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Fluorescence spectroscopic analysis of the structure and dynamics of Bacillus subtilis lipase A governing its activity profile under alkaline conditions

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Abstract

Because of their vast diversity of substrate specificity and reaction conditions, lipases are versatile materials for biocatalysis. Lipase A from Bacillus subtilis (BSLA) is the smallest lipase yet discovered. It has the typical α/β hydrolase fold but lacks a lid covering the substrate cleft. In this study, the pH-dependence of the activity, stability, structure, and dynamics of BSLA was investigated by fluorescence spectroscopy. By use of a fluorogenic substrate it was revealed that the optimum pH for BSLA activity is 8.5 whereas thermodynamic and kinetic stability are maximum at pH 10. The origin of this behavior was clarified by investigation of ANS (8-anilino-1-naphthalenesulfonic acid) binding and fluorescence quenching of the two single tryptophan mutants W31F and W42F. Variations in segmental dynamics were investigated by use of time-resolved fluorescence anisotropy. This analysis showed that the activity maximum is governed by high surface hydrophobicity and high segmental mobility of surface loops whereas the stability optimum is a result of low segmental mobility and surface hydrophobicity.

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Acknowledgments

We thank Dr Ulrich Kraus, Forschungszentrum Jülich, for kindly providing the DNA of BSLA and the purification procedure, and Professor Nickisch-Hartfiel for making it possible to conduct parts of this research in the Biotechnology Laboratories of the Chemistry Department.

Funding sources

The work was supported by the “Professorinnenprogramm des Bundes und der Länder”, 01FP09231B.

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Authors and Affiliations

  1. Department of Chemistry, Niederrhein University of Applied Sciences, Adlerstr. 32, 47798, Krefeld, Germany

    Daniel Kübler, Kim N. Ingenbosch, Anna Bergmann, Monika Weidmann & Kerstin Hoffmann-Jacobsen

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  1. Daniel Kübler
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  2. Kim N. Ingenbosch
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  3. Anna Bergmann
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  4. Monika Weidmann
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  5. Kerstin Hoffmann-Jacobsen
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Correspondence to Kerstin Hoffmann-Jacobsen.

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Kübler, D., Ingenbosch, K.N., Bergmann, A. et al. Fluorescence spectroscopic analysis of the structure and dynamics of Bacillus subtilis lipase A governing its activity profile under alkaline conditions. Eur Biophys J 44, 655–665 (2015). https://doi.org/10.1007/s00249-015-1061-6

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  • DOI: https://doi.org/10.1007/s00249-015-1061-6

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