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


RESULTS
Several coumarin derivatives were found to be more promising than 4-MU, with ethyl-7-hydroxycoumarin-3-carboxylate (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.


CONCLUSIONS
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.
Chilvers
K F
KF
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, UK.
Perry
J D
JD
James
A L
AL
Reed
R H
RH
D001431:Bacteriological Techniques; D015169:Colony Count, Microbial; D003374:Coumarins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D005697:Galactosides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D014871:Water Microbiology; D001616:beta-Galactosidase / Q000378:metabolism*
D003374:Coumarins; D005456:Fluorescent Dyes; D005697:Galactosides; C030123:coumarin; D001616:beta-Galactosidase