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Heme-GroEL complex as well as preparation method and application thereof

A heme and complex technology, applied in the field of biological enzyme detection, can solve the problems of easy inactivation, low recycling rate, high cost, etc., and achieve the effect of good stability and strong activity

Inactive Publication Date: 2019-03-15
QINGDAO GUGAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the defects of natural catalase limit its application, such as easy inactivation during preparation or purification, poor operational stability, high cost, low recycling rate, etc.

Method used

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  • Heme-GroEL complex as well as preparation method and application thereof
  • Heme-GroEL complex as well as preparation method and application thereof
  • Heme-GroEL complex as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The preparation of embodiment 1 heme-GroEL complex

[0023] A preparation method of a heme-GroEL complex, the specific steps are as follows:

[0024] (1) GroEL was prepared by Escherichia coli strain BL21 containing the pTric99 plasmid, with a purity of 95%, dissolved in 10 mM phosphate buffer with pH 7.0 to obtain a 1.25 μM GroEL solution;

[0025] (2) Prepare 1 mM heme stock solution by using 20 mM NaOH containing surfactant Triton X100;

[0026] (3) Dilute the heme stock solution described in step (2) to 125 μM with 10 mM phosphate buffer solution of pH 7.0, and then mix it with the same volume of 1.25 μM GroEL solution described in step (1) to obtain a mixed solution ;

[0027] (4) The mixed solution described in step (3) was dialyzed with 10 mM phosphate buffer solution with pH 7.0 for 48 hours to obtain the heme-GroEL complex.

[0028] That is, heme molecules combine with the hydrophobic inner surface of GroEL to form a heme-GroEL complex, and the results are a...

Embodiment 2

[0029] The catalase-like catalytic activity of embodiment 2 heme-GroEL

[0030] OPD solution (1mM), an equal volume of H 2 o 2 The solution (10 mM) was mixed with the heme-GroEL solution, and the peroxidase catalytic activity of the heme-GroEL complex was studied at 25° C.; the heme was fixed at an initial concentration of 20 μM. All solutions used were prepared in pH 7 10 mM phosphate buffer. Changes in absorbance were monitored using a UV-1700 UV-Vis spectrophotometer.

[0031] The result is as figure 2 As shown, the heme-GroEL complex has peroxidase catalytic activity in oxidizing OPD. In the presence of heme-GroEL, containing H 2 o 2 and OPD solution turns yellow. The UV-Vis spectrum of the reaction mixture changed gradually, and a new absorption band appeared around 450 nm, which was attributed to the charge transfer during the oxidation of OPD. In the absence of H 2 o 2 Or in the case of heme-GroEL, the spectrum remains unchanged. The results showed that the ...

Embodiment 3

[0034] Change the equilibrium molar ratio of heme and GroEL, and determine its influence on the initial rate of heme-GroEL catalyzed OPD oxidation; and test the catalytic activity of TritonX100 solubilized heme; The mixture was kept at 20 μM.

[0035] The result is as image 3 As shown, the catalytic activity increases significantly when heme is transferred from Triton X100 micelles into the GroEL cavity, reaches a maximum when the equilibrium molar ratio is 40:1, and then decreases significantly.

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PUM

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Abstract

The invention discloses a heme-GroEL complex and a preparation method and application thereof. The heme-GroEL complex is provided with two back-to-back cavities. The inner surface at an opening of each cavity is provided with seven hydrophobic grooves perpendicular to the center line of the cavity. Each cavity can be combined with a plurality of monomer heme molecules through hydrophobic interaction. The heme-GroEL complex is good in stability, has the significant catalase activity, can be used instead of natural peroxidase, can be used for chromogenic detection of hydrogen peroxide or glucose, and can be also used for detection or oxidation removal of phenolic pollutants.

Description

technical field [0001] The invention relates to the technical field of biological enzyme detection, in particular to a heme-GroEL complex and its preparation method, as well as its application in color detection and oxidation decontamination. 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 when oxidizing a large number of substrates at normal temperature and pressure; During the catalytic process of hydrogenase, the iron in the heme at the active site is an environmentally friendly metal. However, the defects of natural catalase limit its application, such as easy inactivation during preparation or purification, poor operational stability, high cost, and low recycling rate. [0003] Therefore, it is an urgent problem for those skilled in the art to provide a catalase mimic with good stability, strong activity, low cost and recyclability. Contents o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12Q1/54C12Q1/30C12Q1/26A62D3/02A62D101/28
CPCA62D3/02A62D2101/28C12N9/0065C12Q1/26C12Q1/30C12Q1/54C12Y111/01006G01N2333/904
Inventor 王小强高娟郝锰黄翔宇
Owner QINGDAO GUGAO BIOTECH CO LTD
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