Enzyme immobilization method based on super-branching polymer

Abstract

The invention relates to an enzyme immobilization method which is based on hyper-branched polymer. The method belongs to the biology engineering technology area. The method comprises that the carrieris immersed in the synthesized hyper-branched polymer solution with a large amount of peripheral diazo salt groups or in the diluted solution which dilutes the hyper-branched polymer solution or the organic solvent of N, N-dimethylformamide less than 100 times over 30 minutes; then the carrier is taken out from the diluted solution and washed by water; the carrier is immersed in the buffer which contains the enzyme below 4 degree for over 4 hours to reach saturation adsorption. The method can greatly simplify the operation steps of enzyme immobilization, which requires no modification in priorperiod for the material and the enzyme; the method has high universality, which can be used on a plurality of enzymes and carriers; and the method does not affect catalytic activity of the enzyme.

Description

technical field

[0001] The invention belongs to the technical field of bioengineering, in particular to a method for immobilizing enzymes and other bioactive components on various polymer films and inorganic materials. Background technique

[0002] With the rapid development of biotechnology, bioengineering, as a frontier field of technological revolution, has been widely used in industry, agriculture, and medicine, and plays a decisive role in solving many problems faced by human beings today, such as resources, energy, and environmental protection. effect.

[0003] As one of the components of bioengineering, enzyme engineering has been widely used in food, textile and other sectors in industrial production. However, due to the low stability of natural enzymes, they are extremely sensitive to high temperature and organic solvents, are easily inactivated, and cannot be reused. , the enzyme is mixed in the product after the catalyzed reaction, making it difficult to purify, ...

Examples

Embodiment 1

[0029] Cut the polyethylene film into a 2cm×2cm film, wash it with ultrapure water, dilute the synthesized DAS solution 10 times with water, then soak and absorb it in the diluted DAS solution at 4°C for 30min, and put the polyethylene film Take it out, wash it with water, add it to the phosphate buffer solution of beta-glucosidase at pH=7.0, let it stand at 4°C for 4 hours to make it saturated, take out the film, and wash it.

[0030] The material was immersed in the prepared substrate solution of a certain concentration, and the amount of immobilized enzyme was determined by measuring the ultraviolet absorption of the product. It was found that when the hyperbranched diazonium salt was used as the enzyme immobilization reagent, an obvious UV absorption; when the diazonium salt is not used as the enzyme immobilization reagent, the UV absorption of the product cannot be detected, indicating that the use of diazonium salt as the enzyme immobilization reagent can have a good immo...

Embodiment 2

[0032] Take a certain amount of polypropylene tubular material, wash it with ultrapure water, dilute the synthesized DAS-1 solution 20 times with DMF, then soak and absorb it in the DAS solution at 3°C ​​for 40min, take out the polypropylene, and use ultrapure water Wash it with pure water, then add it to the citric acid-sodium citrate buffer solution of beta-glucosidase at pH=6.5, place it at 2°C for 6 hours to make it saturated, take out the material, wash it, and then put It is immersed in the prepared substrate solution of a certain concentration, and the amount of immobilized enzyme is determined by measuring the ultraviolet absorption of the product.

Embodiment 3

[0034] Cut the nylon 66 film into a 2cm×2cm film, wash it with water, dilute the synthesized DAS-2 solution 90 times with water, then soak and absorb it in the DAS solution at 1°C for 1 hour, take out the nylon 66 film, and use Wash it with ultrapure water, then add it to the beta-glucosidase disodium hydrogen phosphate-citric acid buffer solution at pH=6.0, place it at 4°C for 24 hours to make it saturated, take out the film, wash it, Then immerse it in the prepared substrate solution of a certain concentration, and determine the amount of immobilized enzyme by measuring the ultraviolet absorption of the product.

technical field

[0001] The invention belongs to the technical field of bioengineering, in particular to a method for immobilizing enzymes and other bioactive components on various polymer films and inorganic materials. Background technique

[0002] With the rapid development of biotechnology, bioengineering, as a frontier field of technological revolution, has been widely used in industry, agriculture, and medicine, and plays a decisive role in solving many problems faced by human beings today, such as resources, energy, and environmental protection. effect.

[0003] As one of the components of bioengineering, enzyme engineering has been widely used in food, textile and other sectors in industrial production. However, due to the low stability of natural enzymes, they are extremely sensitive to high temperature and organic solvents, are easily inactivated, and cannot be reused. , the enzyme is mixed in the product after the catalyzed reaction, making it difficult to purify, ...

Examples

Embodiment 1

[0029] Cut the polyethylene film into a 2cm×2cm film, wash it with ultrapure water, dilute the synthesized DAS solution 10 times with water, then soak and absorb it in the diluted DAS solution at 4°C for 30min, and put the polyethylene film Take it out, wash it with water, add it to the phosphate buffer solution of beta-glucosidase at pH=7.0, let it stand at 4°C for 4 hours to make it saturated, take out the film, and wash it.

[0030] The material was immersed in the prepared substrate solution of a certain concentration, and the amount of immobilized enzyme was determined by measuring the ultraviolet absorption of the product. It was found that when the hyperbranched diazonium salt was used as the enzyme immobilization reagent, an obvious UV absorption; when the diazonium salt is not used as the enzyme immobilization reagent, the UV absorption of the product cannot be detected, indicating that the use of diazonium salt as the enzyme immobilization reagent can have a good immo...

Embodiment 2

[0032] Take a certain amount of polypropylene tubular material, wash it with ultrapure water, dilute the synthesized DAS-1 solution 20 times with DMF, then soak and absorb it in the DAS solution at 3°C ​​for 40min, take out the polypropylene, and use ultrapure water Wash it with pure water, then add it to the citric acid-sodium citrate buffer solution of beta-glucosidase at pH=6.5, place it at 2°C for 6 hours to make it saturated, take out the material, wash it, and then put It is immersed in the prepared substrate solution of a certain concentration, and the amount of immobilized enzyme is determined by measuring the ultraviolet absorption of the product.

Embodiment 3

[0034] Cut the nylon 66 film into a 2cm×2cm film, wash it with water, dilute the synthesized DAS-2 solution 90 times with water, then soak and absorb it in the DAS solution at 1°C for 1 hour, take out the nylon 66 film, and use Wash it with ultrapure water, then add it to the beta-glucosidase disodium hydrogen phosphate-citric acid buffer solution at pH=6.0, place it at 4°C for 24 hours to make it saturated, take out the film, wash it, Then immerse it in the prepared substrate solution of a certain concentration, and determine the amount of immobilized enzyme by measuring the ultraviolet absorption of the product.

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