Method for preparing steroid drug intermediate employing bioconversion phytosterol

A technology for phytosterols and steroid drugs, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of harmful bacteria and the environment, difficult recovery of co-solvents, and limited concentration of bacteria. The effect of reducing the risk of bacteria, shortening the transformation reaction time, and convenient separation

Active Publication Date: 2014-04-23
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] The purpose of the present invention is to provide a kind of method that biotransformation phytosterol prepares steroid drug intermediate, thus overcomes the low substrate feeding amount in the prior art, the bacterium concentration is limited, the reaction time is long, the cosolvent reclaims difficulty, and the bacterium Physically and environmentally harmful defects

Method used

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  • Method for preparing steroid drug intermediate employing bioconversion phytosterol
  • Method for preparing steroid drug intermediate employing bioconversion phytosterol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. Seed preparation and cell culture: inoculate Mycobacterium neoaurum NwIB-Ⅴ cryopreserved in glycerol into primary seed medium MYC / 01. Inoculate into secondary seed medium and cultivate to mid-logarithmic phase (2d), then inoculate into growth medium MYC / 03 with 10% inoculum, and collect bacteria after 3d.

[0066] 2. Bacteria collection and treatment process: collect the bacteria in 3 days, centrifuge at 6000rpm for 10min, wash twice with deionized water (pH8), and weigh the bacteria by centrifugation to obtain a wet weight of 35g.

[0067] 3. Resting cell transformation process: set the rated transformation volume in a 1L shake flask to 100mL, accurately measure 50mL of phytosterol mother liquor, and weigh 400g of hydroxypropyl-β-cyclodextrin (the molar ratio to phytosterol is 1: 1) Weigh 10-15g of the wet bacteria, mix the three immediately, set the volume to 100mL, adjust the initial pH to 8, and incubate for 4-5d.

[0068] 4. After the transformation, centrifuge...

Embodiment 2

[0072] Example 2: The bacterial strains and culture method are the same as in Example 1, except that the resting cell transformation experiment is done on a 5L fermenter. The experiment proved that the transformation experiment carried out on the 5L fermenter basically obtained the similar experimental effect as that of the shake flask.

Embodiment 3

[0073] Embodiment 3, 4: Same as Embodiment 1, 2, the difference is that the bacterial strain is Mycobacterium neoaurum NwIB-02. Regardless of whether it is a 1000mL shake flask or a 5L fermenter, the conversion rate of phytosterols measured by HPLC is close to 100%, and the AD yield is 37g / L. Bacteria and cyclodextrin can be reused 8 times.

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Abstract

The invention provides a method for preparing a steroid drug intermediate employing bioconversion phytosterol. The method comprises the following steps: 1) providing turbid liquid of phytosterol; 2) providing a resting cell of a microbial strain for converting the phytosterol into a steroid drug intermediate; 3) mixing the turbid liquid of the phytosterol with the resting cell, and adding beta-cyclodextrin or chemically modified beta-cyclodextrin to evenly mix, and cultivating to finish conversion; 4) centrifuging the conversion liquid obtained in the step 3), separating supernatant from thallus, separating to obtain the steroid drug intermediate from the supernatant by solvent extraction. By adopting the method provided by the invention, the sterol feeding concentration is improved, the target product quantity is increased, simple separation of the thallus, a cosolvent and a product is achieved when the conversion lasting time is shortened, repeated use of the thallus and the cosolvent for a plurality of times is achieved, the production cost is greatly saved, and the method is applicable to industrial production.

Description

technical field [0001] The invention relates to the technical field of sterol biotransformation, in particular to a method for preparing steroid drug intermediates through biotransformation of phytosterols. Background technique [0002] Steroids are a class of compounds with cyclopentane polyhydrophenanthrene as the core and similar structures. They are widely found in animal and plant tissues and some microbial cells. The more common ones are phytosterol, cholesterol, androsterone and diosgenin Wait. Steroids are important raw materials for the production of steroid drugs. Steroid drugs play a very important role in regulating the body and are the second largest class of drugs after antibiotics. The main components of hormone drugs that occupy an important position in medicine, such as hydrocortisone, testosterone, androsterone, progesterone and estradione, etc., are all drugs with a steroidal structure. Androst-4-diene-3,17-dione (AD), androst-1,4-diene-3,17-dione (ADD)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/02C12R1/32
Inventor 王学东高兴强冯建勋徐辛未何伟魏东芝
Owner EAST CHINA UNIV OF SCI & TECH
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