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High-Throughput Screening Assays for Lipolytic Enzymes

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Protein Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1685))

Abstract

Screening is defined as the identification of hits within a large library of variants of an enzyme or protein with a predefined property. In theory, each variant present in the respective library needs to be assayed; however, to save time and consumables, many screening regimes involve a primary round to identify clones producing active enzymes. Such primary or prescreenings for lipolytic enzyme activity are often carried out on agar plates containing pH indicators or substrates as triolein or tributyrin. Subsequently, high-throughput screening assays are usually performed in microtiter plate (MTP) format using chromogenic or fluorogenic substrates and, if available, automated liquid handling robotics. Here, we describe different assay systems to determine the activity and enantioselectivity of lipases and esterases as well as the synthesis of several substrates. We also report on the construction of a complete site saturation library derived from lipase A of Bacillus subtilis and its testing for detergent tolerance. This approach allows for the identification of amino acids affecting sensitivity or resistance against different detergents.

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Author information

Authors and Affiliations

  1. Institute of Molecular Enzyme Technology, Heinrich-Heine - Universität Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany

    Alexander Fulton & Karl-Erich Jaeger

  2. Novozymes A/S, Krogshoejvej 36, 2880, Bagsvaerd, Denmark

    Alexander Fulton

  3. Institute of Bioorganic Chemistry, Heinrich-Heine - Universität Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany

    Marc R. Hayes & Jörg Pietruszka

  4. Lehrstuhl für Biotechnologie, RWTH Aachen University, 52074, Aachen, Germany

    Ulrich Schwaneberg

  5. DWI Leibniz-Institute for Interactive Materials at RWTH Aachen University, 52056, Aachen, Germany

    Ulrich Schwaneberg

  6. Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52428, Jülich, Germany

    Jörg Pietruszka & Karl-Erich Jaeger

Authors
  1. Alexander Fulton
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  2. Marc R. Hayes
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  3. Ulrich Schwaneberg
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  4. Jörg Pietruszka
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  5. Karl-Erich Jaeger
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Corresponding author

Correspondence to Karl-Erich Jaeger .

Editor information

Editors and Affiliations

  1. Department of Biotechnology and Enzyme Catalysis Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Uwe T. Bornscheuer

  2. Protein Biochemistry Institute of Biochemistry, Greifswald University, Greifswald, Germany

    Matthias Höhne

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Fulton, A., Hayes, M.R., Schwaneberg, U., Pietruszka, J., Jaeger, KE. (2018). High-Throughput Screening Assays for Lipolytic Enzymes. In: Bornscheuer, U., Höhne, M. (eds) Protein Engineering. Methods in Molecular Biology, vol 1685. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7366-8_12

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  • DOI: https://doi.org/10.1007/978-1-4939-7366-8_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7364-4

  • Online ISBN: 978-1-4939-7366-8

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