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

A technology of aldolidase and hybrid gel, which is applied in the direction of immobilized on/in organic carriers, hydrolytic enzymes, etc., can solve the problems of poor effect of enzyme molecules, easy leakage of enzyme molecules, and affecting the adsorption effect, etc. Achieve the effects of simple and easy preparation process, good reusability and low swelling degree

Inactive Publication Date: 2009-10-28
TIANJIN UNIV
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  • Summary
  • 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
  • Method for fixing beta-glucuronidase by alginate-calcium carbonate hybrid gel
  • Method for fixing beta-glucuronidase by alginate-calcium carbonate hybrid gel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Accurately weigh 2.5mg of β-glucuronidase, dissolve in 0.05mol / L tris-hydrochloric acid (Tris-HCl) buffer solution, and dilute to 5mL to obtain 0.5mg / mL β-glucuronidase Acidase solution. Then, mix 15 mg of calcium carbonate particles with 1 ml of 0.5 mg / mL β-glucuronidase solution, adsorb for 2 hours, and centrifuge to obtain calcium carbonate particles adsorbing glucuronidase. Dissolve sodium alginate in deionized water to make a 2.0% solution, take 5ml of sodium alginate solution and mix evenly with 15mg of calcium carbonate particles adsorbing glucuronidase, and add dropwise to 0.2M calcium chloride solution with a syringe , stand still for 30min, get alginic acid-calcium carbonate hybrid gel immobilized β-glucuronidase granule, measure the glucuronidase content in the calcium chloride solution, obtain the glucuronidase in the process of gel formation The enzyme leakage rate is 9.4%. The hybrid gel particles of immobilized glucuronidase are soaked in tris-hydrochlor...

Embodiment 2

[0021] Quickly pour 100ml of 0.33M sodium carbonate solution into calcium chloride solution of equal volume and concentration, stir at high speed for 30s at room temperature, then stop stirring, let stand for 15 minutes, and centrifuge the obtained solid calcium carbonate in turn with 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 evenly with 15mg of calcium carbonate particles, and use The syringe was added dropwise into 0.2M calcium chloride solution and aged for 30 minutes to obtain alginic acid-calcium carbonate hybrid gel particles. Dissolve baicalin and anhydrous sodium sulfite into 0.03mol / L Tris-HCl buffer solution to prepare a solution with baicalin concentration of 0.09mol / L and anhydrous sodium sulfite concentration of 0.1% w / v, and add hybrid gel particles , store at roo...

Embodiment 3

[0022] Embodiment 3: the mensuration of cycle use stability

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

[0024] Dissolve baicalin and anhydrous sodium sulfite in 0.03mol / L Tris-HCl buffer solution, make baicalin concentration be 0.09mol / L, anhydrous sodium sulfite concentration be the solution of 0.1% w / v, 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 production amount of baicalin was determined by high performance liquid chromatography to obtain the immobilized β-glucuronidase Enzyme activity, and taking this enzyme activity as the initial enzyme activity, was defined as 100%.

[0025] The reaction solution was filtered, and the particles were washed with deionized water until there was no baicalin and baicalein in the supernatant. Th...

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Abstract

The invention relates to a method for immobilizing beta-glucuronidase by alginic acid-calcium carbonate hybrid gel. Sodium carbonate solution and calcium chloride solution are mixed and stirred at room temperature to generate calcium carbonate powder precipitation, which is left to stand, washed with water and acetone, and dried to obtain micron-sized mesoporous calcium carbonate particles. Adsorb in aminomethane-hydrochloric acid (Tirs-HCl) buffer solution or aqueous solution, and centrifuge to obtain calcium carbonate particles adsorbing glucuronidase and mix them evenly with sodium alginate solution, then add dropwise to calcium chloride solution, Ageing. The preparation condition of the invention is mild, the preparation process is simple and easy, the obtained hybrid carrier has good immobilization ability for enzyme, the leakage rate of the immobilized β-glucuronidase is low, the swelling degree is low, and the repeated use stability is good.

Description

technical field [0001] The invention relates to an enzyme immobilization technology, in particular to a method for immobilizing beta-glucuronidase by alginic acid-calcium carbonate hybrid gel. Background technique [0002] Alginate gel is a commonly used carrier for immobilized enzymes. At present, the alginate gel immobilization method mainly adopts 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 usual problem with alginate gel immobilization of enzymes is that the carrier has a large pore size and the enzyme molecules are easy to leak. During the gel formation process, there is a phenomenon of "syneresis", and a part of the enzyme will be discharged from the carrier with water. The ...

Claims

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Application Information

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Patent Type & Authority Patents(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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