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Updated: Nov 5, 2025
A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Improving the activity and stability of Bacillus clausii alkaline protease using directed evolution and molecular
Jialin Li1, Luying Jiang1, Xue Cao1
1Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, National Engineering Laboratory for Industrial Enzymes, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China.
Researchers engineered a Bacillus clausii alkaline protease (PRO) variant, G95P, with a 9-fold increase in specific activity. This enhanced enzyme also shows improved alkaline stability and thermostability, crucial for detergent applications.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Alkaline proteases are key detergent enzymes, with improvements in specific activity reducing production costs.
- Bacillus clausii alkaline protease (PRO) is a significant component of detergent enzyme sales.
Purpose of the Study:
- To enhance the specific activity and stability of Bacillus clausii alkaline protease (PRO) through protein engineering.
- To investigate the structure-function relationship of PRO variants for industrial applications.
Main Methods:
- Directed evolution using error-prone PCR to create a mutant library of Bacillus clausii alkaline protease (PRO).
- Identification and characterization of a high-activity variant (G95P).
- Molecular Dynamics (MD) simulations to analyze structural changes and stability.
Main Results:
- A variant, G95P, exhibited a 9-fold increase in specific activity compared to wild-type PRO.
- G95P demonstrated enhanced alkaline stability, retaining 67% activity after 70h at pH 11.0.
- G95P showed improved thermostability, maintaining 17% activity after 50h at 60°C, unlike wild-type PRO.
Conclusions:
- The G95P variant offers significant improvements in specific activity, alkaline stability, and thermostability.
- MD simulations confirmed enhanced stability in the Gly95-Gly100 loop and Arg19-Asp265 salt bridge of G95P.
- This study provides a foundation for protein engineering strategies to optimize PRO for industrial detergent use.
