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Immobilized kappa-carrageenan enzyme and method for preparing kappa-carrageenan oligosaccharide by adopting immobilized kappa-carrageenan enzyme

A technology of carrageenan oligosaccharides and carrageenase, which is applied in the direction of immobilization on or in the inorganic carrier, fermentation, etc., can solve the problems of complicated operation, destruction of the high-level structure of the enzyme, and severe reaction conditions, and achieve good stability and high enzyme efficiency. The effect of high recovery rate and simple process

Active Publication Date: 2015-09-09
JIMEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the adsorption method and the embedding method have simple processes, mild conditions, cheap and easy-to-obtain carriers, and a low degree of destruction of the biological activity of the enzyme, but the combination of the enzyme and the carrier is not firm, and it is easy to fall off, and the embedding method is harmful to macromolecular substrates and enzymes. The product is not suitable
In the covalent method and cross-linking method, the enzyme is firmly combined with the carrier, it is not easy to fall off during use, and it has good stability. It can use high-concentration substrates and solutions with ionic strength. higher order structure of enzymes

Method used

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  • Immobilized kappa-carrageenan enzyme and method for preparing kappa-carrageenan oligosaccharide by adopting immobilized kappa-carrageenan enzyme
  • Immobilized kappa-carrageenan enzyme and method for preparing kappa-carrageenan oligosaccharide by adopting immobilized kappa-carrageenan enzyme
  • Immobilized kappa-carrageenan enzyme and method for preparing kappa-carrageenan oligosaccharide by adopting immobilized kappa-carrageenan enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation of immobilized κ-carrageenase and κ-carrageenan oligosaccharide

[0039] Using Fe 2+ and Fe 3+The ferroferric oxide magnetic nanoparticles were prepared by co-precipitation method, oleic acid was added in the presence of ammonia water for embedding, and then 10 mg / mL oxidant potassium permanganate was added to oxidize the double bond on oleic acid to obtain carboxyl functionalized The Fe3O4 magnetic nanoparticles were freeze-dried and stored at room temperature. Carboxyl-functionalized iron ferric oxide magnetic nanoparticles were cross-linked with glutaraldehyde solution, washed several times with 0.05 M pH 7.5 PBS buffer after cross-linking, and immobilized by adding κ-carrageenan enzyme solution after magnetic separation. After immobilization, the unbound free enzyme was washed several times with 0.05 M PBS buffer solution at pH 7.5 to obtain immobilized κ-carrageenase. The mass volume concentration of glutaraldehyde was 3%, the cross-linkin...

Embodiment 2

[0045] Example 2: Characterization of magnetic properties of magnetic nanoparticles, immobilized κ-carrageenase and magnetic separation effect

[0046] Take the carboxyl-functionalized magnetic nanoparticle carrier and immobilized κ-carrageenanase at room temperature, and measure the magnetic hysteresis loop with a vibrating sample magnetometer (VSM), which shows that the prepared carrier and the immobilized κ-carrageenan Enzymes are magnetic, see Picture 1-1 ; Take 20 mg of immobilized κ-carrageenase in a test tube, add 5 mL of distilled water, shake and mix well, and then use an external magnetic field, that is, a permanent magnet, for magnetic separation. It can be seen that the separation effect is good, see Figure 1-2 .

[0047] Example: 3: Effects of different glutaraldehyde concentrations on the activity of immobilized κ-carrageenase

[0048] Take 20 mg of carboxyl-functionalized magnetic nanoparticles, add 5 mL of glutaraldehyde solution with a concentration of 0, 0...

Embodiment 5

[0051] Example 5: Effects of different fixation times on the activity of fixed κ-carrageenase

[0052] Take 20 mg of carboxyl-functionalized magnetic nanoparticles, add 5 mL of 2.5% (w / v) glutaraldehyde solution, and cross-link at 4°C for 0.5, 1, 2, 3, 4, and 5 h, and the cross-linking is completed. Wash several times with 0.05 M pH 7.5 buffer, add 1 mL of κ-carrageenan enzyme solution with an enzyme activity of 13.9 and U / mL, fix at 4°C for 3 h, and finish the fixation with 0.05 M pH 7.5 buffer Washing several times, applying a magnetic field for magnetic separation to obtain immobilized κ-carrageenase. The measured maximum immobilized enzyme activity and enzyme activity recovery rate were obtained when the crosslinking time was 2 h, which were 311.2 U / g nanoparticles and 44.8%, respectively. The results are shown in Figure 4 .

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Abstract

The invention discloses an immobilized kappa-carrageenan enzyme and a method for preparing kappa-carrageenan oligosaccharide by adopting the immobilized kappa-carrageenan enzyme. The method comprises steps as follows: (a), ferroferric oxide magnetic nanoparticles are prepared with a Fe<2+> and Fe<3+> coprecipitation method, oleic acid is added under the alkaline condition to perform embedding, potassium permanganate is added for oxidizing double bonds of the oleic acid, and carboxyl-functionalized ferroferric oxide magnetic nanoparticles are obtained; (b), glutaraldehyde is taken as a crosslinking agent and has a crosslinking reaction with the carboxyl-functionalized ferroferric oxide magnetic nanoparticles, magnetic separation is performed on a carrier by means of an applied magnetic field, a kappa-carrageenan enzyme liquid is added for immobilization after the carrier is cleaned, cleaning is performed for multiple times after immobilization ends, uncombined free enzymes are removed, and the immobilized kappa-carrageenan enzyme is obtained; (c), kappa-carrageenan is hydrolyzed with the adoption of the prepared immobilized kappa-carrageenan enzyme, and the kappa-carrageenan oligosaccharide is prepared. The process is simple, the enzyme activity recovery rate is high, the immobilized enzyme has good stability, can be repeatedly used and is suitable for industrial production, and a product is easy to separate.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme preparation, and in particular relates to an immobilized κ-carrageenan enzyme and a method for preparing κ-carrageenan oligosaccharides. Background technique [0002] Carrageenan, also known as carrageenan, carrageenan, Irish carrageenan, is mainly a natural seaweed polysaccharide extracted from the cell wall of some red algae seaweed, widely used in food, chemical industry, light industry, medicine and other fields. Carrageenan oligosaccharides are formed by the degradation of carrageenan, and have physiological activities such as anti-oxidation, anti-tumor, anti-virus, anti-coagulation, and immune regulation. [0003] The traditional methods for preparing carrageenan oligosaccharides are mainly physical degradation and chemical degradation. However, the preparation of carrageenan oligosaccharides by physical and chemical degradation methods has disadvantages such as difficult control...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12P19/14
Inventor 肖安风蔡慧农倪辉许彩云朱艳冰杨秋明杜希萍陈艳红
Owner JIMEI UNIV
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