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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 their resistance to nicotine toxicity is weak, which limits their application in actual production

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
Comparison scheme
Effect test

Embodiment 1

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

[0066] Collect tobacco plant root soil samples from long-term tobacco planting fields in Weifang, Shandong and other places, take 2-4g and add it to a liquid medium containing 1g / L nicotine, and shake it on a shaker for 2-4 days at 30°C. Then transfer 5mL to the same culture medium for culture, continue to transfer 3 to 5 times in this way, dilute the obtained culture solution and spread it on a solid plate medium containing 1g / L nicotine, culture at 30°C After 1 to 3 days, after the colonies grow, pick a large colony and streak continuously on the same nicotine solid plate medium to confirm that the strain has the ability to degrade nicotine, and it is a pure culture, and then pick a single colony to contain On a solid slant medium with 1g / L nicotine, culture at 30°C for 1 to 3 days to obtain the Agrobacterium tumefaciens wsn1-4 of the present invention, which was preserved in China on No...

Embodiment 2

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

[0073]Collect 2 mL of the culture of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131 that has grown to the stationary phase, centrifuge at 6,000 rpm for 5 minutes, and remove the supernatant; add 565 μL of TE solution to the precipitate, and the formula of the TE solution is : Tris (Tris) of 10mmol / L, ethylenediaminetetraacetic acid (EDTA) of 1mmol / L, adjust the pH to 8.0 with hydrochloric acid; blow and beat repeatedly with a pipette to suspend the precipitate, then add 30 μL of mass-volume ratio Concentration of 10% sodium dodecylsulfonate (SDS) and 5 μL 20 mg / mL proteinase K (Merck, USA), mix well, and put in a water bath at 37 ° C for 1 hour; add 100 μL 5mol / L NaCl, fully Mix well, then add 80 μL of CTAB / NaCl solution, the formula of CTAB / NaCl solution is: the mass volume ratio concentration is 10% cetyltriethylammonium b...

Embodiment 3

[0098] Embodiment 3: Utilize the method for processing tobacco processing solid waste with the complete cell of Agrobacterium tumefaciens (Agrobacterium tumefaciens) wsn1-4CCTCC NO.M206131, wherein the volume of tobacco processing solid waste quality and biocatalyst suspension is in g / mL ratio is 1:8

[0099] (1) Microbial strain: 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 in a mass volume ratio, and culture statically for 28 hours at 20°C;

[0101] (3) Seed culture: the bacterial strain cultivated in step (2), under aseptic conditions, use an inoculation loop to connect 1 loop in 20mL of nicotine liquid medium containing 0.02% mass volume ratio, at 20°C, on a shaker Vibrating culture for 6 hours to obtain first-class seeds;

[0102] (4) Expansion cultivation: with the inoculum amount of 5% by volume, connect the first-stage seeds in 200 mL of nicoti...

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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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