Strain capable of efficiently converting DHEA (dehydroepiandrosterone) into 3 beta, 7 alpha and 15 alpha-trihydroxy androstane-5-alkene-17-ketone obtained by compound mutation screening

A compound mutagenesis, hydroxyandrosteroid technology, applied in fungi, mutant preparation, microorganism-based methods, etc., can solve the problems of many by-products, large amount of organic solvent, low microbial transformation efficiency, etc., to improve work efficiency. Efficiency, the effect of reducing work steps

Inactive Publication Date: 2013-04-24
JIANGNAN UNIV
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also disadvantages such as low microbial conversion efficiency, low substrate concentration, poor conversion reaction specificity, many by-products, and large amount of organic solvent used in the extraction process, causing environmental pollution.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Strain capable of efficiently converting DHEA (dehydroepiandrosterone) into 3 beta, 7 alpha and 15 alpha-trihydroxy androstane-5-alkene-17-ketone obtained by compound mutation screening
  • Strain capable of efficiently converting DHEA (dehydroepiandrosterone) into 3 beta, 7 alpha and 15 alpha-trihydroxy androstane-5-alkene-17-ketone obtained by compound mutation screening
  • Strain capable of efficiently converting DHEA (dehydroepiandrosterone) into 3 beta, 7 alpha and 15 alpha-trihydroxy androstane-5-alkene-17-ketone obtained by compound mutation screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Implementation example 1: the selection of starting bacterial strain, the determination of growth curve, transformation curve and transformation ability

[0069] Selection of starting strains:

[0070] Stretch the existing slant strains to isolate single colonies, pick 100 well-growth single colonies on the plate, inoculate them on the seed medium, cultivate them at 30 ℃, 200 r / min for 4-5 days, and then transfer them for the second time with 10% inoculum amount. Inoculated in the seed medium, cultured for 20-30 h, inoculated in a 250 mL shake flask with 10% inoculum, cultivated at 30 °C, 200 r / min for 24 h, fed with 10 g / L DHEA, 28 °C, 200 r / min Fermentation was terminated after 72 h of min fermentation, samples were extracted with ethyl acetate for 3 to 4 times, substrate conversion and product formation were initially detected by TLC, and substrate conversion and product formation were detected qualitatively and quantitatively by HPLC. Select a strain with relativel...

Embodiment 2

[0076] Implementation example 2: the determination of the minimum inhibitory concentration of ketoconazole and the compound mutagenesis screening of high conversion rate strains

[0077] Take the bacterial strain selected in Example 1 as the starting strain, cultivate to the late logarithmic phase, filter with 4 layers of lens paper to obtain the spore suspension, centrifuge and discard the supernatant, wash with 0.9% normal saline for 2 to 3 times, and wash with normal saline Resuspended, diluted and spread on plates containing different concentrations of ketoconazole, cultured in a 30°C incubator for 4 to 5 days and then counted, the lowest concentration of ketoconazole that did not grow colonies was the minimum inhibitory concentration.

[0078] Take the bacterial strain selected in Example 1 as the starting strain, cultivate to the late logarithmic phase, filter with 4 layers of lens paper to obtain the spore suspension, centrifuge and discard the supernatant, wash with 0.9...

Embodiment 3

[0083] Implementation Example 3: Research on Transformation Ability of Mutant Strain ST-1

[0084] DHEA feeding concentration g / L 4 8 10 12 16 DHEA conversion rate% 98.4 88.3 83.2 75.9 62.8 Product yield % 90.3 81.3 62.3 50.6 37.7

[0085] It can be seen from the table that the substrate conversion ability and product yield of the mutant strain ST-1 have been greatly improved compared with the original strain.

[0086] 18SrDNA

[0087] 1 GTTCGACTTT ACTTCTCTAA TGACCGAGTT TGGATAACTT TCCGGCCCTG AGTGGTAGTT

[0088] 61 GCCCACCTCT CTGGGCCAGT CCGAAAGCCT CACTGAGCCA TTCAATCGGT AGTAGCGACG

[0089] 121 GGCGGTGTGT ACAAAGGGCA GGGACGTAAT CAACGCAAGC TGATGACTTG CGCTTACTAG

[0090] 181 GGATTCCTCG TTGAAGAGCA ATAATTGCAA TGCTCTATCC CCAGCACGAC GGAGTTTAAC

[0091] 241 AAGATTACCC GGGCCTTCCG GCCAAGGGAG TACTCGCTGG CTCCGTCAGT GTAACGCGCG

[0092] 301 TGCGGCCCAG AACATCTAAG GGCATCACAG ACCTGTTATT GCCTCAAACT TCCATCGGCT

[0093] 361 TGAGCCGATA GTCCCTCTAA GAAGCCGGC...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
Login to view more

Abstract

The invention belongs to the field of a fermentation technology in bioengineering, and particularly relates to a high yield mutant strain (Collectotrichumlini) ST-1 which is obtained by compound mutation screening through ultraviolet and nitrogen ion injecting and is capable of efficiently converting dehydroepiandrosterone (DHEA) into 3 beta, 7 alpha and 15 alpha-trihydroxy androstane-5-alkene-17-ketone. The strain which is high in substrate feeding concentration, high in conversion rate and low in by-product production is obtained by compound mutation screening through ultraviolet and nitrogen ion injecting, so that a foundation is provided for the industrialization of microbial conversion based steroid drug.

Description

technical field [0001] The invention relates to a method for obtaining highly transformed DHEA into a 3β, 7α, 15α-trihydroxyandrost-5-en-17-one mutant strain through compound mutagenesis of A. flaxensis, and belongs to the technical field of fermentation in bioengineering. Background technique [0002] Dehydroepiandrosterone (DHEA) is an important active substance in natural organisms, and has an important regulatory effect on the metabolic activities of life. Its derivative 3β, 7α, 15α-trihydroxyandrost-5-en-17-one is The precursor compound of the fourth-generation oral contraceptive droxone has the advantages of high efficiency, low toxicity, and no side effects. It also has non-contraceptive clinical benefits, such as weight control and skin quality improvement. At present, drospirenone is the world's No. 1 female oral contraceptive, with global annual sales of 1.45 billion euros, so 3β,7α,15α-trihydroxyandrost-5-en-17-one is used as its pharmaceutical intermediate , the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N13/00C12N15/01C12P33/06C12R1/645
Inventor 许正宏李传鹏李会史劲松窦文芳
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap