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Preparation method of immobilized enzyme by applying amphiphilic porous hollow carbon microspheres

An immobilized enzyme, amphiphilic technology, applied in the direction of immobilization on or in an inorganic carrier, can solve the problems of high immobilization cost, complicated and complicated operation process, etc., to prevent the damage of the enzyme from external factors, and to have a wide range of raw material sources. , The effect of improving operation stability

Inactive Publication Date: 2012-05-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immobilization of enzymes by covalent bonding makes the combination of enzymes and carriers very firm and stable, but the cost of immobilization is high and the operation process is cumbersome and complicated

Method used

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  • Preparation method of immobilized enzyme by applying amphiphilic porous hollow carbon microspheres
  • Preparation method of immobilized enzyme by applying amphiphilic porous hollow carbon microspheres
  • Preparation method of immobilized enzyme by applying amphiphilic porous hollow carbon microspheres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Application of Amphiphilic Hollow Carbon Microspheres to Immobilize Lipase

[0023] (1) Preparation of amphiphilic hollow carbon microspheres

[0024] Weigh 20g of brewer's yeast (Saccharomyces cerevisiae) (trade name is high-temperature resistant high-activity brewer's yeast, purchased from Angel Yeast Co., Ltd., catalog number 80000012), dispersed in 100ml of 0.01mol / L adipaldehyde aqueous solution, and then transferred Put the reaction kettle in a constant temperature box and heat at 180°C for 14h. After the reaction, collect the precipitate, wash the obtained precipitate with deionized water, and then dry it at 20°C for 24h to obtain amphiphilic hollow carbon microspheres. Scanning electron microscope (SEM) topography photos of amphiphilic hollow carbon microspheres are shown in figure 1 , the particle size is 2-4 microns, and the pore size is >50nm.

[0025] (2) Preparation of immobilized lipase

[0026] Free lipase (protein): the trade name is Lipozym...

Embodiment 2

[0041] Example 2 Application of Amphiphilic Hollow Carbon Microspheres to Immobilize Lipase

[0042] (1) Preparation of amphiphilic hollow carbon microspheres

[0043] With the step (1) of embodiment 1.

[0044] (2) Preparation of immobilized lipase

[0045] Free lipase (protein): purchased from Sigma, model L3126.

[0046] Get 50 mg of amphiphilic hollow carbon microspheres prepared in step (1), and evenly disperse them into 2 mL of lipase solution (composed of free lipase and 0.02 mol / L acetic acid-sodium acetate buffer at pH 6.0; lipase concentration is 4 mg / L mL), shake at 100rpm for 12h at 4°C, centrifuge, wash the carbon microspheres loaded with lipase for 2-3 times with 10mL 0.02mol / L acetic acid-sodium acetate buffer solution of pH 6.0, and the protein loading capacity is 15.89 mg / g of immobilized lipase.

[0047] (3) Catalytic performance

[0048] Dissolve 200mg of nitrophenyl palmitate (p-NPP) in 8mL of 1g / 100mL n-butanol in n-heptane solution, then add 50mg of ...

Embodiment 3

[0050] Example 3, application of amphiphilic hollow carbon microspheres to immobilize catalase

[0051] (1) Preparation of amphiphilic hollow carbon microspheres

[0052] Weigh 20g of brewer's yeast (Saccharomyces cerevisiae) (trade name is high-temperature resistant high-activity brewer's yeast, purchased from Angel Yeast Co., Ltd., catalog number 80000012), dispersed in 100ml of 0.01mol / L adipaldehyde aqueous solution, and then transferred Put the reaction kettle in a constant temperature box and heat it at 240°C for 8h. After the reaction, collect the precipitate, wash the obtained precipitate with deionized water, and then dry it at 20°C for 24h to obtain amphiphilic hollow carbon microspheres (particles) The diameter is 2-4 microns, and the pore diameter is > 50nm).

[0053] (2) Preparation of immobilized catalase

[0054] Free catalase (protein): solid powder, purchased from Sigma, USA, product number is C9322.

[0055] Get 50 mg of the amphiphilic hollow carbon micro...

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Abstract

The invention discloses a preparation method of an immobilized enzyme by applying amphiphilic porous hollow carbon microspheres. The method disclosed by the invention comprises the following steps: mixing amphiphilic porous hollow carbon microspheres and a solution of an enzyme to be immobilized, and reacting for 0.5 to 24 hours at a temperature of between 4 and 45 DEG C to obtain the immobilized enzyme, wherein the amphiphilic porous hollow carbon microspheres are prepared by the following way: carbonizing yeast in an aqueous solution or water at a temperature of between 180 and 240 DEG C to obtain the amphiphilic porous hollow carbon microspheres. The method has the advantages that: (1) enzyme package embedding and carrier preparation are separately carried out to avoid inactivation of the enzyme; (2) non-covalent effect exists between the enzyme and the carrier, the surface group of the carrier is not required to be modified, and the method is simple; (3) damage of strong stirring and other external factors to the enzyme can be effectively avoided, and the operation stability of the immobilized enzyme is improved; (4) the mass transfer resistance is small; (5) the amphiphilic property of the hollow carbon microspheres provides possibility for the application of the hollow carbon microspheres in an organic medium system; (6) the method has universality; and (7) the enzyme carrying capacity is high, and the stability is strong.

Description

technical field [0001] The invention relates to a method for preparing immobilized enzymes using amphiphilic porous hollow carbon microspheres. Background technique [0002] Enzymes are a class of catalytically active proteins that can efficiently and selectively catalyze complex chemical reactions under mild reaction conditions. However, the shortcomings of natural enzymes, such as poor stability, easy inactivation, and difficulty in recycling and reuse, hinder the development and application of enzyme catalysis, and the separation and purification of enzymes in the reaction system also increase the cost of catalysts. If the immobilization of enzymes can be realized and the water-soluble enzyme protein can be turned into an insoluble solid catalyst, not only the stability, activity and specificity of the enzyme can be improved to a certain extent, but more importantly, the recovery and recycling of the enzyme can be easily realized. The utilization and the separation and p...

Claims

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Application Information

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IPC IPC(8): C12N11/14
Inventor 杨森孙运辉倪德志关政荣
Owner CHINA AGRI UNIV
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