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An Esterase with Increased Acetone Tolerance from Bacillus subtilis E9 over Expressed in E. coli BL21 Using pTac
P Soumya1, Jayachandran Kochupurackal2
1School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, 686560, India.
Molecular Biotechnology
|February 9, 2022
Summary
We engineered a stable esterase enzyme from Bacillus subtilis E9 for industrial biocatalysis. The recombinant esterase shows enhanced organic solvent tolerance, making it suitable for synthetic transformations.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Engineering
Background:
- Bacillus subtilis E9 identified as a source of esterase.
- Esterase enzyme characterization is crucial for biocatalysis.
Purpose of the Study:
- To clone, express, and characterize the esterase from B. subtilis E9.
- To evaluate the organic solvent stability of the recombinant esterase for industrial applications.
Main Methods:
- Gene amplification, in silico translation, and protein modeling.
- Golden gate DNA assembly for cloning into pTac vector.
- Heterologous expression in Escherichia coli BL21 (DE3) and protein purification.
- LC-MS/MS for protein identification and activity assays.
Main Results:
- The esterase gene was successfully cloned and expressed in E. coli.
- Purified recombinant esterase exhibited high specific activity (772 U/mg).
- The enzyme demonstrated remarkable stability in organic solvents (95% activity in 30-90% Acetone for 5 days).
Conclusions:
- The recombinant esterase from B. subtilis E9 possesses superior organic solvent stability compared to the native enzyme.
- This engineered esterase is a promising candidate for biocatalytic and synthetic applications in industry.
