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CN-121975789-A - Preparation method of magnetic covalent immobilized enzyme carrier

CN121975789ACN 121975789 ACN121975789 ACN 121975789ACN-121975789-A

Abstract

The invention discloses a preparation method of a magnetic covalent immobilized enzyme carrier, which comprises the steps of adding polyethyleneimine into Fe 3 O 4 microsphere dispersion liquid coated with silicon to obtain modified Fe 3 O 4 microspheres, dispersing the modified Fe 3 O 4 microspheres in a solvent, adding dialdehyde cellulose, and reacting to obtain the magnetic covalent immobilized enzyme carrier. The amino group of the modified Fe 3 O 4 microsphere can be subjected to a condensation reaction with the aldehyde group of dialdehyde cellulose to generate Schiff base groups which are reacted with unreacted aldehyde groups, amino groups and other groups together, so that the enzyme can be effectively immobilized on the surface of the magnetic carrier. The preparation method of the magnetic covalent immobilized enzyme carrier has the advantages of simple process, easy control and operation of reaction, low operation cost, high enzyme loading amount of the immobilized enzyme carrier, high biocompatibility, good dispersion effect, good stability, recovery of immobilized enzyme activity and the like.

Inventors

  • XU CHENBING
  • LIN WEI
  • LI KAI
  • ZHU HONGTAO
  • LIANG GUOBIN
  • WU HAIYAN
  • BAO TIANTIAN

Assignees

  • 江苏万年长药业有限公司
  • 江苏理工学院

Dates

Publication Date
20260505
Application Date
20260209

Claims (10)

  1. 1. A preparation method of a magnetic covalent immobilized enzyme carrier is characterized by comprising the following steps of, Adding polyethyleneimine into the Fe 3 O 4 microsphere dispersion liquid coated with silicon to obtain modified Fe 3 O 4 microspheres; dispersing the modified Fe 3 O 4 microsphere in a solvent, adding dialdehyde cellulose, and reacting to obtain the magnetic covalent immobilized enzyme carrier.
  2. 2. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 1, wherein the concentration of the Fe 3 O 4 microsphere dispersion liquid coated with silicon is 0.02-0.05 g/mL; The addition amount of the polyethyleneimine is 0.1-0.3 times of the mass of the Fe 3 O 4 microsphere coated with silicon; The ratio of the modified Fe 3 O 4 microsphere to the solvent to the dialdehyde cellulose is 10-30 g, 1000-2500 mL and 0.5-1 g.
  3. 3. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 1, wherein the method for preparing the silicon-coated Fe 3 O 4 microsphere comprises the following steps of, Dissolving ferric salt in a mixed solvent of water and alcohol to obtain ferric salt solution; Adding sodium citrate, sodium acetate and a structure regulator into an iron salt solution to obtain a mixed solution, and then carrying out hydrothermal reaction to obtain Fe 3 O 4 microspheres; Dispersing Fe 3 O 4 microsphere in water to obtain Fe 3 O 4 microsphere dispersion, adding sodium silicate aqueous solution, and reacting to obtain Fe 3 O 4 microsphere coated with silicon.
  4. 4. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 3, wherein the ratio of ferric salt, water and alcohol in the ferric salt solution is 10-20 g to 100-300 mL to 500-900 mL; the mass ratio of the ferric salt in the ferric salt solution to the sodium citrate, the sodium acetate and the structure regulator is 1:0.2-0.5:0.8-1.5:0.1-0.3; The structure regulator is formed by mixing carboxyl polyethylene glycol dansyl and disodium ethylenediamine tetraacetate.
  5. 5. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 4, wherein the mass ratio of carboxyl polyethylene glycol dansyl to disodium ethylenediamine tetraacetate in the structure regulator is 1-3:1.
  6. 6. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 3, wherein the volume ratio of the Fe 3 O 4 microsphere dispersion liquid to the sodium silicate aqueous solution is 8-10:1; The concentration of the Fe 3 O 4 microsphere dispersion liquid and the sodium silicate aqueous solution is 0.01-0.03 g/mL.
  7. 7. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 3, wherein the hydrothermal reaction is continued for 6-12 hours at 180-220 ℃.
  8. 8. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 1, wherein the method for preparing dialdehyde cellulose comprises, Dispersing alpha-cellulose in water, adding sodium periodate for oxidation reaction, and collecting insoluble substances to obtain dialdehyde cellulose.
  9. 9. The method for preparing a magnetic covalent immobilized enzyme carrier according to claim 8, wherein the ratio of the alpha-cellulose to water is 3-5 g/120-200 ml.
  10. 10. A magnetic covalent immobilized enzyme carrier characterized in that it is obtained by the preparation method according to any one of claims 1 to 9.

Description

Preparation method of magnetic covalent immobilized enzyme carrier Technical Field The invention relates to the technical field of enzyme carriers, in particular to a preparation method of a magnetic covalent immobilized enzyme carrier. Background The immobilized enzyme is a technology which limits the free enzyme in a certain space area by a physical or chemical method, continuously catalyzes the specific reaction of the free enzyme and can recycle the immobilized enzyme. The choice of a suitable immobilization carrier is also critical for the immobilization of the enzyme, and carriers for immobilizing the enzyme are generally required to be non-toxic, stable and have good biocompatibility. Common immobilization carriers are mesoporous silicon materials, nano carbon materials, magnetic particles, natural organic polymers and the like. The carrier prepared from the magnetic material can simplify the treatment process, efficiently recover the enzyme preparation in the catalytic reaction, particularly the ferric oxide nano particles, has the advantages of excellent physical and chemical properties and no toxicity, and meanwhile, the nano structure and the good biocompatibility of the carrier are easier for surface modification of the enzyme, so that the carrier has been widely used for the carrier of the enzyme. For different enzymes, the suitable magnetic nanomaterials and the magnetic drive fixation methods are also different. The surface functionalization modification (modification of silane groups, organic polymers, mesoporous materials and metal-organic framework materials) can improve the combination efficiency of the magnetic carrier and enzyme molecules and regulate the dispersibility of the magnetic nanoparticles, and is an important step for improving the properties of the magnetic nanoparticles. CN110343693a discloses a magnetic immobilized enzyme carrier and a preparation method thereof, wherein magnetic nano-particle Fe 3O4 is used as a matrix material, low-cost catechol and polyamine (PCPA) binary compound is used for replacing expensive dopamine to modify the surface of the Fe 3O4 carrier, and chain molecule Ethylene Glycol Diglycidyl Ether (EGDE) is used as a flexible spacer arm ligase and carrier, so as to obtain Fe 3O4Dopamine-like PCPAThe immobilized enzyme carrier with the space interval arm EGDE three-layer structure has the advantages of superparamagnetic performance, renewable utilization, good dispersion effect, high enzyme load and immobilized enzyme activity recovery. Catechol, however, is highly toxic and irritating. CN113462680a discloses the preparation of magnetically immobilized penicillin G acylase doped with divalent manganese ions, comprising (1) preparing magnetic nanoparticles ferroferric oxide @ beta doped with divalent manganese ionsCyclodextrin (2) 3Ethanol solution of glycidol ether oxygen propyl trimethoxy silane and magnetic nanoparticle ferroferric oxide @ beta doped with divalent manganese ionsThe cyclodextrin is mixed for reaction to obtain the magnetic nano particle ferroferric oxide @ beta doped with bivalent manganese ionsCyclodextring3Glycidyl ether oxypropyl trimethoxy silane carrier, and (3) magnetic nanometer particle ferroferric oxide @ beta doped with bivalent manganese ion dissolved in phosphate buffer solutionCyclodextring3The glycidol ether oxygen propyl trimethoxy silane carrier is added into the free penicillin G acylase solution for oscillating reaction. This method can improve the carrier recovery rate, as well as the enzyme activity recovery rate and reusability, however the enzyme loading is not high. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a preparation method of a magnetic covalent immobilized enzyme carrier, which comprises the following steps of, Adding polyethyleneimine into the Fe 3O4 microsphere dispersion liquid coated with silicon to obtain modified Fe 3O4 microspheres; dispersing the modified Fe 3O4 microsphere in a solvent, adding dialdehyde cellulose, and reacting to obtain the magnetic covalent immobilized enzyme carrier. The use of Fe 3O4 microspheres is limited to a certain extent because it is subject to oxidation when exposed to air and corrosion in acidic environments. The silicon dioxide has extremely high stability and chemical inertness and is easy to modify, so the basic carrier of the invention is selected as Fe 3O4 microsphere coated with silicon. The polyethyleneimine contains amino groups and is a water-soluble high molecular polymer. And (3) carrying out amination modification on the Fe 3O4 microsphere coated with silicon by using polyethyleneimine. The alpha-cellulose is prepared by immersing a cellulose raw material in 17.5% or 18% sodium hydroxide solution at 20 ℃ for 45 minutes to obtain the insoluble part. Dialdehyde cellulose can be obtained by oxidizing the C2 and C3 bonds in the hydrogen-free glucose units in alpha-cellulose by sodium periodate to generate t