develop protein framework materials self- assembled from enzyme-encapsulated virus-like particles (VLPs) and evaluate their catalytic activities while systematically modulating the structural factors of these frameworks. The scientific objective of this research is to identify the extent to which structural factors, such as substrate accessibility and enzyme spatial arrangement in the frameworks, affect overall catalytic efficiency. We hypothesize that the catalytic efficiency of multi-enzyme protein frameworks can be significantly improved through the rational design of these structural factors. As a model cascade reaction, we will use a pathway from glucose to pyruvate because cell-free multi- enzyme pathway for this cascade reaction has been well established. We will test the hypothesis through three aims: (1) encapsulate three key enzymes involved in the pathway within VLPs and evaluate their catalytic activities and stabilities in comparison with free enzymes, (2) investigate how the structure (random or ordered) and interstitial spacing of single-enzyme frameworks affect catalytic efficiency, and (3) investigate how spatial arrangement of multiple populations of enzymes within the frameworks affect catalytic efficiency. Through these aims, we will identify key structural factors which affect overall catalytic efficiency of multi-enzyme assemblies. Since the assembly of VLPs into the framework structure is independent of the enzymes encapsulated within the VLPs, the findings from this research will have broad applicability for a wide range of cascade reactions, enabling more efficient synthesis of biologically active and pharmaceutical molecules. Furthermore, this project will provide valuable hands-on research training for students at California State University, Fresno (CSU-Fresno), many of whom are from historically underrepresented communities in higher education. Collectively, this project will strengthen both the research capabilities and educational efforts at CSU-Fresno.
