CN1202242C - Pseudomonas offensire smell ZWL 73 and its making method and application - Google Patents
Pseudomonas offensire smell ZWL 73 and its making method and application Download PDFInfo
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- CN1202242C CN1202242C CN 03128122 CN03128122A CN1202242C CN 1202242 C CN1202242 C CN 1202242C CN 03128122 CN03128122 CN 03128122 CN 03128122 A CN03128122 A CN 03128122A CN 1202242 C CN1202242 C CN 1202242C
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- pseudomonas
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- pseudomonas putida
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Abstract
The present invention relates to cacosmia pseudomonas ZWL73, a preparation method thereof and an application thereof. The cacosmia pseudomonas ZWL73 is determined in a mode that 16S rDNA carries out sequence determination and nucleotide homology comparison with the sequence embodied by genbank. The cacosmia pseudomonas is used as the degradation bacterium of 1-chloro-4-nitrobenzene, and the 1-chloro-4-nitrobenzene is used as unique carbon source, nitrogen source and energy source to grow. The preparation method of the cacosmia pseudomonas comprises steps of the preparation of an inorganic salt culture medium, and the enrichment and the separation of strain, and degradation cacosmia pseudomonas which is consistent to the shape of thallus is obtained. The present invention has the advantages of simple preparation method, convenient operation, strong capability of degrading a 1-chloro-4-nitrobenzene compound, etc., and provides a strain resource for biologically controlling the pollution of the compound, so the present invention is favorable to environmental protection and the health of human beings.
Description
Technical field
The present invention relates to microorganism field, particularly a kind of pseudomonas putida ZWL73 and method for making and application.The microorganism that this patent is used is pseudomonas putida ZWL73 (Pseudomonas putida ZWL73), and by China's typical culture collection center preservation, preservation date is on April 4th, 2003, and protecting minus sign is CCTCC NO:M 203021.
Background technology
As everyone knows, chloronitrobenzene compounds (Chloronitrobenzenes, hereinafter to be referred as CNBs) be widely used in producing and making various dyestuffs, agricultural chemicals, medical drugs as starting material and intermediate, aromatics such as chloronitrobenzene have number of ways and enter environment and become pollutent, the waste water of for example residual discharging in process of production, as thoroughly not the transforming of intermediate of production processes such as dyestuff, agricultural chemicals and with waste discharge.The European Community announces that already chloronitrobenzene is a kind of deleterious especially compound and is difficult to degraded (EEC, 1982) in environment, and China determines that it is the priority pollutant in the water.Chloronitrobenzene also is found the methemoglobinemia (methemoglobinemia) (Linch, 1974) that can cause human body and animal, and is a kind of mutagenic compound (Shimizu, 1983) and carcinogen (Weisburger, 1978).Because of than, the biological degradation of research chloronitrobenzene compounds is significant for environment protection and human beings'health.
Biological degradation for the chloronitrobenzene compounds, and microorganism rarely has report to this type of degradation approach, some worker has observed the bio-transformation of CNBs rather than their thorough inorganicization, for example, the resting cell of false pseudomonas bacillus CBS3 bacterial strain changes into things such as 4-chloroaniline with low rate with 1-chloro-4-oil of mirbane (hereinafter to be referred as 1C4NB), but can not continue degraded (Schackmann and Muller, 1991).Livingston (1993) is with mixed culture degraded 1-chloro-3 oil of mirbane, and makes its thorough inorganicization.And Park human two strain bacterial strain actings in conjunction such as (1999), with 1-and 2-C4NBs degraded and inorganicization.Bacterium LW1 (belonging to Comamonadaceae) can utilize 1C4NB as unique carbon source, nitrogenous source and energy growth, infer that its pathways metabolism may be as open loop substrate (Katsivela et al. by 2-amino-5-chlorophenol, 1999), this is at present both at home and abroad about only two reports of the thorough mineralising chloronitrobenzene of pure culture microorganism strains.
Summary of the invention
Technical problem to be solved by this invention is: a kind of pseudomonas putida ZWL73 and method for making and application are provided, this bacterium with 1C4NB as unique carbon source, nitrogenous source and energy growth, thereby provide competent microorganism resource for the pollution of this compounds of biological treating.
The scheme that the present invention solves its technical problem is: pseudomonas putida ZWL73 is by 16S rDNA sequencing and carry out nucleotide homology with the sequence that GenBank has included and relatively determine.
Pseudomonas putida ZWL73, as the degradation bacteria of 1-chloro-4-oil of mirbane, it is grown as unique carbon source, nitrogenous source and the energy with 1-chloro-4-oil of mirbane.
The present invention has following advantage:
One. pseudomonas putida ZWL73 can tolerate the 1C4NB of higher concentration, and can be the growth of unique C, N source.Therefore, this bacterium can thoroughly be degraded environment is poisoned bigger 1C4NB, and its degradation capability is strong, the 1C4NB of 6.3mM if the culture of pressing 1% inoculation can thoroughly be degraded in 48 hours, simultaneously, can be translated into material, or be utilized by thalline to the environment toxicological harmless.
They are two years old. the separation of pseudomonas putida ZWL73 and acquisition, for studying the catabolism approach of microorganism from now on, and disclose degradation pathway and the mechanism of 1C4NB from the molecular biology level to 1C4NB, and material is provided.
They are three years old. and in view of can thoroughly the degrade ability of 1C4NB of pseudomonas putida ZWL73, this bacterium provides microorganism resource for this compounds of biological treating pollutes.
They are four years old. and to the further investigation of pseudomonas putida ZWL73, the biological control and the biological restoration of the environmental problem that causes of pollutant provide the theory and technology basis for this reason.
They are five years old. and the preparation method of pseudomonas putida ZWL73 is simple, and is easy to operate.
Description of drawings
Fig. 1 is the electromicroscopic photograph (* 12000) of pseudomonas putida ZWL73.
Fig. 2 is the agarose gel electrophoresis figure of the plasmid pZWL73 of pseudomonas putida ZWL73.
Fig. 3 is the agarose gel electrophoresis figure of the restriction enzyme EcoRI endonuclease bamhi of plasmid pZWL73.
Fig. 4 is that pseudomonas putida ZWL73 utilizes 1-chloro-4-oil of mirbane growth curve.
Fig. 5 is the 16S rDNA sequence of pseudomonas putida ZWL73
Embodiment
The present invention is a kind of pseudomonas putida ZWL73 (Pseudomonas putida ZWL73) CCTCC M203021 and method for making and application.The invention will be further described below in conjunction with embodiment and accompanying drawing.
One. pseudomonas putida ZWL73
1. morphological specificity:
Bacterium colony is white in color, and is smooth, neat in edge.Thalline is rod-short, extremely gives birth to multiple flagellum (see figure 1), Gram-negative.
The flagellum observational technique: inoculate this bacterium in the LB inclined-plane that contains 0.6% agar, 30 ℃, static cultivation 10 hours, distilled water washes thalline, sample preparation, electron microscopic observation.
2.16S rDNA
1) sequence: as shown in Figure 5, about 1500bp fragment, 16s rRNA is prokaryotic organism ribosomal subunit 16s RNA, has higher conservative property in prokaryotic organism, can provide section, genus, kind, etc. multi-level information.
2) preparation method:
With E.Z.N.A.Bacterial DNA kit (Omega Bio-tek) preparation bacterial genomes DNA.Special primer 27F (5-AGAGTTTGATCCTGGC TCAG-3) with bacterial 16 S rDNA universal primer 1492R (5-GGTTACCTTGTTACGACTT-3) and bacterium is an amplimer; bacterial genomes DNA is a template; 16S rDNA (about 1500bp) fragment of amplification degradation bacteria is delivered Jikang Biotechnology Co Ltd, Shanghai's order-checking.
3) homology is relatively:
Carry out nucleotide homology relatively with the sequence that NCBI (natioal center for biotechnology information) blast program has been included to 16SrDNA sequence and the GenBank of this bacterium, the homology of the 16S rDNA sequence among 16S rDNA sequence and the GenBank is seen attached list, and to reach 99.8～99.9% bacterial classification mainly be Pseudomonas putida bacterial strain to sequence homology with it.So identifying this bacterium is pseudomonas putida, and called after pseudomonas putida ZWL73 (Pseudomonas putida ZWL73).
3. plasmid detects:
By alkaline lysis method of extracting (with reference to molecular cloning experiment guide second edition p19), find that this bacterium has big plasmid pZWL73 (seeing 73 samples of Fig. 2).With the TOL plasmid pWWO (seeing the pWWO sample of Fig. 2) of the pseudomonas putida PaW1 of the 117kb of standard, and the pDTG1 plasmid of the pseudomonas putida NCIB 9816-4 of 81kb (seeing 9816 samples of Fig. 2) relatively, and this bacteria plasmid pZWL73 is about 100kb.Cut the pZWL73 plasmid with restriction enzyme EcoR I enzyme,, obtain the distinctive EcoR I of pZWL73 plasmid endonuclease bamhi electrophorogram (seeing the 2 electrophoresis holes of Fig. 3) through agarose gel electrophoresis.
Among Fig. 31 is the molecular weight contrast: λ-HindIII endonuclease bamhi, descending 23kb, 9.4kb, 6.5kb, 4.3kb, 2.3kb, 2.0kb and the 0.56kb of being respectively of this fragment.
Two. the application of pseudomonas putida ZWL73
Pseudomonas putida ZWL73 is as the degradation bacteria of 1-chloro-4-oil of mirbane, and this bacterium grows as unique carbon source, nitrogenous source and the energy with 1-chloro-4-oil of mirbane.
The characteristic of pseudomonas putida ZWL73 degraded 1-chloro-4-oil of mirbane is as follows:
Inoculation pseudomonas putida ZWL73 is in the 10ml of 0.5mM 1C4NB minimal medium, 28 ℃ of shaking table overnight incubation, transfer in the 100ml minimal medium that contains 1mM 1C4NB, 28 ℃ of shaking table overnight incubation, centrifugal collection thalline, with the fresh resuspended thalline of the minimal medium that contains 0.33mM 1C4NB, 28 ℃ of shaking tables are cultivated, different time is taken a sample at interval, survey 1C4NB concentration with the HPLC method, OD600 measures cell concentration, and ion selective electrode (Ao Lilong) is measured chlorine ion concentration, the Nai Shi method is measured ammonium concentration, the results are shown in Figure 3.
Fig. 4 result shows: along with incubation time increases, the concentration of 1C4NB reduces gradually, and cell concentration increases gradually, and is accompanied by ammonium ion and chlorion generation.Because of not containing carbon in the minimal medium, nitrogenous source, pseudomonas putida ZWL73 bacterium can not utilize its growth, and the minimal medium that has replenished 1C4NB can supply this bacteria growing, shows that this bacterium is to grow in unique C, N source with 1C4NB.The generation of ammonium ion shows that the nitro among the 1C4NB at first thoroughly is reduced to amino in addition, produces ammonium ion through decomposing then, and chlorion also is to produce when 1C4NB decomposes.In Fig. 4,
Curve representation cell concentration (OD600 absorption value),
The concentration of curve representation 1C4NB,
The curve representation ammonium concentration
The curve representation chlorine ion concentration.
By 1% inoculum size, Pseudomonas putida ZWL73 bacterium is inoculated in the 100ml minimal medium of 6.3mM 1C4NB, 28 ℃ of constant temperature, shaking table was cultivated 48 hours, and 1C4NB is degraded fully.
Three. the preparation of pseudomonas putida ZWL73
A. prepare minimal medium
Composition is Na
2HPO
412H
2O 14.3g; KH
2PO
43.0g; MnSO
4H
2O 0.28mg; FeSO
47H
2O0.3mg; MgSO4 0.06mg; CaCL
21mg; CuSO
40.05mg; ZnSO
40.05mg and H
3BO
30.05mg, it is settled to 1000ml with distilled water, transfer pH 7.0, autoclaving.
B. the enrichment of bacterial classification
From Gedian Chemical Plant, Wuhan City's soil, take a sample, soil sample is mixed with 20% suspension, inoculum size with 10% is inoculated in the minimal medium that contains 1-chloro-4-oil of mirbane (0.5mM), 30 ℃ of constant temperature, shaking table is cultivated, after treating that bacterium grows, get 1% bacterium liquid and insert in the same substratum, continue shaking table and cultivate.
C. separate
After obtaining that the enrichment culture thing of remarkable degradation property is arranged, utilize dull and stereotyped culture technique, culture is separated and purifying in the minimal medium that contains 1-chloro-4-oil of mirbane repeatedly, obtain the degradation bacteria of thalli morphology unanimity.
Four. subordinate list
The BLAST of the 16S rDNA of pseudomonas putida ZWL73 analyzes
| Bacterium for alignment GenBank Accession No. Identity |
| Pseudomonas sp.ML2 AF378011 1422/1423(99.9％) Pseudomonas putida KT2440 section 1 of 21 AE016774 1422/1423(99.9％ Pseudomonas sp.HR 13 AY032725 1421/1423(99.8％) Pseudomonas sp.WBC-3 AY040872 1421/1423(99.8％) Pseudomonas putida strain ATCC 17453 AF094746 1421/1423(99.8％) Pseudomonas putida strain ATCC 17514 AF094741 1421/1423(99.8％) |
Claims (2)
1. a pseudomonas putida ZWL73 (Pseudomonas putida ZWL73) CCTCC M203021,
It is characterized in that described pseudomonas putida ZWL73, its 16S rDNA sequence is as follows:
GTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGT
GGG
GGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTA
TCA
GATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGA
TGA
TCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAA
GCC
TGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGC
TAA
TACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGT
GCA
AGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAAC
CTG
GGAACTGCATCCAAAACTGGCAAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAG
ATA
TAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAAC
AGG
ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCT
AAC
GCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGT
GGA
GCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTG
GTG
CCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAAC
GAG
CGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGG
TGG
GGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAA
GCC
GCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATC
GCT
AGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGG
GTT
GCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATGACTGGG
2. the application of pseudomonas putida ZWL73 CCTCC M203021 is characterized in that: pseudomonas putida ZWL73 is as the degradation bacteria of 1-chloro-4-oil of mirbane, and this bacterium grows as unique carbon source, nitrogenous source and the energy with 1-chloro-4-oil of mirbane.
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| CN100376667C (en) * | 2006-03-03 | 2008-03-26 | 山东大学 | Pseudomonas putida capable of resisting organic solvent and application in dual liquid phase reaction |
| CN101475925B (en) * | 2009-01-13 | 2012-05-30 | 北京凯拓三元生物农业技术有限公司 | A strain of quinoline-degrading bacterium, as well as cultivation method and use thereof |
| CN102888378B (en) * | 2012-11-02 | 2013-12-25 | 中北大学 | Culture medium for pseudomonas putida |
| CN104531592A (en) * | 2015-01-15 | 2015-04-22 | 浙江工业大学 | Pseudomonas putida having n-hexylene degradation capacity and application thereof |
| CN109486721B (en) * | 2018-12-18 | 2020-12-22 | 广东海洋大学 | Pseudomonas putida and application thereof |
| CN113957031A (en) * | 2021-12-06 | 2022-01-21 | 上海市农业科学院 | Construction method and application of escherichia coli engineering bacteria capable of completely degrading nitrobenzene |
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