CN103305439B - Stenotrophomonassp. H-4 and preparation method of degrading enzyme preparation thereof as well as application - Google Patents
Stenotrophomonassp. H-4 and preparation method of degrading enzyme preparation thereof as well as application Download PDFInfo
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Abstract
The invention provides stenotrophomonassp. H-4 and a preparation method of degrading enzyme preparation thereof as well as application. The stenotrophomonassp. H-4 has been conserved in the conservation unit specified by the State Intellectual Property Office; the conservation date is February 1, 2013; the name of the conservation unit is China General Microbiological Culture Collection Center; and the conservation number is CGMCC No.7274. The stenotrophomonassp. H-4 can be grown on a culture medium with chlorothalonil as the unique carbon source and energy, and after being cultivated for 7 days, the degradation rate of the stenotrophomonassp. H-4 reaches 83.6%; the enzyme activity is 3.28 U and the degradation rate of the degrading enzyme to the chlorothalonil is 86% when the extraction conditions for crude enzyme in the stenotrophomonassp. H-4 are as follows: ultrasonic powder: 30 W; and ultrasonic time: 5 minutes. Obviously, the degrading enzyme preparation provided by the invention has bright application prospect in clearing away residual pesticides on vegetables and fruits.
Description
Technical field
The invention belongs to microbial technology field, particularly relate to the preparation method and application of a kind of agricultural bactericide m-tetrachlorophthalodinitrile degradation bacteria Stenotrophomonas H-4 and catabolic enzyme preparation thereof.
Background technology
China is large agricultural country, also be agricultural chemicals consumption big country, especially along with the fast development of industrialized agriculture, the high frequency high dosage of a large amount of agricultural chemicals drops into and causes agricultural-food and soil pollution, cause the pesticide residue accident of vegetables and fruits to happen occasionally, have a strong impact on China's vegetables and fruits quality safety.Thus, vegetable and fruit quality security problem has become the important factor of restriction China fruits and vegetables industry development, strengthen fruit vegetables and fruits quality and safety control, to raising fruits and vegetables quality safety level, promote fruits and vegetables industry to develop in a healthy way and ensure that people's life has important practical significance safely.Show according to investigation both at home and abroad, m-tetrachlorophthalodinitrile is the most widely used one in current agricultural bactericide, is widely used in the control of the oidium of facility for prevention and control booth vegetable, Powdery Mildew, gray mold and tomato blight in China.But along with the continuous expansion of facility cultivation area, the residue problem of m-tetrachlorophthalodinitrile in vegetables is more and more outstanding.In order to address this problem, scientific workers have screened some m-tetrachlorophthalodinitrile degradation bacteria from soil, as nitrogen Zymomonas mobilis (Azomonas), Flavobacterium (Flavobacterium), catarrhalis (Moraxella), pseudomonas (Pseudomonas), micrococci (Micrococcus) etc.But, about the Stenotrophomonas germ plasm resource of m-tetrachlorophthalodinitrile of degrading is undiscovered.Report is there is no at present about the degradation effect of Stenotrophomonas catabolic enzyme preparation to vegetables and fruits pesticide residue.In view of this, the present invention separates a strain has good degradation property Stenotrophomonas H-4 to m-tetrachlorophthalodinitrile from soil, have studied the degradation characteristic of this bacterium to m-tetrachlorophthalodinitrile, and the fermentation condition optimization of Stenotrophomonas seed liquor, the optimization of degrading enzyme extracting method and the preparation method of catabolic enzyme preparation in born of the same parents, provide scientific basis and new approaches for developing vegetables and fruits pesticide residue cleaning product further, be widely used potentiality.
Summary of the invention
The technical problem solved: the object of the invention is to for the practical problems in production practice and demand, provide a kind of can the Microbial resources of degrading pesticide fungicide chlorothalonil, this bacterial strain is Stenotrophomonas (Stenotrophomonas sp) H-4(CGMCC No.7247), this bacterium can grow on the substratum taking m-tetrachlorophthalodinitrile as sole carbon source and the energy, and during cultivation 7d, its degradation rate reaches 83.6%; In this mycetocyte, the extraction conditions of crude enzyme liquid is ultrasonic power 30%, and during ultrasonic time 5min, its enzyme activity is 3.28U, and degrading enzyme liquid is 86% to the degradation rate of m-tetrachlorophthalodinitrile.Visible, catabolic enzyme preparation of the present invention has good application prospect to remains of pesticide on cleaning vegetables and fruit.
Technical scheme: a strain Stenotrophomonas (Stenotrophomonas sp.) H-4, this bacterial strain is in depositary institution's preservation that State Intellectual Property Office specifies, preservation date is on February 1st, 2013, depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC No.7247.
The substratum of Stenotrophomonas H-4, often liter of substratum contains in proportion: KH
2pO
40.5g, Na
2hPO
412H
2o 1.2g, NH
4nO
30.5g, MgSO
47H
2o 0.2g, FeSO
40.05g, trace element solution 10mL, agricultural chemicals is 20mg, pH6.8 ~ 7.2.Described agricultural chemicals is m-tetrachlorophthalodinitrile.Trace element often rises containing 50mg Ca (OH)
2, 30mg H
3bO
3, 10mg ZnSO
47H
2o, 5mg MnSO
4h
2o, 5mg CuSO
45H
2o, 5mg Na
2moO
42H
2o.
After bacterial strain H-4 cultivates 48h on LB culture medium flat plate, bacterium colony is faint yellow, circular, clayey is translucent, neat in edge, center slightly projection, moistening smooth (see figure 1).The Physiology and biochemistry qualification result of this bacterial strain is as shown in table 1.As can be seen from Table 1, methyl red, the V-P experiment of bacterial strain H-4 are tested with indoles and are negative, catalase experiment is positive, oxydase experiment is negative, DNA, Vitamin C2 urea and urea can not be hydrolyzed, in process of growth, do not produce hydrogen sulfide and grow and can be suppressed by potassium cyanide, ornithine carboxylase, Methionin carboxylase and arginine decarboxylase react and are positive, and glucose fermentation produces sour aerogenesis.Utilize bacterial 16 S rDNA universal primer to carry out pcr amplification, the length obtaining amplified production after order-checking is 1454bp, and the PCR primer electrophoresis result of the 16S rDNA of bacterial strain H-4 as shown in Figure 2.Compare according to GeneBank sequence homology, bacterial strain H-4 and Stenotrophomonas sp.SeaH-As3w(GenBank accession number No.FJ607350) homology is 99%, in conjunction with its morphological specificity and physio-biochemical characteristics result, be tentatively that Stenotrophomonas belongs to (Stenotrophomonas sp.) by this dientification of bacteria.The phylogenetic tree of the 16S rDNA of this bacterial strain as shown in Figure 3.Bacterial strain H-4 to the degradation dynamic result of m-tetrachlorophthalodinitrile in solution as shown in Figure 4.As can be seen from Figure 4, after access H4 carries out biological degradation, in solution, the content of m-tetrachlorophthalodinitrile significantly reduces, and during cultivation 7d, its degradation rate reaches 83.6%.
The catabolic enzyme preparation of Stenotrophomonas H-4, described zymin is obtained by following method: first by volume Stenotrophomonas is inoculated in beef-protein medium by per-cent 5-10%, and culture temperature is 30-35 DEG C, shaking culture 24-36h; By the centrifugal 10min of Stenotrophomonas suspension 10000r/min after cultivation, the PBS phosphoric acid buffer of gained precipitation pH7.4 washs 2 times, be dissolved in (cell density A600 reaches 15.1) in 20mL PBS phosphoric acid buffer, according to ultrasonic power 10-30W under condition of ice bath, ultrasonic time 5-15min, every ultrasonic 5s interval dwell 5s, then centrifugal, rotating speed 10000r/min, centrifugation time is 10min, and supernatant liquor is degrading enzyme liquid.
The application of described Stenotrophomonas H-4 in degraded vegetables and fruits agricultural bactericide m-tetrachlorophthalodinitrile remains.
As can be seen from Table 4, in this mycetocyte, the extraction conditions of crude enzyme liquid is ultrasonic power 30%, and during ultrasonic time 5min, its enzyme activity is 3.28U, and degrading enzyme liquid is 86% to the degradation rate of m-tetrachlorophthalodinitrile.Visible, catabolic enzyme preparation of the present invention has good application prospect to remains of pesticide on cleaning vegetables and fruits.
Beneficial effect: this degradation bacteria has the ability of degraded m-tetrachlorophthalodinitrile, can be used for pesticide residue on vegetables and fruits and cleans.
Accompanying drawing explanation
Fig. 1 is the colony morphology characteristic of bacterial strain H-4;
Fig. 2 is the PCR primer electrophoresis result of the 16S rDNA of bacterial strain H-4;
Fig. 3 is the phylogenetic tree of the 16S rDNA of bacterial strain H-4;
Fig. 4 is the degradation curve of bacterial strain H-4 to m-tetrachlorophthalodinitrile;
Fig. 5 is the cleaning performance of catabolic enzyme preparation to m-tetrachlorophthalodinitrile in different vegetable.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition and replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
Embodiment 1: the Screening and Identification of m-tetrachlorophthalodinitrile degradation bacteria and degradation characteristic
1.1 materials and methods
1.1.1 for examination material
Basal salt media (degraded substratum, MM): 0.5g KH
2pO
4, 1.2g Na
2hPO
412H
2o, 0.5g NH
4nO
3, 0.2g MgSO
47H
2o, 50mg FeSO
4, (often liter containing 50mg Ca (OH) for 10mL trace element solution
2, 30mg H
3bO
3, 10mg ZnSO
47H
2o, 5mg MnSO
4h
2o, 5mg CuSO
45H
2o, 5mg Na
2moO
42H
2o) ultrapure water is added to 1L.Adjust the pH value of substratum 6.8 ~ 7.2 by 2mol/L HCl and 2mol/L NaOH solution, in water, chlorion is on the impact of experimentation, and substratum adopts ultrapure water preparation.M-tetrachlorophthalodinitrile filtration sterilization adds basal salt media to as sole carbon source;
LB substratum: peptone 10.0g, yeast powder 5.0g, NaCl 10.0g, adds ultrapure water and adjusts the pH value of substratum 6.8 ~ 7.2 to 1L 2mol/L HCl and 2mol/L NaOH solution.
Major chemical: m-tetrachlorophthalodinitrile (Accu Standard) is purchased from Beijing lark prestige Science and Technology Ltd..Anhydrous sodium sulphate (analytical pure) 400 DEG C changes 6h, and it is stand-by that cooling is placed on sealed type storage in full Glass Containers, another standby ammonium sulfate and chromatographically pure normal hexane, and the test kit extracting DNA adopts the E.Z.N.A DNA of bacteria of OMEGA to extract test kit.
1.1.2 the source of degradation bacteria
Take the topsoil of 0-20cm from a certain Experimental Base at Nanjing Soil Inst., Chinese Academy of Sciences's red soil ecological experiment station, load in valve bag and preserve that to take back laboratory stand-by.
1.1.3 the screening of degradation bacteria
The liquid nutrient medium Erlenmeyer flask that it take m-tetrachlorophthalodinitrile as sole carbon source that the topsoil taking 1g is encased in containing 99mL is placed in 30 DEG C, under 120rpm condition after enrichment culture 7d, with the continuous enrichment of the inoculum size of 5%vt, switching 5 times.Getting 200 μ L extent of dilution is 10
-4~ 10
-6bacterium liquid coat on LB solid plate, 30 DEG C of cultivations.After the single bacterium colony of dull and stereotyped upper appearance, picking list bacterium colony is rule and is carried out purifying 3 ~ 4 times, after bacterial strain being inoculated into dibbling method the double-layer plate cultivation 7d that m-tetrachlorophthalodinitrile is sole carbon source after purifying, choosing and occurs that the degradation bacteria that degraded is secretly enclosed analyzes its degradation effect.
1.1.4 the Physiology and biochemistry qualification of bacterial strain H-4
Strain morphology and physio-biochemical characteristics measure reference literature and carry out.Methyl red experiment (M.R. test), voges-Proskauer test (V.P. test), catalase test (catalase test), oxidase test, nitrate reduction test, indole test, liquefy gelatin test, citrate utilization test and glycolysis-test (glucose, lactose).
1.1.5 the 16S rDNA Molecular Identification of bacterial strain
After bacterial strain H-4 being forwarded to LB liquid nutrient medium enlarged culturing 24h, after collected by centrifugation thalline sterilized water washs 3 times, the method provided according to the E.Z.N.A DNA of bacteria extraction test kit of OMEGA extracts thalline STb gene.With bacterial 16 S rDNA amplification universal primer (synthesis of Hua Da gene company limited), pcr amplification is carried out to bacterial strain H4 genome.27F：5′-AGAGTTTGATCCTGGCTCAG-3′；1492R：5′-GGTTACCTTGTTACGACTT-3′。With H-4 STb gene for template, 50 μ L systems are: template 1 μ L, Premix rTaq(comprises Taq enzyme, Buffer, dNTP, Mg
2+) 25 μ L, each 1 μ L of primer (20mmol/L), ultrapure water 22 μ L.Polymerase chain reaction condition: 94 DEG C, 10min; 94 DEG C, 30s; 55 DEG C, 30s; 72 DEG C, 1.5min; Circulate 35 times, 72 DEG C extend 10min.PCR primer through 1.2% sepharose, in 120V electrophoresis 30 minutes.PCR primer adopts TaKaRa Gel Extraction kit by the sequencing fragment (Shanghai Sheng Gong company limited completes) of amplification.16S rDNA sequence in sequencing result and GenBank is carried out BLAST tetraploid rice.
1.1.6 the solution degradation performance analysis of bacterial strain H-4
By the degraded substratum (concentration of m-tetrachlorophthalodinitrile is 20mg/L) of sterilizing, be divided in 100mL Erlenmeyer flask with every bottle of 28.5mL.Single bacterium colony of picking LB flat board activation in 50mL LB liquid nutrient medium, 30 DEG C, 120rpm shaking culture to logarithmic phase, the centrifugal 3min of 5000rpm collects thalline, resuspended after washing 3 times with sterilized water, and regulates OD
600be about 1.0 as the inoculation bacterium liquid of Degrading experiment.To degraded inoculation of medium 1.5mL bacteria suspension, if do not inoculate the process of degradation bacteria for contrast (CK).Centrifugal bottle is placed in 30 DEG C, 120rpm shaking table cultivates 7 days, every day samples, and measures the growing state (OD of concentration that wherein m-tetrachlorophthalodinitrile remains and degradation bacteria
600nm light absorption value).
1.1.7 the abstraction and quantification of m-tetrachlorophthalodinitrile in conversion fluid
Add 0.5g ammonium sulfate after sampling make emulsion splitter and add 2mL(two volumes according in every 1mL sample) chromatographic grade n-hexane extraction, after vortex oscillation 5min being placed in 30 DEG C, the shaking table of 120rpm vibrates 30min, standing 1h is after sample layering is stable, get upper organic phase, extract and merge all organic phases for 3 times afterwards and move on in glass centrifuge tube, and after adding 1.0g anhydrous sodium sulfate dehydration, constant volume carries out GC analysis.Every batch sample establishes 3 groups of panel datas.Detect sample recovery rate with the standard substance of known different concns, its average recovery rate reaches 84% ~ 110%.Show that the method can meet requirement of experiment.
Chromatographic condition: adopt the Varian3800 type chromatographic with electron capture detector and automatic sampler.Chromatographic column: DB-5MS(30m × 0.25mm × 0.25um) temperature programming: initial temperature is 95 DEG C, with 25 DEG C/min gradient increased temperature to 190 DEG C, with 6 DEG C/min gradient increased temperature to 220 DEG C, continue 0.5min, with 30 DEG C/min gradient increased temperature to 280 DEG C, continue 0.7min, detector temperature is 300 DEG C.Splitless injecting-Sample, carrier gas is High Purity Nitrogen, and flow velocity is 4.0mL/min.
Quality control: adopt seven point calibrations to carry out the calibration curve of reference material and external standard method is carried out, carries out data acquisition and processing (DAP) by Starwork Station6.0.
Data processing method: degradation rate (%)=(control treatment strength of solution-experiment process strength of solution)/control treatment strength of solution × 100%.
1.2. result of study
1.2.1 the colony morphology characteristic of bacterial strain H-4
Filter out by separation and purification the degradation bacteria that a strain can be sole carbon source with m-tetrachlorophthalodinitrile, degradation bacteria is numbered H-4.The colony morphology characteristic of bacterial strain H-4 as shown in Figure 1.As can be seen from Figure 1, after H-4 cultivates 48h on LB culture medium flat plate, bacterium colony is faint yellow, circular, clayey is translucent, neat in edge, center are slightly protruding, moistening smooth.
1.2.2 the physiological and biochemical property of bacterial strain H-4
The physio-biochemical characteristics measurement result of bacterial strain H4 is in table 1.1.From table 1.1, methyl red, the V-P experiment of bacterial strain H-4 are tested with indoles and are negative, catalase experiment is positive, oxydase experiment is negative, DNA, Vitamin C2 urea and urea can not be hydrolyzed, in process of growth, do not produce hydrogen sulfide and grow and can be suppressed by potassium cyanide, ornithine carboxylase, Methionin carboxylase and arginine decarboxylase react and are positive, and glucose fermentation produces sour aerogenesis.
The physiological and biochemical property of table 1.1 bacterial strain H-4
Note :-represent reaction result for negative; + represent the reaction result positive;
sour aerogenesis is produced in representative reaction
1.2.3 the Molecular Identification result of bacterial strain H-4
With bacterial strain H4 genomic dna for template, utilize bacterial 16 S rDNA universal primer to carry out pcr amplification, the length obtaining amplified production after order-checking is 1454bp, and the PCR primer electrophoresis result of the 16S rDNA of bacterial strain H4 as shown in Figure 2.Examining order is completed by Shanghai Sheng Gong company limited.Compare according to GeneBank sequence homology, bacterial strain H-4 and Stenotrophomonas sp.SeaH-As3w (GenBank accession number No.FJ607350) homology is 99%, in conjunction with its morphological specificity and physio-biochemical characteristics result, be tentatively that Stenotrophomonas belongs to (Stenotrophomonas sp.) by this dientification of bacteria.Fig. 3 is the phylogenetic tree of the 16S rDNA of this bacterial strain.
1.2.4 the solution degradation performance to m-tetrachlorophthalodinitrile of bacterial strain H-4
Bacterial strain H-4 to the degradation dynamic result of m-tetrachlorophthalodinitrile in solution as shown in Figure 4.As can be seen from Figure 4, after access H-4 carries out biological degradation, find that the content of m-tetrachlorophthalodinitrile in solution significantly reduces, after cultivation 7d, in degraded system, the concentration of m-tetrachlorophthalodinitrile is 3.28mg/L, and its degradation rate reaches 83.6%; Do not inoculating in the control group of degradation bacteria, the concentration of m-tetrachlorophthalodinitrile is 18.41mg/L, and its degradation rate reaches 8.0%.Visible, this bacterial strain H-4 has obvious degradation capability to m-tetrachlorophthalodinitrile.
Embodiment 2: degrading enzyme modifiedly extracted method in the fermentation condition of m-tetrachlorophthalodinitrile degradation bacteria seed liquor and born of the same parents
2.1 materials and methods
2.1.1 for examination material
Strains tested: Stenotrophomonas (being called for short H-4), separation screening obtains [deposit number: CGMCC No.7247] from red soil.First by strain inoculation on LB solid plate substratum, cultivate activation for 28 DEG C, then choose in single bacterium colony access LB liquid nutrient medium and cultivate 24h.
LB substratum (g/L): peptone 10.0, yeast powder 5, NaCl 10, deionized water 1L, pH7.0-7.2,121 DEG C of sterilizing 20min.
PBS phosphoric acid buffer (g/L): NaCl 8, KCl 0.2, Na
2hPO
412H
2o 3.64, KH
2pO
40.24, add ultrapure water to 1L, pH7.4.
2.1.2 seed liquor fermentation orthogonal test
According to Three factors-levels, A: bacterial load (A1=5%vt, A2=8%vt, A3=10%vt); B: culture temperature (B1=30 DEG C, B2=32 DEG C, B3=35 DEG C); C: incubation time (C1=24h, C2=30h, C3=36h), arranges orthogonal table L
9(3
4), adopt the LB liquid nutrient medium of sterilizing, in the mode of shake-flask culture, access Stenotrophomonas seed liquor, is adjusted to 100mL by last volume of culture, cultivates, by measuring cell density, to determine the optimum combination of these factors.
2.1.3 degrading enzyme extracting method in born of the same parents
Adopt the culture condition after optimizing, enlarged culturing Stenotrophomonas, by the centrifugal 10min of bacteria suspension 10000r/min after cultivation, the PBS phosphoric acid buffer of gained precipitation pH7.4 washs 2 times, is dissolved in 20mL PBS phosphoric acid buffer.According to certain condition [A: ultrasonic power (A1=10W, A2=20W, A3=30W) under 4 DEG C of condition of ice bath; B: ultrasonic time (B1=5min, B2=10min, B3=15min)], ultrasonic apparatus smudge cells is used, every ultrasonic 5s interval dwell 5s, then centrifugal (the rotating speed 10000r/min of high speed freezing centrifuge is used, centrifugation time is 10min, 4 DEG C), to remove cell debris, often process repetition 3 times, its supernatant liquor is degrading enzyme liquid in born of the same parents.By thick enzyme after protein content in mensuration crude enzyme liquid and vacuum lyophilization to the degrading enzyme vigor of m-tetrachlorophthalodinitrile, to determine the top condition extracted.
2.1.4 the mensuration of thick enzyme enzyme activity
9.0mL PBS phosphate buffered saline buffer (pH7.4, containing 20mg/L m-tetrachlorophthalodinitrile), adds degrading enzyme liquid in born of the same parents prepared by 1.0mL, after 30 DEG C of reaction 1h, stops enzyme reaction with Tricholroacetic Acid.Often process in triplicate, with the reaction mixture after the prior deactivation of enzyme liquid for contrast, remain m-tetrachlorophthalodinitrile in extraction system, vapor-phase chromatography detects m-tetrachlorophthalodinitrile content in sample, calculates the enzyme activity that different treatment extracts degrading enzyme.Be 1 Ge Meihuo unit (U) with the enzyme amount under 30 DEG C of conditions required for per minute catalysis 1 μm of ol m-tetrachlorophthalodinitrile.
2.2 result of study
2.2.1 the optimization of seed liquor fermentation condition
As can be seen from table 2.1, in 9 test combinations, test 4(A2B2C3) the strain density that produces of fermentation condition maximum, test 3(A1B3C3) basically identical with the strain density of test 4, as can be seen from extreme difference value, fermentation condition to the influence degree of strain density is: C factor >B factor >A factor, and analyzing its best of breed is A2B2C3, namely, under 32 DEG C of conditions, bacterium cultivates 36h by the inoculum size of 8%.
The orthogonal test of table 2.1 seed liquor fermentation
2.2.2 the ultrasonic extraction conditions optimization of degrading enzyme in born of the same parents
Ultrasound condition to the extraction effect of crude enzyme liquid in born of the same parents in table 2.2.From table 2.2, the total protein concentration that 9 methods of testing extract is the highest, for 2.55mg/mL, but to degrading enzyme to the degradation efficiency of m-tetrachlorophthalodinitrile and enzyme activity aspect, the degradation rate of crude enzyme liquid to m-tetrachlorophthalodinitrile that the extracting method of test 7 obtains is up to 86%, its enzyme activity is 3.28U, also higher than other process.Therefore in extraction afterwards, be set to by the ultrasonic extracting method of crude enzyme liquid in born of the same parents: ultrasonic power is set to 30W, ultrasonic time is 5min.
The optimization of thick enzyme ultrasonic extracting method in table 2.2 born of the same parents
Embodiment 3: catabolic enzyme preparation is to the cleaning performance of m-tetrachlorophthalodinitrile in different vegetable
3.1 materials and methods
3.1.1 for examination material
Pollute the preparation of vegetables: adopt the agricultural m-tetrachlorophthalodinitrile pulvis of 75%, cucumber, tomato and green vegetables are immersed molten for 0.5g m-tetrachlorophthalodinitrile pulvis in 10L water, contaminate after 30 minutes, take out vegetables use water and gently rinse, be placed in stink cupboard and dry stand-by.
The preparation of degrading enzyme liquid: adopt the culture condition after optimizing, enlarged culturing Stenotrophomonas, by the centrifugal 10min of bacteria suspension 10000r/min after cultivation, the PBS phosphoric acid buffer of gained precipitation pH7.4 washs 2 times, is dissolved in 20mL PBS phosphoric acid buffer.With ultrasonic apparatus smudge cells (ultrasonic power is 30%) under 4 DEG C of condition of ice bath, ultrasonic time is 5min, every ultrasonic 5s interval dwell 5s, then centrifugal (the rotating speed 10000r/min of high speed freezing centrifuge is used, centrifugation time is 10min, 4 DEG C), to remove cell debris, its supernatant liquor is degrading enzyme liquid.
3.1.2 test method
Test design: crude enzyme liquid 200mL in the born of the same parents of extraction is joined in 1300mL deionized water and is mixed with cleaning solution, then the cucumber polluted by m-tetrachlorophthalodinitrile respectively, green vegetables and tomato sample immerse in scavenging solution, vegetable sample is stirred gently in cleaning process, room temperature with constant is after 30 minutes, and the m-tetrachlorophthalodinitrile after detecting washing in vegetables remains.In contrast, in calculating born of the same parents, crude enzyme liquid is to the degradation rate of m-tetrachlorophthalodinitrile on vegetables for the experimental group of washing with clear water.
3.1.3 the analysis of m-tetrachlorophthalodinitrile in vegetables
After three kinds of vegetables after washing are vacuumized lyophilize, take the sample that 1.00g crosses 60 mesh sieves, add acetone and normal hexane (volume ratio 1:1) the mixed solvent supersound extraction of 30mL, former solvent is replaced with normal hexane by after extracting solution rotary evaporation, extracting solution after replacement, through acidic silica gel column purification, carries out GC analysis by after the sample constant volume after purifying also dilution in certain proportion.Chromatographic condition: adopt the Varian3800 type chromatographic with electron capture detector and automatic sampler.Chromatographic column: DB-5MS(30m × 0.25mm × 0.25um) temperature programming: initial temperature is 95 DEG C, with 25 DEG C/min gradient increased temperature to 190 DEG C, with 6 DEG C/min gradient increased temperature to 220 DEG C, continue 0.5min, with 30 DEG C/min gradient increased temperature to 280 DEG C, continue 0.7min, detector temperature is 300 DEG C.Splitless injecting-Sample, carrier gas is High Purity Nitrogen, and flow velocity is 4.0mL/min.
3.2 result of study
The degrading enzyme liquid of gained of the present invention is compared with clean water, and effect is better, and the m-tetrachlorophthalodinitrile residual quantity after washing on vegetables is lower than clear water washing, and this zymin reaches 86%, 82% and 64% respectively to the degradation rate of m-tetrachlorophthalodinitrile on green vegetables, tomato and cucumber.
Sequence table
<110> Shanghai A Si water enterprise development company limited
Nanjing Soil Inst., Chinese Academy of Sciences
The preparation method and application of <120> Stenotrophomonas H-4 and catabolic enzyme preparation thereof
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> universal primer
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213> universal primer
<400> 2
ggttaccttg ttacgactt 19
Claims (5)
1. a strain Stenotrophomonas (
stenotrophomonas sp.) H-4, this bacterial strain is in depositary institution's preservation that State Intellectual Property Office specifies, and preservation date is on February 1st, 2013, depositary institution's title: China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC No. 7247.
2. the substratum of Stenotrophomonas H-4 according to claim 1, is characterized in that often liter of substratum contains in proportion: KH
2pO
40.5 g, Na
2hPO
412H
2o 1.2g, NH
4nO
30.5 g, MgSO
47H
2o 0.2 g, FeSO
40.05g, trace element solution 10 mL, Chlorothalonil is 20 mg, pH 6.8 ~ 7.2.
3. the substratum of Stenotrophomonas H-4 according to claim 2, is characterized in that: trace element often rises containing 50 mg Ca (OH)
2, 30 mg H
3bO
3, 10 mg ZnSO
47H
2o, 5 mg MnSO
4h
2o, 5 mg CuSO
45H
2o, 5 mg Na
2moO
42H
2o.
4. the catabolic enzyme preparation of Stenotrophomonas H-4 described in claim 1, it is characterized in that described zymin is obtained by following method: first by volume Stenotrophomonas is inoculated in beef-protein medium by per-cent 5-10%, culture temperature is 30-35 DEG C, shaking culture 24-36h; By the centrifugal 10min of Stenotrophomonas suspension 10000r/min after cultivation, the PBS phosphoric acid buffer of gained precipitation pH7.4 washs 2 times, be dissolved in 20mL PBS phosphoric acid buffer, cell density A600 reaches 15.1, according to ultrasonic power 10-30W under condition of ice bath, ultrasonic time 5-15min, every ultrasonic 5s interval dwell 5s, then centrifugal, rotating speed 10000r/min, centrifugation time is 10min, and supernatant liquor is degrading enzyme liquid.
5. the application of Stenotrophomonas H-4 described in claim 1 in degraded vegetables and fruits agricultural bactericide m-tetrachlorophthalodinitrile remains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310199748.4A CN103305439B (en) | 2013-05-27 | 2013-05-27 | Stenotrophomonassp. H-4 and preparation method of degrading enzyme preparation thereof as well as application |
Applications Claiming Priority (1)
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| CN106282070B (en) * | 2016-10-08 | 2019-06-21 | 南开大学 | The screening and application of one plant of polybrominated diphenyl ethers (PBDEs) degradation bacteria |
| CN107916238B (en) * | 2017-10-27 | 2020-06-05 | 青岛农业大学 | Efficient chlorothalonil degrading bacterium and application thereof in greenhouse soil environment |
| CN110791498B (en) * | 2019-08-20 | 2021-09-10 | 中国环境科学研究院 | Immobilization method of stenotrophomonas and composition prepared by same |
| CN116555066B (en) * | 2022-09-06 | 2024-02-20 | 中国科学院南京土壤研究所 | Efficient PBAT agricultural film degrading bacterium and application thereof |
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| CN102115726A (en) * | 2010-12-16 | 2011-07-06 | 中国农业科学院农产品加工研究所 | Stenotrophomonas (sp.) and application thereof |
| CN102250788A (en) * | 2011-05-27 | 2011-11-23 | 清华大学 | Stenotrophomonas maltophilia for degrading high-concentration methylbenzene and application of stenotrophomonas maltophilia |
| CN102296041A (en) * | 2011-08-26 | 2011-12-28 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for efficiently degrading residual pesticide carbendazim and application thereof |
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| CN102115726A (en) * | 2010-12-16 | 2011-07-06 | 中国农业科学院农产品加工研究所 | Stenotrophomonas (sp.) and application thereof |
| CN102250788A (en) * | 2011-05-27 | 2011-11-23 | 清华大学 | Stenotrophomonas maltophilia for degrading high-concentration methylbenzene and application of stenotrophomonas maltophilia |
| CN102296041A (en) * | 2011-08-26 | 2011-12-28 | 中国农业科学院农业资源与农业区划研究所 | Bacterium for efficiently degrading residual pesticide carbendazim and application thereof |
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