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Preparation method of magnetic chitosan microsphere immobilized horse radish peroxidases

A technology of horseradish peroxidase and chitosan microspheres, applied in the direction of immobilization on/in the organic carrier, can solve the problem of difficult long-term continuous operation, unfavorable rapid separation and reuse of immobilized enzymes, Liquid fluidity is not good and other problems, to achieve the effect of good magnetic response, high enzyme activity

Inactive Publication Date: 2012-10-03
SOUTHWEST PETROLEUM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when chitosan is used as a carrier, the fluidity of the liquid is not good, and it is difficult to carry out continuous operation for a long time; chitosan microspheres are also not conducive to the rapid separation and reuse of immobilized enzymes.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: by chitosan: the ratio of magnetic fluid mass ratio=3:1 accurately takes by weighing 0.3g chitosan, be dissolved in the 10ml acetic acid solution that mass percent concentration is 2.5%, add 0.1g magnetic fluid, stir Dissolve, and make a homogeneous suspension by ultrasonication and let it stand for degassing for later use. Take 3 g of the suspension and add it dropwise to 60 ml of liquid paraffin containing 3% emulsifier Span-80 in mass percent concentration, and stir and emulsify at a constant temperature of 35 °C at a speed of 3000 rpm for 30 min. After the emulsification is complete, add 0.1ml of a 25% glutaraldehyde solution to solidify. After 3 hours, transfer the reactant to a small beaker. After the magnetic field is separated, discard the supernatant, and wash the surface of the lower layer of microspheres with ether. organic solvent, washed with distilled water, and after magnetic filtration, the product was dried in a vacuum oven at 60° C. to ob...

Embodiment 2

[0016] Embodiment 2: by chitosan: the ratio of magnetic fluid mass ratio=3:1 accurately takes by weighing 0.3g chitosan, is dissolved in the 10ml acetic acid solution that mass percent concentration is 2.5%, adds 0.1g magnetic fluid, stirs Dissolve, and make a homogeneous suspension by ultrasonication and let it stand for degassing for later use. Take 3 g of the suspension and add it dropwise to 60 ml of liquid paraffin containing 3% emulsifier Span-80 in mass percent concentration, and stir and emulsify at a constant temperature of 35 °C at a speed of 3000 rpm for 30 min. After the emulsification is complete, add 0.1 ml of a 25% glutaraldehyde solution to solidify. After 3 hours, transfer the reactant to a small beaker. After the magnetic field is separated, discard the supernatant, and wash the microsphere surface with ether organic solvent, and then washed with distilled water, and after magnetic filtration, the product was dried in a vacuum oven at 60° C. to obtain magneti...

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Abstract

The invention relates to a preparation method of magnetic chitosan microsphere immobilized horse radish peroxidases. The preparation method is used in the fields of enzyme engineering development application and environmental protection, and enzyme can be recycled after enzyme immobilization is carried out. The technical scheme is dissolving polyethylene glycol 4000in water, adding Fe<2+> salt and Fe<3+> salt according to the mass ratio of 1:4, dropwise adding stronger ammonia water until pH of a system is larger than or equal to 10, adjusting the pH to 7.0 by using acetic acid after 2h, and conducting magnetic field separation and washing to obtain magnetofluid; then preparing the acetic acid solution of chitosan magnetofluid according to mass ratio of the chitosan to the magnetofluid of3:1, ultrasonically blending, adding in atoleine containing emulsifier span 80 for emulsifying, adding glutaraldehyde for solidification for 3h, and conducting separation, washing and drying to obtain magnetic chitosan microspheres; and finally adding the magnetic chitosan microspheres in horseradish peroxide enzyme phosphate buffer, solidifying for 3h at 4DEG C, conducting separation, washing and filtering to prepare the magnetic chitosan microsphere immobilized horse radish peroxidases. Carriers of the method are cheap and easy to obtain, enzyme stability is good, and the preparation methodis favorable for rapid and repeated use of the enzyme.

Description

technical field [0001] The invention relates to a preparation method of magnetic chitosan microsphere immobilized horseradish peroxidase which can be reused after the enzyme is immobilized in the field of enzyme engineering development and application and the field of environmental protection. Background technique [0002] Phenolic compounds are protoplasmic poisons, which can directly enter the blood circulation through contact with skin and mucous membranes without detoxification by the liver, causing cell damage and loss of vitality; they can also invade the human body through the oral cavity, causing cell damage. High-concentration phenol solution can coagulate protein and continue to penetrate into the body, causing deep tissue damage, necrosis and even systemic poisoning. [0003] Enzyme treatment technology has the advantages of mild reaction conditions and fast treatment rate, and has attracted increasing attention in the field of water treatment. Horseradish peroxi...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04
Inventor 陈秀丽陈贵任玉婷程方圆
Owner SOUTHWEST PETROLEUM UNIV
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