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Method for detecting content of phenylalanine and tyrosine by enzyme method, and application thereof

A technology of phenylalanine and phenylalanine dehydrogenase, which is applied in applications, measuring devices, botanical equipment and methods, and can solve the problems of unsuitable high-throughput sample analysis and determination, expensive instruments, and time-consuming costs. advanced questions

Pending Publication Date: 2020-02-21
申友基因组研究院(南京)有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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 phenylalanine and tyrosine requires complex pretreatment or pre-column preparation of the sample. Derivatization, and both instruments are expensive, time-consuming and costly, not suitable for the analysis and determination of high-throughput samples
The traditional spectrophotometric method can only determine the content of phenylalanine or tyrosine by enzymatic method alone

Method used

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  • Method for detecting content of phenylalanine and tyrosine by enzyme method, and application thereof
  • Method for detecting content of phenylalanine and tyrosine by enzyme method, and application thereof
  • Method for detecting content of phenylalanine and tyrosine by enzyme method, and application thereof

Examples

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Embodiment 1

[0100] A method for enzymatically detecting the content of phenylalanine and tyrosine, comprising the following steps:

[0101] (1) Exogenous expression and isolation and purification of protein:

[0102] (1) Combining sequence comparison and analysis methods, select the gene coding sequence of Phenylalanine Dehydrogenase, and then obtain the Phenylalanine Dehydrogenase Phenylalanine Dehydrogenase target gene through the method of whole gene synthesis of the target gene;

[0103] According to the literature "Noriko O , Yasuo H , Yasuhisa A , et al. Cloning and nucleotide sequencing of phenylalanine dehydrogenase gene of Bacillus sphaericus [J].Gene, 1988, 63(2):337-341." report Bacillus sphaericus Phenylalanine Dehydrogenase exists in the SCRC-79a strain, and the gene sequence can be obtained by searching for AAA22646.1 in the NCBI Protein database;

[0104] Phenylalanine Dehydrogenase Phenylalanine Dehydrogenase was commissioned by Biological Company through the whole gene ...

Embodiment 2

[0139] Except the proportion composition of every 190 μ l mixed solution, all the other conditions are consistent with embodiment 1;

[0140] The proportion composition of each 190 μl reaction mixture includes: Glycine-KCl-KOH buffer (0.2 M, pH10.5), 100 μl; NAD + That is, β-nicotinamide adenine dinucleotide aqueous solution (40mM), 15 μl; enzyme solution, 10 μl; ultrapure water to make up to 190 μl.

Embodiment 3

[0142] Except the proportion composition of every 190 μ l mixed solution, all the other conditions are consistent with embodiment 1;

[0143] The proportion composition of each 190 μl reaction mixture includes: Glycine-KCl-KOH buffer (0.2 M, pH10.5), 100 μl; NAD + That is, β-nicotinamide adenine dinucleotide aqueous solution (40mM), 20 μl; enzyme solution, 10 μl; ultrapure water to make up to 190 μl.

[0144] Use a microplate reader to detect the absorbance at 340nm by the endpoint method or the initial velocity method, and draw phenylalanine with the absorbance change value as the ordinate, and the concentration of phenylalanine and tyrosine standard solution as the abscissa and tyrosine concentration standard curve, draw the standard curve by software such as Excel, it is found that there is a good linear relationship between 0-200 μM phenylalanine and tyrosine concentration, so the choice of phenylalanine and tyrosine concentration 0 ~200 μΜ as the main measurement range, ...

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Abstract

The invention relates to a method for detecting the content of phenylalanine and tyrosine by an enzyme method, and an application thereof. The method uses phenylalanine dehydrogenase to quickly and correctly characterize the content of phenylalanine and phenylalanine in a system by the increasing of the content of NADH. The total content of phenylalanine and tyrosine in a sample can be quickly determined by the end point method or the initial velocity method. The method has high sensitivity, rapid and stable detection, good repeatability, simple and convenient operation, relatively low cost and relatively wide 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 the content of phenylalanine and tyrosine and its application. Background technique [0002] Currently, the methods for detecting phenylalanine and tyrosine mainly include liquid chromatography, ion chromatography, and spectrophotometry. 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 phenylalanine and tyrosine requires complex pretreatment or pre-column preparation of the sample. Derivatization treatment, and both instruments are expensive, time-consuming and costly, and are not suitable for the analysis and determination of high-throughput samples. The traditional spectrophotometric method can only determine the content of phenylalanine...

Claims

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Application Information

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IPC IPC(8): C12Q1/32C12N15/53C12N15/70C12N9/06G01N21/31
CPCC12Q1/32C12N9/0016C12N15/70C12Y104/01001G01N21/31G01N2333/90622
Inventor 董辉邓伟伟金维荣花强朱虹
Owner 申友基因组研究院(南京)有限公司
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