WO2006061945A1 - Méthode de dosage quantitatif de l’acide propionique - Google Patents

Méthode de dosage quantitatif de l’acide propionique Download PDF

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Publication number
WO2006061945A1
WO2006061945A1 PCT/JP2005/018750 JP2005018750W WO2006061945A1 WO 2006061945 A1 WO2006061945 A1 WO 2006061945A1 JP 2005018750 W JP2005018750 W JP 2005018750W WO 2006061945 A1 WO2006061945 A1 WO 2006061945A1
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propionic acid
coa
propionyl
quantifying
oxidase
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PCT/JP2005/018750
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English (en)
Japanese (ja)
Inventor
Eranna Rajashekhara
Kazuya Watanabe
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Marine Biotechnology Institute Co., Ltd.
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Publication of WO2006061945A1 publication Critical patent/WO2006061945A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3504—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing gases, e.g. multi-gas analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • the present invention relates to a method for specifically and highly sensitively quantifying propionic acid, and more specifically, by combining propionyl CoA transferase and isyl CoA oxidase, propionic acid in blood, fermentation broth and the like can be simplified. And specific and sensitive method.
  • Biosensors using enzymes or the like are well known as means for easily and sensitively detecting a specific substance.
  • Sode et al. Reported a biosensor for detecting long-chain fatty acids using acyl-CoA synthetase and acyl-Co A oxidase (Non-patent Document 1).
  • Non-special reference 1 Sode, K., Tamiya, ⁇ ⁇ , Karube, I. & Kameaa Y. Sensor for free fatty ac ids based on acyl coenzyme-A synthetase and acyl coenzyme-A oxidase. Anal. Chim Acta 220, 251-255. 1989.
  • the above biosensor uses force S using Cosyl CoA synthetase.
  • CoA must be added to the reaction system.
  • the sensitivity of the biosensor decreases due to the influence of this reaction.
  • the above-mentioned biosensor is capable of detecting only fatty acids with a high concentration (0.5 mM or more).
  • the present invention has been made under the above technical background, and an object thereof is to provide a means for quantitatively determining propionic acid with high sensitivity.
  • the present inventor has obtained a reaction system by using propionyl CoA transferase in place of the asil CoA synthetase used in the biosensor 1. Based on this finding, the inventors have found that propionic acid can be quantified without adding CoA to the present invention.
  • a method for quantifying propionic acid characterized by using propionyl CoA transferase and isacyl CoA oxidase.
  • a method for quantifying propionic acid comprising the following steps (1) to (3): (1) a step of allowing propionyl CoA transferase to act on propionic acid in a sample in the presence of acetyl CoA;
  • [6] The method for quantifying propionic acid according to any one of [1] to [5], which is propionyl CoA transferase S and propionyl CoA transferase specifically reacting with propionic acid.
  • Capillary CoA oxidase power The method for quantifying propionic acid according to any one of [1] to [7], wherein the method is an Arabidopsis derived facyl CoA oxidase or a variant thereof.
  • propionyl CoA transferase quantification kit according to [10], which is a propionyl CoA transferase derived from propionyl CoA transferase clostridium propionica or a variant thereof.
  • a propionic acid quantification apparatus comprising an immobilized propionyl CoA transferase and an immobilized acyl CoA oxidase.
  • propionic acid can be quantified easily on-site (patient's house, food factory or storage, waste disposal site, etc.) or online (for unmanned management of fermenters). For the purpose).
  • the method of the present invention can quantitate propionic acid with higher sensitivity than the conventional gas chromatography method, propionic acidemia and the like can be diagnosed at an early stage.
  • FIG. 1 is a conceptual diagram of a biosensor for quantifying propionic acid.
  • FIG. 2 shows the results of electrophoresis of purified PCT (A: molecular weight marker, B: purified PCT).
  • FIG. 3 is a diagram showing the results of electrophoresis of purified ACO (A: molecular weight marker, B: purified ACO).
  • FIG. 4 is a diagram showing a calibration curve for the determination of propionic acid by the amino aminopyrine method.
  • FIG. 5 is a diagram showing a calibration curve for the determination of propionic acid by the Abreck Red method.
  • FIG. 6 shows a calibration curve for propionic acid quantification by the oxygen electrode method.
  • FIG. 7 shows a calibration curve for propionic acid quantification by electrical detection method.
  • the method for quantifying propionic acid according to the present invention comprises propionyl CoA transferase (hereinafter referred to as “ PCTj and les) and Facil CoA oxidase (hereinafter referred to as “ACO”). Specifically, propionic acid is quantified using the following two enzyme reactions.
  • "quantification of propionic acid” means how much propionic acid is present in the sample. It also includes examining whether propionic acid is contained in the sample just by examining (ie, “detecting propionic acid”).
  • the method for causing the enzyme reaction is not particularly limited.
  • acetylyl CoA, PCT and ACO may be added to a sample containing propionic acid, and these may be mixed.
  • the sample containing propionic acid may be added.
  • Acetyl CoA may be added and contacted with a resin or the like to which PCT and ACO are fixed.
  • Samples include, for example, urine and blood of patients with or suspected of propionic acidemia, fermented liquid in a methane fermentation digester, soy sauce, juice, bread, etc. to which propionic acid has been added as a preservative.
  • the ability to cite foods, etc. are not particularly limited as long as they can quantitate propionic acid in combination with ACO. It is preferable to use one that reacts automatically.
  • “specifically reacts with propionic acid” means that it has almost no reactivity with substances other than propionic acid. For example, reactivity with butyric acid is reactive with propionic acid. Of 5.
  • PCT PCT derived from Clostridium propionicum. This enzyme reacts specifically with propionic acid (C3) to produce propionyl CoA when acetyl CoA is used as the donor of CoA. To do. Reactivity with butyric acid (C4) is only a few percent of propionic acid and does not react with valeric acid (C5). This enzyme can be obtained, for example, as follows.
  • Clostridium propionica (DSMZ 1682) force Genomic DNA can be extracted, and the enzyme gene can be cloned from this genomic DNA by PCR.
  • This enzyme gene is inserted into an expression vector for E. coli (such as pET28a plasmid) and expressed in E. coli cells transformed with the resulting plasmid. In that case, if it is expressed as a fusion protein with His-tag or the like, it can be easily purified by affinity chromatography on His-tag.
  • mutants can also be used.
  • mutant refers to an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of PCT derived from Clostridium propionica, and is specific to propionic acid. It refers to a protein that exhibits PCT activity.
  • the amino acid sequence of PCT derived from Clostridium propionica is known (GenBank Accession on No. CAB77207).
  • PCT that reacts specifically with propionic acid other than PCT derived from Clostridium propionica.
  • examples of such enzymes include Clostridium perfringens-derived PCT (GenBank Accession No. NP_561012).
  • ACO is not particularly limited as long as propionic acid can be quantified in combination with PCT, but it is preferable to use ACO that does not substantially react with acetyl CoA.
  • “substantially does not react with acetyl CoA” means that the reactivity with acetyl CoA is remarkably low compared with the reactivity of acetyleno CoA other than acetyl CoA, for example, with acetyl CoA.
  • the reactivity is 1% or less of the reactivity with propionyl CoA, it can be said that “substantially does not react with acetyl CoA”.
  • Examples of the ACO that does not substantially react with acetyl CoA include ACO derived from Arabidopsis.
  • This enzyme is a force that oxidizes propyleneyl CoA to otatanyl CoA to generate hydrogen peroxide and does not react with acetyl CoA.
  • This enzyme can be obtained, for example, as follows. Genomic DNA can be extracted from Arabidopsis thaliana, and the enzyme gene can be cloned from this genomic DNA by PCR. This Is inserted into an expression vector for E. coli (such as pET28a plasmid) and expressed in E. coli cells transformed with the resulting plasmid. At that time, if it is expressed as a fusion protein with His-tag or the like, it can be easily purified by an affinity matrix for His-tag.
  • mutants thereof can also be used.
  • mutants refers to an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence of ACO derived from Arabidopsis thaliana, and does not substantially react with acetyl CoA. A protein that exhibits ACO activity.
  • the amino acid sequence of ACO derived from Arabidopsis thaliana is known (GenBank Accession No. BAA82478).
  • ACO which does not substantially react with acetyl CoA other than ACO derived from Arabidopsis thaliana.
  • Examples of such an enzyme include ACO (GenBank Accession No. XP_414406) derived from chiabo.
  • the amount of acetyl CoA, PCT and ACO used is not particularly limited.
  • the concentration of acetyl CoA is 50 to 500 nmol / ml
  • the concentration of PCT is preferably 0.5 to 10 U / ml and the ACO concentration is 10 to 200 U / ml.
  • the enzyme reaction generates hydrogen peroxide according to the amount of propionic acid and consumes oxygen. Therefore, propionic acid can be quantified by quantifying produced hydrogen peroxide or consumed oxygen.
  • any method may be used, such as a method using a coloring substance or a fluorescent substance.
  • the coloring substance and the fluorescent substance include substances that develop color by being reduced with hydrogen peroxide by a peroxidase reaction, substances that emit fluorescence, and the like.
  • the coloring substance or the like may be one that emits color or fluorescence by a combination of a plurality of substances, which is not necessarily a single substance.
  • Specific examples of coloring materials and fluorescent materials include a combination of aminobenzoic acid and 4-aminoaminopyrine, Amplex red (registered trademark, 10-acetyl-3-7-dihydroxyphenoxazine), etc. Can be mentioned.
  • the determination of hydrogen peroxide may be performed using an electric current generated by hydrogen peroxide. Fermentation If a platinum electrode or the like is placed in the elemental reaction system, hydrogen peroxide generated as a result of the enzyme reaction reacts with the electrode and passes electrons to the electrode. Therefore, propionic acid can be quantified by measuring the amount of current generated in the electrode after the enzyme reaction.
  • FIG. 1 shows a conceptual diagram of a biosensor for propionic acid quantification using such an electrical quantification method.
  • the biosensor shown in Fig. 1 consists of an electrode 1, an enzyme column 2, a temperature controller 3, a Sampnore inlet 4, an air trap 5, a pump 6, a buffer tank 7, a waste tank 8, and a current measuring instrument 9.
  • the buffer solution is moved from the buffer solution tank 7 to the waste solution tank 8, so that the sample that has entered from the Sampnore inlet 4 reaches the electrode 1 after passing through the enzyme column 2.
  • Enzyme column 2 is fixed with PCT and AC ⁇ , and these enzymes act on propionic acid in the sample to generate hydrogen peroxide.
  • the generated hydrogen peroxide reacts with the electrode to generate an electric current.
  • propionic acid can be quantified.
  • a biosensor not only this type but also a portable type in which an enzyme is immobilized on a chip can be produced.
  • the method for quantifying the consumed oxygen is not particularly limited.
  • the consumed oxygen can be quantified by measuring the oxygen amount before and after the enzyme reaction with an oxygen electrode.
  • Ratatopyranoside (IPTG) was added to induce the expression of the PCT gene and further cultured overnight. After cultivation, the cells are collected by centrifugation, and 20 ml of buffer A (50 mM sodium phosphate, 300 It was suspended in mM NaCl, 10 mM imidazole, H 7.0). This was treated with a French press (12 MPa) to disrupt the bacteria, and the supernatant of centrifugation at 8000 rpm for 20 minutes was collected. This supernatant was mixed with 0.5 ml of NiNTA resin (Qiagen) and left on ice for 30 minutes. This resin was packed in a polystyrene column and washed with a sufficient amount of buffer A to remove unbound protein.
  • buffer A 50 mM sodium phosphate, 300 It was suspended in mM NaCl, 10 mM imidazole, H 7.0. This was treated with a French press (12 MPa) to disrupt the bacteria, and the supernatant of centrifug
  • the His-tag PCT bound to the resin was eluted with buffer B (50 mM sodium phosphate, 300 mM NaCl, 2000 mM imidazole, pH 7.0). This enzyme solution was sufficiently dialyzed against buffer A to remove imidazole, and stored at 4 ° C. The purity of the PCT enzyme was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 2).
  • ACO gene fragments were recovered by PCR using the following primers from plasmid vector DZL containing Arabidopsis thai iana ACO cDNA provided by Dr. Mano of the National Institute of Basic Biology. Restrictions in the primers The enzyme site was cleaved with restriction enzymes M and 2 ⁇ 1.
  • the reaction solution (100 ⁇ 1, 50 mM sodium phosphate buffer pH 7.0) is PCT (7.75 ⁇ g), AC ⁇ (5.6 ⁇ mg), 100 ⁇ acetyl CoA, 50 ⁇ M FAD, 0.04 U horseradish rust.
  • HRP peroxidase
  • 13 mM ⁇ -aminobenzoic acid 1 mM 4-aminoantipyrine.
  • the reaction solution (100 ⁇ 1, 50 mM sodium phosphate buffer, pH 7.0) is composed of PCT (7.75 ⁇ g), AC ⁇ (5.6 ⁇ g), 10 ⁇ acetyl CoA, 50 ⁇ M FAD, 0.04 U HRP, 50 Includes ⁇ M Amplex Red (Molecular probe).
  • the reaction formula for coloring in this method is shown below. The reaction was carried out at 25 ° C for 10 minutes, and the absorbance at 550 mm was measured with a spectrophotometer. The results are shown in Fig. 5. Thus, it was shown that propionic acid at a concentration of 1 / iM or more can be quantified by this method.
  • the reaction solution (1500 ⁇ 1, 50 mM sodium phosphate buffer pH 7.0) is PCT (116 ⁇ g), AC Contains O (84 ⁇ g), 100 ⁇ M acetylyl CoA, 50 ⁇ FAD.
  • This solution was placed in a constant temperature cuvette (25 ° C.) of an oxygen electrode (Oxygraph, Gilson), and when the electrode was stabilized, propionic acid was added to measure oxygen consumption.
  • Figure 6 shows the detection range. As a result, it became clear that quantification was possible at a concentration of 50 ⁇ 200 to 200 ⁇ .
  • PCT and ACO are fixed to enzyme-fixing resin ( ⁇ -aminopropyltriethoxysilane) according to a conventional method, packed in a column for the nanosensor unit BF-5 (Oji Scientific Instruments), and as shown in Fig. 1. It was set in the device so as to be configured.
  • the buffer was 50 mM sodium phosphate buffer pH 7.0 containing 50 ⁇ FAD, and the reaction temperature was 30 ° C. After mixing 100 ⁇ M acetyl CoA into the sample, 30 ⁇ L was injected into the device, and the amount of current generated was measured. As a result, as shown in Fig. 7, it was shown that there was quantitativeity in the range of 20 forces 200 ⁇ .
  • sludge from a methane fermentation digester for livestock manure treatment as a seed and synthetic substrates (sucrose, acetic acid, propionic acid, peptone in a ratio of 4.5: 2.25: 2.25: 1.
  • substrates sucrose, acetic acid, propionic acid, peptone in a ratio of 4.5: 2.25: 2.25: 1.
  • Example 3 to Example 6 The method shown in Example 3 to Example 6 and the method using a gas chromatograph (Imachi H, Sekiguchi Y, Kamagata Y, Onasni A, riarada H. and ultivation and in situ detection of a Quantification was performed by the method described in Appl Environ Microbiol. 66, 3608-3615. 2000.) thermophilic bacteria capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. The results are shown in Table 2.
  • Propionic acid was added to the rabbit and goat sera to a final concentration of 0.5 mM, and the measurement was performed by the Amplex Red method of Example 4. At this time, 10 ⁇ L of the propionate-containing serum diluted 10-fold with pure water was added to the reaction solution (100 zL). The results are shown in Table 3. From these results, it was shown that the method for quantifying propionic acid of the present invention can also be used for the quantification of propionic acid in serum.
  • Soy sauce (Kikkoman) was diluted 500 times with pure water and treated with U / ml ascorbic acid oxidizing enzyme (Wako Pure Chemical Industries) for 10 minutes at room temperature. 10 ⁇ L of this sample was added to the reaction solution (100 ⁇ L), and the propionic acid concentration was measured by the Amplex Red method of Example 4. At this time, the acetyl CoA concentration was changed to 250 ⁇ .
  • Apple juice (mini-maid) was diluted 10-fold with pure water, 10 x L of this sample was added to the reaction solution (100 z L), and the propionic acid concentration was measured by the Amplex Red method of Example 4. . At this time, the acetyl CoA concentration was changed to 250 ⁇ .
  • Table 4 shows the measurement results of the Amplex Red method. For comparison, this table also shows the results of measuring the same sample by the gas-mouth method.
  • the soy sauce sample is known to contain propionic acid with a force of about 0.3% or less, which could not be quantified by the gas chromatographic method, and the detection result (32 mM is equivalent to 0.24%) is considered reasonable. It was. Thus, it was demonstrated that the method for quantifying propionic acid of the present invention can be used for the quantification of propionic acid in food samples.

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Abstract

La présente invention a pour objet une méthode de dosage quantitatif de l’acide propionique qui comprend une étape (1) de réaction d’une propionyl-CoA transférase avec l'acide propionique contenu dans un échantillon en présence d'acétyl-CoA, une étape (2) de réaction d'une acyl-CoA oxydase sur la propionyl-CoA formée par l’action de la propionyl-CoA transférase, et une étape (3) de détermination de la quantité de peroxyde d’hydrogène formée par action de l'acyl-CoA oxydase ou de la quantité d’oxygène consommée par l’action de l’acyl-CoA oxydase. Ladite méthode permet un dosage quantitatif simple de l’acide propionique, avec une sensibilité élevée.
PCT/JP2005/018750 2004-12-08 2005-10-12 Méthode de dosage quantitatif de l’acide propionique WO2006061945A1 (fr)

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JP2004354784A JP2006158297A (ja) 2004-12-08 2004-12-08 プロピオン酸の定量方法
JP2004-354784 2004-12-08

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011108670A1 (fr) * 2010-03-04 2011-09-09 富山県 Procédé d'analyse de la taurine
WO2012147822A1 (fr) * 2011-04-25 2012-11-01 国立大学法人東京農工大学 Procédé de détermination de la détérioration de l'huile et dispositif l'utilisant

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JPH1052299A (ja) * 1996-08-12 1998-02-24 Internatl Reagents Corp 試薬キット及び試薬キットの使用方法
JP2000316600A (ja) * 1999-05-14 2000-11-21 Toyobo Co Ltd 遊離脂肪酸の定量方法及び遊離脂肪酸定量用試薬

Patent Citations (2)

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JPH1052299A (ja) * 1996-08-12 1998-02-24 Internatl Reagents Corp 試薬キット及び試薬キットの使用方法
JP2000316600A (ja) * 1999-05-14 2000-11-21 Toyobo Co Ltd 遊離脂肪酸の定量方法及び遊離脂肪酸定量用試薬

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HAYASHI H. ET AL: "A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes", J. BIOL. CHEM., vol. 274, no. 18, 30 April 1999 (1999-04-30), pages 12715 - 12721, XP002157736 *
MARUO F. ET AL: "Koso Handbook", 10 October 1995, ASAKURA SHOTEN, pages: 413, XP002996878 *
SELMER T. ET AL: "Propionate CoA-transferase from Clostridium propionicum. Cloning of gene and identification of glutamate 324 at the active site", EUR.J. BIOCHEM., vol. 269, no. 1, January 2002 (2002-01-01), pages 372 - 380, XP002996879 *
SODE K. ET AL: "Sensor for free fatty acids based on acyl coenzyme-A synthetase and acyl coenzyme-A oxidase", ANALYTICA CHIMICA ACTA, vol. 220, 1989, pages 251 - 255, XP002996877 *
WATANABE K. ET AL: "Acyl-Coa Sanka Koso o Mochiita Propionic Acid Koso Sensor no Kaihatsu", THE CHEMICAL SOCIETY OF JAPAN KOEN YOSHISHU, vol. 85, no. 2, 11 March 2005 (2005-03-11), pages 1229, XP002996881 *
WATANABE K. ET AL: "Propionic Acid Tokuiteki Koso Sensor no Kaihatsu", ANNUAL MEETING OF JSBBA KOEN YOSHISHU, vol. 2005, 5 March 2005 (2005-03-05), pages 170, XP002996880 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011108670A1 (fr) * 2010-03-04 2011-09-09 富山県 Procédé d'analyse de la taurine
WO2012147822A1 (fr) * 2011-04-25 2012-11-01 国立大学法人東京農工大学 Procédé de détermination de la détérioration de l'huile et dispositif l'utilisant
JP6125423B2 (ja) * 2011-04-25 2017-05-10 国立大学法人東京農工大学 油劣化の判定方法およびこれを用いる装置

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