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A method for detecting the content of gamma-aminobutyric acid with two enzymes

A technology for GABA and enzyme detection, applied in the measurement of color/spectral characteristics, etc., can solve problems such as high detection data and interference, and achieve accurate detection methods, accurate diagnosis and treatment, great practical value and guiding significance Effect

Active Publication Date: 2018-09-25
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that for crude GABA products extracted from animal and plant tissues and human blood, the measurement is prone to the interference of glutamate decarboxylase activity and cytochrome in the body, resulting in high detection data

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) Instrument

[0034] Refrigerated high-speed centrifuge; vacuum dryer; ultraviolet-visible spectrophotometer.

[0035] (2) Reagents

[0036] pH 7.2 Potassium Phosphate Buffer (0.1M); pH 8.6 Tris Buffer (0.2M); NADP + (4mM); α-ketoglutarate (20mM); Glycerol; β-mercaptoethanol; GABA transaminase-succinic semialdehyde dehydrogenase dual enzyme solution (2U / ml, with 0.1M pH7.2 potassium phosphate Dissolved in buffer containing 12.5% ​​glycerol and 5 mM β-mercaptoethanol); methanol; lanthanum chloride (80 mM); KOH (1M).

[0037] (3) Specific steps

[0038] After the fresh leaf materials of normal tomato plants are washed and blotted dry, quickly put them into liquid nitrogen and grind to obtain a powdery solid, take 0.4g of the powdery solid and transfer it to an Eppendorf tube, add 1.6ml of methanol to inactivate glutamate decarboxylase ; After 10 minutes, dry in vacuum for 2h. Then add 4.0ml of lanthanum chloride with a concentration of 80mM to remove pigment inter...

Embodiment 2

[0040] (1) Instrument

[0041] Refrigerated high-speed centrifuge; vacuum dryer; ultraviolet-visible spectrophotometer.

[0042] (2) Reagents

[0043] pH 7.2 Potassium Phosphate Buffer (0.1M); pH 8.6 Tris Buffer (0.2M); NADP + (6mM); α-ketoglutarate (40mM); glycerol; β-mercaptoethanol; GABA transaminase-succinic semialdehyde dehydrogenase dual enzyme solution (4U / ml, buffered with 0.1M pH7.2 potassium phosphate solution, containing 12.5% ​​glycerol and 5mM β-mercaptoethanol); n-butanol; lanthanum bromide (0.1M); NaOH (1M).

[0044] (3) Specific steps

[0045]After the leaves of rice seedlings subjected to anaerobic stress were washed and blotted dry, they were quickly placed in liquid nitrogen and ground to obtain a powdery solid. Take 0.4g of the powdery solid and transfer it to an Eppendorf tube, and add 2.4ml of n-butanol to passivate the grains. amino acid decarboxylase; after 10 minutes, vacuum drying was carried out. Then add 4.0ml of lanthanum bromide with a concen...

Embodiment 3

[0047] (1) Instrument

[0048] Refrigerated high-speed centrifuge; vacuum dryer; ultraviolet-visible spectrophotometer.

[0049] (2) Reagents

[0050] pH 7.2 Potassium Phosphate Buffer (0.1M); pH 8.6 Tris Buffer (0.2M); NADP + (8mM); α-ketoglutarate (60mM); glycerol; β-mercaptoethanol; GABA transaminase-succinic semialdehyde dehydrogenase dual enzyme solution (1U / ml, buffered with 0.1M pH7.2 potassium phosphate solution, containing 12.5% ​​glycerol and 5mM β-mercaptoethanol); ethanol; lanthanum chloride (0.1M); NaHCO 3 (5M).

[0051] (3) Specific steps

[0052] After the white mouse brain was washed and blotted dry, it was quickly put into liquid nitrogen and ground to obtain a powdery solid. Take 0.4 g of the powdery solid and transfer it to an Eppendorf tube, and add 3.2 ml of n-propanol. After 15 minutes, dry in vacuo; add 4.0 ml of lanthanum chloride at a concentration of 0.1M. After shaking for 20 minutes, centrifuge at 13000 g for 5 minutes, and collect the supern...

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Abstract

The invention provides a method for two-enzyme detection of the content of gamma-aminobutyric acid. The method comprises the following steps: grinding a sample to be detected in liquid nitrogen, and adding alcohol; carrying out vacuum drying, adding lanthanum halide, uniformly mixing above materials, centrifuging the obtained mixture, and collecting obtained supernatant; adding an alkali liquid to the supernatant, uniformly mixing the alkali liquid with the supernatant, and centrifuging the obtained mixture to obtain a gamma-aminobutyric acid extract liquid; adding NADP<+>, a Tris buffer liquid and a gamma-aminobutyrate aminotransferase-succinic semialdehyde dehydrogenase two enzyme liquid to the obtained extract liquid in order to obtain a mixed liquid; determining the initial value of the mixed liquid at a wavelength of 340nm by using an ultraviolet-visible spectrophotometer; adding alpha-oxpentanedioic acid to the mixed liquid, reacting, determining the final value of the obtained reaction liquid at the wavelength of 340nm, drawing a standard curve, and acquiring a primary regressive curve; and calculating the content of gamma-aminobutyric acid according to the initial value, the final value and the primary regressive curve. The method effectively reduces interferences, and can simply, rapidly and accurately detect the content of gamm-aminobutyric acid in plant and animal materials.

Description

technical field [0001] The invention relates to the technical field of gamma-aminobutyric acid detection, in particular to a method for dual-enzyme detection of gamma-aminobutyric acid content. Background technique [0002] γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid and an important component of the free amino acid pool in animal and plant cells. The content of GABA in plant tissues is low, about 0.03-2.00 μmol / gFW, but it can increase exponentially in response to different external stimuli, such as mechanical damage, hypoxia, and salt stress. Correct determination of GABA content in animal and plant tissues is an important indicator for treating related diseases and analyzing the response and adaptability of plants to external stimuli. [0003] In order to determine the GABA content of animal and plant tissues, people have developed a variety of detection methods. The common ones are thin-layer chromatography, paper chromatography, paper electrophor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 苏国兴
Owner SUZHOU UNIV
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