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Genetically engineered bacterium for rapidly degrading methyltestosterone and application thereof

A technology of methyl testosterone and genetically engineered bacteria is applied in the field of application of environmental microorganisms to achieve the effects of improving degradability, strong adaptability and high removal efficiency

Inactive Publication Date: 2013-05-08
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, if the inhibitor of the gene encoding 3,17β-hydroxysteroid dehydrogenase can be found, and then the gene encoding the inhibitor can be knocked out, it is likely to improve the degradation ability of Comamonas testosteroni to methyltestosterone, but At present, there are no relevant research and technical application reports on the regulation of 3,17β-hydroxysteroid dehydrogenase gene expression

Method used

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  • Genetically engineered bacterium for rapidly degrading methyltestosterone and application thereof
  • Genetically engineered bacterium for rapidly degrading methyltestosterone and application thereof
  • Genetically engineered bacterium for rapidly degrading methyltestosterone and application thereof

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Embodiment 1

[0029] According to the gene sequence (GenBank accession No. AHIL00000000), the gene map was drawn centering on the 3,17β-hydroxysteroid dehydrogenase gene, and other genes close to the 3,17β-hydroxysteroid dehydrogenase gene were selected to analyze their expression and production The properties of the protein, the final selection PhaR gene is the target gene, the result is as follows figure 1 shown.

Embodiment 2

[0031]Total DNA was extracted from Comamonas testosteroni grown overnight in LB medium by CTAB method. According to the gene sequence in GenBank, an additional base G was added behind the third base at the 5' end of the PhaR gene to design the forward primer, that is, the forward primer was 5'-ATGGCAAGAAAACAAAGAG-3', and the reverse primer was 5' '-CTGCATGGCATGACCATA-3'. Using the total DNA as a template, carry out PCR amplification and use PCR technology to obtain the first 300 bp mutant gene fragment of the PhaR gene from the genome of Comamonas testosteroni (such as figure 2 -a), and then clone the mutated gene fragment into the expression vector pCR2.1-TOPO to obtain the expression vector pPK-8 containing the mutated gene fragment, and prove that the plasmid pPK-8 is the target plasmid by plasmid size and EcoRI fragment (like figure 2 -b), and then use the electroporation introduction method (pulse voltage 1.8kv, time 4.9 milliseconds) to integrate the pPK-8 plasmid in...

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Abstract

The invention provides a genetically engineered bacterium for rapidly degrading methyltestosterone and an application thereof. The process comprises the steps of knocking a PhaR gene of testosterone comamonas to obtain a high-expression engineering strain PK-4 capable of generating 3,17 beta-hydroxysteroid dehydrogenase by a gene homologous recombination technology; preparing bacterial suspension from the engineering strain PK-4, inoculating into waste water generated by livestock and poultry and aquatic product cultivation containing testosterone pollutant; cultivating for 48 hours when the temperature is 25 DEG C and the rotating speed is 150rpm, wherein the degradation rate of the methyltestosterone is 78.36-90.23%; the pH value is 6-8 in the waste water of which the methyltestosterone concentration is 1mg / L; the temperature is 15-25 DEG C; and cultivating for 48 hours at 150rpm in an oscillating manner, so that the degradation rate of the methyltestosterone can achieve 60.15%-90.23%. The genetically engineered bacterium has the beneficial effects that the engineering strain PK-4 of removed testosterone comamonas of the PhaR gene is obtained for the first time; and the genetically engineered bacterium is strong in adaptive capacity in swine wastewater, and fast in removal speed of the methyltestosterone, and has a great application value in the aspects of treatment and disposal of breeding waste and bioremediation of environmental pollution.

Description

technical field [0001] The invention discloses a genetically engineered bacterium for rapidly degrading methyltestosterone and its application, and also provides a degradation method for rapidly removing methyltestosterone in aquaculture wastewater by the genetically engineered bacterium, belonging to the technical field of applied environmental microorganisms. Background technique [0002] Methyltestosterone (Methyltestosterone), also known as methyltestosterone, methyltestosterone, methyltestosterone, is a synthetic androgen that promotes the formation, development, and maturation of male sexual organs, resists estrogen, and inhibits endometrial growth And the function of the ovarian pituitary gland, is widely used in the pharmaceutical industry. The maximum solubility of methyltestosterone in water is 0.5g / L, but because it is difficult to be used by common microorganisms, the effluent of sewage treatment plants, especially those in the pharmaceutical industry, often con...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/09C02F3/34C12R1/01C02F101/34
Inventor 李明堂刘文丛郝林琳王呈玉
Owner JILIN AGRICULTURAL UNIV
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