Method for simultaneously determining DL-ATC content, L-cysteine content and L-cystine content in enzyme catalysis reaction solution

An enzymatic reaction, cysteine ​​technology, applied in the field of high-performance liquid chromatography, can solve the problems of unreported, difficult accurate quantification, difficult L-cysteine ​​detection, etc., to achieve a simple and reproducible method Good performance and high accuracy

Active Publication Date: 2017-04-26
湖北远大生物技术有限公司
View PDF4 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the reaction solution of enzymatic reaction to produce L-cysteine, DL-ATC, L-cysteine ​​and L-cystine exist at the same time, and under the catalysis of the enzyme, DL-ATC is converted into L-cysteine, at the same time, under alkaline conditions, L-cysteine ​​is oxidized by air to generate L-cystine. This dynamic change has brought great benefits to the accurate quantification of the three contents. Difficulties
[0005] At present, the traditional determination methods of L-cysteine ​​include spectrophotometry, paper chromatography, electrochemical determination, etc. However, due to the complex composition of the enzymatic reaction solution, it is difficult to exclude the interference of impurities, so the above methods are difficult to apply Detection of L-cysteine ​​in enzymatic reaction solution
The content of DL-ATC, L-cysteine ​​and L-cystine in the alkaline enzymatic reaction solution is extremely unstable, and it is difficult for ordinary methods to meet the requirements of accurate quantitative detection of the three at the same time. Therefore, a A simple method is used to stabilize the content of DL-ATC, L-cysteine ​​and L-cystine in the enzymatic reaction solution, and it is of great practical significance to quickly and accurately realize the simultaneous determination of the three contents
[0006] According to the literature search, there is no report on the method of simultaneous determination of DL-ATC, L-cysteine ​​and L-cystine in the enzymatic reaction solution by HPLC.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for simultaneously determining DL-ATC content, L-cysteine content and L-cystine content in enzyme catalysis reaction solution
  • Method for simultaneously determining DL-ATC content, L-cysteine content and L-cystine content in enzyme catalysis reaction solution
  • Method for simultaneously determining DL-ATC content, L-cysteine content and L-cystine content in enzyme catalysis reaction solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] This embodiment relates to the detection of mixed contrast solution

[0037] 1. Instruments and testing conditions:

[0038] Instrument: Dionex Ultimate 3000 High Performance Liquid Chromatograph

[0039]Column: Normal phase silica bonded diol column

[0040] Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH=3.0) (80:20)

[0041] Detection wavelength: 200nm

[0042] Flow rate: 1.5mL / min

[0043] Column temperature: 35°C

[0044] Injection volume: 20μL

[0045] 2. Specific steps:

[0046] L-cysteine ​​reference stock solution: Accurately weigh 50 mg of L-cysteine ​​reference substance into a 10 mL measuring bottle, add 0.01M ammonium dihydrogen phosphate solution (pH=3.0) to dissolve, constant volume, shake well to obtain 5.0 mg / mL L-cysteine ​​control stock solution.

[0047] DL-ATC and L-cystine mixed control stock solution: Accurately weigh 50 mg each of DL-ATC and L-cystine reference substance and place it in a 100 mL measuring bottl...

Embodiment 2

[0052] This embodiment relates to the detection of need testing solution

[0053] 1. Instruments and testing conditions:

[0054] Instrument: Dionex Ultimate 3000 High Performance Liquid Chromatograph

[0055] Column: Normal phase silica bonded diol column

[0056] Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH=3.0) (80:20)

[0057] Detection wavelength: 200nm

[0058] Flow rate: 1.5mL / min

[0059] Column temperature: 35°C

[0060] Injection volume: 20μL

[0061] 2. Specific steps:

[0062] Take the enzymatic reaction solution of L-cysteine ​​produced by enzymatic method, immediately add hydrochloric acid to adjust the pH of the solution to 2.0, take 1mL of the above solution into a 10mL measuring bottle, add 0.01M ammonium dihydrogen phosphate solution (pH=3.0) to dissolve and set Take the above solution and filter it with a 0.45 μm water filter membrane, and then take the filtrate as the test solution. Take 20 μL of the test solution, inj...

Embodiment 3

[0065] This embodiment relates to the stability detection of the enzymatic reaction solution at different pH values

[0066] 1. Testing equipment and conditions

[0067] Instrument: Dionex Ultimate 3000 High Performance Liquid Chromatograph

[0068] Column: Normal phase silica bonded diol column

[0069] Mobile phase: acetonitrile-0.01M ammonium dihydrogen phosphate solution (pH=3.0) (80:20)

[0070] Detection wavelength: 200nm

[0071] Flow rate: 1.5mL / min

[0072] Column temperature: 35°C

[0073] Injection volume: 20μL

[0074] Take 50mL of the enzymatic reaction solution of L-cysteine ​​produced by the enzymatic method, immediately add hydrochloric acid to adjust the pH of the solution, and adjust the pH of the solution to 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and place it at room temperature for 0, 4, and 8 hours respectively. Take 1 mL of the above solution into a 10 mL measuring bottle, add 0.01M ammonium dihydrogen phosphate solution (pH=3.0) to dissolve and make to volu...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a method for simultaneously determining the DL-ATC content, the L-cysteine content and the L-cystine content in an enzyme catalysis reaction solution. The method comprises: adjusting the pH value of an enzyme catalysis reaction solution, and separating and simultaneously determining the DL-ATC content, the L-cysteine content and the L-cystine content in the enzyme catalysis reaction solution by using a HPLC method. According to the present invention, by adjusting the pH value of the enzyme catalysis reaction solution, the DL-ATC concentration, the L-cysteine concentration and the L-cystine concentration in the enzyme catalysis reaction solution can maintain the stable states within a certain time, and the rapid and simultaneous determination of the DL-ATC content, the L-cysteine content and the L-cystine content in the enzyme catalysis reaction solution by using the high performance liquid chromatography method can be achieved.

Description

technical field [0001] The invention relates to an HPLC method for simultaneously measuring the contents of DL-ATC, L-cysteine ​​and L-cystine in an enzymatic reaction liquid, belonging to the field of biological analysis. Background technique [0002] L-cysteine ​​is an important sulfur-containing amino acid, and it is also the only α-amino acid containing a sulfhydryl group among more than 20 amino acids that make up proteins. L-cysteine ​​has many important physiological functions, and has been widely used in many fields such as medicine, food, health products, cosmetics and feed industry. [0003] The market demand for L-cysteine ​​is very large. At present, its main production method is hair hydrolysis. Although the process is relatively simple, it consumes a lot of energy and causes serious environmental pollution. In recent years, enzymatic conversion method has attracted more and more attention due to its advantages of good stereoselectivity, single configuration pr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/027
Inventor赵东明欧阳晖
Owner湖北远大生物技术有限公司