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Preparation method of nanometer fiber immobilization beta-D-galactosidase

A technology of galactosidase and nanofibers, which is applied in the fields of immobilization on/in organic carriers, fiber treatment, textiles and papermaking, etc., to achieve the effect of mild immobilization conditions and high vitality maintenance rate

Inactive Publication Date: 2010-10-20
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above enzyme immobilization methods help to improve the stability of the enzyme, the immobilized enzyme with a particle size of nanometer or micrometer scale still cannot be recycled in a convenient way
CN101191782 firstly cross-link β-gal on the surface of aldylated cyclodextrin microspheres, then smear the enzyme-loaded microspheres on two polycarbonate microporous membranes, add sodium alginate solution dropwise on the membranes, and then apply the two polycarbonate microporous membranes The membranes are soaked together in calcium chloride solution and solidified to obtain a β-gal enzyme membrane with a sandwich structure. The half-life of the immobilized enzyme membrane is 15-20 days, the stability is good, and the block membrane is easy to recycle, but the disadvantage of this method is that There are severe diffusion limitations

Method used

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  • Preparation method of nanometer fiber immobilization beta-D-galactosidase
  • Preparation method of nanometer fiber immobilization beta-D-galactosidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Accurately weigh 3.5 g of PSMA and dissolve it in 10 mL of N,N-dimethylformamide, and stir it with magnetic stirring at room temperature to make it fully dissolved to obtain an electrospinning solution with a concentration of 35% (W / V). 5 mL of electrospinning solution was sucked into a syringe equipped with an 8-gauge needle for electrospinning, the distance from the syringe needle to the receiving plate was 25 cm, the flow rate of the electrospinning solution was 250 μL / h, the voltage was 21 kV, and PSMA nanofibers were obtained after electrospinning for 30 min. Leave at room temperature until the solvent evaporates completely.

[0016] 1 mg of PSMA nanofibers were soaked in 1 mL of 25% (V / V) ethanol aqueous solution for 15 min, and washed twice with deionized water and 0.2 mol / L acetic acid-sodium acetate buffer solution (pH 4.0) respectively. A β-gal solution with a concentration of 1.0 mg / mL was prepared with acetic acid-sodium acetate buffer solution (0.2 mol / L) a...

Embodiment 2

[0018] Accurately weigh 3.5 g of PSMA and dissolve it in 10 mL of N,N-dimethylacetamide, and stir it with magnetic stirring at room temperature to make it fully dissolved to prepare an electrospinning solution with a concentration of 35% (W / V). 5 mL of electrospinning solution was sucked into a syringe equipped with an 8-gauge needle for electrospinning, the distance from the syringe needle to the receiving plate was 25 cm, the flow rate of the electrospinning solution was 250 μL / h, the voltage was 21 kV, and PSMA nanofibers were obtained after electrospinning for 30 min. After the solvent was completely evaporated at room temperature, the microscopic morphology of the nanofibers was characterized by a scanning electron microscope (SEM). The SEM pictures showed that the diameter of the fibers was about 300 nm.

[0019] 1 mg of PSMA nanofibers were soaked in 1 mL of 25% (V / V) ethanol aqueous solution for 15 min, and washed twice with deionized water and 0.2 mol / L acetic acid-sod...

Embodiment 3

[0021] The immobilized β-gal prepared in Example 2 was used to catalyze the hydrolysis of 2-nitrophenol β-D-galactopyranoside (ONPG), and 1 mg of immobilized β-gal was added to 5 mL of ONPG preheated to 37 °C In the solution (0.2mol / L acetic acid-sodium acetate buffer solution, pH4.5), place the reaction solution in a 70rpm shaker for 20min, take out 100μL of the reaction solution and add 1mol / LNa 2 CO 3 100 μL to stop the reaction. The absorbance value of the solution was measured at 405 nm to calculate the enzymatic activity. After the reaction was completed, the immobilized enzyme was washed with 5 mL of 0.2 mol / L acetic acid-sodium acetate buffer solution (pH 4.5), and then put into the next reaction. The immobilized β-gal could still maintain 85% catalytic activity after repeated use for 21 times.

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Abstract

The invention relates to a preparation method of nanometer fiber immobilization beta-D-galactosidase, which belongs to the technical field of enzyme immobilization and application thereof. The method comprises the following processes: using phenylethylene-maleic anhydride copolymers for preparing nanometer fiber by a static electric spinning method; carrying out pretreatment on an ethanol water solution; and then, using anhydride groups on the surface of the fiber for carrying out covalence coupling on beta-D-galactosidase to obtain immobilization beta-D-galactosidase. The enzyme load capacity can reach 20 to 30 mg enzyme / g nanometer fiber, the enzyme activity recovery rate is between 45 and 60 percent, and 85 percent catalysis activity of the immobilization enzyme can still be maintained after the immobilization enzyme is repeatedly used for 21 times. The method has the advantages of simplicity, convenience and easy implementation. The obtained immobilization enzyme can be easily recovered and repeatedly used, and can be used in the fields of the synthesis of alkyl galactoside and functional oligomate, the production of low lactose milk and the like.

Description

technical field [0001] The invention relates to a preparation method of nanofiber immobilized beta-D-galactosidase, belonging to the technical field of enzyme immobilization and its application. Background technique [0002] β-D-galactosidase (β-D-galactosidase, β-gal, EC 3.2.1.23) has the dual function of catalyzing the hydrolysis and generation of β-galactosidic bonds, and is used in the fields of dairy products, health products and detergent production. have important uses. The lactose in milk can be decomposed into galactose and glucose by the hydrolysis of β-gal, and milk products that can be consumed by people with lactose intolerance can be obtained; the hydrolysis function of β-gal is related to the isomerization of glucose isomerase. Binding converts lactose solution into galactose sugar syrup, thereby replacing sucrose in food processing. On the other hand, a variety of functional oligosaccharides, such as galacto-oligosaccharides and lactulose oligosaccharides, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08D01D5/00
Inventor 苏志国张羽飞张松平郑国建
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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