Thermostabilizing ketoreductase ChKRED20 by consensus mutagenesis at dimeric interfaces.
Yu-Jie Yang1, Xiao-Qiong Pei2, Yan Liu2
1CAS Key Laboratory of Environmental and Applied Microbiology, Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Enzyme and Microbial Technology
|May 1, 2022
Summary
Researchers enhanced ketoreductase stability using a sequence consensus approach. This identified mutations significantly improving thermostability, with one mutant showing remarkable resistance to heat inactivation for industrial applications.
Area of Science:
- Biochemistry
- Protein Engineering
Background:
- Protein stability is essential for enzymatic catalysis.
- Improving enzyme thermostability is critical for industrial applications.
Purpose of the Study:
- To develop an efficient strategy for identifying thermostabilizing mutations in ketoreductase ChKRED20.
- To enhance the kinetic and thermodynamic stability of ChKRED20.
Main Methods:
- Applied a sequence consensus approach targeting dimeric interface residues of ketoreductase ChKRED20.
- Constructed combinatorial mutants and characterized their thermostability.
- Utilized structural and molecular dynamic simulations for analysis.
Main Results:
- The sequence consensus approach yielded a 43% success rate, identifying 9 beneficial mutations.
- Mutant M8K exhibited significantly enhanced thermostability, with a melting temperature (Tm) of 89°C and a half-inactivation temperature (T50) of 93.4°C.
- M8K demonstrated prolonged stability at optimal temperatures and a substantially increased inactivation half-life compared to the wild-type.
Conclusions:
- The sequence consensus strategy is effective for discovering thermostabilizing mutations.
- Mutant M8K represents a highly thermostable ketoreductase variant with potential for industrial use.
- Structural analysis confirmed increased intramolecular interactions and reduced flexibility contribute to enhanced stability.
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