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Method of utilizing coenzyme regeneration and resin in-situ extraction to promote hydroxylation of DHEA by Colletotrichum lini ST-1

A resin and extraction technology, applied in the biological field, can solve the problem of small increase in conversion rate and achieve high product yield

Inactive Publication Date: 2015-05-13
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These studies have a positive impact on the conversion of DHEA to a certain extent, but the conversion rate of DHEA into 7α, 15α-diOH-DHEA is not greatly improved

Method used

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  • Method of utilizing coenzyme regeneration and resin in-situ extraction to promote hydroxylation of DHEA by Colletotrichum lini ST-1
  • Method of utilizing coenzyme regeneration and resin in-situ extraction to promote hydroxylation of DHEA by Colletotrichum lini ST-1

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Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1 Determination of the type of macroporous adsorption resin

[0018] Add an appropriate amount of substrate or product to 30 ml / 250 mL aqueous solution, equilibrate on a shaker at 220 rpm for 2 h until the solution is saturated, and let stand to separate the supernatant. Then add 1 g of different types of resins, including H103, D101, AB8, HP20, HZ801, and DA201 (selected according to the polarity of the substrate and product), to the substrate and product supernatant, and then place it on a 220 rpm shaker to balance 2 h until the resin reaches adsorption equilibrium. The adsorbed resin was extracted with ethyl acetate, and the adsorption amount of the resin to the substrate and product was calculated after liquid phase analysis, and finally the resin with high affinity to the product and low affinity to the substrate was selected for subsequent conversion experiments.

Embodiment 2

[0019] Example 2 Coenzyme regeneration and resin in-situ extraction "two-in-one" strategy promotion C. lini Hydroxylation of DHEA by ST-1

[0020] (1) Preparation C. lini Cell liquid culture of ST-1 strain

[0021] Pick a loop of the Neptonia flaxensis strain on the solid PDA medium, inoculate it in a 250 mL Erlenmeyer flask containing 30 mL of seed medium, place it on a shaker at 200 r / min at 30 °C Cultivate for 20-24 h to the logarithmic phase, that is, to obtain C. lini Liquid culture of ST-1 cells.

[0022] (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 7H 2 O 0.01-0.10 g / L; pH 6.5-7.5, sterilized under high-pressure steam at 121°C for 20 min.

[0023] (4) Shake flask fermentation: mix the above C. lini Cell liquid culture of ST-1 strain was inoculated at an inoculum size of 8-10% (w / w) in a...

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Abstract

Belonging to the field of biotechnology, the invention relates to a method of utilizing a coenzyme regeneration and resin in-situ extraction integrated strategy to promote hydroxylation of DHEA (dehydroepiandrosterone) by Colletotrichum lini ST-1. The method is characterized in that in the conversion process, a 10g / L cosubstrate is added to conduct NADPH regeneration, and simultaneously D101 type macroporous adsorption resin is employed for in-situ extraction of the product, thus significantly enhancing the conversion rate of C. lini ST-1 on DHEA. Under the condition of a substrate feeding amount of 10g / L, the conversion rate of the substrate DHEA is 93%, and the yield of 7 alpha, 15 alpha-diOH-DHEA can reach 7.12 g / L.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a "two-in-one" strategy of using coenzyme regeneration and resin in-situ extraction to improve the growth rate of Phytophthora flaxensis ( Colletotrichum lini ) ST-1 method for converting dehydroepiandrosterone (DHEA) into 7α, 15α-hydroxy-dhydroepiandrosterone (7α, 15α-diOH-DHEA). Background technique [0002] Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid compound, which mainly has the functions of anti-cancer, anti-aging and immune regulation. As a steroidal drug precursor, it has been widely studied and made some progress in the treatment of menopausal syndrome, scleroderma, coronary heart disease, gout, AIDS and so on. In recent years, researchers at home and abroad have conducted extensive and in-depth research on the biotransformation of DHEA. research shows Colletotrichum lini, Mucorracemosus , Fusarium moniliforme and other strains can further hydrox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/06C12R1/645
Inventor 许正宏吴燕李会李恒史劲松张峥斌张汝金
Owner JIANGNAN UNIV
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