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Preparation method of immobilized enzyme

An enzyme immobilized, time-fixed technology, applied in biochemical equipment and methods, immobilized on or in inorganic carriers, oxidoreductase, etc. , fierce reaction and other problems, to avoid adverse effects of enzyme activity, high recovery rate of enzyme activity, and not easy to leak

Inactive Publication Date: 2018-04-10
宁夏乙征生物工程有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical immobilization of enzymes has the advantages of firm combination and high stability, but the immobilization process is relatively intense, which may easily cause enzyme inactivation and low recovery rate of enzyme activity.
[0006] With the continuous deepening of the research on immobilized enzyme technology, many new immobilization methods are emerging, but they are all improvements or combinations of the above methods, and it is still difficult to balance high enzyme activity recovery and high stability.

Method used

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  • Preparation method of immobilized enzyme
  • Preparation method of immobilized enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The mensuration of embodiment 1 enzyme solution protein concentration

[0044] The protein concentration was determined by the Bradford method. Coomassie Brilliant Blue G-250 dye was light red in acidic ethanol solution, and the blue solution formed after combining with protein had the maximum light absorption at 595nm. The absorbance value is directly proportional to the protein concentration, so detect the light absorbance value of the solution at 595nm to calculate the protein content.

[0045] (1) Bradford stock solution: Weigh 350 mg of Coomassie Brilliant Blue G-250, dissolve it in 100 mL of 95% ethanol, add 200 mL of 85% phosphoric acid, and store it in a refrigerator at 4°C;

[0046] (2) Bradford working solution: Add 15mL 95% ethanol, 30mL 85% phosphoric acid and 30mL Bradford stock solution to 425mL double-distilled water, mix well and store in a refrigerator at 4°C. Before each use, filter with filter paper before use;

[0047] (3) Standard bovine serum alb...

Embodiment 2

[0050] Embodiment 2 Enzyme solution and the determination of the activity of immobilized enzyme

[0051] Enzyme activity, also known as enzyme activity, refers to the ability of an enzyme to catalyze a certain chemical reaction. The unit of enzyme activity is U, which means that under certain conditions, the amount of enzyme required to convert 1 micromole of substrate or generate 1 micromole of product per unit time is an activity unit (U). The certain conditions are usually the optimum temperature and optimum pH conditions of the enzyme. In the actual enzyme activity determination, a certain volume of enzyme solution or a certain mass of immobilized enzyme is used to catalyze a chemical reaction of a certain concentration of substrate under certain conditions. The activity of the enzyme solution is expressed as U / mL, and the activity of the immobilized enzyme is expressed as U / g.

Embodiment 3

[0052] Example 3 Immobilization rate of enzyme and recovery rate of immobilized enzyme enzyme activity

[0053] Enzyme immobilization rate=(protein amount of enzyme solution before immobilization−protein amount in supernatant of immobilization solution after immobilization) / protein amount of enzyme solution before immobilization×100%. Recovery rate of immobilized enzyme enzyme activity = enzyme activity of immobilized enzyme / (enzyme activity of input enzyme solution-enzyme activity of supernatant of immobilized solution after immobilization)×100%.

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Abstract

The invention discloses a preparation method of an adsorption-film covering combined immobilized enzyme. The preparation comprises the following steps: (1) dissolving an enzyme in a buffer solution toprepare an enzyme solution, mixing the enzyme solution with an adsorption material, carrying out oscillating adsorption for a certain time, and filtering out, to obtain an adsorbent immobilized enzyme; (2) dissolving a film covering protective agent in a buffer solution to prepare a protective agent solution, placing the adsorbent immobilized enzyme in the protective agent solution, carrying outoscillation treatment for a certain time, filtering out and draining, to obtain an adsorbent immobilized enzyme treated by the protective agent; (3) dissolving a film covering material in a solvent toprepare a film covering solution, mixing the adsorbent immobilized enzyme treated by the protective agent with the film covering solution, carrying out oscillating film covering for a period of time,and filtering out and drying; and (4) finally, swashing with deionized water or a buffer solution to obtain the adsorption-film covering combined immobilized enzyme. The preparation method has the advantages of mild conditions of the adsorption and film covering processes, high recovery rate of enzyme activity, high stability of the immobilized enzyme, simple operation and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of immobilized enzyme. Background technique [0002] In recent years, modern biotechnology represented by genetic engineering, cell engineering, enzyme engineering, and fermentation engineering has developed rapidly, and is increasingly affecting and changing people's production and lifestyle. Enzyme engineering is an important part of biotechnology. It is a science and technology that uses the biocatalytic function of enzymes to convert corresponding raw materials into useful substances with the help of engineering methods and applies them to social life. It includes the preparation of enzyme preparations, immobilization of enzymes, modification and transformation of enzymes, and enzyme reactors. [0003] As a kind of biocatalyst, enzyme has high efficiency, selectivity, specificity and mild reaction conditions, and has been widely used in pharmaceu...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/12C12N11/08C12N11/04
CPCC12N9/0061C12N9/63C12N11/04C12N11/08C12N11/12C12N11/14C12Y110/03002C12Y304/22002
Inventor 苏二正杨猛孙伊伊陈龙
Owner 宁夏乙征生物工程有限公司
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