Preparation method for dehydroepiandrosterone, and enzyme for preparation thereof

A technology of dehydroepiandrosterone and androstenedione, which is applied in the field of biomedicine, can solve the problems of complicated purification operation, great environmental damage, low efficiency and the like, and achieves the effects of simple process, low cost and high yield

Inactive Publication Date: 2019-02-05
BONTAC BIO ENG SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the synthesis method is completed in more than 3 steps of chemical reactions, the price of raw materials is high, and solvents such as benzene, halogenated alkanes or pyridine are used in the process, which is harmful to the environment.
In addition, the product in the traditional process has low purity and low efficiency, and the subsequent purification operation is cumbersome

Method used

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  • Preparation method for dehydroepiandrosterone, and enzyme for preparation thereof
  • Preparation method for dehydroepiandrosterone, and enzyme for preparation thereof
  • Preparation method for dehydroepiandrosterone, and enzyme for preparation thereof

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preparation example Construction

[0068] The embodiment of the present invention provides a method for preparing ketoreductase and glucose dehydrogenase, the ketoreductase and glucose dehydrogenase are the enzymes used in the preparation method of dehydroepiandrosterone, the specific experiment Steps include:

[0069] (1) Construction of pET22b-KRED3 / 4 / 5-GDH recombinant plasmid

[0070] Upstream primers and downstream primers are provided, and the gene coding sequences of ketoreductase KRED3, ketoreductase KRED4, ketoreductase KRED5 and glucose dehydrogenase are obtained by PCR amplification experiment. Wherein, the gene coding sequence of the ketoreductase KRED3 is shown in SEQ ID NO:1, the gene coding sequence of the ketoreductase KRED4 is shown in SEQ ID NO:2, and the gene coding sequence of the ketoreductase KRED5 As shown in SEQ ID NO:3, the gene coding sequence of the glucose dehydrogenase is shown in SEQ ID NO:4. The base sequence of the upstream primer corresponding to the ketoreductase KRED3 is show...

Embodiment 1

[0078] A preparation method for dehydroepiandrosterone, comprising the following steps:

[0079](1) Under the protection of nitrogen, add 82.5g of potassium tert-butoxide to 388mL of tert-butanol, then raise the temperature to 60°C, stir for 0.5h until the potassium tert-butoxide is completely dissolved, cool down to 30°C, and add 100g of 4-AD was stirred continuously for 0.5h, then cooled to room temperature to obtain a mixture. Under nitrogen protection, 6.0 g of sodium ascorbate was added to 205.3 g of 26.8% acetic acid aqueous solution to obtain an acetic acid solution containing sodium ascorbate. Under the protection of nitrogen, at 30°C, slowly drop the mixture into the acetic acid solution containing sodium ascorbate within 30 minutes, maintain the temperature at 30°C and continue stirring for 0.5h, and cool at room temperature to obtain a reaction containing 5-AD liquid.

[0080] (2) Add 500mL ethyl acetate to the 5-AD reaction solution, stir evenly, add 400mL 1.8g / L...

Embodiment 2

[0083] A preparation method for dehydroepiandrosterone, comprising the following steps:

[0084] (1) Under the protection of nitrogen, add 80.0g of potassium tert-butoxide to 388mL of tert-butanol, then raise the temperature to 55°C, stir for 0.8h until the potassium tert-butoxide is completely dissolved, cool down to 30°C, and add 100g of 4-AD was stirred continuously for 0.6h, then cooled to room temperature to obtain a mixture. Under nitrogen protection, 6.0 g of sodium ascorbate was added to 200.0 g of 26.8% acetic acid aqueous solution to obtain an acetic acid solution containing sodium ascorbate. Under the protection of nitrogen, at 30°C, slowly drop the mixture into the acetic acid solution containing sodium ascorbate within 30 minutes, maintain the temperature at 30°C and continue stirring for 0.5h, and cool at room temperature to obtain a reaction containing 5-AD liquid.

[0085] (2) Add 500mL ethyl acetate to the 5-AD reaction solution, stir evenly, add 400mL 1.5g / ...

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Abstract

A preparation method for dehydroepiandrosterone, comprising: in a protective atmosphere, adding potassium tert-butoxide to tert-butanol, stirring evenly, adding 4-androstenedione to obtain a mixture,and adding the mixture dropwise to a sodium ascorbate-containing acetic acid solution for a reaction to obtain 5-androstenedione; dissolving the 5-androstenedione in an organic solvent, adding a ketone reductase, a glucose dehydrogenase, glucose and a redox coenzyme to obtain a mixture, controlling the pH of the mixture to be 6.0-6.3, stirring and reacting for 1-6 hours at 22-26 DEG C to obtain areaction solution, and performing separation and purification on the reaction solution to obtain dehydroepiandrosterone, the ketone reductase and the glucose dehydrogenase being coexpressed by a microbial strain and added in the form of a crude enzyme solution. The synthesis process of the preparation method has few steps, simple operations, high yields and low costs, and may be widely applied toindustrial scale production. Also provided is an enzyme for preparation.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method of dehydroepiandrosterone and an enzyme for preparation thereof. Background technique [0002] Dehydroepiandrosterone (dehydroepiandrosterone) (dehyroepiandrosterone, DHEA) is a C19 steroid carrier compound, chemical name is 3β-hydroxyandrost-5-en-17-one, molecular formula C 19 h 28 o 2 . DHEA is a large number of adrenal steroid compounds that coexist in human plasma. It has anti-inflammatory, anti-proliferation and anti-depressant activities. Its derivatives can promote animal immune response in experiments and have good neuroprotective effects. In addition, DHEA is also an important intermediate for the synthesis of steroid drugs. In recent years, DHEA has gradually developed into one of the most popular research objects by virtue of its important research value. [0003] The traditional synthesis of DHEA mostly adopts chemical methods, such as us...

Claims

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Application Information

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IPC IPC(8): C12P33/06C12P7/02C12N9/04C12N15/53C12N15/70C12R1/19
CPCC12N9/0006C12N15/70C12P7/02C12P33/06C07K14/195C12N15/64C12R2001/19C12N1/205
Inventor 傅荣昭刘立辉陈小春曹磊刘滔滔
Owner BONTAC BIO ENG SHENZHEN
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