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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

AI Technical Summary

Problems solved by technology

[0007] However, for the application of chitosan as a carrier for immobilizing enzymes, there are problems such as centrifugation, sample dilution and large loss of carrier recovery.

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
Effect test

Embodiment 1

[0047] (1) Take by weighing 0.5g chitosan powder in a 100mL three-neck bottle, add 30ml mass fraction of 5% acetic acid solution, stir at room temperature, after chitosan is fully dissolved, add 30ml liquid paraffin, stir for 10 minutes;

[0048] (2) Warm up the product 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 obtained product of step (2), and stir and react for 1.5 hours;

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

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

[0052](6) Heat the product obtaine...

Embodiment 2

[0086] (1) Accurately weigh 10 mg of chitosan microsphere carrier, and separate after swelling with 10 mL of phosphate buffer solution (PBS, pH=7) for 24 hours;

[0087] (2) Add 10 mL of 5% glutaraldehyde solution to the obtained product 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 obtained product of step (2), shake and fix at 30° C. for 4 hours, filter, and wash the filter residue repeatedly with phosphate buffer to obtain immobilized enzyme. The immobilized enzymes were 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 will be described below.

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

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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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