Construction and application of Comamonas testosteroni 3,17beta-hydroxysteroid dehydrogenase (3,17beta-HSD) gene reinforced strain

A Comamonas and hydroxysteroid technology, applied in the field of applied environmental microorganisms, can solve the problems of unresearched gene structure and function

Inactive Publication Date: 2013-05-01
CHANGCHUN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 3. 17β-HSD is the key enzyme in the series of enzymes that degrade steroids and other substances in Comamonas testosteroni (C. testosteroni). However, no research has been done on its gene structure and function, and there has been no report in China
Especially not reported in C. testosteroni and Pseudomonas

Method used

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  • Construction and application of Comamonas testosteroni 3,17beta-hydroxysteroid dehydrogenase (3,17beta-HSD) gene reinforced strain
  • Construction and application of Comamonas testosteroni 3,17beta-hydroxysteroid dehydrogenase (3,17beta-HSD) gene reinforced strain
  • Construction and application of Comamonas testosteroni 3,17beta-hydroxysteroid dehydrogenase (3,17beta-HSD) gene reinforced strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0106] Example 2.3, Construction of 17β-HSD Gene Enhanced Strain and Its Application to Cholesterol Degradation

[0107] 2.1 The construction of 3,17β-HSD gene enhanced strain refers to Example 1.1-1.6.

[0108] 2.2 Comamonas testosteroni 3, 17β-HSD gene-enhanced strain applied to the research of cholesterol degradation

[0109] 2.2.1 Cholesterol standard

[0110] 25mM cholesterol standard sample: prepared according to the 2010 edition of "Chinese Pharmacopoeia"; the cholesterol standard sample was purchased from the National Institute for the Control of Pharmaceutical and Biological Products.

[0111] 2.2.2 Experimental method

[0112] 2.2.2.1 Blank control group:

[0113] 1) Add 50 μl of 25mM cholesterol standard sample to 5ml of fresh LB liquid medium, set up four parallel control groups, and culture on a shaker at 27°C and 180rpm for 18h.

[0114] 3) Centrifuge at 10,000 rpm for 5 minutes at 4°C to collect the LB medium supernatant;

[0115] 4) Under the following con...

Embodiment 3

[0130] Example 3.3, Construction of 17β-HSD Gene Enhanced Strain and Its Application to the Degradation of Testosterone

[0131] 3.1 The construction of 3,17β-HSD gene enhanced strain refers to Example 1.1-1.6.

[0132] 3.2 Comamonas testosteroni 3, 17β-HSD gene-enhanced strain applied to the research of degrading testosterone

[0133] 3.2.1 Standard sample of testosterone

[0134] 25mM testosterone standard sample: prepared according to the 2010 edition of "Chinese Pharmacopoeia"; testosterone standard product was purchased from China Institute for the Control of Pharmaceutical and Biological Products.

[0135] 3.2.2 Experimental method

[0136] 3.2.2.1 Blank control group:

[0137] 1) Add 50 μl of 25mM testosterone standard sample to 5ml of fresh LB liquid medium, set up four parallel control groups, culture at 27°C, 180rpm on a shaker for 18h.

[0138] 3) Centrifuge at 10,000 rpm for 5 minutes at 4°C to collect the LB medium supernatant;

[0139] 4) Under the following...

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Abstract

The invention relates to a construction of a Comamonas testosteroni 3,17beta-HSD gene reinforced strain, and belongs to the microbial field. The construction of the Comamonas testosteroni 3,17beta-HSD gene reinforced strain is characterized in that a recombinant plasmid is pK-Tacpromoter-3,17beta-HSD; and the construction comprises the following steps: carrying out PCR technological amplification, and target segment recovery and purification of a Comamonas testosteroni genome which is a template to connect with a pUCm-T carrier; 2, carrying out PCR technological amplification, and target segment recovery and purification of a plasmid pAH10 which is a template to connect with the pUCm-T carrier; 3, extracting a recombinant plasmid 3,17beta-HSD-T through adopting a column plasmid DNA small-amount extraction kit; and 4, respectively extracting the recombinant plasmid and pK-3,17beta-HSD through adopting the column plasmid DNA small-amount extraction kit. According to the invention, the Comamonas testosteroni 3,17beta-HSD gene reinforced strain is constructed for the first time and is applied, and the estriol degradation efficiency, the cholesterol degradation efficiency and the stosterone degradation efficiency of the Comamonas testosteroni 3,17beta-HSD gene reinforced strain are 30%, 15% and 150% higher than that of a wild Comamonas testosteroni strain respectively.

Description

technical field [0001] The invention belongs to the field of applied environmental microorganisms. Background technique [0002] Comamonas testosteroni is a Gram-negative bacterium with partial tufted flagella, motile, and widely distributed in nature. The bacterium is an aerobic, non-fermenting, chemoorganotrophic bacterium that can use testosterone, cholesterol, estriol and other steroid hormones as the only carbon source and energy source. [0003] 3. 17β-HSD is the key enzyme in the series of enzymes that degrade steroids and other substances in Comamonas testosteroni (C. testosteroni). However, there is no research on its gene structure and function, and there is no report in China. Especially in C. testosteroni and Pseudomonas have not been reported. [0004] Tac promoter is a functional trp-lac hybrid promoter in Escherichia coli, the DNA sequence of the transcription initiation point upstream of the -20 region is derived from the trp promoter, and the DNA sequence ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/66C12N1/21A62D3/02C12R1/01A62D101/28
Inventor 于源华王保学于化东李艳红王妍
Owner CHANGCHUN UNIV OF SCI & TECH
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