CN108624519A - Application of the chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium - Google Patents
Application of the chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium Download PDFInfo
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- CN108624519A CN108624519A CN201710152060.9A CN201710152060A CN108624519A CN 108624519 A CN108624519 A CN 108624519A CN 201710152060 A CN201710152060 A CN 201710152060A CN 108624519 A CN108624519 A CN 108624519A
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- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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Abstract
The invention discloses application of the chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium.The present invention changes the fungistatic effect to staphylococcus aureus, bacillus subtilis, Escherichia coli and saccharomycete by the concentration of chain alkyl San butyl phosphonium bromides, when concentration is less than 25mg/mL, inhibiting effect unobvious of the tributyl octadecyl bromination Phosphonium to staphylococcus aureus, show the toxicity extremely low to staphylococcus aureus and good tolerance bacterium performance, and tributyl heptadecyl phosphonium bromide shows the toxicity extremely low to E.coli and yeast and good tolerance bacterium performance to the inhibition zone pole unobvious of E.coli and yeast.Tributyl octadecyl bromination Phosphonium and tributyl heptadecyl phosphonium bromide can be used as ionic liquid, tolerance bacterium is filtered out, to establish tolerance bacterium library, determines its ionic liquid resistance mechanism, relevant target gene is found, being resistant to engineering bacteria for subsequent builds ionic liquid lays the foundation.
Description
Technical field
The present invention relates to application of the chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium, belong to ionic liquid
Body technique field.
Background technology
Ionic liquid have liquid temperature range is wide, fixedness, electrochemical window mouth width, thermal stability is good, corrosivity is low,
The unique physicochemical properties such as property is adjustable, with supercritical CO2, aqueous two-phase be listed as three big " green " solvents, material, change
The fields such as work, biomass, electrochemistry present good application prospect.Ionic liquid as a kind of novel highly polar medium,
Energy consumption, material consumption and pollution can be greatly lowered, for energy-saving and emission-reduction and realize that mildly conversion provides new approach to substance.
Ionic liquid can effectively improve industrial production efficiency as green solvent.But ionic liquid all has mostly
Toxicity, inhibit the growth of microorganism, reduce the activity of enzyme.Ionic liquid can destroy cell membrane, endanger the structure and work(of cell
Energy integrality, but tolerance bacterium is adaptable to ionic liquid, it can be saturating by solvent rear pump, quick repair membrane, reduction film
Property, increase film tightness and reduce the modes such as cell surface hydrophobicity and do not influenced by ionic liquid.Since tolerance bacterium shows
The tolerance mechanism of very high flexibility, bacterium especially pseudomonas and Colibacter has been widely studied.Pass through sieve
It selects ionic liquid to be resistant to bacterium, determines its ionic liquid resistance mechanism, find relevant target gene, can finally recombinate out both resistance to
There are living things catalysis or other functional engineering bacterias again by ionic liquid.
Invention content
For the few deficiency of the type of the ionic liquid of existing microorganism tolerance, the present invention provides three fourths of chain alkyl
Application of the base phosphonium bromide in screening ionic liquid tolerance bacterium, the three butyl phosphonium bromide of chain alkyl is ([H (CH2)4]3
[(CH2)nH]P+Br-), n=17 or 18, i.e., described three butyl phosphonium bromide of chain alkyl be tributyl octadecyl bromination Phosphonium or
Tributyl heptadecyl phosphonium bromide.
Further, application of the above-mentioned chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium, specifically
Method be：
The methanol solution of the chain alkyl San butyl phosphonium bromides of gradient concentration is prepared, aseptic filter paper piece, which is placed into culture, to be had
The methanol solution of chain alkyl San butyl phosphonium bromides is added drop-wise to aseptic filter paper piece up to complete by the media surface of bacterial strain to be screened
Full wetting, constant temperature incubation are observed around filter paper whether there is or not inhibition zone generations, measurement antibacterial circle diameter, if antibacterial circle diameter not with
Concentration changes, then bacterial strain to be screened is that ionic liquid is resistant to bacterium, and the chain alkyl San butyl phosphonium bromides are tributyl 18
Alkane base phosphonium bromide or tributyl heptadecyl phosphonium bromide.
The constant temperature incubation time is 24~48h.
Chain alkyl San butyl phosphonium bromides have higher thermal stability, can be as the solvent of high-temperature chemical reaction.This
Invention filters out ionic liquid using chain alkyl San butyl phosphonium bromides and is resistant to bacterium, when concentration is less than 25mg/mL, tributyl 18
Alkane base phosphonium bromide to the inhibiting effect unobvious of staphylococcus aureus, show the toxicity extremely low to staphylococcus aureus and
It is good to be resistant to the inhibition zone pole unobvious of bacterium performance and tributyl heptadecyl phosphonium bromide to E.coli and yeast,
Show the toxicity extremely low to E.coli and yeast and good tolerance bacterium performance.Tributyl octadecyl bromination Phosphonium and three
Butyl heptadecyl phosphonium bromide can be used as ionic liquid, filter out tolerance bacterium, to establish tolerance bacterium library, determine its ionic liquid
Body resistance mechanism, finds relevant target gene, and structure tolerance bacterium genomic library is resistant to work for subsequent structure ionic liquid
Journey bacterium lays the foundation.
Description of the drawings
Fig. 1 be high concentration 4PnBr series bromination phosphonium salts to the antibacterial result figure of microorganism, a is staphylococcus aureus, b
For bacillus subtilis, c is Escherichia coli, and d is saccharomycete.
Fig. 2 be middle low concentration 4PnBr series bromination phosphonium salts to the antibacterial result figure of microorganism, a is staphylococcus aureus,
B is bacillus subtilis, and c is Escherichia coli, and d is saccharomycete.
Specific implementation mode
With reference to embodiment and attached drawing, the present invention will be further described.
The preparation method of chain alkyl San butyl phosphonium bromides in the embodiment of the present invention refers to【Chinese patent application
201510224434.4】。
Embodiment 1
1. the preparation of bacteria culture media
Meat soup (MH) culture medium：Beef extract 0.3g, peptone 1g, NaCl 0.5g, distilled water 100mL.1M NaOH are added dropwise
PH is adjusted to 7.2~7.4 by solution.2.0%~2.5% agar is added in solid medium in above-mentioned culture medium.
2, the preparation of fungi culture medium
Improve Martin's fluid nutrient medium：Glucose 2g, yeast powder 0.2g, peptone 0.5g, K2HPO40.1g,
MgSO40.05g, distilled water 100mL.PH value is adjusted to 6.8.Solid medium is added 2.0%~2.5% in above-mentioned culture medium
Agar.
3, measurement (filter paper enzyme) of the chain alkyl San butyl phosphonium bromide to microorganism inhibition zone
(1) after the solid medium sterilizing of 100mL, 50 DEG C or so is cooled to, OD (light is added thereto with liquid-transfering gun
Density) 100 μ L of bacterium or fungal suspension liquid that value is 0.5, are uniformly poured into 5 culture dishes after mixing, standing waits for its solidification.
(2) preparation of gradient concentration chain alkyl quaternary phosphine salting liquid：0.015g Wan Ji quaternary alkylphosphonium salts are weighed, 300 μ l first are added
Alcohol dissolves, and obtains 50mg/mL alkane base quaternary phosphine salt concentrates.It takes 11 sterile centrifugation tubes and numbers, 300 μ are added into No. 1 centrifuge tube
L methanol and 300 μ L alkane base quaternary phosphine salt concentrates (50mg/mL), mixing.300 μ L methanol are added to 2~No. 11 centrifuge tubes, from No. 1 from
Heart pipe is drawn 300 μ L solution and is added in No. 2 centrifuge tubes, then is added in No. 3 centrifuge tubes from 300 μ L solution are drawn in No. 2 centrifuge tubes,
So continuous doubling dilution is to No. 10 centrifuge tubes.No. 11 centrifuge tubes are the feminine gender of the methanol aqueous solution without chain alkyl quaternary alkylphosphonium salt
Control.In each centrifuge tube alkane base quaternary phosphine concentration of salt solution be 25,12.5,6.25,3.13,1.56,0.78,0.39,0.20,
0.10、0.05、0mg/mL。
(3) first tweezers calcination is sterilized, tweezer 7.6mm aseptic filter paper pieces are attached to solid culture primary surface after cooling, (each
Culture dish can be 5, are uniformly distributed, and numbered in culture dish bottom to correspond to quaternary phosphine concentration of salt solution gradient, centre plus a piece of dropwise addition
Methanol is as a contrast), and ensure that it is close to agar surface, the chain alkyl quaternary phosphine salting liquid for drawing 5 μ L various concentrations slowly drips
It (is slowly added dropwise in circle centre position, liquid is made uniformly to spread in the medium) on filter paper.
(4) addition finishes, and after horizontal rest 20min, is inverted tablet, is put into 37 DEG C of constant incubators and is incubated (fungi for 24 hours
Cultivation temperature is 28 DEG C, 48h), it observes around filter paper whether there is or not inhibition zone generations, measurement antibacterial circle diameter.
(5) two groups of parallel laboratory tests are done again, and measured antibacterial circle diameter is averaged.
Fig. 1 is antibacterial result figure of the high concentration 4PnBr series bromination phosphonium salts to microorganism, and Fig. 2 is middle low concentration 4PnBr systems
Row bromination phosphonium salt is to the antibacterial result figure of microorganism, and a is staphylococcus aureus, and b is bacillus subtilis, and c is Escherichia coli,
D is saccharomycete.As shown in Figure 1, tributyl octadecyl bromination Phosphonium (4P18Br) is when concentration is less than 25mg/mL, to golden yellow
Staphylococcus has extremely low toxicity and well tolerance bacterium performance；Tributyl heptadecyl phosphonium bromide (4P17Br) is small in concentration
When 25mg/mL, there is extremely low toxicity and well tolerance bacterium performance to E.coli and yeast.As can be seen from Figure 2, three fourth
Base hexadecane base phosphonium bromide (4P16Br) is when concentration is less than 1.6mg/mL, to staphylococcus aureus, Escherichia coli and yeast
Bacterium has extremely low toxicity and well tolerance bacterium performance.Tributyl hexadecane base phosphonium bromide (4P16Br), tributyl heptadecane
Base phosphonium bromide (4P17Br), tributyl octadecyl bromination Phosphonium (4P18Br) are when concentration is less than 0.8mg/mL, to withered grass gemma
Bacillus shows smaller toxicity and preferable tolerance bacterium performance.Tributyl heptadecyl phosphonium bromide (4P17Br) is less than in concentration
When 1.6mg/mL, there is extremely low toxicity and well tolerance bacterium performance to staphylococcus aureus.Tributyl pentadecyl bromide
Hua Phosphonium (4P15Br) has smaller toxicity and preferable tolerance bacterium performance when concentration is less than 3.1mg/mL, to Escherichia coli.
In conclusion though chain alkyl San butyl phosphonium bromides have higher thermal stability, high temeperature chemistry can be used as anti-
The solvent answered, but only tributyl heptadecyl phosphonium bromide and tributyl octadecyl bromination Phosphonium shows the pole to microorganism
Low toxicity and good tolerance bacterium performance.When concentration is less than 25mg/mL, tributyl octadecyl bromination Phosphonium is to golden yellow grape
The inhibiting effect unobvious of coccus show the toxicity extremely low to staphylococcus aureus and good tolerance bacterium performance, and
Tributyl heptadecyl phosphonium bromide shows to Escherichia coli and yeast the inhibition zone pole unobvious of E.coli and yeast
The extremely low toxicity of bacterium and good tolerance bacterium performance.Tributyl heptadecyl phosphonium bromide and tributyl octadecyl bromination Phosphonium can
As ionic liquid, tolerance bacterium is filtered out, to establish tolerance bacterium library, its ionic liquid resistance mechanism is determined, finds relevant
Target gene, structure tolerance bacterium genomic library are resistant to engineering bacteria for subsequent structure ionic liquid and lay the foundation.
Claims (3)
1. application of the chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium, which is characterized in that the long-chain
Three butyl phosphonium bromide of alkyl is tributyl octadecyl bromination Phosphonium or tributyl heptadecyl phosphonium bromide.
2. application according to claim 1, which is characterized in that specifically method is：Prepare the chain alkyl of gradient concentration
Aseptic filter paper piece is placed into the media surface that culture needs bacterium, by long-chain by the methanol solution of San butyl phosphonium bromides
The methanol solution of alkyl San butyl phosphonium bromides is added drop-wise to aseptic filter paper piece up to complete wetting, constant temperature incubation, observation filter paper week
It is with no inhibition zone to generate, measures antibacterial circle diameter, if antibacterial circle diameter does not change with concentration, bacterial strain to be screened is ionic liquid
Body is resistant to bacterium.
3. application according to claim 2, which is characterized in that the constant temperature incubation time is 24~48h.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710152060.9A CN108624519A (en) | 2017-03-15 | 2017-03-15 | Application of the chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium |
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| CN201710152060.9A CN108624519A (en) | 2017-03-15 | 2017-03-15 | Application of the chain alkyl San butyl phosphonium bromide in screening ionic liquid tolerance bacterium |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752356A (en) * | 2018-11-27 | 2019-05-14 | 河南师范大学 | A kind of method of quick visualization screening ionic-liquid catalyst |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074412A (en) * | 2012-08-22 | 2013-05-01 | 驻马店华中正大有限公司 | New chlortetracycline production bacterial screening method |
| CN104876961A (en) * | 2015-05-05 | 2015-09-02 | 南京理工大学 | Alkyl quaternary phosphonium salt ionic liquid and preparation method thereof |
-
2017
- 2017-03-15 CN CN201710152060.9A patent/CN108624519A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074412A (en) * | 2012-08-22 | 2013-05-01 | 驻马店华中正大有限公司 | New chlortetracycline production bacterial screening method |
| CN104876961A (en) * | 2015-05-05 | 2015-09-02 | 南京理工大学 | Alkyl quaternary phosphonium salt ionic liquid and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 吴佳婷: "单条长链烷基季鏻盐的性能及其微生物毒性研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109752356A (en) * | 2018-11-27 | 2019-05-14 | 河南师范大学 | A kind of method of quick visualization screening ionic-liquid catalyst |
| CN109752356B (en) * | 2018-11-27 | 2021-02-26 | 河南师范大学 | Method for rapidly and visually screening ionic liquid catalyst |
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