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A reversible dual-enzyme co-immobilization method with adjustable enzyme ratio

A technology for regulating the ratio of enzymes, which is applied to biochemical equipment and methods, and enzymes immobilized on or in inorganic carriers, can solve the problem of affecting the performance and stability of immobilized enzyme systems, difficult to control different enzyme ratios, etc. problems, to achieve the effects of saving preparation costs, maintaining activity, and mild immobilization reaction conditions

Active Publication Date: 2020-10-02
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Traditional methods of immobilizing enzymes are basically based on directly immobilizing enzymes on the surface of modified magnetic nanoparticles through covalent or non-covalent methods. This immobilization method is usually irreversible and will affect the stability of the immobilized enzyme system. performance and stability
In the multi-enzyme immobilization system, when the corresponding enzymes have the same or similar structure, it is still difficult to precisely control the ratio of different enzymes by directly immobilizing enzymes on the surface of multifunctional magnetic nanoparticles by traditional methods.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Establishment of a multi-enzyme immobilization system with adjustable enzyme ratio

[0032] (1) Ultrasonic disperse 25 mg of magnetic nanoparticles (MP) with a particle size of 12-18 nm in 5 mL of anhydrous methanol, add 5.3 mM dopamine (DA) and dopamine maleimide derivatives (MA) , the mixture was ultrasonically reacted at room temperature for 1 h; after the reaction, the product (DM@MP) was washed three times with deionized water, soaked in deionized water, and stored at 4 °C for subsequent use.

[0033] (2) 0.5 OD 5’ carboxyl-modified single-stranded DNA (P1) was dissolved in 25 mM MES (pH 6.0) buffer solution, and then 500 μL of 20 mg mL was added -1 NHS and 40 mg mL -1 EDC solution, the mixture was reacted on a shaker at 29°C for 20 min; after the reaction, the above reaction solution was added to 5 mL DM@MP (5 mg mL -1 ) dispersion solution, placed in a shaking table for reaction at 29°C for 6 h; after the reaction, washed 3 times with 10 mM PBS (0.1 ...

Embodiment 2

[0039] Example 2: Regulating the ratio of enzymes in the multi-enzyme immobilization system.

[0040] (1) Preparation of immobilized multi-enzyme system with different enzyme ratios: the specific steps are the same as in Example 1. Adjust the molar ratios of DA and MA, P1 and P2, C1-GOx and C2-HRP, and prepare GOx:HRP molar ratios as 1:4, 3:4, 5:4, 8:4, 10:4, 13: 4 GOx-HRP@DM@MP.

[0041] (2) Prepare 5 mM β-Glucose substrate solution, 0.5 mg GOx-HRP@DM@MP synthesized in Example 1, add 1 mL β-Glucose solution (containing 1 mM TMB), and react on a shaking table at 37°C for 5 min . After the reaction, GOx-HRP@DM@MP was separated by using a magnetic field, and 20 μL of 2 M sulfuric acid solution was added to the supernatant, and its absorbance at 450 nm was detected by a UV spectrophotometer, and the relative activity of the enzymes was compared according to the absorbance. active.

[0042] After investigation, the GOx-HRP multi-enzyme immobilization system prepared by the pre...

Embodiment 3

[0043] Example 3: Enzymatic properties of the multi-enzyme immobilization system

[0044] (1) Preparation of the immobilized multi-enzyme system: same as Example 1.

[0045] (2) Prepare a series of concentrations of β-Glucose substrate solution, 0.5 mg GOx-HRP@DM@MP synthesized in Example 1, add 1 mL β-Glucose solution (containing 1 mM TMB), shake at 37 °C bed reaction for 5 min. Add 20 μL of 2 M sulfuric acid solution to the supernatant, and detect its absorbance at 450 nm. The enzymatic properties of free GOx-HRP and GOx-HRP@APTES@MP were determined by the same method, and compared with GOx-HRP@DM@MP.

[0046] The analysis results showed that the Michaelis constant of GOx-HRP@DM@MP was 1.41 mM, that of free GOx-HRP was 2.63 mM, and that of GOx-HRP@APTES@MP was 1.92 mM. Compared with other GOx-HRP multi-enzyme immobilization systems and free GOx-HRP, the Michaelis constant value of the GOx-HRP multi-enzyme immobilization system prepared by the present invention is smaller,...

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Abstract

The invention relates to a reversible double enzyme co-immobilization method capable of regulating and controlling enzyme ratio. By the method, various kinds of enzyme are fixed on the surfaces of multifunctional magnetic nanoparticles modified by dopamine and derivative thereof through a DNA mediated immobilization technology, the method is mild in synthesis condition, and the immobilization quantity of different enzyme in a multi-enzyme reaction system can be controlled by adjusting the number of the functional groups on the surfaces of the magnetic nanoparticles. The immobilized multi-enzyme system established by the method has the advantages of high activity, high stability and the like.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme preparation, and in particular relates to a reversible double-enzyme co-immobilization method with adjustable enzyme ratio, especially on the surface of multifunctional magnetic nanoparticles modified by dopamine and dopamine derivatives through DNA complementation-mediated immobilization multienzyme method. Background technique [0002] Enzyme is a kind of macromolecule substance produced in organisms with biocatalytic function, which can effectively control specific chemical reactions in vivo or in vitro. In recent years, enzymes have replaced many traditional chemical catalysts and have been widely used in research and production because of their high catalytic efficiency, wide variety, mild reaction conditions, high selectivity, and environmental protection. [0003] However, due to the lack of long-term stability of free enzymes in the reaction process, the high cost of recovery a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N11/18
CPCC12N9/0006C12N11/14C12N11/18C12Y101/03004
Inventor 杨屹杨烨苏萍
Owner BEIJING UNIV OF CHEM TECH
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