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Application and preparation method of protein mimic enzyme based on heme and gold nanoclusters

A technology of nano-gold clusters and protein simulation, which is applied in the direction of material analysis by chemical reaction of materials, material analysis by observing the influence on chemical indicators, measurement devices, etc. Effect

Active Publication Date: 2016-06-15
QUFU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, gold nanoparticles have been used to modify horseradish peroxidase to improve its catalytic activity, but with little success

Method used

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  • Application and preparation method of protein mimic enzyme based on heme and gold nanoclusters
  • Application and preparation method of protein mimic enzyme based on heme and gold nanoclusters
  • Application and preparation method of protein mimic enzyme based on heme and gold nanoclusters

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Embodiment 1

[0049] A preparation method of protein mimic enzyme based on heme and gold nano clusters, the steps are:

[0050] 1) At room temperature, mix bovine serum albumin, urea, EDTA and water, and stir for 20 minutes to obtain a mixture of 8.0mg / mL bovine serum albumin, 6.0M urea and 2.0m MEDTA, and then add to the mixture 1,4-Dimercaptothreitol to a concentration of 0.10mM, then continue to stir for 30min under the protection of nitrogen, and then dialyzed in deionized water with a 14KDa dialysis bag for 12h to obtain a sulfhydryl-containing chain bovine serum albumin solution ,spare;

[0051] 2) At room temperature, mix 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hemin and water to obtain the respective concentrations A mixture of 100mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 80mMN-hydroxysuccinimide and 2.0mg / mL hemin, stirred and activated at 22°C for 1h, Add the sulfhydryl-containing chain bovine serum albumin solution pre...

Embodiment 2

[0054] A preparation method of protein mimic enzyme based on heme and gold nano clusters, the steps are:

[0055] 1) At room temperature, mix bovine serum albumin, urea, EDTA and water, and stir for 20 minutes to obtain a mixture of 7.0mg / mL bovine serum albumin, 5.0M urea and 1.8m MEDTA, and then add to the mixture 1,4-Dimercaptothreitol to a concentration of 0.08mM, then continue to stir for 30min under nitrogen protection, and then dialyzed with a dialysis bag of 14KDa in deionized water for 10h to obtain a sulfhydryl-containing chain bovine serum albumin solution ,spare;

[0056] 2) At room temperature, mix 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hemin and water to obtain the respective concentrations A mixture of 90mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 70mMN-hydroxysuccinimide and 1.5mg / mL hemin, stirred and activated at 20°C for 1h, Then add the sulfhydryl-containing chain bovine serum albumin solution pre...

Embodiment 3

[0059] A preparation method of protein mimic enzyme based on heme and gold nano clusters, the steps are:

[0060] 1) At room temperature, mix bovine serum albumin, urea, EDTA and water, and stir for 20 minutes to obtain a mixture of 10.0mg / mL bovine serum albumin, 8.0M urea and 3.2m MEDTA, and then add to the mixture 1,4-Dimercaptothreitol to a concentration of 0.20mM, then continue to stir for 30min under the protection of nitrogen, and then dialyzed in deionized water with a dialysis bag of 14KDa for 14h to obtain a chain bovine serum albumin solution containing sulfhydryl groups ,spare;

[0061] 2) At room temperature, mix 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, hemin and water to obtain the respective concentrations A mixture of 120mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 100mMN-hydroxysuccinimide and 3.0mg / mL hemin, stirred and activated at 25°C for 1h, Then add the sulfhydryl-containing chain bovine serum alb...

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Abstract

The invention provides application and a preparation method of a protein mimic enzyme based on heme and gold nanoclusters.The method includes: using carbamide for denaturation of bovine serum albumin, using 1,4-dithiothreitol for recovering the bovine serum albumin to obtain sulfydryl-containing chain bovine serum albumin, crosslinking with hemin to obtain sulfydryl-containing chain bovine serum albumin and hemin scaffold composite, and taking the composite as a stabilizer and a reducing agent to synthesize the gold nanoclusters to further obtain the protein mimic enzyme based on the heme and the gold nanoclusters.The protein mimic enzyme prepared according to the method has the advantages of low cost, high catalytic activity, high stability and the like.The defects of low catalytic activity, insolubility in water, proneness to clustering in water solutions, proneness to structural damages in oxidation media and the like of the heme are overcome, and application range of the heme is widened.In addition, the protein mimic enzyme is application to quick and high-sensitivity detection of H2O2 content in common foods, avoids complex photoelectric instruments in a detection process and is simple and easy in operation, thereby being expected to be widely applied to the field of biocatalysis.

Description

Technical field [0001] The invention belongs to the technical field of enzyme catalysis and sensor detection, and relates to a method for preparing protein mimic enzymes, in particular to a method for preparing protein mimic enzymes based on heme and nano-gold clusters and its application in common foods. 2 O 2 Application in rapid testing. Background technique [0002] As a highly efficient and specific biocatalyst, protease can catalyze many biological reactions in the body, and is widely used in functional catalysis and biological detection fields. However, natural proteases also have many shortcomings, such as being easily degraded, high cost, difficult to store, especially its activity is easily affected by external environmental conditions (pH, temperature, inhibitors, etc.) and denatured and inactivated. At the same time, the catalytic active center of common proteases (such as catalase, peroxidase, myoglobin and hemoglobin) is heme. In recent years, researchers have devo...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N21/31G01N21/33G01N27/416
CPCG01N21/31G01N21/33G01N21/78G01N27/416
Inventor 王桦张立燕李帅董敏敏冯路平
Owner QUFU NORMAL UNIV
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