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Pseudomonas Methods and Protocols pp 111–134Cite as

Determination of Lipolytic Enzyme Activities

Part of the Methods in Molecular Biology book series (MIMB,volume 1149)

Abstract

Pseudomonas aeruginosa is a versatile human opportunistic pathogen that produces and secretes an arsenal of enzymes, proteins and small molecules many of which serve as virulence factors. Notably, about 40 % of P. aeruginosa genes code for proteins of unknown function, among them more than 80 encoding putative, but still unknown lipolytic enzymes. This group of hydrolases (EC 3.1.1) is known already for decades, but only recently, several of these enzymes have attracted attention as potential virulence factors. Reliable and reproducible enzymatic activity assays are crucial to determine their physiological function and particularly assess their contribution to pathogenicity. As a consequence of the unique biochemical properties of lipids resulting in the formation of micellar structures in water, the reproducible preparation of substrate emulsions is strongly dependent on the method used. Furthermore, the physicochemical properties of the respective substrate emulsion may drastically affect the activities of the tested lipolytic enzymes. Here, we describe common methods for the activity determination of lipase, esterase, phospholipase, and lysophospholipase. These methods cover lipolytic activity assays carried out in vitro, with cell extracts or separated subcellular compartments and with purified enzymes. We have attempted to describe standardized protocols, allowing the determination and comparison of enzymatic activities of lipolytic enzymes from different sources. These methods should also encourage the Pseudomonas community to address the wealth of still unexplored lipolytic enzymes encoded and produced by P. aeruginosa.

Key words

  • Pseudomonas aeruginosa
  • Lipase
  • Esterase
  • Phospholipase A
  • Lysophospholipase
  • Agar-plate assay
  • Colorimetric assay
  • Fluorometric assay
  • Titrimetric assay
  • Lipase fingerprinting
  • Enantioselectivity assay

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Authors and Affiliations

  1. Institute of Molecular Enzyme Technology, Research Centre Juelich Heinrich-Heine-University of Duesseldorf, D-52426, Juelich, Germany

    Karl-Erich Jaeger & Filip Kovacic

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  1. Karl-Erich Jaeger
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  2. Filip Kovacic
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Correspondence to Karl-Erich Jaeger .

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Editors and Affiliations

  1. MRC Centre for Molecular Bacteriology and Infection (CMBI) Department of Life Sciences, Imperial College, London, UK

    Alain Filloux

  2. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain

    Juan-Luis Ramos

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Jaeger, KE., Kovacic, F. (2014). Determination of Lipolytic Enzyme Activities. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0473-0_12

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