Directed Immobilization of Horseradish Peroxidase Using a Covalently Attached Competitive Inhibitor
Anne Muschter1,2, Sophia Rosencrantz1,3, Takwa Chouki1,2
1Division of Life Science & Bioprocesses, Fraunhofer Institute for Applied Polymer Research IAP, Geiselbergstr. 69, 14476 Potsdam, Germany.
Molecules (Basel, Switzerland)
|August 13, 2026
Summary
Researchers developed a novel enzyme immobilization method using Remazol Brilliant Blue R (RB) to orient horseradish peroxidase (HRP). This directed approach significantly enhances enzyme activity for biosensor applications.
Area of Science:
- Biochemistry
- Materials Science
- Chemical Engineering
Background:
- Enzyme immobilization is crucial for developing stable and reusable biocatalytic systems.
- Controlled enzyme orientation enhances specific activity and performance in biosensors and biocatalysis.
- Current methods often lack precision or require genetic modification, limiting enzyme structure and function.
Purpose of the Study:
- To develop a site-directed immobilization strategy for horseradish peroxidase (HRP) using a competitive inhibitor.
- To achieve controlled enzyme orientation on functionalized surfaces without genetic manipulation.
- To evaluate the impact of directed immobilization on enzyme specific activity and compare it to random immobilization.
Main Methods:
- Site-directed immobilization using Remazol Brilliant Blue R (RB) as a competitive inhibitor.
- Covalent binding of RB to poly(ethylene-alt-maleic anhydride) (PEMA)-coated glass substrates.
- Enzyme loading via active site interaction followed by secondary surface functionalization for covalent attachment.
- Characterization using Atomic Force Microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS), Nuclear Magnetic Resonance (NMR), and Attenuated Total Reflectance Infrared (ATR-IR) spectroscopy.
Main Results:
- Successful covalent immobilization of HRP with controlled orientation on PEMA-coated surfaces.
- Characterization confirmed interfacial layer assembly and successful RB acetylation.
- Directed immobilization yielded HRP loadings with approximately twofold higher specific activity compared to non-directed methods.
- The inhibitor-mediated method preserved the native enzyme structure.
Conclusions:
- The developed inhibitor-mediated strategy provides a straightforward platform for enhancing enzyme performance through controlled immobilization.
- This method offers a non-genetic approach to improve enzyme activity and stability.
- The technique shows significant promise for applications in biosensors, biocatalysis, and enzyme-based devices.
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