CN104312946B - Pusillimonas sp. T2 and application thereof in microbial degradation of nicotine - Google Patents
Pusillimonas sp. T2 and application thereof in microbial degradation of nicotine Download PDFInfo
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Abstract
The invention provides a novel high-efficiency nicotine degradation bacterium strain-Pusillimonas sp. T2 and application thereof in microbial degradation of nicotine. The Pusillimonas sp. T2 can be used for degrading nicotine in the water body and soil by direct addition, and can safely, efficiently and quickly degrade the residual nicotine in the water body, soil and other objects. The microbial inoculant containing the strain has the advantages of simple preparation technique and low cost, is convenient to use, and has favorable application prospects.
Description
(1) technical field
The present invention relates to one plant of new and effective nicotine degradation bacterium --- minimum Zymomonas mobiliss (Pusillimonas sp.) T2,
And its application in microbial degradation nicotine.
(2) background technology
Nicotine (nicotine), is commonly called as nicotine, is a kind of amine substance collectively constituted by pyridine ring and pyrrole ring, point
Minor is C10H14N2, position difference of the nicotine due to N- methyl nafoxidines on pyridine ring generate α-nicotine, β-nicotine,
The stereoisomers such as γ-nicotine, are mainly β-nicotine present in Nicotiana tabacum L., and structural formula is as shown in Figure 8 respectively.Pure Ni Gu
Fourth is at normal temperatures colourless or pale yellow transparent oily liquids, has strong volatility, special acid and hygroscopy, in sunlight
Under be easily oxidized to lead.At 20 DEG C, density is 1.0097g/mL, and boiling point is 274.5 DEG C under 760mm Hg.Ni Gu
Fourth is in alkalescence, when temperature is less than 60 DEG C, can be dissolved each other with arbitrary proportion with water, is highly soluble in alcohol, ether, chloroform etc. organic molten
Agent, when being bonded at skin surface, it is easy to penetrate in vivo and be absorbed by organisms.
Nicotine is a kind of psychotropic substances, with additive as ***e, heroin, mainly with Nicotiana tabacum L. and its useless
The various ways such as gurry, nicotine type nicotinic pesticide, the medicament containing nicotine and the rubber containing nicotine are widely present in environment.Buddhist nun
Ancient fourth and its metabolite have the various diseases of induction, the life for deteriorating associated disease symptom, stronger carcinogenecity, affecting organism
The bio-toxicity such as grow and develop, usually cause various harm.At present, the bio-toxicity of nicotine has caused Chinese scholars
Extensive concern, and obtained multi-angle, multi-level research.
Remove the nicotine in Nicotiana tabacum L. and its garbage than the method for physics, chemistry, microbial method have it is simple to operate,
Easily unique advantage such as transformation, is increasingly paid close attention to by people.Early in the forties in 20th century, microorganism drop has just been related to
The report of solution nicotine.At present researcher both domestic and external has been isolated to many plants of nicotine degradation bacterium, mainly has：Pseudomonass
Category, straw Pseudomonas, Cellulomonas, Ochrobactrum, bacillus etc..These microorganism majorities can be using nicotine
Sole carbon source, nitrogen source and the energy are grown, and they are mainly by pyridine approach, pyrroles's approach, pyridine and pyrrolidine approach
The growth of cross-pathway, demethylation approach by nicotine degradation and for microorganism provides carbon source, nitrogen source and the energy, so as to reach drop
The purpose of solution toxic waste.
The present invention obtains one plant of nicotine height from separation screening in the activated sludge of Qingfeng Agrochemical Co., Ltd's collection
Effect degradation bacteria, by the research that strain identification and degradation characteristic are carried out to it, it is found that the bacterial strain there may be brand-new degraded way
Footpath, this provides foundation for the degradation pathway for more fully understanding nicotine, also has higher degradation efficiency and ability to build
Basis is established by the engineering bacteria of higher nicotine concentration.
Microorganism is that a class species is more, breeds the strong organism of fast, strong adaptability, metabolic capacity.If can screen and separate
Go out the microorganism of effectively degrading nicotine, by the paired human body of nicotine degradation and the material of environment nonhazardouss, this is to safeguarding the mankind
Health and ecological safety have profound significance.
(3) content of the invention
It is an object of the present invention to provide one plant of new and effective minimum zygosaccharomycess nicotine degradation bacterium-minimum Zymomonas mobiliss T2 and
Its application in degraded nicotine.
The technical solution used in the present invention is：
Minimum Zymomonas mobiliss (Pusillimonas sp.) T2, is preserved in China typical culture collection center, address：In
State, Wuhan, Wuhan University, postcode 430072, preservation date is on June 23rd, 2014, and deposit number is CCTCC No:M
2014272。
The Genbank numbers of logging in of the 16S rDNA of the minimum Zymomonas mobiliss T2 be KM054873, the primary biological of the bacterial strain
Be characterized as：Thalline is shaft-like, the raw flagellum in end, and without spore, size is about (0.5～1.0) × (1.5～2.0) μm, and bacterium colony is flat,
Intermediate projections, edge-diffusion, in faint yellow, Gram’s staining is negative, anti-ampicillin, kanamycin, gentamycin, chlorine
Mycin, streptomycin.The optimum growth conditionss of the bacterial strain：PH value is 7.0,30 DEG C of temperature.Bacterial strain Jing 16S rDNA sequences point
Analysis is accredited as Pusillimonas category, therefore is named as minimum Zymomonas mobiliss (Pusillimonassp.) T2.
The invention further relates to applications of the described minimum Zymomonas mobiliss T2 in microbial degradation nicotine.
Specifically, described application is：It is centrifuged what the fermented cultures of minimum Zymomonas mobiliss T2 were obtained containing fermented liquid, precipitation
Suspended with the phosphate buffer that pH value is 7.0 and make bacteria suspension as enzyme source, with nicotine as substrate, in minimal medium
In, degradation reaction, (bacterial strain after reaction completely are carried out under 25～45 DEG C, pH value 5.5～8.5,100～200rpm, dark condition
Can make the quality residual quantity of nicotine in reactant liquor less than 0.1%) after increased activity in 10～16h, obtain nicotine
Culture fluid of the quality residual quantity less than 0.1%, realizes the degraded to nicotine.Preferably, the degraded is at 30 DEG C, pH 7.0,
Carry out under 150rpm, dark condition.
Final concentration of 100～1500mg/L the culture medium (preferred 100mg/L) of the nicotine, enzyme source consumption is with containing bacterium
Cell number is calculated as 2 × 106The culture medium of individual/mL.
Enzyme source of the present invention prepares as follows：
(1) slant culture：Minimum Zymomonas mobiliss T2 is inoculated in into slant medium, 20～40 DEG C are cultivated 3～5 days, obtain bacterium
Body inclined-plane；The final concentration of the slant medium is consisted of：Yeast powder 10.0g/L, peptone 5.0g/L, Sodium Chloride 10.0g/L,
Agar 20.0g/L, solvent is ultra-pure water；
(2) seed culture：It is seeded in minimal medium from the inoculating loop thalline of picking one on step (1) thalline inclined-plane,
25～40 DEG C are cultivated 3～5 days, obtain seed liquor；Every liter of minimal medium is consisted of：NaCl 1g, K2HPO41.5g,
KH2PO40.5g, (NH4)2SO41.5g, MgSO40.1g, 1ml trace element solution, solvent is ultra-pure water, and natural ph, high pressure steams
Vapour sterilizing (121 DEG C, 20min) is obtained afterwards, wherein every liter of trace element solution is consisted of：MnSO4·H2O 0.13g,
ZnCl20.23g, CuSO4·H2O 0.03g, CoCl2·6H2O 0.42g, Na2MoO4·2H2O 0.15g, AlCl3·6H2O
0.05g, solvent is ultra-pure water；
(3) fermentation culture (amplification culture)：The seed liquor that step (2) is obtained is with the inoculum concentration of volumetric concentration 10～20%
In being seeded to LB fluid mediums, 30 DEG C, 150rpm shaken cultivation to OD600 be 0.15, obtain fermentation liquid, by fermentation liquid from
The heart, abandons supernatant, and precipitation pH value is that 7.0 phosphate buffer suspends and makes bacteria suspension and (obtain thin containing minimum Zymomonas mobiliss T2
Born of the same parents' suspension) enzyme source is, the bacteria suspension can add the degraded for being used for nicotine in water body or soil；Every liter of LB liquid training
Support basis set becoming：Yeast powder 10.0g, peptone 5.0g, Sodium Chloride 10.0g, solvent is ultra-pure water, natural ph.
Thalli growth amount of the present invention is detected using ultraviolet spectrophotometer, by measuring thalline (i.e. mycetocyte training
Nutrient solution) absorbance at 600nm to be representing.
Ultra-pure water of the present invention refers to that resistivity reaches the water of 10M Ω * cm (25 DEG C).
The present invention detects the residual quantity of nicotine in minimal medium using reversed phase high-performance liquid chromatography.Reversed phase high efficiency
Liquid chromatographic detection condition：Mobile phase is methanol：H2O=10:90 (volume ratios), analytical column is Grace Alltima C18 colors
Spectrum post (4.6 × 250mm, 5 μm), flow velocity is 0.8ml/min, and sample size is 20 μ l, and column temperature is 30 DEG C.
Compared with prior art, the beneficial effects are mainly as follows：
Minimum Zymomonas mobiliss T2 of the present invention can be applied to nicotine in water body and soil by way of directly adding
It is prepared by degraded, the nicotine remained on the objects such as water body, soil that can safely, efficiently, fastly degrade, the microbial inoculum containing the bacterial strain
Process is simple, it is with low cost, it is easy to use, with good application prospect.
(4) illustrate
Fig. 1 is the electron microscope of minimum Zymomonas mobiliss T2 of the invention；
Fig. 2 is the canonical plotting of nicotine standard concentration and peak area；
Fig. 3 be the present invention minimum Zymomonas mobiliss T2 under the conditions of pure culture to concentration for 100mg/L nicotine degraded
Curve chart；
Fig. 4 is growth curves of the minimum Zymomonas mobiliss T2 of the present invention under the conditions of nicotine concentration is 100mg/L pure cultures
Figure.
Fig. 5 is impact figure of the temperature to degradation rate.
Fig. 6 is impact figure of the pH value to degradation rate.
Fig. 7 is impact figure of the nicotine initial concentration to degradation rate.
Fig. 8 is nicotine structure chart.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：The screening of bacterial strain and identification
1) culture medium
The preparation of minimal medium：NaCl 1.0g, K2HPO41.5g, KH2PO40.5g, (NH4)2SO41.5g,
MgSO40.1g, 1ml trace element solution, ultra-pure water complements to 1000ml, stirs after mixing, natural ph, high steam
Sterilizing (121 DEG C, 20min) is obtained afterwards, wherein containing MnSO in every liter of trace element solution4·H2O 0.13g, ZnCl20.23g,
CuSO4·H2O0.03g, CoCl2·6H2O 0.42g, Na2MoO4·2H2O 0.15g, AlCl3·6H2O 0.05g, use ultra-pure water
It is settled to 1000ml.
Enrichment culture liquid：Nicotine is added in minimal medium so that the final concentration of 100mg/L of nicotine.
The preparation of LB fluid mediums：Yeast powder 10g, peptone 5.0g, Sodium Chloride 10.0g, ultra-pure water is settled to
1000ml, stirs after mixing, natural ph, and high pressure steam sterilization (121 DEG C, 20min) is obtained afterwards.
The preparation of LB solid mediums：Yeast powder 10.0g, peptone 5.0g, Sodium Chloride 10.0g, agar 20.0g is ultrapure
Water is settled to 1000ml, stirs after mixing, natural ph, and high pressure steam sterilization (121 DEG C, 20min) is obtained afterwards.
2) strains separation purification
Mud sample picks up from Qingfeng Agrochemical Co., Ltd, takes 5ml mud samples and is placed in 250ml conical flasks, adds
100ml enrichment culture liquid, dark shaken cultivation (30 DEG C, 150rpm) 1 week, takes the turbid liquid in 5ml upper stratas in fresh enrichment culture liquid
In, continue dark shaken cultivation (30 DEG C, 150rpm) 1 week, repeat aforesaid operations process 3 times, every time the inoculum of culture takes
Culture fluid obtained by cultivated in last time.
Taking the culture fluid 5ml obtained by last time culture carries out gradient dilution ((10-3、10-4、10-5、10-6), take each dilute
The μ l of culture fluid 100 after releasing are coated on the inorganic salt solid medium flat board containing 100mg/L nicotine, are placed in constant incubator
Culture in (30 DEG C, 150rpm), after bacterium colony is grown on flat board, each bacterium colony of picking is trained in the LB solids containing 100mg/L nicotine
Purification repeatedly on foster base flat board, until bacterium colony is single, each bacterium colony after purification is respectively connected to shake in LB fluid medium test tubes
Swing culture (30 DEG C, 150rpm) overnight, the centrifugation of cultured bacterium solution is followed by cultivating 3d into enrichment culture liquid, by anti-phase height
Effect liquid phase chromatogram method detects the residual quantity of nicotine in each enrichment culture liquid, and finally screening obtains one plant can effectively degrading nicotine
Bacterial strain, be designated as bacterial strain T2.
3) identification of strains
The bacterial strain T2 of above-mentioned acquisition is carried out into morphological characteristic and molecular biology identification, electromicroscopic photograph such as Fig. 1 of the bacterial strain
It is shown.The main biological property of the bacterial strain is：Thalline is shaft-like, the raw flagellum in end, without spore, size about (0.5～1.0) ×
(1.5～2.0) μm, bacterium colony is flat, intermediate projections, edge-diffusion, and in faint yellow, Gram’s staining is negative, anti-ammonia benzyl penicillium sp
Element, kanamycin, gentamycin, chloromycetin, streptomycin.The optimum growth conditionss of the bacterial strain：PH value is 7.0,30 DEG C of temperature.
Bacterial strain Jing 16S rDNA sequence analysis (the Genbank numbers of logging in are KM054873) are accredited as Pusillimonas sp. category, because
This is named as minimum Zymomonas mobiliss (Pusillimonas sp.) T2, is preserved in China typical culture collection center, address：In
State, Wuhan, Wuhan University, postcode 430072, preservation date is on June 23rd, 2014, and deposit number is CCTCC No:M
2014272。
Embodiment 2：The preparation of mycetocyte suspension
(1) slant culture：Minimum Zymomonas mobiliss T2 is inoculated in into slant medium, 30 DEG C are cultivated 3～5 days, obtain thalline oblique
Face；The final concentration of every liter of slant medium is consisted of：Yeast powder 10.0g, peptone 5.0g, Sodium Chloride 10.0g, agar
20.0g, ultra-pure water 1000ml, natural pH；
(2) seed culture：It is seeded in minimal medium from the inoculating loop thalline of picking one on step (1) thalline inclined-plane,
30 DEG C are cultivated 3～5 days, obtain seed liquor；The minimal medium final concentration is constituted with embodiment 1；
(3) amplification culture：The seed liquor that step (2) is obtained is seeded to the training of LB liquid with the inoculum concentration of volumetric concentration 20%
In foster base (100ml), 30 DEG C, 150rpm shaken cultivation to OD600 be 0.1～0.15, obtain bacterium solution, bacterium solution is centrifuged
(6000rpm, 5min), abandons supernatant, and precipitation pH value is that 7.0 phosphate buffer suspends, and obtains and contains minimum Zymomonas mobiliss T2 cells
Minimum Zymomonas mobiliss T2 concentration in suspension 100mL, wherein cell suspension is 4 × 107Individual/ml；The LB fluid mediums are dense eventually
Degree composition is with embodiment 1；PH value is that the formula of the phosphate buffer of 7.0 0.2mol/L is：Take the biphosphate of 0.2mol/L
The disodium hydrogen phosphate 61ml of sodium 39ml and 0.2mol/L, with ultra-pure water 1000ml is settled to, high pressure steam sterilization (121 DEG C,
Obtain final product after 20min).
Embodiment 3：Degradation experiment to nicotine
1) in minimal medium cell concentration and nicotine content detection：
Thalli growth amount is detected using ultraviolet spectrophotometer, by suction of the thalline at 600nm in measurement culture fluid
Shading value is representing.
This experiment detects the residual quantity of nicotine in minimal medium using reversed phase high-performance liquid chromatography, ancient according to Buddhist nun
Fourth standard curve calculates the content of nicotine in culture medium to be measured.Reversed-phase high-performance liquid chromatography testing conditions：Mobile phase is first
Alcohol：H2O=10:90 (volume ratios), analytical column is GraceAlltima C18 chromatographic columns (4.6 × 250mm, 5 μm), and flow velocity is
0.8ml/min, sample size is 20 μ l, and column temperature is 30 DEG C.
By the aseptic water dissolution of nicotine standard substance, in reversed-phase liquid chromatography testing standard curve, nicotine standard curve
As shown in Fig. 2 calibration curve equation is y=20342x+1.6E+5, R2=0.9989, y are peak area, and x is nicotine concentration,
mg/L)。
2) nicotine degradation experiment：
A, 4 250ml conical flasks are taken, be separately added into 100ml minimal mediums, high pressure steam sterilization (121 DEG C,
Nicotine is added after 20min), makes nicotine final concentration be 100mg/L, the method for each Example 2 obtain containing minimum unit cell
Bacterium T2 cell suspension 5ml, during this minimal medium is inoculated in respectively, make minimum Zymomonas mobiliss T2 final concentration of 2 × 106Individual/ml
Minimal medium, respectively at 25,30,35,40 DEG C of culture shaking tables (pH value is 7.0,150rpm, dark), at regular intervals
Be measured by sampling residual nicotine concentration, experiment find, in certain temperature range, in culture medium the residual concentration of nicotine with
The prolongation of time and be gradually lowered.It is that in the range of 10h～15h, the degradation rate of nicotine is maximum, and 30 DEG C or so for most in the time
Good degradation temperature, as a result as shown in Figure 5.
B, 6 250ml conical flasks are taken, be separately added into 100ml minimal mediums, high pressure steam sterilization (121 DEG C,
Nicotine is added after 20min), makes nicotine final concentration be 100mg/L, the method for each Example 2 obtain containing minimum unit cell
Bacterium T2 cell suspension 5ml, during this minimal medium is inoculated in respectively, make minimum Zymomonas mobiliss T2 final concentration of 2 × 106Individual/ml
Minimal medium, respectively adjust medium pH 5.5,6.5,7.0,7.5,8.0,8.5, (30 DEG C, 150rpm is black for shaking table culture
Secretly), residual nicotine concentration is measured by sampling at regular intervals, and experiment shows that pH value is too high or too low to the degraded of bacterial strain
Efficiency has an impact, and optimum pH is 7.0, as a result as shown in Figure 6.
C, 3 250ml conical flasks are taken, add 100ml minimal mediums, high pressure steam sterilization (121 DEG C, 20min)
After add nicotine, make nicotine final concentration be 100mg/L, it is thin containing minimum Zymomonas mobiliss T2 that the method for each Example 2 is obtained
Born of the same parents suspension 5ml, during this minimal medium is inoculated in respectively, makes minimum Zymomonas mobiliss T2 final concentration of 2 × 106Individual/ml inorganic salts
Culture medium, corresponding to arrange 3 experiments without the strain as blank, (30 DEG C, pH value is then together to be placed in shaking table
It is 7.0,150rpm, dark) in dark shaken cultivation.Sample at regular intervals in the training period, according to above-mentioned detection method come
The increment and the residual quantity of nicotine of thalline in detection minimal medium, as a result as shown in Fig. 3, Fig. 4.
Bacterial strain of the present invention is to the degradation curve of the nicotine of 100mg/L concentration as shown in figure 3, the growth curve of thalline is as schemed
Shown in 4, Fig. 4 can be seen that OD600Increase to 0.18 from 0.07, illustrate that thalli growth is good, observe Fig. 3, it is found that culture
After 14h, the nicotine degradation bacterium of the present invention is close to 100% to the degradation rate of the nicotine of 100mg/L, nicotine in reactant liquor
Quality residual quantity be 0, and it is all plus the percent hydrolysiss of the blank after 22h of bacterium are respectively less than 5%.
D, 5 250ml conical flasks are taken, be separately added into 100ml minimal mediums, high pressure steam sterilization (121 DEG C,
Nicotine is added after 20min), make nicotine final concentration be respectively 100mg/L, 200mg/L, 500mg/L, 1000mg/L and
1500mg/L, the method for each Example 2 obtain containing minimum Zymomonas mobiliss T2 cell suspension 5ml, the training of this inorganic salt is inoculated in respectively
In foster base, minimum Zymomonas mobiliss T2 final concentration of 2 × 10 is made6Individual/ml minimal mediums, adjust respectively medium pH 7.0, shake
Bed culture (30 DEG C, 150rpm is dark), is measured by sampling at regular intervals residual nicotine concentration, as a result as shown in Figure 7.
Test result indicate that the strain centering, the nicotine of high concentration (see Fig. 7) have extraordinary degradation capability, but Buddhist nun
When ancient fourth concentration is higher than 1500mg/L, the degradation capability of nicotine is remarkably decreased, and degradation cycle is longer, and this strain is new
Type nicotine degradation bacterium, therefore, the bacterium has very big facilitation to the degradation pathway for studying nicotine with degrading genes,
Degraded to nicotine in environment is especially repaired with larger positive effect to the concentration of nicotine.
The nicotine degradation of embodiment 4
250ml conical flasks are taken, adds 100ml minimal mediums, high pressure steam sterilization (121 DEG C, 20min) to add Buddhist nun afterwards
Gu Ding, makes nicotine final concentration be respectively 100mg/L, the method for Example 2 obtain containing minimum Zymomonas mobiliss T2 cell suspension
5ml, in being inoculated in this minimal medium, makes minimum Zymomonas mobiliss T2 final concentration of 2 × 106Individual/ml minimal mediums, are adjusted
Medium pH 7.0, shaking table culture (30 DEG C, 150rpm is dark) 15h, taking culture fluid measure residual nicotine concentration is
0.09mg/L, as a result shows that nicotine quality residual quantity is 0.09%.
Claims (6)
1. minimum Zymomonas mobiliss (Pusillimonas sp.) T2, is preserved in China typical culture collection center, address：China,
Wuhan, Wuhan University, postcode 430072, preservation date is on June 23rd, 2014, and deposit number is CCTCC No:M
2014272。
2. applications of the minimum Zymomonas mobiliss T2 in microbial degradation nicotine as claimed in claim 1.
3. application as claimed in claim 2, it is characterised in that described application is：The fermented cultures of minimum Zymomonas mobiliss T2 are obtained
What is obtained is centrifuged containing fermented liquid, and precipitation pH value is that 7.0 phosphate buffer suspension makes bacteria suspension as enzyme source, with nicotine
For substrate, in minimal medium, dropped under 25～45 DEG C, pH value 5.5～8.5,100～200rpm, dark condition
Solution reaction, after reaction completely, realizes the degraded to nicotine.
4. application as claimed in claim 3, it is characterised in that the final concentration of 100～1500mg/L culture medium of the nicotine,
Enzyme source consumption is calculated as 1 × 10 with mycetocyte number6～5 × 106Individual/mL culture medium.
5. application as claimed in claim 3, it is characterised in that contain NaCl 1g in every liter of minimal medium,
K2HPO41.5g, KH2PO40.5g, (NH4)2SO41.5g, MgSO40.1g, 1ml trace element solution, solvent is ultra-pure water, natural
PH value；Contain MnSO in every liter of trace element solution4·H2O 0.13g, ZnCl20.23g, CuSO4·H2O 0.03g, CoCl2·
6H2O 0.42g, Na2MoO4·2H2O 0.15g, AlCl3·6H2O 0.05g, solvent is ultra-pure water.
6. application as claimed in claim 3, it is characterised in that the enzyme source prepares as follows：
(1) slant culture：Minimum Zymomonas mobiliss T2 is inoculated in into slant medium, 20～40 DEG C are cultivated 3～5 days, obtain thalline oblique
Face；The final concentration of the slant medium is consisted of：Yeast powder 10.0g/L, peptone 5.0g/L, Sodium Chloride 10.0g/L, agar
20.0g/L, solvent is ultra-pure water, pH value nature；
(2) seed culture：It is seeded in minimal medium from the inoculating loop thalline of picking one on step (1) thalline inclined-plane, 25～
40 DEG C are cultivated 3～5 days, obtain seed liquor；Every liter of minimal medium is consisted of：NaCl 1g, K2HPO41.5g,
KH2PO40.5g, (NH4)2SO41.5g, MgSO40.1g, 1ml trace element solution, solvent is ultra-pure water, natural ph；Per liter micro-
Secondary element solution composition is：MnSO4·H2O 0.13g, ZnCl20.23g, CuSO4·H2O 0.03g, CoCl2·6H2O
0.42g, Na2MoO4·2H2O 0.15g, AlCl3·6H2O 0.05g, solvent is ultra-pure water；
(3) fermentation culture：The seed liquor that step (2) is obtained is seeded to the training of LB liquid with the inoculum concentration of volumetric concentration 10～20%
In foster base, 30 DEG C, 150rpm shaken cultivation to OD600 be 0.1～0.15, obtain fermentation liquid, fermentation liquid is centrifuged, abandon supernatant
Liquid, precipitation pH value is that bacteria suspension, as enzyme source are made in 7.0 phosphate buffer suspension；Every liter of LB liquid cultures are basis set
Become：Yeast powder 10.0g, peptone 5.0g, Sodium Chloride 10.0g, solvent is ultra-pure water, natural ph.
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| CN201410505852.6A CN104312946B (en) | 2014-09-28 | 2014-09-28 | Pusillimonas sp. T2 and application thereof in microbial degradation of nicotine |
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| CN201410505852.6A CN104312946B (en) | 2014-09-28 | 2014-09-28 | Pusillimonas sp. T2 and application thereof in microbial degradation of nicotine |
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| CN104312946B true CN104312946B (en) | 2017-04-12 |
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Non-Patent Citations (2)
| Title |
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| A sirA-like gene,sirA2,is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading Pseudomonas sp. HZN6 strain;Qiu J,et al;《Appl Microbiol Biotechnol》;20111231 * |
| Pusillimonas sp.5HP degrading 5-hydroxypicolinic acid;Karvelis L,et al;《Biodegradation》;20140228;第25卷(第1期);11-19 * |
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