Robust ω-Transaminases by Computational Stabilization of the Subunit Interface.

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Protein Stability

Background:

• Transaminases are valuable biocatalysts for producing enantiopure amines.
• Enzyme stability is a major limitation in their industrial application.

Purpose of the Study:

• To enhance the stability of a ω-transaminase from *Pseudomonas jessenii* using computational design.
• To identify key mutations for improving enzyme performance.

Main Methods:

• Employed a computational framework (FRESCO) for enzyme stability engineering.
• Generated libraries of surface and subunit interface mutations.
• Screened mutations experimentally for stabilizing effects.
• Determined crystal structures of engineered variants.

Main Results:

• Subunit interface mutations were significantly more successful (56%) than surface mutations (6%) in stabilizing the enzyme.
• Engineered variants showed increased melting temperatures (up to 85 °C) and 5-fold higher activity.
• Optimized variants achieved high yield (92%) and enantiomeric excess (>99%) for (S)-1-phenylethylamine production.
• Identified steric strain removal as a stabilization mechanism.

Conclusions:

• Computational redesign of the subunit interface is a powerful strategy for transaminase stabilization.
• Enhanced enzyme variants offer improved performance for industrial biocatalysis.
• This approach accelerates the development of robust biocatalysts.