WO2014092050A1 - Procédé de mesure de l'adénosine triphosphate dérivée de cellules - Google Patents

Procédé de mesure de l'adénosine triphosphate dérivée de cellules Download PDF

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Publication number
WO2014092050A1
WO2014092050A1 PCT/JP2013/082972 JP2013082972W WO2014092050A1 WO 2014092050 A1 WO2014092050 A1 WO 2014092050A1 JP 2013082972 W JP2013082972 W JP 2013082972W WO 2014092050 A1 WO2014092050 A1 WO 2014092050A1
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atp
adenosine triphosphate
microcapsule
derived
sample
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PCT/JP2013/082972
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English (en)
Japanese (ja)
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昭宏 栗田
山岸　豊
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東洋ビーネット株式会社
東洋インキＳｃホールディングス株式会社
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Publication of WO2014092050A1 publication Critical patent/WO2014092050A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
    • C12Y306/01005—Apyrase (3.6.1.5), i.e. ATP diphosphohydrolase

Definitions

  • the present invention relates to a method for measuring cell-derived adenosine triphosphate, and more specifically, after removing free adenosine triphosphate in a sample using a microcapsule containing adenosine triphosphate degrading enzyme,
  • the present invention relates to a method for measuring cell-derived adenosine triphosphate, which comprises detecting cell-derived adenosine triphosphate present in a sample.
  • a method of measuring microbial contamination in beverages culture for 2 to 3 days using an agar medium, filter the sample using an agar plate method for measuring the number of colonies produced, or a membrane filter, and attach the filter to the agar medium.
  • a membrane filter method of culturing on a filter is generally used. The above method takes time to obtain a measurement result, and lacks rapidity as a process control method.
  • a bioluminescence method using luciferase is used as a rapid detection method of adenosine triphosphate (hereinafter referred to as ATP) derived from microbial cells.
  • ATP adenosine triphosphate
  • This method is based on the principle that the amount of luminescence generated by the oxidation of luciferin is proportional to the amount of ATP in the presence of luciferin, luciferase, and ATP.
  • ATP is required to be measured accurately at a concentration of several nanomoles / L.
  • Patent Document 1 The first is a method in which a nonionic surfactant is allowed to act on a sample to extract non-microbial cell-derived ATP, followed by filtration to extract concentrated microbial cell ATP and estimating the amount of ATP by bioluminescence. This method requires time for filtration and is complicated.
  • non-ionic surfactant is allowed to act, non-microbe-derived ATP is extracted, the supernatant is removed by centrifugation, microbial cell-derived ATP is extracted from a precipitate containing microbial cells, and bioluminescence is used. It is a measurement method. In this method, separation of microorganisms is ensured by repeating the centrifugation operation, but it takes a considerable amount of time, and the recovery of microorganism cells becomes worse as the number of operations increases.
  • a non-ionic surfactant or the like is allowed to act on the measurement sample to extract non-microbe-derived ATP, which is then decomposed by an ATP hydrolase such as apyrase, and after the decomposition is completed, the apyrase is fixed with glass beads.
  • an ATP hydrolase such as apyrase
  • this method when removal or inactivation of apyrase is incomplete, there is a risk that ATP derived from microbial cells is degraded by residual apyrase. As a result, the variation in the ATP value increases, and only about 10 6 CFU / ml can be expected.
  • the present invention removes ATP derived from raw materials in a simple operation and in a short time, thereby increasing the contamination of microorganisms.
  • An object of the present invention is to provide a technique for sensitive and accurate determination.
  • microcapsule containing ATP hydrolyzing enzyme meets the above-mentioned purpose.
  • ATP hydrolase is encapsulated in a semi-permeable microcapsule, and ATP that has entered the capsule through the microcapsule thin film is decomposed by an enzymatic reaction.
  • the microcapsules are removed, the ATP derived from the microorganism is extracted, and the amount thereof is detected by a bioluminescence reaction. It was removed in time, and it was found that the contamination of microorganisms could be determined with high sensitivity and accuracy, and the present invention was completed (FIG. 1).
  • the method of the present invention can be used not only for microorganisms but also for measurement of cells such as animal cells and plant cells.
  • the gist of the present invention is as follows. (1) Using a microcapsule encapsulating adenosine triphosphate degrading enzyme, after decomposing free adenosine triphosphate in the sample, adenosine triphosphate is extracted from cells present in the sample not containing the microcapsule. A method for measuring adenosine triphosphate derived from a cell, comprising measuring the extracted adenosine triphosphate.
  • the adenosine triphosphate degrading enzyme is at least one enzyme selected from the group consisting of apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P and deaminase.
  • the film of the microcapsule is a semipermeable membrane that allows permeation of ions having a molecular weight of 1000 or less and does not permeate adenosine triphosphate degrading enzyme.
  • the coating of the microcapsule includes at least one material selected from the group consisting of alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan and wax, according to any one of (1) to (3) the method of.
  • a reagent for measuring adenosine triphosphate comprising a microcapsule encapsulating adenosine triphosphate degrading enzyme.
  • An adenosine triphosphate measurement kit comprising the adenosine triphosphate measurement reagent according to (7).
  • ATP derived from a raw material can be removed with a simple operation in a short time, and contamination of microorganisms can be determined with high sensitivity and accuracy.
  • This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2012-270933, which is the basis of the priority of the present application.
  • the present invention uses a microcapsule encapsulating adenosine triphosphate degrading enzyme to decompose free adenosine triphosphate in a sample, and then extracts adenosine triphosphate from cells present in the sample not containing the microcapsule. And providing a method for measuring adenosine triphosphate derived from a cell, comprising measuring the extracted adenosine triphosphate.
  • adenosine triphosphate (ATP) degrading enzyme examples include, but are not limited to, apyrase, acid phosphatase, alkaline phosphatase, hexokinase, nuclease P, deaminase and the like.
  • Microcapsules are inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
  • inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
  • the microcapsule film only needs to contain ATP-degrading enzyme and have semi-permeability to allow ATP to pass through.
  • ions such as ATP, ADP, AMP, pyrophosphate and the like having ions with a molecular weight of 1000 or less
  • a semipermeable membrane that permeates and does not permeate ATP-degrading enzyme is suitable.
  • the average particle size (d 50 ) of the microcapsules is suitably 10 ⁇ m to 1 mm, preferably 50 ⁇ m to 500 ⁇ m, more preferably 100 ⁇ m to 300 ⁇ m.
  • the value of standard deviation (particle size distribution), which is an index representing the particle size distribution, is suitably 1% to 300%, preferably 1% to 100%, more preferably 1% to 30%.
  • the average particle size and the particle size distribution can be determined by actually measuring the diameter of the capsule with a microscope or the like under a microscope.
  • the specific gravity of the microcapsules is suitably 1.0 or more, preferably 1.0 to 3.0, more preferably 1.0 to 2.0. Specific gravity can be measured according to the pipette method (JIS Z8820-2: 2004).
  • the content of ATP-degrading enzyme in one capsule is preferably 0.1 ng to 1 mg, preferably 1 ng to 500 ⁇ g, more preferably 1 ng to 1 ⁇ g.
  • the amount of ATP-degrading enzyme to be encapsulated in the capsule can be adjusted by the amount of enzyme introduced into the solution before capsule formation.
  • the microcapsule film may contain materials such as alginic acid, methoxy pectin, cellulose sulfate, gelatin, chitosan, carrageenan, and wax.
  • Samples include tea-based beverages, fruit liquor, juice (fruit juice beverages, plant-based beverages), cider, cola, carbonated water, syrup, coffee beverages (including milk), tea beverages (including milk), and nourishing beverages , Mineral water, beer, sake refined sake, refined sake sake, grilled sake, synthetic refined sake, whiskey, miso (including straightening), gin, vodka, brandy, condiment liquor, liqueur, rum, old liquor, plum wine, marine products
  • processed foods such as processed meat products, processed milk products, processed vegetable products, processed fruit products, oil and fat foods, taste foods, seasonings, confectionery, frozen foods, retort foods, canned foods, bottled foods, and instant foods
  • processed foods such as processed meat products, processed milk products, processed vegetable products, processed fruit products, oil and fat foods, taste foods, seasonings, confectionery, frozen foods, retort foods, canned foods, bottled foods, and instant foods
  • the reaction is preferably carried out at a temperature of 15 to 45 ° C. under a pH of 4.0 to 10.0 for 1 to 60 minutes.
  • the reaction between ATP and ATP-degrading enzyme is preferably carried out in a reaction system in which the concentration of ATP-degrading enzyme is 1 ng / ml to 100 mlmg / ml.
  • the amount of ATP present in the reaction system may be an amount that can be completely decomposed by the ATP-degrading enzyme present in the reaction system.
  • an appropriate diluent for example, Tris buffer, HEPES buffer
  • Solution MES buffer solution, TES buffer solution, pure water, etc.
  • a suitable amount of the diluted solution may be reacted with ATP-degrading enzyme.
  • the sample may be in a state that does not include microcapsules.
  • the sample not containing microcapsules include a sample obtained by separating the microcapsule by an operation such as filtration and centrifugation, and a supernatant obtained by precipitating the microcapsule in the sample by natural precipitation or coagulation precipitation.
  • the present invention is not limited to this. Or you may isolate
  • a charged bead can be obtained by immersing the completed alginate beads (microcapsules) in a solution containing an amino group or a carboxyl group.
  • the solution containing an amino group include a solution containing a substance such as methylamine, piperidine, spermine, spermidine, aniline, pyridine, triethanolamine and the like.
  • solutions containing carboxyl groups include unsaturated carboxylic acids (linoleic acid, oleic acid, etc.), hydroxy acids (malic acid, citric acid, etc.), aromatic carboxylic acids (phthalic acid, benzoic acid, etc.), dicarboxylic acids (malonic acid) And a solution containing a substance such as succinic acid).
  • the microcapsule and the sample can be separated by collecting the magnetic material in the microcapsule and collecting it by magnetic force.
  • the magnetic material include iron oxide, cobalt, ferrite, chromium oxide, and the like, and these may be enclosed inside a bead (microcapsule). That is, a magnetic substance can be enclosed inside a bead by dropping together with a substance that becomes an enzyme, a dye (colorant), and a film.
  • a commercially available ATP extraction reagent may be used.
  • ATP extracted from cells can be measured by a luciferin / luciferase reaction, but is not limited to this method.
  • Detection of ATP by the luciferin / luciferase reaction can be performed by emitting light using a commercially available luciferin / luciferase luminescence reagent (LL luminescence reagent) and measuring the luminescence with a luminometer.
  • the luciferin / luciferase reaction is a reaction that depends on the amount of ATP. Luciferin and ATP react to form adenylate luciferin, and this adenylate luciferin and oxygen are decomposed by oxidative decarboxylation in the presence of the luciferase enzyme, and a part of the energy obtained in the process of this reaction is luminescence. appear.
  • ATP By quantifying this luminescence, ATP can be quantified.
  • concentration of luciferase in the luminescent reaction system luciferin / luciferase 0.1 [mu] g / mL ⁇ 100 [mu] g / mL are suitable, preferably from 1 ⁇ g / mL ⁇ 20 ⁇ g / mL.
  • Luciferase is a firefly luciferin derived from a beetle, that is, a multi-heterocyclic organic acid D-( ⁇ )-2- (6′hydroxy-2′-benzothiazolyl) - ⁇ 2-thiazoline-4-carboxylic acid (hereinafter specifically described) Unless otherwise indicated, it is expressed as “luciferin”). It is an enzyme that catalyzes oxidation and emits photons. It is an enzyme that emits photons. Contains all enzymes. This includes enzymes in which the stability and luminescence properties of the enzyme protein itself have been artificially altered by recombinant DNA technology or mutation technology.
  • the concentration of luciferin is suitably 0.001 mM to 100 mM, preferably 0.01 mM to 10 mM.
  • Luciferin is a beetle-derived firefly luciferin (Coleoptera luciferin), that is, a multi-heterocyclic organic acid D-( ⁇ )-2- (6′hydroxy-2′-benzothiazolyl) - ⁇ 2-thiazoline-4-carboxylic acid) Often this includes those extracted and purified directly from beetles and those that are chemically synthesized.
  • a luminescent substrate which is a derivative of beetle luciferin and has luminescence activity after digestion with an enzyme.
  • Such beetle luciferin derivatives include 4-methyl-D-luciferin, D-luciferyl-L-methionine, 6-O-galactopyranosyl-luciferin, DEVD-luciferin, luciferin-6'methyl ester, luciferin 6 Examples include '-chloroethyl ester, 6'-deoxyluciferin, and luciferin 6'benzyl ester.
  • Luciferin and its derivatives may be in the form of a salt. Examples of the salt include potassium salt and sodium salt.
  • quantification of cell-derived ATP quantification of cells (quantity of living cells, if known cells can be quantified with high sensitivity and accuracy, and unknown cells can also be detected), etc.
  • the cell may be any cell such as a microbial cell, an animal cell, or a plant cell.
  • the method of the present invention can be used for detection and quantification of microorganisms required for food hygiene management and the like, and inspection of the degree of cleaning. For example, by using the luciferin / luciferase reaction, 10 -16 moles of ATP can be detected, and high-sensitivity detection such as about 10 3 in bacteria and several tens in yeast and fungi is possible. Furthermore, it is possible to quickly measure that the time required from the ATP removal to the light emission amount measurement / judgment is within one hour.
  • the present invention also provides a microcapsule that encapsulates ATP-degrading enzyme.
  • Microcapsules are inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
  • inorganic pigments such as titanium dioxide, zinc white (zinc oxide), iron oxide, chromium oxide, iron black, cobalt blue, alumina white, iron oxide yellow, viridian, zinc sulfide, lithopone, cadmium yellow, vermilion, cadmium.
  • Red, yellow lead, molybdate orange, zinc chromate, strontium chromate, agate white carbon, clay, talc, ultramarine, precipitated barium sulfate, barite powder, calcium carbonate, lead white ferrocyanide (bituminous), phosphate (manganese violet) , Carbon (carbon black), organic pigments, rhodamine lake, methyl violet lake, quinoline yellow lake, malachite green lake, alizarin lake, carmine 6B, lakeette C, disazo yellow, lakelet 4R, chromophthalo yellow 3G, black Phthascarlet RN, Nickel Azo Yellow, Permanent Orange HL, Phthalocyanine Blue, Phthalocyanine Green, Flavanthrone Yellow, Thioindigo Bordeaux, Perinone Red, Dioxadone Violet, Quinacridone Red, Naphthol Yellow S, Pigrant Green B, Lumogen Yellow, Signal Red, Bovine serum albumin, gelatin, trehalose or the like may
  • microcapsule The material and physical properties of the microcapsule have been described above.
  • Microcapsules containing ATP-degrading enzyme can be any known method such as phase separation, submerged drying, melt dispersion cooling, spray drying, pan coating, interfacial polymerization, in-situ polymerization, submerged curing coating. You may manufacture by the method.
  • ATP can be decomposed and removed using microcapsules containing ATP-degrading enzyme.
  • ATP derived from the ingredients in the beverage is removed, and then ATP derived from cells such as microorganisms present in the sample is extracted and measured to measure ATP derived from cells in the sample. be able to. Therefore, the present invention provides an ATP measurement reagent comprising a microcapsule that encapsulates an ATP-degrading enzyme.
  • the ATP-degrading enzyme-encapsulating capsule may be included in any form such as a lyophilized product or a suspension (capsule suspension).
  • a suspension the capsule may be dispersed in an appropriate solvent (for example, Tris buffer, HEPES buffer, MES buffer, TES buffer, pure water, etc.).
  • the ATP measurement reagent of the present invention may further contain a pH adjuster, an antioxidant, a preservative, an antifungal agent and the like.
  • the ATP measurement reagent of the present invention can be used for ATP measurement, for example, measurement of cell-derived ATP present in a sample (described above). Therefore, the present invention provides an ATP measurement kit containing the above ATP measurement reagent.
  • the ATP measurement kit of the present invention further converts a luciferin / luciferase luminescence reagent, a luminescence reagent solution, an ATP extractant, an ATP standard reagent, a calibration curve that associates the luminescence amount with the ATP amount, an instruction manual, and an ATP amount.
  • Software etc. may be included.
  • Example 1 Method for preparing ATP-erased beads 1.1 ml of apyrase potato-derived Grade III (Sigma Aldridge), 2 g of sodium alginate (Wako Pure Chemical Industries), and 200 ⁇ l of pigment ink (aqueous pigment ink blue for graph pen ink pen) were dissolved in 200 ml of pure water. 2. The lysate was set in a microbead manufacturing apparatus (Nippon Büch Encapsulator B-390). 3. A droplet having a diameter of about 150 ⁇ m was generated on the microbead manufacturing apparatus. 4).
  • the droplet was dropped into a 2% calcium chloride solution. 5.
  • the microbeads containing apyrase and pigment ink whose surface was gelled were filtered using a 0.2 ⁇ m pore size filter (Nalgen filter unit). 6).
  • the filtrate (microcapsule) on the filter was washed with 10 mM HEPES buffer. 7).
  • the microcapsules were resuspended in an appropriate amount of 10 mM HEPES buffer. 8).
  • 10 ⁇ l of a 10 ⁇ 7 mole / l ATP standard solution Toyo Benet
  • the physical property values of the microbeads are as follows. ⁇ Average particle size: 155.2 um ⁇ Particle size distribution: 16.4% (The microscopic observation (magnification 40) measured the capsule diameter to determine the average particle size and particle size distribution. 100 particles were measured in one observation, and this was repeated three times. The average particle size was the average value of 300 particles. The particle size distribution is indicated by its standard deviation.) Specific gravity: 1.6 (Measured according to the pipette method (JIS Z8820-2: 2004).) -Apyrase content in one capsule: 11.6 ng (The amount of liquid contained inside a capsule having a diameter of 150 um is about 1.8 ul, so it is calculated that 11.6 ng of enzyme is contained in the conditions of this experiment.
  • the amount of enzyme contained in the capsule can be adjusted by the amount of enzyme added to the solution before capsule formation.In this experiment, 1.1 mg of apyrase was mixed with 200 ml of solution. When mixed, the amount of encapsulated enzyme is estimated to be 110 ng.>
  • ATP-erased beads Necessity of washing operation 6. (Method for preparing ATP-erased beads) Microcapsule suspension (indicated as “microcapsule-containing liquid” in Table 1) and I.8. 10 ⁇ l of a 10 ⁇ 7 mole / l ATP standard solution (Toyo Benet Corporation) was mixed with 100 ⁇ l of each of the supernatants, and reacted for 30 minutes. Then, after mixing 100 microliters of LL luminescent reagent (Fujiro ATP luminescent reagent LL100-1 Toyo Beenet) and making it react for 30 seconds, the luminescence was measured with the luminometer (LB9507 Berthold). The integration time for measurement was 10 seconds.
  • LL luminescent reagent Flujiro ATP luminescent reagent LL100-1 Toyo Beenet
  • microcapsule-containing liquid in Table 1 contains apyrase in the capsule, it has ATP degradation activity.
  • Table 2 shows the measurement results of III of ATP degradation in green tea by bead treatment III (specimen: green tea).
  • ATP contained in green tea was attempted to be decomposed by the microcapsule method and the conventional method (method according to the procedure of the Mycosis ATP measurement kit (Toyo B-Net)).
  • the microcapsule method 200 ⁇ l of microcapsule solution was added to 200 ⁇ l of green tea, and the mixture was allowed to stand at room temperature for the treatment time shown in Table 2. After the elapse of time, 100 ⁇ l of the sample solution was fractionated, and an equal amount of 100 ⁇ l of ATP luminescence reagent (Toyo Benet) was added to measure the amount of luminescence.
  • Table 3 shows the results of detecting the presence of 5 ⁇ 10 5 E. coli cells in 1 ml of the sample (green tea) by detecting the change in the amount of ATP.
  • microcapsule microbe contamination test 200 ⁇ l of green tea beverage mixed with E. coli was dispensed into a tube, mixed with 200 ⁇ l of 200 ⁇ l of apyrase-containing microcapsule suspension, and allowed to stand at room temperature for 30 minutes. 100 ⁇ l of the supernatant was collected taking care not to contaminate the microcapsules. Next, an equal amount of 100 ⁇ l of ATP extraction reagent (Fujiro Shiro ATP extraction reagent LL100-2, Toyo Benet) was added to the collected solution, and ATP extraction from microorganisms was performed for 10 seconds.
  • ATP extraction reagent Frujiro Shiro ATP extraction reagent LL100-2, Toyo Benet
  • the conventional method followed the procedure of the Mycosis ATP measurement kit. That is, 200 ⁇ l of green tea beverage mixed with Escherichia coli was collected in a tube, mixed with 200 ⁇ l of ATP removal reagent (Fujiro ATP removal reagent LL100-3), and allowed to stand at room temperature for 30 minutes. Collect 100 ⁇ l of the supernatant and add 100 ⁇ l of an equal amount of ATP extraction reagent (Fujiro ATP extraction reagent LL100-2, Toyo Benet) to the solution, and perform ATP extraction from microorganisms for 10 seconds. It was.
  • the present invention can be used for measuring microbial contamination in foods such as beverages.

Abstract

L'invention concerne une technique permettant de retirer l'ATP dérivée de cellules d'une matière brute par une procédure simple, en peu de temps, et d'évaluer avec précision la contamination microbienne avec une haute sensibilité, pour un contrôle de qualité efficace et une inspection d'échantillonnage de produits alimentaires, tels que des boissons, et autres produits, contenant de grandes quantités d'ATP. Le procédé de mesure de l'adénosine triphosphate dérivée de cellules consiste à procéder à la dégradation de l'adénosine triphosphate libre dans un échantillon au moyen de microcapsules qui encapsulent une enzyme dégradant l'adénosine triphosphate, puis extraire l'adénosine triphosphate de cellules présentes dans un échantillon sans microcapsule, et mesurer l'adénosine triphosphate extraite.
PCT/JP2013/082972 2012-12-12 2013-12-09 Procédé de mesure de l'adénosine triphosphate dérivée de cellules WO2014092050A1 (fr)

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JP2012270933A JP5405650B1 (ja) 2012-12-12 2012-12-12 細胞由来のアデノシン三リン酸を測定する方法
JP2012-270933 2012-12-12

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6156076A (ja) * 1984-08-25 1986-03-20 Agency Of Ind Science & Technol 固定化酵素及びその製造方法
JP2003210197A (ja) * 2002-01-22 2003-07-29 Unitika Ltd 試料溶液中のａｔｐを除去する方法
JP2009178091A (ja) * 2008-01-31 2009-08-13 National Institute Of Advanced Industrial & Technology 酵素を内包した無機マイクロカプセル、その製造方法及び使用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1156393A (ja) * 1997-08-21 1999-03-02 Kikkoman Corp Ａｔｐの測定法およびａｔｐ測定用試薬キット

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6156076A (ja) * 1984-08-25 1986-03-20 Agency Of Ind Science & Technol 固定化酵素及びその製造方法
JP2003210197A (ja) * 2002-01-22 2003-07-29 Unitika Ltd 試料溶液中のａｔｐを除去する方法
JP2009178091A (ja) * 2008-01-31 2009-08-13 National Institute Of Advanced Industrial & Technology 酵素を内包した無機マイクロカプセル、その製造方法及び使用

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