CN102796681B - Pseudomonas sp.HZN6 and application thereof to nicotine degradation - Google Patents
Pseudomonas sp.HZN6 and application thereof to nicotine degradation Download PDFInfo
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Abstract
The invention discloses Pseudomonas sp.HZN6 and application thereof to nicotine degradation. In application, with nicotine as a substrate, an inorganic salt nutrient solution as a reaction medium and Pseudomonas sp.HZN6 as an enzyme, the reaction is carried out for 8-15h under the conditions that temperature is 25-45 DEG C and a Ph value is 5.5-9.0 to ensure that the content of nicotine in the reaction solution is less than 0.1 percent by mass, thus the purpose of degrading nicotine is reached. The Pseudomonas sp.HZN6 can be directly added to a water body and soil to degrade nicotine in the water body and soil, and can be used for safely, efficiently and rapidly degrading residual nicotine on objects such as water and soil. A bactericide containing the Pseudomonas sp.HZN6 is prepared by a simple process with low cost, is convenient to use, and has a good application prospect.
Description
(1) technical field
The present invention relates to the new and effective nicotine degradation bacterium of a strain, the particularly new and effective Pseudomonas nicotine degradation of strain bacterium, i.e. Rhodopseudomonas HZN6 and application thereof.
(2) background technology
Nicotine (nicotine), is commonly called as nicotine, is peculiar in multiple tobacco, most important alkaloid, accounts for the 1%-2% of tobacco dry weight, is one of important factor affecting quality of tobacco, is also one of main objectionable constituent of tobacco leaf and cigarette simultaneously.The molecular formula of Nicotine is C
10h
14n
2, structural formula is as shown in formula I.
Because Nicotine is a kind of psychotropic substances, the mankind are keeping the custom of smoking tobaccos for a long time.But Nicotine is the important as precursors thing of Analyses of major carcinogens in mainstream composition tobacco-specific nitrosamine (TSNA) in tobacco leaf and flue gas, long-term smoking not only can cause the dependency of human body to Nicotine, and excessive suction can suppress human central nervous, paralysis heart, severe patient has fatal danger; Meanwhile, Nicotine is also a kind of environment toxic substance, and pure Nicotine is colourless, bitter, the oily liquids that has intense stimulus at normal temperatures, is very easily oxidized to lead in air.In flue gas environment, just contain a large amount of Nicotines, in China's part tobacco leaf, nicotine content is too high at present, and especially the content in upper tobacco leaf is generally too high, and this brings very large challenge to leaf tobacco production.Meanwhile, tobacco can produce the higher Nicotine waste material of concentration in the course of processing, and this waste material is considered to " poisonous Hazardous wastes ", and environment is caused to very large harm.Therefore in continuous control and reduction cigarette, the content of Nicotine is the inexorable trend of international tobacco development, is also one of important channel reducing harmfulness of smoking; Reduce in environment nicotine content simultaneously and reduce tobacco waste to the pollution of environment for safeguarding that human health has profound significance.
Chemical structure and the chemical property of Nicotine are more stable, if remove by the method for physics, chemistry, cost is higher, and harmful byproduct is more, also can have influence on the original fine quality of tobacco; And microorganism has unique effect to the metabolism of Nicotine in flue-cured tobacco, utilize microbial metabolism method to remove Nicotine, not only cost is low, and harmful byproduct is few, and does not affect the original good characteristic of tobacco, therefore has broad application prospects.
Microorganism is that a class kind is many, breeding is fast, strong adaptability, organism that metabolic capacity is strong.If the microorganism that energy screening and separating goes out energy effectively degrading nicotine, becomes the material to human body and environment toxicological harmless such as carboxylic acid, amino acid by nicotine degradation, this has profound significance to safeguarding human health.
(3) summary of the invention
The object of the invention is to provide a strain new and effective Pseudomonas nicotine degradation bacterium HZN6 and application thereof.
The technical solution used in the present invention is:
Rhodopseudomonas HZN6(Pseudomonas sp.HZN6), be preserved in Chinese Typical Representative culture collection center, address: Luojia Mountain, Wuhan, Hubei Province Wuhan University, 430072, preservation date is on August 9th, 2010, preserving number is CCTCC No:M2010196.
The Genbank number of logging in of the 16S rDNA of described Rhodopseudomonas HZN6 is HQ108345.
Rhodopseudomonas HZN6(Pseudomonas sp.HZN6) screening and the qualification of bacterial strain:
1) substratum
Minimal medium final concentration consists of: in every liter of nutrient solution, contain NaCl 1g, K
2hPO
41.5g, KH
2pO
40.5g, (NH4)
2sO
41.5g, MgSO
40.1g, 1ml trace element solution, solvent is water, natural pH value makes after high pressure steam sterilization (121 DEG C, 20min), wherein in every liter of trace element solution, contains MnSO
4h
2o 0.13g, ZnCl
20.23g, CuSO
4h
2o0.03g, CoCl
26H
2o 0.42g, Na
2moO
42H
2o 0.15g, AlCl
36H
2o 0.05g, solvent is water.
Enrichment culture liquid: add Nicotine in inorganic salt nutrient solution, the final concentration that makes Nicotine is 200mg/L.
LB liquid nutrient medium: in every liter of nutrient solution, contain yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, solvent is water, natural pH value makes after high pressure steam sterilization (121 DEG C, 20min).
LB solid medium: in every liter of cultivation, contain yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, agar 15.0g, solvent is water, natural pH value makes after high pressure steam sterilization (121 DEG C, 20min).
2) strains separation purifying
Mud sample picks up from Hangzhou insecticide factory, get 5ml mud sample and be placed in 250ml Erlenmeyer flask, add 100ml enrichment culture liquid, dark shaking culture (30 DEG C, 150rpm) 1 week, gets the turbid liquid in 5ml upper strata in fresh enrichment culture liquid 100ml, continue (30 DEG C of dark shaking culture, 150rpm) 1 week, repeat aforesaid operations process 3 times, each inoculum of cultivating is all taken from the nutrient solution of cultivating gained last time.
The nutrient solution 5ml that gets last cultivation gained carries out gradient dilution (10
-4, 10
-5, 10
-6), the nutrient solution 150 μ l that get after each dilution coat on the LB solid medium flat board containing 500mg/L Nicotine, being placed in constant incubator (30 DEG C) cultivates, until growing on flat board after bacterium colony, the each bacterium colony of picking is in containing purifying repeatedly on the LB solid medium flat board of 500mg/L Nicotine, until bacterium colony is single, each bacterium colony after purifying is connected to respectively in LB liquid nutrient medium test tube to (30 DEG C of shaking culture, 150rpm) spend the night, by the centrifugal (8000rpm of cultured bacterium liquid, 5min), be connected to 25 ~ 45 DEG C of cultivation 3d in enrichment culture liquid, detect the residual quantity of Nicotine in each enrichment culture liquid by high performance liquid chromatography (HPLC), finally screening obtains the bacterial strain of a strain energy effectively degrading nicotine, called after HZN6.
3) identification of strains
The bacterial strain of above-mentioned acquisition is carried out to morphological specificity and molecular biology identification, and the electromicroscopic photograph of this bacterial strain as shown in Figure 1.The main biological property of this bacterial strain is: gramstaining reaction negative, and thalline is shaft-like, the raw flagellum of end, without gemma, size is about (0.25 ~ 0.5) × (0.8 ~ 1.0) μ m, and bacterium colony is smooth, intermediate projections, edge-diffusion, is faint yellow, the catalase positive, oxidase positive, the methyl red test positive, can utilize beta-cyclodextrin, starch, glucose, polysorbate40, acetate, voges-Proskauer test feminine gender, V.P. reaction negative.The optimum growth conditions of this bacterial strain is pH value 7.0,30 DEG C of temperature.This bacterial strain is accredited as Pseudomonas through 16S rDNA sequential analysis and belongs to, and is therefore Rhodopseudomonas HZN6(Pseudomonas sp.HZN6).
The invention provides the application of a kind of described Rhodopseudomonas HZN6 in degraded Nicotine.
Further, the application of described Rhodopseudomonas HZN6 in degraded Nicotine is taking Nicotine as substrate, taking inorganic salt nutrient solution as reaction medium, taking Rhodopseudomonas HZN6 as degradation bacteria (being enzyme), under the condition that 25 ~ 45 DEG C, pH value are 5.5 ~ 9.0, react 8 ~ 15h make reaction solution in the mass content of Nicotine be less than 0.1%, reach the object of degraded Nicotine.
Further, described is applied as: inorganic salt nutrient solution is mixed with Nicotine, making Nicotine final concentration is the preferred 200mg/L of 100 ~ 3000mg/L(), add again containing the cell suspension of Rhodopseudomonas HZN6 and form reaction system, the dark shaking culture of condition that is 5.5 ~ 9.0 at 25 ~ 45 DEG C, pH value until in reaction solution the quality residual quantity of Nicotine be less than 0.1%(cultivation 8 ~ 15h conventionally); It is 1 × 10 that the described cell suspension add-on containing Rhodopseudomonas HZN6 makes Rhodopseudomonas HZN6 final concentration in reaction system
7~ 5 × 10
9individual/ml.
Further, described Rhodopseudomonas HZN6 in degraded in the application in Nicotine the preparation method containing the cell suspension of Rhodopseudomonas HZN6 be:
(1) slant culture: Rhodopseudomonas HZN6 is inoculated in to slant medium, cultivates 5 ~ 7 days for 25 ~ 45 DEG C, obtain thalline inclined-plane; The final concentration of described slant medium consists of: yeast powder 10g/L, and peptone 5.0g/L, sodium-chlor 10.0g/L, agarose 2.0g/L, solvent is water;
(2) seed culture: picking one transfering loop thalline is seeded in minimal medium from step (1) thalline inclined-plane, cultivates 5 ~ 7 days for 25 ~ 45 DEG C, obtains seed liquor; Described minimal medium final concentration consists of: in every liter of nutrient solution, contain NaCl 1g, K
2hPO
41.5g, KH
2pO
40.5g, (NH4)
2sO
41.5g, MgSO
40.1g, 1ml trace element solution, solvent is water, natural pH value makes after high pressure steam sterilization (121 DEG C, 20min), wherein in every liter of trace element solution, contains MnSO
4h
2o 0.13g, ZnCl
20.23g, CuSO
4h
2o 0.03g, CoCl
26H
2o0.42g, Na
2moO
42H
2o 0.15g, AlCl
36H
2o 0.05g, solvent is water;
(3) enlarged culturing: the seed liquor that step (2) is obtained is seeded in LB liquid nutrient medium with the inoculum size of volumetric concentration 10 ~ 20%, 30 DEG C, 150rpm vibration are supported to logarithmic phase, obtain bacterium liquid, by centrifugal bacterium liquid, abandon supernatant, the phosphoric acid buffer that precipitation is 7.0 by pH value suspends, and obtains the cell suspension containing Rhodopseudomonas HZN6; Described LB liquid nutrient medium final concentration consists of: in every liter of cultivation, contain yeast powder 10g, and peptone 5.0g, sodium-chlor 10.0g, solvent is water, natural pH value.
Further, described Rhodopseudomonas HZN6 being applied as in degraded Nicotine: inorganic salt nutrient solution is mixed with Nicotine, making Nicotine final concentration is 200mg/L, add again the described cell suspension containing Rhodopseudomonas HZN6 to form reaction system, at 30 DEG C, dark shaking culture 8h under pH7.0,150rpm condition, makes the quality residual quantity of Nicotine in reaction solution be less than 0.1%, reaches the object of degraded Nicotine; It is 1 × 10 that the add-on of the described cell suspension containing Rhodopseudomonas HZN6 makes Rhodopseudomonas HZN6 final concentration in reaction system
7~ 5 × 10
9individual/ml, preferably 1 × 10
7~ 5 × 10
7individual/ml.
Thalli growth amount of the present invention adopts ultraviolet spectrophotometer to detect, and represents at the absorbance at 600nm place by measuring thalline (being mycetocyte nutrient solution).
The present invention adopts reversed-phased high performace liquid chromatographic to detect the residual quantity of Nicotine in inorganic salt nutrient solution.RPLC testing conditions: moving phase is methyl alcohol: 1mM H
2sO
4=10:90(volume ratio), analytical column be Grace Alltima C18 chromatographic column (4.6 × 250mm, m), flow velocity is 0.6ml/min to 5 μ, sample size is 20 μ l, column temperature is 30 DEG C.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
Rhodopseudomonas HZN6 of the present invention can be applied to by the mode directly adding the degraded of Nicotine in water body and soil, the residual Nicotine on the object such as water body, soil of safely, efficiently, fastly degrading, the microbial inoculum preparation technology who contains this bacterial strain is simple, with low cost, easy to use, there is good application prospect.
(4) brief description of the drawings
Fig. 1 is the Electronic Speculum figure of Rhodopseudomonas HZN6 of the present invention;
Fig. 2 is the canonical plotting of Nicotine;
Fig. 3 is Rhodopseudomonas HZN6 of the present invention degradation curve figure to Nicotine under differing temps: square (■) is 30 DEG C, and circular (●) is 25 DEG C, and equilateral triangle (▲) is 37 DEG C, del
it is 45 DEG C;
Fig. 4 is that Rhodopseudomonas HZN6 of the present invention affects figure to nicotine degradation under different pH;
Fig. 5 is the degradation curve figure of the Rhodopseudomonas HZN6 of the present invention Nicotine that is 200mg/L to final concentration under pure culture condition;
Fig. 6 is that Rhodopseudomonas HZN6 of the present invention is the growth curve chart under 200mg/L pure culture condition at Nicotine final concentration;
Fig. 7 is the degradation curve figure of Rhodopseudomonas HZN6 of the present invention to different concns Nicotine.
(5) embodiment
Below in conjunction with 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 qualification
1) substratum
The preparation of minimal medium: NaCl 1g, K
2hPO
41.5g, KH
2pO
40.5g, (NH
4)
2sO
41.5g, MgSO
40.1g, 1ml trace element solution, distilled water complements to 1000ml, after mixing, stirs, and natural pH value makes after high pressure steam sterilization (121 DEG C, 20min), wherein in every liter of trace element solution, contains MnSO
4h
2o 0.13g, ZnCl
20.23g, CuSO
4h
2o0.03g, CoCl
26H
2o 0.42g, Na
2moO
42H
2o 0.15g, AlCl
36H
2o 0.05g, complements to 1000ml with distilled water.
Enrichment culture liquid: add Nicotine in inorganic salt nutrient solution, the final concentration that makes Nicotine is 200mg/L.
The preparation of LB liquid nutrient medium: yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, distilled water complements to 1000ml, after mixing, stirs, and natural pH value makes after high pressure steam sterilization (121 DEG C, 20min).
The preparation of LB solid medium: yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, agar 15.0g, distilled water complements to 1000ml, after mixing, stirs, and natural pH value makes after high pressure steam sterilization (121 DEG C, 20min).
2) strains separation purifying
Mud sample picks up from Hangzhou insecticide factory, get 5ml mud sample and be placed in 250ml Erlenmeyer flask, add 100ml enrichment culture liquid, dark shaking culture (30 DEG C, 150rpm) 1 week, gets the turbid liquid in 5ml upper strata in fresh enrichment culture liquid, continue (30 DEG C of dark shaking culture, 150rpm) 1 week, repeat aforesaid operations process 3 times, each inoculum of cultivating is all taken from the nutrient solution of cultivating gained last time.
The nutrient solution 5ml that gets last cultivation gained carries out gradient dilution ((10
-4, 10
-5, 10
-6), the nutrient solution 150 μ l that get after each dilution coat on the LB solid medium flat board containing 500mg/L Nicotine, being placed in constant incubator (30 DEG C) cultivates, until growing on flat board after bacterium colony, the each bacterium colony of picking is in containing purifying repeatedly on the LB solid medium flat board of 500mg/L Nicotine, until bacterium colony is single, each bacterium colony after purifying is connected to respectively in LB liquid nutrient medium test tube to (30 DEG C of shaking culture, 150rpm) spend the night, cultivate 3d by being connected in enrichment culture liquid after centrifugal cultured bacterium liquid, detect the residual quantity of Nicotine in each enrichment culture liquid by reversed-phased high performace liquid chromatographic, finally screening obtains the bacterial strain of a strain energy effectively degrading nicotine, called after HZN6.
3) identification of strains
The bacterial strain of above-mentioned acquisition is carried out to morphological specificity and molecular biology identification, and the electromicroscopic photograph of this bacterial strain as shown in Figure 1.The main biological property of this bacterial strain is: thalline is shaft-like, and the raw flagellum of end, without gemma, size is about (0.25 ~ 0.5) × (0.8 ~ 1.0) μ m, and bacterium colony is smooth, intermediate projections, edge-diffusion, is faint yellow, gramstaining reaction negative, the catalase positive, oxidase positive, the methyl red test positive, can utilize beta-cyclodextrin, starch, glucose, polysorbate40, acetate, voges-Proskauer test feminine gender, V.P. reaction negative.The optimum growth conditions of this bacterial strain is pH value 7.0,30 DEG C of temperature.This bacterial strain is accredited as Pseudomonas through 16S rDNA sequential analysis and belongs to, and is therefore Rhodopseudomonas HZN6(Pseudomonas sp.HZN6).
Embodiment 2: the preparation of mycetocyte suspension
(1) slant culture: Rhodopseudomonas HZN6 is inoculated in to slant medium, cultivates 6 days for 30 DEG C, obtain thalline inclined-plane; The final concentration of described slant medium consists of: yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, agarose 2.0g, water 1000ml;
(2) seed culture: picking one transfering loop thalline is seeded in minimal medium from step (1) thalline inclined-plane, cultivates 6 days for 30 DEG C, obtains seed liquor; Described minimal medium final concentration forms with embodiment 1;
(3) enlarged culturing: the seed liquor that step (2) is obtained is seeded in LB liquid nutrient medium (100mL) with the inoculum size of volume dense 15%, 30 DEG C, 150rpm shaking culture are to logarithmic phase, obtain bacterium liquid, by centrifugal bacterium liquid (8000rpm, 5min), abandon supernatant, the phosphoric acid buffer that precipitation is 7.0 by pH value suspends, obtain containing Rhodopseudomonas HZN6 cell suspension 100mL, wherein the Rhodopseudomonas HZN6 concentration in cell suspension is 1 × 10
9individual/ml; Described LB liquid nutrient medium final concentration forms with embodiment 1; PH is that the formula of the phosphoric acid buffer of 7.0 0.2mol/L is: get the SODIUM PHOSPHATE, MONOBASIC 39ml of 0.2mol/L and the Sodium phosphate dibasic 61ml of 0.2mol/L, be settled to 1000ml with ultrapure water, after high pressure steam sterilization (121 DEG C, 20min) and get final product.
Embodiment 3: nicotine degradation experiment
1) detection of cell concentration and nicotine content in inorganic salt nutrient solution:
Thalli growth amount adopts ultraviolet spectrophotometer to detect, and represents at the absorbance at 600nm place by measuring thalline in nutrient solution.
This experiment adopts reversed-phased high performace liquid chromatographic to detect the residual quantity of Nicotine in inorganic salt nutrient solution.RPLC testing conditions: moving phase is methyl alcohol: 1mM H
2sO
4=10:90(volume ratio), analytical column be Grace Alltima C18 chromatographic column (4.6 × 250mm, m), flow velocity is 0.6ml/min to 5 μ, sample size is 20 μ l, column temperature is 30 DEG C.
2) nicotine degradation experiment:
Nicotine standard substance are dissolved to the reference liquid that is mixed with 100mg/L with sterilized water, at reversed-phase liquid chromatography testing standard curve, as shown in Figure 2, typical curve equation is y=5.4964x+6.8301 to Nicotine typical curve, R
2=0.9929, y is peak area, and x is nicotine concentration.
A, the impact of differing temps on degraded: get 250ml Erlenmeyer flask, add respectively 100ml inorganic salt nutrient solution, (121 DEG C of high pressure steam sterilizations, 20min), add Nicotine, make Nicotine final concentration be 200mg/L, respectively get the mycetocyte suspension 5ml that embodiment 2 methods obtain, be inoculated in respectively in this inorganic salt nutrient solution, making Rhodopseudomonas HZN6 final concentration is 5 × 10
7individual/ml, respectively at 25,30,37,45 DEG C of cultivation shaking tables (pH7.0,150rpm), timing sampling per hour is measured residual nicotine concentration, and as shown in Figure 3,30 DEG C is the suitableeest degradation temperature to result.
B, the different pH impact on degraded: get 250ml Erlenmeyer flask, add respectively 100ml inorganic salt nutrient solution, (121 DEG C of high pressure steam sterilizations, 20min), add Nicotine, make Nicotine final concentration be 200mg/L, respectively get the mycetocyte suspension 5ml that embodiment 2 methods obtain, be inoculated in respectively in this inorganic salt nutrient solution, making Rhodopseudomonas HZN6 final concentration is 5 × 10
7individual/ml, regulates respectively pH to 5.5,6.5,7.0,7.5,8.0 and 9.0, and under 30 DEG C, 150rpm, shaking table is cultivated, and timing sampling per hour is measured residual nicotine concentration, and as shown in Figure 4, pH7.0 is the suitableeest degraded pH to result.
C, the impact of different time on degraded: get 250ml Erlenmeyer flask, add 100ml inorganic salt nutrient solution, after high pressure steam sterilization (121 DEG C, 20min), add Nicotine, making Nicotine final concentration is 200mg/L, and 3 Duplicate Samples are set altogether.Respectively get the mycetocyte suspension 5ml that embodiment 2 methods obtain, be inoculated in respectively in this inorganic salt nutrient solution, making Rhodopseudomonas HZN6 final concentration is 5 × 10
7individual/ml, arranges 3 parallel laboratory tests that do not contain this bacterial classification accordingly as blank, is then together placed in dark shaking culture in shaking table (30 DEG C, pH7.0,150rpm).Be 0,2,4,6 at incubation time, timing sampling when 8h, detect the increment of thalline and the residual quantity of Nicotine in inorganic salt nutrient solution according to above-mentioned detection method, the results are shown in Figure shown in 5.
As shown in Figure 5, as shown in Figure 6, Fig. 6 can find out OD to the growth curve of thalline to the degradation curve of the Nicotine of bacterial strain of the present invention to 200mg/L concentration
600be increased to 0.5 from 0.25, illustrate that thalli growth is good, observe Fig. 5, can find, cultivate after 8h, the degradation rate of the Nicotine of nicotine degradation bacterium of the present invention to 200mg/L is close to 100%, and the percent hydrolysis of all blanks that do not add bacterium after 8h is all less than 5%.
D, impact on different concns nicotine degradation:
Get 250ml Erlenmeyer flask, add respectively 100ml inorganic salt nutrient solution, (121 DEG C of high pressure steam sterilizations, 20min), add Nicotine, make that Nicotine final concentration is respectively 200,500,1000,2000,3000mg/L, respectively get the mycetocyte suspension 5ml that embodiment 2 methods obtain, be inoculated in respectively in this inorganic salt nutrient solution, making Rhodopseudomonas HZN6 final concentration is 5 × 10
7individual/ml, under pH7.0,30 DEG C, 150rpm, shaking table is cultivated, and timing sampling per hour is measured residual nicotine concentration, and result is as shown in Figure 7.
The degradation rate of the Nicotine that bacterial strain of the present invention is 100-3000mg/L to concentration as shown in Figure 7, result shows all have extraordinary degradation capability, and this bacterial classification is novel nicotine degradation bacterium, therefore, this bacterium has very large promoter action to degradation pathway and the degrading genes of research Nicotine, and the degraded of Nicotine in environment is especially had to certain positive effect to the concentrated reparation of Nicotine.
Claims (6)
1. Rhodopseudomonas HZN6(Pseudomonas sp.HZN6), be preserved in Chinese Typical Representative culture collection center, address: Luojia Mountain, Wuhan, Hubei Province Wuhan University, preservation date is on August 9th, 2010, deposit number is CCTCC No:M2010196.
2. the application of Rhodopseudomonas HZN6 as claimed in claim 1 in degraded Nicotine.
3. the application of Rhodopseudomonas HZN6 in degraded Nicotine as claimed in claim 2, it is characterized in that described being applied as: taking Nicotine as substrate, taking inorganic salt nutrient solution as reaction medium, taking Rhodopseudomonas HZN6 as degradation bacteria, under the condition that 25～45 DEG C, pH value are 5.5～9.0, react 8～15h make reaction solution in the mass content of Nicotine be less than 0.1%, reach the object of degraded Nicotine.
4. the application of Rhodopseudomonas HZN6 in degraded Nicotine as claimed in claim 2, it is characterized in that described being applied as: inorganic salt nutrient solution is mixed with Nicotine, making Nicotine final concentration is 100～3000mg/L, add containing the cell suspension of Rhodopseudomonas HZN6 again and form reaction system, the condition dark shaking culture that is 5.5～9.0 at 25～45 DEG C, pH value to the quality residual quantity of Nicotine in reaction solution is less than 0.1%; It is 1 × 10 that the described cell suspension add-on containing Rhodopseudomonas HZN6 makes Rhodopseudomonas HZN6 final concentration in reaction system
7～5 × 10
9individual/ml.
5. the application of Rhodopseudomonas HZN6 in degraded Nicotine as claimed in claim 4, is characterized in that the preparation method of the described cell suspension containing Rhodopseudomonas HZN6 is:
(1) slant culture: Rhodopseudomonas HZN6 is inoculated in to slant medium, cultivates 5～7 days for 25～45 DEG C, obtain thalline inclined-plane; The final concentration of described slant medium consists of: yeast powder 10g/L, and peptone 5.0g/L, sodium-chlor 10.0g/L, agarose 2.0g/L, solvent is water;
(2) seed culture: picking one transfering loop thalline is seeded in minimal medium from step (1) thalline inclined-plane, cultivates 5～7 days for 25～45 DEG C, obtains seed liquor; Described minimal medium final concentration consists of: in every liter of nutrient solution, contain NaCl1g, K
2hPO
41.5g, KH
2pO
40.5g, (NH
4)
2sO
41.5g, MgSO
40.1g, 1ml trace element solution, solvent is water, natural pH value makes after 121 DEG C of high pressure steam sterilization 20min, wherein in every liter of trace element solution, contains MnSO
4h
2o0.13
g, ZnCl
20.23g, CuSO
4h
2o0.03g, CoCl
26H
2o0.42g, Na
2moO
42H
2o0.15g, AlCl
36H
2o0.05g, solvent is water;
(3) enlarged culturing: the seed liquor that step (2) is obtained is seeded in LB liquid nutrient medium with the inoculum size of volumetric concentration 10～20%, 30 DEG C, 150rpm vibration are supported to logarithmic phase, obtain bacterium liquid, by centrifugal bacterium liquid, abandon supernatant, the phosphoric acid buffer that precipitation is 7.0 by pH value suspends, and obtains the cell suspension containing Rhodopseudomonas HZN6; Described LB liquid nutrient medium final concentration consists of: in every liter of cultivation, contain yeast powder 10g, and peptone 5.0g, sodium-chlor 10.0g, solvent is water, natural pH value.
6. the application of Rhodopseudomonas HZN6 in degraded Nicotine as claimed in claim 5, it is characterized in that described being applied as: inorganic salt nutrient solution and Nicotine are mixed and made into mixed solution, making Nicotine final concentration is 200mg/L, add again containing the cell suspension of Rhodopseudomonas HZN6 and form reaction system, dark shaking culture 8h under 30 DEG C, pH7.0,150rpm condition, make the quality residual quantity of Nicotine in reaction solution be less than 0.1%, reach the object of degraded Nicotine; It is 1 × 10 that the add-on of the described cell suspension containing Rhodopseudomonas HZN6 makes Rhodopseudomonas HZN6 final concentration in reaction system
7～5 × 10
9individual/ml.
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