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Immobilized enzyme and preparation method thereof

A technology for immobilizing enzymes and lipases, applied in the fields of immobilized enzymes, immobilized enzymes and their preparation, and preparation of immobilized enzymes, which can solve the problems of lipase immobilized enzymes that need to be improved and few studies on co-immobilized lipases.

Inactive Publication Date: 2019-07-12
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the co-immobilization research at home and abroad mainly focuses on glucoamylase, and there are few co-immobilization studies on lipase.
Thus, the lipase-immobilized enzyme for preparing functional oil needs to be improved

Method used

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  • Immobilized enzyme and preparation method thereof
  • Immobilized enzyme and preparation method thereof
  • Immobilized enzyme and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] This example is a method for preparing functional oils rich in phytosterol esters, diglycerides and linolenic acid based on magnetically immobilized double enzymes, the method is as follows:

[0074] (1) PDA@Fe 3 o 4 Preparation of NPs: Co-precipitation method to prepare Fe 3 o 4 , take 250mg Fe 3 o 4 Dissolve in 100mL of Tris buffer solution (0.01M pH 8.5), and sonicate for 15min. 125mg of dopamine was added, and the reaction was stirred at room temperature for 4h. The reaction product was separated with a magnet, and after being washed three times with ultrapure water, the product was resuspended in ultrapure water.

[0075] (2) Dual-enzyme co-immobilization: Dissolve CRL and TLL lipase (m / m 1:7) in PBS buffer (0.01M pH7.0) solution, add the PDA@Fe prepared above 3 o 4 After NPs were stirred and reacted at 4°C for 4 hours, the unreacted enzyme solution was washed with pure water for magnetic separation, and then resuspended in ultrapure water, and stored at 4°...

Embodiment 2

[0080] This example is a method for preparing functional oils rich in phytosterol esters, diglycerides and linolenic acid based on magnetically immobilized double enzymes, the method is as follows:

[0081] (1) PDA@Fe 3 o 4 Preparation of NPs: Co-precipitation method to prepare Fe 3 o 4 , take 250mg Fe 3 o 4 Dissolve in 100mL of Tris buffer solution (0.01M pH 8.5), and sonicate for 15min. 125mg of dopamine was added, and the reaction was stirred at room temperature for 4h. The reaction product was separated with a magnet, and after being washed three times with ultrapure water, the product was resuspended in ultrapure water.

[0082] (2) Dual-enzyme immobilization: CRL and TLL lipase (m / m 1:7) were dissolved in PBS buffer (0.01M pH 7.0), and the PDA@Fe prepared above was added 3 o 4 After NPs were stirred and reacted at 4°C for 4 hours, the unreacted enzyme solution was washed with pure water for magnetic separation, and then resuspended in ultrapure water, and stored ...

Embodiment 3

[0087] This example is a method for preparing functional oils rich in phytosterol esters, diglycerides and linolenic acid based on magnetically immobilized double enzymes, the method is as follows:

[0088] (1) PDA@Fe 3 o 4 Preparation of NPs: Co-precipitation method to prepare Fe 3 o 4 , take 250mg Fe 3 o 4 Dissolve in 100mL of Tris buffer solution (0.01M pH 8.5), and sonicate for 15min. 125mg of dopamine was added, and the reaction was stirred at room temperature for 4h. The reaction product was separated with a magnet, and after being washed three times with ultrapure water, the product was resuspended in ultrapure water.

[0089] (2) Dual-enzyme immobilization: CRL and TLL lipase (m / m 1:7) were dissolved in PBS buffer (0.01M pH 7.0), and the PDA@Fe prepared above was added 3 o 4 After NPs were stirred and reacted at 4°C for 4 hours, the unreacted enzyme solution was washed with pure water for magnetic separation, and then resuspended in ultrapure water, and stored ...

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Abstract

The invention discloses an immobilized enzyme and a preparation method thereof. The preparation method of the immobilized enzyme comprises the following steps: dissolving ferroferric oxide in a firstbuffer solution to obtain a base material solution, and adding a dopamine salt for coating treatment so as to obtain a polydopamine coated ferroferric oxide nanocomposite; dissolving nonspecific lipase and sn-1,3 specific lipase in a second buffer solution so as to obtain an enzyme buffer solution; enabling the polydopamine coated ferroferric oxide nanocomplex to be in contact with the enzyme buffer solution, and obtaining the immobilized double enzyme through covalent fixation. According to the method, the ferroferric oxide nanocomposite is used as a fixed carrier, and the sn-1,3 specific lipase and the nonspecific lipase are subjected to co-immobilization treatment to obtain the immobilized double enzyme. The preparation process is simple, the conditions are mild, meanwhile the stabilityof the prepared immobilized double enzyme is good, the catalytic effect is high, the separation and recovery are easy, the repeated use effect is good, and the enzyme is convenient for industrial production.

Description

technical field [0001] The present invention relates to the field of biology, specifically, optionally, an immobilized enzyme and a preparation method thereof, more specifically, a method for preparing an immobilized enzyme and an immobilized enzyme. Background technique [0002] As a special biocatalyst, lipase can not only promote the hydrolysis of oil, but also accelerate the synthesis of oil under certain conditions and undergo transesterification. It is widely used in the fields of food, medicine and biodiesel. However, in the actual use process, the use of free enzymes has great limitations, such as enzymes are not stable enough under conditions such as heat, strong acid, strong alkali, organic solvents, etc., easily lose their catalytic activity, and there are difficulties in separation and purification, and difficult to recycle Repeated use will cause product pollution and increase production costs. Immobilization technology can solve the above problems. The immobi...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/08
CPCC12N9/20C12N11/08C12N11/14C12Y301/01003
Inventor 张峰姚桂红凌云杨敏莉
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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