Method for preparing testolactone by microbial transformation

A technology of microbial transformation and testosterone, applied in the biological field, can solve the problems of many by-products and low conversion efficiency, and achieve the effects of less by-products, low production cost and high substrate conversion rate.

Inactive Publication Date: 2014-08-20
SICHUAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to aim at the deficiencies such as low conversion efficiency and more by-products existing in the preparation process of testolactone by microbial transformation, aiming to provide a new method for preparing testolactone by microbial transformation, to reduce production costs, while reducing environmental pollution

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  • Method for preparing testolactone by microbial transformation

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Embodiment 1

[0028] 1) Culture of Fusarium oxysporum cells. Inoculate Fusarium spores on a solid medium (20% potato, 2% glucose, 2% agar, pH 6.0), culture at about 30°C for about 5 days, wash the spores with sterile water, filter with sterile gauze, and make a spore suspension solution with a spore concentration of 10 7 individual / mL. Take a concentration of 1×10 with a 5% inoculum 7 A / ml spore suspension was inoculated in 500ml liquid medium (0.5% soybean powder, 0.5% yeast extract powder, 2% glucose, 0.5% NaCl, 0.5% K 2 HPO 4 , pH=6.0-6.5), 200rpm, cultured at 28-30°C for 3 days, centrifuged to collect the cells, which are resting cells for transformation.

[0029] 2) Preparation of transformation solution. The transformation solution is a phosphate buffer solution with a pH value of 6.0. The preparation is as follows: respectively prepare NaH with a concentration of 0.2mol / L 2 PO 4 and 0.2mol / L Na 2 HPO 4 Mother liquor, according to need, the two are mixed according to the rat...

Embodiment 2

[0034] 1) Culture of Fusarium oxysporum cells. Inoculate Fusarium spores on a solid medium (20% potato, 2% glucose, 2% agar, pH 6.0), culture at 28°C for 6 days, wash the spores with sterile water, filter them with sterile gauze, and make a spore suspension. Spore concentration is 10 7 individual / mL. Take a concentration of 1×10 with a 5% inoculum 7 Individual / ml spore suspension was inoculated in 500ml liquid medium (1% corn steep liquor, 2% glucose, 0.5% NaCl, 0.5% K 2 HPO 4 , pH 6.5), 200rpm, cultured at 28-30°C for 3 days, centrifuged to collect the cells, which were resting cells for transformation.

[0035] 2) Preparation of transformation solution. The transformation solution is sterile water, natural pH. Take a certain amount of dehydroepiandrosterone and dissolve it in 0.1% Tween-80, and ultrasonically break it for 25 minutes. Added to 200ml of sterilized transformation solution, the substrate concentration was 10g / L.

[0036] 3) Inoculate resting cells of Fus...

Embodiment 3

[0040] 1) Culture of Fusarium oxysporum cells. Inoculate Fusarium spores on a solid medium (20% potato, 2% glucose, 2% agar, pH 6.0-6.5), culture at 30°C for 7 days, wash the spores with sterile water, filter with sterile gauze, and make a spore suspension solution with a spore concentration of 10 7 individual / mL. Take a concentration of 1×10 with a 5% inoculum 7 1 / ml spore suspension was inoculated in 1L liquid medium (0.5% soybean powder, 0.5% yeast extract powder, 2% glucose, 0.5% NaCl, 0.5% K 2 HPO 4 , pH=6.0-6.5), culture at 200rpm, 30°C for 3 days, and collect the cells by centrifugation, which are resting cells for transformation.

[0041] 2) Configuration of transformation solution. The conversion solution is a phosphate buffer solution with a pH value of 5.5. The configuration is as follows: respectively prepare NaH with a concentration of 0.2mol / L 2 PO 4 and 0.2mol / L Na 2 HPO 4 Mother liquor, the two are mixed at a ratio of 92:8 to form a phosphate buffer s...

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Abstract

The invention discloses a method for preparing testolactone by microbial transformation, which mainly comprises the following steps: dissolving dehydroepiandrosterone in a solubilizer, adding the mixture in a transformation solution to prepare a transformation substrate solution; adding the transformation substrate solution into a transformation reaction device, adding fusarium oxysporum, transforming to obtain an intermediate of 3beta-hydroxy-17alpha-oxo-D-hexocyclo-androst-5-ene-17-ketone under the fusarium oxysporum resting cell transformation action through a Baeyer-Villiger oxidation reaction, adding gordonia after the transformation reaction is completed, performing a transformation reaction of the intermediate under the gordonia resting cell action to obtain testolactone. The method for preparing testolactone of the invention has the advantages of simple process route, easily available raw materials, cheap auxiliary materials, high substrate transformation rate, few by products, low production cost, and the like, overcomes the deficiencies of original production technology, and is suitable for industrial production.

Description

technical field [0001] The invention relates to a method for preparing dehydroepiandrosterone into steroid raw material testolactone by using a microbial conversion method, and belongs to the field of biotechnology. technical background [0002] Testolactone (Testolactone, 17α-oxo-D-expanded-androst-1,4-diene-3,17-dione), also known as dehydrotestolactone, is an endogenous steroid It is an aromatase inhibitor that can bind aromatase non-competitively, thus effectively inhibiting the conversion of androgens into estrogens. Testolactone is usually used as an antineoplastic drug. Clinically, it is mainly used to treat certain types of breast cancer in postmenopausal women. In addition, it can also be used to treat girls' precocious puberty and male idiopathic oligospermia. [0003] Testolactone can be prepared by chemical synthesis and microbial transformation. Because the oxidant used in the chemical synthesis method is poisonous and explosive, it pollutes the environment gr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P39/00C12R1/77C12R1/01
Inventor 李维葛方兰陈贵英张光祥
Owner SICHUAN NORMAL UNIV
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