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Silk nano granular of immobilized enzyme, and prepn. process thereof

A technology of silk fibroin nanoparticles and immobilized enzymes, which is applied in the directions of immobilization on/in organic carriers, inactive components of polymer compounds, and capsule delivery, etc., which can solve the problem of low immobilized enzyme activity and poor crystallinity of silk fibroin powder. , No more reports and other issues, to achieve good biocompatibility, low cost, good stability

Inactive Publication Date: 2008-10-22
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are no more reports at home and abroad about the immobilized enzyme research using silk fibroin powder as a carrier.
Trace it to its cause mainly because the silk fibroin powder prepared by above-mentioned these methods has poor crystallinity, poor stability, small specific surface area, low activity of immobilized enzyme

Method used

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  • Silk nano granular of immobilized enzyme, and prepn. process thereof
  • Silk nano granular of immobilized enzyme, and prepn. process thereof
  • Silk nano granular of immobilized enzyme, and prepn. process thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] After cleaning the seed cocoons, cocoon clothes, waste silk or waste silk cloth in sericulture, seed cocoon production, silk reeling, and textile production, add 30 times the amount of 0.5% sodium carbonate aqueous solution or other alkaline solutions or add surface active Boil for 1 hour, change the liquid once more, and boil for 1 hour to ensure that all the sericin is removed. The silk fiber from which the sericin has been removed is washed with water repeatedly and then dried for use.

[0028]Take the above-mentioned silk fiber from which sericin has been removed and mix it with 5-20 times the amount (W / V) of 8-9M lithium bromide aqueous solution or lithium bromide-methanol-water ternary mixed solvent, and dissolve at 40°C for 2-40 hours; or Mix the silk fiber with 5-20 times the amount of calcium chloride / ethanol / water ternary mixed solvent (molar ratio 1:2:8), and dissolve it at 50°C for 1-5 hours; or mix the above-mentioned silk fiber Dissolved in other concentr...

Embodiment 2

[0038] The preparation method is the same as that in Example 1. After mixing 1.5-3.5% water-soluble silk fibroin solution with superoxide dismutase, add it to excess acetone (final volume above 70%) that is stirred rapidly, and the rest of the preparation steps are the same. The acetone treatment method was used to prepare immobilized superoxide dismutase silk fibroin nanoparticle suspension or freeze-dried powder. The activity determination method and the activity recovery rate of the immobilized enzyme are shown in Table 2. The values ​​in the table are the average value of 5 repeated determinations.

[0039] In this experiment, the superoxide dismutase (SOD) assay kit (produced by Nanjing Jiancheng Bioengineering Institute Branch 1) was used to measure the activity of solution enzyme or immobilized enzyme. Its determination method is to produce O through the reaction system of xanthine and xanthine oxidase. 2 - , the latter oxidizes hydroxylamine to form nitrite, which is...

Embodiment 3

[0043] The preparation method is the same as that in Example 1. After mixing 1.5-3.5% water-soluble silk fibroin solution with L-asparaginase, add it to excess acetone (final volume above 70%) that is stirred rapidly, and the rest of the preparation steps are the same. The immobilized L-asparaginase silk fibroin nanoparticle suspension or freeze-dried powder were respectively prepared by this acetone treatment method, and the activity determination method and the activity recovery rate of the immobilized enzyme are shown in Table 3. The values ​​in the table are the average value of 5 repeated determinations.

[0044] L-asparaginase activity assay method (Mashburn, L.T., Wriston, J.C., jr.: Tumorinhibitory effect of L-Asparaginase. Biochen. Biophys. Res. Comm. 12: 50, 1963): Pipette several branches 0.2 ml Tris-HCl buffer solution (pH 8.6) and 1.7 ml substrate solution were dropped into the centrifuge tube, and placed in a water bath to adjust the temperature to 37°C. Add 0.1...

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Abstract

This invention discloses a method for manufacturing silk fibroin nanoparticles fixed with enzyme from silk fibroin, which comprises the steps of: (1) completely mixing the enzyme and regenerated silk fibroin solution; (2) injecting into a water-soluble organic solvent under high speed stirring to obtain white crystalline silk fibroin nanoparticles fixed with the enzyme; and (3) centrifuging or filtrating to remove the organic solvent to obtain crystalline silk fibroin nanoparticles fixed with the enzyme. The average particle size of the nanoparticles is 35-125 nm, and the activity recovery is as high as 70%. The fixed enzyme has a high thermal stability, and is not easy to be decomposed by proteinase, thus can largely reduce or even eliminate the immunogenicity of the enzyme. The nanoparticle fixed with the enzyme has important applications in sustained drug release, industrial enzyme reactor, food additives and cosmetics.

Description

technical field [0001] The invention discloses a carrier for immobilized enzymes and a preparation method thereof, in particular to a silk fibroin nanoparticle for immobilized enzymes produced by silk fibroin and a preparation method thereof, which belongs to the field of biotechnology enzyme engineering and also belongs to the field of nanotechnology and polymer chemistry technology. Background technique [0002] Introducing enzymes or drugs into the body in the form of particles, microcapsules, liposomes, albumin, red blood cell carriers, etc. can prolong the half-life of enzymes or drugs, make them release slowly, and improve the effect of enzymes or drugs on hereditary enzyme deficiency and metabolic disorders. Therapeutic efficacy of diseases, tumors and cardiovascular diseases. There are many studies on the use of natural biological materials as enzyme immobilization carriers or drug slow-release carriers because of their safety, stability, biocompatibility and abilit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/04
CPCA61K47/42C12N11/02A61K9/5169A61K9/5192
Inventor 张雨青
Owner SUZHOU UNIV
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