Activation of Lysozyme with Robust Flavoenzyme Activity by Amyloid Fibrillation

Area of Science:

• Biocatalysis
• Materials Science
• Protein Engineering

Background:

• Natural enzymes have limitations hindering practical use.
• Developing artificial enzymes with green processes, low cost, high stability, and biocompatibility is challenging.
• Amyloid fibrils offer unique properties for electron transfer and biocatalysis due to their ordered structure.

Purpose of the Study:

• To design and create a stable, metal-free artificial enzyme using amyloid fibrils.
• To investigate the catalytic activity and stability of the engineered amyloid fibril complex.
• To explore a novel strategy for converting proteins into functional artificial enzymes.

Main Methods:

• Lysozyme amyloid fibril (LAF) formation.
• Noncovalent binding of flavin mononucleotide (FMN) to LAF to form a nanofibril complex (^FMNLAF).
• Assays to determine NADH oxidase and Fe³⁺-cyt c reductase activities, and stability under various conditions (temperature, organic solvents).

Main Results:

• ^FMNLAF was successfully formed with stable noncovalent interactions.
• ^FMNLAF demonstrated significant NADH oxidase activity (K_m = 55.3 μM, K_cat = 0.65 min^-1) generating NAD⁺.
• The artificial enzyme exhibited enhanced stability at 60 °C, tolerance to organic solvents, and Fe³⁺-cyt c reductase-like activity.

Conclusions:

• Amyloid fibrillation provides a viable strategy for creating metal-free artificial enzymes.
• ^FMNLAF serves as a stable and active biocatalyst with potential applications in various chemical transformations.
• This approach offers a green, cost-effective, and rational method for protein-based artificial enzyme design.