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Method of using surface-carboxyl-modified magnetic spheres to prepare immobilized enzyme to screen aromatase inhibitors

A technology of aromatase and carboxyl modification, applied to biochemical equipment and methods, oxidoreductases immobilized on or in inorganic carriers, etc., can solve problems such as relatively high cost, medical ethics issues, and substrate competition. Achieve the effects of improved storage stability, high research value, fast and convenient recycling and reuse

Active Publication Date: 2017-08-29
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many problems: for example, there are medical ethics issues in the source of placenta; placental microsomes are a mixture of various enzymes, and there is a problem of substrate competition, which is easy to cause false positive results
In recent years, patent CN103149186B has invented a method using homogeneous time-resolved fluorescence technology to achieve high-throughput screening of aromatase inhibitory activity, but the relative cost is also high

Method used

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  • Method of using surface-carboxyl-modified magnetic spheres to prepare immobilized enzyme to screen aromatase inhibitors
  • Method of using surface-carboxyl-modified magnetic spheres to prepare immobilized enzyme to screen aromatase inhibitors
  • Method of using surface-carboxyl-modified magnetic spheres to prepare immobilized enzyme to screen aromatase inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of Surface Carboxyl Modified Magnetic Ball Carrier

[0035] (1) Fe 3 o 4 Preparation of magnetic core: Weigh a certain amount of ferric chloride (FeCl 3 ·6H 2O), trisodium citrate, and ammonium acetate were placed in a dry round bottom flask. Add a certain amount of ethylene glycol, stir to fully dissolve. Heat and stir in an oil bath at 170-200°C for 1-2h. After the solution was slightly cooled, the black reaction solution was poured into a high-pressure reactor with polytetrafluoroethylene, and reacted in a sealed oven at 200° C. for 8-16 hours. Then take out the reaction kettle, cool it down to room temperature naturally, pour the magnetic ball solution that has finished the reaction into a beaker for magnetic sedimentation, and after obvious solid-liquid separation occurs, discard the supernatant, and dehydrate the solid magnetic balls with absolute ethanol. Washed three times with deionized water. Then put it into a vacuum drying oven a...

Embodiment 2

[0040] Example 2 Preparation of surface carboxyl-modified magnetic sphere immobilized aromatase

[0041] (1) According to Example 1, surface carboxyl-modified magnetic spheres (Fe 3 o 4 @SiO 2 -COOH).

[0042] (2) Preparation of potassium phosphate buffer (100mmol / L, pH 7.4): Weigh 4.56g K 2 HPO 4 Dissolve in 200mL deionized water to make liquid A, weigh 2.72g KH 2 PO 4 Dissolve in 200mL deionized water to form B solution, and mix A and B at a ratio of 80.2:19.8.

[0043] (3) Preparation of 0.1mol / L 2-(N-morpholine)ethanesulfonic acid monohydrate (MES) buffer solution: Weigh 1.9524g MES and dissolve it in 100mL deionized water, adjust the pH to 4.0-7.0 with 50% NaOH, Store at 4°C for later use.

[0044] (4) Weigh 7.68mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 11.5mg N-hydroxysuccinimide (NHS) in 5mL Add 2 mL of 0.1 mol / L MES buffer solution to the EP tube, vortex and mix well, and use it as an activation solution.

[0045] (5) take b...

Embodiment 3

[0047] Example 3 Determination of Surface Carboxyl Modified Magnetic Ball Immobilized Enzyme Enzyme Activity

[0048] (2) Preparation of nicotinamide adenine dinucleotide phosphate (NADPH) working solution: Weigh 8.33mg of NADPH (BIOSHARP company) into a 1mL volumetric flask, dilute with potassium phosphate buffer (100mmol / L, pH 7.4) To the mark, mix well, and store in a refrigerator at 4°C.

[0049] (3) The method for measuring the activity of the immobilized enzyme is as follows: draw 20 μL of the immobilized enzyme prepared in step (5) of Example 2 into a 2mLEP tube, and after magnetically absorbing the supernatant, add 175 μL of potassium phosphate buffer (100 mmol / L , pH 7.4), 5 μL androstenedione working solution, mix well, after shaking at 37°C for 5 minutes, add 20 μL NADPH working solution to start the reaction, react for 20 minutes, then collect the supernatant by magnetic force, take 50 μL of the supernatant and inject it into the high performance liquid phase spec...

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Abstract

The invention provides a method of using surface-carboxyl-modified magnetic spheres to prepare an immobilized enzyme to screen aromatase inhibitors; surface-carboxyl-modified magnetic spheres are used herein as a carrier, and aromatase is covalently immobilized by means of EDC / NHS (1-ethyl-3-(3-dimethylaminepropyl)carbodiimide and N-hydroxysuccinimide) process; compared with free enzymes, the immobilized enzyme has greatly improved properties of thermal stability at 37 DEG C and storage stability at 4 DEG C, and the weakness that aromatase is easily inactivated during use and storage is solved; by introducing the magnetic spheres, the enzyme may be recycled quickly and conveniently by the aid of a magnetic field after participating in the reaction; afterwards, the immobilized enzyme is used to screen potential inhibitors for various compound monomers in Tripterygium wilfordii, test under a built screening system discovers that celastrol is able to affinitively bind with aromatase, and its IC50 reaches 23.625 Mu g / Ml. The immobilized enzyme has a promising application prospect in the field of medicine research.

Description

technical field [0001] The invention relates to a method for screening aromatase inhibitors by using surface carboxyl-modified magnetic balls to immobilize enzymes. The specific technology used is to use immobilized aromatase to screen potential aromatase inhibitors, which belongs to enzymology and enzyme engineering field. Background technique [0002] Aromatase (also known as CYP19) is a key enzyme in the synthesis of estrogen in the body. It belongs to one of the cytochrome P450 enzymes. With the help of nicotinamide adenine dinucleotide phosphate (NADPH) The A-ring aromatization of enedione and testosterone is converted into estrone and estradiol respectively; in vivo, estrogen can promote the proliferation of breast cancer cells, and aromatase is an important target for the treatment of estrogen-dependent breast cancer , especially for postmenopausal women, the estrogen in the body is mainly converted from the androgen in the surrounding tissues, the use of aromatase i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12Q1/26
CPCC12N9/0071C12N11/14C12Q1/26C12Y114/14G01N2333/90245
Inventor 苏梦翔张倩柏印狄斌
Owner CHINA PHARM UNIV
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