Method for quantifying target substance

A substance and substrate technology, applied in the field of quantitative pyrophosphate, which can solve the problem that ATP cannot respond to the regeneration of pyrophosphate

Inactive Publication Date: 2014-11-05
TOYAMA PREFECTURE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, by this method, ATP cannot be regenerated in response to pyrophosphate depletion

Method used

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  • Method for quantifying target substance
  • Method for quantifying target substance
  • Method for quantifying target substance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0430] 1. Example of Enzyme Preparation

[0431] 1-1. Construction of PPDK expression plasmid

[0432] Propionibacterium freudennerii subsp. shermanii PPDK (PfPPDK) derived from NBRC 12426 strain and plasmid attached to PPDK (TtPPDK) derived from Thermoproteus NBRC 100435 strain. Genomic DNA was prepared from cells of each strain.

[0433] The PfPPDK gene was amplified by PCR using the genomic DNA obtained above as a template and primers (SEQ ID NO: 2 and 3) designed based on the PPDK gene sequence (SEQ ID NO: 1) obtained from the database. The amplified product was inserted into pET-28a to obtain a plasmid for expressing PfPPDK. The TtPPDK gene was PCR amplified by using the genomic DNA obtained above as a template and primers (SEQ ID NO: 5 and 6) designed based on the PPDK gene sequence (SEQ ID NO: 4) obtained from the database. The amplified product was inserted into pET-28a to obtain a plasmid for expressing TfPPDK.

[0434] Each expression plasmid was sequenced...

Embodiment 2

[0447] 2. Example of Quantification of Pyrophosphate

[0448] 2-1. Example of reaction conditions for quantification of pyrophosphoric acid (1)

[0449] A test sample containing pyrophosphoric acid and a reaction mixture for quantifying pyrophosphoric acid having the following components were mixed. The mixture was maintained at 30°C to allow the reaction to progress and the decrease in absorbance at 340 nm was measured. The volume of the mixture is 1 ml or 200 μl. Absorbance measurements were performed with cuvettes and absorptiometers with an optical path length of 1 cm (for the former volume) or with microplates and a microplate reader (with a variable optical path length) (for the latter volume). In the graphs shown in the accompanying drawings, the absorbance measured with a cuvette with an optical path length of 1 cm is represented by Abs, and the absorbance measured with a microplate is represented by AU.

[0450] Reaction mixture for quantification of pyrophosph...

Embodiment 3

[0475] 3. Example of quantification of methionine

[0476] 3-1. Examples of reaction conditions for quantifying methionine

[0477] A test sample containing methionine and a reaction mixture for quantifying methionine having the following components were mixed. A 200 μl volume of the mixture was maintained at 30°C to allow the reaction to progress and the decrease in absorbance at 340 nm was measured with a microplate reader.

[0478] Reaction mixture for quantification of methionine: 20 mM imidazole-HCl (pH 7.0), 5 mM MgCl 2 , 1 mM ATP, 0.5 mM PEP, 0.4 mM AMP, 0.25 mM NADH, 0.1 U / ml PfPPDK, 0.5 U / ml lactate dehydrogenase and 0.2 U / ml AdoMetS (the concentration is the final concentration after mixing with the test sample).

[0479] 3-2. Example of preparation of calibration curve for quantification of methionine

[0480] As test samples, a standard methionine solution and a standard pyrophosphate solution were used which were mixed with the reaction mixture to give a ...

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Abstract

Provided is a method for quantifying a target substance typified by amino acid. This method includes: a step in which an enzyme capable of converting the target substance and also of generating a pyrophosphate using an adenosine tri-phosphate (ATP) as a base material therefor is caused to act on the target substance, and pyrophosphate is generated; a step in which a pyruvate phosphate dikinase (PPDK) is caused to act on the generated pyrophosphate, in the presence of adenosine monophosphate (AMP) and phosphoenolpyruvic acid (PEP), and ATP, phosphoric acid, and pyruvic acid is generated; and a step in which the generated pyruvic acid is quantified. The amount of target substance is then determined on the basis of the obtained pyruvic acid amount. As a result of this invention, amino acid in a sample derived from an organism including a large quantity of a variety of impurities such as inorganic phosphoric acid and urea can be readily and quickly quantified without being affected by the impurities.

Description

[0001] Cross references to related applications [0002] This application claims a convention preference based on Japanese Patent Application No. 2012-026534 filed at the Japan Patent Office on February 9, 2012 and Japanese Patent Application No. 2012-069625 filed at the Japan Patent Office on March 26, 2012 right. The entire disclosure content of these applications is incorporated into the disclosure content of the present application. technical field [0003] The present invention relates to a method for quantifying pyrophosphate comprising allowing pyruvate to react with pyruvate pyrophosphate dikinase (PPDK) and quantifying pyruvate as a product, which is suitable for a reaction system comprising adenosine triphosphate (ATP) and inorganic phosphate. The invention also relates to a method for the quantification of methionine, citrulline or arginine, comprising allowing the synthesis of methionine with adenosylmethionine synthetase (AdoMetS), citrulline with argininosuccina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/25C12Q1/26C12Q1/32C12N15/09
CPCG01N33/6815G01N33/6812C12Q1/48C12Q1/32C12Q1/25C12Q1/485C12Y207/09001C12Y601/01002C12Q1/34
Inventor 浅野泰久龟谷将史
Owner TOYAMA PREFECTURE
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