Premium
Synthesis and evaluation of novel fluorogenic substrates for the detection of bacterial β‐galactosidase
Author(s) -
Chilvers K.F.,
Perry J.D.,
James A.L.,
Reed R.H.
Publication year - 2001
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2001.01484.x
Subject(s) - coumarin , galactoside , substrate (aquarium) , chemistry , fluorescence , bacteria , chromatography , enzyme , biochemistry , stereochemistry , biology , organic chemistry , ecology , physics , genetics , quantum mechanics
Aims: A widely used coumarin derivative is 7‐hydroxy‐4‐methylcoumarin‐β‐ D ‐galactoside (4‐methylumbelliferone‐β‐ D ‐galactoside; 4‐MU‐GAL). This galactoside is utilized as a substrate for the detection of the β‐galactosidase activity of coliform bacteria in water analysis. The intense fluorescence of coumarin‐based molecules has enabled them to be incorporated into enzyme‐based tests for the quantitative assay of indicator bacteria. The aim of this present study was to evaluate the potential of other coumarin derivatives, by synthesis of a selection of core coumarin molecules. Methods and Results: Several coumarin derivatives were found to be more promising than 4‐MU, with ethyl‐7‐hydroxycoumarin‐3‐caboxylate (EHC) giving a combination of greater fluorescence over a broad pH range and reduced growth inhibition with 12 representative coliform strains. On conversion to a β‐galactoside derivative, EHC‐GAL generated a more rapid fluorescence than any other tested substrate. Conclusions: When tested in a broth assay format, based on most probable number (MPN), low numbers of coliforms were detected with EHC‐GAL around 1 h earlier than with 4‐MU‐GAL. Significance and Impact of the Study: The present study suggests that EHC‐GAL should be evaluated as a substrate for the detection of coliforms in water analysis, due to a combination of the following favourable features: (i) reduced toxicity; (ii) increased fluorescence; (iii) pH stability of fluorescence; and (iv) rapid detection.