Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

5 alpha-reductase mutant, genetic engineering bacterium and application of genetic engineering bacterium to realizing high-efficiency catalysis of 5 alpha-AD production

A technology of genetically engineered bacteria and reductase, applied in the field of genetic engineering, can solve problems such as unsatisfactory catalytic efficiency, and achieve the effect of solving low catalytic activity and improving catalytic efficiency

Active Publication Date: 2019-12-31
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
View PDF11 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the molecular modification of 5α-reductase is mainly to study the enzymatic activity of the substrate testosterone, and its catalytic efficiency is not ideal; 5α-reductase is the key enzyme that catalyzes the production of 5α-AD from AD. The transformation of 5α-reductase gene has not been reported

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
  • 5 alpha-reductase mutant, genetic engineering bacterium and application of genetic engineering bacterium to realizing high-efficiency catalysis of 5 alpha-AD production
  • 5 alpha-reductase mutant, genetic engineering bacterium and application of genetic engineering bacterium to realizing high-efficiency catalysis of 5 alpha-AD production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Construction of mutant enzyme gene and recombinant expression vector

[0043] Using the synthetic recombinant plasmid pUC57-5α with the Treponema smegmatis 5α-reductase gene as a template, amplify the 5α-reductase gene whose nucleotide sequence is shown in SEQ ID NO.1, and amplify the amplified product It was connected with the Escherichia coli-mycobacterium shuttle plasmid pMV261, transformed into E.coli DH5α, and subjected to resistance screening, plasmid PCR, double enzyme digestion verification and DNA sequencing. The recombinant plasmid with correct sequencing and stable inheritance was selected and named pMV261-5α.

[0044] Using the pMV261-5α recombinant plasmid as a template, using F1 primer (sequence shown in SEQ ID NO.5) and R1primer (sequence shown in SEQ ID NO.6) as primers, site-directed mutagenesis was carried out by overlapping extension PCR to obtain The recombinant plasmid of the 5α-reductase mutant gene, named pMV261-5α Y187F .

[0045...

Embodiment 2

[0047] Embodiment 2: Construction of 5α-reductase mutant mycobacterium engineering bacteria

[0048] The recombinant plasmid pMV261-5α obtained in Example 1 Y187F Electroporation to MNR M3△ksdd competent cells, screening strains that can be stably inherited is the 5α-reductase mutant mycobacterium engineering bacteria; the specific method is as follows:

[0049] 1) Take 10 μL of the plasmid that was sequenced correctly in the previous stage and add it to the melted MNR M3Δksdd competent cells, gently blow and mix with a gun, and treat in ice bath for about 15 minutes;

[0050] 2) Completely transfer the pre-cooled plasmid and competent cell mixed solution into a 1mm electric transfer cup (clean the electric transfer cup with absolute ethanol, place it on a sterile operating table, dry it, and place it on ice for pre-cooling for 5 minutes);

[0051] 3) Place the electroporation cup containing the mixture of plasmids and competent cells on a high-voltage pulse electroporation i...

Embodiment 3

[0055] Embodiment 3: 5α-reductase mutant engineering bacteria MNR M3△ksdd / 261-5α Y187F Transformation of PS to produce 5α-AD

[0056] 1) Bacteria activation culture:

[0057] The 5α-reductase mutant engineered strain MNR M3△ksdd / 261-5α Y187F Transfer to fresh slant medium, culture at 30°C for 3 days, wash off the strains on the slant medium with 20mL 0.5% Tween 80 sterile aqueous solution, mix well to obtain eluate, absorb 1mL eluate and add to 30mL seed In the culture medium, cultured on a shaker at 30°C and 200r / min for 36h to obtain a seed culture solution;

[0058] Slant medium composition: K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, ferric ammonium citrate 0.05g / L, citric acid 2g / L, ammonium nitrate 2g / L, glycerin 20g / L, glucose 5g / L, CaCO 3 10g / L, agar 20g / L, the rest is water, pH7.2;

[0059] Seed medium composition: K 2 HPO 4 0.5g / L, MgSO 4 ·7H 2 O 0.5g / L, ferric ammonium citrate 0.05g / L, citric acid 2g / L, ammonium nitrate 2g / L, glycerin 20g / L, glucose 5g / L, ...

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

No PUM Login to View More

Abstract

The invention provides a 5 alpha-reductase mutant. The 5 alpha-reductase mutant is obtained through site-specific mutation and reconstruction of 5 alpha-reductase, a genetic engineering bacterium of the 5 alpha-reductase mutant is constructed and applied to high-efficiency catalysis of 5 alpha-AD production, in the 5 alpha-reductase subjected to site-specific mutation and reconstruction, 187th tyrosine is mutated into phenylalanine, 5 alpha-reductase mutant recombinant plasmids are electrically transferred into mycobacterium neoaurum to be subjected to heterologous expression, then enzymatic activity and production efficiency are determined, the inventor finds that the enzyme activity of a mycobacterium neoaurum genetic engineering bacterium of the 5 alpha-reductase mutant is increased by2.3 times, the catalysis efficiency is improved by 22.8%, and the 5 alpha-AD production is increased to 1.78g / L from the original 1.45g / L; and when the 5 alpha-reductase mutant is used as a key enzymefor production of an important sterides medicine intermediate, the bioconversion efficiency and the yield of products can be obviously improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a 5α-reductase mutant and its application in high-efficiency catalytic 5α-AD production. Background technique [0002] Steroidal 5α-reductase belongs to the reduced coenzyme II (NADPH)-dependent enzyme, which can catalyze the reduction of a series of steroid substrates at the 4,5 double bond, and add the hydrogen at the C-5 position to the α Position becomes the corresponding 5α-reduction product. For example, testosterone (TS) is reduced to a more active steroid hormone under the action of 5α-reductase: dihydrotestosterone (DHT), which plays an important role in the physiological regulation of androgen and the differentiation of human sexes. Extremely important role; androst-4-ene-3,17dione (AD) can be reduced to an important steroid drug intermediate by the catalysis of steroid 5α-reductase: 5α-androstenedione (5α- AD) Intermediates. [0003] Some chemical and ...

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
IPC IPC(8): C12N9/02C12N15/53C12N15/74C12N1/21C12P33/00C12R1/32
CPCC12N15/74C12P33/00C12N9/0006C12N9/1205C12N9/0004C12Y101/01049C12Y207/01023
Inventor 王敏申雁冰任小贤赵云秋骆健美夏梦雷
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com