A method for constructing an immobilized multi-enzyme system based on the interaction between DNA, graphene oxide and metal-organic framework materials

A metal-organic framework and enzyme system technology, applied in the field of immobilized multi-enzyme system preparation, can solve the problems of low efficiency of double-enzyme cascade reaction and easy enzyme shedding, etc., achieve simple, gentle and efficient immobilization method, and improve easy enzyme shedding , the effect of improving the catalytic efficiency of enzymes

Active Publication Date: 2022-05-10
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of Janus particle property based on ssDNA, utilize the π-π stacking effect of its nucleic acid base and graphene oxide (GO) and the coordination of phosphoric acid skeleton and metal zirconium cluster in PCN-222 to combine enzyme The method of immobilizing on the dual carrier to overcome the problems of the traditional adsorption method that the enzyme is easy to fall off and the efficiency of the double enzyme cascade reaction is not high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Synthesis of Metal Organic Framework - PCN-222 Nanoparticles.

[0027] Add DMF (0.84mol) to ultrasonically dissolve zirconium oxychloride octahydrate (0.47mmol) and meso-tetrakis(4-carboxyphenyl) porphine (32.8μmol), then add dichloroacetic acid (12.0mmol) and use Glass rod to stir well. The solution was transferred to the reaction kettle, and reacted at 130°C for 18h to obtain deep purple crystals and yellow mother liquor; The ratio is 76:13:30800:780; the nanocrystals are collected by centrifugation (11500rpm, 20min), and then solvent exchanged three times with DMF, the obtained PCN-222 nanoparticles are alternately washed twice with ethanol and buffer A, centrifuged (11500rpm , 20min) to remove the waste liquid, add buffer A to disperse evenly, and store at 4°C for use.

Embodiment 2

[0028] Example 2: Enzyme-ssDNA complex (GO X - Preparation of ssDNA, HRP-ssDNA).

[0029] will go X Solution (10mg mL -1 , 500μL) and suflo-SMCC solution (5mg mL -1 , 500μL) were mixed and incubated in a shaker for 2h (37°C, 400rpm); the mass ratio of suflo-SMCC and GOx was 1:2; after the reaction, the mixture was filtered and washed 6 times with a 10K ultrafiltration tube (10min / time, 8100xg), remove unreacted suflo-SMCC. After centrifuging 1OD ssDNA (24base) (6min, 12000rpm), add buffer A and vortex until completely dissolved; ssDNA (0.11mg mL -1 , 300μL) and TCEP (0.87mg mL -1 , 120μL) and incubate in a shaker for 2h (37°C, 400rpm); the mass ratio of ssDNA to TCEP was 33:104; after the reaction, the mixture was filtered and washed 6 times with a 3K ultrafiltration tube (20min / time, 8100xg) , to remove TCEP that did not participate in the reaction. Filtered GO X The solution and the ssDNA solution were mixed and incubated on a shaker for 12h (29°C, 400rpm). After the ...

Embodiment 3

[0030] Example 3: Construction of an immobilized multi-enzyme system based on the interaction between DNA, graphene oxide and PCN-222.

[0031] (1) Synthesis and GO of PCN-222 X -Preparation of ssDNA and HRP-ssDNA: Same as Example 1 and Example 2.

[0032] (2) PCN-222 prepared in Example 1 and GO prepared in Example 2 X - ssDNA complex, HRP-ssDNA complex and GO solution were mixed and incubated in a shaker for 3h (29°C, 400rpm); PCN-222, GO X - The mass ratio of ssDNA complex, HRP-ssDNA complex and GO is 200:17:17:180; after the reaction, the reaction solution is centrifuged (11500 rpm, 25 min), and the supernatant is removed. The product was washed twice with buffer A (3 mL / time), and centrifuged (11500 rpm, 20 min) to obtain the immobilized double enzyme (PCN-222@GO@GOx@HRP), which was evenly dispersed in buffer A and stored in 4 ℃ for later use.

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Abstract

A method for constructing an immobilized multi-enzyme system based on the interaction between DNA, graphene oxide and metal-organic framework materials belongs to the field of immobilized enzyme preparation. Steps of the present invention: first prepare metal-organic framework material PCN-222 nanoparticles and single-stranded DNA (ssDNA) functionalized glucose oxidase (GO X ) and horseradish peroxidase (HRP); followed by preparing a uniformly dispersed graphene oxide (GO) solution; then PCN‑222 nanoparticles, GO X ‑ssDNA complexes, HRP‑ssDNA complexes were incubated with GO solution; through the Janus particle properties of ssDNA, that is, the π‑π stacking effect of nucleic acid bases and GO aromatic domains and the coordination of phosphate backbones and metal zirconium clusters on PCN‑222 The function is to immobilize the dual enzymes on the dual carrier to construct an immobilized multi-enzyme system. The invention overcomes the problems of low adsorption efficiency and weak binding of traditional immobilized enzymes, and has simple preparation process and high cascade efficiency.

Description

technical field [0001] The invention belongs to the technical field of immobilized multi-enzyme system preparation, and specifically relates to the Janus particle properties of single-stranded DNA (ssDNA), utilizing the π-π stacking action of its nucleic acid base and graphene oxide (GO) and the phosphoric acid skeleton and PCN- 222 The method of immobilizing the enzyme by the coordination of metal zirconium clusters. Background technique [0002] Enzyme is a natural and efficient catalyst with high selectivity, low pollution and mild reaction conditions. In nature, we can often observe a large number of enzyme cascade reactions in the process of cell metabolism. Because this multi-enzyme cascade system can directly reduce production costs and improve production efficiency by eliminating the tedious separation and purification of reaction intermediates, it is widely used in industrial production such as food processing, pharmaceutical synthesis, bioenergy, and pollutant deg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N9/08C12N11/089C12N11/14
CPCC12N9/0006C12N9/0065C12Y101/03004C12Y111/01007C12N11/14C12N11/08
Inventor 杨屹贺雯婷苏萍宋佳一沈昊周梓昕
Owner BEIJING UNIV OF CHEM TECH
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