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  11. Activity And Stability Improvement: Structure-function Insights Into Cota From Bacillus Subtilis

Activity and stability improvement: structure-function insights into CotA from Bacillus subtilis

Wei He1, Yiru1, Wang1

  • 1Beijing Key Laboratory of Bioactive Substances and Functional Foods, College of Biochemical Engineering, Beijing Union University, Beijing 100023, China.

International Journal of Biological Macromolecules
|June 12, 2025

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View abstract on PubMed

Summary
This summary is machine-generated.

Researchers engineered a bacterial multicopper oxidase (CotA) variant with 6-fold higher activity and improved thermal stability. Molecular dynamics simulations revealed structural changes linked to enhanced enzyme function and stability for industrial applications.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzyme Engineering

Background:

  • Bacterial multicopper oxidase (CotA) is a versatile enzyme with potential in industrial biocatalysis and environmental remediation.
  • Existing methods to enhance CotA's properties include directed evolution and protein design, but structural insights remain limited.

Purpose of the Study:

  • To investigate the structural basis for enhanced activity and stability in CotA mutants.
  • To identify specific mutations that improve CotA's performance for industrial applications.

Main Methods:

  • Site-directed mutagenesis was used to create CotA single and double mutants from Bacillus subtilis.
  • Molecular dynamics (MD) simulations were employed to analyze the structural features of the mutants.
  • Enzyme activity and thermal stability were experimentally assessed.
Keywords:
Catalytic efficiencyCotAMD simulationSite-directed mutagenesis

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Main Results:

  • The T377I/T418G CotA variant showed a 6.12-fold increase in ABTS-specific activity and 50% improved thermal stability at 80°C.
  • MD simulations indicated that increased activity correlated with an expanded enzyme binding pocket.
  • Enhanced thermal stability was associated with a higher proportion of random coils in the enzyme's secondary structure.

Conclusions:

  • Specific mutations can significantly enhance CotA's catalytic activity and thermal stability.
  • Structural analysis via MD simulations provides insights into the mechanisms underlying enzyme improvement.
  • This study provides a foundation for optimizing CotA for diverse industrial and environmental applications.
Thermal stability