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A shell-core structure immobilized enzyme and its preparation method and application

An immobilized enzyme, nuclear structure technology, applied in immobilized enzymes, biochemical equipment and methods, immobilized on/in organic carriers, etc., can solve the problem of low immobilized enzyme activity, difficult to control network size, and reaction conditions. Violent and other problems, to achieve the effect of improving immobilization efficiency, improving structural stability, and high environmental stability

Active Publication Date: 2021-07-27
SOUTH CHINA INST OF COLLABORATIVE INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its own disadvantages, traditional methods, such as immobilized enzymes by physical adsorption, have limited immobilization capacity for enzymes and poor stability; covalent binding methods require that the group at the binding site must not be the active center of the enzyme, and the immobilized enzymes usually obtained The recovery rate of enzyme activity is low; the cross-linking method requires the combination of the enzyme and the carrier material, the reaction conditions are relatively severe, and the activity of the immobilized enzyme is low; the encapsulation method is difficult to control the network size of the carrier material, and the enzyme is easy to leak

Method used

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  • A shell-core structure immobilized enzyme and its preparation method and application
  • A shell-core structure immobilized enzyme and its preparation method and application
  • A shell-core structure immobilized enzyme and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0037] (1) Accurately weigh a certain amount of organophosphate hydrolase (OPH), and prepare an enzyme solution with a concentration of 1 mg / mL with a 50 mM, pH=9.0 phosphate buffer solution;

[0038] (2) Double-bond modification of the protein: For the enzyme solution prepared in step (1), pipette 20mL of the enzyme solution into three 25mL glass vials, and follow the N-acryloyloxysuccinimide (NAS) : Enzyme = [5:1; 100:1; 200:1] (molar ratio) for feeding, put the solution in a water bath at 30°C for 1 hour, and obtain double bond modified enzyme solutions OPH-NAS5, OPH-NAS100, OPH -NAS200;

[0039] (3) Subsequently, pipette 10 mL of the double bond-modified enzyme solutions OPH-NAS5, OPH-NAS100, OPH-NAS200 in step (2) respectively, put them into three 25 mL glass vials, add monomer acrylamide (AAM ), nitrogen gas was slowly passed into the solution for 3min; then, the cross-linking agent N,N-methylenebisacrylamide (BIS), the initiator ammonium persulfate (APS) and the cataly...

Embodiment 2

[0044] (1) Accurately weigh a certain amount of organophosphate hydrolase, and prepare an enzyme solution with a concentration of 10 mg / mL with a 50 mM, pH=8.0 phosphate buffer solution;

[0045] (2) Double bond modification of protein: feed the enzyme solution prepared in step (1) according to NAS: enzyme = 10:1 (molar ratio), put the solution in a refrigerator at 4°C for 10 hours to obtain double bond modification Enzyme solution OPH-NAS10;

[0046] (3) Subsequently, pipette 10 mL of the enzyme solution OPH-NAS10 modified by the double bond in step (2), place it in four 25 mL glass vials, add N-(3-aminopropyl)-methacrylamide Hydrochloride (APM), nitrogen gas was slowly passed into the solution for 3 minutes, and then the following mass ratios of the cross-linking agent BIS (OPH: BIS = 1: 0.1 / 0.2 / 0.5 / 1) were added to the 4 vials respectively;

[0047] (4) then, in the enzyme solution of step (3), add initiator ammonium persulfate (APS) and catalyzer tetramethylethylenediamin...

Embodiment 3

[0050] (1) Accurately weigh a certain amount of organophosphate hydrolase, and prepare an enzyme solution with a concentration of 5 mg / mL with a phosphate buffer solution of 50 mM and pH=7.5;

[0051] (2) Double-bond modification of the protein: feed the enzyme solution prepared in step (1) according to sodium acrylate (AAS): enzyme = 200:1 (molar ratio), and place the solution at room temperature at 25°C for 5 hours. Obtain double bond modified enzyme solution OPH-NAS50;

[0052] (3) Subsequently, pipette 10 mL of the enzyme solution OPH-AAS200 modified by the double bond in step (2), place it in three 25 mL glass vials, and add mixed monomer 1 (mass ratio AAM:APM=1: 3), mixed monomer 2 (mass ratio AAM:APM=2:2), mixed monomer 3 (mass ratio AAM:APM=3:1), slowly feed nitrogen into the solution for 3min; then, add nitrogen to the enzyme solution Add the cross-linking agent N, N-methylenebisacrylamide (BIS), the initiator ammonium persulfate (APS) and the catalyst tetramethyleth...

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Abstract

The invention belongs to the field of immobilized enzymes, in particular to a shell-core structure immobilized enzyme and its preparation method and application. The preparation method comprises the following steps: preparing the enzyme protein into an enzyme solution, adding a modifier to the enzyme solution, mixing and reacting for 2-10 hours to obtain an enzyme protein modified with a double bond on the surface; then adding a mass ratio of After vortexing and mixing, remove dissolved oxygen, add cross-linking agent, initiator and catalyst in sequence, place the solution at room temperature for 1-5 hours, then dialyze and purify to obtain an immobilized enzyme with a shell-core structure. Compared with the traditional method of immobilizing enzymes, the present invention realizes the immobilization of enzymes on a smaller scale, improves the efficiency of enzyme immobilization, and makes the final "shell" structure realize the embedding and immobilization of all enzyme molecules , and the immobilized enzyme system obtained at the same time has many excellent properties such as high enzyme activity retention rate, environmental stability and wider application fields.

Description

technical field [0001] The invention belongs to the field of immobilized enzymes, in particular to a shell-core structure immobilized enzyme and its preparation method and application. Background technique [0002] Enzymes are a class of highly efficient and specific biocatalysts, which are widely used in many fields such as biopharmaceuticals, food processing, environmental protection, and clean energy development. Under the new situation of vigorously advocating the concept of sustainable development, developing green and energy-saving industrial production methods and using renewable resources and energy, enzyme catalysis will play an increasingly important role in the production and processing of bulk commodities and fine chemicals. . [0003] Enzymes are special organic substances (proteins, RNA) with catalytic activity and high selectivity produced by living cells, and the chemical nature of most enzymes is protein. Limited by the characteristics of the protein itsel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/098C12N11/082C12N11/087C12N11/04
CPCC12N9/0065C12N9/16C12N9/20C12N11/04C12N11/08C12Y111/01007C12Y301/01003C12Y301/08001
Inventor 罗志刚陈永志程建华
Owner SOUTH CHINA INST OF COLLABORATIVE INNOVATION