Agrobacterium tumefaciens capable of metabolizing nicotine and application thereof

An Agrobacterium tumefaciens, nicotine technology, applied in bacteria, chemical instruments and methods, water/sludge/sewage treatment, etc., can solve the problems of limited application, weak nicotine toxicity resistance, and weak metabolism.

Inactive Publication Date: 2007-08-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these microorganisms can metabolize nicotine, their metabolic ability is weak and the

Method used

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  • Agrobacterium tumefaciens capable of metabolizing nicotine and application thereof
  • Agrobacterium tumefaciens capable of metabolizing nicotine and application thereof

Examples

Experimental program
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Example Embodiment

[0065] Example 1: Screening of Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 strain

[0066] Collect tobacco plant root soil samples from the long-term tobacco growing fields in Weifang, Shandong, and add 2 to 4 g to a liquid medium containing 1 g / L nicotine, and shake culture on a shaker for 2 to 4 days at 30°C. Then transfer 5 mL to the same medium for culture. Follow this method to transfer continuously 3 to 5 times. Dilute the obtained culture solution and spread it on a solid plate medium containing 1g / L nicotine, and cultivate at 30°C After 1 to 3 days, after the bacteria have grown, pick large colonies on the same solid plate medium with the same nicotine and streak continuously to confirm that the strain has the ability to degrade nicotine and is pure culture, and then pick a single colony to contain On a solid slant medium with 1g / L nicotine and cultured at 30°C for 1 to 3 days, the Agrobacterium tumefaciens wsn1-4 of the present invention was obtained. This strain was...

Example Embodiment

[0072] Example 2: Obtaining the 16S rDNA of the above Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 strain

[0073]Collect 2 mL of the culture of Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131 that has grown to the stable stage, centrifuge at 6,000 rpm for 5 minutes, and remove the supernatant; add 565 μL of TE solution to the precipitate. The formula of TE solution is :10mmol / L tris (Tris), 1mmol / L ethylenediaminetetraacetic acid (EDTA), adjust the pH to 8.0 with hydrochloric acid; use a pipette to repeatedly pipette to suspend the precipitate, and then add 30μL mass volume ratio Sodium dodecyl sulfonate (SDS) at a concentration of 10% and 5μL of 20mg / mL proteinase K (Merck, USA), mix well, and incubate in a water bath at 37°C for 1 hour; add 100μL of 5mol / L NaCl to fully Mix well, then add 80μL of CTAB / NaCl solution. The formula of CTAB / NaCl solution is: dissolve cetyltriethylammonium bromide (CTAB) with a concentration of 10% by mass to volume in 0.7mol / L NaCl In the solut...

Example Embodiment

[0098] Example 3: A method for treating tobacco processing solid waste using the intact cells of Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131, wherein the ratio of the mass of the tobacco processing solid waste to the volume of the biocatalyst suspension is calculated in g / mL 1:8

[0099] (1) Microbial strains: use Agrobacterium tumefaciens wsn1-4CCTCC NO.M206131;

[0100] (2) Slant culture: Inoculate the above-mentioned strains on a solid slant medium containing 0.02% nicotine by mass and volume, and culture at 20°C for 28 hours;

[0101] (3) Seed culture: Connect the strains cultured in step (2) with an inoculum loop under aseptic conditions in 20 mL of nicotine liquid medium containing 0.02% by mass and volume, and place it on a shaker at 20°C. Shake culture for 6 hours to obtain first-grade seeds;

[0102] (4) Expansion culture: inoculation amount of 5% by volume, followed by first-level seeds in 200mL liquid medium containing 0.02% by mass and volume of nicotine, and shak...

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Abstract

The invention discloses a pseudomonas putida to metabolite nicotine, which is characterized by the following: preserving in China Typical Culture Preservation Center (Wuhan university, china Wuhan) with keeping number as CCTCC NO.M 206131; possessing stronger nicotine metabolic ability and nicotine toxin immunity ability; growing highest at 5g/L nicotine density; culturing a great deal of a strain; using intact cell as biological activator; co-suspending in phosphoric acid buffer with solid waste matter from tobacco industry; oscillating; degrading nicotine completely. This bacterium possesses simple operation, which can be used to dispose tobacco waste matter widely.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of Agrobacterium tumefaciens capable of metabolizing nicotine and its application. Background technique [0002] Nicotine is the main alkaloid in many tobaccos, with a content of 0.5-12%. It plays an important role in people's tobacco smoking behavior and makes people highly dependent on tobacco. Yet nicotine is also a kind of " poisonous dangerous substance " simultaneously, and when one-time inhalation amount reaches 40ml / person, can suppress central nervous system, cause heart paralysis, have fatal danger; When neutralizing smoking, it will be nitrosated to generate tobacco-specific N-nitrosamines (tobacco-specific nitrosamines, TSNA), such as nitrosamine4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which Is a strong lung cancer carcinogen. [0003] Tobacco processing produces a large amount of solid waste, which can no longer be used due to its ph...

Claims

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Application Information

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IPC IPC(8): C12N1/20C02F3/34
Inventor 王书宁许平
Owner SHANDONG UNIV
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