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Joel, EB, Aberuagba, A, Bello, AJ, Akanbi-Gada, M, Igunnu, A, Malomo, SO and Olorunniji, FJ (2024) Role of the C-Terminal β Sandwich of Thermoanaerobacter tengcongensis Thermophilic Esterase in Hydrolysis of Long-Chain Acyl Substrates. International Journal of Molecular Sciences, 25 (2). p. 1272. ISSN 1661-6596
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Role of the C-Terminal β Sandwich of Thermoanaerobacter tengcongensis Thermophilic Esterase in Hydrolysis of Long-Chain Acyl Substrates.pdf - Published Version Available under License Creative Commons Attribution. Download (1MB) | Preview |
Abstract
To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain.
| Item Type: | Article |
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| Uncontrolled Keywords: | Thermoanaerobacter; Esterases; Bacterial Proteins; Cloning, Molecular; Enzyme Stability; Amino Acid Sequence; Substrate Specificity; Hydrolysis; Firmicutes; C-terminal domain; Thermoanaerobacter tengcongensis; esterase; substrate preference; thermophilic esterases; Esterases; Amino Acid Sequence; Hydrolysis; Bacterial Proteins; Thermoanaerobacter; Enzyme Stability; Substrate Specificity; Cloning, Molecular; Firmicutes; 0399 Other Chemical Sciences; 0604 Genetics; 0699 Other Biological Sciences; Chemical Physics |
| Subjects: | R Medicine > RS Pharmacy and materia medica |
| Divisions: | Pharmacy & Biomolecular Sciences |
| Publisher: | MDPI |
| SWORD Depositor: | A Symplectic |
| Date Deposited: | 05 Feb 2024 11:16 |
| Last Modified: | 05 Feb 2024 11:16 |
| DOI or ID number: | 10.3390/ijms25021272 |
| URI: | https://researchonline.ljmu.ac.uk/id/eprint/22509 |
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