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Preparation method of immobilized lipase

A technology of immobilizing lipase and enzyme solution, applied in biochemical equipment and methods, methods based on microorganisms, immobilized on or in inorganic carriers, etc., can solve the problems of enzyme molecules falling off and easy leaching, and achieve production The effect of cost reduction, immobilized enzyme protein loading and enzyme activity improvement

Active Publication Date: 2017-05-03
XIAMEN UNIV
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  • Abstract
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Problems solved by technology

[0004] There are many methods for immobilizing protein enzymes on mesoporous materials. For example, adsorption and cross-linking are commonly used traditional methods. However, proteases immobilized by physical methods are easy to leach during the catalysis process, and the enzyme molecules are easy to fall off from the carrier. down, and the immobilized enzyme obtained by forming a covalent bond between the carrier and the enzyme does not have these defects

Method used

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  • Preparation method of immobilized lipase
  • Preparation method of immobilized lipase
  • Preparation method of immobilized lipase

Examples

Experimental program
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Effect test

preparation example Construction

[0063] (2) Hollow mesoporous SiO 2 preparation of

[0064] Measure 250-300ml of absolute ethanol, 20-40ml of deionized water, mix well under magnetic stirring, add 10-20ml of ammonia water, mix well again, quickly add 20-30ml of TEOS, stir vigorously at room temperature for 1-2h, centrifuge Collect the white precipitate and wash twice with absolute ethanol and deionized water respectively. Place the obtained product in a blast drying oven at 70-80°C for 24 hours; ultrasonically disperse 2 g of the above-mentioned dried product in 350-400 ml of deionized water, add 35~45ml (12.5mg / ml) CTAB solution, stirred at room temperature for 30~60min, added 6~10g Na 2 CO 3 , stirred vigorously at 35°C for 24-36h, collected the precipitate by centrifugation, washed twice with absolute ethanol and deionized water, and dried in a blast drying oven at 70-80°C for 24-48h to obtain HMSS.

[0065] (3) Modification of HMSS

[0066] Ultrasonic dispersion of 1-2g HMSS in 20-50ml toluene, magnet...

Embodiment 1

[0075] Example 1: Characterization of HMSS

[0076] figure 1 The XRD diffraction pattern of the prepared HMSS is given. Detection conditions: the starting angle is 5°, the ending angle is 60°, and the scanning step is 0.016711. from figure 1 It can be seen that the product has a broad peak between 15° and 34° at 2°, while almost no impurity peaks are generated at other angles, indicating that the obtained product is exactly a high-purity standard amorphous silica.

Embodiment 2

[0077] Embodiment 2: SEM scanning electron microscope characterization

[0078] figure 2 For the SEM image of HMSS, from figure 2 It can be seen from the figure that most of the particles of the mesoporous silica have a particle size of about 300-400nm, and the shape is very uniform and regular spherical.

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Abstract

The preparation method of immobilized lipase relates to immobilized lipase. After activation and cultivation of the strains, the free enzyme liquid is fermented in a tank; mix absolute ethanol and deionized water, then add ammonia water, after mixing, add TEOS, then centrifuge to collect the white precipitate, wash and dry, and then ultrasonically disperse in the deionized Add CTAB solution and Na2CO3 to ionized water, centrifuge to collect the precipitate, wash and dry to obtain HMSS; ultrasonically disperse HMSS in toluene, stir magnetically, condense and reflux, add (EtO)3SiPrCN, reflux, collect the product by centrifugation, wash and After drying, the modified product HMSS-CN is obtained; then the modified product is refluxed in sulfuric acid, and the product is collected by centrifugation. After washing and drying, the product HMSS-COOH is obtained, and then mixed with the free enzyme solution, stirred in an ice bath, and centrifuged The precipitate was collected, washed with potassium phosphate buffer, and freeze-dried to obtain the immobilized lipase.

Description

technical field [0001] The invention relates to immobilized lipase, in particular to a preparation method of immobilized lipase. Background technique [0002] Lipase (EC 3.1.1.3), also known as triacylglyceride hydrolase, is a general term for a class of enzymes that catalyze the decomposition, synthesis, transesterification and other reactions of ester compounds [1] . Antarctica lipase B (Candida antarctica lipaseB, CALB) is an important lipase with many excellent characteristics, and has strong catalytic ability for aqueous and non-aqueous phase reactions, especially in the ammonia hydrolysis reaction been widely applied. However, the application of lipase is often also affected by factors such as low enzyme activity, poor stability, and difficulty in recycling, but if the enzyme is immobilized before use, these problems can be avoided to a large extent. The currently commercialized immobilized lipase is Novozymes' Novozym 435, but due to its high price, it cannot be us...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/16C12N11/14C12R1/84
CPCC12N1/16C12N9/20C12N11/14C12N1/165C12R2001/84
Inventor 卢英华何彩云姚传义林旺锦
Owner XIAMEN UNIV
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