Abstract
In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0–8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, β-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.
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Abbreviations
- 4-NP:
-
4-nitrophenyl
- 4-NPC12:
-
4-nitrophenyl laurate
- 4-NPC2:
-
4-nitrophenyl acetate
- 4-NPC4:
-
4-nitrophenyl butyrate
- 4-NPC8:
-
4-nitrophenyl caprylate
- DTT:
-
Dithiothreitol
- DMF:
-
Dimethylformamide
- DMSO:
-
Dimethyl sulfoxide
- EDTA:
-
Ethylenediaminetetraacetic acid
- EstGtA3:
-
esterase from G. thermodenitrificans NG80-2
- EtOH:
-
Ethanol
- MeOH:
-
Methanol
- MES:
-
2-(N-morpholino)ethanesulfonic acid
- PMSF:
-
phenylmethane sulfonyl fluoride
- SDS–PAGE:
-
Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis
- SDS:
-
Sodium Dodecyl Sulphate
- TCV:
-
inhibitor tris-catechol vector
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Acknowledgements
We thank Francesco La Cara and collaborators at the Research Institute on Terrestrial Ecosystems (IRET) from the National Research Council of Italy for the gift of Geobacillus thermodenitrificans NG80-2 genome. We are grateful to Chiara Nobile and Marco Petruzziello at the Institute of Biosciences and BioResources (IBBR) from the National Research Council of Italy for administrative and technical assistance. This work was supported by a grant from the Italian Ministry of Research (MIUR) PON03PE_00107_1 BIOPOLIS.
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Nicola Curci and Andrea Strazzulli contributed equally to the work
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Curci, N., Strazzulli, A., De Lise, F. et al. Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2. Extremophiles 23, 407–419 (2019). https://doi.org/10.1007/s00792-019-01093-9
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DOI: https://doi.org/10.1007/s00792-019-01093-9