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Covalent immobilized cell as well as preparation method and application thereof

An immobilized cell and covalent technology, applied in the field of bioengineering, can solve the problems of inapplicability to large-scale production, difficulty in separation and purification, and difficulty in repeated use, and achieve good biodegradability, good biocompatibility, and guaranteed enzyme The effect of the activity

Pending Publication Date: 2021-09-28
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the free enzyme obtained through separation and purification is used for biocatalysis, there are disadvantages such as poor stability, difficulty in recovery, and difficulty in repeated use, and the residual enzyme causes difficulties in the separation and purification of subsequent products.
In the process of biocatalysis, although free cells can be recycled by high-speed centrifugation or filtration, the operation is complicated and the mechanical properties are poor, which is not suitable for large-scale production.

Method used

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  • Covalent immobilized cell as well as preparation method and application thereof
  • Covalent immobilized cell as well as preparation method and application thereof
  • Covalent immobilized cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of covalently immobilized cells

[0035] (1) Preparation of alkyne-functionalized magnetic nanoparticles:

[0036] Fe 2+ and Fe 3+ Preparation of Fe by co-precipitation method at a ratio of 1:3 3 o 4 Magnetic nanoparticles, take 1g Fe 3 o 4 Nanoparticles 3 mL tetraethyl silicate, stirred for 5 hours to obtain Fe 3 o 4 @SiO 2 nanoparticles.

[0037] to 0.5 mg Fe 3 o 4 @SiO 2 Add 10 mL of 3-aminopropyltriethoxysilane to the nanoparticles, stir vigorously for 24 hours in the anaerobic environment, then add 400 μL of propiolic acid, and react overnight in the anaerobic condition to obtain alkyne-functionalized magnetic Nanoparticles, denoted as Fe 3 o 4 @SiO 2 -NH 2 -alkyne.

[0038] (2) Preparation of recombinant Escherichia coli after azido modification:

[0039] Recombinant Escherichia coli containing the glycerol dehydrogenase gene in KDO-N 3 Cultured in culture medium, adding IPTG with a final concentration of 0.1 mM to induce ex...

Embodiment 2

[0043] Example 2: Preparation of covalently immobilized cells

[0044] (1) Preparation of alkyne-functionalized magnetic nanoparticles:

[0045] Fe 2+ and Fe 3+ Preparation of Fe by co-precipitation method at a ratio of 1:3 3 o 4 Magnetic nanoparticles, take 3 g Fe 3 o 4 Nanoparticles 9 mL tetraethyl silicate, stirred for 5 hours to obtain Fe 3 o 4 @SiO 2 nanoparticles.

[0046] to 7.5 mg Fe 3 o 4 @SiO 2 Add 15 mL of 3-aminopropyltriethoxysilane to the nanoparticles, stir vigorously for 24 hours in the anaerobic environment, then add 600 μL of propiolic acid, and react overnight in the anaerobic condition to obtain alkyne-functionalized magnetic Nanoparticles, denoted as Fe 3 o 4 @SiO 2 -NH 2 -alkyne.

[0047] (2) Preparation of recombinant Escherichia coli after azido modification:

[0048] Recombinant Escherichia coli containing the glycerol dehydrogenase gene in KDO-N 3 cultured in culture medium, adding IPTG at a final concentration of 0.5 mM to induce e...

Embodiment 3

[0052] Example 3: Preparation of covalently immobilized cells

[0053] (1) Preparation of alkyne-functionalized magnetic nanoparticles:

[0054] Fe 2+ and Fe 3+ Preparation of Fe by co-precipitation method at a ratio of 1:3 3 o 4 Magnetic nanoparticles, take 5 g Fe 3 o 4 Nanoparticles 15 mL tetraethyl silicate, stirred for 5 hours to obtain Fe 3 o 4 @SiO 2 nanoparticles.

[0055] to Fe 3 o 4 @SiO 2 Add 3-aminopropyltriethoxysilane to the nanoparticles, stir vigorously for 24 hours in anaerobic environment, then add 800 μL propiolic acid, and react overnight in anaerobic conditions to obtain alkyne-functionalized magnetic nanoparticles , denoted as Fe 3 o 4 @SiO 2 -NH 2 -alkyne.

[0056] (2) Preparation of recombinant Escherichia coli after azido modification:

[0057] Recombinant Escherichia coli containing the glycerol dehydrogenase gene in KDO-N 3 cultured in culture medium, adding IPTG with a final concentration of 1 mM to induce expression at 25°C for 6 ...

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Abstract

The invention provides a covalent immobilized cell as well as a preparation method and application thereof, and belongs to the field of bioengineering. According to the method, firstly, a covalent immobilized cell is prepared, and the covalent immobilized cell is constructed by taking Fe3O4@SiO2-NH2-alkyne as a carrier and connecting with azido-modified recombinant escherichia coli; and then the covalent immobilized cell is applied to biotransformation of 1, 3-dihydroxyacetone, and the biotransformation process is mild in reaction condition, high in specificity, environmentally friendly and high in substrate utilization rate.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a covalently immobilized cell and its preparation method and application. Background technique [0002] 1,3-Dihydroxyacetone (DHA) is the simplest ketose sugar and is widely used in medicine, chemical industry, food, cosmetics and other fields. At present, the production methods of 1,3-dihydroxyacetone include chemical synthesis and biotransformation. The chemical synthesis method has disadvantages such as high raw material cost, harsh reaction conditions, high equipment requirements, low product yield and environmental pollution, which limit the application of 1,3-dihydroxyacetone. Although the biotransformation method has the advantages of mild reaction conditions, strong reaction specificity, low environmental pollution, and high substrate utilization, there are still problems in the production process such as low strain transformation efficiency, substrate and produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N1/21C12P7/26C12R1/19
CPCC12N11/14C12N9/0006C12P7/26C12Y101/01006
Inventor 范小蔓李瑞芳黄佳滢朱晨媛徐媛媛张业旺
Owner JIANGSU UNIV
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