A kind of method for preparing immobilized aromatase

An aromatase, immobilized enzyme technology, applied in biochemical equipment and methods, immobilized on or in inorganic carriers, oxidoreductase, etc. Evaluate problems such as drugs to be screened, and achieve high research value and good stability

Active Publication Date: 2019-03-26
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The free aromatase present in placental microsomes as a screening model has the following obvious defects: (1) The enzyme source is not easy to come by, and each screening test needs to find fresh human placenta to prepare microsomes as the enzyme source of aromatase , there are medical ethical issues in the acquisition of raw materials; (2) The microsomes extracted from human placenta are a mixture of various hormone-converting enzymes, and various enzymes will compete with each other to catalyze the conversion of substrates into different products, and the mixed species obtained from each extraction are aromatic. There are differences in enzyme activity, which cannot effectively evaluate the drug to be screened; (5) It is difficult to separate the enzyme from the product after the reaction, and it cannot be recycled and reused
However, due to the individual differences of enzymes, there is no universal immobilization technology applicable to all enzymes. Up to now, no immobilization technology of aromatase has been published in the world, and aromatase is a very important immobilization technology. The therapeutic target of breast cancer can be immobilized to construct a rapid screening technology platform for new aromatase inhibitors, providing a powerful tool for the development of new drugs

Method used

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  • A kind of method for preparing immobilized aromatase
  • A kind of method for preparing immobilized aromatase
  • A kind of method for preparing immobilized aromatase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Weigh 4g of mesoporous silicon spheres (sepax@HP-silica; 5μm, ), add 100mL of 10% hydrochloric acid, reflux at 90°C for 8 hours with stirring, then wash with a large amount of deionized water until neutral, and vacuum dry at 150°C. An activated mesoporous silica sphere carrier is obtained.

[0032] (2) Weigh 500 mg of the activated mesoporous silicon spheres obtained in step (1) and disperse them in 20 mL of anhydrous toluene, add 250 μL of γ-glycidyl etheroxypropyltrimethoxysilane (KH-560) dropwise, and stir at 90 °C Refluxed for 12 h, then washed three times with toluene and acetone, dried in vacuo at 60 °C, and stored at 4 °C to obtain epoxy-modified functionalized mesoporous silicon spheres (Exopy-SiO 2 ).

[0033] (3) Preparation of 100mmol / L potassium phosphate buffer (pH7.4): 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 liquid, and mix A and B at (A:B) 80.2:1...

Embodiment 2

[0041] (1) Prepare the enzyme solution for immobilization according to step (5) of Example 1, with a concentration of 10 pmol / mL.

[0042] (2) Weigh 5 mg of the epoxy-modified functionalized mesoporous silica spheres obtained in step (2) of Example 1 and wash them three times with 0.5 mL of potassium phosphate buffer (100 mmol / L, pH 7.4), then add step (1) The prepared enzyme solution was 0.5mL, rotated and shaken at 4°C for more than 30min, centrifuged at low temperature and high speed, and the precipitate was washed 3 times with 0.5mL potassium phosphate buffer (100mmol / L, pH7.4), and the enzyme activity was measured.

[0043] (3) The relationship between the enzymatic reaction of free aromatase and time: 170 μL potassium phosphate buffer solution (100 mmol / L, pH 7.4), 5 μL androstenedione working solution, 5 pmol aromatase, After vortexing to mix well, add 20 μL NADPH to start the reaction after 5 minutes in a water bath at 37°C, and react each tube in a water bath for 10 m...

Embodiment 3

[0047] (1) According to step (1) (2) of embodiment 1, functionalized mesoporous silicon spheres modified by epoxy group are obtained;

[0048] (2) Preparation of 100mmol / l potassium phosphate buffer solution (pH7.4): 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 liquid, and mix A and B at (A:B) 80.2:19.8.

[0049] (3) Preparation of androstenedione stock solution and working solution: Weigh 2 mg androstenedione into a 1 mL volumetric flask, dilute to the mark with DMSO, and store at -20°C, which is a 2 mg / mL stock solution; take 5 μL The stock solution was mixed with 95 μL of potassium phosphate buffer (100 mmol / l, pH 7.4) to obtain a 100 μg / mL androstenedione working solution.

[0050] (4) Preparation of enzyme solution for immobilization: Aromatase from the baculovirus-insect cell expression system (Corning, USA) was diluted to 30 pmol / mL with potassium phosphate buffered saline...

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Abstract

The invention provides a method for preparing immobilized aromatase. According to the method, epoxy group functionalization modified mesoporous silicon balls are used for adsorbing and immobilizing aromatase at low temperature; in the continuous catalysis process, the immobilized enzyme is inactivated, and the half life is prolonged; and the catalytic reaction rate of the free aromatase decreases after 40 minutes, and the catalytic reaction rate of the immobilized enzyme decreases after 80 minutes. The immobilization rate can reach 90% or above, and the immobilized enzyme can be reutilized repeatedly, thereby overcoming the defect that the free aromatase can easily become inactivated in the continuous catalysis process, and can not be recovered after participating in the reaction; and thus, the immobilized enzyme has favorable application prospects for establishing a novel aromatase inhibitor fast screening technique platform in the field of pharmaceutical research.

Description

technical field [0001] The invention relates to a method for preparing immobilized aromatase, and the technology specifically utilized is a mesoporous silicon sphere adsorption method modified by surface epoxy group functionalization. Background technique [0002] Aromatase (aromatase), also known as CYP19, is one of the cytochrome P450 enzymes and is the key enzyme for the conversion of estrogen and androgen in the body. In the presence of NADPH, it can convert androstenedione and testosterone into estrone and estradiol respectively, and a variety of estrogen-dependent tumors such as breast cancer, endometrial cancer, and malignant ovarian tumors have aromatization Enzyme overexpression. Among them, the relationship between aromatase and breast cancer is particularly close, and the growth of about 60% to 70% of breast cancer depends on estrogen. Aromatase inhibitors disable the enzyme aromatase, which prevents the conversion of androgens into estrogens, thereby blocking t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12P33/06
CPCC12N9/0071C12N11/14C12P33/06C12Y114/14Y02P20/50
Inventor 苏梦翔柏印靖雅狄斌
Owner CHINA PHARM UNIV
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