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Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof

A technology of magnetic microspheres and plasmin, applied in directions such as being fixed on/in an organic carrier, can solve the problems of complicated purification process, difficult industrial production, low economic benefit, etc. Ease of operation and simple preparation method

Active Publication Date: 2015-12-30
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] At present, there is no report on the immobilization of earthworm fibrinolytic enzymes by coupling chitosan magnetic microspheres with trypsin inhibitor

Method used

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  • Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof
  • Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof
  • Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof

Examples

Experimental program
Comparison scheme
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 Fe 3 o 4 , ultrasonically dispersed for 15 min; prepared into a chitosan solution with a concentration of 8.1 mg / ml, then added to the oil phase, stirred at 500 rpm for half an hour to make the solution form an emulsion system, and slowly added 10 mL 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, continue to react for 1.5h, stop the reaction, separate with a magnet, wash with petroleum ether, ethanol and deionized water, and dry in vacuum to obtain magnetic chitosan sugar 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 earthworm fibrinolytic enzyme immobilization and a preparation method thereof. Enzymatic activity of the magnetic microsphere is 40-60 U / mg. The preparation method comprises the following steps: firstly dissolving a sagittaria sagittifolia trypsin inhibitor in a buffer solution of pH 7-9 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 4-7 h so as to obtain sagittaria sagittifolia trypsin inhibitor-coupled magnetic chitosan microspheres; adding the sagittaria sagittifolia 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 earthworm fibrinolytic enzyme immobilization. According to the invention, the sagittaria sagittifolia trypsin inhibitor used as ligand is coupled with the magnetic microspheres, and the magnetic microspheres are used in separation and purification of earthworm fibrinolytic enzyme to enhance separation and purification selectivity of earthworm fibrinolytic enzyme. In addition, the magnetic microspheres are 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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IPC IPC(8): C12N11/08
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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