A method for separating and immobilizing multiple enzymes

A mass percentage content and coating technology, which is applied to multi-enzyme systems, fixed on/in organic carriers, etc., can solve the problems that the reaction cannot be carried out as scheduled, so as to maintain activity, realize simple recycling, and react The effect of mild conditions

Active Publication Date: 2018-12-25
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such as protease, when it works together with other enzymes, it tends to decompose other enzymes (essentially proteins) so that the reaction cannot proceed as expected
However, there are relatively few reports on the separate immobilization of multiple enzymes that inhibit each other on the same substrate

Method used

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  • A method for separating and immobilizing multiple enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Spread 40 μL, 3 mmol / mL of isopropylthioxanthone acetone solution evenly on 3 × 3 cm 2 One side of the low-density polyethylene film, and then the film coated with the solution was placed between two quartz plates, and then the system was placed in a high-pressure mercury lamp (wavelength 254nm, 9mW / cm 2 ) at room temperature for 3 minutes. After the ultraviolet irradiation, the low-density polyethylene film was extracted with acetone for 24 hours, and dried at room temperature.

[0034]Dissolve a certain amount of trypsin in 0.1M Tris-HCl (pH=7.0) buffer solution, shake and dissolve to form a 11.5 mg / mL trypsin solution, and mix this solution with polyethylene glycol diacrylate according to 75: After mixing at 45 (v / v), take 60 μL and apply it on one side of the low-density polyethylene film grafted with isopropylthioxanthone, and then place the film coated with the solution on two pieces with patterned Between the masks in the light-transmitting area, the system was...

Embodiment 2

[0043] Spread 600 μL, 5 mmol / mL isopropylthioxanthone acetone solution evenly on 3×3 cm 2 One side of the polypropylene non-woven fabric, and then the solution-coated non-woven fabric is arranged between two quartz plates, and then the system is placed in a high-pressure mercury lamp (light intensity is 5mW / cm 2 ) at room temperature for 5 minutes. After the ultraviolet irradiation, the non-woven fabric was extracted with acetone for 24 hours, and dried at room temperature.

[0044] Dissolve a certain amount of papain in 0.1M Tris-HCl (pH=7.0) buffer, shake and dissolve to form a 10 mg / mL papain solution, and mix this solution with polyethylene glycol dimethacrylate according to 1 : 1 (v / v) after mixing, take 800μL and apply it on one side of the non-woven fabric grafted with isopropylthioxanthone, and then arrange the non-woven fabric coated with the solution between two quartz plates time, and then put the system under LED lamp (light intensity 0.5mW / cm 2 ) under irradiat...

Embodiment 3

[0051] Spread 300 μL, 2 mmol / mL isopropylthioxanthone acetone solution evenly on 4×4 cm 2 cotton cloth, and then the cotton coated with the solution was arranged between two quartz plates, and then the system was placed in a high-pressure mercury lamp (the light intensity was 1mW / cm 2 ) at room temperature for 10 minutes. After the ultraviolet irradiation, the cotton cloth was extracted with acetone for 24 hours and dried at room temperature.

[0052] Dissolve a certain amount of glucose oxidase in 0.1M Tris-HCl (pH=7.0) buffer solution, shake and dissolve to form a 10 mg / mL glucose oxidase solution, and mix this solution with methylenebisacrylamide according to 2: 1 (v / v) after mixing, take 500 μL and apply it on one side of the cotton cloth grafted with isopropylthioxanthone, then arrange the cotton coated with the solution between two quartz plates, and then place the The system was placed in a xenon lamp (light intensity 2mW / cm 2 ) under irradiation for 60 minutes. Aft...

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Abstract

Belonging to the field of immobilized enzyme preparation, the invention provides a method for separation and fixation of multiple enzymes. The method includes the steps of: firstly conducting in-situ embedding and fixing of enzyme A in a three-dimensional network on a polymer substrate surface; then under the irradiation of visible light, letting light induced dormant species on the surface of the three-dimensional network layer fixed with the enzyme A regenerate surface free radicals, initiating an enzyme B mixed monomer solution to conduct secondary reactive grafting and crosslinking polymerization, subjecting the enzyme B to in-situ embedding and fixation in the three-dimensional network on a first layer surface to form a second layer cross-linking network; and repeating the above steps to embed different enzymes respectively in more network layers. The immobilization process can preserve the activity of most enzymes. Different enzymes are separated and fixed in different network layers, thus avoiding mutual interference suppression, and also completing a complex chemical reaction together. The polymer substrate can improve the mechanical strength of a composite membrane, enhances the stability of the enzyme membrane, and is more conducive to recycling of enzyme membranes.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme preparation, and in particular relates to a method for separating and immobilizing various enzymes on the surface of a polymer substrate through active graft crosslinking polymerization. Background technique [0002] As a kind of mild and efficient catalyst, biological enzyme has a very bright prospect in laboratory research and industrial application. Compared with chemical reactions, enzymatic reactions have milder conditions, simpler operations, higher regioselectivity, fewer by-products in the reaction, and many reactions that are difficult to synthesize by chemical methods can be achieved by one or more enzymes. To be done. However, the cost of the enzyme itself is high, and the stability of the free enzyme is poor, which has become a bottleneck hindering its popularization. In order to further broaden the scope of application of the enzyme, immobilizing the biological enzyme on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/18C12N11/08
Inventor 杨万泰朱兴赵长稳马育红
Owner BEIJING UNIV OF CHEM TECH
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