Enzyme engineering of choline oxidase for improving stability

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Choline oxidase is a flavoprotein that converts choline to glycine betaine.
• Glycine betaine has broad industrial applications.
• The enzyme's industrial utility is limited by thermal instability.

Purpose of the Study:

• To enhance the thermal stability of *A. globiformis* choline oxidase.
• To identify residues contributing to thermal instability.
• To improve enzyme function at elevated temperatures (60°C).

Main Methods:

• Molecular Dynamics (MD) simulations to identify unstable residues (Trp 331, Val 464, Ser 101).
• Site-directed mutagenesis replacing unstable residues with phenylalanine.
• MD simulations of the mutated enzyme at 60°C.
• Re-docking and MM/GBSA analyses to assess binding affinity and activity.

Main Results:

• MD simulations pinpointed Trp 331, Val 464, and Ser 101 as key to thermal instability.
• Mutating these residues to phenylalanine resulted in a thermostable enzyme.
• The mutated enzyme showed insignificant fluctuations at 60°C.
• Re-docking and MM/GBSA confirmed maintained binding affinity and catalytic activity.

Conclusions:

• Specific residue mutations can significantly enhance choline oxidase thermostability.
• The engineered enzyme retains catalytic function at higher temperatures.
• This study provides a computational strategy for improving enzyme industrial applicability.