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Enzyme engineering using computational methods significantly improved the stability and activity of ω-transaminase, enabling efficient production of enantiopure amines for biocatalysis.

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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.