Structure-Guided Immobilization of an Evolved Unspecific Peroxygenase

Area of Science:

• Biocatalysis
• Protein Engineering
• Immobilization Techniques

Background:

• Unspecific peroxygenases (UPOs) are versatile enzymes with inherent mono(per)oxygenase capabilities.
• Enzyme immobilization is crucial for enhancing biocatalyst stability and reusability in industrial applications.
• Controlling enzyme orientation during immobilization is key to preserving catalytic activity.

Purpose of the Study:

• To develop a method for covalently immobilizing a laboratory-evolved yeast-secreted UPO.
• To ensure enzyme activity and proper orientation through a specific surface mutation (S221C).
• To evaluate the distribution and biochemical characteristics of the immobilized UPO.

Main Methods:

• Site-directed mutagenesis to introduce the S221C mutation for single disulfide bridge formation.
• Covalent immobilization of the engineered UPO onto activated carriers via one-point attachment.
• Fluorescence confocal microscopy for assessing enzyme distribution.
• Biochemical characterization of the immobilized biocatalyst.

Main Results:

• Successful one-point covalent immobilization of the engineered UPO was achieved.
• The S221C mutation facilitated controlled orientation and disulfide bond formation with the support.
• Homogeneous enzyme distribution was confirmed across different carrier materials.
• The immobilized UPO retained its biochemical activity, demonstrating its potential for practical use.

Conclusions:

• The developed immobilization strategy effectively preserves UPO activity and ensures controlled orientation.
• This method offers a robust platform for creating stable and reusable UPO biocatalysts.
• The immobilized UPO presents a promising tool for advancements in applied synthetic chemistry.