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Preparation method and application of protein mimetic enzyme based on heme and nano-gold clusters

A kind of nano-gold cluster and protein simulation technology, which can be applied in the direction of material analysis and instrumentation by making materials undergo chemical reactions, and by observing the impact on chemical indicators, which can solve problems such as micro-efficiency.

Active Publication Date: 2018-11-02
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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  • Preparation method and application of protein mimetic enzyme based on heme and nano-gold clusters
  • Preparation method and application of protein mimetic enzyme based on heme and nano-gold clusters
  • Preparation method and application of protein mimetic enzyme based on heme and nano-gold clusters

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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 8.0 mg / mL bovine serum albumin, 6.0 M urea and 2.0 m MEDTA, and then add them to the mixture Add 1,4-dimercaptothreitol to a concentration of 0.10 mM, then continue to stir for 30 minutes under nitrogen protection, and then dialyzed in deionized water with a dialysis bag of 14KDa for 12 hours to obtain chain bovine serum albumin containing sulfhydryl groups Solution, standby;

[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 100 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 80 mMN-hydroxysuccinimide and 2.0 mg / mL hemin, stirred at 22°C Activate for 1 hour, add the sulfhydryl-containing chain bovin...

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 7.0 mg / mL bovine serum albumin, 5.0 M urea and 1.8 m MEDTA, and then add them to the mixture Add 1,4-dimercaptothreitol to a concentration of 0.08 mM, then continue to stir for 30 minutes under the protection of nitrogen, and then dialyze with a dialysis bag of 14KDa in deionized water for 10 hours to obtain chain bovine serum albumin containing sulfhydryl groups Solution, standby;

[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 90 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 70 mMN-hydroxysuccinimide and 1.5mg / mL hemin, stirred at 20°C Activate for 1 hour, then add the sulfhydryl-containing ch...

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 10.0 mg / mL bovine serum albumin, 8.0 M urea and 3.2 m MEDTA, and then add them to the mixture Add 1,4-dimercaptothreitol to a concentration of 0.20 mM, then continue to stir for 30 minutes under nitrogen protection, and then dialyzed in deionized water with a dialysis bag of 14KDa for 14 hours to obtain chain bovine serum albumin containing sulfhydryl groups Solution, standby;

[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 120 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 100 mMN-hydroxysuccinimide and 3.0mg / mL hemin, stirred at 25°C Activate for 1 hour, then add the sulfhydryl-containing chain...

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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 sensing 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 dev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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