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Method for detecting glycine content by enzymic method and application thereof

A technology of glycine and glycine oxidase is applied in the field of enzymatic detection of glycine content, and can solve the problems of inability to obtain accurate results of glycine content, limitations, and the inability of the reaction to proceed normally.

Pending Publication Date: 2020-02-07
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, the enzyme activity of HRP is inhibited by the blood environment, the reaction cannot be carried out normally, and accurate results of glycine content in the sample cannot be obtained.
This limits the application of glycine oxidase-horseradish peroxidase and its kit in the above samples

Method used

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  • Method for detecting glycine content by enzymic method and application thereof
  • Method for detecting glycine content by enzymic method and application thereof
  • Method for detecting glycine content by enzymic method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0085] A method for enzymatic detection of glycine content, comprising the following steps:

[0086] (1) Exogenous expression and isolation and purification of protein:

[0087] (1) Combining with sequence comparison and analysis means, select the gene coding sequence of glycine oxidase Glycine Oxidase and the gene coding sequence of glyoxylate reductase Glyoxylate Reductase, and then obtain the target gene of glycine oxidase Glycine Oxidase through the method of whole gene synthesis Gene and target gene of glyoxylate reductase Glyoxylate Reductase;

[0088] According to the literature "Mayumi K , Sumitaka H , Kazushi F , et al. Whole-GenomeSequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods[J]. PLOS ONE, 2015, 10(10):e0141369-. "reported Bacillus subtilis strain HJ0-6 Glycine Oxidase (thiO) exists in the strain, and the gene sequence can be obtained by searching for thiO in the Gene database of NCBI;

[...

Embodiment 2

[0149] Except the ratio composition of every 175 μl mixed solution, all the other conditions are consistent with embodiment 1;

[0150] The proportion composition of each 175 μl reaction mixture includes: TEA-HCl solution, 100 μl; flavin adenine dinucleotide disodium aqueous solution, 20 μl; NADH solution, 8 μl; double enzyme mixture, 12 μl; Make up to 175 μl with pure water.

Embodiment 3

[0152] Except the ratio composition of every 175 μl mixed solution, all the other conditions are consistent with embodiment 1;

[0153] The proportion composition of each 175 μl reaction mixture includes: TEA-HCl solution, 100 μl; flavin adenine dinucleotide disodium aqueous solution, 10 μl; NADH solution, 10 μl; double enzyme mixture, 12 μl; Make up to 175 μl with pure water.

[0154] Use a microplate reader to detect the absorbance at 340nm by the endpoint method or the initial velocity method, draw the standard curve of glycine concentration with the absorbance change value as the ordinate, and the concentration of the glycine standard solution as the abscissa, and draw the standard by Excel and other software As a result, it is found that there is a good linear relationship under the glycine concentration of 0-150 μM, so the glycine concentration of 0-150 μM is selected as the main measurement range, such as figure 2 Shown is the standard curve under the concentration of...

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Abstract

The invention relates to a method for detecting glycine content by an enzymic method and an application thereof, and the method comprises the following steps: determining the glycine content by utilizing a double-enzyme coupling system of glycine oxidase and glyoxylate reductase; according to the method, the defect that a traditional method for coupling glycine oxidase with horseradish peroxidase(HRP) is not suitable for a blood environment is overcome, a double-enzyme coupling method of glycine oxidase and glyoxylate reductase is creatively adopted, glycine is oxidized to form glyoxylic acid, and the content of glycine in the system is rapidly and correctly represented by reducing the content of NADH through a reduction reaction of glyoxylic acid. The method is high in sensitivity, rapidand stable in detection, good in repeatability, simple and convenient to operate, relatively low in cost and relatively 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 glycine content and its application. Background technique [0002] At present, the methods for detecting glycine 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, the liquid chromatography method for the determination of glycine requires pre-column derivatization of the sample, and the instruments of the two methods are expensive and time-consuming. The 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 glycine content is mainly based on the coupling reaction of glycine oxidase G...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26G01N21/31
CPCC12Q1/26G01N21/31G01N2333/90638G01N2333/902
Inventor 朱虹花强董辉金维荣邓伟伟
Owner 申友基因组研究院(南京)有限公司
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