Probing Selective Self-Assembly of Putrescine Oxidase with Controlled Orientation Using a Genetically Engineered...

Area of Science:

• Biocatalysis and Surface Chemistry
• Nanotechnology and Materials Science
• Enzyme Engineering and Immobilization

Background:

• Enzyme orientation and location on surfaces are critical for biosensor and lab-on-a-chip device functionality.
• Solid-binding peptides offer versatile immobilization of biomolecules onto inorganic materials.
• Previous work demonstrated a gold-binding peptide (AuBP) enabling self-assembly of putrescine oxidase (PutOx-AuBP) on gold.

Purpose of the Study:

• To explore the selective self-assembly of PutOx-AuBP on various surfaces.
• To assess the impact of surface material on enzyme coverage, distribution, size, shape, and functional activity.
• To provide insights into material-selective enzymatic assembly for nanoscale material fabrication.

Main Methods:

• Utilized atomic force microscopy (AFM) to analyze enzyme self-assembly.
• Investigated enzyme behavior on diverse surfaces: metal (gold), oxide (SiO2), minerals (mica, graphite), and self-assembled monolayers (SAMs).
• Compared immobilized PutOx-AuBP with native putrescine oxidase (PutOx) to understand peptide-directed interactions.

Main Results:

• Material-selective binding with controlled orientation was observed exclusively for PutOx-AuBP on templated-stripped gold (TSG).
• Surface coverage, enzyme dimensions, and distribution varied significantly across different substrates.
• Enzymatic activity measurements correlated with observed differences in surface interactions, ranging from self-assembly to aggregation.

Conclusions:

• The gold-binding peptide tag facilitates controlled, orientation-specific enzyme immobilization on gold surfaces.
• Substrate properties critically influence enzyme-surface interactions and assembly behavior.
• This study provides a foundation for fabricating enzyme patterns and functional nanoscale materials using self-assembling peptide tags.