Tetracycline antibiotic degrading bacterium at low temperature and application thereof

A technology of tetracyclines and degrading bacteria, applied in the fields of applications, bacteria, microorganisms, etc.

Pending Publication Date: 2022-01-28

SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI

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AI-Extracted Technical Summary

Problems solved by technology

At present, most studies only focus on the degradation of oxytetracycline at medium and high temperatures, and there are few s...

Abstract

The invention belongs to the technical field of biological treatment of antibiotic pollution, and particularly relates to pseudomonas putida capable of degrading oxytetracycline at low temperature (5-15 DEG C) and application of the pseudomonas putida. The degrading bacterium is gram-negative pseudomonas putida TU-3 which is preserved in the culture collection center of the Institute of Microbiology of Chinese Academy of Sciences on July 22, 2021, and the number of the degrading bacterium is CGMCC (China General Microbiological Culture Collection Center) No. 22940. The strain can be applied to preparation of oxytetracycline degrading bacteria, the strain resources of the oxytetracycline degrading bacteria are enriched, and a scientific basis is provided for degradation of oxytetracycline in soil.

Application Domain

BacteriaWater contaminants +5

Technology Topic

Laboratory cultureTetracycline antibiotics +8

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Examples

Experimental program(3)

Example Embodiment

[0035] Example 1: Isolation of Pseudomonas putida Pseudomonas putida

[0036] Soil in Shenyang City, a livestock farm soil Shenbei Liaoning Province, after enrichment, separation and purification to obtain specific, comprising the steps of:

[0037] Step a: Strain Enrichment

[0038] Weigh 5g of soil was added 250mL enriched media containing 20mg / L of oxytetracycline, at 15 ℃, 160r / min shaker incubated in the dark 7d. Take 5mL enrichment broth added to the new enriched media and cultured according to the culture conditions, and then switching again, the concentration of antibiotics added to the medium enrichment of each adapter to improve 20mg / L. Repeating the above steps adapter culture until the concentration of the antibiotic enriched media 100mg / L.

[0039] Step Two: Isolation and purification of the strains

[0040] The enrichment broth sterile water at 10 -3 10 -4 10 -5 10 -6 Dilutions, and the diluted solution after different gradients are uniformly applied on an LB solid medium, the colony morphology were observed, picked colony morphology different single colony isolated on a solid medium further purified, pure picked colonies on the plate It was deposited with glycerin tube upon activation.

[0041] Step three: Degradation Strains

[0042] The separated respective single strain of 5%, respectively, was inoculated to selection medium containing 20mg / L of oxytetracycline, 15 ℃, shaking at 160rpm shaking dark conditions using high performance liquid chromatography after 7d - series Quantitative Determination of residual mass concentration of oxytetracycline, oxytetracycline degradation rate obtained degrading bacteria, the degradation rate of the highest strain was named TU-3.

[0043] Liquid chromatography - tandem mass spectrometry

[0044]High Performance Liquid Chromatography - Series Mass Spectrometry (HPLC-MS / MS) Quantitative Analytical Analysis of the concentration of gentamicin. Atlantis T3 column (3μm, 2.1 * 150mm), column temperature of 30 ° C, flow phase with 98% (v / v) acetate + 0.1% formic acid aqueous solution (A) and 2% (v / v) acetonitrile (B) Composition, the injection speed is 0.4 ml / min, and the amount of injection is 10 μL.

[0045] The strain has a degradation effect at low temperatures, which has the following characteristics:

[0046] The purified strain Tu-3 was pulled out on the LB solid medium, and 1d was cultured at 28 ° C. The colony was circular, pale yellow, and the surface was smooth, and the edge is neatly. figure 1 Indicated.

Example Embodiment

[0047] Example 2: Strain 16S Identification

[0048] (1) Genome extraction

[0049] The genomic DNA of strain Tu-3 was extracted in accordance with the specification operation procedure using the Tianamp Bacteria DNA Kit bacteria genomic DNA extraction kit.

[0050] (2) PCR amplification

[0051] 16S general primers are: 27f: agagttgatcctggctcag 1492r: TacggctacctTgttacgActt, PCR length is about 1500 bp.

[0052] PCR reaction system:

[0053]

[0054] PCR response procedure:

[0055] The predetermined 95 ° C is 4 min, 94 ° C deformation 30s, 55 ° C, and extends 40s at 72 ° C, 35 cycles, 72 ° C extension 7 min, 4 ° C until termination.

[0056] (3) Gel electrophoresis

[0057] Finally, the resulting PCR product was observed with 1% agarose gel electrophoresis, and the electrophoretic conditions were 80V for 30 min. The PCR product of the amplified amplification was sent to Huada Genetic Technology Co., Ltd., the sequencing primer is the same as the amplification primer. The nucleotide sequence of the 16S gene of strain Tu-3 was obtained, and the nucleotide sequence of the 16S gene of strain Tu-3 was as follows.

[0058] Strain Tu-3 gene sequence list

[0059]

[0060]

[0061] The above sequence consists of 1396 base (BP). The measured 16S gene sequence was performed on the US National Biological Information Center (NCBI) website to obtain a strain similar to a higher similarity. The results showed that strains TU-3 were identified as malodous pseudogen.

[0062] The strain Tu-3 is the apepomonas PuTIDA, which has been prepared on July 22, 2021, and the general microbial center of China's Microbial Square Safety Management Committee, the preservation number CGMCC No.22940.

Example Embodiment

[0063] Example 3: TU-3 Degradation Effect of Hymodyncin

[0064] After oscillating the Tu-3 at 37 ° C, 160 rpm under the conditions of 37 ° C, 160 rpm was oscillated with 5% inoculation, and the inoculation amount was cultured in a screening medium containing hydrocarcin 20 mg, from 5 ° C, 10 ° C, respectively. Side of 15 ° C, 160 rpm was protected from light oscillating culture, and three parallel per group were set as a blank control. The resulting cultured was 0.22 um organic filtration, and the concentration of hydrocarcin residue was detected.

[0065] Calculated as follows:

[0066]

[0067] The results showed that the initial content of oxytetracycline was 20 mg / L, the bacterium was at 15 ° C, and the degradation rate was 66.6% under 160 rpm conditions, the blank control degradation rate was 32%; at 10 ° C, the degradation rate of 20D under 160 rpm can reach 45.5. %, Whitening control degradation rate of 20%; 20D degradation rate at 5 ° C, 160 rpm can reach 37%, and blank control degradation rate is 18.3%. The difference between the samples and the blank control group in the latexecomicin degradation period is small due to oxytetracyclines.

PUM

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