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Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing
Catarina Barbosa1, Célia M Silveira1, Diogo Silva1
1Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa, Av. da República, 2780-157, Oeiras, Portugal.
Biosensors & Bioelectronics
|February 15, 2020
Summary
The wild-type and 29E4 variant of dye-decolorizing peroxidase (PpDyP) show high sensitivity and stability, making them excellent candidates for hydrogen peroxide (H₂O₂) biosensor development. These PpDyP variants offer superior performance compared to traditional horseradish peroxidase (HRP) biosensors.
Area of Science:
- Biochemistry and Biotechnology
- Biosensor Technology
- Enzyme Engineering
Background:
- Hydrogen peroxide (H₂O₂) detection is crucial in various fields, necessitating sensitive and stable biosensors.
- Dye-decolorizing peroxidase (PpDYP) from Pseudomonas putida MET94 is explored as an alternative to conventional peroxidases.
- Directed evolution (DE) was employed to engineer PpDYP variants with potentially enhanced biosensing capabilities.
Purpose of the Study:
- To investigate the structural and electrocatalytic properties of wild-type (wt) PpDYP and its DE variants.
- To evaluate their suitability for developing highly sensitive and stable H₂O₂ biosensors.
- To compare the performance of PpDYP-based biosensors with existing horseradish peroxidase (HRP) counterparts.
Main Methods:
- Enzyme immobilization and characterization of wild-type (wt) PpDYP and three directed evolution (DE) variants.
- Resonance Raman (RR) and surface-enhanced RR (SERR) spectroscopy for structural analysis in solution and immobilized states.
- Electrochemical analysis to determine electrocatalytic properties and biosensor performance.
Main Results:
- Wt PpDYP and the 29E4 variant demonstrated excellent dynamic response range (1-200 μM H₂O₂), rapid response times (2 s), and high sensitivity (1.3-1.4 A·M⁻¹·cm⁻²).
- These variants exhibited good selectivity and long-term stability, outperforming HRP-based biosensors by 1-4 orders of magnitude in sensitivity.
- The 6E10 and 25F6 variants showed reduced activity and inhibition by H₂O₂ upon electrode adsorption, indicating limitations for biosensing applications.
Conclusions:
- Wt PpDYP and the 29E4 variant are highly promising for H₂O₂ biosensor development due to their superior sensitivity, stability, and response characteristics.
- PpDYP-based biosensors represent a significant advancement over traditional HRP-based devices.
- The study supports the development of wt or 29E4 PpDYP-based biosensors as a valuable alternative for H₂O₂ detection.
