Method for constructing artificial multi-enzyme system based on lanthanide series nucleotide compound and DNA (Deoxyribonucleic Acid) oriented immobilization technology

Abstract

The invention provides a method for constructing an artificial multi-enzyme system based on a lanthanide series nucleotide compound and a DNA (Deoxyribonucleic Acid) oriented immobilization technologyand belongs to the field of preparation of immobilized enzymes. The method comprises the following steps: firstly, preparing single-stranded DNA modified magnetic nanoparticles and complementary single-stranded DNA functionalized glucose oxidase; then mixing the magnetic nanoparticles with the enzyme; carrying out DNA complementary hybridization to realize the immobilization of the enzyme; commonly hatching the immobilized enzyme, cerium nitrate hexahydrate and disodium adenosine 5'-phosphate; carrying out self-assembling on a lanthanide series nucleotide coordinated polymer to realize the encapsulation of the immobilized enzyme and constructing the artificial multi-enzyme system. An encapsulation vector can realize the effect of protecting the immobilized enzyme and is also used as an analogue enzyme to form an enzyme cascade system with the immobilized natural enzyme. A preparation process of the artificial multi-enzyme system, provided by the invention, is simple, has moderate conditions and achieves high enzyme activity; the artificial multi-enzyme system is easy to separate from a reaction system and has excellent stability and reusability; meanwhile, the analogue enzyme is introduced and the cost is extremely reduced.

Description

technical field

[0001] The invention belongs to the technical field of immobilized multi-enzyme system preparation, and in particular relates to a method for encapsulating the enzyme with a lanthanide nucleotide coordination polymer immobilizing the enzyme through a double-stranded DNA complementation-mediated technology. Background technique

[0002] Enzymes are efficient biocatalysts with high substrate specificity and mild reaction conditions. In organisms, a multi-enzyme system is usually composed of a variety of different enzymes to achieve a variety of physiological processes through cascade catalytic reactions. Inspired by nature, researchers have devoted themselves to the development of artificial multi-enzyme systems in which natural enzymes or nanomimetic enzymes are co-immobilized to achieve complex functions. However, enzymes immobilized on carriers are usually difficult to be protected by the carrier, limited by their rapid inactivation in non-physiological env...

Examples

Embodiment 1

[0033] (1) Weigh 0.5g Fe 3 o 4 @SiO 2 Add 48 mL of methanol and 2 mL of (3-aminopropyl) triethoxysilane (APTES) to a 100 mL flask of nanoparticles, and react at room temperature for 12 h. After the reaction, the product was ultrasonically cleaned three times with ethanol, and baked in a vacuum oven at 50°C for 3 hours to obtain Fe 3 o 4 @SiO 2 @APTES Magnetic nanoparticles.

[0034] (2) 25 mg of magnetic nanoparticles synthesized in (1) were incubated with 4.5 mL of phosphate buffer (PBS, 20 mM, pH 8.0) and 0.5 mL of glutaraldehyde solution (50% wt) at 25° C. for 2.5 h. Magnetic separation was performed after the reaction, and the product was washed once with buffer A (10 mM PBS, pH 7.4, 0.1 M NaCl, 0.05% Tween-20), and then washed twice with 20 mM PBS to obtain MNPs.

[0035] (3) Preparation of single-stranded DNA modified magnetic nanoparticles (MNPs@sspDNA)

[0036] Add 300 μL buffer B to 1OD sspDNA, and vortex until the DNA is completely dissolved. 150 μL of DNA solu...

Embodiment 2

[0040] Example 2: Condition optimization and kinetic investigation of artificial multi-enzyme system

[0041] (1) The thickness, temperature and pH of nucleotide coordination polymer supramolecular nano-coating all have influence on the activity and the stability of artificial multi-enzyme system, so the present invention investigates the reaction conditions of the artificial multi-enzyme system prepared .

[0042] (2) Prepare artificial multi-enzyme systems (0.5 mg) with coordination polymer self-assembly reaction times of 2, 4, 6, and 8 hours, and enzymatically catalyze substrates 100 mM glucose and 0.5 mM 2,2'- Azizo-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was reacted on a shaking table for 10min, 400rpm, and the effect of the ratio of the two enzymes on the immobilized enzyme system was investigated. The absorbance of the product at 415 nm was measured with a UV-Vis spectrophotometer. Meanwhile, the package thickness of the multi-enzyme system was analyzed by t...

Embodiment 3

[0046] Embodiment 3: Artificial multi-enzyme system reusability and stability test

[0047] (1) Preparation of artificial multi-enzyme system: Same as Example 1.

[0048] (2) Reusability investigation: prepare 1mL of 100mM glucose and 0.5mM ABTS substrate solution, add 0.5mg of the artificial multi-enzyme system synthesized in Example 3, and react on a shaking table for 10min at 37°C and 400rpm.

[0049] (3) After the reaction, measure the absorbance of the product supernatant at 415nm with a UV-Vis spectrophotometer, and the artificial multi-enzyme system in (2) is fully washed with 10mM PBS (pH 7.4, 0.1M NaCl) to remove its surface Add 1mL of 100mM glucose and 0.5mM ABTS substrate solution to the sticky substrate solution, react on a shaking table for 10min, 37°C, 400rpm, measure the absorbance of the product at 415nm with a UV-Vis spectrophotometer. 1mL 100mM glucose and 0.5mM ABTS substrate solution were repeatedly catalyzed in batches to investigate the reusability of th...

technical field

[0001] The invention belongs to the technical field of immobilized multi-enzyme system preparation, and in particular relates to a method for encapsulating the enzyme with a lanthanide nucleotide coordination polymer immobilizing the enzyme through a double-stranded DNA complementation-mediated technology. Background technique

[0002] Enzymes are efficient biocatalysts with high substrate specificity and mild reaction conditions. In organisms, a multi-enzyme system is usually composed of a variety of different enzymes to achieve a variety of physiological processes through cascade catalytic reactions. Inspired by nature, researchers have devoted themselves to the development of artificial multi-enzyme systems in which natural enzymes or nanomimetic enzymes are co-immobilized to achieve complex functions. However, enzymes immobilized on carriers are usually difficult to be protected by the carrier, limited by their rapid inactivation in non-physiological env...

Examples

Embodiment 1

[0033] (1) Weigh 0.5g Fe 3 o 4 @SiO 2 Add 48 mL of methanol and 2 mL of (3-aminopropyl) triethoxysilane (APTES) to a 100 mL flask of nanoparticles, and react at room temperature for 12 h. After the reaction, the product was ultrasonically cleaned three times with ethanol, and baked in a vacuum oven at 50°C for 3 hours to obtain Fe 3 o 4 @SiO 2 @APTES Magnetic nanoparticles.

[0034] (2) 25 mg of magnetic nanoparticles synthesized in (1) were incubated with 4.5 mL of phosphate buffer (PBS, 20 mM, pH 8.0) and 0.5 mL of glutaraldehyde solution (50% wt) at 25° C. for 2.5 h. Magnetic separation was performed after the reaction, and the product was washed once with buffer A (10 mM PBS, pH 7.4, 0.1 M NaCl, 0.05% Tween-20), and then washed twice with 20 mM PBS to obtain MNPs.

[0035] (3) Preparation of single-stranded DNA modified magnetic nanoparticles (MNPs@sspDNA)

[0036] Add 300 μL buffer B to 1OD sspDNA, and vortex until the DNA is completely dissolved. 150 μL of DNA solu...

Embodiment 2

[0040] Example 2: Condition optimization and kinetic investigation of artificial multi-enzyme system

[0041] (1) The thickness, temperature and pH of nucleotide coordination polymer supramolecular nano-coating all have influence on the activity and the stability of artificial multi-enzyme system, so the present invention investigates the reaction conditions of the artificial multi-enzyme system prepared .

[0042] (2) Prepare artificial multi-enzyme systems (0.5 mg) with coordination polymer self-assembly reaction times of 2, 4, 6, and 8 hours, and enzymatically catalyze substrates 100 mM glucose and 0.5 mM 2,2'- Azizo-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was reacted on a shaking table for 10min, 400rpm, and the effect of the ratio of the two enzymes on the immobilized enzyme system was investigated. The absorbance of the product at 415 nm was measured with a UV-Vis spectrophotometer. Meanwhile, the package thickness of the multi-enzyme system was analyzed by t...

Embodiment 3

[0046] Embodiment 3: Artificial multi-enzyme system reusability and stability test

[0047] (1) Preparation of artificial multi-enzyme system: Same as Example 1.

[0048] (2) Reusability investigation: prepare 1mL of 100mM glucose and 0.5mM ABTS substrate solution, add 0.5mg of the artificial multi-enzyme system synthesized in Example 3, and react on a shaking table for 10min at 37°C and 400rpm.

[0049] (3) After the reaction, measure the absorbance of the product supernatant at 415nm with a UV-Vis spectrophotometer, and the artificial multi-enzyme system in (2) is fully washed with 10mM PBS (pH 7.4, 0.1M NaCl) to remove its surface Add 1mL of 100mM glucose and 0.5mM ABTS substrate solution to the sticky substrate solution, react on a shaking table for 10min, 37°C, 400rpm, measure the absorbance of the product at 415nm with a UV-Vis spectrophotometer. 1mL 100mM glucose and 0.5mM ABTS substrate solution were repeatedly catalyzed in batches to investigate the reusability of th...