Method of manufacturing a probe for determining the activity of pectinolytic enzymes and method of determining the activity of pectinolytic enzymes using said probe

Abstract

The subject of the present invention is a method of obtaining a fluorescent probe for determining the activity of pectinolytic enzymes, a method of determining the activity of pectinolytic enzymes using the obtained probe, and a use of this probe for determining the activity of pectinolytic enzymes.

Description

[0001] METHOD OF MANUFACTURING A PROBE FOR DETERMINING THE ACTIVITY OF PECTINOLYTIC ENZYMES AND METHOD OF DETERMINING THE ACTIVITY OF PECTINOLYTIC ENZYMES USING SAID PROBE

[0002] DESCRIPTION

[0003] The present invention relates to a method of manufacturing a probe for determining the activity of pectinolytic enzymes, a probe for determining the activity of pectinolytic enzymes, and a method of determining the activity of pectinolytic enzymes using said probe.

[0004] STATE OF THE ART

[0005] Currently used methods for determining the activity of pectinolytic enzymes in a sample are based on the detection of the effects of the fragmentation process of the native pectin chain.

[0006] One of the methods used is based on measuring A230 (absorbance at a wavelength of 230 nm) of the sample during incubation with a pectinolytic enzyme preparation. Enzymatic digestion of pectin leads to an increase in absorbance due to the formation of unsaturated products (in particular, 4,5-unsaturated oligogalacturonates) [1], This method, despite the relative simplicity of performance and the possibility of tracking the digestion reaction progress in real time, has a number of drawbacks that hinder its use; these include the need to conduct the reaction in a quartz cuvette, the inapplicability for turbid samples and those characterized by high absorbance in the UV spectrum (e.g., preparations containing high concentrations of proteins or many extracts from fruits and vegetables). The instability of unsaturated products of the digestion reaction is also a problem, making it impossible to read the result more than a few minutes after the digestion is completed. Other methods are based on titration of the released acidic products of enzymatic digestion of pectins and polygalacturonates; for example, the method of titration with sodium thiosulfate solution according to the Kertesz protocol [2], A limitation of the above-mentioned method is the need to conduct titrations until the pale yellow color of the test solution disappears, which, in addition to the possibility of errors on the part of the analyst, prevents its use for turbid or colored samples. An alternative to this method is the Miller protocol, which is based on the determination of reducing sugars released by enzymatic digestion. These sugars (in this case, mainly galacturonate) react with 3,5-dinitrosalicylic acid to produce a colored product, the concentration of which can be determined spectrophotometrically by measuring the absorbance at a wavelength of about 545 nm [3], [4], However, this method requires the use of harmful substances (such as phenol or its derivatives including nitro derivatives), as well as due to the lack of specificity, it is prone to falsification (false-positive or overestimated results) related to the presence of reducing sugars in the sample not formed by digestion.

[0007] The numerous limitations of currently available methods for determining the activity of pectinolytic enzyme preparations indicate the usefulness of developing a method of determining pectinolytic enzyme activity characterized by simplicity of performance, low susceptibility to interferents and high specificity. An effective and frequently used method in the biological sciences to determine enzymatic activity is the use of fluorescently labeled probes. They are used, for example, in quantifying the progress of PCR. reactions [5], but can also be used to determine the activity of polysaccharide-digesting enzymes (e.g., cellulases) [6], There are numerous methods for conducting coupling of fluorophores to sugar substrates, depending mainly on the chemical property of the labeled substrate. Methods for obtaining fluorescently labeled acidic saccharides and their derivatives (e.g., glucuronic acid [7], or glycosaminoglycans [8]) are available in the literature, but they differ from the synthesis method described herein in the substrate to be labeled (in this case pectin, pectinate or polygalacturonic acid), the fluorescent dye used (a mixture of 6- aminofluorescein and rhodamine B), as well as the reaction conditions and method of purification of the final product.

[0008] AIM OF THE INVENTION

[0009] The numerous limitations of currently available methods for determining the activity of pectinolytic enzyme preparations indicate the usefulness of developing a new method of determining the activity of pectinolytic enzymes, characterized by simplicity of performance, low susceptibility to interferents and high specificity. The aim of the invention was to obtain a molecule or a mixture of molecules that can be used as a specific probe, allowing the determination of pectinolytic enzyme activity in a sample, as well as its use to carry out the above-mentioned determinations. SUMMARY OF THE INVENTION

[0010] Thus, the subject matter of the present invention is a method for obtaining a probe for determining pectinolytic enzyme activity, comprising steps in which:

[0011] - the substrate to be labeled, selected from pectin, pectinate, polygalacturonic acid or a mixture thereof, is suspended in pyridine buffer or other aqueous-organic buffer with a pH of 2 to 6;

[0012] - a fluorescent dye, preferably containing amino groups, and a coupling reagent that activates the carboxyl groups of the substrate are added to the resulting mixture,

[0013] - the reaction mixture is incubated without light at room temperature, with stirring for a period of 2 to 20 hours, preferably 8 to 16 hours,

[0014] - the resulting product is separated from the reaction mixture by precipitation with a polar organic solvent of increased ionic strength and centrifugation,

[0015] - additional purification of the product is carried out by washing with an organic solvent, preferably dichloromethane, followed by ethanol.

[0016] Preferably, EDAC is used as a coupling agent to activate the carboxyl groups of the substrate.

[0017] Preferably, the reaction is carried out in a pyridine buffer with a pH of 4 to 5, more preferably 4.5.

[0018] Preferably, the probe is precipitated from the reaction mixture using a 0.1 M solution of sodium acetate in 96% ethanol.

[0019] Further subject matter of the invention is a method of determining the activity of pectinolytic enzymes, comprising the steps in which:

[0020] - the probe solution obtained by the method defined in any of the claims 1-4 is subjected to digestion with a pectinolytic activity-exhibiting test sample over a period of 1 hour to 48 hours, preferably 2 hours to 24 hours;

[0021] - after incubation, separation of the digested and undigested fractions of the probe is carried out by precipitation, preferably by adding an aqueous solution of a divalent metal salt, such as a calcium salt, until a final salt concentration in the range of 10- 500 mM, preferably 50 to 100 mM is obtained;

[0022] - the precipitate is separated, preferably by centrifugation or filtration;

[0023] - the liquid fraction is collected and the absorbance or fluorescence signal is measured, from which the pectinolytic activity of the preparation under test is determined.

[0024] Preferably, the separation of the digested and undigested fractions of the probe by precipitation is carried out by adding a calcium chloride solution.

[0025] The subject matter of the invention is also a use of a probe obtained by the method defined above for the determination of pectinolytic enzyme activity.

[0026] The developed innovation is based on the coupling of pectin, pectinate or polygalacturonic acid with a fluorescent dye using an EDAC condensing agent in a pyridine buffer environment, resulting in a fluorescently labeled probe for the determination of pectinolytic activity. The specific, fluorescently labeled probe obtained in this way is used to carry out determinations of pectinolytic activity in the sample by subjecting the probe to digestion and then separating the digested and undigested fractions by precipitation and centrifugation or filtration, followed by measurement of the absorbance or fluorescence signal of the supernatant or filtrate.

[0027] DESCRIPTION OF THE DRAWINGS

[0028] The subject matter of the invention is shown in the drawings:

[0029] Fig. 1. Appearance of probe variants at different stages of purification by precipitation with ethanol.

[0030] Fig. 2. Visual comparison of probes at a concentration of 1 mg / mL undigested (K-) and digested with a commercially available pectin lyase enzyme preparation (PL2), followed by precipitation with calcium chloride and centrifugation. Both the sample digested for 2 h and 24 h relative to the control show an increase in fluorescence intensity in the supernatant and a decrease in the volume of the pellet containing the precipitated, undigested probe. Visualization under UV light (280-400 nm). Fig. 3. Standard curve illustrating the increase in fluorescence signal in the supernatant as a function of digestion time using a commercially available pectin lyase enzyme preparation (PL2).

[0031] Fig. 4. Use of the invention to determine the temperature optimum of pectin lyase enzyme (PL2) activity. The curve illustrates the fluorescence signal in the supernatant after 2 h digestion with a commercially available protein preparation under analogous reaction conditions (excluding incubation temperature). Error bars indicate standard deviation.

[0032] Fig. 5. Use of the invention to determine the pH optimum for pectin lyase enzyme (PL2) activity. The curve illustrates the fluorescence signal in the supernatant after 2 h digestion with a commercially available protein preparation under analogous reaction conditions (excluding buffer pH). Error bars indicate standard deviation.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] The subject matter of the invention is a method of obtaining a probe and its use for determining pectinolytic enzyme activity. The above-mentioned probe is a fluorescently labeled derivative of galacturonic polyacid or pectin and is obtained by coupling reaction of a substrate (galacturonic polyacid and / or pectin, for example, selected from such as substances with CAS numbers: 9000-69-5, 9005-88-3, 503591-24-0, 220424-61-3, 9047-18-1, 9049-37- 0, 25990-10-7, 25249-06-3, or oligo- and polymers based on structures with CAS numbers 685-73-4, 552-12-5, 14982-50-4, 6294-16-2, 9046-38-2, 25990-10-7, 9046-40-6, 18968-14-4, 9000-69-5) with a selected fluorescent dye having the appropriate reactive functional groups (amino groups), allowing the reaction to take place. The coupling reaction is carried out through activation of the carboxyl groups of pectin / galacturonic acid with 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC) and incubating with the selected dye(s) (containing amino groups) in a pyridine buffer medium with a pH in the range of 4-5.

[0035] After an 8-16 hour incubation of the reaction mixture with stirring and without light at room temperature, the product is precipitated, e.g., by adding to the reaction mixture twice the volume of a solution of 100 mM sodium acetate in 96% ethanol and separated by centrifugation, filtration or other separation techniques. After this, the product undergoes pre-cleaning by washing three times with dichloromethane (DCM). Then, the product is further purified by washing with 96% ethanol until a supernatant showing no significant absorbance signal characteristic of the dye used in the synthesis is obtained. After this, the product is incubated in an open vessel to remove residual ethanol until the product is completely dry. The probe thus obtained can be stored dry under refrigeration or an aqueous solution of 10 mg / mL can be prepared and used for further studies.

[0036] The probe obtained as described above can be used to determine the activity of pectinolytic enzymes in the sample.

[0037] The probe obtained in this way can be subjected to enzymatic digestion with a selected preparation containing pectinolytic enzymes. Such a preparation can be, among others: a purified enzyme preparation; an enzyme-containing mixture, supernatant or metabolized medium from a culture of microorganisms secreting pectinolytic enzymes; a culture or suspension of microorganisms secreting pectinolytic enzymes; or commercially available preparations that are mixtures of enzymes. It is important that the prepared probe solution has chemical properties (such as, for example, the type of buffer and its concentration, pH, buffer strength, salt concentration, presence of additional cofactors, e.g., calcium cations or other divalent metals) that are optimal for the particular enzyme preparation chosen to do the digestion; it is also important to carry the reaction on at a temperature that is optimal for the activity of the enzyme / enzyme mixture. The optimal conditions depend on the enzyme / enzymatic preparation in question. Thus, for example, for pectate lyase 2 (PL2), derived from the microorganism Pectobacterium carotovorum, the optimal conditions for activity are temperature 37 °C and pH 8.5 [9]; pectin lyase of the same family, but derived from the microorganism Yersinia enterocolitica has an optimum of activity at 37 °C, but the optimum pH value for its activity is 9.6

[0010] ; while pectin lyase 9 (PL9), derived from the microorganism Clostridium cellulovorans, has an optimum activity at a pH of 8.0, but at a temperature of 50 °C

[0011] ,

[0038] It is also possible to carry out enzymatic digestion reactions under suboptimal conditions, for example, if such conditions are planned for use in further technological processes for which tests or determinations are carried out using the described invention. The probe can also be used to determine the optimal conditions of a preparation with enzymatic activity having uncharacterized properties. This is possible, for example, using a gradient of the selected factor or factors; such an approach makes it possible to determine the points of optimal activity and, consequently, optimize the conditions for conducting the enzymatic reaction and / or determine the optimum of the characterized preparation with pectinolytic activity.

[0039] To obtain results of a quantitative nature of pectinolytic enzyme activity in the preparation (i.e., for example, determination of universal units of enzymatic activity (U), understood as the amount of enzyme capable of converting 1 pmole of product in 1 minute) using the described invention, it is necessary to use a standard curve plotted from a pectinolytic enzyme preparation of known activity.

[0040] After incubation for a fixed time with a preparation showing pectinolytic activity, the test should be developed by adding a precipitating agent, for example an aqueous solution of calcium salts. The fixed time depends on the conditions for conducting the reaction, among other things, the activity of the given preparation, the enzyme and substrate concentrations used, etc. The time should be constant in a given test and may need to be optimized before the actual determination.

[0041] The precipitation process takes advantage of the solubility difference in the presence of high concentrations of divalent metal salts, described in the literature, occurring between undigested, polymeric pectin and the oligo- and monomers of galacturonic acid formed by enzymatic digestion. Under the described conditions, pectin precipitates out of solution in the form of pectinate; the potential for precipitation is dependent on the polymer chain length, which means that only polymeric pectin undergoes efficient precipitation and sedimentation, while oligo- and monomers remain soluble, allowing simple separation of the products of enzymatic digestion from the undigested probe. At this stage, separation efficiency can be improved by centrifugation or filtration, facilitating the collection of the soluble fraction for further measurements.

[0042] It is characteristic of the described invention that, due to the presence of fluorescent dyes covalently bound to the pectin backbone, the precipitation carried out in this way affects the distribution of the fluorescent signal in the sample — the signal remaining in the liquid fraction (supernatant, filtrate) is strictly dependent on the degree of digestion of the probe by pectinolytic enzymes. This means that for a sample incubated with a preparation that does not show pectinolytic activity, after the addition of a precipitating agent and separation, the resulting liquid fraction will be essentially devoid of signal from the fluorescent dye. For samples incubated with a preparation showing pectinolytic activity, the signal from the fluorescent dye will be proportional to the concentration of labeled oligo- and monomers in the sample. This concentration will in turn be derived from the degree of probe digestion, which depends on the amount of pectinolytic activity in the enzyme preparation used.

[0043] Precise measurement of the signal from the digested fraction of the probe can be made by spectrophotometric or spectrofluorometric methods, of which fluorescence measurement is preferred due to higher sensitivity and precision of measurement, as well as lower susceptibility to interferents such as color compounds. Once measured, comparative analysis between samples can be performed to determine relative activity, or a standard curve can be used to determine the absolute value of enzymatic activity in a sample.

[0044] EXAMPLES

[0045] General description of the synthesis

[0046] The pyridine buffer is prepared by mixing in equal proportions of 3 M HO solution and pyridine, then the pH of the mixture is brought to about 4.5. An amount corresponding to about 0.1 pmole of substrate for labeling is suspended in 15 mL of the previously prepared pyridine buffer; EDAC (72.5 times the substrate concentration, i.e., 7.25 pmole) and the chosen fluorescent dye or fluorescent dye mixture (at least 20 times the substrate concentration) are added. The reaction mixture thus obtained is incubated without light, with stirring for 8-16 h. After completion of the reaction, the product of the coupling reaction is separated by precipitation with 2 volumes of 0.1 M sodium acetate solution in 96% ethanol. The precipitate is separated by centrifugation (4350 rpm, 3 minutes). The supernatant is then removed, the precipitate is washed three times with 10 mL DCM and centrifuged again. Before further use, the product is further purified by rinsing with 96% ethanol until the supernatant shows no significant absorbance / fluorescence signal from the dye used in the synthesis. After rinsing, the probe is dried until the ethanol is completely evaporated, and an aqueous solution of 10 mg / mL is prepared for further use. Unless otherwise noted, in vitro digestions were carried out at 37 °C using pectin lyase 2 enzyme (PL2, from Dickeya dadantii bacteria, commercially available preparation, NZYtech, Portugal). Digestions were carried out in Tris buffer, 100 mM, pH 8.5 supplemented with 0.1 mM CaCL. Results were further verified using pectin lyases 1A and 9 (PL1A, PL9) (NZYtech, Portugal) and the industrial preparation Pectinex® XXL (Novozymes, Denmark).

[0047] Example 1

[0048] Probe preparation. Rhodamine B and aminofluorescein were coupled to pectin. Rhodamine B (RB6), 6-aminofluorescein (6-AF, fluorescein isomer II) and 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC) were obtained from Thermo Fisher Scientific. AG366 pectin with a minimum mass of 22 kDa was obtained from Dipecta, Agidia Biofords. The carboxyl groups of pectin were activated by EDAC in pyridine buffer, pH 4-5.

[0049] The pyridine buffer was prepared from one part of 3 M HCI solution and one part of pyridine and brought to pH 4-5 (approximately pH 4.5). Pectin (0.1 pmole, 220 mg) was suspended in 15 mL of pyridine buffer, and 6-AF (0.2 molar equivalents to pectin), RB6 (20 molar equivalents to pectin) and EDAC were added. The suspension was stirred overnight in the dark. After conjugation, two volumes of 0.1 M sodium acetate solution in 96% ethanol were added. The mixture was centrifuged at 4350 rpm for about 3 minutes.

[0050] The supernatant was removed. The residue was washed three times with 10 mL DCM and centrifuged at 4350 rpm for about 3 min. The residue was collected. The probe was further purified for analysis:

[0051] 200 mg of lyophilizate was suspended in 96% ethanol (1.5 mL). The suspension was mixed using a vortex mixer, after which the mixture was centrifuged at 13,300 rpm for about 5 minutes. The supernatant was removed. The procedure was repeated until the intensity of the fluorophore in the spectrophotometric spectra was negligible. After the last washing procedure, the residue was left in a dry bath incubator at 40 °C to remove the remaining ethanol. The dried fluorescently labeled probe was suspended in water or Tris buffer (100 mM, pH=8.5) to a concentration of 10 mg / mL. The stock was stored at 4 °C. Example 2

[0052] Scaling up to 1 g

[0053] The procedure for using scale-up synthesis was investigated. Pectin (0.45 pmole, 1 g) was suspended in 100 mL of pyridine buffer, and 6-AF, R.B6 and EDAC were added. The suspension was stirred overnight in the dark. After conjugation, two volumes of 0.1 M sodium acetate in 96% ethanol were added. The mixture was filtered using a Buchner funnel. The filtrate was removed. The residue was washed 3 times with 50 mL DCM. The residue was collected.

[0054] Example 3

[0055] Probe preparation. 6-aminofluorescein (6-AF, fluorescein isomer II) was coupled with pectin. 6-aminofluorescein and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) were obtained from Thermo Fisher Scientific. Pectin with a minimum mass of 22 kDa was obtained from Dipecta. The carboxyl groups of pectin were activated by EDAC in pyridine buffer at pH 4-5.

[0056] The pyridine buffer was prepared from one part of 3 M HCI solution and one part of pyridine and brought to pH 4-5 (approximately pH 4.5). Pectin (0.1 pmole, 220 mg) was suspended in 15 mL of pyridine buffer, and 6-AF (100 molar equivalents relative to pectin) and EDAC (72.5 molar equivalents relative to pectin) were added. The suspension was stirred overnight in the dark. After conjugation, two volumes of 0.1 M sodium acetate solution in 96% ethanol were added. The mixture was centrifuged at 4350 rpm for about 3 minutes.

[0057] The supernatant was removed. The residue was washed three times with 10 mL of DCM and centrifuged at 4350 rpm for about 3 minutes. The residue was collected. The probe was further purified for analysis:

[0058] 200 mg of lyophilizate was suspended in 96% ethanol (1.5 mL). The suspension was mixed using a vortex mixer, after which the mixture was centrifuged at 13,300 rpm for about 5 minutes. The supernatant was removed. The procedure was repeated until the intensity of the fluorophore in the spectrophotometric spectra was negligible. After the last washing procedure, the residue was left at room temperature until the ethanol evaporated. The dried fluorescently labeled probe was suspended in water or Tris buffer (100 mM, pH=8.5) to a concentration of 10 mg / mL. The stock was stored at 4 °C.

[0059] Example 4

[0060] Probe preparation. Rhodamine B and 6-aminofluorescein (6-AF, fluorescein isomer II) were coupled to pectin. Rhodamine B (RB6), 6-aminofluorescein (6-AF, fluorescein isomer II) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) were obtained from Thermo Fisher Scientific. Pectin isolated from citrus peel with a minimum mass of 22 kDa was obtained from Thermo Fisher Scientific (Cat. No. J61021). The carboxyl groups of pectin were activated by EDAC in pyridine buffer, pH 4-5.

[0061] The pyridine buffer was prepared from one part of 3 M HCI solution and one part of pyridine and brought to pH 4-5 (approximately pH 4.5). Pectin (0.1 pmole, 220 mg) was suspended in 15 mL of pyridine buffer, and 6-AF (0.5 molar equivalents to pectin), RB6 (50 molar equivalents to pectin) and EDAC were added. The suspension was stirred overnight in the dark. After conjugation, two volumes of 0.1 M sodium acetate solution in 96% ethanol were added. The mixture was centrifuged at 4350 rpm for about 3 minutes.

[0062] The supernatant was removed. The residue was washed three times with 10 mL DCM and centrifuged at 4350 rpm for about 3 min. The residue was collected. The probe was further purified for analysis:

[0063] 200 mg of lyophilizate was suspended in 96% ethanol (1.5 mL). The suspension was mixed using a vortex mixer, after which the mixture was centrifuged at 13,300 rpm for about 5 minutes. The supernatant was removed. The procedure was repeated until the intensity of the fluorophore in the spectrophotometric spectra was negligible. After the last washing procedure, the residue was left at room temperature until the ethanol evaporated. The dried fluorescently labeled probe was suspended in water or Tris buffer (100 mM, pH=8.5) to a concentration of 10 mg / mL. The stock was stored at 4 °C. Example 5

[0064] Determination of kinetic constants and affinity of PL2 enzyme for fluorescently labeled probe compared to AG366 pectin

[0065] In order to determine the affinity of the probe for the PL2 enzyme and compare it to the affinity of PL2 for native pectin, an experiment was conducted to determine the kinetic constants for both pectinase substrates.

[0066] For this purpose, digestion was carried out on an uDrop plate in a volume of 5 pL; the experiment used solutions of probe and AG366 pectin at five concentrations (1 to 0.012 mg / mL) with a constant concentration of PL2 enzyme. The digestion reaction was monitored every 15 seconds by measuring absorbance at 230 nm. The kinetic parameters thus obtained were used to determine Vmax (based on measurements 2 to 5) and the Michaelis-Menten constant (Km) based on the Lineweaver-Burk linearization method. The fit measure of the linearization model for both substrates was R.2 = 0.995. The results of the kinetic constants were obtained: for AG366 pectin Vmax = 0.03750 and Km = 0.2249 mg / mL; for the test probe Vmax = 0.01705 and Km = 0.2221 mg / mL.

[0067] The similar Km values for both substrates confirmed earlier observations on the preservation of chemical and biological properties of the obtained probes similar to AG366 pectin, a phenomenon that is extremely favorable from the point of view of the probe susceptibility to digestion by enzymes. The experiment also ruled out the possibility of a strong preference of digestion of native pectins by pectinases in comparison to the obtained probes, as well as the occurrence of the phenomenon of pectinase inhibition by the obtained fluorescently labeled probes.

[0068] Example 6

[0069] To confirm the suitability of differential precipitation of fluorescently labeled probes in 50 mM CaCL solution for detection of pectinolytic activity, 12 h digestion of probes labeled with 6AF or R.B at a concentration of 0.5 mg / mL was carried out with PL2 enzyme at a concentration of 1 U / mL (digestion buffer 100 mM Tris with 0.1 mM CaCL, pH 8.5). A calcium chloride solution was then added to the samples to a final concentration of 50 mM, mixed, centrifuged (5 min, 13,300 rpm) and the supernatant thus obtained was subjected to fluorescence measurements at excitation and emission wavelengths characteristic of fluorescein (Ex 490 nm, Em 525 nm) and rhodamine (Ex 550 nm, Em 580 nm) to compare the differences in signal obtained for digested and undigested samples.

[0070] The experiment was also repeated with the modification of placing 250 pL samples in 1 mL syringes with a disk of Whatman blotting paper placed at the end of the syringe. Such samples were differentially precipitated in situ before calcium chloride solution was added to the syringe, gently stirred and filtered into another tube. Fluorescence measurement of the filtrates obtained in such a way was carried out analogously as described above. For all fluorescently labeled probes tested, results were obtained that allowed easy and unambiguous detection of pectinolytic activity in the sample. The signal differences obtained between samples showing and not showing pectinolytic activity, depending on the probe variant used, ranged from a 3.9-fold increase (relative to the undigested control) to a 13.95- fold increase in signal.

[0071] Literature

[0072] [1] V. E. Shevchik, G. Condemine, J. Robert-Baudouy, and N. Hugouvieux-Cotte-Pattat, “The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in Erwinia chrysanthemi 3937,” j Bacterial, vol. 181, no. 13, pp. 3912-3919, Jul. 1999, doi: 10.1128 / JB.181.13.3912-3919.1999.

[0073] [2] Z. I. Kertesz, Methods in Enzymology, Volume I, 162-164. 1955.

[0074] [3] G. L. Miller, “Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar,” Anal Chem, vol. 31, no. 3, pp. 426-428, Mar. 1959, doi: 10.1021 / ac60147a030.

[0075] [4] M. T. M. Jalil and D. Ibrahim, “Partial purification and characterisation of pectinase produced by Aspergillus niger LFP-1 grown on pomelo peels as a substrate,” Trap Life Sci Res, vol. 32, no. 1 , pp. 1- 22, 2021, doi: 10.21315 / tlsr2021.32.1.1.

[0076] [5] H. Liu et al., “TaqMan probe array for quantitative detection of DNA targets,” Nucleic Acids Res, vol. 34, no. 1, Jan. 2006, doi: 10.1093 / nar / gnj006.

[0077] [6] S. Armand, S. Drouillard, M. Schulein, B. Henrissa, and H. Driguez, “A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases,” Journal of Biological Chemistry, vol. 272, no. 5, pp. 2709-2713, 1997, doi: 10.1074 / jbc.272.5.2709.

[0078] [7] V. Vreeland and W. M. Laetsch, “Identification of associating carbohydrate sequences with labelled oligosaccharides : Localization of alginate-gelling subunits in cells walls of a brown alga.,” Planta, vol. 177, no. 4, pp. 423-34, Apr. 1989, doi: 10.1007 / BF00392610.

[0079] [8] A. Ogamo, K. Matsuzaki, H. Uchiyama, and K. Nagasawa, “Preparation and properties of fluorescent glycosamino-glycuronans labeled with 5-aminofluorescein,” Carbohydr Res, vol. 105, no. 1, pp. 69-85, Jul. 1982, doi: 10.1016 / 50008-6215(00)81855-8.

[0080] [9] J. C. D. Hinton, J. M. Sidebotham, D. R. Gill, and G. P. C. Salmond, “Extracellular and periplasmic isoenzymes of pectate lyase from Erwinia carotovora subspecies carotovora belong to different gene families,” Mo / icrobio / , vol. 3, no. 12, pp. 1785-1795, Dec. 1989, doi: 10.1111 / j.1365-

[0081] 2958.1989.tb00164.x.

[0082]

[0010] D. W. Abbott and A. B. Boraston, “A Family 2 Pectate Lyase Displays a Rare Fold and Transition Metal-assisted [3-Elimination,” Journal of Biological Chemistry, vol. 282, no. 48, pp. 35328-35336, Nov. 2007, doi: 10.1074 / jbc.M705511200.

[0083]

[0011] Y. Tamaru and R. H. Doi, “Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome,” Proceedings of the National Academy of Sciences, vol. 98, no. 7, pp. 4125-4129, Mar. 2001, doi: 10.1073 / pnas.071045598.

Claims

CLAIMS1. A method for obtaining a probe for determining the activity of pectinolytic enzymes, characterized in that it comprises steps in which: the substrate to be labeled, selected from pectin, pectinate, polygalacturonic acid or a mixture thereof, is suspended in pyridine buffer or other aqueous-organic buffer with a pH of 2 to 6; a fluorescent dye, preferably containing amino groups, and a coupling agent that activates the carboxyl groups of the substrate are added to the resulting mixture, the reaction mixture is incubated without light with stirring for a period of 2 to 20 hours, preferably 8 to 16 hours, the resulting product is separated from the reaction mixture by precipitation with an organic solvent and centrifugation, additional purification of the product is carried out by washing with an organic solvent, preferably with ethanol.

2. The method according to claim 1, characterized in that EDAC is used as a coupling agent to activate the carboxyl groups of the substrate.

3. The method according to claim 1 or 2, characterized in that the reaction is carried out in a pyridine buffer with pH 4 to 5, preferably 4.5.

4. The method according to claims 1 or 2 or 3, characterized in that the probe is precipitated from the reaction mixture using a 0.1 M solution of sodium acetate in 96% ethanol.

5. A method of determining the activity of pectinolytic enzymes, characterized in that it comprises steps in which:the probe solution obtained by the method defined in any of the claims 1-4 is subjected to digestion with a pectinolytic activity-exhibiting test preparation over a period of 1 hour to 48 hours, preferably 2 hours to 24 hours;- after incubation, separation of the digested and undigested fractions of the probe is carried out by precipitation, preferably by adding an aqueous solution of a divalent metal salt, such as a calcium salt, until the final salt concentration in the range of 10-500 mM, preferably 50 to 100 mM is obtained;- the precipitate is separated, preferably by centrifugation or filtration;- the liquid fraction is collected and the absorbance or fluorescence signal is measured, from which the pectinolytic activity of the preparation under test is determined.

6. The method according to claim 5, characterized in that the separation of the digested and undigested fractions of the probe by precipitation is carried out by adding a solution of a soluble calcium salt, preferably calcium chloride.

7. A use of a probe obtained by the method defined in any of the claims 1-4 for the determination of pectinolytic enzyme activity.

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