FR2831888A1 - Generating an idiosyncratic catalytic fingerprint of a sample from chemical, biological, microbiological animal, vegetable, and human sources - Google Patents

Abstract

Method (M1) of generating an idiosyncratic catalytic fingerprint of a sample involves: (i) contacting the sample, which is capable of having a catalytic activity, with an assembly of substrates; and (ii) capturing the signals generated by this contact.

Description

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 METHOD FOR GENERATING THE IDIOSYNCRASIC CATALYTIC FOOTPRINT OF A SAMPLE AND SYSTEMS FOR ITS IMPLEMENTATION

 The present invention relates to a method for generating the idiosyncratic catalytic imprint of a sample. The invention also relates to the systems for implementing such a method and their use.

 A sample may have various catalytic characteristics. They are expressed, for example, by the different enzymatic classes.

 It is known in the prior art the use of the same type of substrates for revealing a given enzyme.

 Those relating to the determination of the best substrate of a specific protease (PNAS 97 (14), 7754-7759 and Nature Biotechnology 28, 187-193) can be cited. This technique involves testing libraries of several thousand to several hundred thousand peptide-only substrates with a known protease. This is limited to the case of proteases because the protein substrates are synthesized by combinatorial chemistry.

 The detection of microorganisms can also be mentioned (US Pat. No. 4,623,108, Patent EP 451,775, J. Clin Microbiol (1982), 16 (3), 417-21). These techniques are based on the detection of enzymes to identify microorganisms whose characteristics are already known.

 The PCT patent application published under No.

W001 / 60986 relates to esterases having a particular activity. The identification of these esterases is done by comparison with reference esterase profiles previously made on esterase substrates. It's about looking for the best enzyme for a type of

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 given substrate. These substrates correspond only to ester functions.

 The present invention aims to overcome the disadvantages of the prior art techniques by providing a method for generating the idiosyncratic catalytic imprint of a sample, ie its specific identity card.

 The subject of the invention is therefore a method for generating the idiosyncratic catalytic imprint of a sample, characterized in that it comprises the following steps: the sample likely to possess the catalytic activity is brought into contact with a set of ordered substrates, the signals generated after said contacting are captured.

 The process of the invention is remarkable in that this imprint can be obtained without knowing the catalytic activities of the sample.

 The term "idiosyncratic catalytic impression" according to the invention is understood to mean the image or the identity card, which is an individual of the catalytic activities. This footprint is specific to a sample. It is obtained after contacting said sample with a set of substrates. The idiosyncratic catalytic footprint combines the measurements made on different substrates. The idiosyncratic catalytic imprint certifies the identity of a sample as a representation of a sample reacted with a set of substrates.

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 By idiosyncratic is meant the individual ability of a sample to react with a set of substrates.

 Catalytic activity is understood to mean any activity of the sample capable of modifying a substrate. This activity may correspond for example to that of one or more proteins, DNA molecules, RNA molecules or a mixture thereof. Advantageously, this activity corresponds to that of an enzyme or a mixture of enzymes. These activities can also be those of recombinant products obtained by genetic engineering.

 These activities may correspond for example to hydrolases, oxidoreductases, transferases, lyases, isomerases, ligases.

 The catalytic activity defined in the meaning of the invention may correspond to a mixture of activities, it may be known or unknown.

 The sample can come from different origins. It can be for example chemical, biological, microbiological, animal, plant, human. It can come from all types of environments and samples.

 The sample may be simple or complex, prepared from standard extraction techniques and then optionally purified or used as such.

 A substrate used in the process of the invention is any molecule comprising at least one functional group sensitive to the presence of a catalytic activity and allowing the generation of a signal.

 The functional groups correspond as non-limiting examples: ester group, suitable for lipases, esterases, peptidases; carbonate group, suitable for lipases, esterases, peptidases;

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 carbamate group, adapted to peptidases; amide group, adapted to peptidases; - lactam group, adapted to peptidases, lactamases; epoxide group, adapted to hydrolases, in particular epoxide hydrolases; - phosphate group, adapted to phosphatases, phytases; glycoside group, suitable for glycosidases; ether, alkene and alkane groups, adapted to monoxygenases, hydroxylases; alcohol group suitable for alcohol dehydrogenases; aldol group adapted to aldolases.

 In addition to the above functional groups defining catalytic activities, each substrate may comprise a chemical group capable of conferring on it a specificity for an activity defined by the functional group. In a preferred embodiment, these chemical groups are constituted by substitution variations. The nature of the variations will preferably be of order: - steric, such as, for example, different alkyl or aryl groups, or - stereochemical, or else - electronic, such as, for example, electron donor or electroattractor groups.

 Thus, variations of stereochemical order make it possible to separately use each enantiomer of a pair of enantiomers, more generally each of the stereoisomers of a group of stereoisomers.

 The substrates furthermore make it possible to generate a signal directly or indirectly. We hear by

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 a direct signal any signals obtained directly after contacting the substrate with the sample.

 Indirect signals are understood to mean any signals obtained after one or more reactions, especially chemical reactions, after contacting the substrate with the sample.

 The signals emitted are a function of the nature of the substrate and / or of the detection system chosen. They can be of different types, in particular spectrophotometric (absorbance or fluorescence of light at a given wavelength), electrical or chemical. Chromogenic or fluorogenic substrates are preferably used. But it is also possible to record the progress of the reaction on any substrate by appropriate external sensors, such as a thermometric probe, a pM indicator, a protein product sensor (anti-product antibody) or a synthetic chemosensor, or measurement coupled with secondary enzymes. As an example, alcohol dehydrogenase can be used to detect the formation of ethanol by oxidation and formation of NADH or by the direct detection of the product after analytical separation (CE, HPLC, TLC).

 The signals are measured by any suitable sensor capable of measuring the generated signals such as, for example, a fluorometer, a spectrophotometer, an ammeter, a pH meter, a thermometer or a thermographic probe. The idiosyncratic catalytic footprint generated is collected by an analyzer suitable for signal recognition.

 Advantageously, the substrates are such that the generated signals are of the same type.

 They can be measured on the same type of sensor.

 The idiosyncratic catalytic footprint corresponding to the signals generated can be observed from

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 directly or indirectly after treatment of raw values. This treatment will be considered according to the analyzed signals.

 The substrates advantageously have the characteristic of reacting on a short reaction time, for example less than 2 hours, preferentially less than 20 minutes. This characteristic has the advantage of quickly obtaining the idiosyncratic catalytic impression of a sample. It also allows a study of its evolution over time. This facilitates the analysis of quality control.

 The substrates used in the process of the invention advantageously each have a specific reactivity for a given type of catalytic activity. They are little or not sensitive to degradation or non-specific activities. In addition, when stable substrates are used, they can be stored for long periods.

 The method of the invention makes it possible to follow a sample over time through its idiosyncratic catalytic imprint by using substrates from which several measurements can be made.

 Visualization of the effect of catalytic activities on a set of substrates is reproducible.

 The substrates described advantageously correspond to those described in International Patent Application PCT / FR01 / 01686 and in the article by G. Klein and JL. Reymond, Helvetica Chimica Acta, vol. 82.1999.

These substrates correspond to formulas (I) to (V) below:

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- la liaison Cl-C2 est insensible à une coupure par une réaction d'oxydation chimique. in which :

the Cl-C2 bond is insensitive to cleavage by a chemical oxidation reaction.

 - Di represents the precursor of a detectable product, - X and Y, identical or different, are chosen from an oxygen atom, a sulfur atom, an amine of formula-NRR, Ru is chosen from: an atom of hydrogen, an alkyl group, an aryl group, substituted or unsubstituted, and R12 is not a hydrogen atom.

 - R1 to R3, identical or different are selected from a hydrogen atom, a substituted or unsubstituted alkyl group or a chemical group as defined above.

 - Pi and P2, identical or different are functional groups as defined above, and at most one of the groups P1 and P2 is a hydrogen atom.

 P 3 is chosen from a carbonyl group, a group -PO 2 R 11 or a group R 11 PO-, where Ru has the same meaning as above, a group -SO 2, a group -CHOR 13 where R 13 represents an aryl, alkyl or glycosyl group, a group SiRR, where Ra and RIs, identical or

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 different, represent an aryl, alkyl, aryloxy or alkoxy group and an AsO 2 H- group.

 - G is selected from an oxygen atom, a sulfur atom, an amine group of formula NRRo where R and R12 have the same meaning as above.

 These substrates are detected after their chemical transformation by a sample to give products that will be chemically oxidized to obtain directly or indirectly a detectable compound corresponding to one or more compounds capable of presenting alone or in combination one or more variations of their biophysical, biological or chemical properties before and after the oxidation step, such as in particular a spectral type variation or a solubility variation. By way of example, FIG. 2 represents a principle of detection of fluorescent substrates.

 A second example of substrates useful according to the method of the invention comprises substrates (S) whose transformation to product (P) can be detected by measuring a change in pM resulting from the chelating power difference of the substrate (S) and the product (P) with respect to a metal ion. Such substrates are described in French Patent Application No. 01/05574 and in the articles of Jean-Louis Reymond et al. (G. Klein, D.

Kaufmann, S. Schürch and JL. Reymond, Chem. Commun., 2001, 561-562; G. Klein and JL. Reymond, Angew. Chem., 2001, 113, Nr. 9). These substrates allow the detection of the transformation of a substrate (S) into a product (P) by measuring the change in the spectral properties of a chemical detector (L), chelating a metal ion, resulting from exchanges of metal ions according to the following equilibrium:

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ML (MP and / or MS) + L where:
S represents a substrate, which may be any chemical molecule, whose chelating power is different from that of the product resulting from the chemical transformation of the substrate. These include specific substrate like whole proteins for proteases. This substrate may have enantioselective properties.

The substrates have no structural constraint other than that of having a chelating power difference with the corresponding products.

 P represents the product resulting from the chemical transformation of S and having a chelating power different from S. As the substrates, the products have no structural constraint other than that of having a chelating power difference with the corresponding substrates.

 L represents a chemical detector, also referred to as a pM sensor, corresponding to any ligand capable of chelating the metal ions in solution such that the chelation of the metal by the chemical detector gives a measurable change in pM by any appropriate method such as a change of property spectral detector. The sensor pM has a chelating power such that there can be exchanges of metal ions between the product (P) and the ligand (L) or the substrate (S) and the ligand (L) according to the product ( P) or the substrate (S) has the strongest chelating power.

As indicated above, the chelating power of the chemical detector (L) is advantageously greater than that of the substrate (S) or the product (P) so as to carry out the method of the invention with small amounts of chemical detectors. The chemical detector (L) can

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 correspond to a fluorescein derivative or any other fluorescent nucleus substituted with a chelating group.

 ML represents the metal-ligand complex with spectral properties different from L.

 MS represents the metal-substrate complex.

 MP represents the metal-product complex.

 Said complexes ML, MS and MP do not necessarily have a stoichiometry of 1/1.

 These substrates allow the detection of the transformation of a substrate (S) into a product (P) by measuring the change in the spectral properties of a chemical detector (L), chelating a metal ion, resulting from exchanges of metal ions: - between the product (P) and the chemical detector (L) when the product (P) has a higher chelating power than the substrate (S), or - between the substrate (S) and the chemical detector (L) ), when the substrate (S) has a higher chelating power than the product (P).

 A combination of different substrates could be used to make the idiosyncratic catalytic imprints.

 The invention also relates to a set of substrates for generating the idiosyncratic catalytic imprint according to the method of the invention.

 In one embodiment of the invention, substrates having different functional groups or different chemical groups or a combination of these two possibilities, in particular substrates having identical functional groups and different chemical groups, will be chosen.

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 In another implementation, the substrates will be identical. The contacting of the substrates with the sample will then be done under different reaction conditions. The reaction conditions are by way of non-limiting example in terms of pH, temperature, incubation time, substrate concentration or a sample amount or a mixture thereof. These conditions may also correspond to other parameters that can be taken both individually or in combination. These conditions may also correspond to the addition of one or more additional reagents.

 The substrates may also be a combination of different and identical substrates.

 In a particular embodiment of the invention, the idiosyncratic catalytic imprint of a sample can be achieved using substrates capable of revealing different subclasses of at least one class of catalytic activity.

 By way of example, substrates of the epoxide, ester, carbonate, amide, cyano, lactam, carbamate, glycoside, alkyl halide, mono and diester phosphate, enol ether and alkyl ether type may make it possible to reveal the idiosyncratic catalytic hydrolysis of a sample. Similarly, substrates of ethanol, methanol, isopropanol, butanol, glycerol, ethylene glycol, lactic acid, malic acid, citric acid, formic acid, acetic acid, propionic acid, benzoic acid, benzaldehyde, acetaldehyde, propionaldehyde may allow to reveal an idiosyncrasic dehydrogenase catalytic imprint of a sample.

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 The number of substrates is advantageously at most 1000. It is generally of the order of 1 to 200.

 The substrates used in the process of the invention may be ordered on one or more supports, such as, for example, on a microtiter plate or a microsystem. This support having substrates may constitute a system used to generate the idiosyncratic catalytic imprint according to the method of the invention.

 The reaction of the substrates with the sample can be followed in real time or in end point.

 The resulting idiosyncratic catalytic imprints can be stored to create sample fingerprint databases.

 The idiosyncratic catalytic imprint of a sample may correspond to the combination of several imprints obtained according to different forms of implementation or over time.

 The method of the invention may also comprise, after the collection of idiosyncratic catalytic imprints, signals generated by a sample, an analysis of the idiosyncratic catalytic imprint. This analysis makes it possible to characterize or monitor the evolution of the sample. It is not necessary to know exactly all activities to perform this analysis.

 The signal is a function of the modification made to the activity. Thus in a sample, catalytic activities belonging to the same class are

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 ability to react on the same substrates with a different reaction rate. The generated signal will be different.

 The footprint of a sample is likely to change, for example, because the sample has been exposed to contaminants or because the sample is degrading or deteriorating over time. For monitoring sample evolution such as quality control, the result obtained can be considered by comparing it with a fingerprint that is part of a database or with a fingerprint made before or after the sample analyzed.

 The idiosyncratic catalytic imprint obtained can be superimposed on another imprint obtained by way of example: with the same set of substrates in the presence of a mixture of samples or of a reference sample. with the same sample but with a different set of substrates.

 - with the same set of substrates revealed with the same sample at a different time.

 with the same set of substrates revealed with the same sample taken at a different time.

 with the same set of substrates revealed under different reaction conditions.

 This comparison of idiosyncratic catalytic imprints can be carried out by any technique, in particular: by visual comparison of the catalytic modification imprints or generated generated signals by computer graphic analysis.

 The modification of an idiosyncratic catalytic footprint of a sample is the indicator of a

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 variation or appearance or disappearance of one or more catalytic activities.

 The use of the method of the invention and the superposition of impressions is entirely appropriate for the monitoring of processes or products implementing catalytic activities for quality control in particular.

. alosyncrasique caractéristique < -n= signaux et signaux et l'interprétation de ladite empreinte idiosyncrasique pour caractériser l'échantillon. The method of the invention is also remarkable in that it can be automated. Indeed, such automation consists in the possible computer processing of the transformation results of the substrates to obtain the catalytic digital impression.

. characteristic alosyncrasic <-n = signals and signals and the interpretation of said idiosyncratic imprint to characterize the sample.

 The invention also relates to a system comprising a support on which are ordered a plurality of substrates as defined above, identical or different to implement the method of the invention.

 This system may comprise, in addition to the set of substrates, one or more following accessories: a device for controlling the contacting of the sample with at least one substrate; sensors for measuring the signals generated from the transformation of the substrates by the sample. These sensors may correspond by way of non-limiting example to a fluorimeter, a spectrophotometer, an ammeter, a pH meter.

 An analyzer for generating the idiosyncratic catalytic imprint of a sample.

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The footprint will for example be stored as a series of gray scale bits.

 - A memory allowing to store in a database the original idiosyncratic catalytic fingerprints.

 An analyzer allowing the superimposition between the newly obtained idiosyncratic catalytic impression and all the imprints stored in the memory. This analyzer will be able to perform statistical tests known to those skilled in the art to perform analyzes of differences and similarities between the newly obtained fingerprint and the set of fingerprints stored in the memory.

 These statistical tests may involve, for example, discrepancy or correlation tests.

 The system of the invention can be used to track the evolution of a sample. The invention thus also relates to the use of a system as defined above for monitoring the evolution of a sample, characterized in that: at least one sample is brought into contact with a set of substrates ordered on said system, each having at least one functional group sensitive to the presence of catalytic activity of the sample, - the generated signals resulting from said contacting of the substrates with the catalytic activity of a sample are captured.

 - The impression is compared for one sample to another established footprint.

 Other advantages and characteristics of the invention will become apparent in the examples which follow. These

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 Examples show that the method of the invention makes it possible to obtain the idiosyncratic imprints of a sample.

 The embodiment of these examples can easily be applied to any type of sample.

 In these examples, reference will be made to the accompanying drawings in which: - Figure 1 shows examples of fluorogenic substrates formulas (Figure la) and chromogenic (Figure lb) useful according to the invention.

 FIG. 2 represents a principle of detection of fluorescent substrates.

 FIG. 3 represents the kinetics of the absorbance signal (OD, at A. = 405 nm) for the idiosyncratic catalytic imprint of the signals generated by a sample with the grid of chromogenic substrates.

 FIG. 4a shows the idiosyncratic catalytic imprints obtained with fluorescent substrates. The distribution of the substrates has been given in FIG. The different samples tested by imprint are shown in Table 1 below. Figure 4b shows the idiosyncratic catalytic imprints obtained with chromogenic substrates. The distribution of the substrates was given in FIG. 1b. The various samples tested by imprinting are indicated in Table 1 below.

Table 1

<Tb>
<tb> 1 <SEP> A <SEP> = <SEP> Aspergillus <SEP> niger <SEP> lipase <SEP> (ANL), <SEP> B <SEP> = <SEP> Aspergillus
<tb> niger <SEP> lipase <SEP> (ANL) <SEP> lU / mg, <SEP> C <SEP> = <SEP> Aspergillus <SEP> oryzae
<tb> lipase <SEP> (AOL), <SEP> D <SEP> = <SEP> Candida <SEP> antartica <SEP> lipase <SEP> (CAL), <SEP> E <SEP> =
<tb> Candida <SEP> Cylindracea <SEP> Lipase <SEP> (CCL) <SEP> F <SEP> = <SEP> Candida
<tb> lipolytica <SEP> lipase <SEP> (CLL), <SEP> G <SEP> = <SEP> hog <SEP> pancreatic <SEP> lipase
<tb> (HPL), <SEP> H <SEP> = <SEP> Mucor <SEP> javanicus <SEP> lipase <SEP> (MJL)
<tb> 2 <SEP> A <SEP> = <SEP> Mucor <SEP> miehei <SEP> lipase <SEP> (MML), <SEP> B <SEP> = <SEP> Penicillium
<tb> roquefort <SEP> lipase <SEP> (PRL), <SEP> C <SEP> = <SEP> Pseudomonas <SEP> cepacia
<tb> lipase <SEP> (PCL) <SEP> 40U / mg, <SEP> D <SEP> = <SEP> Pseudomonas <SEP> cepacia <SEP> lipase
<tb> (PCL) <SEP> 50U / mg, <SEP> E <SEP> = <SEP> Pseudomonas <SEP> fluorescens <SEP> lipase
<tb> (PFL), <SEP> F <SEP> = <SEP> Rhizomucor <SEP> miehei <SEP> lipase <SEP> (RML), <SEP> G <SEP> =
<tb> Rhizopus <SEP> arrhizus <SEP> lipase <SEP> (RAL), <SEP> H <SEP> = <SEP> Rhizopus <SEP> niveus
<tb> lipase <SEP> (RNL)
<Tb>

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<Tb>
<tb> 3 <SEP> A <SEP> = <SEP> wheat <SEP> germ <SEP> lipase <SEP> (WGL), <SEP> B <SEP> = <SEP> Bacillus <SEP> sp. <SEP> esterase
<tb> (BSE), <SEP> C <SEP> = <SEP> Bacillus <SEP> stearothermophilus <SEP> esterase
<tb> (BStE), <SEP> D <SEP> = <SEP> Bacillus <SEP> thermoglucosidasius <SEP> esterase
<tb> (BTE), <SEP> E <SEP> = <SEP> Candida <SEP> lipolytica <SEP> esterase <SEP> (KEY), <SEP> F <SEP> = <SEP> hog
<tb> liver <SEP> esterase <SEP> (HLE) <SEQ> 100J-lg / ml, <SEP> G <SEP> = <SEP> hog <SEP> liver <SEP> esterase
<tb> (HLE) <SEP> 10J-1g / ml, <SEP> H <SEP> = <SEP> horse <SEP> liver <SEP> Esterase <SEP> (HrLE)
<tb> 4 <SEP> A <SEP> = <SEP> Aspergillus <SEP> niger <SEP> epoxide <SEP> hydrolase <SEP> (AEH)
<tb> 10OJ-1g / ml, <SEP> B <SEP> = <SEP> Chromobacterium <SEP> viscosum <SEP> lipoprotein
<tb> lipase <SEP> (CVL), <SEP> C <SEP> = <SEP> Pseudomonas <SEP> sp. <SEP> lipoprotein <SEP> lipase
<tb> (PSL) <SEP> 1500U / mg, <SEQ> D <SEP> = <SEP> Pseudomonas <SEP> sp. <SEP> lipoprotein <SEP> lipase
<tb> (PSL) <SEP> 50000U / mg, <SEQ> E <SEP> = <SEP> Pseudomonas <SEP> sp. <SEP> Type <SEP> B
<tb> lipoprotein <SEP> lipase <SEP> (PSBL), <SEP> F <SEP> = <SEP> Mucor <SEP> miehei <SEP> esterase
<tb> (MME), <SEP> G <SEP> = <SEP> Saccharomyces <SEP> cerevisiae <SEP> esterase <SEP> (SCE), <SEP> H
<tb> = <SEP> Thermoanaerobium <SEP> brockii <SEP> Esterase <SEP> (TBE)
<tb> 5 <SEP> A <SEP> = <SEP> Aspergillus <SEP> niger <SEP> epoxide <SEP> hydrolase <SEP> (SEH) <SEP> 10J-1g / ml,
<tb> B <SEP> = <SEP> Aspergillus <SEP> melleus <SEP> acylase <SEP> I <SEP> (AMA), <SEP> C <SEP> =
<tb> Escherichia <SEP> coli <SEP> penicillin <SEP> G <SEP> acylase <SEP> (PGA), <SEP> D <SEP> =
<tb> Bacillus <SEP> thermoproteolyticus <SEP> thermolysin <SEP> (The), <SEP> E <SEP> =
<tb> swine <SEP> stomach <SEP> mucosa <SEP> pepsin <SEP> (Pep), <SEP> F <SEP> = <SEP> Clostridium
<tb> histolyticum <SEP> clostripain <SEP> (Clos), <SEP> G <SEP> = <SEP> Papaya <SEP> latex
<tb> papain <SEP> (Pap), <SEP> H <SEP> = <SEP> Electrophorus <SEP> electricus
<tb> acetylcholine <SEP> esterase <SEP> (AChE)
<tb> 6 <SEP> A <SEP> = <SEP> Rhodotorula <SEP> glutinis <SEP> epoxide <SEP> hydrolase <SEP> (REH), <SEP> B <SEP> =
<tb> hog <SEP> kidney <SEP> acylase <SEP> I <SEP> (HKA), <SEP> C <SEP> = <SEP> Bacillus <SEP> licheniformis
<tb> subtilisin <SEP> (Sub), <SEP> D <SEP> = <SEP> hog <SEP> pancreas <SEP> trypsin <SEP> (Try), <SEP> E <SEP> =
<tb> porcine <SEP> pancreatic <SEP> elastase <SEP> (Ela), <SEP> F <SEP> = <SEP> bovine
<tb> pancreatic <SEP> a-chymotrypsin <SEP> (Chy) <SEP> 37U / mg, <SEP> G <SEP> = <SEP> bovine
<tb> pancreatic <SEP> a-chymotrypsin <SEP> (Chy) <SEP> 70U / mg, <SEP> H <SEP> = <SEP> blank
<Tb>

Example 1: Idiosyncratic Catalytic Impregnations of Samples

 1) The different fluorescent and chromogenic substrates are arranged in the wells of microtitre plates respectively according to the arrangement of FIG. 1a and 1b, in the form of 5 microliters of a 2 mM stock solution in DMF / 50/50 water. The grid includes solutions of the reference products that will serve as a positive control in the measurement.

 2) A solution of a prediluted sample in a buffer containing the developer (1 mM NaIO 4, BSA 2 mg / ml) is then added to each well, and the reaction is monitored as a function of time for 2 hours.

The list of samples tested is shown on the

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 Figure 5. Only the data of the first 30 minutes are necessary for the subsequent calculation of the signals generated to realize the corresponding idiosyncratic catalytic imprints.

pM. s-1, (S)-10 2'300 pM. s-1, 14 1'600 pM. s-1, (S)-13 1'400 pM. s-l, (R) -13 1 r 300 pM. s-1. b) Détermination des vitesses maximum apparentes à partir de la pente maximum apparente dans l'empreinte de vitesse, qui se situe toujours avant 30 minutes de réaction (figure 3). c) Soustraction des vitesses enregistrées pour la mesure de référence sans échantillon dans les même conditions. d) Division de toutes les vitesses par la vitesse maximum observée dans la grille pour un échantillon donné. e) Coloration de la case correspondante au substrat en échelle de gris en proportion de la vitesse relative. 0 = blanc. Maximum = noir. 3) Fingerprints are obtained as follows: a) Conversion of fluorescence or absorbance data at 405 nm to umbelliferone or nitrophenol concentration according to calibration curves. A typical reaction fingerprint is exemplified in Figure 3. Conditions: 0.1 mg / mL enzyme sample, 100 M substrate, 20 mM aq. borate pH 8.8, 2.5% v / v DMF, 26 C, 2 mg. mL-1 BSA, 1 mM NaIO4. The velocities determined from these curves are as follows: 209,900 μM. s-1, 16 130,800 μM. s-1, (S) -12a 31,800 μM. Sl, (sub 25,500 μM, sl (R) -12a 25,000 μM, (R) -12b 22,000 μM s-1, 4,000 μM s-1, (R) -10 2'600

pM. s-1, (S) -10 2300 μM. s -1, 1'600 μM. s-1, (S) -13400 μM. sl, (R) -13 1 300 μM. s-1. b) Determination of apparent maximum velocities from the apparent maximum slope in the velocity fingerprint, which is always before 30 minutes of reaction (Figure 3). c) Subtraction of recorded velocities for the reference measurement without sample under the same conditions. d) Division of all speeds by the maximum speed observed in the grid for a given sample. e) Coloring of the box corresponding to the gray scale substrate in proportion to the relative speed. 0 = white. Maximum = black.

 The representation of the analyzed fingerprint, which is necessary for the visual analysis, may advantageously take the form of a square or rectangular grid or each square of the grid is tinted with

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 gray scale in proportion to the value assigned to a given substrate in the series, ranging from white (no activity) to black (maximum activity).

 The idiosyncratic catalytic imprint of the generated signals is then displayed as well as the value of the apparent maximum velocity in pM / s.

 We have verified that these measurements are reproducible, namely that the same sample gives the same idiosyncratic catalytic footprint when the measurement is repeated under the same conditions. 0.1 mg / mL sample, 2 mg / mL BSA (beef serum albumin), 20 mM borate buffer pH 8.8, 0.1 mM each substrate, 1 mM NabiO4 incubation 26 C.

 For FIGS. 4a and b, 47 samples (see FIG. 6) were tested. The corresponding idiosyncratic footprints were obtained (Figures 4a and 4b). They illustrate the individual ability of each sample to react with a set of substrates.

 Example 2 Combination of substrates that can be used to generate the idiosyncratic catalytic imprint of a sample.

 The substrates and 4 control substrates, the formulas of which are given in FIG. 5, were used.

All substrates are tested at a concentration of 0.1 mM, pH 7.4, in two types of conditions:
1) in direct time: sample (0.1 mg / mL), BSA (2 mg / mL), aqueous inorganic buffer (phosphate, borate, ammonium carbonate) (10-100 mM) with 7 <pH <11. Temperature between 15 and 45 OC.

 2) end point: a) incubation of the sample (0.1 mg / mL) at a given pH / temperature in

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 inorganic buffer; b) addition of buffer stabilizer to obtain a final pH between 7 and 9, BSA (2 mg / mL), then recording the signal either directly or after 20 min.

 Substrates A-F and controls K: addition of NaI04 (1 mM).

 Substrates G: addition of NAD + (1 mM) and NADP + (1 mM).

 The signals generated after the contact of this set of substrates with a sample will be fluorescent.

These different substrates make it possible to reveal the following classes of enzymes:
Lipases, esterases, proteases and amidases: AlA22, B1-B22, C1-C22, D1-D22, E1-E4 - Phosphatases: A23-D23 - Sulfatases: A24-D24 - Urease: A25-D25 - Argininases: A26-D26 - Dehalogenases: A27-D27 - Aldolases: A28-D28 without NaI04 - Bayer-villigerases: A28-D28 with lipase and NaIO4 - Ether-hydrolases: A29-D29, A30-D30 - Epoxide hydrolases: F1-F4 - Alcohol dehydrogenases: G1- G4 without Nais4 with NAD + / NADP + - and hydroxylases: H1-H8 without Nais4 - Glycosidases: J1-J8 without NaIO4
In another embodiment, the same substrate grid will be made with paranitrophenol replacing the coumarin (Coum) as a leaving group. The signals measured after contacting the substrates with the sample will be chromogenic.

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 Example 3: Idiosyncratic imprint using substrates requiring the presence of a sensor.

 The idiosyncratic of a sample can be achieved by a grid of non-chromogenic or non-fluorogenic substrates if the reaction can be indirectly followed, for example by means of a sensor, such as a thermographic probe, or a pH or pM sensor. As an example, a grid composed of esters will be used to obtain the lipase and esterase sample fingerprint using a pH indicator for the measurement. A series of amino acid derivatives such as the N-acyl amino acids (N-acetyl amino natural L-amino + N-acetyl amino D-amino corresponding) will advantageously serve to obtain the idiosyncratic sample containing acylases by following the reaction using a pM sensor.

Claims (22)

1) Process for generating the idiosyncratic catalytic impression of a sample, characterized in that it comprises the following steps: the sample likely to possess the catalytic activity is brought into contact with a set of ordered substrates; captures the signals generated after said contacting.  2) Process according to claim 1, characterized in that each substrate comprises at least one functional group sensitive to the presence of a catalytic activity of a sample and allows the generation of a signal directly or indirectly.  3) Process according to claim 2, characterized in that each substrate comprises a chemical group capable of conferring a specificity for an activity defined by the functional group.  4) Process according to claim 3, characterized in that the chemical groups capable of conferring on the substrate a specificity for an activity defined by the functional group are constituted by substitution variations.  5) Method according to any one of claims 1 to 4, characterized in that the signals generated by contacting the substrates with the sample can be measured on the same type of sensor. <Desc / Clms Page number 23>  6) Process according to any one of the preceding claims, characterized in that the signals generated by bringing the substrates into contact with the sample are of the same type.  7) Process according to any one of the preceding claims, characterized in that the substrates are capable of reacting on a short reaction time, for example less than 2 hours, preferably less than 20 minutes.  8) Method according to any one of the preceding claims, characterized in that the substrates are little or not sensitive to degradation or non-specific activities.  9) Process according to any one of the preceding claims, characterized in that the catalytic activities of the sample are unknown.  10) Process according to any one of the preceding claims, characterized in that the catalytic activity of the sample corresponds to at least one enzyme or a mixture of enzymes. <Desc / Clms Page number 24>  11) Process according to any one of claims 1 to 10, characterized in that the substrates are chosen from those corresponding to one of the following formulas (I) to (V):

 in which: the C1-C2 bond is insensitive to a cleavage by a chemical oxidation reaction; Di represents the precursor of a detectable product; X and Y, which are identical or different, are chosen from an oxygen atom; , a sulfur atom, an amine of formula-NRllR, Ru is chosen from: a hydrogen atom, an alkyl group, an aryl group, substituted or unsubstituted, and R12 is not a hydrogen atom, - R1 at R3, which are identical or different, are chosen from a hydrogen atom, a substituted or unsubstituted alkyl group or a chemical group as defined above; and Pi and P2, which are identical or different, are functional groups as defined above, and at most one of Pi and P2 groups is a hydrogen atom, <Desc / Clms Page number 25>  - P3 is selected from a carbonyl group, a PORn group or an RPO- group, where Ru has the same meaning as above, a group -CHOR13 group where R represents an aryl, alkyl or glycosyl group, a group SiR14R15 where R14 and R, which may be identical or different, represent an aryl, alkyl, aryloxy or alkoxy group and an AsO 2 H- group. G is chosen from an oxygen atom, a sulfur atom and an amine group of formula NR 11 R 12, where R 11 and R 12 have the same meaning as above, and in that said substrates of formulas (I) to (V) are transformed by the sample into products which will be oxidized to obtain directly or indirectly a detectable compound corresponding to one or more compounds capable of presenting alone or in combination one or more variations of their biophysical, biological or chemical properties before and after the oxidation step.  12) Process according to any one of claims 1 to 10, characterized in that the substrates (S) are chosen from those whose transformation into product (P) can be detected by measuring a change in pM resulting from the difference in chelating power of the substrate (S) and the product (P) with respect to a metal ion, and in that the transformation of a substrate (S) into a product (P) is carried out by measuring the change in the spectral properties a chemical detector (L), chelating a metal ion, resulting from metal ion exchanges according to the following equilibrium:

 ML Ob (MP and / or MS) + L <Desc / Clms Page number 26> S represents a substrate, which may be any chemical molecule, whose chelating power is different from that of the product resulting from the chemical transformation of the substrate. These include specific substrate, such as phytic acid for phytases or whole proteins for proteases. This substrate may have enantioselective properties. The substrates have no structural constraint other than that of having a chelating power difference with the corresponding products.  or :  P represents the product resulting from the chemical transformation of S and having a chelating power different from S. As the substrates, the products have no structural constraint other than that of having a chelating power difference with the corresponding substrates.  L represents a chemical detector, also referred to as a pM sensor, corresponding to any ligand capable of chelating the metal ions in solution such that the chelation of the metal by the chemical detector gives a measurable change in pM by any appropriate method such as a change of property spectral detector. The sensor pM has a chelating power such that there can be exchanges of metal ions between the product (P) and the ligand (L) or the substrate (S) and the ligand (L) according to the product ( P) or the substrate (S) has the strongest chelating power. As indicated above, the chelating power of the chemical detector (L) is advantageously greater than that of the substrate (S) or the product (P) so as to carry out the method of the invention with small amounts of chemical detectors. The chemical detector (L) may be a fluorescein derivative or any other fluorescent nucleus substituted with a chelating moiety. <Desc / Clms Page number 27>  ML represents the metal-ligand complex with spectral properties different from L.  MS represents the metal-substrate complex.  MP represents the metal-product complex.  13) Method according to any one of the preceding claims, characterized in that the number of substrates is advantageously at most 1000.  14) Method according to any one of the preceding claims, characterized in that the number of substrates is of the order of 1 to 200.  15) Method according to any one of claims 2 to 14, characterized in that the substrates have different functional groups.  16) Method according to any one of claims 4 to 15, characterized in that the substrates have different chemical groups.  17) Method according to any one of the preceding claims, characterized in that after the capture of the signals generated by a sample, an analysis of the idiosyncratic catalytic footprint is carried out.  18) Method according to claim 17, characterized in that said analysis consists in characterizing or monitoring the evolution of the sample.  19) Method according to one of claims 17 or 18, characterized in that said analysis comprises the comparison of the idiosyncratic catalytic footprint <Desc / Clms Page number 28>  obtained with another idiosyncratic catalytic impression.  20) Application of a method according to any one of the preceding claims to the quality control.  21) A substrate assembly as defined in any one of claims 1 to 19.  22) A system for carrying out a method according to any one of claims 1 to 19, characterized in that it comprises a support on which are ordered a plurality of substrates as defined in any one of claims 1 to 19 and one or more accessories: - a device for controlling the contacting of the sample with at least one substrate, - sensors for measuring the signals generated from the transformation of the substrates by the sample, - a first analyzer for generating the idiosyncratic catalytic imprint of a sample, - a memory for storing in a database the original idiosyncratic catalytic imprints, a second analyzer allowing the superimposition between the newly obtained idiosyncratic catalytic imprint and the whole imprints stored in the memory.