A method for preparing 11α, 17α-hydroxyprogesterone by conversion of immobilized hydroxylase

A technology for immobilizing hydroxylase and hydroxyprogesterone, which can be applied to immobilized enzymes, microorganism-based methods, biochemical equipment and methods, etc., can solve the complex extraction and purification process of products, large amount of fermentation wastewater, and final product yield. Low problems, to achieve the effect of simple and clear reaction system components, simple extraction and purification process, and improved reaction rate

Active Publication Date: 2021-11-19
SHANGHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problems to be solved by the present invention are: the existing fermentation method for preparing 11α, 17α-hydroxyprogesterone has low feed concentration, large amount of fermentation waste water, complex extraction and purification process of the product, and low final yield of the product, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Aspergillus ochraceus was cultured on a slant to obtain the strains required for fermentation.

[0033] 1. Preparation of Aspergillus ochraceus slant strain and seed solution

[0034] (1) Incline cultivation

[0035] Incline medium: peeled potato 200g / L, glucose 20g / L, agar 20g / L, natural pH, sterilized at 121℃ for 30min.

[0036] The bacterial species preserved in the glycerol tube is aseptically drawn a certain amount of bacterial liquid and spread evenly on the slant medium, and cultured at 28°C for 4-6 days.

[0037] (2) Seed cultivation

[0038] The weight portion composition of primary medium comprises (g): glucose 5, yeast extract 2, K 2 HPO4 1.0, NaCl 0.1, water 1000, and adjust the pH to 5.0, sterilize by high-pressure steam at 121°C for 30 minutes, and cool down for use.

[0039] With the strain cultivated in step (1), under sterile conditions, use an inoculation loop to pick 6 ring spores into 100mL seed medium, inoculate 4 bottles in the same way, and c...

Embodiment 2

[0054] Aspergillus ochraceus was cultured on a slant to obtain the strains required for fermentation.

[0055] 1. Preparation of Aspergillus ochraceus slant strain and seed solution

[0056] (1) Incline cultivation

[0057] Incline medium: peeled potato 200g / L, glucose 20g / L, agar 20g / L, natural pH, sterilized at 121℃ for 30min.

[0058] The bacterial species preserved in the glycerol tube is aseptically drawn a certain amount of bacterial liquid and spread evenly on the slant medium, and cultured at 28°C for 4-6 days.

[0059] (2) Seed cultivation

[0060] The weight portion composition of primary medium comprises (g): glucose 7, yeast extract 5, K 2 HPO 4 1.5, NaCl 0.4, water 1000, and adjust the pH to 5.5, sterilize by high-pressure steam at 121°C for 30 minutes, and cool for later use.

[0061] With the strain cultivated in step (1), under aseptic conditions, use an inoculation loop to pick 6 ring spores into 100mL seed medium, inoculate 4 bottles in the same way, an...

Embodiment 3

[0076] Aspergillus ochraceus was cultured on a slant to obtain the strains required for fermentation.

[0077] 1. Preparation of Aspergillus ochraceus slant strain and seed solution

[0078] (1) Incline cultivation

[0079] Incline medium: peeled potato 200g / L, glucose 20g / L, agar 20g / L, natural pH, sterilized at 121℃ for 30min.

[0080] The bacterial species preserved in the glycerol tube is aseptically drawn a certain amount of bacterial liquid and spread evenly on the slant medium, and cultured at 28°C for 4-6 days.

[0081] (2) Seed cultivation

[0082] The weight portion composition of primary medium comprises (g): glucose 10, yeast extract 8, K 2 HPO 4 2.0, NaCl 1.0, water 1000, and adjust the pH to 6.8, sterilize by high-pressure steam at 121°C for 30 minutes, and cool for later use.

[0083] With the strain cultivated in step (1), under aseptic conditions, use an inoculation loop to pick 6 ring spores into 100mL seed medium, inoculate 4 bottles in the same way, a...

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Abstract

The invention discloses a method for preparing 11α, 17α-hydroxyprogesterone by conversion of immobilized hydroxylase, which is characterized in that 11α, 17α-hydroxyprogesterone is prepared by conversion of immobilized hydroxylase with 17α-hydroxyprogesterone as a substrate Hydroxyprogesterone includes preparation of medium, cultivating Aspergillus ochrae in the medium, induction of hydroxylase, preparation of immobilized hydroxylase, preparation of transformation system, conversion of substrate, extraction and purification of products and other steps. The invention has the advantages of high feeding concentration, high conversion rate of substrate, short conversion cycle, easy separation of products and repeated use of immobilized hydroxylase. The feeding concentration of 17α-hydroxyprogesterone in a single enzyme reaction is 40g / L, and the molar conversion rate can reach 93%. At the same time, the immobilized hydroxylase can be used repeatedly for more than 3 times. The invention has simple operation process, low production cost and high industrial application prospect.

Description

technical field [0001] The invention relates to a method for preparing 11α, 17α-hydroxyprogesterone by conversion of immobilized hydroxylase, and belongs to the technical field of preparation of steroid intermediates. Background technique [0002] Steroids, also known as steroids, are a class of compounds containing cyclopentane polyhydrophenanthrene nuclei. Its basic structure consists of three six-membered rings and one five-membered ring, which are called A, B, C, and D rings respectively. Generally, there are angular methyl groups (-CH 3 ), there may be a hydroxyl group (-OH) or a keto group (-C=O) at the 3rd and 11th positions, some double bonds exist in the A ring or the B ring, and there are side chains of different lengths at the 17th position. A series of compounds with unique physiological functions are formed due to the differences in substituents, double bond positions or stereo configurations on the steroidal core. Steroid drugs are the second largest class of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10C12N11/084C12N9/02C12N1/14C12P33/10C12R1/66
CPCC12N1/14C12N9/0083C12N11/10C12P33/10C12N11/084
Inventor 管世敏荣绍丰蔡宝国李茜茜
Owner SHANGHAI INST OF TECH
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