JP3994353B2 - Method and apparatus for analyzing hydroplankton / microbe-produced toxin - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an analysis method and analysis apparatus for aquatic plankton / microbe-produced toxins. More specifically, the present invention relates to a protein phosphatase activity-inhibiting toxin contained in microkistis, nodularia, black sea sponge, lake water, river water, seawater and the like. The present invention relates to a method and an analyzer for analyzing concentration easily and quickly.
[0002]
[Prior art]
Large amounts of cyanobacteria and black sponge are generated in eutrophied lakes and seas. Some of these aquatic plankton and microorganisms produce toxins that inhibit the catalytic activity of protein phosphatase, an enzyme that exists in animal tissues and dephosphorylates from phosphorylated proteins. . A typical example is microcystin, which is a microcystis-producing toxin and is known as liver toxin.
[0003]
Considering that lake water is used for drinking water, livestock drinking water, and fish farming, and that seawater is used for fish farming and shellfish cultivation, the dynamic concentration of produced toxins in these lake water and sea water is determined. Understanding and managing water quality is an important issue.
For these reasons, concentration analysis of protein phosphatase activity-inhibiting toxins present in cyanobacteria and water has recently started.
[0004]
The analysis procedure in this case includes a step of first extracting a toxin produced in aquatic plankton or a microorganism cell and then quantitatively analyzing the extracted toxin.
As for toxin extraction, for example, a method of extracting a toxin by applying ultrasonic vibration to a sample to break a cell membrane, a method of repeatedly extracting and freezing and thawing a sample, and the like are generally performed. .
[0005]
Moreover, as an analysis method of the extracted toxin, the following method is generally performed. For example, by reacting an extract toxins and protein phosphatase, the reaction system labeled substrate is reacted with a radioactive isotope P ^32, the measured radiation dose at that time, based on the measured values, have been prepared in advance Extracted toxins in the reaction system and concentration analysis are performed from the calibration curve.
[0006]
In addition, specific toxins such as microcystines are analyzed by a method of isolation and analysis by liquid chromatography, or by an enzyme immunosorbent assay (ELISA method) using a monoclonal antibody that reacts specifically. The method is known.
[0007]
[Problems to be solved by the invention]
However, in the case of the conventional toxin extraction method described above, there is a problem that, for example, a special ultrasonic oscillator is required, and the toxin extraction by freeze-thawing requires a lot of time.
Regarding the conventional analytical methods, for example, when using a P ^32, in view of the radiation exposure prevention, it is necessary to develop a special analysis environment. When applying liquid chromatography or ELISA, there is a problem that only a specific toxin can be analyzed and a long time is required for the analysis.
[0008]
The present invention solves the above-mentioned problems in conventional toxin extraction and quantitative analysis thereof, and can easily extract the produced toxin from aquatic plankton and microorganisms, and accurately and rapidly analyze the concentration of the extracted toxin. An object of the present invention is to provide an analysis method and an analysis apparatus for a novel aquatic plankton / microorganism-produced toxin.
[0009]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have obtained new knowledge that the produced toxins can be easily extracted simply by heating aquatic plankton and microorganisms. It came.
Moreover, the following knowledge was also obtained.
[0010]
That is, when the extracted toxin reacts with protein phosphatase, depending on the quantity relationship between the extracted toxin and protein phosphatase, if the amount of the extracted toxin to the protein phosphatase is small, a part of the protein phosphatase is produced by the extracted toxin. Is inhibited in its dephosphorylation activity, but when a predetermined concentration of p-nitrophenyl phosphate (transparent colorless) using protein phosphatase as a substrate is added to the reaction system, due to the enzymatic action of the remaining protein phosphatase, The fact is that the p-nitrophenyl phosphate decomposes into phosphoric acid and p-nitrophenol. The reaction system at this time exhibits a yellow color due to the generated p-nitrophenol. Therefore, by measuring the absorbance thereof, the abundance of p-nitrophenol, conversely, the catalyst is not poisoned by the extracted toxin. The inventors have obtained the knowledge that the amount of protein phosphatase retaining activity, more specifically, the poisoning amount of the extracted toxin, that is, the concentration of the extracted toxin can be detected.
[0011]
Based on these findings, the present inventors have developed the analysis method and analysis apparatus of the present invention.
That is, the method analyzes the hydrosphere plankton microbial toxins of the invention, toxin extraction in hydrosphere plankton microorganisms producing micro cis Ting subjected to heat treatment in 20 to 60 minutes at a temperature 50-70 ° C. for extracting the micro-cis Tin Process;
An enzyme reaction step of reacting the microcystine with protein phosphatase and then adding p-nitrophenyl phosphate to the reaction system to produce p-nitrophenol; and
Measuring the absorbance of the reaction system, and reading the concentration of the microcystine from a calibration curve based on the measured value, preferably, the toxin extraction step, the enzyme reaction step, In this method, a toxin concentration step for selectively concentrating the extracted microcystine is interposed.
[0012]
In the present invention, a toxin extraction means for extracting the microcystine by subjecting the hydroplankton / microorganism producing microcystine to a heat treatment at a temperature of 50 to 70 ° C. for 20 to 60 minutes ;
An enzyme reaction means connected to the toxin extraction means for contacting the microcystine , protein phosphatase and p-nitrophenyl phosphate to produce p-nitrophenol; and
An absorptiometer for measuring the absorbance of the effluent from the enzyme reaction means is provided. An analyzer for aquatic plankton / microorganism-produced toxin is provided.
[0013]
Preferably, the enzyme reaction means comprises an enzyme column packed with a carrier carrying protein fosterase, a reservoir of p-nitrophenyl phosphate, and a reservoir of the microcystine , Is connected through a three-way cock, and a toxin enrichment means for selectively concentrating the microcystine is interposed between the toxin extraction means and the enzyme reaction means. An analyzer for produced toxins is provided.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
First, the analysis method of the present invention will be described in detail.
The analysis method of the present invention is characterized in that a toxin extraction step of subjecting hydrospheric plankton and microorganisms to heat treatment to extract microcystine , which is a produced toxin , is essential.
[0015]
Specifically, only heats the water that contains the blue-green algae, such as Microcystis, is to extract the micro cis Ting.
Temperature to be applied to this case, Ru 50-70 ° C. der.
For example, when microcystis is heated, the extraction amount of microcystine, which is a produced toxin, rapidly increases from around 40 ° C., and the extraction amount tends to saturate around 60 ° C. For this reason, the heating temperature is set in the above range in consideration of realizing effective microcystine extraction.
[0016]
In addition, the heating time of that time Ru Oh in 20 to 60 minutes.
For example, when microcystine extraction is performed at a temperature of 50 ° C., the extraction amount increases as the heating time increases, but even if the heating time is 20 minutes or more, the extraction amount shows a saturation tendency, so that the extraction amount is too long. Heating is useless. For this reason, the heating time is set in the above range.
[0017]
In this toxin extraction step, it is preferable to add hydrolase to the reaction system because microcystine extraction is promoted. Preferred examples of the hydrolase used include α-amylase and lysozyme. However, the enzyme is not limited to these, and any enzyme can be used as long as it can degrade the target plankton or the cell membrane of the microorganism. Further, the amount added is not particularly limited.
[0018]
The extracted toxin thus obtained is then transferred to an enzyme reaction step and subjected to a reaction with protein phosphatase.
Although it does not specifically limit as protein phosphatase to be used, For example, protein phosphatase type-2A is made into a suitable example. In addition, any biological extract containing protein phosphatase can be used.
[0019]
The concentration of protein phosphatase in this reaction system is not particularly limited.
In this reaction system, a part of the protein phosphatase is deactivated by the dephosphorylation activity due to the poisoning action of microcystine . However, other parts are not deactivated. The reaction system conditions are preferably set to a temperature of around 37 ° C. and a reaction time of about 15 to 30 minutes. This is because the sensitivity at the time of measuring the absorbance described later is improved.
[0020]
Subsequently, p-nitrophenyl phosphate is added to the reaction system. At this time, p-nitrophenyl phosphate is decomposed into phosphate and p-nitrophenol by the enzymatic action of non-poisoned protein phosphatase.
When the concentration of microcystine in the reaction system is high, the amount of protein phosphatase poisoning (deactivation degree of catalytic activity) increases, so the amount of degradation of added p-nitrophenyl phosphate is small. As a result, the amount of p-nitrophenol produced decreases. Conversely, when the concentration of microcystine in the reaction system is low, the amount of protein phosphatase poisoning is small, so the amount of added p-nitrophenyl phosphate is large, resulting in the formation of p-nitrophenol. The amount increases. That is, the decrease amount of the added p-nitrophenyl phosphate and the production amount of p-nitrophenol change corresponding to the concentration of microcysteine in the reaction system.
[0021]
The solution of p-nitrophenyl phosphate is colorless and transparent, and p-nitrophenol has an absorption wavelength of 405 to 410 nm. That is, the decrease in p-nitrophenyl phosphate described above and the formation of p-nitrophenol paired therewith can be grasped as a color change in the reaction system. In other words, by measuring the absorbance of the reaction system, the above-described microcystin concentration analysis can be performed.
[0022]
The concentration of p-nitrophenyl phosphate in this reaction system is not particularly limited. In order to facilitate the enzymatic decomposition of the above-described p-nitrophenyl phosphate, it is preferable to set the temperature of the reaction system to around 37 ° C. and set the reaction time to 30 to 45 minutes.
The effluent thus prepared in the enzymatic reaction step and producing p-nitrophenol is then transferred to an absorptiometer where the absorbance is measured. The measurement wavelength at this time is preferably set to 405 nm in order to obtain high sensitivity.
[0023]
On the other hand, with respect to the microcystine to be analyzed, in the above-described reaction system, the relationship between it and the absorbance is measured using a microcystine having a known concentration, and a calibration curve for both is prepared in advance.
Then, using the calibration curve, the concentration of microcysteine in the reaction system is read from the actual measurement value.
[0024]
In the present invention, the object can be achieved sufficiently even after the above steps. However, if the following steps are interposed between the toxin extraction step and the enzyme reaction step, the measurement sensitivity is improved. Since it can raise further, it is suitable. That is, it is a step of selectively concentrating microcystine .
In the toxin extraction process described above, not only microcystine but also impurities such as cell debris such as cell membranes are by-produced. When the above-described absorbance measurement is performed in a state where the impurities coexist, the measurement sensitivity is greatly reduced due to light scattering or the like. Therefore, in order to perform highly sensitive measurement, it is preferable to remove these contaminants and simultaneously concentrate microcystine after the toxin extraction step.
[0025]
Therefore, in the present invention, the extract from the toxin extraction step is separated from microcystin and impurities using, for example, an ultrafiltration membrane, and only the microcystine that has passed through the ultrafiltration membrane is selectively collected. The enzyme reaction step and absorbance measurement described above are performed using this.
Next, the analyzer of the present invention will be described with reference to the drawings.
[0026]
FIG. 1 is a basic configuration diagram showing a preferred example of the device of the present invention capable of performing the above-described selective concentration of microcystine .
This apparatus is provided with a toxin extraction means A, an enzyme reaction means C, and an absorptiometer D, and has a structure in which a toxin concentration means B is interposed between the toxin extraction means A and the enzyme reaction means C.
[0027]
The toxin extraction means A includes a heating tank 1 provided with a heating means (not shown), and this heating tank 1 is connected to a path W1 for introducing a sample via a three-way cock 21. The heating tank 1 is provided with a path W2 for deriving an extract from the heating tank 1, which is connected to a membrane separation device 31 of the toxin concentration means B described later via a pump P1. Yes. The membrane separation device 31 and the three-way cock 21 are connected by a path W3. Further, the heating tank 1 is provided with a path W4 for discharging the sample in the tank, and is connected to the drain path W13 of the entire apparatus via valves V1 and V2.
[0028]
Here, as the membrane separation device 31, it is preferable to use an ultrafiltration membrane device that can pass microcystine (molecular weight of about 1000) but cannot pass other impurities having a larger molecular weight.
In this toxin extraction means A, the sample collected in the heating tank 1 with the valve V1 closed is heated at a temperature of 50 to 70 ° C. for 20 to 60 minutes to extract the microcystine . And after operating the three-way cock 21 and forming the closed circuit between the path | route W2 and the path | route W3, the extract in the heating tank 1 is circulated to the said closed circuit by operating the pump P1.
[0029]
Of the extract introduced into the membrane separation device 31 from the path W2, the microcystine passes through the membrane and flows out to the path W4. However, impurities in the extract are refluxed from the three-way cock 21 to the heating tank 1 via the path W3. That is, among the extracts, the target microcystine is selectively separated and stored in the extracted toxin storage tank 4 of the enzyme reaction means C described later from the path W4.
[0030]
The toxin concentration means B includes another membrane separation device 32 in addition to the membrane separation device 31 described above, and the membrane separation device 32 and the extracted toxin storage tank 4 are connected by a path W5. . The membrane separation device 32 is connected to the toxin storage tank 4 through a path W6 via the pump P2 and the three-way cock 22, and further connected to the path W4 via a path W7 and connected to the entire drain path W13. is doing. The three-way cock 22 is connected to the entire drain path W13 via a path W8.
[0031]
Here, contrary to the case of the membrane separation device 31 described above, another membrane separation device 32 is an ultrafiltration membrane device that cannot pass microcystine but can pass other contaminants having a smaller molecular weight. It is preferable to use it.
In this toxin concentration means B, when a certain amount of microcystine from the path W4 is stored in the storage tank 4, the valve V1 is closed, the valve V2 is opened, the path W8 is blocked by the three-way cock 22, and further Then, the three-way cock 23 of the enzyme reaction means C described later is closed to form a closed circuit of the path W5-reservoir 4-path W6-membrane separation device 32. And the microcystine of the storage tank 4 is circulated by operating the pump P2.
[0032]
From the microcystin that has flowed into the membrane separation device 32 from the storage tank 4, the remaining impurities are removed, and in a more concentrated state, the microcystine returns to the storage tank through the path W5. The separated and removed impurities are discharged from the entire drain path W13 through the path W7.
Thus, in the storage tank 4, the microcystine is stored in a concentrated state.
[0033]
In this toxin concentration means B, an example of using an ultrafiltration membrane device for separation of microcystine and contaminants has been shown, but the device used for separation is not limited to this, for example, a gel filtration device Any material can be used as long as it can separate microcystin and impurities.
The microcystin obtained by the toxin concentration means B is then transferred to the enzyme reaction means C.
[0034]
The enzyme reaction means C is composed of the extracted toxin storage tank 4, the p-nitrophenyl phosphate storage tank 5, and the enzyme column 6. And the path | route W9 from the storage tank 4 and the path | route W10 from the storage tank 5 merge by the three-way cock 23, become the path | route W11, and are connected to the enzyme column 6 via the pump P3. The enzyme column 6 is connected to the absorptiometer D through the path W12.
[0035]
The storage tank 5 stores p-nitrophenyl phosphate and a buffer solution having a predetermined concentration. As the buffer, a 40 mM Tris-HCl buffer containing 43 mM MgCl2, 4 mM EDTA and 4 mM DTT is suitable. Moreover, it is preferable to adjust and use to pH 8.9-9.0.
On the other hand, the enzyme cell 6 is a cell in which protein phosphatase is supported on a carrier such as alumina. For example, a material in which protein phosphatase Type-2A is supported on silica gel is suitable.
[0036]
In this enzyme reaction means C, by operating the three-way cock 23 and the pump P3, a predetermined amount of the microcystine in the storage tank 4 and the p-nitrophenyl phosphate buffer in the storage tank 5 is supplied to each path W9. , W10 flows into the path W11 to be mixed and injected into the enzyme cell 6.
The mixture injected into the enzyme cell 6 flows down the enzyme cell 6, and in the process, the decomposition of p-nitrophenyl phosphate proceeds by protein phosphatase to produce p-nitrophenol.
[0037]
Since this reaction system effectively proceeds at a temperature of about 37 ° C., a heating means is provided around the enzyme cell 6 so that the temperature of the protein phosphatase carried is maintained at the above-described temperature. . Further, the reaction time at the above temperature is preferably 15 to 30 minutes in terms of sensitivity at the time of absorbance measurement, so that the time required for the mixture to flow out after being injected is the above-described time. In addition, the packing density of the carrier in the enzyme column is adjusted.
[0038]
The effluent flowing out of the enzyme cell 6 is then transferred to an absorptiometer D where the absorbance is measured.
Then, by using a calibration curve prepared in advance, the concentration of microcystine is quantified from the measured value.
[0039]
【Example】
(1) Creation of calibration curve A calibration curve was created as follows.
First, in each well of a known 96-well microplate, 5 μL of 40 mM nickel chloride solution, 5 μL of 5 mg / L bovine serum albumin solution, 5 μL of 0.2 unit protein phosphatase Type-2A solution, and 0.1 mM of pH 7.0. 75 μL of a solution prepared by dissolving calcium chloride to a concentration of 0.1 mM in Tris-HCl buffer was injected.
[0040]
Here, 5 μL of a sample solution in which a known amount of standard microcystine was dissolved in water and the microcystine concentration was changed was added, stirred for 1 minute with a micromixer, and further reacted in a constant temperature bath at a temperature of 37 ° C. for 30 minutes. I let you.
On the other hand, a solution was prepared by dissolving p-nitrophenyl phosphate in 0.1 mM Tris-HCl buffer at pH 7.0 to a concentration of 1.5 mg / L.
[0041]
And 120 microliters of this solution was added to the above-mentioned reaction system, and the whole was heated for 45 minutes in a 37 degreeC thermostat, and reaction was advanced.
Next, 20 μL of 13% dipotassium hydrogen phosphate solution was added thereto to stop the reaction, and the absorbance of the obtained solution was measured at a wavelength of 405 nm using a microplate reader.
[0042]
The absorbance was plotted against the microcystine concentration. The results are shown in FIG.
As is apparent from FIG. 2, the measured absorbance changes linearly with respect to the microcystine concentration. That is, this straight line can sufficiently function as a calibration curve.
[0043]
(2) Comparison between the analysis method of the present invention and the ELISA method In order to confirm the reliability of the measurement results obtained by the analysis method of the present invention, the microcystin sample of the same concentration was subjected to the analysis procedure described above according to the analysis procedure described above. Analysis was performed, and analysis was performed by a conventional ELISA method.
The relationship between the microcystine concentrations analyzed from each method is shown in FIG.
[0044]
As is apparent from FIG. 3, the microcystine concentration was detected at a slightly higher value in the analysis method of the present invention, but a good linear relationship with the ELISA method was shown.
That is, the analysis method of the present invention can be employed as a microcystine concentration quantification method instead of the conventional ELISA method.
[0045]
(3) In a plastic centrifugal sedimentation tube having an actually measured volume of 5 mL, 1 mL of test water having a different content of microkistis was collected, and the whole was heated at a temperature of 50 ° C. for 20 minutes. Subsequently, it filtered with the ultrafiltration membrane with an average hole diameter of 0.2 micrometer. The obtained solution was used as a sample for analysis.
Thereafter, the absorbance was measured using this analytical sample according to the analytical procedure described in (1). The results are shown in FIG.
[0046]
The straight line in FIG. 4 is the calibration curve shown in FIG. As is apparent from FIG. 4, the concentration of microcystine extracted from the microcystis in the test water by the extraction method of the present invention can be quantified.
[0047]
【The invention's effect】
As is clear from the above description, according to the present invention, the produced toxin can be extracted simply by heating aquatic plankton and microorganisms, and quantitative analysis thereof is possible.
Therefore, the present invention has a great industrial value as a method and apparatus for rapidly and simply analyzing a protein phosphatase activity-inhibiting toxin such as microcystine present in cyanobacteria and water.
[Brief description of the drawings]
FIG. 1 is a basic configuration diagram showing an example of a preferred analyzer according to the present invention.
FIG. 2 is a calibration curve in the analysis method of the present invention.
FIG. 3 is a graph showing a comparison between the result of the analysis method of the present invention and the result of the ELISA method.
FIG. 4 is a graph showing actual measurement results in the analysis method of the present invention for microcystis.
[Explanation of symbols]
A Toxin extraction means B Toxin concentration means C Enzyme reaction means D Absorption photometer 1 Heating tank 4 Extracted toxin reservoir 5 p-nitrophenyl phosphate buffer reservoir 6 Enzyme columns 21, 22, 23 Three-way cock 31, 32 Membrane separators P1, P2, P3 Pumps W1-W12 Path W13 Overall drain path V1, V2 Valve

Claims (6)

A toxin extraction step of extracting the microcystine by subjecting the aquatic plankton / microorganism producing microcystine to a temperature of 50 to 70 ° C. for 20 to 60 minutes;
An enzyme reaction step of reacting the microcystine with protein phosphatase and then adding p-nitrophenyl phosphate to the reaction system to produce p-nitrophenol; and
Measuring the absorbance of the reaction system, and reading the concentration of the microcystine from a calibration curve based on the measured value; and a method for analyzing aquatic plankton / microorganism-produced toxin.   The method for analyzing aquatic plankton / microorganism-produced toxins according to claim 1, wherein a toxin concentration step for selectively concentrating the extracted microcystines is interposed between the toxin extraction step and the enzyme reaction step. A toxin extraction means for extracting the microcystine by subjecting the aquatic plankton / microorganism producing microcystine to a temperature of 50 to 70 ° C. for 20 to 60 minutes ;
An enzyme reaction means connected to the toxin extraction means for contacting the microcystine, protein phosphatase and p-nitrophenyl phosphate to produce p-nitrophenol; and
An absorptiometer for measuring the absorbance of the effluent from the enzyme reaction means; a hydroplankton / microbe-produced toxin analyzer.   The enzyme reaction means comprises an enzyme column packed with a carrier carrying protein fosterase, a reservoir of p-nitrophenyl phosphate, and a reservoir of the microcystine, each having a three-way cock. The apparatus for analyzing aquatic plankton / microorganism-produced toxins according to claim 3, which is connected via   4. The aquatic plankton / microorganism-produced toxin analyzing apparatus according to claim 3, wherein a toxin concentrating means for selectively concentrating the microcystine is interposed between the toxin extracting means and the enzyme reaction means.   6. The apparatus for analyzing aquatic plankton / microorganism-produced toxin according to claim 5, wherein the toxin concentration means comprises an ultrafiltration membrane device.

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