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Reversible double enzyme co-immobilization method capable of regulating and controlling 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: 2018-03-06
BEIJING UNIV OF CHEM TECH
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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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  • Reversible double enzyme co-immobilization method capable of regulating and controlling enzyme ratio

Examples

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Effect test

Embodiment 1

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

[0032] (1) 25 mg of magnetic nanoparticles (MP) with a particle size of 12-18 nm were ultrasonically dispersed in 5 mL of anhydrous methanol, and 5.3 mM dopamine (DA) and dopamine maleimide derivatives (MA) were added. , the mixture was sonicated for 1 h at room temperature; 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, followed by adding 500 μL of 20 mg mL -1 of NHS and 40 mg mL -1 After the reaction was completed, the above reaction solution was added to 5 mL DM@MP (5 mg mL -1 ) dispersion, placed on a shaker for reaction at 29°C for 6 h; after the reaction, washed 3 times with 10 mM PBS (0.1 M NaCl, pH 7.4), and added 0.5 OD 5' thiol-modified single-s...

Embodiment 2

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

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

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

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

Embodiment 3

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

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

[0045] (2) Prepare a series of concentrations of β-Glucose substrate solution, 0.5 mg of GOx-HRP@DM@MP synthesized in Example 1, add 1 mL of β-Glucose solution (containing 1 mM TMB), and shake at 37 °C. bed reaction for 5 min. 20 μL of 2 M sulfuric acid solution was added to the supernatant, and its absorbance at 450 nm was detected. 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 show that the Michaelis constant of GOx-HRP@DM@MP is 1.41 mM, the Michaelis constant of free GOx-HRP is 2.63 mM, and the Michaelis constant of GOx-HRP@APTES@MP is 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 sys...

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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 dual-enzyme co-immobilization method capable of regulating the ratio of enzymes, in particular to the surface of multifunctional magnetic nanoparticles modified by dopamine and dopamine derivatives through DNA complementary mediated immobilization method of polyenzymes. Background technique [0002] Enzyme is a kind of macromolecular substance with biocatalytic function produced in vivo, 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 due to 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 the free enzyme during the reaction process, the high cost...

Claims

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Application Information

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Patent Type & Authority Applications(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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