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Two high-temperature-resistant engineering bacteria capable of effectively degrading nitroalkane-type compounds

A nitroalkane, engineering bacteria technology, applied in microorganism-based methods, bacteria, microorganisms, etc., can solve problems such as mammalian carcinogenicity and human toxicity

Active Publication Date: 2020-02-28
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some nitroalkanes are considered toxic to humans and carcinogenic to mammals

Method used

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  • Two high-temperature-resistant engineering bacteria capable of effectively degrading nitroalkane-type compounds
  • Two high-temperature-resistant engineering bacteria capable of effectively degrading nitroalkane-type compounds
  • Two high-temperature-resistant engineering bacteria capable of effectively degrading nitroalkane-type compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] The clones (including the promoter part) of the full-sequence genes encoding nitroalkane oxidase GTNG-0930 and GTNG-1755 were constructed.

[0089] 1. Extraction of total DNA from Agrobacillus NG80-2

[0090] In this example, the Geobacillus thermodenitrificansFor NG80-2, take 3 mL of the fresh culture cultured overnight, collect the cells by centrifugation, suspend the cells in 250 μL of 50 mM Tris buffer (pH8.0), add 10 μL of 0.4M EDTA (pH8.0), and mix well at 37 °C Incubate for 20 minutes, then add 30 μL of 20 mg / L lysozyme, mix well and then incubate at 37 °C for another 20 min, then add 5 μL of 20 mg / L proteinase K, mix gently, then add 20 μL of 10% SDS, keep warm at 50 °C until the solution is clear, and use equal volumes of Phenol: chloroform: isoamyl alcohol extraction twice, chloroform: isoamyl alcohol extraction once, the last supernatant solution, add 2.5 times the volume of pre-cooled absolute ethanol, recover DNA, wash with 70% ethanol, precipitate dissol...

Embodiment 2

[0103] Geobacillus thermodenitrificans NG80-2 competent preparation and transformation process is as follows:

[0104] 1) Inoculate NG80-2 in 20 ml TGP liquid medium, culture overnight at 60°C in a shaker at 180 rpm.

[0105] 2) According to the proportion of 1% (v / v), inoculate the overnight cultured bacterial liquid into 50 mL of L TGP liquid medium, shake and cultivate on a shaker at 60 ℃ and 180 rpm until the OD600 is about 1.0~1.6.

[0106] 3) Place the bacterial solution on ice for 10 min.

[0107] 4) Transfer the bacterial solution into a 50 ml Beckmam high-speed centrifuge tube in an ultra-clean bench, centrifuge at 9000 g, 4 °C for 20 min.

[0108] 5) Discard the supernatant, add about 25 ml of competent cell washing buffer [0.5 M sorbitol, 0.5 M mannitol, 10% glycerol (v / v)] pre-cooled at 4 °C to resuspend the cells by shaking, Then make up to 50ml with washing buffer, centrifuge at 9000g, 4°C for 20min. This step was repeated 3 times, and the volume of washing...

Embodiment 3

[0116] Degradation experiment of 2-nitropropane

[0117] The engineered bacteria (NG-S1, NG-S2) and NG80-2 were inoculated into LB medium containing kanamycin and LB medium without kanamycin, respectively, and cultured at 60°C for 12 hours. Cells were harvested by centrifugation, washed twice with thermophilic minimal medium, and the cell pellet was resuspended. Under aerobic conditions, add 25 ml minimal medium containing appropriate antibiotics to a 100 ml headspace vial to a final concentration of 1.08 mmol 2-nitropropane. The anaerobic conditions were as follows: the minimal medium was boiled for 30 minutes to eliminate most of the dissolved oxygen, and placed in an anaerobic incubator to remove oxygen. Add L-cysteine ​​and resazurin as oxygen reducing agent and indicator to the culture medium at final concentrations of 0.1% and 0.01 g / L, respectively, until the oxygen indicator in the culture medium turns colorless . Recombinant strains and NG80-2 were inoculated into ...

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Abstract

The invention discloses geobacillus thermodenitrificans engineering bacteria capable of efficiently degrading nitroalkanes. The geobacillus thermodenitrificans engineering bacteria capable of efficiently degrading the nitroalkanes are obtained by using an NOEs (nitroalkane-oxidizing enzymes) gene that overexpresses a nitroalkane degradation pathway. The modified engineering bacteria are named NG-S1 and NG-S2. The nucleotide sequence of the NOEs gene is represented by SEQ ID NO. 1-2. Compared with wild-type geobacillus thermodenitrificans, the obtained engineering bacteria have the advantages that the degradation efficiency is obviously improved, and the degradation rates of the NG-S1 (under anaerobic conditions) and the NG-S2 (under aerobic conditions) are increased by 2 times and 2.8 times respectively. The two high-temperature-resistant strains capable of efficiently degrading toxic pollutants respectively in an aerobic environment and in an anaerobic environment are provided, and have great significance and application value in treatment of environmental pollution; new methods and examples are provided for modification of thermophilic engineering bacteria for biodegradation.

Description

technical field [0001] The invention belongs to the field of microbial strain transformation, and relates to the construction of engineering strains NG-S1 and NG-S2 and their application to the degradation of 2-nitropropane, in particular to thermophilic denitrification soil for efficiently degrading nitroalkanes Bacillus engineering bacteria and its construction method and application. Background technique [0002] Nitro compounds are widely used as solvents, fuels and organic synthesis intermediates in the chemical industry, and are widely used as herbicides, insecticides and fungicides in agriculture, which have important industrial significance. Among them, nitroalkane compounds mainly include nitromethane, nitroethane, 1-nitropropane and 2-nitropropane. 2-Nitropropane is widely used in chemical intermediates, solvents and components of inks, paints, varnishes and other coatings. However, some nitroalkanes are considered toxic to humans and carcinogenic to mammals. Ni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A62D3/02C12R1/01A62D101/04A62D101/47
CPCC12N9/0046A62D3/02C12Y107/03001A62D2101/04A62D2101/47Y02A50/20
Inventor 王威张秉玲宋晓如孙林博
Owner NANKAI UNIV
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