Chitosan microspheres for enzyme immobilization and preparation method and application thereof

A chitosan microsphere and enzyme immobilization technology, which is applied in the preparation of microspheres, immobilization on/in organic carriers, microcapsule preparations, etc., can solve the problems of sample dilution and large loss of carrier recovery, etc. Achieve the effect of easy separation and recovery, high recovery rate of enzyme activity and good heat resistance

Inactive Publication Date: 2016-06-22
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, for the application of chitosan as a carrier for immobilizing enzymes, the

Method used

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  • Chitosan microspheres for enzyme immobilization and preparation method and application thereof
  • Chitosan microspheres for enzyme immobilization and preparation method and application thereof
  • Chitosan microspheres for enzyme immobilization and preparation method and application thereof

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0046] Example 1

[0047] (1) Weigh 0.5g of chitosan powder into a 100mL three-necked flask, add 30ml of 5% acetic acid solution, and stir at room temperature. After the chitosan is fully dissolved, add 30ml of liquid paraffin and stir for 10 minutes;

[0048] (2) Heat the resultant of step (1) to 50°C, add 2-3 drops of emulsifier Span80 dropwise, and emulsify for 10 minutes to form fine chitosan droplets;

[0049] (3) Add 1.0 mL of formaldehyde solution to the resultant of step (2), and stir and react for 1.5 hours;

[0050] (4) Raise the temperature of the resultant of step (3) to 70°C, add 5-6 drops of 10% NaOH solution with a mass fraction of 10% dropwise to keep the pH of the solution alkaline, and slowly add 2.0 mL of epoxy chloride dropwise Propane, react for 5 hours;

[0051] (5) Filter the resultant of step (4), wash with distilled water, and then wash with petroleum ether, absolute ethanol, and water successively until the pH value of the washing liquid reaches neutral;

[005...

Example Embodiment

[0085] Example 2

[0086] (1) Weigh accurately 10 mg of chitosan microsphere carrier, swell it with 10 mL of phosphate buffer (PBS, pH=7) for 24 hours and then separate;

[0087] (2) Add 10 mL of 5% glutaraldehyde solution to the resultant of step (1), shake and activate at 25°C for 5 hours and filter, and wash off excess glutaraldehyde with distilled water;

[0088] (3) Add 2.0 mg of cellulase enzyme solution to the resultant of step (2), shake and fix at 30°C for 4 hours, filter, and wash the residue repeatedly with phosphate buffer to obtain immobilized enzyme. The immobilized enzyme is stored in the refrigerator (4°C) for later use.

[0089] The method for measuring cellulase activity and the method for measuring the enzyme activity of the immobilized enzyme obtained in Example 2 are described below.

[0090] Determination of free enzyme activity: Take an appropriate amount of cellulase and dissolve it in HAc-NaAc buffer to prepare a certain concentration of cellulase solution. Ta...

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Abstract

The invention discloses chitosan microspheres for enzyme immobilization and a preparation method and application thereof.The preparation method comprises the steps that an acetic acid solution with the mass fraction of 5% is added to 0.5 g of chitosan powder, liquid paraffin is added after the chitosan powder is sufficiently dissolved, and stirring is conducted for 10 min; the temperature is increased to 50 DEG C, an emulsifier Span80 is dropwise added, and emulsification is conducted for 10 min; 0.5-2.0 mL of formaldehyde solution is added, and a stirring reaction is conducted for 1.5 h; the temperature is increased to 70 DEG C, a NaOH solution with the mass fraction of 10% is dropwise added, 1.0-2.5 mL of epoxy chloropropane is slowly and dropwise added on the condition that the pH value of the solution is kept alkaline, and filtering is conducted after a reaction is conducted for 5 h; an obtained product is washed with distilled water and then sequentially washed with petroleum ether, absolute ethyl alcohol and water, the product is subjected to heating treatment with hydrochloric acid of l mol/L for 9 h and then sequentially subjected to alkali washing, acid washing and water washing till the pH value of washing liquid is neutral, the product is dried to a constant weight, and the chitosan microspheres (NCTS) for enzyme immobilization are obtained.The chitosan microspheres are good carriers for immobilizing enzymes.

Description

technical field [0001] The invention relates to the field of chitosan microsphere preparation and application research, in particular to a chitosan microsphere for enzyme immobilization, a preparation method and application thereof. Background technique [0002] Cellulose is the most widely distributed, most abundant, and renewable natural organic substance, which can continuously provide energy for human beings. However, in nature, most of the cellulose will be decomposed and transformed by various microorganisms, and only about 11% of the cellulose raw materials are used in crop products, feed, pharmaceuticals, textiles, papermaking and construction, which not only causes a huge waste of resources , and pollute the environment, the harm is quite large. [0003] Enzymes are extremely sensitive to environmental conditions and are unstable to acids, alkalis, heat, metal ions, and some organic solvents. Generally, enzymes act on substrates in a water-soluble state, and there...

Claims

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Application Information

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IPC IPC(8): C08B37/08B01J13/04C12N11/10
Inventor 熊春华缪喜喜皮蕾蕾李松范卓莹
Owner ZHEJIANG GONGSHANG UNIVERSITY
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