Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-11-15
2002-01-22
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S283100, C435S975000, C435S024000, C435S004000
Reexamination Certificate
active
06340573
ABSTRACT:
The present invention relates to the detection of enzymes of hydrolase type, in particular peptidases, by using effective chromogenic substrates. The present invention also relates to a process and a device for identifying microorganisms which are both simple and reliable.
Specific substrates have been used for many years to determine the presence or absence of enzymatic activities characteristic of bacteria. By the choice of substrates, depending on whether or not there is a reaction, it is possible to characterize the nature of a genus of bacteria or to differentiate between the species of a given bacterial genus.
Synthetic substrates of enzymes consist of two portions, a first portion which is specific for the enzymatic activity to be detected, and a second portion which acts as a label, and which is referred to herein-below as the labeling portion.
These specific substrates can be fluorescent or chromogenic substrates. In point of fact, it is the second portion or labeling portion which is fluorescent or chromogenic, when it is not combined with the first portion.
Fluorescent substrates may be of diverse composition.
First of all, substrates based on umbelliferone or aminocoumarin, and the derivatives thereof substituted in position 2, which allow the release of a fluorescent compound whose color ranges from blue to green under an ultraviolet (UV) lamp (&lgr;
ex
=365 nm).
Next, substrates based on resorufin (and derivatives), in which there is release of a compound which is fluorescent pink under natural light (&lgr;
ex
=530 nm).
Finally, substrates based on fluorescein (and derivatives) which, after degradation, releases a compound which is fluorescent yellow under natural light (&lgr;
ex
=485 nm).
These substrates are unsuitable for use in agar media, and are used more in liquid medium.
The chromogenic substrates may also be of diverse nature.
Firstly, there are substrates based on indoxyl and its derivatives which, in the presence of oxygen, produce a precipitate ranging from blue to pink.
Their applications are essentially limited to osidases and esterases and do not concern the detection of a peptidase activity. Whereas they are well suited to use on a solid support (filter, agar, electrophoresis gel, etc.), they are less well suited to the use in liquid aqueous medium (formation of a precipitate).
Secondly, there are substrates based on hydroxyquinoline or esculetin and their derivatives, which produce a brown precipitate in the presence of iron salts.
In this case also, their applications are limited to osidases and esterases. They are suitable for use on a solid support, and relatively unsuitable for use in liquid aqueous medium.
Thirdly, there are substrates based on nitrophenol and nitroaniline and derivatives, which lead to the formation of a yellow compound.
They make it possible to detect osidase and esterase activities in the case of nitrophenol-based substrates, and peptidase activities in the case of nitroaniline-based substrates. However, in the case of detecting peptidase activities, the nitroaniline released is toxic to the bacteria which it is desired to identify or characterize, which may have a negative impact on current or subsequent analyses. Moreover, they are relatively unsuitable for use on a solid support, and are better suited to use in liquid medium. Furthermore, they are not particularly chromogenic on account of the relatively low extinction coefficient of the color (yellow) which gives a relatively weak contrast in biological media.
Fourthly, there are substrates based on naphthol and naphthylamine and its derivatives. In this case, the reaction is carried out in two stages; the naphthol or naphthylamine released by the enzymatic activity undergoes an “azo-coupling” in the presence of a diazonium salt which is added at the detection stage, leading to the formation of a colored insoluble compound.
They make it possible to detect osidase and esterase activities by means of naphthol, and peptidase activities by means of naphthylamine. The azo-coupling reaction is carried out in a medium which is often chemically corrosive and toxic to bacteria, making the sample unusable for other analyses, and, what is more, naphthylamines are carcinogenic.
To detect naphthylamine and thus a peptidase activity, it is also possible to add p-dimethylamino-cinnamaldehyde in acidic medium at the end of the enzymatic reaction, instead of a diazonium salt, although this still has toxicity drawbacks with respect to the sample analyzed.
Patent application FR-A-2 708 286 proposes the use of a mixture of chromogenic substrates, each chromogen giving a particular coloration for a specific enzyme which is different from the coloration and enzyme associated with the other chromogen. When the two colorations and thus the two enzymes are present, there is formation of a “tertiary coloration”.
However, this technique is unsatisfactory since, as a function of the low concentration of one of the two enzymes, it is not possible to detect this enzymatic activity which is thus masked by the coloration associated with the enzyme whose concentration is predominant.
Finally, patents U.S. Pat. No. 4,681,841 and U.S. Pat. No. 4,588,836 describe an indirect method for detecting a single enzymatic activity using a coupling between an aminobenzene and a hydroxyaromatic derivative (for example &agr;-naphthol), this coupling leading to the formation of a chromogenic indicator in the presence of oxidase. One of the two compounds (the aminobenzene) forms part of the composition of the starting substrate; if the desired enzymatic activity is present, this compound will be released and will be able to react with the other compound.
Nevertheless, in order for the detection of the desired enzyme to be possible, the presence of oxidase in the reaction medium is an absolute necessity. However, although this information discourages a person skilled in the art from looking for a solution which does not use oxidase, the Applicant has proved, by numerous tests carried out in its laboratories, that it is possible to detect the desired enzyme by means of using, for example, aminobenzene and &agr;-naphthol, without the addition of oxidase.
It may thus be readily appreciated that no chromogenic substrate which is particularly effective and advantageous as regards detecting at least one peptidase activity currently exists.
As regards the identification process and device, the state of the art consists of an identification process which involves a three-step manipulation:
taking a sample of the colony to be identified,
carrying out an orinetation test, and
looking for colonies similar to those observed and preparing an inoculum.
The orientation test should be performed before using an identification system. This is especially the case for Gram staining which requires an observation by microscope.
However, this coloration is not always easy to carry out and above all to interpret. Moreover, the cost of this test is far from negligible.
One of the aims of the present invention is thus to create a link between a culture medium and antibiotic assay and identification systems, offering biologist the possibility of performing a simple one-step test both for confirming the result of the Gram staining and for preparing the inoculum. Thus, the choice of antibiotic assay and identification tests is made reliable.
Patent application EP-A-0 122 028 proposes a colorimetric method for detecting the presence of at least one enzyme suspected of being present in a biological sample. It recommends the preparation of an absorbent brush, absorbing in this material at least one susbstrate which is specific for the enzyme which it is desired to detect. The absorbent material containing the substrate(s) is dried before use.
The invention allows the colormetric detection of enzymatic activities of hydrolase (osidase, esterase, phosphatase or peptidase) type with the aid of synthetic substrates based on two compounds, as well as a novel process which can be applied to both liquid and solid reaction media.
The
Armstrong Lyle
James Arthur
Monget Daniel
Orenga Sylvain
Bio Merieux
Leary Louise N.
Oliff & Berridg,e PLC
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