Extraction and purification of pyocyanin: a simpler and more reliable method

Abstract

Pseudomonas aeruginosa is an aerobic Gram negative, rod shaped bacterium with special features making it one of the most commercially and biotechnologically valuable microorganisms.1,2 One of the hallmarks of P. aeruginosa is its ability to produce the blue-green pigment pyocyanin. Pyocyanin belongs to the family of phenazine which are, by definition, heterocyclic compounds that are produced naturally, but with side chains substituted at different points around their rings by different bacterial species.3 The biosynthesis of pyocyanin starts from a phenazine produced naturally: the deep red 5-methyl-7-amino-1-carboxyphenazium betaine that is converted to phenazine-1-carboxilic acid, which is lemon yellow in color. This is the last precursor of the bright blue 1-hydroxy-5-methyl phenazine or pyocyanin.4 Genetic analysis and gene expression assays showed that in P. aeruginosa, 7 genes are responsible for the synthesis of pyocyanin. These genes include phz C, D, E, F, G, M and S, but it was shown that phzM and phzS were the primary genes responsible for converting phenazine-1-carboxylic acid to pyocyanin through the production of S-adenosyl methionine-dependent N-methyltransferase and flavin-dependent hydroxylase respectively.4 The biosynthetic loci of the phenazine were found in all P. aeruginosa species (Figure 1).4–6 Pyocyanin is characterized by its blue color, but, its color and characteristic absorption spectrum are pH-sensitive. In fact, the strong blue color is only detected in neutral or basic pH, whereas under acidic conditions, pyocyanin becomes red in color.7 Although pyocyanin is a blue pigment when isolated in neutral pH, its presence in agar plates is detectable by the appearance of a blue-greenish color. This is due to the interference between the blue pigment and the color of the medium.