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Method for detecting glutamic acid content by enzyme method and application of method

A technology of glutamic acid and content, applied in measurement devices, biochemical equipment and methods, biological tests, etc., can solve the problems of inability to obtain glutamic acid content and limited application, etc.

Pending Publication Date: 2020-02-11
申友基因组研究院(南京)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when this method is used to detect blood samples, urine samples, and other samples containing high-concentration ammonia, the normal progress of the reaction is often inhibited by the presence of high-concentration ammonia in the system, and accurate results of glutamic acid content in the sample cannot be obtained.
This limits the application of glutamate dehydrogenase and its kit in the above samples

Method used

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  • Method for detecting glutamic acid content by enzyme method and application of method
  • Method for detecting glutamic acid content by enzyme method and application of method
  • Method for detecting glutamic acid content by enzyme method and application of method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] A method for enzymatic detection of glutamic acid content, comprising the following steps:

[0087] (1) Exogenous expression and isolation and purification of protein:

[0088] (1) Combining with sequence comparison analysis methods, select glycine aminotransferase AT 2 The gene coding sequence of the gene coding sequence and the gene coding sequence of 3-phosphoglycerate dehydrogenase SerA, and then the method of obtaining the target gene through whole gene synthesis and PCR respectively (wherein the template of serA gene cloning is Escherichia coli E. coli k-12 strain genome), respectively, to obtain glycine aminotransferase AT 2 Target gene and 3-phosphoglycerate dehydrogenase SerA target gene;

[0089] According to the literature "Masafumi Kameya, et al. (2010) Purification of threeaminotransferases from Hydrogenobacter thermophilus TK-6 – novel types of alanine or glycine aminotransferase. FEBS Journal, 277:1876-1885.” report Hydrogenobacter thermophilus Gly...

Embodiment 2

[0151] Except the proportion composition of every 75 μl mixed solution, all the other conditions are consistent with embodiment 1;

[0152] The ratio composition of each 75 μl reaction mixture includes: TEA-HCl solution, 25 μl; sodium glyoxylate aqueous solution, 20 μl; NADH solution, 4 μl; double enzyme mixture, 10 μl; ultrapure water, 16 μl.

Embodiment 3

[0154] Except the proportion composition of every 75 μl mixed solution, all the other conditions are consistent with embodiment 1;

[0155]The ratio composition of each 75 μl reaction mixture includes: TEA-HCl solution, 25 μl; sodium glyoxylate aqueous solution, 10 μl; NADH solution, 5 μl; double enzyme mixture, 5 μl; ultrapure water, 30 μl.

[0156] After using a microplate reader to detect the absorbance at 340 nm by the endpoint method or the initial velocity method, the absorbance change value is used as the ordinate, and the glutamic acid standard solution concentration is used as the abscissa to draw the glutamic acid concentration standard curve. Excel and other software draw the standard curve, and it is found that there is a good linear relationship under the concentration of 0-100 μΜ glutamic acid, so the concentration of glutamic acid 0-100 μΜ is selected as the main measurement range, such as figure 2 Shown is the standard curve under the concentration of 0~100 μ ...

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Abstract

The invention relates to a method for detecting glutamic acid content by an enzyme method and application of the method. The method for detecting glutamic acid content by the enzyme method includes the steps that the glutamic acid concent is determined by using a coupled enzymatic reaction system of glycine aminotransferase AT2 and 3-phosphoglycerate dehydrogenase SerA. The method for detecting the glutamic acid content by the enzyme method overcomes the defect that a traditional glutamic acid dehydrogenase method is not suitable for a high-concentration ammonia environment, and through a coupled enzymatic reaction method of the glycine transaminase AT2 and 3-phosphoglycerate dehydrogenase SerA, glutamic acid is deammoniated to form alpha-ketoglutarate, and through a reduction reaction ofthe alpha-ketoglutarate, the glutamic acid content in the system is quickly and correctly characterized by the reduction of nicotinamide adenine dinucleotide (NADH) content. The method for detecting the glutamic acid content by the enzyme method is high in sensitivity, fast and stable in detection, good in repeatability, simple in operation, low in cost and wide in application range.

Description

technical field [0001] The invention relates to the technical field of enzymatic detection, in particular to a method for enzymatic detection of glutamic acid content and its application. Background technique [0002] At present, the methods for detecting glutamate mainly include liquid chromatography, ion chromatography, spectrophotometry and so on. Liquid chromatography and ion chromatography have high analytical sensitivity and good separation effect, and are suitable for the analysis and determination of complex samples. However, some liquid chromatography methods for the determination of glutamic acid require derivatization of the sample, and the instruments of the two methods are expensive and costly. The time cost is high, and it is not suitable for the analysis and determination of high-throughput samples. [0003] The traditional spectrophotometric detection system for the determination of glutamic acid content is mainly based on glutamic acid dehydrogenase. This m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/32C12Q1/52G01N33/68
CPCC12Q1/32C12Q1/52G01N33/6812
Inventor 花强朱虹董辉金维荣邓伟伟
Owner 申友基因组研究院(南京)有限公司