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Method for fixing beta-glucuronidase by alginate-calcium carbonate hybrid gel

A technology of aldolidase and hybrid gel, applied in the direction of immobilized on/in organic carrier, hydrolytic enzyme, etc., can solve the problems of poor effect of enzyme molecules, affecting adsorption effect, easy leakage of enzyme molecules, etc. Achieve the effect of simple and easy preparation process, good repeated use and good stability

Inactive Publication Date: 2008-06-25
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The usual problem with alginate gel immobilized enzymes is that the carrier has a large pore size and the enzyme molecules are easy to leak
[0004] Mesoporous silica materials are the most commonly used adsorbents for immobilizing enzymes, but studies have shown that although such mesoporous materials have large specific surface areas and pore volumes, they generally have small pore diameters and can adsorb large-sized enzyme molecules. Ineffective
In addition, due to the limitation of the synthesis method, the general mesoporous silica is negatively charged, and there is electrostatic repulsion with the negatively charged enzyme molecules, which affects the adsorption effect.

Method used

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  • Method for fixing beta-glucuronidase by alginate-calcium carbonate hybrid gel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Accurately weigh 2.5 mg of β-glucuronidase, dissolve it in 0.05mol / L tris-hydrochloric acid (Tris-HCl) buffer solution, and dilute to 5 mL to obtain 0.5 mg / mL β-glucuronic acid glycosidase solution. Then, 15 mg of calcium carbonate particles were mixed with 1 ml of 0.5 mg / mL beta-glucuronidase solution, adsorbed for 2 hours, and centrifuged to obtain calcium carbonate particles adsorbing glucuronidase. Dissolve sodium alginate in deionized water to make a 2.0% solution, mix 5ml of sodium alginate solution with 15mg of calcium carbonate particles that adsorb glucuronidase, and add dropwise to 0.2M calcium chloride solution with a syringe , let stand for 30 min to obtain alginate-calcium carbonate hybrid gel-immobilized β-glucuronidase particles, measure the content of glucuronidase in the calcium chloride solution, and obtain the glucuronidase during the gel formation process. The enzyme leakage rate was 9.4%. The hybrid gel particles with immobilized glucuronidase were...

Embodiment 2

[0021] Pour 100ml of 0.33M sodium carbonate solution into an equal volume and equal concentration of calcium chloride solution rapidly, stir at high speed for 30s at room temperature, then stop stirring, let stand for 15 minutes, and after centrifugation, the solid calcium carbonate obtained is successively deionized. Wash with water and acetone, and dry naturally in the air to obtain micron-sized mesoporous calcium carbonate particles; dissolve sodium alginate in deionized water to make a 2.0% solution, take 5ml of sodium alginate solution and mix 15mg of calcium carbonate particles evenly, and use Syringe was added dropwise to 0.2M calcium chloride solution, and aged for 30min to obtain alginic acid-calcium carbonate hybrid gel particles. Dissolve baicalin and anhydrous sodium sulfite into 0.03 mol / L Tris-HCl buffer solution to prepare a solution with a baicalin concentration of 0.09 mol / L and an anhydrous sodium sulfite concentration of 0.1% w / v, and add hybrid gel particles...

Embodiment 3

[0022] Example 3: Determination of Cyclic Stability

[0023] The cycling stability of the β-glucuronidase-immobilized hybrid gel particles prepared in Example 1 of the present invention was determined:

[0024] Dissolve baicalin and anhydrous sodium sulfite in a 0.03mol / L Tris-HCl buffer solution to prepare a solution with a baicalin concentration of 0.09mol / L and an anhydrous sodium sulfite concentration of 0.1% w / v, and add the solution prepared in Example 1. The granules of immobilized β-glucuronidase were subjected to the conversion reaction of baicalin under stirring conditions at 37 °C, and the amount of baicalin was determined by high performance liquid chromatography to obtain the immobilized β-glucuronidase. The enzyme activity was taken as the initial enzyme activity, which was defined as 100%.

[0025] The reaction solution was filtered, and the particles were washed with deionized water until the supernatant was free of baicalin and baicalein. The above reaction ...

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Abstract

The invention relates to a method for using alginic acid-calcium carbonate hybrid gel to immobilize beta-glucuronidase, wherein, calcium carbonate powdered precipitation is generated by mixing and stirring sodium carbonate solution and calcium chloride solution at room temperature, and then kept, washed by water and acetones and dried, and micron mesoporous calcium carbonate particles are obtained which are added into trihydroxymethyl aminomethane-hydrochloric acid (Tirs-HCl) buffer of beta-glucuronidase or aqueous solution thereof for absorption, and calcium carbonate particles absorbing glucuronidase are obtained after centrifugal separation, then uniformly mixed with sodium alginate solution and added into calcium chloride solution by dropping for aging. The invention has the advantages of mild preparation conditions, simple and easy preparation technologies, good immobilization ability of obtained hybrid vectors on enzymes, low leakage rate of immobilized beta-glucuronidase, low swelling capacity and good reusage stability.

Description

technical field [0001] The invention relates to an enzyme immobilization technology, in particular to a method for immobilizing β-glucuronidase on an alginic acid-calcium carbonate hybrid gel. Background technique [0002] Alginate gel is a commonly used immobilized enzyme carrier. At present, alginate gel-immobilized enzymes mainly adopt the embedding method. The embedding method has mild conditions and good biocompatibility, which is conducive to maintaining the structural integrity of the enzyme, thereby improving the activity maintenance rate of the enzyme, and the carrier has a large pore size, which is conducive to the transfer of reactants and products. [0003] The common problem with alginate gel immobilized enzymes is that the pore size of the carrier is large, and the enzyme molecules are easy to leak. There is a phenomenon of "syneresis" during the gel formation process, and a part of the enzyme will be expelled from the carrier with the water. The gel is easy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/04C12N9/24
Inventor 姜忠义吴洪李健张羽飞杨冬
Owner TIANJIN UNIV
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