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A method for promoting the regeneration of reducing power and improving the conversion efficiency of microbial hydroxylated dhea

A DHEA and seed technology, applied in the biological field, can solve the problems of providing sufficient reducing power for the hydroxylation reaction, affecting the normal progress of the hydroxylation reaction, and NADPH consumption.

Active Publication Date: 2019-04-23
TIANJIN TIANYAO PHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the steroidal hydroxylation process, NADPH is continuously consumed, which cannot provide enough reducing power for the hydroxylation reaction, thus directly affecting the normal progress of the hydroxylation reaction

Method used

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  • A method for promoting the regeneration of reducing power and improving the conversion efficiency of microbial hydroxylated dhea
  • A method for promoting the regeneration of reducing power and improving the conversion efficiency of microbial hydroxylated dhea
  • A method for promoting the regeneration of reducing power and improving the conversion efficiency of microbial hydroxylated dhea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Research on the Hydroxylation of DHEA by Colletotrichum lini ST-1 under the Condition of High Substrate Feeding in Example 1

[0021] (1) Preparation of C.lini ST-1 cell liquid culture

[0022] C. lini ST-1 on the PDA solid medium was picked and inoculated into a 250mL Erlenmeyer flask filled with 30mL of fresh liquid seed medium, and cultured at 30°C and 120-220r / min for 24h. Then, the inoculation amount of 8-10% (v / v) is inserted into a 5L fermenter equipped with a fermentation medium, and the liquid filling amount is 3L. In the growth stage, the culture temperature is 25-40°C, the tank pressure is set to 0.05MPa, the rotation speed is set to 220-300r / min, the ventilation rate is 0.4-1.0vvm, and the dissolved oxygen level is maintained at 10-20%. After 12 hours of growth, That is, a liquid culture solution suitable for DHEA conversion is obtained;

[0023] (2) Hydroxylation of DHEA

[0024] Accurately weigh 15g / L powdered DHEA, and drop it into the bacterial cell c...

Embodiment 2

[0029] Example 2 Substrate Batch Adding Process to the Study of the Hydroxylation of DHEA

[0030] (1) Preparation of C.lini ST-1 cell liquid culture

[0031] C. lini ST-1 on the PDA solid medium was picked and inoculated into a 250mL Erlenmeyer flask filled with 30mL of fresh liquid seed medium, and cultured at 30°C and 120-220r / min for 24h. Then, the inoculation amount of 8-10% (v / v) is inserted into a 5L fermenter equipped with a fermentation medium, and the liquid filling amount is 3L. In the growth stage, the culture temperature is 25-40°C, the tank pressure is set to 0.05MPa, the rotation speed is set to 220-300r / min, the ventilation rate is 0.4-1.0vvm, and the dissolved oxygen level is maintained at 10-20%. After 12 hours of growth, That is, a liquid culture solution suitable for DHEA conversion is obtained;

[0032] (2) Substrate batch feeding process

[0033] The feeding method of three different substrates in batches is as follows: A) Accurately weigh three parts ...

Embodiment 3

[0038] Example 3: Study on the Hydroxylation of DHEA by Combining Substrate Batch Feeding and Promoting Reducing Power Regeneration Process

[0039] (1) Preparation of C.lini ST-1 cell liquid culture

[0040] C. lini ST-1 on the PDA solid medium was picked and inoculated into a 250mL Erlenmeyer flask filled with 30mL of fresh liquid seed medium, and cultured at 30°C and 120-220r / min for 24h. Then, the inoculation amount of 8-10% (v / v) is inserted into a 5L fermenter equipped with a fermentation medium, and the liquid filling amount is 3L. In the growth stage, the culture temperature is 25-40°C, the tank pressure is set to 0.05MPa, the rotation speed is set to 220-300r / min, the ventilation rate is 0.4-1.0vvm, and the dissolved oxygen level is maintained at 10-20%. After 12 hours of growth, That is, a liquid culture solution suitable for DHEA conversion is obtained;

[0041] (2) Substrate batch dosing process

[0042] Accurately weigh three parts of 5g / L powdered DHEA, drop i...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a 5L fermentation tank technique for efficiently converting DHEA (dehydroepiandrosterone) by using Colletotrichum lini ST-1. The technical scheme is as follows: the method comprises the steps of strain activation, seed culture, DHEA bioconversion, product detection and the like. Substrate consumption, residual sugar content and other indexes in the conversion process are monitored, and the batch-by-batch substrate addition is combined with reducing force regeneration promotion to solve the problems of lower substrate feed concentration and lower conversion efficiency. The method increases the product yield, shortens the conversion period, is simple to operate, has high feed amount, and is suitable for large-scale production of steroid compound bioconversion.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method of promoting the regeneration of reducing power under the condition of feeding high-concentration dehydroepiandrosterone (DHEA) to improve the 7α, 15α- Hydroxy-dehydroepiandrosterone (7α,15α-diOH-DHEA) method. Background technique [0002] Dehydroepiandrosterone (DHEA) is a precursor of the adrenal hormone DHEA sulfate (DHEAS) secreted by the adrenal gland. It has a wide range of pharmacological activities in the treatment of nervous system diseases and immune regulation. DHEA can be used as an intermediate to synthesize many steroid compounds with important physiological activities, such as 7α, 15α-diOH-DHEA is the precursor compound of drospirenone, the active ingredient of the new oral contraceptive "Yasmin". Since 7α,15α-diOH-DHEA has important pharmacological effects and medicinal value, its market prospect is very promising, so how to increase the feeding ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/06
CPCC12P33/06
Inventor 许正宏史劲松李会李聪
Owner TIANJIN TIANYAO PHARM CO LTD
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