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Synthesis and evaluation of novel fluorogenic substrates for the detection of bacterial beta-galactosidase

Lookup NU author(s): Dr John Perry

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Abstract

Aims: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-beta -D-galactoside (4-methylumbelliferone-beta -D-galactoside; 4-MU-GAL). This galactoside is utilized as a substrate for the detection of the beta -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 beta -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.

Publication metadata

Author(s): Chilvers KF, Perry JD, James AL, Reed RH

Publication type: Article

Publication status: Published

Journal: Journal of Applied Microbiology

Year: 2001

Volume: 91

Issue: 6

Pages: 1118-1130

ISSN (print): 1364-5072

ISSN (electronic): 1365-2672

Publisher: Wiley-Blackwell Publishing Ltd.

URL: http://dx.doi.org/10.1046/j.1365-2672.2001.01484.x

DOI: 10.1046/j.1365-2672.2001.01484.x

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