Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1998-04-27
2000-04-04
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 4, 435 19, 435 29, 435 34, 436 63, 436164, 436166, 436172, C12Q 102, C12Q 137, C12N 500
Patent
active
060460164
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for demonstrating an enzymatic activity of microorganisms. Such a method can be used for the identification of microorganisms which may or may not express this enzymatic activity.
The detection and identification of micro-organisms are very important especially in medicine, in the agrifoods industry, for environmental control (for example for controlling water, etc.). Micro-organisms may be desired for their pathogenicity, as contamination indicators, or alternatively for controlling manufacturing processes.
The techniques for detecting and identifying microorganisms are currently based on the search for characteristic nucleotide sequences, the search for antigens or antibodies, culturing in a selective or non-selective medium, or alternatively the search for metabolic and especially enzymatic activities (for example osidase, esterase, peptidase, oxidase etc. activities).
Usually, the methods for detecting and identifying microorganisms combine several of these techniques. Thus, culturing is used to multiply and select the desired microorganisms. In order to simplify their detection, it has been proposed to demonstrate biochemical activities by introducing molecules which produce a coloration or a fluorescence, directly into the culture medium. Such media are referred to as detection media. The biochemical activities can be demonstrated by various methods such as: coloured or fluorescent indicator (methylumbelliferone), (tetrazolium salt) or a fluorescent indicator, present in the medium, leading to a coloration, (naphthol, coumarin).
The hydrolyses detected are generally the result of the action of an enzyme produced by the microorganism on a natural or synthetic enzymatic substrate. These enzymatic activities are, for example, those of the following enzymes: esterases (for example lipases, phosphatases), osidases (.beta.-galactosidase, .beta.-glucuronidase, N-acetyl-hexosaminidase), peptidases (alanine-aminopeptidase, trypsinase, gelatinase), DNAses, decarboxylases, deaminases, ureases, tryptophanases, oxidases, catalases, etc.
It is known that gelled media are particularly suitable for culturing and isolating microorganisms from a sample, as well as for detecting "target" microorganisms in a mixture of microorganisms. On these media, the microorganisms form colonies that can be detected with the naked eye, and it is highly desirable for the products of the biochemical activities studied to remain localized at their site of production. This effectively makes it possible to distinguish one colony from its neighbours if they do not express the same activities. Various detection methods can thus be used, for example changes in pH (FR-A-2,671,100), esterase activities (FR-2-457,323), osidase activities (FR-A-2,684,110), etc. Needless to say, it is possible to use several of these methods in conjunction, in order to demonstrate several species or strains, and/or in order to increase the sensitivity and/or specificity of the detection.
There are currently no means available, which are suitable for gelled media, for demonstrating the activities of L-alanine-aminopeptidases, D-alanine-aminopeptidases and L-leucine-aminopeptidases of microorganisms. The reason for this is that the enzyme substrates used to date release coloured or fluorescent molecules which diffuse into the gelled media and/or which are only revealed by UV irradiation (in the case of naphthylamine or aminocoumarin) and/or after the action of reagents (in the case of naphthylamine), or whose coloration is of relatively poor contrast in the reaction media used in microbiology (in the case of nitroaniline).
It is known that L-leucine-aminopeptidase has been demonstrated in mammalian histological sections by means of an enzyme substrate, L-leucine-3-(5-bromoindolamine), known as L-Leu-BIA for short, which produces a coloured compound after hydrolysis; see Pearson et al., 1963, Lab. Invest., 12: 712, who called this compound L-N-(5-bromoindol-3-yl) (leucinamide hydrobromide). In 1967, Yarborough et al., J. Re
REFERENCES:
patent: 4278763 (1981-07-01), Berger et al.
patent: 5336600 (1994-08-01), Monget
patent: 5434056 (1995-07-01), Monget et al.
Logda, Z. et al. "The Histochemical Demonstration of Aminopeptidase with Bromoindolyl Leucinamide." Histochemistry, vo. 43, 1975, pp. 355-366.
Rath, J. et al. "Ectoenzymatic Activity and Bacterial Dynamics along a Tropic Gradient in the Caribbean Sea." Marine Ecology Progress Series, vol. 102, 1993, pp. 89-96.
Yarborough et al., Histochemistry of Macrophage Hydrolases, III. Studies on .beta.-Galactosidase, .beta.-Glucuronidase and Aminopeptidase with Indolyl and Naphthyl Substrates, Journal of the Reticuloendothelial Society 4, pp. 390-408 (1967).
Lojda et al., The Histochemical Demonstration of Aminopeptidase With Bromoindolyl Leueinamide, Histochemistry 43, pp. 355-366 (1975).
Pearson et al., The Histochemical Demonstration of Leucine Aminopeptidase by Means of a New Indolyl Compound, Histochemistry of Leucine Aminopeptidase vol. 12, No. 7 pp. 712-720 (1963).
Bio Merieux
Lankford , Jr. Leon B.
Tate Christopher R.
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