Determination of Lipolytic Enzyme Activities

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.

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

“…However, 80 putative are predicted for P . aeruginosa (Jaeger and Kovacic 2014) and about half as many for P . putida (Nelson et al 2002; Winsor et al 2016).…”

A membrane‐bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli

“…Enzymatic activities were determined in 96‐well microplates by adding 5 μL of enzyme sample to 200 μL of substrate with p ‐nitrophenyl hexanoate ( p NPC 6 ) for esterase and p ‐nitrophenyl palmitate ( p NPP) for lipase activity .…”

“…Carboxyl esterases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/1/1/1.html) and lipases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/1/1/3.html), usually referred to as lipolytic enzymes, hydrolyse ester bonds of a wide range of lipidic substrates . They exert various cellular functions in animals, plants and microorganisms .…”

Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community

“…Clones with hydrolytic activities were identified by the formation of clear halos surrounding the colonies after growth for 24 h at 37 °C on indicator plates followed by storage at 4 °C for several days, if necessary. Phospholipase activity was examined with an egg yolk LB agar plate assay78.…”