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Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini

A technology of Coccidioides flax and dehydroepiandrosterone, which is applied in the biological field, can solve the problems of low water solubility of the substrate, little improvement of the conversion rate, poor biocompatibility, etc., and achieves a high substrate conversion rate. , the effect of high product yield and high feed concentration

Active Publication Date: 2012-10-03
ZHEJIANG XIANJU JUNYE PHARM CO LTD +1
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AI Technical Summary

Problems solved by technology

In the process of microbial transformation of steroids, the low water solubility and poor biocompatibility of the substrate are the key factors limiting the transformation rate, which directly become the bottleneck in the industrial production of microbial transformation of steroids
In response to this situation, many researchers have begun to improve the feeding methods of substrates, such as ultrasonic refinement, organic solvent solubilization, and aqueous two-phase catalysis. degree of inhibition, so the increase in conversion rate is not large
Therefore, it is of great significance to adopt an efficient substrate feeding method, and the current research on the use of methyl-β-cyclodextrin (M-β-CD) for Spirospora flaxensis ( Colletotrichum lini ) The study of hydroxylated DHEA has not been reported, and it significantly improves the water solubility and conversion rate of the substrate, which is a major advantage of industrial production

Method used

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  • Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini
  • Method for hydroxylating dehydroisoandrosterone by using colletotrichumlini

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 The clathrate DHEA / M-β-CD was prepared by lyophilization.

[0018] (1) The phase equilibrium curve determines the inclusion ratio.

[0019] Weigh an appropriate amount of M-β-CD, put 30 mL of deionized water in a 250 mL Erlenmeyer flask to prepare a concentration gradient of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, and 60 mM, and add excess base After adding DHEA, place it on a shaker at 20-40°C and 120-200 r / min for 24 h until inclusion equilibrium. The inclusion solution was centrifuged at 1000 r for 3 min to remove unincluded substrates, and the supernatant was diluted by appropriate multiples and filtered through a 0.22 μm water membrane to remove impurities, and then the substrate concentration was detected by high performance liquid chromatography. Take the concentration of M-β-CD as the abscissa and the concentration of DHEA as the ordinate to draw the phase equilibrium curve to determine the inclusion ratio of DHEA and M-β-CD.

[0020] (2) The inclusion co...

Embodiment 2

[0022] Embodiment 2 utilizes Trichospora flaxensis ( Colletotrichum lini ) Inclusion complex of ST-1 hydroxylated DHEA / M-β-CD.

[0023] (1) Preparation of Trichospora flaxensis ( Colletotrichum lini ) Liquid culture of cells from the ST-1 strain

[0024] Pick the spiny sporophyta on the solid PDA medium ( Colletotrichum lini ) A loop of ST-1 strain, inoculated in a 250 mL Erlenmeyer flask containing 30 mL of seed medium, placed on a shaker at 200 r / min at 30°C and cultivated for 20-24 h to the logarithmic phase , that is, to prepare a liquid cell culture of Spirocystis flaxensis strain.

[0025] (3) The composition and ratio of the fermentation medium are: glucose 20-50 g / L; yeast powder 10-30 g / L; peptone 5-20 g / L; corn steep liquor 3-10 g / L; NaCl 0.2-2 g / L; K 2 HPO 4 0.1-1.0 g / L; MgSO 4 0.1-1 g / L; FeSO 4 7 h 2 O 0.01-0.10 g / L; pH 6.5-7.5, sterilized under high-pressure steam at 121°C for 20 min.

[0026] (4) Shake flask fermentation: the above-mentioned Spirocyst...

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Abstract

The invention relates to a method for hydroxylating dehydroisoandrosterone (DHEA) by using colletotrichumlini ST-1. The method for hydroxylating the dehydroisoandrosterone by using the colletotrichumlini ST-1 is characterized in that: in a conversion process, a way of pre-including the DHEA and methyl-beta-cyclodextrin with an equimolar ratio first and then feeding is adopted, so that the water solubility and the conversion efficiency of the DHEA are significantly improved. Under a condition that a substrate feeding amount is 10g / L, the conversion rate is as high as 95% and the total yield of products 7 alpha-OH-dehydroepiandrosterone and 7,15 alpha-OH-dehydroepiandrosterone is 80.94%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently utilizing Spirospora flaxensis ( Colletotrichum lini ) ST-1 hydroxylated dehydroepiandrosterone (DHEA) is 7α-hydroxy-dehydroepiandrosterone (7α-OH-DHEA) and 7,15α-hydroxy-dehydroepiandrosterone (7,15α-OH- DHEA) method. Background technique [0002] 7α-hydroxy-dehydroepiandrosterone (7α-OH-DHEA) and 7,15α-hydroxy-dehydroepiandrosterone (7,15α-OH-DHEA) are important active substances in organisms and have important roles in metabolism Regulatory effect. At present, studies have shown that 7α-OH-DHEA can prevent hypoxic cell death of neurons in vitro, and has anti-oxidation and anti-tumor effects. 7,15α-hydroxy-dehydroepiandrosterone (7,15α-OH-DHEA), is an important steroid drug intermediate, which can be used to synthesize a variety of steroid hormone drugs, such as the synthesis of "fourth generation" The main ingredient of oral contraceptives ...

Claims

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Application Information

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IPC IPC(8): C12P33/06C12R1/645
Inventor 许正宏吴燕朱晓宏李会史劲松李恒
Owner ZHEJIANG XIANJU JUNYE PHARM CO LTD
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