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Cloning, recombinant expression and biochemical characterisation of novel esterases from Bacillus sp. associated with the marine sponge Aplysina aerophoba

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Abstract

Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity (26–44%) with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73–74%) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards p-nitrophenyl and methyl esters and the respective kinetic parameters Km and Vmax were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30–50°C and 20–35°C, respectively. The pH optima were found to be in the range of 6.5–7.5 and 6.5–8.0, respectively. Both enzymes showed the highest stability in up to 50% (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10–50 mM Zn2+ and 50 mM Mg2+ and Ca2+ ions was observed for both esterases. One to five millimolar PMSF deactivated the enzymes, whereas β-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.

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Acknowledgements

We thank Fatma Kabaoglu for providing the novel Bacillus strain used in that project. Part of this work (F.B.) was supported by the German Federal Ministry of Education and Research (BMBF) through the Screening Center project and the project Centre of Excellence BIOTECmarin (FO345D) and by the Ministry of Science, Research and Arts of the State of Baden-Württemberg and the University of Stuttgart.

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Authors and Affiliations

  1. Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    A. Karpushova, S. Barth, S. Lange & R. D. Schmid

  2. Institut für Biologie, Zoologie, Universität Stuttgart, Pfaffenwaldring 57, 70569, Stuttgart, Germany

    F. Brümmer

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  1. A. Karpushova
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  2. F. Brümmer
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  3. S. Barth
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  4. S. Lange
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  5. R. D. Schmid
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Correspondence to R. D. Schmid.

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Karpushova, A., Brümmer, F., Barth, S. et al. Cloning, recombinant expression and biochemical characterisation of novel esterases from Bacillus sp. associated with the marine sponge Aplysina aerophoba. Appl Microbiol Biotechnol 67, 59–69 (2005). https://doi.org/10.1007/s00253-004-1780-6

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  • DOI: https://doi.org/10.1007/s00253-004-1780-6

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