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CN-121991363-A - Double-amino-sequence functionalized graphene oxide immobilized enzyme and preparation method and application thereof

CN121991363ACN 121991363 ACN121991363 ACN 121991363ACN-121991363-A

Abstract

The invention discloses a double-amino-sequence functionalized graphene oxide immobilized enzyme, and a preparation method and application thereof, and relates to the technical field of graphene oxide immobilized enzymes. The method comprises the steps of grafting polydopamine onto the surface of graphene oxide to obtain a GO@PDA material, reacting the GO@PDA material with an amino functionalization reagent in a first dispersion system, centrifuging, cleaning and drying to obtain a diamino sequential functionalized graphene oxide material, wherein the diamino sequential functionalized graphene oxide material is used for fixing target enzymes, and the amino functionalization reagent is polyethylenimine and/or polyethylenimine. The preparation process of the diamino sequential functionalized graphene oxide material provided by the invention is simple, and the process condition is mild. The immobilized enzyme prepared by the material not only has good reusability, but also has better catalytic performance and stability than free enzyme when the catalytic reaction product is prepared in practical application.

Inventors

  • YU LONGJIANG
  • Gui Yifan
  • XU HANG
  • SUN CHENGYU
  • LU GEN
  • WU YA
  • HU YAOFENG

Assignees

  • 华中科技大学

Dates

Publication Date
20260508
Application Date
20241101

Claims (10)

  1. 1. The preparation method of the diamino sequential functionalized graphene oxide material is characterized by comprising the following steps of: Grafting polydopamine to the surface of graphene oxide to obtain a GO@PDA material; And reacting the GO@PDA material with an amino functional reagent in a first dispersion system, centrifuging, cleaning and drying to obtain the double-amino sequence functionalized graphene oxide material, wherein the double-amino sequence functionalized graphene oxide material is used for fixing target enzymes, and the amino functional reagent is polyethylenimine and/or polyethylenimine.
  2. 2. The preparation method of the diamino sequential functionalized graphene oxide material according to claim 1 is characterized in that graphene oxide is dispersed in a second dispersion system, dopamine hydrochloride is added, the reaction is carried out for 4-20 hours at 25-45 ℃, and the precipitate is centrifugally taken for later use, wherein the second dispersion system is Tris-HCl buffer solution.
  3. 3. The preparation method of the diamino sequential functionalized graphene oxide material according to claim 2, wherein in the preparation method of the GO@PDA material, the reaction condition is 25-35 ℃ for 4-8h.
  4. 4. The preparation method of the diamino sequentially functionalized graphene oxide material according to claim 2, wherein the mass ratio of the graphene oxide to the dopamine hydrochloride is 1 (0.4-1.6).
  5. 5. The preparation method of the diamino sequentially functionalized graphene oxide material according to claim 1, wherein the mass ratio of the GO@PDA material to the amino functionalizing agent is 1 (0.5-2).
  6. 6. A diamino sequential functionalized graphene oxide material prepared by the method of any one of claims 1-5.
  7. 7. Use of a diamino sequential functionalized graphene oxide material according to claim 6 for immobilizing an enzyme of interest.
  8. 8. The use of a bisamino sequentially functionalized graphene oxide material according to claim 7, wherein the enzyme of interest is an enzyme with isoelectric point <7, preferably the enzyme of interest is one or more of xylose dehydrogenase, carbonic anhydrase, lactate dehydrogenase.
  9. 9. An immobilized enzyme is obtained by immobilizing a target enzyme by using the diamino sequential functionalized graphene oxide according to claim 7, preferably the target enzyme is an enzyme with isoelectric point <7, more preferably the target enzyme is one or more of xylose dehydrogenase, carbonic anhydrase and lactate dehydrogenase.
  10. 10. Use of an immobilized enzyme of claim 9 for the catalytic obtainment of a corresponding catalytic reaction product using said immobilized enzyme.

Description

Double-amino-sequence functionalized graphene oxide immobilized enzyme and preparation method and application thereof Technical Field The invention relates to the technical field of graphene oxide immobilized enzymes, in particular to a diamino sequential functionalized graphene oxide immobilized enzyme, and a preparation method and application thereof. Background Enzymes are widely used as bioconversion catalysts because of their high efficiency, specificity, and freedom from contamination. However, free enzymes are limited in industrial applications because of their high cost, poor stability, and difficulty in recovery from the substrate. Immobilization strategies aimed at minimizing limitations, thereby improving the stability and reusability of biocatalysts, are key technologies for most industrial applications. Currently, immobilization of enzymes can be performed by physical adsorption, covalent bonding, entrapment, crosslinking, and the like. The physical adsorption method can keep the natural structure of the enzyme to the greatest extent, has wide application range and is considered as one of the simplest and most economical immobilization methods. Graphene Oxide (GO) is considered as an ideal enzyme immobilization carrier due to its large specific surface area and abundant surface active groups. However, excessive accumulation of negative charge on GO reduces its binding affinity for enzymes. Many industrial enzymes such as formate dehydrogenase, lipase, pectinase, laccase, etc., are enriched in acidic amino acids on their surface, usually by immobilization on positively charged amino-functional materials, to maintain enzyme activity and structural stability. To improve the surface charge properties of GO, its ability to adsorb enzymes can be enhanced by grafting molecules or polymers with specific functional groups. Polydopamine (PDA) is commonly used for material surface modification or as a post-functionalization platform, co-deposition with amine-based functionalizing agents such as polyethylenimine, can increase the functionality of the material surface. In the prior studies, as disclosed in literature "Improved adenylate cyclase activity via affinity immobilization onto co-modified GO with bio-inspired adhesive and PEI"(https://doi.org/https://doi.org/10.1016/j.colsurfb.2021.111888), adenylate cyclase was immobilized on the surface of GO modified by co-deposition of PDA and polyethylenimine, and its catalytic activity was improved. However, this copolymerization process requires consumption of a large amount of active amine groups, resulting in a substantial reduction in the density of surface active amine groups, limiting adsorption of negatively charged enzyme molecules, and affecting the amount of protein immobilized. Therefore, developing an immobilized carrier material with high-density surface active amino groups has important value for improving the immobilized amount and catalytic stability of enzymes, reducing industrial production cost and being better applied to the biological manufacture of large-scale chemicals such as xylonic acid, gluconic acid, lactic acid and the like. Disclosure of Invention The invention provides a double amino sequence functionalized graphene oxide immobilized enzyme, a preparation method and application thereof, wherein PDA is used as an anchoring agent, amino functionalized polymer grafting is carried out on the surface of GO nano composite material, and finally, a large number of surface active amino groups are introduced through sequential modification of the PDA and the amino functionalized polymer, so that the target enzyme is more efficiently immobilized, and the catalytic stability and reusability of the enzyme are improved. Specifically, the method is realized by the following technology. The invention provides a preparation method of a diamino sequential functionalized graphene oxide material, which comprises the following steps: Grafting polydopamine to the surface of graphene oxide to obtain a GO@PDA material; And reacting the GO@PDA material with an amino functional reagent in a first dispersion system, centrifuging, cleaning and drying to obtain the double-amino sequence functionalized graphene oxide material, wherein the double-amino sequence functionalized graphene oxide material is used for fixing target enzymes, and the amino functional reagent is polyethylenimine and/or polyethylenimine. Further, the preparation method of the GO@PDA material comprises the steps of dispersing graphene oxide in a second dispersion system, adding dopamine hydrochloride, reacting for 4-20 hours at 25-45 ℃, and centrifuging to obtain a precipitate for later use, wherein the second dispersion system is Tris-HCl buffer solution. Further, in the preparation method of the GO@PDA material, the reaction condition is that the reaction is carried out for 4-8 hours at 25-35 ℃. Further, the mass ratio of the graphene oxide to the dopamine hydrochloride is 1 (0.4-1.6).