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Streptomycete 2-nitropropane dioxyenase gene and its application

A technology of dioxygenase and nitropropane, applied in application, genetic engineering, oxidoreductase and other directions, can solve the problem of not many structural genes, and achieve the effect of improving the environment

Inactive Publication Date: 2004-08-11
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been found in microorganisms such as Mrakhansenula, Bacillus subtilis and Streptomyces, and their optimal substrates are 2-nitropropane, nitroethane, etc. These enzymes were isolated and purified and deduced Amino acid sequence, but there are not many reports about the structural genes of these enzymes, especially in Streptomyces and even Actinomyces.

Method used

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  • Streptomycete 2-nitropropane dioxyenase gene and its application
  • Streptomycete 2-nitropropane dioxyenase gene and its application
  • Streptomycete 2-nitropropane dioxyenase gene and its application

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Embodiment 1

[0025] Example 1: Cloning and expression of npD gene

[0026] 1. Cloning of npD gene

[0027] 1.1 Preparation of probes

[0028] In the cloned Streptomyces coelicolor A3 (2) development and differentiation promoter pTH270 upstream part of the gene inside there is a SmaI restriction site, using SmaI and HindIII on the multiple cloning site for plasmid pIJ4478 [Tan H & Chater K.J.Bacteriol .1993, 175 (4): 933-940] (the gene sequence containing pTH270 and its upstream and downstream partial sequences) to perform double enzyme digestion to obtain a DNA fragment of about 320bp.

[0029] Take 10ng-3μg DNA to denature in boiling water for 10 minutes, immediately put it in ice / NaCl for 3 minutes; add 2μl hexanucleotide mixture, 2μl dNTP mixture containing digoxigernin-11-dUTP, add distilled water to 19μl, finally add 1μl Klenow enzyme After incubation at 37°C for 20 hours, 2 μl of 0.2M EDTA (pH 8.0) was added to terminate the reaction. Then add 2.5 μl 4M LiCl, 75 μl pre-cooled abso...

Embodiment 2

[0056] Embodiment 2: Separation, purification and biological activity of 2-nitropropane dioxygenase

[0057] The obtained cell fragments were subjected to Sephadex column chromatography, eluted with 0.01M phosphate buffer (pH 7.0), the active fractions were combined, and subjected to DEAE Sepharose Fast Flow column chromatography. Wash with 2-3 column volumes of 0.2M and 0.3M sodium chloride, and finally elute with 0.4M sodium chloride. The active part is subjected to repeated column chromatography on DEAE Sepharose Fast Flow, and the purity is detected by SDS-PAGE . The results showed that after the above purification process, the target protein NPD with good purity and activity was obtained.

[0058] After mixing 0.05 μg of enzyme solution, 100 μl of 0.4 mM substrate and 800 μl of 0.2M phosphate buffer (pH 7.0), they were reacted at 37° C. for 5 minutes, and the reaction was terminated with 200 μl of 5 mM copper sulfate. Add 0.5ml of p-aminobenzenesulfonic acid (0.33%) and...

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Abstract

The present invention relates to the application of environmental microbe. Especially, by means of molecular technology, one new gene is cloned from Streptomyces ansochromogenes 7100 and the gene encodes one kind of 2-streptomycete dioxyenase. The encoded product has great value in biologically degrading industrial pollutant nitroalkane and in improving environment.

Description

technical field [0001] This invention belongs to the field of applied environmental microorganisms, specifically, a new gene is cloned from Streptomyces chromogenes by molecular biology techniques, and the product encoded by it is a kind of 2-nitropropane dioxygenase, It has great application value in the biodegradation of industrial pollutants nitroalkanes and can be used to improve the environment. technical background [0002] Nitroalkane oxidase is a large class of enzymes whose substrates are mainly nitroalkanes, denitrates these compounds and generates corresponding aldehydes and ketones, accompanied by the generation of nitrous acid. Among them, 2-nitropropane dioxygenase (2-Nitropropane Dioxygenase) is an important nitroalkane oxidase. It has been found in microorganisms such as Mrakhansenula, Bacillus subtilis and Streptomyces, and their optimal substrates are 2-nitropropane, nitroethane, etc. These enzymes were isolated and purified and deduced However, there are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N15/70
Inventor 谭华荣张集慧马文勃
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI