CN104312946A - 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 PDF

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CN104312946A
CN104312946A CN201410505852.6A CN201410505852A CN104312946A CN 104312946 A CN104312946 A CN 104312946A CN 201410505852 A CN201410505852 A CN 201410505852A CN 104312946 A CN104312946 A CN 104312946A
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nicotine
minimum
ultrapure water
zymomonas mobilis
solvent
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CN104312946B (en
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马云
张豆
刘猛
温荣提
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Heze Jianshu Intelligent Technology Co Ltd
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Zhejiang University of Technology ZJUT
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    • A62—LIFE-SAVING; FIRE-FIGHTING
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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

Minimum Zymomonas mobilis T2 and the application in microbiological deterioration Nicotine thereof
(1) technical field
The present invention relates to a strain new and effective nicotine degradation bacterium---minimum Zymomonas mobilis (Pusillimonas sp.) T2, and the application in microbiological deterioration Nicotine.
(2) background technology
Nicotine (nicotine), is commonly called as nicotine, and be a kind of amine substance be jointly made up of pyridine ring and pyrrole ring, molecular formula is C 10h 14n 2, Nicotine creates the steric isomers such as α-nicotine, β-nicotine, γ-nicotine due to the position difference of N-methyl Pyrrolidine on pyridine ring, and what exist in tobacco is mainly β-nicotine, and structural formula respectively as shown in Figure 8.Pure Nicotine is oily liquids that is colourless or pale yellow transparent at normal temperatures, has strong volatility, special sharp flavor and deliquescence, is easily oxidized to lead in the sun.20 DEG C time, density is 1.0097g/mL, and under 760mm Hg, boiling point is 274.5 DEG C.Nicotine is weakly alkaline, when temperature is lower than 60 DEG C, can dissolves each other, be very easily dissolved in the organic solvents such as alcohol, ether, chloroform with water with arbitrary proportion, when being bonded at skin surface, is easy to infiltrate in body and absorbed by body.
Nicotine is a kind of psychotropic substances, has the same with Cocaine, heroine additive, is mainly extensively present in environment with various ways such as tobacco and waste, nicotine type nicotinic agricultural chemicals, the medicament containing Nicotine and the rubber containing Nicotine.Nicotine and meta-bolites thereof have and bring out various diseases, worsen associated disease symptom, stronger carinogenicity, affect the bio-toxicity such as reproduction and development of organism, usually cause multiple harm.At present, the bio-toxicity of Nicotine has caused the extensive concern of Chinese scholars, and obtains multi-angle, multi-level research.
Method than physics, chemistry removes the Nicotine in tobacco and waste thereof, and microbial method has unique advantage such as simple to operate, easy transformation, more and more receives the concern of people.As far back as the forties in 20th century, just there is the report about microbiological deterioration Nicotine.Current investigator both domestic and external has been isolated to many strains nicotine degradation bacterium, mainly contains: Rhodopseudomonas, stalk Pseudomonas, Cellulomonas, Ochrobactrum, bacillus etc.These microorganism majorities can utilize that Nicotine is sole carbon source, nitrogenous source and the energy grow, they provide carbon source, nitrogenous source and the energy mainly through the cross-pathway of pyridine approach, pyrroles's approach, pyridine and tetramethyleneimine approach, demethylation approach by nicotine degradation and for microbial growth, thus reach the object of degraded Toxic waste.
The present invention's separation screening from the active sludge that Qingfeng Agrochemical Co., Ltd gathers obtains a strain Nicotine efficient degrading bacteria, by carrying out the research of strain identification and degradation characteristic to it, find that this bacterial strain may exist brand-new degradation pathway, this is that the degradation pathway more fully understanding Nicotine provides foundation, also establishes basis for structure has higher degradation efficiency with the engineering bacteria that can tolerate higher nicotine concentration.
Microorganism is the organism that a class kind is many, breeding is fast, strong adaptability, metabolic capacity are strong.If can screen and isolate the microorganism of effectively degrading nicotine, by the material of paired for nicotine degradation human body and environment toxicological harmless, this is to safeguarding that human health and ecological safety have profound significance.
(3) summary of the invention
The object of the invention is to provide the new and effective minimum zygosaccharomyces nicotine degradation bacterium-minimum Zymomonas mobilis T2 of a strain and the application in degraded Nicotine thereof.
The technical solution used in the present invention is:
Minimum Zymomonas mobilis (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.
The Genbank number of logging in of the 16S rDNA of described minimum Zymomonas mobilis T2 is KM054873, the main biological property of this bacterial strain is: thalline is shaft-like, and the raw flagellum of end, without gemma, size is about (0.5 ~ 1.0) × and (1.5 ~ 2.0) μm, bacterium colony is smooth, intermediate projections, edge-diffusion, in faint yellow, gramstaining is negative, anti-penbritin, kantlex, gentamicin, paraxin, Streptomycin sulphate.The optimum growth conditions of this bacterial strain: pH value is 7.0, temperature 30 DEG C.This bacterial strain is accredited as Pusillimonas through 16S rDNA sequential analysis and belongs to, the therefore minimum Zymomonas mobilis of called after (Pusillimonassp.) T2.
The invention still further relates to the described application of minimum Zymomonas mobilis T2 in microbiological deterioration Nicotine.
Concrete, described is applied as: by minimum Zymomonas mobilis T2 through fermentation culture obtain containing fermented liquid centrifugal, precipitation pH value be 7.0 phosphoric acid buffer suspend make bacteria suspension as enzyme source, take Nicotine as substrate, in minimal medium, at 25 ~ 45 DEG C, pH value 5.5 ~ 8.5, 100 ~ 200rpm, DeR is carried out under dark condition, after reacting completely (strain activity can make the quality residual quantity of Nicotine in reaction solution be less than 0.1% after strengthening in 10 ~ 16h), the nutrient solution that the quality residual quantity obtaining Nicotine is less than 0.1%, realize the degraded to Nicotine.Preferably, described degraded, at 30 DEG C, is carried out under pH 7.0,150rpm, dark condition.
Described Nicotine final concentration is 100 ~ 1500mg/L substratum (preferred 100mg/L), and described enzyme source consumption counts 2 × 10 with mycetocyte number 6the substratum of individual/mL.
Enzyme source of the present invention is prepared as follows:
(1) slant culture: minimum Zymomonas mobilis T2 is inoculated in slant medium, cultivates 3 ~ 5 days for 20 ~ 40 DEG C, obtains thalline inclined-plane; The final concentration of described slant medium consists of: yeast powder 10.0g/L, peptone 5.0g/L, sodium-chlor 10.0g/L, agar 20.0g/L, and solvent is ultrapure water;
(2) seed culture: picking one transfering loop thalline is seeded in minimal medium from step (1) thalline inclined-plane, cultivates 3 ~ 5 days for 25 ~ 40 DEG C, obtains seed liquor; Described often liter of minimal medium consists of: NaCl 1g, K 2hPO 41.5g, KH 2pO 40.5g, (NH4) 2sO 41.5g, MgSO 40.1g, 1ml trace element solution, solvent is ultrapure water, natural ph, and high pressure steam sterilization (121 DEG C, 20min) obtains afterwards, and wherein often liter of trace element solution consists of: MnSO 4h 2o 0.13 g, ZnCl 20.23g, CuSO 4h 2o 0.03g, CoCl 26H 2o 0.42g, Na 2moO 42H 2o 0.15g, AlCl 36H 2o 0.05g, solvent is ultrapure water;
(3) fermentation culture (enlarged culturing): seed liquor step (2) obtained is seeded in LB liquid nutrient medium with the inoculum size of volumetric concentration 10 ~ 20%, 30 DEG C, 150rpm shaking culture is 0.15 to OD600, obtain fermented liquid, fermented liquid is centrifugal, abandon supernatant liquor, precipitation pH value be 7.0 phosphoric acid buffer suspend and make the bacteria suspension cell suspension of minimum Zymomonas mobilis T2 (obtain containing) and be enzyme source, this bacteria suspension can add the degraded for Nicotine in water body or soil; Described often liter of LB liquid nutrient medium consists of: yeast powder 10.0g, peptone 5.0g, sodium-chlor 10.0g, and solvent is ultrapure water, natural ph.
Thalli growth amount of the present invention adopts ultraviolet spectrophotometer to detect, and represents by measuring thalline (the i.e. mycetocyte nutrient solution) absorbance at 600nm place.
Ultrapure water of the present invention refers to that resistivity reaches the water of 10M Ω * cm (25 DEG C).
The present invention adopts reversed-phased high performace liquid chromatographic to detect the residual quantity of Nicotine in minimal medium.RPLC testing conditions: moving phase is methyl alcohol: H 2o=10:90 (volume ratio), analytical column is Grace Alltima C18 chromatographic column (4.6 × 250mm, 5 μm), and flow velocity is 0.8ml/min, and sample size is 20 μ l, and column temperature is 30 DEG C.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
Minimum Zymomonas mobilis T2 of the present invention is applied to the degraded of Nicotine in water body and soil by the mode directly added, safely, efficiently, fastly to degrade Nicotine residual on the object such as water body, soil, microbial inoculum preparation technology containing this bacterial strain is simple, with low cost, easy to use, there is good application prospect.
(4) accompanying drawing explanation
Fig. 1 is the Electronic Speculum figure of the minimum Zymomonas mobilis T2 of the present invention;
Fig. 2 is the canonical plotting of Nicotine standard concentration and peak area;
The degradation curve figure of Fig. 3 to be minimum Zymomonas mobilis T2 of the present invention to concentration be under pure culture condition Nicotine of 100mg/L;
Fig. 4 is the growth curve chart of minimum Zymomonas mobilis T2 of the present invention under nicotine concentration is 100mg/L pure culture condition.
Fig. 5 is the effect diagram of temperature to degradation rate.
Fig. 6 is the effect diagram of pH value to degradation rate.
Fig. 7 is the effect diagram of Nicotine starting point concentration to degradation rate.
Fig. 8 is Nicotine structure iron.
(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 selection systems of bacterial strain
1) substratum
The preparation of minimal medium: NaCl 1.0g, K 2hPO 41.5g, KH 2pO 40.5g, (NH 4) 2sO 41.5g, MgSO 40.1g, 1ml trace element solution, ultrapure water complements to 1000ml, stirs after mixing, natural ph, and high pressure steam sterilization (121 DEG C, 20min) obtains afterwards, wherein contains MnSO in often liter of trace element solution 4h 2o 0.13 g, ZnCl 20.23g, CuSO 4h 2o0.03g, CoCl 26H 2o 0.42g, Na 2moO 42H 2o 0.15g, AlCl 36H 2o 0.05g, is settled to 1000ml with ultrapure water.
Enrichment culture liquid: add Nicotine in minimal medium, makes the final concentration of Nicotine be 100mg/L.
The preparation of LB liquid nutrient medium: yeast powder 10g, peptone 5.0g, sodium-chlor 10.0g, ultrapure water is settled to 1000ml, stirs after mixing, natural ph, and high pressure steam sterilization (121 DEG C, 20min) obtains afterwards.
The preparation of LB solid medium: yeast powder 10.0g, peptone 5.0g, sodium-chlor 10.0g, agar 20.0g, ultrapure water is settled to 1000ml, stirs after mixing, natural ph, and high pressure steam sterilization (121 DEG C, 20min) obtains afterwards.
2) strains separation purifying
Mud sample picks up from Qingfeng Agrochemical Co., Ltd, 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 dark shaking culture (30 DEG C, 150rpm) 1 week, repeat aforesaid operations process 3 times, each inoculum cultivated all is taken from the nutrient solution cultivating gained last time.
Get the last nutrient solution 5ml cultivating gained and carry out gradient dilution ((10 -3, 10 -4, 10 -5, 10 -6), the nutrient solution 100 μ l got after each dilution coats on the inorganic salt solid medium flat board containing 100mg/L Nicotine, be placed in constant incubator (30 DEG C, cultivate 150rpm), after flat board grows bacterium colony, the each bacterium colony of picking is in containing purifying repeatedly on the LB solid medium flat board of 100mg/L Nicotine, until bacterium colony is single, each bacterium colony after purifying is connected to respectively shaking culture (30 DEG C in LB liquid nutrient medium test tube, 150rpm) spend the night, 3d is cultivated by being connected in enrichment culture liquid after centrifugal for cultured bacterium liquid, the residual quantity of Nicotine in each enrichment culture liquid is detected by reversed-phased high performace liquid chromatographic, finally screen the bacterial strain of acquisition one strain energy effectively degrading nicotine, be designated as bacterial strain T2.
3) identification of strains
The bacterial strain T2 of above-mentioned acquisition is carried out 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, the raw flagellum of end, without gemma, size is about (0.5 ~ 1.0) × and (1.5 ~ 2.0) μm, bacterium colony is smooth, intermediate projections, edge-diffusion, in faint yellow, gramstaining is negative, anti-penbritin, kantlex, gentamicin, paraxin, Streptomycin sulphate.The optimum growth conditions of this bacterial strain: pH value is 7.0, temperature 30 DEG C.This bacterial strain is accredited as Pusillimonas sp. through 16S rDNA sequential analysis (the Genbank number of logging in is KM054873) and belongs to, therefore the minimum Zymomonas mobilis of called after (Pusillimonas sp.) T2, be 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.
Embodiment 2: the preparation of mycetocyte suspension
(1) slant culture: minimum Zymomonas mobilis T2 is inoculated in slant medium, cultivates 3 ~ 5 days for 30 DEG C, obtains thalline inclined-plane; The final concentration of described often liter of slant medium consists of: yeast powder 10.0g, peptone 5.0g, sodium-chlor 10.0g, agar 20.0g, ultrapure water 1000ml, natural pH;
(2) seed culture: picking one transfering loop thalline is seeded in minimal medium from step (1) thalline inclined-plane, cultivates 3 ~ 5 days for 30 DEG C, obtains seed liquor; Described minimal medium final concentration composition is with embodiment 1;
(3) enlarged culturing: seed liquor step (2) obtained is seeded in LB liquid nutrient medium (100ml) with the inoculum size of volumetric concentration 20%, 30 DEG C, 150rpm shaking culture is 0.1 ~ 0.15 to OD600, obtain bacterium liquid, by centrifugal for bacterium liquid (6000rpm, 5min), abandon supernatant, precipitation pH value is the phosphoric acid buffer suspension of 7.0, obtain containing minimum Zymomonas mobilis T2 cell suspension 100mL, the minimum Zymomonas mobilis T2 concentration wherein in cell suspension is 4 × 10 7individual/ml; Described LB liquid nutrient medium final concentration composition is with embodiment 1; PH value is the formula of the phosphoric acid buffer of the 0.2mol/L of 7.0: 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: to the degradation experiment of Nicotine
1) detection of cell concentration and nicotine content in minimal medium:
Thalli growth amount adopts ultraviolet spectrophotometer to detect, and represents by measuring the absorbance of thalline at 600nm place in nutrient solution.
This experiment adopts reversed-phased high performace liquid chromatographic to detect the residual quantity of Nicotine in minimal medium, calculates the content of Nicotine in substratum to be measured according to Nicotine typical curve.RPLC testing conditions: moving phase is methyl alcohol: H 2o=10:90 (volume ratio), analytical column is GraceAlltima C18 chromatographic column (4.6 × 250mm, 5 μm), and flow velocity is 0.8ml/min, and sample size is 20 μ l, and column temperature is 30 DEG C.
Dissolved by Nicotine standard substance sterilized water, at reversed-phase liquid chromatography testing standard curve, as shown in Figure 2, typical curve equation is y=20342x+1.6E+5, R to Nicotine typical curve 2=0.9989, y is peak area, and x is nicotine concentration, mg/L).
2) nicotine degradation experiment:
A, get 4 250ml Erlenmeyer flasks, add 100ml minimal medium respectively, high pressure steam sterilization (121 DEG C, Nicotine is added 20min), Nicotine final concentration is made to be 100mg/L, what each Example 2 method obtained contains minimum Zymomonas mobilis T2 cell suspension 5ml, is inoculated in respectively in this minimal medium, makes minimum Zymomonas mobilis T2 final concentration be 2 × 10 6individual/ml minimal medium, respectively at 25,30,35, (pH value is 7.0 to 40 DEG C of cultivation shaking tables, 150rpm, dark), sampling and measuring remains nicotine concentration at regular intervals, and experiment finds, in certain temperature range, in substratum, the residual concentration of Nicotine reduces gradually along with the prolongation of time.Be within the scope of 10h ~ 15h in the time, the degradation rate of Nicotine is maximum, and about 30 DEG C is best degradation temperature, the results are shown in Figure shown in 5.
B, get 6 250ml Erlenmeyer flasks, add 100ml minimal medium respectively, high pressure steam sterilization (121 DEG C, Nicotine is added 20min), Nicotine final concentration is made to be 100mg/L, what each Example 2 method obtained contains minimum Zymomonas mobilis T2 cell suspension 5ml, is inoculated in respectively in this minimal medium, makes minimum Zymomonas mobilis T2 final concentration be 2 × 10 6individual/ml minimal medium, regulates medium pH 5.5,6.5 respectively, 7.0,7.5,8.0,8.5, shaking table is cultivated (30 DEG C, 150rpm, dark), sampling and measuring remains nicotine concentration at regular intervals, and experiment shows, pH value is too high or too low all has impact to the degradation efficiency of bacterial strain, optimum pH is 7.0, the results are shown in Figure shown in 6.
C, get 3 250ml Erlenmeyer flasks, all add 100ml minimal medium, high pressure steam sterilization (121 DEG C, Nicotine is added 20min), Nicotine final concentration is made to be 100mg/L, what each Example 2 method obtained contains minimum Zymomonas mobilis T2 cell suspension 5ml, is inoculated in respectively in this minimal medium, makes minimum Zymomonas mobilis T2 final concentration be 2 × 10 6individual/ml minimal medium, arranges 3 experiments not containing this bacterial classification accordingly as blank, is then together placed in dark shaking culture in shaking table (30 DEG C, pH value is 7.0,150rpm, dark).Sample at regular intervals in the training period, detect the increment of thalline and the residual quantity of Nicotine in minimal medium according to above-mentioned detection method, the results are shown in Figure 3, shown in Fig. 4.
To the degradation curve of the Nicotine of 100mg/L concentration as shown in Figure 3, as shown in Figure 4, Fig. 4 can find out OD to the growth curve of thalline to bacterial strain of the present invention 6000.18 is increased to from 0.07, explanation thalli growth is good, observe Fig. 3, can find, after cultivating 14h, the degradation rate of nicotine degradation bacterium of the present invention to the Nicotine of 100mg/L is close to 100%, and in reaction solution, the quality residual quantity of Nicotine is 0, and all percent hydrolysis of blank after 22h not adding bacterium are all less than 5%.
D, get 5 250ml Erlenmeyer flasks, add 100ml minimal medium respectively, high pressure steam sterilization (121 DEG C, Nicotine is added 20min), Nicotine final concentration is made to be respectively 100mg/L, 200mg/L, 500mg/L, 1000mg/L and 1500mg/L, what each Example 2 method obtained contains minimum Zymomonas mobilis T2 cell suspension 5ml, is inoculated in respectively in this minimal medium, makes minimum Zymomonas mobilis T2 final concentration be 2 × 10 6individual/ml minimal medium, regulates medium pH 7.0 respectively, shaking table is cultivated (30 DEG C, 150rpm, dark), sampling and measuring remains nicotine concentration at regular intervals, the results are shown in Figure shown in 7.
Experimental result shows this bacterial classification centering, the Nicotine of high density (see Fig. 7) has extraordinary degradation capability, but when nicotine concentration is higher than 1500mg/L, the degradation capability of Nicotine significantly declines, and degradation cycle is longer, and this bacterial classification is novel nicotine degradation bacterium, therefore, this bacterium has very large promoter action to the degradation pathway of research Nicotine and degrading genes, especially has larger positive effect to the concentrated reparation of Nicotine to the degraded of Nicotine in environment.
Embodiment 4 nicotine degradation
Get 250ml Erlenmeyer flask, add 100ml minimal medium, high pressure steam sterilization (121 DEG C, Nicotine is added 20min), Nicotine final concentration is made to be respectively 100mg/L, what Example 2 method obtained contains minimum Zymomonas mobilis T2 cell suspension 5ml, is inoculated in this minimal medium, makes minimum Zymomonas mobilis T2 final concentration be 2 × 10 6individual/ml minimal medium, regulates medium pH 7.0, shaking table is cultivated (30 DEG C, 150rpm, dark) 15h, getting the residual nicotine concentration of nutrient solution mensuration is 0.09mg/L, and result shows that Nicotine quality residual quantity is 0.09%.

Claims (6)

1. minimum Zymomonas mobilis (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. the application of minimum Zymomonas mobilis T2 in microbiological deterioration Nicotine as claimed in claim 1.
3. apply as claimed in claim 2, it is characterized in that described being applied as: by minimum Zymomonas mobilis T2 through fermentation culture obtain containing fermented liquid centrifugal, precipitation pH value be 7.0 phosphoric acid buffer suspend make bacteria suspension as enzyme source, take Nicotine as substrate, in minimal medium, 25 ~ 45 DEG C, pH value 5.5 ~ 8.5,100 ~ 200rpm, carry out DeR under dark condition, after reacting completely, realize the degraded to Nicotine.
4. apply as claimed in claim 3, it is characterized in that described Nicotine final concentration is 100 ~ 1500mg/L substratum, described enzyme source consumption counts 1 × 10 with mycetocyte number 6~ 5 × 10 6individual/mL substratum.
5. apply as claimed in claim 3, it is characterized in that in described often liter of minimal medium containing NaCl 1g, K 2hPO 41.5g, KH 2pO 40.5g, (NH 4) 2sO 41.5g, MgSO 40.1g, 1ml trace element solution, solvent is ultrapure water, natural ph; Containing MnSO in often liter of trace element solution 4h 2o 0.13 g, ZnCl 20.23g, CuSO 4h 2o 0.03g, CoCl 26H 2o 0.42g, Na 2moO 42H 2o 0.15g, AlCl 36H 2o 0.05g, solvent is ultrapure water.
6. apply as claimed in claim 3, it is characterized in that described enzyme source is prepared as follows:
(1) slant culture: minimum Zymomonas mobilis T2 is inoculated in slant medium, cultivates 3 ~ 5 days for 20 ~ 40 DEG C, obtains thalline inclined-plane; The final concentration of described slant medium consists of: yeast powder 10.0g/L, peptone 5.0g/L, sodium-chlor 10.0g/L, agar 20.0g/L, and solvent is ultrapure water, pH value nature;
(2) seed culture: picking one transfering loop thalline is seeded in minimal medium from step (1) thalline inclined-plane, cultivates 3 ~ 5 days for 25 ~ 40 DEG C, obtains seed liquor; Described often liter of minimal medium consists of: NaCl 1g, K 2hPO 41.5g, KH 2pO 40.5g, (NH 4) 2sO 41.5g, MgSO 40.1g, 1ml trace element solution, solvent is ultrapure water, natural ph; Often liter of trace element solution consists of: MnSO 4h 2o 0.13 g, ZnCl 20.23g, CuSO 4h 2o 0.03g, CoCl 26H 2o 0.42g, Na 2moO 42H 2o 0.15g, AlCl 36H 2o 0.05g, solvent is ultrapure water;
(3) fermentation culture: seed liquor step (2) obtained is seeded in LB liquid nutrient medium with the inoculum size of volumetric concentration 10 ~ 20%, 30 DEG C, 150rpm shaking culture is 0.1 ~ 0.15 to OD600, obtain fermented liquid, fermented liquid is centrifugal, abandon supernatant liquor, precipitation pH value be 7.0 phosphoric acid buffer suspend make bacteria suspension, be enzyme source; Described often liter of LB liquid nutrient medium consists of: yeast powder 10.0g, peptone 5.0g, sodium-chlor 10.0g, and solvent is ultrapure water, natural ph.
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