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Penicillium simplicissimum capable of biologically synthesizing 1,2-testololactone and synthesis method of 1,2-testololactone

A simple penicillium and microorganism technology, applied in the field of bioengineering, to achieve the effect of less by-products, high feed concentration and strong transformation ability

Active Publication Date: 2015-03-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on the direct synthesis of 1,2-dihydrotestolactone by one-step transformation of Penicillium simpleum at home and abroad. By optimizing the culture conditions of the strain and the transformation conditions, a higher substrate feeding concentration can be achieved, and a high yield can be obtained. target product

Method used

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  • Penicillium simplicissimum capable of biologically synthesizing 1,2-testololactone and synthesis method of 1,2-testololactone
  • Penicillium simplicissimum capable of biologically synthesizing 1,2-testololactone and synthesis method of 1,2-testololactone
  • Penicillium simplicissimum capable of biologically synthesizing 1,2-testololactone and synthesis method of 1,2-testololactone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Screening and identification of strains

[0027] Soil samples were collected from Qingcheng Mountain in Dujiangyan, Sichuan Province, Jixian Mountain in Tianjin City, Weifang, Shandong and other places, and 5g of each soil sample was weighed and suspended in sterile water, oscillated and mixed, and then left to stand. The supernatant was diluted and applied on the PDA solid medium, the mold on the plate was separated, and the obtained single bacteria were transferred to the slant prepared by the PDA medium for preservation, and used for the transformation experiment.

[0028] Inoculate the individual molds obtained above into liquid transformation medium (glucose 30g / L, corn steep liquor dry powder 10g / L, NaNO 3 2g / L, KCl0.5g / L, K 2 HPO 4 ·3H 2 O2g / L, KH 2 PO 4 1g / L, MgSO 4 ·7H 2 O0.5g / L, FeSO 4 ·7H 2 (00.02g / L, pH6.2), 30°C, 200r / min shaking on a shaker for 24-48h, adding 0.5g / L17α-hydroxyprogesterone to start the reaction, and taking samples for...

Embodiment 2

[0030] Embodiment 2: The method for synthesizing 1,2-dihydrotestolactone (testololactone) with pregnane-4-ene-3,20-dione as substrate

[0031] 1 Preparation of seed solution

[0032] The strain of Penicillium simplicissimum WY134-2 was inoculated in the primary seed culture medium, cultured at 30°C for 16 hours, and the shaker speed was 300r / min to obtain the seed culture solution.

[0033] The composition of the seed medium is: glucose 30g / L, corn steep liquor dry powder 10g / L, NaNO 3 2g / L, KCl0.5g / L, K 2 HPO 4 ·3H 2 O2g / L, KH 2 PO 4 1g / L, MgSO 4 ·7H 2 O0.5g / L, FeSO 4 ·7H 2 O0.02g / L, pH6.2.

[0034] 2 secondary fermentation culture

[0035] The seed culture solution obtained in step 1 was transferred to a fresh fermentation medium according to the inoculum amount of 3%. The culture conditions were: 30°C, the rotating speed of the shaker was 250r / min, and the fermentation time was 20h, and the cell fermentation solution was obtained.

[0036] The composition of the...

Embodiment 3

[0042] Embodiment 3: The method for the synthesis of 1,2-dihydrotestolactone (testololactone) with 17β-hydroxyl-androst-4-en-3-one as substrate

[0043] 1 Preparation of seed solution

[0044] The strain of Penicillium simplicissimum WY134-2 was inoculated in the primary seed culture medium, cultured at 30°C for 20 hours, and the shaker speed was 200r / min to obtain the seed culture solution.

[0045] The composition of the seed medium is as shown in Example 2.

[0046] 2 secondary fermentation culture

[0047] The seed culture solution obtained in step 1 was transferred to fresh fermentation medium according to the inoculum amount of 4%. The culture conditions were: 30°C, the rotating speed of the shaker was 220r / min, and the fermentation time was 18h, and the cell fermentation solution was obtained.

[0048] The composition of the fermentation medium is as shown in Example 2.

[0049] 3 Transformation reactions

[0050] 17β-Hydroxy-androst-4-en-3-one II, which was solubil...

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Abstract

The invention discloses penicillium simplicissimum WY134-2 and a method for producing 1,2-testololactone by converting C21 or C19 steroids through fermenting penicillium simplicissimum. The strain is collected in China General Microbiological Culture Collection Center (CGMCC) on August 12, 2013 and has the collection number of CGMCC No. 8017. The production method comprises the following steps: by adopting penicillium simplicissimum WY134-2 as a strain, carrying out primary seed culture and secondary fermentation enlargement culture, putting the crushed steroids into a thallus fermentation broth, acidifying, extracting with an organic solvent, and separating by virtue of silica gel column chromatography to obtain the product, namely, 1,2-testololactone of which the yield is 56-96%. The method is simple and convenient and has the advantages of mild conditions, less byproducts and no environmental pollution.

Description

technical field [0001] The invention relates to the field of bioengineering, and the invention relates to a bacterial strain and a synthesis method for biosynthesizing 1,2-dihydrotestolactone (testololactone). Background technique [0002] [0003] 1,2-Dihydrotestolactone, the English name is testololactone, the chemical formula is C 19 h 24 o 3 , molecular weight 302.41, white or off-white powder, CAS4416-57-3. 1,2-dihydrotestolactone, as a class of hormone drugs, is the first generation aromatase inhibitor. It can inhibit the synthesis of estrogen such as estradiol and estrone in the body, reduce the production of estrogen, and reduce the level of estrogen in the blood, and is used for the treatment of pseudoprecocious puberty. In addition, its derivatives are clinically used as cellular anticancer drugs and in the treatment of diseases such as male infertility (Dao T L. Estrogen synthesis in human breast tumor and its inhibition by testololactone and bromoandrostene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12P33/20C12R1/80
CPCC12N1/14C12P33/12C12P33/20C12N1/145C12R2001/80
Inventor 吴洽庆王玉杨贝贝陈曦朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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