CN106591194A - Variovorax sp., cell fraction and its composition for degrading benzylpenicillin - Google Patents
Variovorax sp., cell fraction and its composition for degrading benzylpenicillin Download PDFInfo
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Abstract
The invention relates to Variovorax sp., cell fraction and its composition for degrading benzylpenicillin. The Variovorax sp. is preserved in the China general microbiological culture collection center (CGMCC) and has a preservation number CGMCC No. 12495. The Variovorax sp., cell fraction and its composition for degrading benzylpenicillin can realize innocent treatment on waste benzylpenicillin hyphae and has the advantages of environmental protection, energy saving and cost saving.
Description
Technical field
The present invention relates to field of microbial culture technology, and in particular to a kind of variovorax of degradable penicillin, cell grade
Point and combinations thereof.
Background technology
Penicillin (Benzylpenicillin) is the general name of a family antibiotic, and it is by suppressing bacteria cell wall tetrapeptide side
The combination of chain and pentapeptide cross-bridge and the synthesis of block cell wall and play bactericidal action, which belongs to wide spectrum anti-infectives.
The production of industrial penicillin at present mainly uses starch, Semen Maydis pulp, soybean cake powder etc. for raw material, Jing penicillium chrysogenums
Fermentation, extraction, purification are formed., Jing after solid-liquid separation obtains benzylpenicillin sodium solution, remaining solid slag is as useless for the fermentation liquid
Mycelia.The useless mycelia main component of penicillin is crude protein, cellulose and crude fat etc., wherein also still have a small amount of Penicillin Residues,
Its residual content is about 0.2g/kg-0.5g/kg.Due to the stable in physicochemical property for remaining penicillin in mycelia, with long-term residual
The property stayed, bioconcentration, which easily causes the various potential safety hazards such as biology drug resistance into after biological chain.According to current international and
Domestic law regulation, useless mycelia can only be processed penicillin according to solid dangerous waste standard, and so not only environment can be made
Into secondary pollution, while the waste of grain resource and the raising of relevant enterprise production cost can be caused.Domestic annual generation
Penicillin mushroom dregs can reach more than 100,000 tons, it is seen then that the practicality in the urgent need to developing a kind of useless mycelia harmless treatment of penicillin
New technique.
However, the method for existing harmless treatment penicillin mycelia mainly has hot steaming method, a soda acid edman degradation Edman, these methods or
Person's power consumption is big, or causes secondary pollution, and can produce unpleasant waste gas.How the means of environmental protection are utilized to remaining in bacteria residue
Penicillin carries out appropriate harmless treatment and carries out effectively utilizes, becomes the technical barrier of relevant industries.
The content of the invention
To solve the deficiencies in the prior art, the invention provides the bacterial strain KDSPL- of one plant of energy degradation selectivity penicillin
04, so as to lay a good foundation to develop the useless mycelia harmless biological treatment technology of penicillin.KDSPL-04 bacterial strains are variovorax, should
Variovorax is preserved in China Committee for Culture Collection of Microorganisms's common micro-organismss with deposit number CGMCC No.12495
The heart.
Wherein, the colony characteristicses that the variovorax is formed on enrichment medium are：Circle, milk yellow, projection, surface light
Sliding, opaque, neat in edge；
Thalline is characterized as：Gram-negative, thalline in shaft-like, size between 0.9-2.3 μm of 0.4-0.5 μ ms, it is single or
Arrange in pairs.
Wherein, the L- pyrrolidinyl arylamine enzymes of the variovorax, glutamy arylamine enzyme pNA, gamma glutamyltransferase, L-
Proline arylamine enzyme, L-Tyrosine arylamine enzyme, urease, ellman's reagent experiment are positive；Lipase, phosphatase, ornithine decarboxylation
Enzyme, lysine deacidification enzyme, histidine assimilation experiment are negative；It is PEARLITOL 25C, D-Glucose, D-MANNOSE, D-Maltose, ancient
Sugared fermenting experiment is negative.
Wherein, the variovorax can be grown by sole carbon source of penicillin.
Wherein, whole base sequences of described variovorax 16S rDNA are as shown in SEQUENCE LISTING.
Invention additionally provides a kind of variovorax of degradable penicillin, which passes through mutation or genetic transformation is above-mentioned greedy bites
Bacterium obtains.
Wherein, whole base sequences of described variovorax 16S rDNA are as shown in SEQUENCE LISTING.
Invention also provides a kind of cell fraction of degradable penicillin, including：From the culture of above-mentioned variovorax
In the DNA prepared products that obtain or cell wall preparation, the culture fluid obtained from above-mentioned variovorax, the supernatant of these culture fluid
The culture fluid of the cell grade of liquid, the fraction of the supernatant and above-mentioned variovorax.
The present invention also provides a kind of compositionss, and which includes above-mentioned variovorax and/or above-mentioned cell fraction.
The variovorax of degradable penicillin that the present invention is provided, cell fraction and combinations thereof, can be to the useless mycelia of penicillin
Harmless treatment is carried out, with environmental protection, energy saving, cost-effective advantage.
Specific embodiment
Further understand to have to technical scheme and beneficial effect, the following detailed description of the present invention's
Technical scheme and its beneficial effect of generation.
The invention provides a kind of Classification And Nomenclature of degradable penicillin is variovorax (Variovorax sp.), this is greedy to bite
It is general that bacterium is preserved in China Committee for Culture Collection of Microorganisms on May 24th, 2016 with deposit number CGMCC No.12495
Logical microorganism center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the strain number is KDSPL-04.
Preferably, the colony characteristicses that the variovorax is formed on enrichment medium are：Circle, milk yellow, projection, surface
Smooth, opaque, neat in edge；
Thalline is characterized as：Gram-negative, thalline in shaft-like, size between 0.9-2.3 μm of 0.4-0.5 μ ms, it is single or
Arrange in pairs.
Preferably, the L- pyrrolidinyl arylamine enzymes of the variovorax, glutamy arylamine enzyme pNA, gamma glutamyltransferase,
L-PROLINE arylamine enzyme, L-Tyrosine arylamine enzyme, urease, ELLMAN reagents are positive；Lipase, phosphatase, ODC Ornithine decarboxylase, rely
Propylhomoserin deacidification enzyme, histidine assimilation are negative；PEARLITOL 25C, D-Glucose, D-MANNOSE, D-Maltose, ancient sugar fermentation are negative.
Preferably, the variovorax can be grown by sole carbon source of penicillin.
Preferably, whole base sequences of described variovorax 16S rDNA are as shown in SEQUENCE LISTING.
The variovorax (Variovorax sp.) of the degradable penicillin that the present invention is provided, with efficient degradation penicillin
Ability, the penicillin in the mycelium that can be produced to fermentation industry are effectively degraded, to eliminate in the useless mycelia of penicillin
Antibiotic remainss, the harmless treatment of the bacteria residue that ferments in can be used for penicillin commercial production, the harmless treatment for mycelia are provided
Effective approach.
Invention additionally provides a kind of variovorax of degradable penicillin, which passes through mutation or genetic transformation is above-mentioned greedy bites
Bacterium obtains.Specifically, the present invention obtains another kind of degraded penicillium sp by mutation or genetic transformation screening are carried out to above-mentioned variovorax
The mutation variovorax of element, the bacterial strain at least remain the characteristic of opportunistic pathogen strain degradable penicillin, the variovorax bacterial strain bag of the mutation
Include the mutation variovorax bacterial strain obtained by insertion mutation, integration mutation by former variovorax bacterial strain.
Preferably, whole base sequences of described variovorax 16S rDNA are as shown in SEQUENCE LISTING.
Invention also provides a kind of cell fraction of degradable penicillin, including：(include former greedy from above-mentioned variovorax
The mutation variovorax that phagocytosis and Jing mutation or genetic transformation are obtained) culture in the DNA prepared products that obtain or cell wall system
Standby thing, the culture fluid obtained from above-mentioned variovorax, the supernatant of these culture fluid and above-mentioned greedy are bitten the fraction of the supernatant
The culture fluid of the cell grade of bacterium.
The present invention also provides a kind of compositionss, and which includes above-mentioned variovorax and/or above-mentioned cell fraction.
The variovorax that the present invention is provided, specifically can be carried out screening, be identified by following methods, and determine which to penicillin
Degradation capability and gene order：
Embodiment 1：The screening of variovorax bacterial strain, identification, degradation capability and sequencing
Step one：From certain antibiotic enterprise periphery collection sludge 10g, the 100mL containing 30mg penicillins is inoculated into inorganic
In salt culture medium, in 30 DEG C, 160rpm shaking table concussion and cultivates.Culture was transferred once per 7 days after starting, with 10% (V/ during switching
V inoculum concentration) is transferred in the culture medium containing penicillin of higher concentration, penicillin containing concentration be followed successively by 10mg/L,
30mg/L, 60mg/L, 90mg/L, 120mg/L, the like, to 900mg/L, obtain the enrichment containing thalline of variable concentrations
Culture fluid.
Wherein, the composition and proportioning of the minimal medium in the present invention is (g/L)：K2HPO41.6g, KH2PO4
0.4g, MgSO4·7H2O 0.2g, CaCl2·2H2O 0.025g, FeCl3·6H2O 0.0023g, NH4NO30.5g, agar
20.0g, adjusts pH value to 7.0 with 1mol/L HCl or NaOH, 121 DEG C of autoclaving 30min.
The composition and proportioning of the enrichment medium of a certain concentration in the present invention is (g/L)：K2HPO41.6g, KH2PO4
0.4g, MgSO4·7H2O 0.2g, CaCl2·2H2O 0.025g, FeCl3·6H2O 0.0023, NH4NO30.5g, yeast extract
0.5g, glucose 1g, peptone 1g, agar 20.0g, adjust pH value to 7.0 with 1mol/L HCl or NaOH, and 121 DEG C of high pressure go out
Bacterium 30min.
Step 2：The corresponding selection containing concentration is applied to containing germy enrichment culture liquid in taking 0.1mL steps one to put down
On plate, cultivate 72 hours at 30 DEG C.The bacterial strain that obvious transparent circle occurs in one plant of speed of growth most fast and periphery of bacterial colonies is obtained, should
To on the minimal medium containing 30mg/L penicillins, periphery of bacterial colonies may occur in which clearly transparent circle to inoculation, by the bacterium
Strain numbering is KDSPL-04, the variovorax that as present invention is provided.
The Main Biological of KDSPL-04 bacterial strains is：The colony characteristicses that the bacterial strain is formed on enrichment medium are：
Circle, milk yellow, projection, surface are smooth, opaque, neat in edge；
The thalline of the bacterial strain is characterized as：Gram-negative, thalline is in shaft-like, size about 0.4-0.5 μ m 0.9-2.3 μ
M, single or paired arrangement.
Determine through test, the L- pyrrolidinyl arylamine enzymes of KDSPL-04 bacterial strains, glutamy arylamine enzyme pNA, gamma-glutamyl
Transferring enzyme, L-PROLINE arylamine enzyme, L-Tyrosine arylamine enzyme, urease, ELLMAN reagents are positive；Lipase, phosphatase, ornithine take off
Carboxylic acid, lysine deacidification enzyme, histidine assimilation are negative；PEARLITOL 25C, D-Glucose, D-MANNOSE, D-Maltose, ancient sugar are sent out
Ferment is negative.
Step 3：By liquid chromatography for measuring in aseptic water, degradation capability of the bacterial strain to penicillin.
In culture fluid, the content of thalline is 10 in every milliliter of culture fluid7Individual, the concentration of penicillin is 30mg/L, in 30 DEG C,
160rpm shaking tables concussion degraded 6h, Jing are determined, and the degradation rate of penicillin is 100%.Meanwhile, the bacterial strain is co-cultured with fermentation bacteria residue
One day, using liquid chromatography for measuring, the bacterial strain can reach 100% to the degradation rate of penicillin.It is demonstrated experimentally that degraded solutions body
It is for tap water, KDSPL-04；, in pH 7.0-8.0,30 to 40 DEG C of well-growns of temperature can be with penicillin as sole carbon for bacterial strain
Source grows, and is one plant of very promising degradation bacteria.
Step 4：Determine KDSPL-02 bacterial strains 16S rDNA sequence, by the 16S rDNA sequences of the measure with
Genebank accession number carries out homology analysis for the gene order of the 16S rDNA of D88006.
First, the STb gene of KDSPL-04 bacterial strains is extracted using DNA a small amount of extracting method.Secondly, it is general using 16S rRNA
Primer 2 7F-1492R expands its 16S rRNA gene order, and the PCR primer that amplification is obtained directly is sequenced (sequencing result
See SEQUENCE LISTING in sequence table), by the 16SrRNA gene orders input GeneBank for obtaining, by Blastn journeys
All sequences in ordered pair data base are compared analysis.
The 16SrRNA gene orders of the bacterial strain KDSPL-04 of the present invention have higher with the existing type strain that variovorax belongs to
Similarity.Itself and Variovorax paradoxusLAM12373TSequence similarity be 99.42%.The bacterial strain belongs to
Variovorax sp., in combination with its physiological and biochemical property, identify that the bacterial strain is Variovorax sp., i.e. variovorax bacterium
Strain.
Embodiment 2：The screening of variovorax bacterial strain, identification and the degradation capability to penicillin are determined
Step one：Take 10g sludge to be contained in the 250mL conical flasks of sterilizing, 100mL concentration is added for the penicillin of 0.3g/L
In solution, in 30 DEG C of rotating speeds be 160r/min shaking table cultures.After culture starts, transferred once per 7 days, with 10% (V/ during switching
V inoculum concentration) is inoculated in fresh penicillin solution domestication culture medium, and steps up the content of penicillin.Detection penicillium sp
The degradation rate of element, picks out best one group so as to used by isolated strains.
Step 2：By the bacteria suspension (1 × 10 of above-mentioned bacterial strains in 1mL7Individual/mL) it is linked into 100mL penicillins containing 30mg/L
Tap water in, take three times it is parallel, and with not inoculated bacteria but the penicillin containing same concentrations tap water as control.30℃、
160rpm shaking table cultures, are cultivated 2,4,6 hours respectively, and liquid chromatography detects the content of penicillin, and experimental result is shown in Table one.Drop
Solution 6 hours, selected bacterial strain can be almost degradable by penicillin.It is KDSPL-04 by the strain number for obtaining.
Degradation rate of the one KDSPL-04 bacterial strains of table in tap water to penicillin
Step 3：Determine KDSPL-04 bacterial strains 16S rDNA sequence, by determine 16S rDNA sequences with
Genebank accession number carries out homology analysis for the gene order of the 16S rDNA of D88006, it is believed that KDSPL-04 bacterial strains belong to
Variovorax sp., in combination with its physiological and biochemical property, identify that the bacterial strain is Variovorax sp., i.e. variovorax.
Embodiment 3：Variovorax bacterial strain screening and the degradation capability to penicillin in bacteria residue are determined
Penicillin wet mycelia of giving up is crossed into 0.2mm sieves, the useless mycelia after 10g sieves is weighed, and is put into 100mL tap waters
In, in the wet mycelia solution that gives up add Jing to cultivate variovorax thalline 6g (weight in wet base) that culture in 48 hours is obtained in 30 DEG C or so,
If 3 groups of parallel tests, sampling in 0,2,4,6 hours is acted on as control, respectively to add penicillin mycelia but be not added with variovorax body, efficiently
The residual quantity of penicillin in liquid phase method detection mycelia.As a result show, variovorax thalline, is made with penicillin mycelia under the conditions of 30 DEG C
After with 6 hours, 100% can reach to the degradation rate of penicillin.
Degradation effect of the two KDSPL-04 bacterial strains of table to penicillin in bacteria residue
The beneficial effects of the present invention is：
1st, the variovorax of the degradable penicillin that the present invention is provided, the ability with efficient degradation penicillin can be to sending out
Penicillin in the mycelium that ferment industry is produced effectively is degraded, and to eliminate antibiotic remainss in the useless mycelia of penicillin, can use
The harmless treatment of fermentation bacteria residue in penicillin commercial production, the harmless treatment for mycelia provide effective approach.
2nd, the variovorax of the degradable penicillin that the present invention is provided, which can be grown for sole carbon source using penicillin, should
Bacterial strain can reach 100% to the degradation capability of 1g/L penicillins within 6 hours.The bacterial strain can be used in penicillin commercial production
The harmless treatment of fermentation bacteria residue, the processing method efficiently, environmental protection, economize on resources, processing cost it is low, completely eliminate green grass or young crops
Useless adverse effect of the mycelia to environment of mycin.
3rd, the variovorax of the invention gene related to degraded and key enzyme are in biological restoration and purification and industry and biology
Field of medicaments has a wide range of applications.
Although the present invention is illustrated using above-mentioned preferred embodiment, so which is not limited to the protection model of the present invention
Enclose, any those skilled in the art carry out various changes with respect to above-described embodiment within without departing from the spirit and scope of the present invention
It is dynamic still to belong to the scope protected by the present invention with modification, therefore protection scope of the present invention is by being defined that claims are defined.
SEQUENCE LISTING
<110>Hebei University of Science and Technology
<120>A kind of variovorax of degradable penicillin
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1392
<212> DNA
<213> Variovorax sp.
<400> 1
acggcagcgc gggagcaatc ctggcggcga gtggcgaacg ggtgagtaat acatcggaac 60
gtgcccaatca gtgggggata acgcagcgaa agctgtgcta ataccgcata cgatctacgg 120
atgaaagcag gggatcgcaa gaccttgcgc gaatggagcg gccgatggca gattaggtag 180
ttggtgaggt aaaggctcac caagccttcg atctggagct ggtctgagag gacgaccagc 240
cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaattttgga 300
caatgggcgc aagcctgatc cagccatgcc gcgtgcagga tgaaggcctt cgggttgtaa 360
actgcttttg tacggaacga aacggccttt tctaataaag agggctaatg acggtaccgt 420
aagaataagc accggctaac tacgtgccag cagccgcggt aatacgtagg gtgcaagcgt 480
taatcggaat tactgggcgt aaagcgtgcg caggcggtaa tgtaagacag ttgtgaaatc 540
cccgggctca acctgggaac tgcatctgtg actgcattgc tggagtacgg cagaggggga 600
tggaattccg cgtgtagcag tgaaatgcgt agatatgcgg aggaacaccg atggcgaagg 660
caatcccctg ggcctgtact gacgctcatg cacgaaagcg tggggagcaa acaggattag 720
ataccctggt agtccacgcc ctaaacgatg tcaactggtt gttgggaatt cactttctca 780
gtaacgaagc taacgcgtga agttgaccgc ctggggagta cggccgcaag gttgaaactc 840
aaaggaattg acggggaccc gcacaagcgg tggatgatgt ggtttaattc gatgcaacgc 900
gaaaaacctt acccaccttt gacatgtacg gaattcgcca gagatggctt agtgctcgaa 960
agagaaccgt aacacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1020
taagtcccgc aacgagcgca acccttgtca ttagttgcta cattcagttg ggcactctaa 1080
tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaagtcct catggccctt 1140
ataggtgggg ctacacacgt catacaatgg ctggtacaaa gggttgccaa cccgcgaggg 1200
ggagctaatc ccataaaacc agtcgtagtc cggatcgcag tctgcaactc gactgcgtga 1260
agtcggaatc gctagtaatc gtggatcaga atgtcacggt gaatacgttc ccgggtcttg 1320
tacacaccgc ccgtcacacc atgggagcgg gttctgccag aagtagttag cttaaccgca 1380
aggagggcga ta 1392
Claims (9)
1. a kind of variovorax of degradable penicillin, it is characterised in that the variovorax is protected with deposit number CGMCC No.12495
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. the variovorax of degradable penicillin according to claim 1, it is characterised in that
The colony characteristicses that the variovorax is formed on enrichment medium are：Circle, milk yellow, projection, surface are smooth, impermeable
Bright, neat in edge；
Thalline is characterized as：Gram-negative, in shaft-like, size is between 0.9-2.3 μm of 0.4-0.5 μ ms, single or paired for thalline
Arrangement.
3. the variovorax of degradable penicillin according to claim 1, it is characterised in that the L- pyrrolidines of the variovorax
Base arylamine enzyme, glutamy arylamine enzyme pNA, gamma glutamyltransferase, L-PROLINE arylamine enzyme, L-Tyrosine arylamine enzyme, urease,
Ellman's reagent experiment is positive；Lipase, phosphatase, ODC Ornithine decarboxylase, lysine deacidification enzyme, histidine assimilation experiment are in the moon
Property；PEARLITOL 25C, D-Glucose, D-MANNOSE, D-Maltose, ancient sugared fermenting experiment are negative.
4. the variovorax of degradable penicillin according to claim 1, it is characterised in that the variovorax can be with penicillin
Grow for sole carbon source.
5. the variovorax of degradable penicillin according to claim 1, it is characterised in that described variovorax 16S rDNA
Whole base sequences as shown in SEQUENCE LISTING.
6. a kind of variovorax of degradable penicillin, it is characterised in which is appointed in passing through mutation or genetic transformation claim 1-5
Variovorax described in one is obtained.
7. variovorax according to claim 6, it is characterised in that whole base sequences of described variovorax 16S rDNA
As shown in SEQUENCE LISTING.
8. a kind of cell grade of degradable penicillin, it is characterised in that include：From any one of claim 1-7
The DNA prepared products obtained in the culture of variovorax or cell wall preparation, bite from greedy any one of claim 1-7
The cell grade of the culture fluid obtained in bacterium, the supernatant of these culture fluid, the fraction of the supernatant and above-mentioned variovorax
Culture fluid.
9. a kind of compositionss, which includes thin described in variovorax and/or claim 8 any one of claim 1-7
Born of the same parents' fraction.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787129A (en) * | 2012-07-20 | 2012-11-21 | 南京师范大学 | Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide |
| CN105819442A (en) * | 2016-03-23 | 2016-08-03 | 河北科技大学 | Method for preparing activated carbon by means of denitrification of waste penicillin mycelia |
-
2016
- 2016-12-27 CN CN201611223442.8A patent/CN106591194B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787129A (en) * | 2012-07-20 | 2012-11-21 | 南京师范大学 | Amidase gene of Variovorax boronicumulans CGMCC 4969, and its application in biological degradation of acrylamide |
| CN105819442A (en) * | 2016-03-23 | 2016-08-03 | 河北科技大学 | Method for preparing activated carbon by means of denitrification of waste penicillin mycelia |
Non-Patent Citations (2)
| Title |
|---|
| 徐晨光: ""茶园土壤抗生素抗性细菌及抗性基因研究"", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 顾超: "土壤中以抗生素为单一碳源的抗性细菌", 《浙江大学学报（农业与生命科学版）》 * |
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