Double-immobilized enzyme and preparation method and application thereof

An immobilized enzyme, dual technology, applied in the direction of immobilization on or in an inorganic carrier, can solve the problems of large difference in immobilization effect, unsatisfactory immobilized enzyme activity, etc., and achieve high environmental stability and repeatability. Use performance, high enzyme activity, high activity effect

Active Publication Date: 2019-01-25
SOUTH CHINA INST OF COLLABORATIVE INNOVATION
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Problems solved by technology

[0005] The special structure and properties of carbon nanomaterials make them ideal enzyme-immobilized materials. The use of carbon nanomaterials to immobilize enzymes has broad application prospects in chemical production, clean energy development, targeted drug delivery, and biosensor preparation. However, How to further improve the immobilization capacity and stability of enzyme molecules on carbon m...

Method used

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  • Double-immobilized enzyme and preparation method and application thereof
  • Double-immobilized enzyme and preparation method and application thereof
  • Double-immobilized enzyme and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048](1) Accurately weigh a certain amount of graphene oxide (GO), use 50mmol / L, pH=6.0 phosphate buffer solution to prepare a dispersion solution with a concentration of 10mg / mL, mechanical stirring (1000r / min) combined with ultrasonic ( 100W) for 1h;

[0049] (2) Prepare an organophosphate hydrolase (OPH) solution with a concentration of 10 mg / mL in 50 mmol / L, pH=6 phosphate buffer solution, and add modifier: N-acryloyloxysuccinimide to the enzyme solution (NAS), after mixing, react the solution at 4°C for 10h, then add monomers to the enzyme solution in sequence: acrylamide (AAM) and N-(3-aminopropyl)-methacrylamide hydrochloride (APM), cross-linking agent: N-methylenebisacrylamide (BIS), after passing through nitrogen for 3 minutes, add initiator: ammonium persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED), initiate In-situ free radical polymerization in water phase, the addition ratio (mass ratio) of the above-mentioned components is OPH:NAS:AAM:APM:BIS:A...

Embodiment 2

[0058] (1) Accurately weigh a certain amount of graphene (GN), use 50mmol / L, pH=9.0 phosphate buffer solution to configure a dispersion solution with a concentration of 5mg / mL, mechanical stirring (100r / min) combined with ultrasonic (200W ) for 2.5 hours;

[0059] (2) Prepare an organophosphate hydrolase (OPH) solution with a concentration of 5 mg / mL in 50 mmol / L, pH=9.0 phosphate buffer solution, and add modifier: N-acryloyloxysuccinimide to the enzyme solution (NAS), after mixing, react the solution at 4°C for 5h, then add monomers to the enzyme solution in sequence: acrylamide (AAM) and N-(3-aminopropyl)-methacrylamide hydrochloride (APM), cross-linking agent: N-methylenebisacrylamide (BIS), after passing nitrogen for 3 minutes, add initiator: ammonium persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED), trigger water Phase in-situ radical polymerization, the addition ratio (mass ratio) of the above-mentioned components is OPH:NAS:AAM:APM:BIS:APS:TEMED=1:0.02...

Embodiment 3

[0064] (1) Accurately weigh a certain amount of single-walled carbon nanotubes (CNTs), use 50mmol / L, pH=7.0 phosphate buffer solution to prepare a dispersion solution with a concentration of 2mg / mL, and mechanically stir (600r / min) to combine Ultrasonic (150W) treatment for 2 hours;

[0065] (2) Prepare an organophosphate hydrolase (OPH) solution with a concentration of 2 mg / mL in a phosphate buffer solution with pH=7.0, and add a modifier: N-acryloyloxysuccinimide (NAS) to the enzyme solution, After mixing, react the solution at 4°C for 2.5 hours, then add monomers to the enzyme solution in sequence: acrylamide (AAM) and N-(3-aminopropyl)-methacrylamide hydrochloride (APM) , Cross-linking agent: N-methylene bisacrylamide (BIS) After passing nitrogen gas for 3 minutes, add initiator: ammonium persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED), trigger the in-situ free Based polymerization reaction, the addition ratio (mass ratio) of the above-mentioned componen...

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Abstract

The invention belongs to the field of immobilized enzyme and discloses a method for double solidification of enzyme by using in-situ free radical polymerization technology and carbon nanometer material. According to the method, firstly, that carbon nano material is dispersed in a phosphate buffer solution to obtain a carbon nano tube dispersion; enzyme nano-capsule solution was prepared by in situradical polymerization to modify and protect enzyme molecules; then adding enzyme nano-capsule solution into the carbon nano-material dispersion, uniformly mixing and standing and, finally, the mixedsolution was separated and washed to obtain a precipitate which was called double immobilized enzyme. Compared with the traditional immobilized enzyme, the double immobilized enzyme system is based on nanocapsules. Compared with the traditional immobilized enzyme, the surface of the enzyme molecule is modified with a polymer layer, which greatly improves the stability of the enzyme molecule. Thedouble-immobilized enzyme obtained by the invention retains high enzyme activity and has higher environmental stability and reusability than free enzyme and traditional immobilized enzyme.

Description

technical field [0001] The invention belongs to the field of immobilized enzymes, in particular to a method and application for preparing double immobilized enzymes by utilizing in-situ radical polymerization technology and carbon nanometer materials. Background technique [0002] Enzymes are special organic substances with catalytic activity and high selectivity, and the chemical nature of most enzymes is protein. Limited by the characteristics of the protein itself, the enzyme is very sensitive to changes in the environment. High temperature, high pressure, heavy metal ions, and too high or too low pH may cause the enzyme to lose its activity. Therefore, it is necessary to immobilize the natural enzyme molecules to improve the structural stability of the enzyme so that it can stably play a catalytic role. [0003] Traditional enzyme immobilization methods mainly include physical adsorption, covalent binding, cross-linking and embedding. To obtain an ideal immobilized enz...

Claims

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Application Information

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IPC IPC(8): C12N11/14
CPCC12N11/14
Inventor 罗志刚陈永志
Owner SOUTH CHINA INST OF COLLABORATIVE INNOVATION
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