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Method for preparing immobilized lipase by utilizing amphipathy chitosan microsphere carrier

A technology for immobilizing lipase and chitosan microspheres, which is applied in the direction of immobilization on/in the organic carrier, can solve the problem that chitosan microspheres are not connected, affect immobilized lipase, and do not take microspheres into account. The surface provides hydrophobic areas and other issues to achieve strong catalytic activity and strong binding effects

Inactive Publication Date: 2013-05-01
SHENYANG INSTITUTE OF CHEMICAL TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few reports on the covalent immobilization of lipase using chitosan, especially the modified chitosan microsphere carrier. These studies either did not prepare chitosan into micron-sized microspheres that can be mass-produced, or The prepared chitosan microspheres are not connected with arm molecules, nor are they considered to provide appropriate hydrophobic regions on the surface of the microspheres, which will inevitably affect the preparation of high-activity immobilized lipases, so the development of chitosan-based lipases with excellent performance Immobilization carrier is still necessary

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1. Preparation of amphiphilic chitosan microsphere carrier:

[0020] Fully mix 120ml of chitosan solution with a mass fraction of 5% and 3.5g of PEG2000, then add 120ml of liquid paraffin, 18 drops of span-80, stir at high speed for 30min to make the system into an emulsion, then add 14ml of formaldehyde, stir for 1h and pour the emulsion Put it into the mixed solution of 10% NaOH / dehydrated ethanol (V(NaOH):V(dehydrated ethanol)=1:1) and let it stand for 1h, fully wash with xylene, dehydrated ethanol and deionized water, and sieve to obtain Chitosan microspheres with a particle size of 100-300 μm are used as the matrix;

[0021] Weigh 5g of CS microspheres into 100mL of methanol, add 30mL of benzaldehyde dropwise, react with constant temperature shaking at 60°C for 16h, then add sodium borocyanide and react at room temperature for 12h, filter and wash, soak the microspheres in an appropriate amount of sodium hydroxide aqueous solution, slowly Add 65mL of ep...

Embodiment 2

[0023] Embodiment 2. Preparation of immobilized lipase:

[0024] Add 10 mL of pH 7.5 phosphate buffer to a 50 mL Erlenmeyer flask with a stopper containing 0.15 g of glutaraldehyde-activated carrier, then add 6 mg of Candida antarctica lipase B, react in a shaker at room temperature for 3 h, and absorb the enzyme Wash the immobilized enzyme with phosphate buffer until the washing solution does not contain protein, and store the immobilized enzyme in a refrigerator at 4°C. The activity of the immobilized lipase was 481 U / g, and the activity recovery rate was 197%.

Embodiment 3

[0025] Embodiment 3. Preparation of immobilized lipase:

[0026]Add 10 mL of pH7.5 phosphate buffer to a 50 mL Erlenmeyer flask with a stopper containing 0.15 g of glutaraldehyde-activated carrier, then add 12 mg of Candida antarctica lipase B, react in a shaker at room temperature for 3 h, and absorb the enzyme Wash the immobilized enzyme with phosphate buffer until the washing solution does not contain protein, and store the immobilized enzyme in a refrigerator at 4°C. The activity of the immobilized lipase was 509 U / g, and the activity recovery rate was 130%.

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Abstract

The invention provides a method for preparing immobilized lipase by utilizing an amphipathy chitosan microsphere carrier, and relates to a method for preparing immobilized lipase. The method comprises the following steps: taking micron-order chitosan microsphere resin prepared by an inverse suspension crosslinking method as an immobilization carrier matrix, performing C2-bit benzene formolatiom on the chitosan microsphere matrix, leading in hydrophobic interaction groups, then using epoxy chloropropane to activate C6-bit hydroxyl, and coupling tetraethylenepentamine to prepare chitosan microspheres with hydrophobic phenyl and hydrophilic polyamine molecules, and taking the chitosan microspheres as the covalent immobilization carrier of lipase after coupling glutaraldehyde. According to the invention, the carrier is adopted to immobilize the antarctic candida lipase B, and the hydrophobic groups and hydrophilic arm molecules are led in, so that the immobilization efficiency of chitosan microspheres to the lipase is improved. When the quality of added lipase of the dry carrier per gram is 40 mg, and the antarctic candida lipase is immobilized for 3 h at room temperature under the condition that the pH is 7.5, the obtained immobilized lipase has the activity recovery rate of 197%, and has the activity of 481.6 U / g.

Description

technical field [0001] The invention relates to a method for preparing immobilized lipase, in particular to a method for preparing immobilized lipase by using amphiphilic chitosan microsphere carrier. Background technique [0002] Lipase (EC.3.1.1.3) is a special class of acyl hydrolase, which is one of the most widely used enzyme catalysts. In addition to catalyzing the hydrolysis of glyceride compounds, it can also be used to catalyze Transesterification, polymer synthesis, peptide synthesis, stereoisomeric resolution and synthesis of chiral drugs, etc. Candida Antarctic lipase B (CALB) is an important lipase, which has high stereoselectivity in hydrolysis and organic synthesis reactions, and has strong catalysis for water-insoluble and water-soluble substances. active. Research results in recent years have shown that CALB has shown better catalytic performance than other lipases in various types of reactions. Lipase is expensive. Although the free enzyme catalyzed rea...

Claims

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Application Information

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IPC IPC(8): C12N11/10
Inventor 朱建星王红艳
Owner SHENYANG INSTITUTE OF CHEMICAL TECHNOLOGY
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