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Applications of heme/chaperonin artificial enzyme in detection of substance content in solution

A technology of heme and chaperone, applied in the field of biological enzyme detection, can solve the problems of low operation stability, elevated temperature, difficult recovery, etc., and achieve the effects of low price, little environmental pollution, and easy handling

Inactive Publication Date: 2018-06-08
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, some deficiencies of natural catalases limit their wide application, such as easy inactivation during preparation, or elevated temperature, low operational stability, and difficulty in recovery, etc.

Method used

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  • Applications of heme/chaperonin artificial enzyme in detection of substance content in solution
  • Applications of heme/chaperonin artificial enzyme in detection of substance content in solution
  • Applications of heme/chaperonin artificial enzyme in detection of substance content in solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation of embodiment 1 heme / chaperone artificial enzyme

[0050] Disperse 1mM heme into 20mM sodium hydroxide deionized water containing 1.5mM TritonX-100 to obtain 1mM heme micellar mother solution;

[0051]Dilute the heme micelle mother solution to 100 μM, and mix it with 1 μM chaperone solution in equal volumes to obtain a mixed solution (1);

[0052] Put the mixed solution (1) into a 50kDa dialysis bag, put it into a buffer and dialyze for 48 hours to obtain the heme / chaperone artificial enzyme;

[0053] The heme / chaperone artificial enzyme was quantified at a heme concentration of 20 μM.

[0054] Such as figure 1 As shown, the TEM picture on the left is a transmission electron microscope picture of the heme / chaperin artificial enzyme prepared in this example, compared with the crystal structure of chaperone (the data on the right is from the protein database 1SS8), as can be seen from the figure The artificial enzyme has an intermediate cavity with a di...

Embodiment 2

[0055] Embodiment 2 heme / chaperone artificial enzyme detects the content of hydrogen peroxide in the hydrogen peroxide solution

[0056] Mix equal volumes of 2mM o-phenylenediamine solution, 20μM heme / chaperone artificial enzyme solution and 1-750μM hydrogen peroxide solution to obtain mixed solution I;

[0057] Place the obtained mixture I in a 37°C water bath and incubate for 30 minutes;

[0058] Add 3.5 μM sulfuric acid to the incubated mixture to terminate the reaction to obtain mixture II;

[0059] Measure the absorbance of the mixed solution II at 492nm;

[0060] Prepare a standard curve from the absorbance of the mixture II and the concentration of hydrogen peroxide, and calculate the amount of hydrogen peroxide.

[0061] Such as figure 2 As shown, after adding different concentrations of hydrogen peroxide, the heme / chaperone artificial enzyme has good sensitivity, indicating that the artificial enzyme has a good catalytic effect on hydrogen peroxide and can be used...

Embodiment 3

[0062] Example 3 Heme / chaperone artificial enzyme detects the content of glucose in the glucose solution

[0063] Mix 5 μl of glucose oxidase (200 units / mL) and 50 μL of glucose solution (0-1.8 mM), and incubate in a water bath at 37°C for 30 min;

[0064] Add an equal volume of 2mM o-phenylenediamine and 20μM heme / chaperone artificial enzyme to the incubated mixture, and incubate in a 37°C water bath for 30min;

[0065] Add 3.5 μM sulfuric acid to the incubated solution to terminate the reaction;

[0066] Measure the absorbance of the mixture at 492nm;

[0067] A standard curve was prepared from the absorbance and glucose concentration, and the amount of glucose was calculated.

[0068] Such as image 3 As shown, the absorbance at 492 nm after quenching the reaction with sulfuric acid exhibits a typical glucose concentration response curve. The linear range of glucose detection is 15 μM-200 μM (correlation coefficient is 0.99768), and the limit of detection is 11 μM.

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Abstract

The invention provides applications of heme / chaperonin artificial enzyme in detection of substance content in solution, and belongs to the technical field of biological enzyme detection. Compared withnatural heme enzyme, the heme / chaperonin artificial enzyme possesses more active sites and a larger reactor, and can be used for sewage monitoring and biological diagnosis applications with high efficiency. According to the invention, o-phenylenediamine is taken as the substrate of the heme / chaperonin artificial enzyme, and reaction with hydrogen peroxide is adopted so as to realize direct / indirect detection of substance content in solution. The heme / chaperonin artificial enzyme can be used for hydrogen peroxide monitoring in industrial / municipal sewage, and detection of glucose in serum.

Description

technical field [0001] The invention belongs to the technical field of biological enzyme detection, and relates to an application of using a heme / chaperin artificial enzyme to detect the content of a substance in a solution, in particular to an application of using a heme / chaperin artificial enzyme with o-phenylenediamine as a substrate, through The application of reacting with hydrogen peroxide to detect the content of substances in the solution. Background technique [0002] Catalase is widely distributed in nature and uses iron(III) porphyrin IX (heme) as a redox cofactor, which exhibits excellent catalytic ability in oxidizing a large number of substrates at normal temperature and pressure, especially in Areas requiring clean oxidation under mild conditions. In the catalytic process of catalase, iron in heme at the active site is an environmentally friendly metal, and the reaction conforms to the current concept of green chemistry. On the other hand, some deficiencies ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/75G01N21/31
CPCG01N21/31G01N21/75
Inventor 王小强王超潘美红
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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