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Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination
Chia-Jung Chang1, Cheng-Chung Lee2,3, Yueh-Te Chan2,3
1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.
Plos One
|March 18, 2016
Summary
Researchers engineered a highly thermostable chimeric cellulase using SCHEMA recombination. This novel enzyme exhibits enhanced activity and stability, offering potential for biofuel and animal feed applications.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Cellulases from Bacillus and Geobacillus bacteria are valuable for biofuel and animal feed industries due to their thermostability.
- GsCelA from Geobacillus sp. 70PC53 is significantly more thermostable than its Bacillus homolog, BsCel5A, making them ideal for studying thermostability mechanisms.
Purpose of the Study:
- To investigate the mechanism of high-temperature activity retention in cellulases.
- To engineer novel chimeric cellulases with improved thermostability and activity using structure-guided recombination.
Main Methods:
- Applied the SCHEMA non-contiguous recombination algorithm to create chimeric proteins from GsCelA and BsCel5A.
- Analyzed the activity and thermostability of the generated chimeric library.
- Performed structural determinations and mutagenesis analyses to identify key structural features.
- Assessed the performance of the optimized chimera under various conditions, including the presence of calcium and crown ether (CR).
Main Results:
- Identified a specific block contributing to the higher thermostability of GsCelA.
- A highly thermostable chimeric cellulase, C10, containing this block showed 22%-43% higher activity and enhanced thermostability compared to parental enzymes.
- A 310 helix within the identified block was found to be responsible for the improved thermostability.
- The chimeric C10 retained 40% residual activity after heat treatment at 90°C in the presence of ionic calcium and CR.
Conclusions:
- Elucidated the mechanism underlying the high thermostability of GsCelA through structure-guided SCHEMA recombination.
- Successfully generated a novel recombinant enzyme with significantly enhanced activity and thermostability.
- The engineered cellulase holds promise for industrial applications in biofuel and animal feed production.
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