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Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid

A technology of ketoglutaric acid and enzymatic reaction, which is applied in the field of biological analysis, can solve the problems of inability to simultaneously detect the content of α-ketoglutaric acid, poor specificity of acid-base titration, and poor accuracy of test results, so as to reduce the cost of enterprises burden, reduce the error rate of experimental operation, and the effect of easy operation

Pending Publication Date: 2021-06-08
湖北远大生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the specificity of the acid-base titration method is poor, and the measured results cannot correctly reflect the content of α-ketoglutaric acid in the sample, so it is gradually eliminated.
The detection results of spectrophotometry have poor accuracy, low precision, long detection time and can only detect the content of α-ketoglutaric acid in the fermentation broth
The established HPLC method has good detection accuracy, but it cannot simultaneously detect the contents of L-glutamic acid and α-ketoglutarate in the fermentation broth
Patent ZL201610395430.7 (invention name is electrochemical biosensor for detecting α-ketoglutarate, preparation method and its application) uses electrochemical principles to detect α-ketoglutarate content, which is difficult to implement in practical applications

Method used

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  • Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid
  • Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid
  • Method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in enzymatic reaction liquid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The equipment and testing conditions used in this embodiment are as follows:

[0063] Instrument: Dionex Ultimate 3000 high performance liquid chromatography; VWD ultraviolet detector;

[0064] Chromatographic column: Thermo Fisher Syncronis C18 (5μm, 4.6×250mm);

[0065] Column temperature: 30°C;

[0066] Wavelength: 215nm;

[0067] Flow rate: 1.0mL / min;

[0068] Injection volume: 10μL;

[0069] Mobile phase: Mobile phase A is a mixed solution of 0.05mol / L dipotassium hydrogen phosphate and 0.005mol / L sodium decanesulfonate, the pH value is 2.3, and mobile phase B is absolute ethanol;

[0070] The volume ratio of mobile phase A and mobile phase B: 1000:70.

[0071] The specific steps for determining the content of standard α-ketoglutaric acid and L-glutamic acid are as follows:

[0072] 1. Preparation of reference substance mixed stock solution: Accurately weigh 0.2g of α-ketoglutarate reference substance and 1.2g of L-glutamic acid reference substance into the s...

Embodiment 2

[0076] The equipment and testing conditions used in this embodiment are as follows:

[0077] Instrument: Dionex Ultimate 3000 high performance liquid chromatography; VWD ultraviolet detector;

[0078] Chromatographic column: Thermo Fisher Syncronis C18 (5μm, 4.6×250mm);

[0079] Column temperature: 30°C;

[0080] Wavelength: 215nm;

[0081] Flow rate: 1.0mL / min;

[0082] Injection volume: 10μL;

[0083] Mobile phase: Mobile phase A is a mixed solution of 0.05mol / L dipotassium hydrogen phosphate and 0.005mol / L sodium decanesulfonate, the pH value is 2.3, and mobile phase B is absolute ethanol;

[0084] The volume ratio of mobile phase A and mobile phase B: 1000:70.

[0085] The specific operation steps for the content determination of α-ketoglutaric acid and L-glutamic acid in the enzymatic reaction solution are as follows:

[0086] 1. Prepare the test solution: Take 1mL of the enzymatic reaction solution in a 10mL volumetric flask, dilute to volume with distilled water, ...

Embodiment 3

[0089] The equipment and testing conditions used in this embodiment are as follows:

[0090] Instrument: Dionex Ultimate 3000 high performance liquid chromatography; VWD ultraviolet detector;

[0091] Chromatographic column: Thermo Fisher Syncronis C18 (5μm, 4.6×250mm);

[0092] Column temperature: 30°C;

[0093] Wavelength: 215nm;

[0094] Flow rate: 1.0mL / min;

[0095] Injection volume: 10μL;

[0096] Mobile phase: Mobile phase A is a mixed solution of 0.05mol / L dipotassium hydrogen phosphate and 0.005mol / L sodium decanesulfonate, the pH value is 2.3, and mobile phase B is absolute ethanol;

[0097] The volume ratio of mobile phase A and mobile phase B: 1000:70.

[0098] The specific operation steps of α-ketoglutarate linearity test are as follows:

[0099] 1. Prepare the test solution: before use, take 0.1mL, 0.3mL, 0.5mL, 0.7mL, 0.9mL of the reference substance mixed stock solution of α-ketoglutaric acid and L-glutamic acid in a 10mL volumetric flask , diluted with ...

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Abstract

The invention relates to the field of biological analysis, in particular to a method for simultaneously detecting alpha-ketoglutaric acid and L-glutamic acid in an enzymatic reaction liquid. A high performance liquid chromatography method is adopted, the enzymatic reaction liquid is injected into a high performance liquid chromatograph, a C18 column is adopted for separation, and an ultraviolet absorption detector is adopted for detection. The content of alpha-ketoglutaric acid and the content of L-glutamic acid in the enzymatic reaction liquid are detected at the same time, reliable data support can be provided for real-time monitoring of alpha-ketoglutaric acid production, pre-column or post-column derivatization treatment is not needed for sample detection, the detection time is greatly shortened, and the detection result is more accurate.

Description

technical field [0001] The invention relates to the field of biological analysis, in particular to a method for simultaneously detecting α-ketoglutaric acid and L-glutamic acid in an enzymatic reaction solution. Background technique [0002] Ketoglutaric acid (Ketoglutaric acid) is two ketone derivatives of glutaric acid, divided into two types: α-type and β-type. Most of α-ketoglutarate refers to the α form. α-Ketoglutaric acid is one of the key intermediates of the basic metabolic tricarboxylic acid cycle in the living system. It regulates many basic metabolic processes and can provide biosynthesis for L-glutamic acid, glutamine, ornithine, etc. carbon skeleton. Studies have found that α-ketoglutarate, as the precursor of glutamine (Gln), can provide energy for gastrointestinal cells, promote gastrointestinal cell proliferation, and maintain gastrointestinal health synergistically. It is also a pharmaceutical preparation, dietary supplement and a special medicinal food ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8809G01N2030/8818
Inventor 赵东明刘晓蓉曹亚杰欧阳晖林添雄
Owner 湖北远大生物技术有限公司
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