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An Esterase with Increased Acetone Tolerance from Bacillus subtilis E9 over Expressed in E. coli BL21 Using pTac Bs-est Vector

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Abstract

Bacillus subtilis E9 was identified as a potential strain producing esterase. The gene coding esterase from B. subtilis E9 was amplified using esterase-specific primers and the sequence was translated in silico. The presence of conserved catalytic triad amino acid residues (His-Ser-Asp/Glu) confirmed the functional nature of the esterase enzyme. Docking studies conducted with modeled protein and the ligand p-nitrophenyl acetate showed that the amino acid residues interacting with the ligand were Ser77, His76, and Gly103. The gene coding for esterase from B. subtilis E9 was cloned into an assembled vector having Tac promoter (pTac), pUC origin of replication, Ni-Histidine residues, ampicillin cassette, and T7 terminator using Golden gate DNA assembly method. The generated pTac Bs-est (4598 bp) recombinant plasmid was transformed and heterologously expressed in Escherichia coli BL21 (DE3) strain. The tagged recombinant protein was purified to yield 43.4% pure protein with specific activity of 772 U/mg. The purified recombinant protein was subjected to peptide sequencing and the identity was confirmed as esterase by peptide tandem mass fragmentation method using the LC-MS/MS analysis. The purified recombinant esterase was found to be organic solvent stable and tolerant up to 5 days retaining around 95% residual activity in 30–90% v/v Acetone. The recombinant esterase expressed in our study was found to exhibit better organic solvent stability and tolerance than compared to the original bacterial esterase from B. subtilis E9, a property which could be explored in the biocatalytic and synthetic transformation reactions for industrial applications.

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Acknowledgements

This work was supported by funding from Department of Science and Technology (DST) under INSPIRE Fellowship scheme.

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  1. School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, 686560, India

    P. Soumya & Jayachandran Kochupurackal

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Soumya, P., Kochupurackal, J. An Esterase with Increased Acetone Tolerance from Bacillus subtilis E9 over Expressed in E. coli BL21 Using pTac Bs-est Vector. Mol Biotechnol 64, 814–824 (2022). https://doi.org/10.1007/s12033-022-00458-4

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