A magnetic microsphere of immobilized earthworm fibrinolytic enzyme and its preparation method

A technology of magnetic microspheres and plasmin, applied in directions such as being fixed on/in an organic carrier, can solve the problems of difficult industrial production, complicated purification procedures, low economic benefits, etc., and achieves easy operation and stable pH. The effect of good performance and low equipment requirements

Active Publication Date: 2018-04-06
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, the extraction of fibrinolytic enzyme is separated and purified through a series of steps such as earthworm homogenate, salting out, gel chromatography, ion exchange chromatography and high performance liquid chromatography (HPLC). The purity of fibrinolytic enzyme extracted by this method is High, but due to the complicated purification process and high cost, long time-consuming and low economic benefits, it can only be used for laboratory research, and it is difficult to be used in industrial production. How to obtain stable and highly active earthworm fibrinolytic enzymes can open up opportunities for the utilization of earthworm resources. a road
[0005] There is no report about mung bean trypsin inhibitor coupled chitosan magnetic microspheres for immobilization of earthworm plasmin

Method used

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  • A magnetic microsphere of immobilized earthworm fibrinolytic enzyme and its preparation method
  • A magnetic microsphere of immobilized earthworm fibrinolytic enzyme and its preparation method
  • A magnetic microsphere of immobilized earthworm fibrinolytic enzyme and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Measure 100mL of liquid paraffin and 2mL of span-80 into a 250mL three-neck flask, heat and stir in a water bath at 45°C for 1 hour to make an oil phase; weigh 0.2g of chitosan, dissolve it with 20mL of 3% acetic acid solution, and wait for chitosan After the sugar is completely dissolved, add 0.6g of Fe 3 o 4 , ultrasonically dispersed for 10 min; prepare a chitosan solution with a concentration of 5 mg / ml, then add it to the oil phase, stir at 300 rpm for half an hour to make the solution form an emulsion system, slowly add 3 mL of glutaraldehyde with a mass of 25%, and continue Stir for 1 hour, adjust the pH to about 10 with 1.0 mol / L NaOH solution, continue the reaction for 1 hour, stop the reaction, separate with a magnet, wash with petroleum ether, ethanol and deionized water, and dry in vacuum to obtain magnetic chitosan microspheres .

[0042] The particle size of the magnetic chitosan microspheres is shown in Table 1.

Embodiment 2

[0044] Measure 100mL of liquid paraffin and 3mL of span-80 into a 250mL three-neck flask, heat and stir in a water bath at 30°C for 2 hours to make an oil phase; weigh 0.8g of chitosan, dissolve it with 20mL of 5% acetic acid solution, and wait for chitosan After the sugar is completely dissolved, add 0.6g of Fe 3 o 4 , ultrasonically dispersed for 20min; prepared into a chitosan solution with a concentration of 6.5mg / ml, then added to the oil phase, stirred at 400rpm for half an hour to make the solution form an emulsion system, and slowly added 5mL of glutaraldehyde with a mass of 25%. Continue stirring for 2 hours, adjust the pH to about 9 with 1.0mol / L NaOH solution, stop the reaction after continuing the reaction for 2 hours, separate with a magnet, wash with petroleum ether, ethanol and deionized water, and dry in vacuum to obtain magnetic chitosan microparticles. ball.

[0045] The particle size of the magnetic chitosan microspheres is shown in Table 1.

Embodiment 3

[0047] Measure 100mL of liquid paraffin and 2.5mL of span-80 into a 250mL three-neck flask, heat and stir in a 40°C water bath for 2 hours to make an oil phase; weigh 1g of chitosan, dissolve it with 20mL of 3% acetic acid solution, and wait for chitosan After the sugar is completely dissolved, add 0.6g of Fe3O4 and ultrasonically disperse for 15 minutes; prepare a chitosan solution with a concentration of 8.1mg / ml, then add it to the oil phase, stir at 500rpm for half an hour to make the solution form an emulsion system, and then slowly add 10mL of glutaraldehyde with a mass of 25%, continue to stir for 1.5h, adjust the pH to about 9.5 with 1.0mol / L NaOH solution, stop the reaction after 1.5h, separate with a magnet, wash with petroleum ether, ethanol and deionized water , and vacuum-dried to obtain magnetic chitosan microspheres.

[0048] The particle size of the magnetic chitosan microspheres is shown in Table 1.

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Abstract

The invention discloses a magnetic microsphere for immobilizing earthworm fibrinolytic enzyme and a preparation method thereof. Enzymatic activity of the magnetic microsphere is 25-35 U / mg. The preparation method comprises the following steps: firstly dissolving a mung bean trypsin inhibitor in a buffer solution of pH 3-9 so as to obtain a protease buffer solution, adding the protease buffer solution into activated magnetic chitosan microspheres, carrying out water-bath shaking and coupling for 2-14 h so as to obtain mung bean trypsin inhibitor-coupled magnetic chitosan microspheres; adding the mung bean trypsin inhibitor-coupled magnetic chitosan microspheres into a crude enzyme solution of earthworm fibrinolytic enzyme and shaking in a thermostatic shaking bath of 30-60 DEG C and carrying out a curing reaction for 0.5-3 h; repeatedly washing after the reaction until no protein is detected from the washing liquid, and carrying out magnetic separation and collection so as to obtain the magnetic microsphere for immobilizing earthworm fibrinolytic enzyme. According to the invention, the mung bean trypsin inhibitor used as ligand is coupled with the magnetic microsphere, and the magnetic microsphere is used in separation and purification of earthworm fibrinolytic enzyme to enhance separation and purification selectivity of earthworm fibrinolytic enzyme. In addition, the magnetic microsphere is easy to separate from earthworm fibrinolytic enzyme.

Description

technical field [0001] The invention belongs to the technical field of magnetic microspheres, and in particular relates to a preparation method of magnetic microspheres for immobilizing earthworm fibrinolytic enzyme. Background technique [0002] Magnetic polymer microspheres are a new type of magnetic material developed in recent years. They are composite microspheres with certain magnetic properties and special structures formed by combining magnetic inorganic particles and organic polymers by appropriate methods. Magnetic composite microspheres not only have many characteristics of ordinary polymer microspheres but also have magnetic responsiveness, so not only can they be endowed with surface functional groups (such as -OH, -COOH, -CHO, -NH 2 , etc.), it can also have a guiding function under the action of an external magnetic field. Magnetic microspheres have broad application prospects in the fields of immobilized enzymes, cell separation, protein separation and purif...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10
Inventor 赵钟兴雷敬玲王朝阳苗剑王欣辉陶萌良谢美萱
Owner GUANGXI UNIV
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