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Immobilized penicillin acylated enzyme with silicon gel rubber as carrier and preparation method

A technology of penicillin acylase and silica gel, which is applied in the direction of immobilization on or in the inorganic carrier, which can solve the problems of high price, difficult handling of polymer carriers, and high cost of immobilized enzymes

Inactive Publication Date: 2009-08-19
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The four methods have their own advantages and disadvantages. In industrial applications, in order to reduce the cost of immobilized enzymes, it is generally required that the immobilized enzymes have good operational stability and can be reused many times.
CN1279287A has invented a multi-component copolymerized porous microbead as a carrier for immobilizing penicillin acylase, but the structure and properties of the polymer itself have determined that it has the following weaknesses: controlling the pore structure of the polymer and the number of surface functional groups and Its distribution is relatively difficult, and the mass diffusion efficiency is low; the thermal stability, mechanical stability and solvent resistance of the high polymer carrier are weak; the disposal of the high polymer carrier is difficult
Domestic 6-APA manufacturers mostly use imported acrylic resin (Eupergit C), which is relatively expensive, making the cost of immobilized enzymes very high

Method used

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  • Immobilized penicillin acylated enzyme with silicon gel rubber as carrier and preparation method
  • Immobilized penicillin acylated enzyme with silicon gel rubber as carrier and preparation method
  • Immobilized penicillin acylated enzyme with silicon gel rubber as carrier and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Dissolve 20ml of silica sol with a mass fraction of 30% in 20ml of ethanol, stir and dissolve evenly, add 23ml of silane coupling agent γ-aminopropyltriethoxysilane (APTES) dropwise into the above solution at room temperature, Stop stirring, let the gel stand for 3 hours, use mechanical agitation to crush the agglomerates, add 20ml ethanol to rinse, and centrifuge, the product is rinsed with a large amount of distilled water, centrifuged, and the carrier (N-SG) used for immobilization is obtained. Store at 4-7°C under state. (Dry carrier specific surface area: 378m2 / g, average pore diameter 17nm)

[0034]2. Add 1 g of the above-mentioned wet carrier to 20 ml of glutaraldehyde solution with a mass fraction of 0.5%, stir at room temperature for 4 hours, centrifuge, rinse with 20 ml of distilled water for 3 times, and obtain the activated carrier (GA-SG) wet 3. Add 0.5g of the activated carrier to 15ml of phosphate buffer solution (0.2M) with pH 7.9, add 900μl of penic...

Embodiment 2

[0036] 1. Same as Example 1, the resulting product was freeze-dried, then vacuum-dried at 100° C. for 3 hours, and subjected to electron microscopy and surface adsorption tests. The specific surface area of ​​the product measured is 320m / g, and the aperture is 17nm, and the electron microscope SEM figure is (a) among accompanying drawing 1.

[0037] 2. Rinse 1 g of the wet carrier obtained in the above step 1 with 20 ml of acetone, then add 20 ml of 0.6% N, N-carbonyldiimidazole (CDI) in acetone solution, stir at room temperature for 4 hours, and centrifuge Separation, rinsed 3 times with 10ml acetone, and the activated carrier (CDI-SG) obtained was suspended in acetone and stored at 4-7°C. The obtained product was treated in the same way as in step 1 to obtain a specific surface area of ​​246m2 / g and a pore size of 24nm. Electron microscopy The SEM image is (b) in Figure 1.

[0038] 3. Rinse 0.5g of the carrier activated in step 2 with 10ml of pH7.9 phosphate buffer solution...

Embodiment 3

[0046] 1. Dissolve 20ml of silica sol with a mass fraction of 30% in 20ml of ethanol, stir and dissolve evenly, and drop 30ml of silane coupling agent γ-(2,3-glycidyloxy)propyltrimethoxysilane at room temperature Add it into the above solution, stop stirring, let the gel stand for 3 hours, break up the agglomerates with mechanical stirring, add 20ml of ethanol to rinse, and centrifuge, the product is rinsed with a large amount of distilled water, centrifuged to obtain a carrier for immobilization, wet Stored at 7°C in a cold state.

[0047] 2. Add 1 g of the above-mentioned wet carrier to 20 ml of 1,4-diglycidyl ether solution with a mass fraction of 1.0%, stir at room temperature for 24 hours, centrifuge, rinse with 20 ml of distilled water for 3 times, and obtain the activated carrier wet Store at 4-7°C under state

[0048] 3. Add 0.5g of the activated carrier to 15ml, pH8.1 phosphate buffer solution (0.25M), add 2000μl penicillin acylase enzyme solution, stir slowly at roo...

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Abstract

The invention relates to an immobilized penicillin acylase using silicone gel as a carrier and a preparation method thereof. The immobilized penicillin acylase is porous immobilized penicillin acylase with grain diameter between 13 and 15mum and aperture between 14 and 30nm, which is prepared by covalent binding between porous silicone gel with a functional group on the surface obtained by copolymerization of silicone sol and a silane coupling agent and penicillin acylase. The preparation method comprises the following steps: first, preparing the carrier; then, activating the carrier; and preparing the immobilized penicillin acylase using the silicone gel as the carrier. The immobilized penicillin acylase using the silicone gel as the carrier prepared by the method has proper grain diameter and aperture, has larger specific surface area (300 to 600m2 / g), can not be contaminated by bacteria easily, has strength superior to agarose 6B microballons and acrylic resin microballs commonly adopted overseas, has the advantages of low price of raw materials, good stability, simple preparation method and stable reaction process, and is applicable to industrial mass production.

Description

technical field [0001] The invention relates to an immobilized enzyme biotechnology, in particular to an immobilized penicillin acylase with silica gel as a carrier and a preparation method thereof. Background technique [0002] Immobilized enzyme (immobilized enzyme) technology is the mechanism of action of Katchallski-Katzir et al. in the 1960s to study the enzyme attached to the biofilm in the natural state. First, some proteolytic enzymes were immobilized on incompatible carriers or wrapped in In the artificial membrane, a new type of biotechnology developed to study its stability and heterogeneous catalytic properties (Annu. Rev. Biochem. 35, 773-908, 1966). It is defined as "an enzyme that is physically confined or located in a specific space area, maintains catalytic activity, and can be used repeatedly and continuously". Natural enzymes are widely used in food, chemical and pharmaceutical industries, but enzymes are soluble proteins that are easily inactivated and d...

Claims

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Application Information

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IPC IPC(8): C12N11/14
Inventor 杨志民董伟兵龚俊波
Owner TIANJIN UNIV
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