Method for preparing selenium substitution papain

A papain and selenium generation technology, which is applied in the field of biochemistry, can solve the problems of low GPx activity of selenized subtilisin protease, and the process is not simple enough, and achieves the effects of excellent antioxidant properties, simple preparation method and good stability.

Inactive Publication Date: 2003-07-23
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technique of the background technology is still not simple enough, and the GPx activity of the prepared subtilisin selenide is far lower than that of natural enzymes

Method used

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  • Method for preparing selenium substitution papain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: the preparation of sodium selenium hydride

[0017] 81mg NaBH 4 Dissolve in a vial with 1mL of water, then slowly add 79mg of selenium powder to NaBH 4 At this moment, a large amount of gas is seen to emerge in the water, and the reaction is carried out at room temperature for 5 minutes. Then cover a plastic cover that inserts the needle until the reaction does not overflow the gas, remove the needle, seal it, and suck it out with the needle when it is used. The reaction equation is

[0018]

[0019] due to NaBH 4 with H + reaction, with a small amount of H 2 Se gas is produced. To prevent poisoning, the reaction was carried out in a fume hood.

Embodiment 2

[0020] Embodiment 2: the preparation of selenopapain

[0021] Papain (2 mg) was dissolved in 2 mL of 50 mM PBS, pH 7.0, and 200 μL of 1 M NaHSe solution prepared in Example 1 was added with a syringe, kept at 40° C. for 36 hours under nitrogen protection, then taken out and freeze-dried, and the dry powder was washed with 10 mM PBS (pH 7.0). 0) dissolving, centrifuging to remove selenium, desalting with Sephadex G-25, collecting protein peaks, and freeze-drying to obtain selenopapain. The specific activity of the obtained selenopapain can reach 44770.3U / μmol.

Embodiment 3

[0022] Embodiment 3: the preparation of selenopapain (re-selenization through activation)

[0023] Papain (2mg) was dissolved in 2mL 50mM PBS, pH7.0, 50μL PMSF (20mg / mL acetonitrile solution) was added to the above buffer solution twice every 30 minutes, incubated at room temperature for 3 hours, and then passed high-purity nitrogen for about 20 minutes to eliminate oxygen. Add 200 μL of 1M NaHSe solution prepared in Example 1 with a syringe, and keep warm at 30° C. for 36 hours under nitrogen protection. Afterwards, the aftertreatment process is the same as in Example 2. The specific activity of the obtained selenopapain can reach 54018.7U / μmol.

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Abstract

A process for preparing selenopapain from papain and sodium selenohydride through dissolving papain in phosphate solution, adding excessive sodium selenohydride, seleno substitution under protection of N2, freeze drying, dissolving in phosphate solution centrifugal removal of Se, removing salt, collecting protein peaks, and freeze drying. Its advantages are simple process high activity of glutathion peroxidase, and excellent antioxidizing power.

Description

technical field [0001] The invention belongs to the field of biochemistry, in particular to a method for preparing an artificial enzyme with high glutathione peroxidase activity. Background technique [0002] Glutathione peroxidase (GPx) is an important selenium-containing enzyme in organisms. Because of its excellent antioxidant properties, it has great potential for the treatment and prevention of Keshan disease, cardiovascular disease, inflammation and cancer. However, due to the instability and limited source of the enzyme, the large molecular weight will cause the human immune response and other shortcomings, which greatly limit the development and application of this enzyme in medicine. Therefore, more and more attention has been paid to the simulation of this enzyme. [0003] The technology closest to the present invention is the selenized subtilisin produced by the Hilvert group, which is a semi-synthetic selenium-containing enzyme with GPx activity. The preparatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N9/50
Inventor 俞慧君罗贵民刘俊秋任晓君沈家骢
Owner JILIN UNIV
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