JP2001299390A - Method for amplifying atp in chainlike manner and method for testing trace atp using the amplification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for amplifying ATP in a chainlike manner, and a method for testing trace ATP by attaining sensitivity and accuracy of the measurement for bioluminescence using the method. SOLUTION: The first reaction which converts ATP to 2 molecules of ADP by reacting the ADP with an adenylate kinase in the presence of trace ATP and the second reaction which converts the 2 molecules of ADP to 2 molecules of ATP and polyphosphoric acid compound by reacting the 2 molecules of ADP with a polyphosphoric acid kinase in the presence of polyphosphoric acid compound are paired to form a reaction system. The paired reaction system is repeated plural times, and thereby, ATP is amplified at a factor of the power of 2 according to the number of the repetitions of the reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for chain-wise increasing the amount of ATP produced in a metabolism / biosynthesis system of animals and plants, and by detecting bioluminescence using the method, Because it can detect a trace amount of ATP, which could not be detected conventionally, it is applied to the inspection of cleanliness by detecting invisible microorganisms in food factories, etc., and to the measurement of freshness of food such as meat, fresh fish, vegetables, etc. You can do it.

[0002]

2. Description of the Related Art ATP is an indicator of living organisms. Therefore, by subjecting ATP derived from microorganisms to bioluminescence, a hygiene inspection of microorganisms using light as an indicator is performed.
(For example, Japanese Patent Publication No. 6-34757, Registration No. 1911659)
In the case of a small amount of microorganisms, bioluminescence derived from ATP is not sufficiently performed.

[0003] Japanese Patent Application Laid-Open No. 8-47399 discloses that "in a method for measuring bioluminescence generated by luciferase, a bioluminescence reaction is carried out in the presence of a polyphosphate compound or a salt thereof and a sulfhydryl compound." A featured technology is disclosed. As a result, a novel effect of enhancing bioluminescence can be obtained, and measurement of a bioluminescence reaction can be performed by using a general luminometer, which has an effect of increasing the penetration rate.

However, the method disclosed in Japanese Patent Application Laid-Open No. 8-47399 is a technique for stabilizing bioluminescence,
It is not a technique of increasing the amount, but has a disadvantage that the luminescence attenuates with time as ATP is consumed.

Japanese Patent Application Laid-Open No. 9-234099 discloses a method for quantifying cyclic AMP by a bioluminescence method represented by the following reaction formula.

Embedded image

In this method, cyclic AMP is hydrolyzed with cyclic 3 ', 5'-nucleotide phosphodiesterase to generate AMP in the reaction system (reaction 1), and the reaction is carried out in the presence of magnesium ions and a small amount of ATP. Then, the AMP is reacted with adenylate kinase to produce AD.
When converted to P (reaction 2), magnesium ion,
The ADP is reacted with pyruvate kinase in the presence of phosphoenolpyruvate to convert it to ATP and pyruvate (reaction 3), and then to luciferin, magnesium ions (or other metal ions) and dissolved oxygen. Then, ATP is reacted with luciferase to generate luminescence (Reaction 4), and a method for quantifying cyclic AMP by measuring the amount of luminescence generated in (Reaction 4) (ME
THODS IN ENZYMOLOGY 38, 62-65; 1974).

However, in this method, AMP converted from cyclic AMP is converted into ADP, and the ADP is recycled as ATP by the reaction of pyruvate kinase in the presence of phosphoenolpyruvate. However, the amount of ATP is not necessarily increasing.

[0008]

SUMMARY OF THE INVENTION The present invention invents a method for increasing the amount of ATP in a chain from a completely different point of view from the prior art, and detects bioluminescence by using the method. A trace amount of ATP that could not be detected conventionally
It is an object of the present invention to provide a method for detecting a scalar.

[0009]

Means for Solving the Problems In order to solve the above-mentioned problems, the invention of claim 1 is a method for converting AMP to adenylate kinase in the presence of a trace amount of ATP to convert it into two molecules of ADP. A reaction and a second reaction in which the two molecules of ADP are reacted with polyphosphate kinase to convert them into two molecules of ATP and a polyphosphate compound in the presence of the polyphosphate compound are formed as a pair of reaction systems. The reaction was repeated a plurality of times to amplify ATP by a power of 2 according to the number of reactions.

In the first reaction of the reaction system, ATP and AMP immediately react with adenylate kinase and are converted into two molecules of ADP (first reaction). Next, the two molecules of ADP react with polyphosphate by polyphosphate kinase and are converted into two molecules of ATP and polyphosphate (second reaction). Then, the process proceeds to the second reaction of the reaction system. At this time, the two molecules of ATP generated in the first reaction of the reaction system react with two molecules of AMP and are converted into four molecules of ADP (first reaction). ). Next, the four molecules of ADP
Reacts with polyphosphate by polyphosphate kinase,
The molecules are converted into ATP and polyphosphate (second reaction). Hereinafter, the ATP is increased by repeating the third time of the reaction system, the fourth time of the reaction system, the fifth time of the reaction system, etc. a plurality of times. This reaction is triggered by a very small amount of ATP added to the reaction system for the first time, and subsequently occurs in a chain, and AMP occurs.
And polyphosphoric acid are present for a long time, and ATP increases by a power of 2 according to the number of reactions in the reaction system.

The polyphosphate compound may be a polyphosphate compound produced by chemical synthesis, wherein at least 10 to 100 phosphoric acids are linearly polymerized. The polyphosphate compounds (PolyP _n) which was connected are n phosphoric acid, in the invention of claim 2, led phosphate (M I ^{3 _P)} of at least 10 to 100 in one molecule of the polyphosphate compound Therefore, the conversion of ADP to ATP in the second reaction of claim 1 is easily performed, and the supply of phosphoric acid when regenerating ATP is small, which is economical. Further, the reaction time of the reaction system is continued by a large number of phosphoric acids.

Further, the polyphosphate compound is a polyphosphate compound derived from bacteria and has at least 10
It is preferable to use one in which about 1000 phosphoric acids are polymerized linearly. In the invention of claim 3, phosphoric acid in a molecule polyphosphate compound _{^(M I} 3 P) of at least 10 to 1000
Because they are connected, the conversion of ADP to ATP can be performed more easily, and the supply of phosphoric acid is small and economical. In addition, the reaction time of the reaction system continues for a long time due to the large number of phosphoric acids.

When the polyphosphate compound is biosynthesized from ATP by the catalysis of polyphosphate synthase, the yield of the polyphosphate compound can be improved and the polyphosphate compound can be produced at low cost. Become.

[0014] A method of amplifying ATP in a chain
A small amount of ATP is amplified, and luciferin and dissolved oxygen
AMP and luminescence by reacting with luciferase in the presence
When the amount of generated light is measured, the increased amount
ATP equivalent to ATP
A trace amount of ATP that has not been detected can be detected. principle
Specifically, one molecule of ATP present in the first reaction system is also detected.
It can be issued.

[0015]

Embodiments of the present invention will be described.
The theoretical reaction formula of the present invention is shown below.

Embedded image

Adenylate kinase (ADK), which serves as a catalyst for the reaction, converts two molecules of adenosine diphosphate (ADP) when adenosine monophosphate (AMP) and adenosine triphosphate (ATP) are reacted. It is the enzyme that produces it.
In addition, polyphosphate kinase (PPK) reacts ADP with polyphosphate (PolyP) to form ATP and polyphosphate (PK).
olyP _n-2 ).

In the present invention, A is used in the presence of a trace amount of ATP.
MP is reacted with adenylate kinase to produce two molecules of AD
A first reaction (formula 1) for conversion to P and a second reaction for converting the two molecules of ADP with polyphosphate kinase to convert them into two molecules of ATP and polyphosphate compound in the presence of the polyphosphate compound (2) is used as a pair of reaction systems, and the pair of reaction systems is repeated a plurality of times to amplify ATP by a power of two according to the number of reactions.

As a result, in the first reaction of the reaction system, ATP and AMP immediately react with adenylate kinase and are converted into two molecules of ADP (Equation 1).
Next, the two molecules of ADP react with polyphosphate by polyphosphate kinase and are converted into two molecules of ATP and polyphosphate (Equation 2). Then, the process proceeds to the second reaction of the reaction system. At this time, the two molecules of ATP generated in the first reaction of the reaction system react with two molecules of AMP and are converted into four molecules of ADP (Equation 3). The four molecules of ADP are then reacted with polyphosphate by polyphosphate kinase to produce four molecules of ADP.
It is converted to TP and polyphosphoric acid (Equation 4). Hereinafter, the ATP is increased by repeating the third time of the reaction system, the fourth time of the reaction system, the fifth time of the reaction system, etc. a plurality of times. This reaction is triggered by a small amount of ATP added in the first reaction of the reaction system, and thereafter occurs in a chain, and continues for a long time as long as AMP and polyphosphoric acid are present.
ATP will increase with powers of.

Polyphosphoric acid (PolyP) is a product in which n phosphoric acids are connected. For example, chemically synthesized polyphosphoric acid is a product in which about 100 phosphoric acids are connected. In addition, those extracted from the bacteria are those in which nearly 1000 phosphoric acids are connected.

For example, the polyphosphate compound (PolyPn) used in the present invention is represented by the following structural formula. Here, (PolyPn) preferably has a range of 10 ≦ n ≦ 1000.

Embedded image

The polyphosphate compound is a bacterial polyphosphate compound, and is at least 10 to 100.
0 phosphate (M I ^{3 _P)} is improved reactivity because it polymerized.

By the catalytic action of polyphosphate synthase, A
When biosynthesized from TP, the yield of the polyphosphate compound can be improved, and the polyphosphate compound can be produced at low cost. For example, a polyphosphate compound is biosynthesized from ATP as shown in the following reaction formula.

Embedded image

For the biosynthesis of the polyphosphate compound according to the above reaction formula, for example, the conventional method for producing polyphosphoric acid disclosed in JP-A-5-153993 may be used. In the present embodiment, a polyphosphate compound is biosynthesized by reacting polyphosphate synthase with ATP and a metal ion such as magnesium for the purpose of suppressing inactivation of the enzyme, using the polyphosphate synthase as a catalyst. The polyphosphate synthase used in the present embodiment may be any that can biosynthesize polyphosphate synthase.

[0024] The present inventors have proposed that the ATP chain amplification reaction
Instead of polyphosphate and polyphosphate kinase
Using creatine kinase and creatine phosphate
It was confirmed that a reaction occurred. That is, two molecules of AD
A phosphorylation compound capable of converting P into two molecules of ATP
Product and enzyme, in principle, ATP is amplified in a chain
It is thought to wake up. However, polyphosphoric acid does not
By combining with phosphorylase kinase, one molecule
Because it has the ability to synthesize many ATPs,
This is advantageous for the ATP amplification reaction.

[0025]

[Example 1] Conditions under which ATP was reacted without addition, and AT
In order to compare with the conditions in which P was added and allowed to react, the change with time in luminescence was examined under the following conditions.

[0026] The samples (a) to (f) were mixed, sampled (5 μl) at each measurement time, and the ATP capacity was measured using an ATP measurement kit manufactured by Boehringer Mannheim.

[0027] In the same manner as described above, the samples (a) to (g) were mixed, sampled (5 μl) at each measurement time, and the ATP capacity was measured with an ATP measurement kit manufactured by Boehringer Mannheim.

The above results and the results are shown in FIG.
From this result, when ATP was added and reacted (reaction), the amount of ATP rapidly increased 30 minutes after the reaction,
The peak reaches after 0 minutes. On the other hand, when the reaction is performed without the addition of ATP (reaction), the amount of ATP does not increase and remains at a low level. Therefore, according to the present invention, the addition of a trace amount of ATP triggers the AT
It can be seen that a cascading increase in P has occurred. Therefore, even a small amount of ATP can be detected with high sensitivity by increasing the amount of ATP, and the accuracy of food inspection and hygiene inspection can be improved. Further, ATP can be detected by a cheap and simple luminometer.

[0029]

As described above, according to the present invention, a small amount of A
A first reaction in which AMP is reacted with adenylate kinase in the presence of TP to convert it into two molecules of ADP, and a reaction in which two molecules of ADP are reacted with polyphosphate kinase in the presence of a polyphosphate compound. The two reactions of ATP and the second reaction to be converted into a polyphosphate compound are formed as a pair of reaction systems, and the pair of reaction systems are repeated a plurality of times, whereby ATP is raised to the power of 2 according to the number of the reactions. A small amount of ATP added at the first time of the reaction system
Is triggered, and thereafter, ATP increases in a chain. Then, the chain increase of ATP continues for a long time as long as AMP and polyphosphoric acid are present, and ATP increases by a power of 2 according to the number of reactions in the reaction system. Thereafter, when the increased ATP is detected by bioluminescence, the amount of light is greatly increased compared to the small amount of ATP added at the first time in the reaction system.

The reaction of polyphosphoric acid and ADP to form ATP
When synthesizing, it is possible to increase ATP in a chain. Thus, the effect of increasing the amount of bioluminescence can be obtained, and the emission time can be maintained.

The polyphosphate compound is preferably a polyphosphate compound produced by chemical synthesis, wherein at least 10 to 100 phosphoric acids are linearly polymerized. Thus, the phosphoric acid in the molecule polyphosphate compound _{^(M I} 3 P) is contained at least 10 to 100, a continuous conversion of ADP to ATP is easily performed.

The polyphosphate compound is a polyphosphate compound derived from bacteria, and has at least 10
When a product obtained by linearly polymerizing １０００1000 phosphides is used, the conversion from ADP to ATP is more easily performed, and the supply of phosphoric acid is reduced in a small amount, which is economical. In addition, the reaction time of the reaction system continues for a long time due to the large number of phosphoric acids.

Furthermore, if the polyphosphate compound is biosynthesized from ATP by the catalysis of polyphosphate synthase, it is possible to improve the yield of the polyphosphate compound and to produce the polyphosphate compound at low cost. Become.

[0034] A method of amplifying ATP in a chain
A small amount of ATP is amplified, and luciferin and dissolved oxygen
AMP and luminescence by reacting with luciferase in the presence
When the amount of generated light is measured, the increased amount
ATP equivalent to ATP
A trace amount of ATP that could not be detected can now be detected.
1000 times more than the method without ATP amplification
Can be detected with the brightness of
The degree and accuracy are particularly improved.

In the present invention, since ATP is amplified, a very small amount of ATP which cannot be detected by the conventional method can be detected. Therefore, it can be applied to the detection of cleanliness by detecting invisible microorganisms at a food factory or the like, or to the measurement of freshness of food such as meat, fresh fish, and vegetables. As described above, in addition to application to hygiene management by detecting trace harmful microorganisms, the present invention can also be applied to inspection of general biochemical reactions that generate ATP or consume ATP. Further, by detecting ATP, application to forensic investigations and the like in place of the luminol reaction can be considered. Further, it can be applied to the synthetic production of ATP and the like.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram comparing a case where a reaction is performed without addition of ATP and a case where a reaction is performed with addition of ATP in a chain amplification reaction of ATP.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA28 AA40 CB21 DA15 FB01 FB13 GC15 2G054 AA06 AA10 AB07 BA04 CA21 CE08 EA02 GB01 4B063 QA01 QQ16 QQ63 QR02 QR07 QR42 QR58 QS36 QX02

Claims (5)

[Claims] 1. A first reaction in which AMP is reacted with adenylate kinase in the presence of a trace amount of ATP to convert it into two molecules of ADP, and the first reaction is carried out in the presence of a polyphosphate compound.
A second reaction for reacting a molecule of ADP with a polyphosphate kinase to convert two molecules of ATP to a polyphosphate compound is formed as a pair of reaction systems, and the pair of reaction systems is repeated a plurality of times to cause a reaction. 2 depending on the number of reactions
ATP characterized by amplifying ATP by a power of
To amplify in a chain. 2. The polyphosphate compound according to claim 1, wherein the polyphosphate compound is a polyphosphate compound produced by chemical synthesis.
2. The method for amplifying ATP in a chain according to claim 1, wherein a linear polymerization of 0 to 100 phosphoric acids is used. 3. The polyphosphate compound according to claim 1, wherein the polyphosphate compound is a bacterial-derived polyphosphate compound, and is at least 10 to 10.
2. The method for amplifying ATP in a chain according to claim 1, wherein the phosphoric acid is obtained by polymerizing 00 phosphoric acids linearly. 4. The method according to claim 3, wherein the polyphosphate compound is biosynthesized from ATP by the catalysis of a polyphosphate synthase. 5. A method for amplifying ATP in a chain according to any one of claims 1 to 4, wherein a very small amount of ATP is used.
Amplifying and reacting with luciferase in the presence of luciferin and dissolved oxygen to produce AMP and luminescence,
A method for testing a trace amount of ATP by measuring the amount of generated light.