Self-Immobilized Putrescine Oxidase Biocatalyst System Engineered with a Metal Binding Peptide

Area of Science:

• Biocatalysis and Enzyme Engineering
• Surface Science and Nanotechnology

Background:

• Flavin oxidases are efficient biocatalysts using molecular oxygen, ideal for biosensor development.
• Functional enzyme immobilization on surfaces is key for expanding applications.
• Genetically engineered peptides enable controlled biomolecule assembly on material interfaces.

Purpose of the Study:

• To explore the bioassembly of putrescine oxidase using a gold binding peptide tag.
• To investigate the functional activity, expression, and binding selectivity of the peptide-tagged enzyme.
• To demonstrate controlled orientation of immobilized enzymes for enhanced performance.

Main Methods:

• Genetic engineering of putrescine oxidase with a gold binding peptide.
• Characterization using protein electrophoresis, enzyme activity assays, microscopy, and spectroscopy.
• Binding studies with quartz crystal microbalance (QCM) and atomic force microscopy (AFM).

Main Results:

• The peptide-tagged enzyme showed selective binding to gold surfaces.
• QCM studies indicated increased affinity of the tagged enzyme compared to the native enzyme.
• AFM revealed controlled orientation of the immobilized enzyme, preserving near-native activity.

Conclusions:

• Bioassembly using a gold binding peptide tag enables selective and oriented immobilization of putrescine oxidase.
• This approach enhances enzyme functionality for applications in biocatalysis and biosensors.
• Controlled enzyme orientation is critical for optimizing performance in diverse biotechnological applications.