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Method for immobilizing enzymes based on biomimetic mineralization process of two-aqueous-phase system

A two-phase system, biomimetic mineralization technology, applied in the direction of fixing on or in the inorganic carrier, fixing on/in the organic carrier, etc., can solve the problems of enzyme shedding and low encapsulation efficiency, and achieve easy Recycling, high encapsulation rate, good mechanical strength

Active Publication Date: 2019-06-28
SOUTHWEST JIAOTONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the adsorption equilibrium in the adsorption method leads to low encapsulation efficiency, and the enzyme is easily detached from the carrier during use (Zheng Lu, et al. Biomimetic mineralization of calcium carbonate / carboxymethylcellulose microspheres for lysozyme immobilization[J]. Materials Science and Engineering: C, 2012,32(7):1982-1987.)

Method used

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  • Method for immobilizing enzymes based on biomimetic mineralization process of two-aqueous-phase system
  • Method for immobilizing enzymes based on biomimetic mineralization process of two-aqueous-phase system
  • Method for immobilizing enzymes based on biomimetic mineralization process of two-aqueous-phase system

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Embodiment 1

[0045]A method for immobilizing enzymes based on a two-phase system biomimetic mineralization process, comprising the following steps:

[0046] Step 1: Methoxypolyethylene glycol-urease copolymer particles

[0047] Weigh 16 mg of methoxypolyethylene glycol acetaldehyde with a molecular weight of 5 kDa, weigh 16 mg of urease with a molecular weight of 540 kDa and place them in a screw bottle; add 1 mL of deionized water (pH=5) to form a mixture aqueous solution The aqueous solution of the mixture was left to react for 100 hours, and dried in a vacuum dryer (DZF-6050, Shanghai Yiheng Scientific Instrument Co., Ltd.) at a pressure of less than 0.01 MPa to form methoxypolyethylene glycol-urease copolymer particles.

[0048] Step 2: Aqueous two-phase system containing the target enzyme

[0049] Prepare 10mmol / L Tris-HCl buffer solution with a pH of 7.4; weigh 10mg of catalase with a molecular weight of 250kDa, add it to 6.68mL Tris-HCl buffer solution, and dissolve it completely t...

Embodiment 2

[0060] A method for immobilizing enzymes based on a two-phase system biomimetic mineralization process, comprising the following steps:

[0061] Step 1: Methoxypolyethylene glycol-urease copolymer particles

[0062] Weigh 16 mg of methoxypolyethylene glycol acetaldehyde with a molecular weight of 5 kDa, weigh 16 mg of urease with a molecular weight of 540 kDa, and place them in a screw bottle; add 1 mL of deionized water (pH=5) to form a mixture Aqueous solution; the aqueous solution of the mixture was left to react for 100 hours, and dried in a vacuum dryer (DZF-6050, Shanghai Yiheng Scientific Instrument Co., Ltd.) at a pressure of less than 0.01MPa to form methoxypolyethylene glycol-urease copolymer particles.

[0063] Step 2: Aqueous two-phase system containing the target enzyme

[0064] Prepare 10mmol / L Tris-HCl buffer solution with a pH of 7.4; weigh 100mg of catalase with a molecular weight of 250kDa; dissolve it in 10mL Tris-HCl buffer solution and dissolve completely...

Embodiment 3

[0073] A method for immobilizing enzymes based on a two-phase system biomimetic mineralization process, comprising the following steps:

[0074] Step 1: Methoxypolyethylene glycol-urease copolymer particles

[0075] Weigh 16 mg of methoxypolyethylene glycol acetaldehyde with a molecular weight of 5 kDa, weigh 16 mg of urease with a molecular weight of 540 kDa and place them in a screw bottle; add 1 mL of deionized water (pH=5) to form a mixture aqueous solution The aqueous solution of the mixture was left to react for 100 hours, and dried in a vacuum dryer (DZF-6050, Shanghai Yiheng Scientific Instrument Co., Ltd.) at a pressure of less than 0.01 MPa to form methoxypolyethylene glycol-urease copolymer particles.

[0076] Step 2: Aqueous two-phase system containing the target enzyme

[0077] Prepare 10mmol / L Tris-HCl buffer solution with a pH of 7.4; weigh 10mg of catalase with a molecular weight of 250kDa, add it to 6.68mL Tris-HCl buffer solution, and dissolve it completely ...

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Abstract

The invention discloses a method for immobilizing enzymes based on a biomimetic mineralization process of a two-aqueous-phase system. The method comprises the following steps of 1, preparation of methoxy polyethylene glycol-urease copolymer particles; 2, preparation of the two-aqueous-phase system containing the target enzymes, wherein an enzyme solution with the mass fraction of 0.1-0.4 wt% is prepared; glucan and polyethylene glycol are added into the enzyme solution, and then the materials are fully mixed to obtain the required two-aqueous-phase system containing the target enzymes; 3, preparation of calcite calcium carbonate for immobilizing the target enzymes, wherein the methoxy polyethylene glycol-urease copolymer particles are added into the two-aqueous-phase system containing thetarget enzymes, and the materials are fully mixed to obtain a mixed solution A; a mixed solution B of urea and calcium chloride is prepared; the mixed solution A and the mixed solution B are mixed fora sufficient reaction, and the required calcite calcium carbonate for immobilizing the target enzymes is obtained after centrifugation. The prepared calcite calcium carbonate for immobilizing the target enzymes is high in mechanical strength, good in shear force resistance, high in encapsulation rate, and high in enzyme activity retention rate.

Description

technical field [0001] The invention relates to the technical field of immobilized enzymes, in particular to a method for immobilizing enzymes based on a two-phase system biomimetic mineralization process. Background technique [0002] The immobilization of enzymes promotes the separation of enzymes, substrates and products, realizes the reuse of enzymes, and reduces operating costs. Currently, enzyme immobilization methods mainly include cross-linking, embedding and carrier binding. The cross-linking method is to use organic cross-linking agents to cross-link the enzyme molecules to form immobilized enzyme particles. However, organic cross-linking agents often lead to changes in the conformation of the enzyme and cause enzyme inactivation; the embedding method is to embed the enzyme in the microgel In the grid of the ball, but the gel is easy to swell when it encounters water, which leads to the leakage of the enzyme. The carrier binding method is to combine the enzyme an...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/10
Inventor 孟涛陈佳琳唐启明郭婷王垚磊
Owner SOUTHWEST JIAOTONG UNIV
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