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Preparation method of immobilized lipase

A technology for immobilizing lipase and lipase, which is applied in the direction of immobilization on or in an inorganic carrier, can solve the problems of loss of catalytic activity, mildness, and inability to meet the needs of industrial production, and achieves good stability and cycle times. Multiple, mild conditions

Active Publication Date: 2014-07-23
BIOTALICON (SHENZHEN) CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although there are magnetic Fe3O4, Fe3O4SiO2 , Fe3O4chitosan nanoparticles immobilized lipase (Chem. Asian J., 2013, 8, 1447-1454; J. Mater. Chem., 2012, 22, 8385-8393; Catal. Commun., 2011, 12, 717-720.), but the current literature reports that the lipase immobilized by magnetic nanoparticles lost part of its catalytic activity during the immobilization process, resulting in the immobilization of fat The recovery rate of enzyme activity is less than 100%, and its immobilization preparation process is not gentle, which cannot meet the needs of industrial production

Method used

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  • Preparation method of immobilized lipase

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preparation example Construction

[0032] In the preparation method of immobilized lipase provided by the invention, the amino functionalized magnetic Fe 3 o 4 C nanoparticles were synthesized by the following methods:

[0033] Weighed 2 mmol of iron acetylacetonate and added it to 60 mL of polyethylene glycol 200, reacted at 270 °C for 3 h in a nitrogen atmosphere, cooled naturally to room temperature, added 4 mmol of glucose, and ultrasonicated for 5 min to obtain a mixed solution; The mixed solution was transferred to an autoclave, and 1 mL of ammonia (NH 3mass percent content, 28%), tighten the hot-pressed reactor, and react at 240 °C for 24 h; after the reaction is completed, it is naturally cooled to room temperature, and the obtained precipitate is magnetically separated, washed, and dried to obtain amino-functionalized magnetic Fe 3 o 4 C nanoparticles, figure 1 For the prepared amino-functionalized magnetic Fe 3 o 4 High-resolution transmission electron microscopy of C nanoparticles.

Embodiment 1

[0035] Step 1. Prepare a 1 mg / mL lipase solution with lyophilized lipase powder derived from Candida rugosa with pH=7.0 phosphate buffer solution, and store it in a refrigerator at 4 °C for later use;

[0036] Step 2. Weigh out 100 mg of amino-functionalized magnetic Fe 3 o 4 C nanoparticles were added to 5 ml of 100g / L glutaraldehyde solution, reacted at 45 °C for 3 h, and the obtained product was magnetically separated, washed, and vacuum-dried;

[0037] Step 3. Take 10 mL of the lipase solution prepared in step 1, add the product obtained in step 2 to it, react at 25 °C for 3 h, and magnetically separate and wash the obtained product to obtain the immobilized fat enzyme.

[0038] Detect lipase in the product in magnetic Fe 3 o 4 The immobilized amount on C nanoparticles is 192.7 mg / g; the enzyme activity of the immobilized lipase is 1895.6 U / g; the recovery rate of the enzyme activity of the immobilized lipase is 164%; After standing for 10 h, if figure 2 Shown, the ...

Embodiment 2

[0040] Step 1. Prepare a 1 mg / mL lipase solution from the freeze-dried lipase powder derived from porcine pancreas with pH=7.0 phosphate buffer solution, and store it in a refrigerator at 4 °C for later use;

[0041] Step 2. Weigh out 100 mg of amino-functionalized magnetic Fe 3 o 4 Add C nanoparticles to 10 ml 10g / L glutaraldehyde solution, react at 25 °C for 24 h, and the obtained product is magnetically separated, washed, and vacuum-dried;

[0042] Step 3. Take 50 mL of the lipase solution prepared in step 1, add the product obtained in step 2 to it, react at 35 °C for 0.5 h, and magnetically separate and wash the obtained product to obtain the immobilized fat enzyme.

[0043] Detect pancrelipase in the product on magnetic Fe 3 o 4 The immobilized amount on the C nanoparticles is 68.3 mg / g; the enzyme activity of the immobilized lipase is 3839 U / g; the recovery rate of the enzyme activity of the immobilized lipase is 172%; the immobilized lipase After 8 cycles of lipas...

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Abstract

The invention provides a preparation method of immobilized lipase. The preparation method is characterized by using amino-functionalized magnetic Fe3O4@C nanoparticles as carriers and immobilizing lipase on the surfaces of the nanoparticles through covalent binding. The method is specifically divided into two methods. The first method is characterized by firstly binding an aldehyde group at one terminal of glutaraldehyde with the amino groups on the surfaces of the amino-functionalized magnetic Fe3O4@C nanoparticles by utilizing nucleophilic reaction and then binding an aldehyde group at the other terminal of glutaraldehyde immobilized on the surfaces of the magnetic Fe3O4@C nanoparticles with the amino groups of lipase. The second method is characterized by converting the amino groups on the surfaces of the magnetic Fe3O4@C nanoparticles to carboxy groups by utilizing succinic anhydride and immobilizing lipase on the surfaces of the Fe3O4@C nanoparticles by utilizing amidation after activation with a mixture of N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide in a molar ratio of 1:(0.5-2). The preparation method has the beneficial effects that the preparation method is simple and mild; the lipase immobilization amount is large; the immobilized lipase has high enzyme activity and enzyme recovery and good stability, can be simply and conveniently recycled, effectively reduces the real cost of production and has wide application prospect.

Description

technical field [0001] The invention relates to a preparation method of immobilized lipase. Background technique [0002] Lipase is a kind of widely used acyl hydrolase, which can catalyze the hydrolysis, alcoholysis, synthesis and exchange reactions of esters, and has been widely used in pharmaceutical, chemical, food processing, sewage treatment and other industries. Compared with traditional industrial catalysts, lipase also has many defects, such as: the extraction and purification process is cumbersome and expensive; it is unstable to heat, strong acid, strong alkali, organic solvents, etc.; Recycling and reuse; lipase often contains miscellaneous proteins and colored substances, which affect the separation and purification of reaction products. In order to overcome the above-mentioned deficiencies of lipase, people combine free lipase with an insoluble carrier to make it an immobilized lipase, which realizes the recovery and reuse of lipase. Therefore, the industrial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14
Inventor 陈志明吴之传徐文惠
Owner BIOTALICON (SHENZHEN) CO LTD
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