JP4288053B2 - Enzyme activity control method, molecular switch, metal measurement method, and metal measurement device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for controlling enzyme activity in a metal enzyme, a molecular switch using the controlled enzyme activity as an index, a metal measuring method, and a metal measuring device.
[0002]
[Prior art]
Currently, as interest in environmental issues is increasing, the measurement method for heavy metals, one of the pollution factors, is drawing attention. Since many metals are essential for the human body, if the amount of intake is small, it affects the living body, and conversely, if it is excessively consumed, it can be harmful.
[0003]
Colorimetric methods and atomic absorption spectrometry are used for general metal quantification, and metals are further quantified by atomic absorption analysis and high-frequency inductively coupled plasma emission analysis for a very small amount of sample.
[0004]
On the other hand, an electrochemical sensor capable of detecting a compound such as a substrate using a specific reaction between an enzyme protein and its substrate has been developed (see Patent Document 1). A sensor in which a polymer such as a protein is connected to at least two electrodes has also been proposed (see Patent Document 2).
[0005]
The sensor described in Patent Document 1 has a protein immobilized on a support material via an anchor group, and an operation for changing the activity of the immobilized protein is not performed. The sensor described in Patent Document 2 is also not operated to change the activity of the protein bound to the electrode.
[0006]
As a result of performing isoelectric focusing on a solution containing alkaline phosphatase, which is a kind of metal enzyme, as a report that metal ions were desorbed from the metal enzyme by using an electrochemical technique, the enzyme activity disappeared, There is a case where it recovered by giving (refer nonpatent literature 1).
[0007]
This isoelectric focusing is one of the commonly used techniques in protein purification, and as a result, reports that the enzyme activity has been affected. The enzyme activity is intentionally lost or recovered. It wasn't something you tried to use.
[0008]
As a prior art technique for electrochemically controlling enzyme activity, coenzyme NADH (reduced NAD) produced by an oxidation reaction in an enzyme reaction system is subjected to NAD by an electrochemical method. ⁺ There is a report of regenerating (nicotinamide dinucleotide) to promote the oxidation reaction (see Non-Patent Document 2).
[0009]
In addition, many similar methods for electrochemically regenerating an enzyme substrate and controlling enzyme activity have been reported. However, there has been no report that the activity is controlled by electrically detaching one element constituting the enzyme itself, not the substrate.
[0010]
It is widely known that a metalloenzyme must retain a metal specific to the enzyme in order to exhibit activity. When the metal is removed using a chelating agent, the enzyme activity disappears, but the lost enzyme activity is restored by the application of metal ions.
[0011]
There has been reported a method in which enzyme activity is controlled by removing and applying such a metal, and the metal is measured using the enzyme activity as an index (see Non-Patent Document 3). In this case, the elimination of the metal for controlling the activity is performed using a chelating agent.
[0012]
[Patent Document 1]
JP-A-8-233773
[Patent Document 2]
Japanese National Patent Publication No. 10-507521
[Non-Patent Document 1]
A. Latner, ME Parsons, AW Skillen, Biochem. J, 118 , 299 (1970)
[Non-Patent Document 2]
O. Miyawaki and T. Yano, Enzyme Microb. Technol., 14 , 474 (1992)
[Non-Patent Document 3]
I. Satoh, “Biosensing of heavy metal ions based on specific interactions with apoenzymes”, In Advances in Molecular and Cell Biology, eds., B. Danielsson and L. B ▲ u ▼ low, 15B , Greenwich, CT, USA JAI Press Inc., 1996, pp. 461-471.
[0013]
[Problems to be solved by the invention]
Conventional methods use the action of drugs, such as the addition of chelating agents and denaturing agents, to remove metals in metalloenzymes. However, in order to completely remove the metal from the metal enzyme by adding a drug such as a chelating agent, it is necessary to prepare a solution containing a drug at a predetermined concentration.
[0014]
In addition, in order to recover the activity of a metal enzyme deactivated by the addition of a drug, it is necessary to replace the solution with a solution that does not contain a chelating agent and the like, and then add metal to the metal enzyme. In addition, the operation is complicated. In addition, when the amount of the solution containing the drug is large or when the drug in the solution is at a high concentration, there is a problem that the drug places a burden on the environment.
[0015]
A specific enzyme reaction occurs as required, and an enzyme that inactivates the enzyme when not needed can be used as a molecular switch. In particular, it is necessary to reversibly change the enzyme activity in an enzyme used for such applications.
[0016]
For example, it is known that the activity of various metalloenzymes such as thermolysin used for protein fragmentation is controlled by the presence or absence of a coordinated metal. When it is desired to reversibly remove a metal from such a metal enzyme and control the enzyme activity, it is not preferable to use a denaturant that irreversibly denatures the protein depending on the amount added.
[0017]
In addition, as described above, atomic absorption analysis and high frequency inductively coupled plasma emission analysis can be cited as methods for analyzing a trace amount of metal in a sample, but these methods are large and expensive instruments or expensive equipment. Requires maintenance costs. Therefore, a method capable of measuring a target metal more easily, quickly and inexpensively than before is desired.
[0018]
The present invention has been made in view of the above problems. Therefore, the present invention provides a method for controlling enzyme activity, which can accurately control enzyme activity by a simple, inexpensive and safe method. The The purpose is to provide.
Another object of the present invention is to provide a metal measuring method and measuring apparatus that can detect a metal simply, quickly and inexpensively using an enzyme reaction.
[0019]
[Means for Solving the Problems]
In order to achieve the above object, the method for controlling enzyme activity of the present invention electrochemically desorbs a metal essential for the expression of enzyme activity in a metal enzyme from the metal enzyme, thereby eliminating or reducing the enzyme activity. It has the process. The method for controlling enzyme activity of the present invention preferably further includes a step of restoring the enzyme activity by imparting the metal to the metal enzyme whose enzyme activity has disappeared or decreased.
[0021]
In order to achieve the above object, the method for measuring a metal of the present invention comprises a step of electrochemically desorbing a metal essential for expression of enzyme activity from a metal enzyme in the metal enzyme to eliminate or reduce the enzyme activity. And a step of supplying a sample to the metalloenzyme in which the enzyme activity has disappeared or decreased, and a step of measuring the amount of the metal in the sample using the enzyme activity recovered by the supply of the sample as an index. It is characterized by.
[0022]
In order to achieve the above object, the metal measuring device of the present invention includes a carrier on which a metal enzyme is fixed, a column packed with the carrier, a liquid passing means for passing the liquid through the column, and the column. A constant current or a constant potential is applied to the metal enzyme, the metal enzyme essential for expression of enzyme activity is electrochemically desorbed from the metal enzyme, and the enzyme activity is lost or reduced, and the column is applied to the column. Substrate supply means for supplying a substrate for the metal enzyme, sample supply means for supplying a sample to the column, and the enzyme activity of the metal enzyme recovered in accordance with the amount of the metal contained in the sample, And detecting means for detecting a product generated from the substrate.
Thereby, it becomes possible to detect a metal simply, quickly and inexpensively using an enzyme reaction and its control.
[0023]
DETAILED DESCRIPTION OF THE INVENTION
Below, the method for controlling the enzyme activity of the present invention, Money An embodiment of a genus measuring method and a metal measuring device will be described with reference to the drawings.
[0024]
The metalloenzyme whose enzyme activity is controlled in the present invention and can be used for metal measurement includes alkaline phosphatase using zinc as a cofactor, thermolysin, carboxypeptidase, and the like. The metal enzymes to which the present invention can be applied include ascorbate oxidase using not only zinc but also copper as a cofactor, galactose oxidase, laccase, or the like, or an enzyme using cobalt or the like as a cofactor.
[0025]
In addition, even for enzymes that use calcium as a cofactor, such as amylase, enzymes that are known to show activity even when a metal is bound instead of calcium are the same as for the above-mentioned metal enzymes. The invention can be applied. There are various kinds of metal coenzyme cofactors, and the present invention can remove cofactors from these metal enzymes.
[0026]
In the present invention, a metal enzyme-containing system is placed in an electric field instead of a chemical treatment agent such as a chelating agent or a denaturing agent that has been used in the past, so that the metal can be removed more easily from a metal enzyme that is difficult to remove the metal. . In particular, proteins generally have a higher-order structure, and in some cases where the metal is inside the enzyme, the metal can be removed more effectively than chemical treatment.
[0027]
According to the chemical treatment, the three-dimensional structure and size of the chelating agent become a problem, and the chelating agent cannot access the metal part embedded in the protein, or even if the chelating agent approaches, it forms a metal complex. It may be difficult.
[0028]
In contrast, the electrochemical method simply applies an electric field and can extract metal ions that are clearly smaller than a chelating agent or the like from a metal binding site of a protein without causing steric hindrance. is there.
[0029]
The enzyme in the present invention may be dissolved, suspended, or supported on a solid support, and the present invention can be applied. In any case, the metal ions desorbed from the metal can be separated from the enzyme by using a semipermeable membrane, a filter, a column, or the like.
[0030]
The current and voltage applied to the dissolved enzyme and the immobilized enzyme are effective as long as the enzyme is not irreversibly denatured, and should be easily performed with a constant current / constant voltage device for protein electrophoresis or electrochemical measurement. Can do. The electric field to be applied is about 100 μA to 10 A in terms of current and about 1 mV to 1000 V in terms of voltage.
[0031]
However, this current or voltage is not a value that is substantially applied to the enzyme, but is applied to the entire system. The resistance of the entire system increases due to the influence of the composition of the solution and bubbles. The applied current or voltage is determined in consideration of the fine environment of the metal binding site of the metal enzyme, the conductivity of the solution, and the like.
[0032]
The present invention can be applied to enzymes by various immobilization methods. Examples of the method for immobilizing an enzyme on a carrier include a crosslinking method using glutaraldehyde, a physical adsorption method using hydroxyapatite, an ion binding method using an ion exchange resin, and a covalent bond using a diazonium salt. Any method generally known as an immobilization method, such as a method, a comprehensive method using alginic acid or a membrane, can be used.
[0033]
As the enzyme immobilization carrier according to the present invention, microporous glass particles can be used. The particle size is effective at about 100 μm to several mm, and the pore size is about 10 nm to 200 nm.
[0034]
In addition to glass, an electrode made of platinum or gold that can be used for immobilization of enzymes, agar, reverse micelles, or the like can be used as the carrier. When using platinum, gold, or the like, it is possible to use platinum black or gold black, or to chemically modify the platinum surface to immobilize the enzyme by chemical bonding.
[0035]
In the present invention, the enzyme activity can be recovered by adding the metal ion-containing liquid again to the system in which the metal ions have been removed from the metal enzyme. A metal that restores the activity of a metalloenzyme is, in principle, a metal that the metalloenzyme originally serves as a cofactor, but is not limited to the metal originally contained.
[0036]
Specifically, alkaline phosphatase can be recovered by adding cobalt instead of zinc, which is the original cofactor, and amylase can be recovered by adding calcium or strontium. .
[0037]
In the present invention, the enzyme activity can be controlled by removing the metal from the metal enzyme and applying the metal to the metal enzyme from which the metal has been removed. The enzyme activity is controlled by the presence / absence of metal addition or by adjusting the concentration of the metal to be applied. On the contrary, the activity can be eliminated from the active state by an electrochemical method. By this control, the present invention can be used as a molecular switch.
[0038]
A molecular switch refers to playing a role of a switch with one molecule, and a molecule in the present invention refers to an enzyme. The state having the enzyme activity is the switch on state, and the state having no enzyme activity is the off state. When a metal is desorbed by applying a potential to the metalloenzyme, the metal enzyme is turned off with no activity. The activity is restored by adding an appropriate amount of metal to the metal enzyme in the off state, and the metal enzyme can be turned on.
[0039]
According to the present invention, an immobilized product of thermolysin, which is a protease having zinc at the active center, can be used as the molecular switch. Thermolysin is an enzyme often used in determining the sequence of a protein with a protein sequencer. The present enzyme is used for fragmenting a protein. By applying the present invention to turn on / off the activity of the enzyme, it is possible to perform protein fragmentation or not.
[0040]
According to the present invention, an immobilized product of alkaline phosphatase having zinc at the active center can be used as the molecular switch as described above. Alkaline phosphatase whose activity has been lost by removal of zinc recovers its enzymatic activity by the addition of zinc, and the enzymatic activity to be recovered depends on the amount of zinc present.
[0041]
The immobilized alkaline phosphatase can be used, for example, to detect the movement or leakage of zinc ions from the protoplast of animal cells or plant cells to the outside of the cells (or protoplasts). This can be applied to confirm the success or failure of cell fusion.
[0042]
In cell fusion, when zinc ions that have been taken into the cell in advance are leaked due to cell destruction, they are taken into the immobilized product of alkaline phosphatase and the switch is turned on. A substrate for alkaline phosphatase with the switch on p When -nitrophenyl phosphate (PNPP) is added, p -Nitrophenol is produced.
[0043]
p Since the absorbance at a wavelength of 405 nm increases with the generation of -nitrophenol, the product can be detected using this absorbance as an index. Through detection of such a product, the movement of zinc out of the cell can be easily recognized. That is, the success or failure of cell fusion can be confirmed by detecting zinc ions as a labeling agent.
[0044]
By using the enzyme activity according to the present invention as an index, the metal concentration can be measured. Enzymes applicable to the measurement of metal concentration are alkaline phosphatase, thermolysin, carboxypeptidase and the like described above, but are not limited thereto. As described above, any metal can be used as long as it exhibits the activity of the metalloenzyme as described above.
[0045]
When measuring a metal, it is possible to perform more selective measurement by combining with an ionophore film or the like that can select a target metal. Moreover, even if the same metal is detected by selecting an enzyme, the sensitivity and accuracy can be changed.
[0046]
【Example】
Hereinafter, the present invention will be described in detail by way of specific examples, but the embodiments of the present invention are not limited thereto.
(1) Immobilization method of metalloenzyme
Metalloenzymes include alkaline phosphatase (ALP; orthophosphoric monoester phosphohydrolase, EC 3.1.3.1, Escherichia coli ( Escherichia coli ) Origin: Asahi Kasei Co., Ltd.).
[0047]
As the immobilization support, 3 g of microporous glass (CPG; pore size 24.2 nm, particle size 120-200 mesh, manufactured by Electronucleonics) was weighed and adjusted to pH 3.45, 10% γ-aminopropyltriethoxysilane (γ- APTES) was added and reacted at 75 ° C. for 3 hours to carry out alkylamination of the glass. 2.5 g glutaraldehyde solution prepared with 10 mM phosphate buffer (pH 7.0) was added to 1.0 g of the resulting solution to form an aldehyde, which was then dried and the alkaline phosphatase solution adjusted to 10 mg / ml with the above buffer solution. In addition, immobilization was performed. This immobilizate is used as a reducing agent (NaBH _Four ).
[0048]
The enzyme reduced after immobilization was packed in a microcolumn and set in a water jacket for keeping the column at a constant temperature. Hereinafter, this is referred to as an immobilized enzyme column unit. FIG. 1 is a block diagram showing an immobilized enzyme column unit and an apparatus for applying a constant current / constant potential thereto.
[0049]
As shown in FIG. 1, a platinum tube 2 and a Teflon tube 3 (“Teflon” is a registered trademark of DuPont) or a flare-equipped Teflon tube 3 a are connected by a Tygon tube 1. The flare-equipped Teflon tube 3 a is embedded in the cap 6 of the water jacket by screws 4 and washers 5. The water jacket 7 covers an immobilized enzyme particle packed layer 9 sandwiched between filters 8. The immobilized enzyme particle packed layer 9 is a layer filled with glass particles on which an enzyme is immobilized. The cap 6 of the water jacket is sealed with a packing 10.
[0050]
A constant temperature circulates in the water jacket 7 via a constant temperature water outlet 11 and a constant temperature water inlet 12, whereby the inside of the immobilized enzyme particle packed bed 9 is maintained at a constant temperature. The cap 13 of the water jacket is a cap on the upstream side of the immobilized enzyme column 14 and is sealed using the packing 10 in the same manner as the downstream side. The cap 13 is connected to the Teflon tube 3 and the flare-equipped Teflon tube 3a using the screw 4 and the washer 5 similarly to the downstream side of the immobilized enzyme column 14.
[0051]
A carrier is supplied to the immobilized enzyme particle packed bed 9 in the immobilized enzyme column 14 from the carrier inlet 15, and the carrier is discharged from the carrier outlet 16. The constant current / constant potential application device 17 is connected to the platinum tube 2 provided on the upstream side and the downstream side in the immobilized enzyme column 14 respectively. Thereby, a constant current or a constant potential is applied to the immobilized enzyme.
[0052]
(2) FIA (Flow Injection Analysis) system
FIG. 2 is a configuration diagram of the FIA system of this embodiment. As shown in FIG. 2, the FIA system includes a carrier C in the tank, a pump P for feeding liquid, a damper Da for preventing pulsating flow, a rotary valve with a sample injection loop (a metal injection valve MV and a substrate injection valve SV). ), An immobilized enzyme column unit IE, a constant current / constant potential application device PG, a detector D (ultraviolet / visible spectroscopic detector), and a recorder R.
[0053]
A 100 mM Tris-HCl buffer (pH 8.0) was used for carrier C, and a plunger pump P was used for feeding carrier C (1.0 ml / min). The FIA system passes through two rotary valves with loops, which are an enzyme cofactor (metal) injection valve MV and an enzyme substrate injection valve SV.
[0054]
The sample injected from these valves reaches the immobilized enzyme column unit IE together with the carrier C. The immobilized enzyme column unit IE is kept at 30 ° C. by a water jacket (see FIG. 1), and the sample is catalyzed by the immobilized enzyme column unit IE.
[0055]
The carrier C that has passed through the column unit IE is introduced into a flow cell installed in the ultraviolet / visible spectroscopic detector D. The absorbance change at a specific wavelength (405 nm) is continuously monitored by the ultraviolet / visible spectroscopic detector D and recorded in the recorder R.
[0056]
(3) Preparation of reagents
The carrier used for measuring enzyme activity was one in which NaCl and citric acid were dissolved in 1.0 mM and 0.1 μM, respectively, in 100 mM Tris-HCl buffer, and the pH was adjusted to 8.0. . A 0.1 M citrate buffer (pH 4.5) was used as a carrier for electroremoval of the cofactor. In addition, 2.0 mM PNPP ( p -nitrophenyl phosphate (manufactured by Sigma) was used. In the preparation, it was diluted with a carrier. As a chelating agent for comparison, 100 mM PDC (2,6-pyridinedicarboxylic acid) was used. For preparing a calibration curve for cofactors, zinc standard solutions (for atomic absorption analysis) were diluted to various concentrations and used.
[0057]
(4) Measurement of alkaline phosphatase column activity
Using the FIA system described in (2), the activity of the column on which alkaline phosphatase was immobilized was measured. After confirming that the carrier was flowing at a flow rate of 1.0 ml / min using a carrier for measuring enzyme activity, 100 μl of 2.0 mM PNPP was injected from the substrate injection valve. The change in absorbance associated with the catalytic reaction of the substrate was measured. This operation was performed 10 times to obtain reproducibility. The average of 10 absorbances was 1.65, and the relative standard deviation was 0.10%. This indicates that this measurement system is very reproducible.
[0058]
The change in absorbance when injecting various concentrations of the substrate was also measured in the same manner. As a result of injecting various concentrations (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0 mM) of the substrate, the absorbance increased as the concentration of the substrate increased. When the substrate concentration was up to 0.5 mM, a proportional relationship was obtained between the concentration and absorbance, indicating that the reaction was quantitative.
[0059]
(Example 1)
Zinc (II) ions, which are cofactors of alkaline phosphatase, were removed from the immobilized enzyme column by the following electrochemical method.
First, it was passed through a carrier for activity measurement, and after confirming enzyme activity, it was switched to a carrier for electrochemical removal of cofactor (pH 4.5), passed for 10 minutes, and then passed through the system shown in FIG. An electric current was applied to platinum tubes installed above and below the immobilized enzyme column unit while performing the liquid. The applied current was 2.0 mA with a constant current device, and the application time was 1 hour.
[0060]
Thereafter, the enzyme activity was again returned to the carrier for enzyme activity, and 2.0 mM PNPP was injected to measure enzyme activity. As a result, as shown in FIG. 3, the activity having an absorbance ΔA of 1.649 before current application decreased to 0.017 after application. This value was almost the same value as when only the substrate was injected without passing through the immobilized enzyme column. Thus, it was shown that the enzyme activity was lost by applying a current and the activity could be controlled.
[0061]
The current application time was in the range of 1 millisecond to 1 hour, but the same result as in the case of 1 hour could be obtained even at 5 minutes. In addition, a significant decrease in enzyme activity was confirmed even after application for several seconds.
[0062]
Zinc ions that were removed from the enzyme when current was applied to the alkaline phosphatase column and flowed out of the column were quantified by ICP-AES (high frequency inductively coupled plasma emission analysis). The application of current was performed under the same conditions as in the case of the above enzyme activity measurement (measurement shown in FIG. 3), and the current application time was 5 minutes.
[0063]
5 minutes is sufficient to remove zinc from the enzyme and allow zinc to flow out of the column with the column of this embodiment, but allow 5 minutes before and after applying current, 5 minutes after applying current, and before and after applying current. The solution that flowed out of the column in 1 minute was collected.
[0064]
With respect to the collected solution, zinc was quantified using a sequential plasma emission spectrometer (ICPS-7000, Shimadzu Corporation). In the determination of zinc, the emission intensity at 213.856 nm, which is the absorption wavelength of zinc, was compared by a calibration curve method.
[0065]
The result of measuring zinc in the above Example 1 is shown in FIG. Here, the estimated zinc content in the alkaline phosphatase column was 3.587 μg. The theoretical zinc content in the alkaline phosphatase column was 3.66 μg, almost the same as the value obtained in the experiment. Here, the theoretical zinc content in the alkaline phosphatase column was calculated using the amount of enzyme immobilized on the carrier, the amount of the carrier loaded into the column, and the zinc content of alkaline phosphatase. From the above, it was shown that zinc can be desorbed from alkaline phosphatase by applying an electric current.
[0066]
(Comparative Example 1)
FIG. 5 shows the results obtained when the carrier for removing the cofactor was changed to 100 mM Tris-HCl buffer (pH 8.0) in Example 1 above. In Comparative Example 1, after applying an electric current to a carrier having a pH of 8.0, the enzyme activity was measured while passing the same carrier as it was. As is clear from comparison between FIG. 3 and FIG. 5, in Comparative Example 1, no change was observed in the enzyme activity, indicating that zinc as a cofactor could not be removed.
[0067]
The pH 4.5 of the carrier for cofactor removal used in Example 1 is the isoelectric point of alkaline phosphatase, and the pH 8.0 of the carrier for cofactor removal used in Comparative Example 1 is the optimum pH of alkaline phosphatase. From the above, it can be said that the carrier used for removing the cofactor in the method of the present invention is preferably adjusted to a pH which is the isoelectric point of the enzyme.
[0068]
(Example 2)
According to the same procedure as in Example 1, zinc in alkaline phosphatase was electrically desorbed and the enzyme activity was measured. Thereafter, enzyme solutions were measured by injecting zinc solutions of various concentrations from the metal injection valve shown in FIG.
[0069]
When a calibration curve was prepared by injecting a 1.0 nM to 1.0 mM zinc solution, the results shown in FIG. 6 were obtained. That is, the desired enzyme activity could be expressed by electrochemically desorbing zinc from the immobilized alkaline phosphatase and then adding an appropriate zinc solution.
[0070]
As mentioned above, according to the system of the present Example, it was shown that alkaline phosphatase activity can be controlled. In addition, after creating a calibration curve showing the relationship between zinc concentration and enzyme activity, measuring the enzyme activity by injecting an unknown sample and comparing it with the calibration curve indicates that the zinc concentration in the unknown sample can be quantified. It was. That is, the apparatus of the present embodiment can be used as a biosensing system.
[0071]
(Example 3)
Alkaline phosphatase was used as a metal enzyme in the same manner as in Examples 1 and 2, and zinc as a cofactor was electrochemically removed from alkaline phosphatase. Then, various divalent metal solutions were added to measure enzyme activity.
[0072]
Specifically, after electrochemically desorbing zinc from alkaline phosphatase, divalent cobalt, calcium, copper, magnesium, nickel, manganese, lead, iron, and cadmium solutions are all separated at a concentration of 1.0 mM. The enzyme activity was measured. Each metal ion was diluted to a target concentration by a carrier.
[0073]
As shown in FIG. 7, significant recovery of activity was observed in zinc and cobalt. For zinc, as can be seen with reference to FIG. 3, the enzyme activity is restored to about the same level as before removing zinc from the metalloenzyme. The recovery of activity was also observed in magnesium and manganese, but the recovery was slight. By measuring the concentration of the cobalt solution in more detail than zinc, it was suggested that it can be applied not only to zinc but also to measurement of cobalt.
[0074]
According to the enzyme activity control method and molecular switch of the embodiment of the present invention described above, the activity of the metalloenzyme can be controlled by electrochemically desorbing the cofactor in the metalloenzyme. . Moreover, it is not necessary to use a chelating agent that imposes a burden on the environment, and the safety is high. Desorption and application of cofactors can be performed reversibly, and application in various fields is expected.
[0075]
Moreover, according to the metal measuring method and metal measuring apparatus of the above-described embodiment of the present invention, it is possible to measure the metal concentration in the solution using the enzyme activity controlled by the control method of the present invention as an index. Become. In the current analysis method, a large-scale measuring device is required for the determination of a trace amount of metal. If the method or device of the present invention is applied, such a device itself or the cost required for maintenance and use of the device is reduced. Can be reduced.
[0076]
Embodiments of the enzyme activity control method, molecular switch, metal measurement method, and metal measurement device of the present invention are not limited to the above description. For example, in the system of FIG. 2, the metal injection valve MV is disposed upstream of the substrate injection valve SV, but the substrate injection valve SV may be disposed upstream by replacing them. In addition, the temperature of the liquid circulated in the water jacket can be appropriately changed according to the optimum temperature of the enzyme to be immobilized. In addition, various modifications can be made without departing from the scope of the present invention.
[0077]
【The invention's effect】
According to the enzyme activity control method and molecular switch of the present invention, the enzyme activity can be accurately controlled by a simple, inexpensive, and safe method.
Moreover, according to the metal measuring method and metal measuring apparatus of the present invention, it is possible to detect a metal simply, quickly and inexpensively using an enzyme reaction.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing an immobilized enzyme column unit according to an embodiment of the present invention and a device for applying an electric current thereto.
FIG. 2 is a block diagram in which an immobilized enzyme column unit according to an embodiment of the present invention is introduced into a flow system.
FIG. 3 relates to Example 1 of the present invention, and shows the evaluation of the activity of an immobilized enzyme column before and after current application.
FIG. 4 is a graph showing the results of measuring the amount of zinc desorbed from the immobilized enzyme column according to Example 1 of the present invention by ICP-AEM.
FIG. 5 is a diagram related to Comparative Example 1 in which the activity of the immobilized enzyme column before and after current application was evaluated.
FIG. 6 is a graph showing the activity of an immobilized enzyme column when zinc solutions having various concentrations are added according to Example 2 of the present invention.
FIG. 7 is a diagram showing the activity of an immobilized enzyme column when various metal solutions are added according to Example 3 of the present invention.
[Explanation of symbols]
1 ... Tygon tube, 2 ... Platinum tube, 3 ... Teflon tube, 3a ... Teflon tube with flare, 4 ... Screw, 5 ... Washer, 6 ... Water jacket cap, 7 ... Water jacket, 8 ... Filter, 9 ... Immobilization Enzyme particle packed bed, 10 ... packing, 11 ... constant temperature water outlet, 12 ... constant temperature water inlet, 13 ... water jacket cap, 14 ... immobilized enzyme column, 15 ... carrier inlet, 16 ... carrier outlet, 17 ... constant current・ Constant potential application device, C ... carrier, P ... liquid pump, Da ... damper, MV ... metal injection valve, SV ... substrate injection valve, IE ... immobilized enzyme column, PG ... constant current / constant potential application device , R ... recorder, D ... detector, W ... effluent.

Claims (7)

In the metal enzyme, the state of the metal enzyme is a solution or suspension system, or a system supported on a solid support,
Apply a constant current of 100 μA to 10 A or a constant voltage of 1 mV to 1000 V to the entire system,
A method for controlling enzyme activity, comprising a step of electrochemically desorbing a metal essential for expression of enzyme activity from the metal enzyme at a pH of an isoelectric point of the enzyme to eliminate or reduce the enzyme activity. In the metal enzyme, the state of the metal enzyme is a solution or suspension system, or a system supported on a solid support,
Apply a constant current of 100 μA to 10 A or a constant voltage of 1 mV to 1000 V to the entire system,
A step of electrochemically desorbing a metal essential for expression of enzyme activity from the metal enzyme at the pH of the isoelectric point of the enzyme to eliminate or reduce the enzyme activity;
Supplying a sample to the metalloenzyme whose enzyme activity has disappeared or decreased;
And measuring the amount of the metal in the sample using the enzyme activity recovered by the supply of the sample as an index. Measuring the enzyme activity recovered when the metal is applied to the metal enzyme whose enzyme activity has disappeared or decreased in advance under a plurality of conditions in which the amount of metal applied is different from each other;
Creating a calibration curve based on the result of the measurement,
The method for measuring a metal according to claim 2 , wherein the step of measuring the amount of the metal using the enzyme activity as an index includes a step of referring to the calibration curve. The method for measuring a metal according to claim 2 , wherein the metal enzyme is immobilized on a carrier. The metal measuring method according to claim 2 , wherein the metalloenzyme is alkaline phosphatase, and the metal is one of zinc and cobalt. A carrier on which a metal enzyme is immobilized;
A column packed with the carrier;
A liquid passing means for passing through the column;
An applying means for applying a constant current or a constant potential to the column, electrochemically desorbing a metal essential for expression of enzyme activity in the metal enzyme from the metal enzyme, and eliminating or reducing the enzyme activity;
Substrate supply means for supplying the metalloenzyme substrate to the column;
Sample supply means for supplying a sample to the column;
A metal measuring apparatus comprising: a detecting unit that detects a product generated from the substrate by the enzyme activity of the metal enzyme recovered according to the amount of the metal contained in the sample. The metal measuring apparatus according to claim 6 , wherein the metalloenzyme is alkaline phosphatase, and the metal is one of zinc and cobalt.

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