CN102796681A - Pseudomonas sp.HZN6 and application thereof to nicotine degradation - Google Patents

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

Pseudomonas HZN6 and its application in the degradation of nicotine

(1) Technical field

The invention relates to a novel high-efficiency nicotine-degrading bacterium, in particular to a novel high-efficiency Pseudomonas nicotine-degrading bacterium, that is, Pseudomonas HZN6 and application thereof.

(2) Background technology

Nicotine, commonly known as nicotine, is the unique and most important alkaloid in various tobaccos, accounting for about 1%-2% of the dry weight of tobacco, and is one of the important factors affecting the quality of tobacco leaves. One of the main harmful components of cigarettes. The molecular formula of nicotine is C ₁₀ H ₁₄ N ₂ , and the structural formula is shown in formula (I).

Because nicotine is a psychotropic drug, human beings have maintained the habit of smoking tobacco for a long time. However, nicotine is an important precursor of tobacco-specific nitrosamines (TSNA), the main carcinogenic component in tobacco leaves and smoke. At the same time, nicotine is also an environmentally toxic substance. Pure nicotine is a colorless, bitter, and strongly irritating oily liquid at room temperature, and is easily oxidized into dark gray in the air. The flue gas environment contains a large amount of nicotine. At present, the nicotine content in some tobacco leaves in my country is too high, especially in the upper tobacco leaves, which brings great challenges to tobacco leaf production. At the same time, tobacco will produce high-concentration nicotine waste during the processing process. This waste is considered to be "toxic and dangerous waste" and causes great harm to the environment. Therefore, it is an inevitable trend for the development of the international tobacco industry to continuously control and reduce the nicotine content in cigarettes, and it is also one of the important ways to reduce the harm of smoking; at the same time, reducing the nicotine content in the environment and reducing the pollution of tobacco waste to the environment have far-reaching implications for maintaining human health. meaning.

The chemical structure and chemical properties of nicotine are relatively stable. If it is removed by physical and chemical methods, the cost will be higher, and there will be more harmful by-products, which will also affect the original good quality of tobacco; The unique effect of using microbial metabolism to remove nicotine not only has low cost and fewer harmful by-products, but also does not affect the original excellent characteristics of tobacco, so it has broad application prospects.

Microorganisms are a class of organisms with many types, rapid reproduction, strong adaptability, and strong metabolic capabilities. If we can screen and isolate microorganisms that can efficiently degrade nicotine, and degrade nicotine into carboxylic acids, amino acids and other non-toxic substances to human body and environment, this will have far-reaching significance for maintaining human health.

(3) Contents of the invention

The purpose of the present invention is to provide a novel high-efficiency Pseudomonas nicotine-degrading bacterium HZN6 and its application.

The technical scheme adopted in the present invention is:

Pseudomonas sp.HZN6 (Pseudomonas sp.HZN6), preserved in the China Center for Type Culture Collection, address: Wuhan University, Luojia Mountain, Wuhan City, Hubei Province, 430072, the preservation date is August 9, 2010, and the preservation number is CCTCC No: M2010196.

The Genbank accession number of the 16S rDNA of the Pseudomonas HZN6 is HQ108345.

Screening and identification of Pseudomonas sp.HZN6 strains:

1) culture medium

The final concentration of the inorganic salt medium is composed of: 1 g of NaCl, 1.5 g of K ₂ HPO ₄ , 0.5 g of KH ₂ PO ₄ , 1.5 g of (NH4) ₂ SO ₄ , 0.1 g of MgSO ₄ , and 1 ml of trace element solution per liter of culture fluid , the solvent is water, the natural pH value, made after high-pressure steam sterilization (121°C, 20min), and each liter of trace element solution contains 0.13g of MnSO ₄ ·H ₂ O, 0.23g of ZnCl ₂ , CuSO ₄ ·H ₂ O0.03g, CoCl ₂ ·6H ₂ O 0.42g, Na ₂ MoO ₄ ·2H ₂ O 0.15g, AlCl ₃ ·6H ₂ O 0.05g, and the solvent was water.

Enriched culture solution: Add nicotine to the inorganic salt culture solution so that the final concentration of nicotine is 200mg/L.

LB liquid medium: Each liter of culture medium contains 10g of yeast powder, 5.0g of peptone, and 10.0g of sodium chloride, the solvent is water, the natural pH value, and it is prepared after high-pressure steam sterilization (121°C, 20min).

LB solid medium: Each liter of culture contains 10g of yeast powder, 5.0g of peptone, 10.0g of sodium chloride, and 15.0g of agar.

2) Isolation and purification of strains

Sludge samples were collected from Hangzhou Pesticide Factory. Take 5ml of sludge samples and place them in a 250ml Erlenmeyer flask, add 100ml of enriched culture medium, and culture with shaking in the dark (30°C, 150rpm) for 1 week. Collect the culture medium into 100ml, continue dark shaking culture (30°C, 150rpm) for 1 week, repeat the above operation process 3 times, and the inoculum for each culture is taken from the culture medium obtained from the previous culture.

Take 5ml of the culture solution obtained from the last culture for gradient dilution (10 ^-4 , 10 ^-5 , 10 ^-6 ), and take 150 μl of each diluted culture solution and spread it on the LB solid medium plate containing 500 mg/L nicotine. Place it in a constant temperature incubator (30°C) for cultivation. After colonies grow on the plate, pick each colony and repeat purification on the LB solid medium plate containing 500 mg/L nicotine until the colony is single. Connect them to LB liquid medium test tubes for shaking culture (30°C, 150rpm) overnight, centrifuge the cultured bacteria solution (8000rpm, 5min) and transfer them to the enriched culture medium for 3 days at 25~45°C. Chromatography (HPLC) was used to detect the residual amount of nicotine in each enriched culture solution, and finally a strain capable of efficiently degrading nicotine was obtained through screening, which was named HZN6.

3) Strain identification

The morphological characteristics and molecular biological identification of the strain obtained above were carried out, and the electron micrograph of the strain is shown in FIG. 1 . The main biological characteristics of this strain are: negative Gram staining, rod-shaped bacteria, terminal flagella, no spores, the size is about (0.25~0.5)×(0.8~1.0) μm, the colony is flat, and the middle is raised , diffuse at the edge, pale yellow, positive for contact enzymes, positive for oxidase, positive for methyl red test, able to use β-cyclodextrin, starch, glucose, Tween 40, acetate, negative for acetylmethylmethanol test, V.P. The response was negative. The most suitable growth conditions for the strain are pH 7.0 and temperature 30°C. The strain was identified as Pseudomonas genus by 16S rDNA sequence analysis, and thus Pseudomonas sp. HZN6 (Pseudomonas sp. HZN6).

The invention provides an application of the pseudomonas HZN6 in degrading nicotine.

Further, the application of Pseudomonas HZN6 in degrading nicotine uses nicotine as a substrate, inorganic salt culture solution as a reaction medium, and Pseudomonas HZN6 as a degrading bacterium (i.e. an enzyme). Under the conditions of 45°C and pH value of 5.5-9.0, react for 8-15 hours so that the mass content of nicotine in the reaction liquid is less than 0.1%, so as to achieve the purpose of degrading nicotine.

Further, the application is: mixing the inorganic salt culture solution with nicotine so that the final concentration of nicotine is 100-3000 mg/L (preferably 200 mg/L), and then adding a cell suspension containing Pseudomonas HZN6 to form a reaction system , at 25-45°C, pH value 5.5-9.0, shake culture in the dark until the residual mass of nicotine in the reaction solution is less than 0.1% (usually cultured for 8-15 hours); the cell suspension containing Pseudomonas HZN6 The amount of solution added is to make the final concentration of Pseudomonas HZN6 in the reaction system be 1×10 ⁷ ~5×10 ⁹ cells/ml.

Furthermore, the preparation method of the cell suspension containing Pseudomonas HZN6 in the application of Pseudomonas HZN6 in degrading nicotine is:

(1) Slant culture: inoculate Pseudomonas HZN6 on the slant medium, and culture at 25-45°C for 5-7 days to obtain the bacterial slant; the final concentration of the slant medium is composed of: yeast powder 10g/L , peptone 5.0g/L, sodium chloride 10.0g/L, agarose 2.0g/L, solvent is water;

(2) Seed culture: Pick an inoculation loop from the slant of the bacteria in step (1) and inoculate the bacteria into the inorganic salt medium, cultivate at 25-45°C for 5-7 days, and obtain the seed liquid; the inorganic salt medium The final concentration is composed of: each liter of culture solution contains 1g of NaCl, 1.5g of K ₂ HPO ₄ , 0.5g of KH ₂ PO ₄ , 1.5g of (NH4) ₂ SO ₄ , 0.1g of MgSO ₄ , 1ml of trace element solution, and the solvent is water , natural pH value, prepared after high-pressure steam sterilization (121°C, 20min), wherein each liter of trace element solution contains 0.13g of MnSO ₄ ·H ₂ O, 0.23g of ZnCl ₂ , 0.03g of CuSO ₄ ·H ₂ O, CoCl ₂ 6H ₂ O 0.42g, Na ₂ MoO ₄ 2H ₂ O 0.15g, AlCl ₃ 6H ₂ O 0.05g, the solvent is water;

(3) Expansion culture: inoculate the seed solution obtained in step (2) into LB liquid medium with an inoculum volume concentration of 10-20%, and shake it at 30°C and 150rpm until the logarithmic growth phase to obtain the bacterial solution. Bacterial solution is centrifuged, supernatant is discarded, and the precipitate is suspended with phosphate buffer solution with a pH value of 7.0 to obtain a cell suspension containing Pseudomonas HZN6; the final concentration of the LB liquid medium is composed of: each liter of culture contains yeast Powder 10g, peptone 5.0g, sodium chloride 10.0g, solvent is water, natural pH value.

Furthermore, the application of the Pseudomonas HZN6 in degrading nicotine is as follows: mixing the inorganic salt culture solution with nicotine so that the final concentration of nicotine is 200mg/L, and then adding the Pseudomonas HZN6 containing The cell suspension constitutes a reaction system, which is shaken and cultivated in the dark for 8 hours at 30°C, pH 7.0, and 150 rpm, so that the residual mass of nicotine in the reaction solution is less than 0.1%, and the purpose of degrading nicotine is achieved; the Pseudomonas spp. The amount of HZN6 cell suspension is added so that the final concentration of Pseudomonas HZN6 in the reaction system is 1×10 ⁷ ~5×10 ⁹ cells/ml, preferably 1×10 ⁷ ~5×10 ⁷ cells/ml.

In the present invention, the growth amount of the bacteria is detected by an ultraviolet spectrophotometer, which is expressed by measuring the absorbance value of the bacteria (that is, the culture solution containing bacteria cells) at 600nm.

The invention adopts reverse phase high performance liquid chromatography to detect the residual amount of nicotine in the inorganic salt culture solution. Reverse-phase high-performance liquid chromatography detection conditions: mobile phase is methanol: 1mM H ₂ SO ₄ =10:90 (volume ratio), analytical column is Grace Alltima C18 column (4.6×250mm, 5μm), flow rate is 0.6ml/min , the injection volume was 20 μl, and the column temperature was 30°C.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

The Pseudomonas HZN6 of the present invention can be applied to the degradation of nicotine in water and soil by direct dosing, and can safely, efficiently and quickly degrade residual nicotine on objects such as water and soil. The preparation process of the agent is simple, the cost is low, the use is convenient, and the application prospect is good.

(4) Description of drawings

Fig. 1 is the electron micrograph of Pseudomonas HZN6 of the present invention;

Fig. 2 is the standard curve figure of nicotine;

为45℃；Figure 3 is the degradation curve of Pseudomonas HZN6 of the present invention on nicotine at different temperatures: the square (■) is 30°C, the circle (●) is 25°C, the equilateral triangle (▲) is 37°C, and the inverted triangle

45°C;

Fig. 4 is the figure of influence of Pseudomonas HZN6 of the present invention on the degradation of nicotine at different pH;

Fig. 5 is the degradation curve figure of the nicotine that Pseudomonas HZN6 of the present invention is 200mg/L to final concentration under pure culture condition;

Fig. 6 is the growth curve figure of Pseudomonas HZN6 of the present invention under the pure culture condition of 200mg/L in nicotine final concentration;

Fig. 7 is a graph showing degradation curves of different concentrations of nicotine by Pseudomonas HZN6 of the present invention.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Embodiment 1: Screening and identification of strains

1) culture medium

Preparation of inorganic salt medium: NaCl 1g, K ₂ HPO ₄ 1.5g, KH ₂ PO ₄ 0.5g, (NH ₄ ) ₂ SO ₄ 1.5g, MgSO ₄ 0.1g, 1ml trace element solution, distilled water to make up to 1000ml, mix After stirring evenly, the natural pH value is prepared after high-pressure steam sterilization (121°C, 20min), and each liter of trace element solution contains 0.13g of MnSO ₄ ·H ₂ O, 0.23g of ZnCl ₂ , CuSO ₄ ·H ₂ O0 .03g, CoCl ₂ ·6H ₂ O 0.42g, Na ₂ MoO ₄ ·2H ₂ O 0.15g, AlCl ₃ ·6H ₂ O 0.05g, make up to 1000ml with distilled water.

Enriched culture solution: Add nicotine to the inorganic salt culture solution so that the final concentration of nicotine is 200mg/L.

Preparation of LB liquid medium: Yeast powder 10g, peptone 5.0g, sodium chloride 10.0g, distilled water to make up to 1000ml, mix and stir evenly, natural pH value, high pressure steam sterilization (121°C, 20min).

Preparation of LB solid medium: yeast powder 10g, peptone 5.0g, sodium chloride 10.0g, agar 15.0g, distilled water to make up to 1000ml, mix and stir evenly, natural pH value, after autoclaving (121°C, 20min) be made of.

2) Isolation and purification of strains

Sludge samples were collected from Hangzhou Pesticide Factory. Take 5ml of sludge samples and place them in a 250ml Erlenmeyer flask, add 100ml of enriched culture medium, and culture with shaking in the dark (30°C, 150rpm) for 1 week. Collect the culture medium, continue the dark shaking culture (30°C, 150rpm) for 1 week, repeat the above operation process 3 times, and the inoculum for each culture is taken from the culture medium obtained from the previous culture.

Take 5ml of the culture solution obtained from the last culture for gradient dilution ((10 ^-4 , 10 ^-5 , 10 ^-6 ), take 150 μl of each diluted culture solution and spread it on the LB solid medium plate containing 500 mg/L nicotine , placed in a constant temperature incubator (30°C) for cultivation, after colonies grow on the plate, pick each colony and repeatedly purify it on the LB solid medium plate containing 500mg/L nicotine until the colony is single, and each purified The colonies were respectively connected to LB liquid medium test tubes for shaking culture (30°C, 150rpm) overnight, and the cultured bacterial liquid was centrifuged and then transferred to the enriched culture medium for 3 days, and each enrichment was detected by reversed-phase high-performance liquid chromatography. The remaining amount of nicotine in the culture medium was finally screened to obtain a strain that can efficiently degrade nicotine, named HZN6.

3) Strain identification

The morphological characteristics and molecular biological identification of the strain obtained above were carried out, and the electron micrograph of the strain is shown in FIG. 1 . The main biological characteristics of the strain are: rod-shaped bacteria, terminal flagella, no spores, about (0.25~0.5)×(0.8~1.0) μm in size, flat colony, raised in the middle, diffuse at the edge, pale yellow in color , Negative Gram stain reaction, positive contact enzyme, positive oxidase, positive methyl red test, can use β-cyclodextrin, starch, glucose, Tween 40, acetate, negative acetylmethylmethanol test, V.P. The response was negative. The most suitable growth conditions for the strain are pH 7.0 and temperature 30°C. The strain was identified as Pseudomonas genus by 16S rDNA sequence analysis, and thus Pseudomonas sp. HZN6 (Pseudomonas sp. HZN6).

Embodiment 2: the preparation that contains bacterial cell suspension

(1) Slant culture: Inoculate Pseudomonas HZN6 on the slant medium and culture at 30°C for 6 days to obtain the slant; the final concentration of the slant medium consists of: yeast powder 10g, peptone 5.0g, chlorine Sodium chloride 10.0g, agarose 2.0g, water 1000ml;

(2) Seed culture: Pick an inoculation loop from the slant of the bacteria in step (1) and inoculate it into the inorganic salt medium, and cultivate it at 30°C for 6 days to obtain the seed liquid; the final concentration of the inorganic salt medium is the same as Embodiment 1;

(3) Expansion culture: Inoculate the seed solution obtained in step (2) into LB liquid medium (100mL) with an inoculum volume concentration of 15%, culture at 30°C and 150rpm until the logarithmic growth phase, and obtain the bacterial solution. Centrifuge the bacterial solution (8000rpm, 5min), discard the supernatant, and suspend the precipitate with phosphate buffer solution with a pH value of 7.0 to obtain 100 mL of a cell suspension containing Pseudomonas genus HZN6, in which the Pseudomonas genus in the cell suspension The concentration of HZN6 is 1× ¹⁰⁹ /ml; the composition of the final concentration of the LB liquid medium is the same as in Example 1; the formula of the 0.2mol/L phosphate buffer solution with a pH of 7.0 is: take 0.2mol/L dihydrogen phosphate Add 39ml of sodium and 61ml of 0.2mol/L disodium hydrogen phosphate, dilute to 1000ml with ultrapure water, and sterilize with high pressure steam (121°C, 20min).

Embodiment 3: Nicotine degradation experiment

1) Detection of bacterial concentration and nicotine content in inorganic salt culture solution:

The growth of bacteria is detected by ultraviolet spectrophotometer, which is expressed by measuring the absorbance value of bacteria in the culture solution at 600nm.

In this experiment, reversed-phase high-performance liquid chromatography was used to detect the residual amount of nicotine in the inorganic salt culture solution. Reverse-phase high-performance liquid chromatography detection conditions: mobile phase is methanol: 1mM H ₂ SO ₄ =10:90 (volume ratio), analytical column is Grace Alltima C18 column (4.6×250mm, 5μm), flow rate is 0.6ml/min , the injection volume was 20 μl, and the column temperature was 30°C.

2) Nicotine degradation experiment:

The nicotine standard substance is dissolved in sterile water to prepare a 100mg/L standard solution, and the standard curve is tested by reversed-phase liquid chromatography. The nicotine standard curve is shown in Figure 2, and the standard curve equation is y=5.4964x+6.8301, R ² =0.9929, y is the peak area, and x is the nicotine concentration.

a. Effects of different temperatures on degradation: Take 250ml Erlenmeyer flasks, add 100ml of inorganic salt culture solution, and add nicotine after high-pressure steam sterilization (121°C, 20min) so that the final concentration of nicotine is 200mg/L. 5ml of the bacterium-containing cell suspension obtained by the method of Example 2 was inoculated in this inorganic salt culture solution respectively, so that the final concentration of Pseudomonas HZN6 was 5× ¹⁰⁷ /ml, and cultivated at 25, 30, 37, and 45°C respectively Shaking table (pH7.0, 150rpm), regularly sampling every hour to measure the residual nicotine concentration, the results are shown in Figure 3, 30°C is the optimum degradation temperature.

b. The effect of different pH on degradation: take 250ml Erlenmeyer flask, add 100ml inorganic salt culture solution respectively, add nicotine after high-pressure steam sterilization (121℃, 20min), so that the final concentration of nicotine is 200mg/L, and implement each 5ml of the bacterium-containing cell suspension obtained by the method of Example 2 was inoculated in this inorganic salt culture solution respectively, so that the final concentration of Pseudomonas HZN6 was 5× ¹⁰⁷ /ml, and the pH was adjusted to 5.5, 6.5, 7.0, and 7.5 respectively. , 8.0 and 9.0, cultivated on a shaker at 30°C and 150 rpm, and took samples every hour to measure the residual nicotine concentration. The results are shown in Figure 4, and pH 7.0 is the optimum pH for degradation.

c. The effect of different time on degradation: Take a 250ml Erlenmeyer flask, add 100ml of inorganic salt culture solution, add nicotine after high-pressure steam sterilization (121°C, 20min), so that the final concentration of nicotine is 200mg/L, and set up 3 parallel Sample. Each get 5ml of the bacterium-containing cell suspension obtained by the method of Example 2, and inoculate them in this inorganic salt culture solution respectively, so that the final concentration of Pseudomonas HZN6 is ⁵ ×10 Parallel experiments of the strains were used as blank controls, and then placed together in a shaker (30°C, pH 7.0, 150rpm) for dark shaking culture. Samples were taken regularly when the culture time was 0, 2, 4, 6, and 8 hours, and the growth amount of bacteria and the residual amount of nicotine in the inorganic salt culture solution were detected according to the above detection method. The results are shown in Figure 5.

The degradation curve of bacterial strain of the present invention to the nicotine of 200mg/L concentration is as shown in Figure 5, and the growth curve of thalline is as shown in Figure 6, and Fig. 6 can find out that _OD600 increases to 0.5 from 0.25, illustrates that thalline growth is good, observes In Fig. 5, it can be found that after 8 hours of cultivation, the degradation rate of 200 mg/L nicotine by the nicotine-degrading bacteria of the present invention is close to 100%, and the hydrolysis rate of all blank controls without bacteria added is less than 5% after 8 hours.

d. Effects on the degradation of nicotine with different concentrations:

Take a 250ml Erlenmeyer flask, add 100ml of inorganic salt culture solution, and add nicotine after high-pressure steam sterilization (121°C, 20min), so that the final concentration of nicotine is 200, 500, 1000, 2000, 3000mg/L, respectively, according to the examples 5ml of the bacterial cell suspension obtained by the method 2, inoculated in this inorganic salt culture solution respectively, so that the final concentration of Pseudomonas HZN6 was 5× ¹⁰⁷ /ml, shake the table at pH 7.0, 30°C, 150rpm After culturing, samples were regularly taken every hour to measure the residual nicotine concentration, and the results are shown in Figure 7.

The bacterial strain of the present invention is that the degradation rate of the nicotine of 100-3000mg/L to concentration is as shown in Figure 7, and the result shows that all has very good degradability, and this bacterial classification is novel nicotine degrading bacterium, therefore, this bacterium is to research nicotine The degradation pathway and the degradation gene have a very large promotion effect, and have certain positive significance for the degradation of nicotine in the environment, especially for the concentrated restoration of nicotine.

Claims (6)

1. Rhodopseudomonas HZN6 (Pseudomonas sp.HZN6) is preserved in Chinese typical culture collection center, the address: Luojia Mountain, Wuhan, Hubei Province Wuhan University, and 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 the degraded Nicotine.

3. like the application of the said Rhodopseudomonas HZN6 of claim 2 in the degraded Nicotine; It is characterized in that described being applied as: be substrate with the Nicotine; With the inorganic salt nutrient solution is reaction medium; With Rhodopseudomonas HZN6 is degradation bacteria, under 25 ~ 45 ℃, pH value are 5.5 ~ 9.0 condition, reacts mass content that 8 ~ 15h makes Nicotine in the reaction solution less than 0.1%, reaches the purpose of degraded Nicotine.

4. like the application of the said Rhodopseudomonas HZN6 of claim 2 in the degraded Nicotine; It is characterized in that described being applied as: the inorganic salt nutrient solution is mixed with Nicotine; Making the Nicotine final concentration is 100 ~ 3000mg/L; Adding the cell suspension contain Rhodopseudomonas HZN6 again and constitute reaction system, is that the quality residual quantity of Nicotine in 5.5 ~ 9.0 condition dark shaking culture to the reaction solution is less than 0.1% at 25 ~ 45 ℃, pH value; The said cell suspension add-on that contains Rhodopseudomonas HZN6 makes that Rhodopseudomonas HZN6 final concentration is 1 * 10 in the reaction system ⁷~ 5 * 10 ⁹Individual/ml.

5. like the application of the said Rhodopseudomonas HZN6 of claim 3 in the degraded Nicotine, it is characterized in that the described preparation method who contains the cell suspension of Rhodopseudomonas HZN6 is:

(1) slant culture: HZN6 is inoculated in slant medium with Rhodopseudomonas, cultivates 5 ~ 7 days for 25 ~ 45 ℃, obtains the thalline inclined-plane; The final concentration of said slant medium consists of: yeast powder 10g/L, and peptone 5.0g/L, sodium-chlor 10.0g/L, agarose 2.0g/L, solvent are water;

(2) seed culture: picking one transfering loop thalline is seeded to the minimal medium from step (1) thalline inclined-plane, cultivates 5 ~ 7 days for 25 ~ 45 ℃, obtains seed liquor; Said minimal medium final concentration consists of: contain NaCl 1g, K in every liter of nutrient solution ₂HPO ₄1.5g, KH ₂PO ₄0.5g, (NH4) ₂SO ₄1.5g, MgSO ₄0.1g 1ml trace element solution, solvent are water, natural pH value makes behind 121 ℃ of high pressure steam sterilization 20min, wherein contains MnSO in every liter of trace element solution ₄H ₂O 0.13g, ZnCl ₂0.23g, CuSO ₄H ₂O 0.03g, CoCl ₂6H ₂O 0.42g, Na2MoO ₄2H ₂O 0.15g, AlCl ₃6H ₂O 0.05g, solvent are water;

(3) enlarged culturing: the seed liquor that step (2) is obtained is seeded in the LB liquid nutrient medium with the inoculum size of volumetric concentration 10 ~ 20%; 30 ℃, 150rpm vibration are supported to logarithmic phase; Obtain bacterium liquid, bacterium liquid is centrifugal, abandon supernatant; Deposition use pH value is 7.0 phosphoric acid buffer suspension, obtains to contain the cell suspension of Rhodopseudomonas HZN6; Said LB liquid nutrient medium final concentration consists of: contain yeast powder 10g in every liter of cultivation, and peptone 5.0g, sodium-chlor 10.0g, solvent are water, natural pH value.

6. like the application of the said Rhodopseudomonas HZN6 of claim 5 in the degraded Nicotine; It is characterized in that described being applied as: inorganic salt nutrient solution and Nicotine are mixed and made into mixed solution; Making the Nicotine final concentration is 200mg/L, adds the cell suspension contain Rhodopseudomonas HZN6 again and constitutes reaction system, dark shaking culture 8h under 30 ℃, pH7.0,150rpm condition; The quality residual quantity that makes Nicotine in the reaction solution reaches the purpose of degraded Nicotine less than 0.1%; The said add-on that contains the cell suspension of Rhodopseudomonas HZN6 makes that Rhodopseudomonas HZN6 final concentration is 1 * 10 in the reaction system ⁷~ 5 * 10 ⁹Individual/ml.

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