CN107446856A - Occupy the application of larva Providence bacterial strain, enzyme preparation and its degrading pesticide residues - Google Patents
Occupy the application of larva Providence bacterial strain, enzyme preparation and its degrading pesticide residues Download PDFInfo
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- CN107446856A CN107446856A CN201710759539.9A CN201710759539A CN107446856A CN 107446856 A CN107446856 A CN 107446856A CN 201710759539 A CN201710759539 A CN 201710759539A CN 107446856 A CN107446856 A CN 107446856A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
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Abstract
The invention discloses the application for occupying larva Providence bacterial strain, enzyme preparation and its degrading pesticide residues.The invention provides the bacterial strains of larva Providence (Providencia vermicola) FY 4 are occupied, its deposit number is CGMCC No.13520.Present invention also offers the screening of bacterial strain, induction, domestication and agricultural chemicals pesticide residual degradation organized enzyme preparation method, i.e., carries out purpose bacterium screening by carbon source of chlopyrifos, and purpose bacterium is carried out into inducing and acclimating in single agricultural chemicals and blended.Bacterial strain is fermented, collects thalline, breaks born of the same parents, centrifugation, supernatant can prepare pesticide residual degradation enzyme preparation, available for the degraded of vegetables and fruits residues of pesticides, have application potential at the degraded residual aspect of vegetables and fruits agriculture.
Description
Technical field
The present invention relates to biotechnology degrading pesticide residues field, more particularly to occupy larva Providence
(Providencia vermicola) bacterial strain screening, inducing and acclimating and the application in removal vegetables and fruits Pesticide Residues.
Background technology
As the kind of agricultural chemicals, yield and usage amount increase severely in recent years, mixing is using a variety of agricultures in agricultural production process
The phenomenon of medicine happens occasionally and more seriously, causes Multiple Pesticides to appear in identical product.The residual exceeded vegetables water of edible agriculture
Fruit can endanger nerve center, make Nerve conduction disorderly；Can Mutation induction, cause deformity, influence offspring health；Liver can be induced
The change of dirty enzyme, liver enlargement is made so that necrosis；Kidney can be invaded and cause lesion.Remains of pesticide is accumulated in human body, exceedes
Some chronic diseases can be caused after certain limit, such as muscle is numb, cough, or even can induce vascular diseases, diabetes and cancer
Disease etc..
Bioremediation technology has occurred since being the eighties and the removing developed and the biotechnology curbed environmental pollution,
It mainly using the ability of biospecific decomposition poisonous and harmful substance, the pollutant gone in depollution environment such as soil, reaches
Remove the purpose of environmental pollution.Microorganism has stronger adaptation and the ability tamed, by certain adaptation process, micro- life
Thing produces corresponding enzyme system, or by the new enzyme system of the foundation such as gene mutation come new agricultural chemicals of degrading.
Food security is improved to be significant, but at present, there is not yet utilizing bacterial strain or the enzyme preparation being made from it
The report of blended residual in degraded vegetables and fruits.
The content of the invention
It is an object of the present invention to provide through screening, agricultural chemicals induction, domestication obtain one plant occupy larva Providian this
Bacteria strain.
It is provided by the invention to occupy larva Providence (Providencia vermicola) bacterial strain (being designated as FY-4),
Its deposit number is CGMCC No.13520.
It is described occupy larva Providence (Providencia vermicola) bacterial strain through screen purpose bacterial strain and
Purpose bacterial strain is obtained through single agricultural chemicals and blended induction, domestication, comprised the following steps：
1) by the purpose bacterial strain rejuvenation of screening；
2) culture medium containing different active ingredient concentration agricultural chemicals is prepared using agricultural chemicals chlopyrifos, by the purpose of step 1) rejuvenation
Bacterial strain streak inoculation, culture and by continuous induction culturing (by improving constantly pesticide concentration), cultivate be resistant to it is higher
Chlopyrifos concentration aimed strain；
3) pesticide carbendazim, carbofuran and lambda-cyhalothrin isoconcentration are mixed, and added after being diluted to finite concentration
Enter in culture medium, be configured to the culture medium containing different effective ingredient concentration agricultural chemicals.The aimed strain that step 2) is obtained again is rule
Inoculation, culture and by continuous induction culturing (by improving constantly pesticide concentration), cultivation obtain being resistant to higher agricultural chemicals dense
The bacterial strain of degree, final separation obtain one plant and occupy larva Providence (Providencia vermicola) bacterial strain (being designated as
FY-4)。
Second object of the present invention is to provide a kind of method for producing biodegradable enzyme.
The method of production biodegradable enzyme provided by the invention, comprises the following steps：By it is above-mentioned occupy larva Providian this
After bacterium (Providencia vermicola) bacterial strain FY-4 fermentations, thalline, broken born of the same parents, centrifugation are collected, supernatant is extracted, is given birth to
Thing degraded enzyme solutions.
In the method for above-mentioned production biodegradable enzyme, the condition of the fermentation is to be cultivated in 34-39 DEG C, 180-200rpm
16-24 hours.
In the method for above-mentioned production biodegradable enzyme, the component for the fermentation medium that the fermentation uses is as follows：Every liter institute
Stating fermentation medium includes tryptone 15-19g, phytone 1-5g, sodium chloride 2-5g, dipotassium hydrogen phosphate 2-5g and grape
Sugared 2-5g, volume is supplied with water；The pH value of fermentation medium is 6.5-7.5, preferably 6.8-7.2.
In the method for above-mentioned production biodegradable enzyme, the fermentation specifically includes following steps：Larva Providian will be occupied
This bacterium (Providencia vermicola) bacterial strain FY-4 strain obtains seed by expanding culture, seed is seeded to described
Cultivated in fermentation medium under conditions of the fermentation.
The condition for expanding culture is to cultivate 16-24h in 34-39 DEG C, 180-200rpm.
The strain is by the above-mentioned bacterium for occupying larva Providence (Providencia vermicola) bacterial strain FY-4
Body is suspended in the solution obtained in phosphate buffer.
In the method for above-mentioned production biodegradable enzyme, after the fermentation, by tunning in 12000-14000rpm, temperature
4-6 DEG C of degree, 15-25min is centrifuged, collect thalline, height crushes born of the same parents after thalline is resuspended, then in 8000-10000rpm, temperature 4-
6 DEG C, 50-65min is centrifuged, supernatant is collected, by supernatant liquid filtering, obtains biodegradable enzyme solutions.
Third object of the present invention is to provide a kind of biodegradable enzyme preparation.
Biodegradable enzyme preparation provided by the invention, its active component occupy larva Providence to be above-mentioned
(Providenciavermicola) bacterial strain FY-4 or above-mentioned biodegradable enzymes.
Fourth object of the present invention, which is to provide, above-mentioned occupies larva Providence (Providencia vermicola)
Bacterial strain FY-4, biodegradable enzyme and biodegradable enzyme preparation answering in pesticide residual degradation, the particularly degraded of vegetables and fruits residues of pesticides
With.
The concentration of the biodegradable enzyme or biodegradable enzyme preparation in vegetables and fruits chemical residual degradation is 2.5-
15ppm, optimal is 2.5-7.5ppm, different because of specific vegetables and fruits.
Beneficial effects of the present invention are embodied in：
The experiment proves that present invention induction, domestication obtain occupying larva Providence (Providencia
Vermicola) bacterial strain FY-4, extraction endocellular enzyme can be used as biodegradable enzyme, the biodegradable enzyme after fermented and cultured is carried out to it
The residues of pesticides that may act in vegetables and fruits, make Multiple Pesticides residue degrading, reach the effect for removing residues of pesticides, in residues of pesticides
Degraded field has application potential.
Brief description of the drawings
Fig. 1 is the development dendrogram for occupying larva Providence (Providencia vermicola) bacterial strain FY-4.
Fig. 2 is the chlopyrifos tolerance for occupying larva Providence (Providencia vermicola) bacterial strain FY-4
Curve.
Fig. 3 is the blended tolerance for occupying larva Providence (Providencia vermicola) bacterial strain FY-4
Linearity curve.
Fig. 4 is based on the liquid biological for occupying larva Providence (Providencia vermicola) bacterial strain FY-4
The most suitable action pH curve of catabolic enzyme preparation.
Fig. 5 is based on the liquid biological for occupying larva Providence (Providencia vermicola) bacterial strain FY-4
Catabolic enzyme preparation optimum temperature curve.
Fig. 6 is based on the liquid biological for occupying larva Providence (Providencia vermicola) bacterial strain FY-4
Catabolic enzyme preparation temperature stabilization linearity curve.
Fig. 7 is based on the liquid biological for occupying larva Providence (Providencia vermicola) bacterial strain FY-4
The result of the residues of pesticides of catabolic enzyme preparation processing pakchoi.
Embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.Embodiment facilitates a better understanding of this hair
It is bright, but do not limit the present invention.Test material used, is routine biochemistry reagent unless otherwise specified in embodiment.Implement
% in example, is weight/mass percentage composition unless otherwise specified.Quantitative test in embodiment, it is respectively provided with and repeats to test three times,
Results averaged.Rotating speed in embodiment, it is the rotating speed centrifuged under radius in radius for 5.5cm.
Colorimetric method for determining bacterial number, i.e. turbidity counting method are used in embodiment；With the light absorption value OD of culture600Table
Show the increment of bacterium.
Example 1, occupy the identification of larva Providence strain isolation
First, the acquisition of bacterial strain
1st, the collection of pedotheque
It is collected in insecticide factory's floss hole sludge.
2nd, the separation screening of bacterial strain
Sludge 10g is weighed in 100mL inorganic salt liquid culture mediums, inorganic salt liquid medium component is as follows：
MgSO4·7H2O 0.2g；K2HPO40.1g；(NH4)2SO40.1g；CaSO40.04g；FeSO4·7H2O
0.001g；Deionized water 1L；pH 7.0.121 DEG C of sterilizing 30min.
The chlopyrifos that concentration is 100mg/L is added in inorganic salt liquid culture medium, nutrient solution is obtained, in 37 DEG C, 180r/
Min shaking table cultures, it is so anti-in 10% inoculum concentration access fresh medium, to transfer weekly once later after cultivating one week
Multiple repeatedly domestication.Finally, isolated and purified, be inoculated into the LB culture mediums containing 100ppm chlopyrifos with plate dilution method,
Select and grow best inoculation inclined-plane, numbering, which preserves, to be used to tame.
2nd, the identification of bacterial strain
Specific authentication step is as follows：Extraction numbering preserves the STb gene of bacterial strain and is used as template, using universal primer ITS1
(5 '-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATG-3 '), PCR expand to obtain it
ITS, 718bp nucleotide sequence (referring to SEQ.ID.NO.1), sequence analysis analysis shows are obtained through sequencing：Numbering
Preserve bacterial strain and occupy larva Providence (Providencia vermicola) homology highest, Phylogenetic
As shown in Figure 1.
Example 2, occupy larva Providence bacterial strain inducing, domestication
First, chlopyrifos induction, domestication
1. the active constituent content section that chlopyrifos is prepared：300mg/L-800mg/L.Prepare the chlopyrifos containing various concentrations
Tryptone agar.Different amounts of chlopyrifos is added in tryptone agar respectively the culture medium containing chlopyrifos is made.
Specific steps：1) chlopyrifos is configured to 4500mg/L pesticide standard liquid.The pesticide standard liquid of 4500mg/L concentration is taken respectively
13.3mL, 17.8mL, 22.2mL, 26.6mL, 31.1mL, 35.6mL are added in 200mL tryptone agars, are configured to contain and are poisoned with poison
Tick 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L and 800mg/L tryptone agar culture medium.Flat board system
Make：By the tryptone agar pour plate of the chlopyrifos containing 300mg/L-800mg/L prepared, 15mL/ wares, solidification to be cooled
After use.
Tryptone agar composition is：Per L tryptones containing 16g, 5g phytones, 3.5g sodium chloride and 15g fine jades
Fat (pH is 7.3 ± 0.2).
2. all purposes bacterial strain is distinguished into streak inoculation in middle 300mg/L flat board.
3. the flat board being inoculated with is placed in 37 DEG C of constant incubators, 24h is cultivated.
4. then by the flat board of cultured bacterial strain renewed vaccination to 400mg/L.The flat board being inoculated with is placed in 37 DEG C of perseverances
In warm incubator, cultivate 24 hours.
5. progressively continuing to cultivate in 500mg/L, 600mg/L, 700mg/L and 800mg/L flat board, finally obtain and be resistant to
The aimed strain of 800mg/L organophosphorus pesticides (chlopyrifos).Agricultural chemicals chlopyrifos tolerance is as shown in Figure 2.
2nd, blended induction, domestication
1. blended dilution process
Carbendazim, carbofuran and lambda-cyhalothrin are configured to phosphate buffer (0.03mol/L, pH7.2) respectively
The solution of 6.2% concentration.
2. being 6.2% three kinds of the pesticide solutions by active constituent content, respectively take 3.60mL to be well mixed, obtain mixing agriculture
Medicine.
3. blended 0.97mL, 1.30mL, 1.61mL, 1.94mL, 2.26mL, 2.58mL is taken to be separately added into 200mL pancreases
In peptone agar, it is well mixed, is configured to 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L and 800mg/L
Blended tryptone agar culture medium, and flat board is made.
4. the aimed strain for being resistant to 800mg/L organophosphorus pesticides (chlopyrifos) is transferred to the flat of 300mg/L blendeds
In plate.The flat board being inoculated with is placed in 37 DEG C of constant incubators, cultivated 24 hours.
5. stepping up blended concentration in flat board, continue to induce, tame, obtain one plant and be resistant to the prominent of high blended
Become bacterial strain, be designated as FY-4.The tolerance of blended is as shown in Figure 3.
It is common that above-mentioned bacterial strains FY-4 has been preserved in China Committee for Culture Collection of Microorganisms on December 30th, 2016
(abbreviation CGMCC, address are at microorganism center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number CGMCC
No.13520, Classification And Nomenclature are to occupy larva Providence (Providencia vermicola).
6. rinsed with sterile phosphate buffer by obtained FY-4 thalline are tamed from culture dish, with repeatedly from
The method washing thalline of heart resuspension 3 times, weighs, by thalline and pH7.2 phosphate buffer according to 0.1g:500 μ L ratio
Again suspension thalline.By 50% glycerine water solution of sterilizing and bacterium solution by volume 1:Dispensed after 1 mixing, often the μ L of pipe 500,4 DEG C
Refrigerated in refrigerator.
Example 3, prepare biodegradable enzyme preparation using occupying larva Providence bacterial strain FY-4
First, liquid biological catabolic enzyme preparation obtains
1. strain amplification cultivation：The strain of refrigeration is taken to be inoculated into the sterile TSB culture mediums of 100mL, 37 DEG C, 180rpm trainings
Support 24h, as first order seed；First order seed is forwarded in 1L TSB culture mediums by 10% inoculum concentration, 37 DEG C, 180rpm trainings
Support 24 hours, as secondary seed.Secondary seed is forwarded in 10L industrial culture medium by 10% inoculum concentration, 37 DEG C,
180rpm cultivates 24h, as three-level seed.
2. three-level seed is inoculated into fermentation tank, inoculum concentration is the 5% of fermentation medium.It is 2.5m to control throughput3/
H, temperature be 37 DEG C, pH be 7.2 ± 0.4, rotating speed 180rpm, every when sample, determine OD600And pH, centre is according to foam situation
Sterile defoamer is added in right amount.Lower tank after fermenting 18 hours.
3. by zymotic fluid high speed centrifugation (14000rpm, 4 DEG C of temperature) 15-20min in fermentation tank, liquid is abandoned, collects bacterium
Body.Thalline is pressed 1 with phosphate buffer:10 (g/mL) suspend again, obtain bacteria suspension.
4. using high pressure cell cracker, in 4-6 DEG C of 2.5-5.0MPa, temperature, the resuspension thalline in bacteria suspension is crushed, is crushed
Twice, broken liquid is obtained.
5. broken liquid is in 8000rpm, 4 DEG C of centrifugation 50min of temperature, supernatant is biodegradable enzyme crude enzyme liquid.
6. filtering：First with 6-8 layers filter paper filtering crude enzyme liquid, then with aperture it is 0.45 μm of membrane filtration, you can must clarify
Biodegradable enzyme solutions.
7. stabilization processes：Biodegradable enzyme solutions are diluted 100 times with phosphate buffer (pH7.2), add 0.01-
0.02% polyhexamethylene guanide, then be thoroughly mixed together in equal volume with the glycerine after sterilizing, 4 DEG C of preservations.
The component of the industrial culture medium (i.e. fermentation medium) is as follows：Fermentation medium is by tryptone described in per L
16g、
Phytone 5g, sodium chloride 3.5g, dipotassium hydrogen phosphate 4.5g, glucose 4.5g and water composition, body is supplied with water
Product.
2nd, biodegradable enzyme enzymatic property is studied
1. Optimun pH
Detect the difference of enzyme activity under condition of different pH：Respectively with different pH phosphate buffer (pH 4.0,5.0,
6.0th, 7.0 100ppm chlopyrifos solution, 8.0,9.0,10.0) is prepared, above-mentioned biodegradable enzyme under each pH value is determined at 37 DEG C
The enzyme activity of solution.Enzyme activity is defined as：Chlopyrifos, the enzyme amount that 1min degrades needed for 1 μm of ol chlopyrifos are hydrolyzed under the conditions of 37 DEG C
For an enzyme activity unit (U).
Using highest enzyme activity as 100%, the ratio of enzyme activity and highest enzyme activity under other pH is enzyme activity, with
PH value is abscissa, and enzyme activity is that ordinate maps (Fig. 4).As a result show, occupy larva Providence bacterial strain FY-4
The biodegradable enzyme solutions of gained are 7.0 to the most suitable action pH of chlopyrifos as liquid biological catabolic enzyme preparation after fermentation.
2. optimum temperature
Detect the difference of enzyme activity under condition of different temperatures：Respectively different reaction temperature (30 DEG C, 35 DEG C, 40 DEG C, 45
DEG C, 50 DEG C, 55 DEG C) and pH 7.0 under the conditions of determine enzyme activity.
Using highest enzyme activity as 100%, the ratio of enzyme activity and highest enzyme activity at other temperature is relative enzyme activity
Power, using temperature as abscissa, enzyme activity is that ordinate maps (Fig. 5).As a result show, occupy larva Providence FY-4
The biodegradable enzyme solutions of gained are 40 to the optimum temperature of chlopyrifos as liquid biological catabolic enzyme preparation after strain fermentation
℃。
3. temperature stability
To occupy the biodegradable enzyme solutions of gained after larva Providence bacterial strain FY-4 fermentations be respectively placed in 30 DEG C, 40
DEG C, held for some time (1h, 2h, 4h, 6h) in 50 DEG C of water-baths, then after measure insulation under conditions of 7.0,40 DEG C of pH
The enzyme activity of biodegradable enzyme solutions, to preserve the enzyme activity (highest enzyme activity) of biodegradable enzyme solutions at 4 DEG C as 100%,
The enzyme activity of biodegradable enzyme solutions and highest enzyme activity ratio are enzyme activity after warm, using soaking time as horizontal seat
Mark, enzyme activity are that ordinate maps (Fig. 6).As a result show, occupy gained after larva Providence bacterial strain FY-4 fermentations
Biodegradable enzyme solutions gradually reduce with the increase enzyme activity of soaking time.
The application of example 4, biodegradable enzyme preparation in vegetables (fruit) residues of pesticides of degrading
More representational vegetables pakchoi is bought from wholesale vegetable market (Shouguang) to be tested, and first obtains example 3
The experimental liquid that enzyme preparation concentration is 2.5-5ppm is configured to pure water to biodegradable enzyme solutions (enzyme preparation), in 37-43 DEG C of perseverance
Pakchoi is soaked in 3-5min in experimental liquid under tepidarium, sending third party testing agency, (promise peace strength can commodity detection (green grass or young crops
Island) Co., Ltd) detected.Pakchoi chemical residual degradation species (carbendazim, carbofuran, 3- hydroxyls carbofuran, chlorine fluorine cyanogen
Chrysanthemum ester, Difenoconazole, dimethomorph, imidacloprid etc.) and effect it is as shown in Figure 7.
Sequence table
<110>Xi'an Rutgers bio tech ltd
<120>Occupy the application of larva Providence bacterial strain, enzyme preparation and its degrading pesticide residues
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 718
<212> DNA
<213> Providencia vermicola
<400> 1
gcctatcaag tggtagcgcc ctcccgaagg ttaagctacc tacttctttt gcaacccact 60
cccatggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgt agcattctga 120
tctacgatta ctagcgattc cgacttcatg gagtcgagtt gcagactcca atccggacta 180
cgacgtactt tatgagttcc gcttgctctc gcgaggtcgc ttctctttgt atacgccatt 240
gtagcacgtg tgtagcccta ctcgtaaggg ccatgatgac ttgacgtcat ccccaccttc 300
ctccggttta tcaccggcag tctcctttga gttcccgacc gaatcgctgg caacaaagga 360
taagggttgc gctcgttgcg ggacttaacc caacatttca caacacgagc tgacgacagc 420
catgcagcac ctgtctcaga gttcccgaag gcaccaaagc atctctgcta agttctctgg 480
atgtcaagag taggtaaggt tcttcgcgtt gcatcgaatt aaaccacatg ctccaccgct 540
tgtgcgggcc cccgtcaatt catttgagtt ttaaccttgc ggccgtactc cccaggcggt 600
cgatttaacg cgttagctcc gaaagccact cctcaaggga acaaccttca aatcgacatc 660
gtttacagcg tggactacca ggggtatcta atcctgtttg ctccccacgc tttcgcaa 718
<210> 2
<211> 19
<212> DNA
<213>Artificial synthesized ()
<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 19
<212> DNA
<213>Artificial synthesized ()
<400> 3
tcctccgctt attgatatg 19
Claims (10)
1. one kind occupies larva Providence (Providencia vermicola) bacterial strain, it is characterised in that：The guarantor of the bacterial strain
It is CGMCC No.13520 to hide numbering.
A kind of 2. biodegradable enzyme, it is characterised in that：The biodegradable enzyme is to utilize to occupy larva Providence
(Providencia vermicola) bacterial strain extracts thalline endocellular enzyme and manufactured, the guarantor of the bacterial strain after carrying out fermented and cultured
It is CGMCC No.13520 to hide numbering.
A kind of 3. preparation method of biodegradable enzyme, it is characterised in that：Comprise the following steps：Larva Providence will be occupied
After (Providencia vermicola) bacterial strain is fermented, thalline, broken born of the same parents, centrifugation are collected, supernatant is extracted, obtains biology
Degraded enzyme solutions, the deposit number of the bacterial strain is CGMCC No.13520.
A kind of 4. preparation method of biodegradable enzyme according to claim 3, it is characterised in that：The culture that the fermentation uses
Base includes tryptone 15-19g/L, phytone 1-5g/L, sodium chloride 2-5g/L, dipotassium hydrogen phosphate 2-5g/L and glucose
2-5g/L, Medium's PH Value 6.5-7.5.
A kind of 5. preparation method of biodegradable enzyme according to claim 3, it is characterised in that：The condition of the fermentation be in
34-39 DEG C, 180-200rpm culture 16-24 hours.
A kind of 6. preparation method of biodegradable enzyme according to claim 3, it is characterised in that：After the fermentation, it will send out
Ferment product centrifuges 15-25min in 12000-14000rpm, collects thalline, height crushes born of the same parents after thalline is resuspended, then in 8000-
10000rpm centrifuges 50-65min, collects supernatant, by supernatant liquid filtering, obtains biodegradable enzyme solutions.
A kind of 7. preparation method of biodegradable enzyme according to claim 6, it is characterised in that：The specific steps of the filtering
For by supernatant, successively through filter paper and 0.45 μm of membrane filtration, it is biodegradable enzyme solutions to obtain supernatant liquid.
A kind of 8. biodegradable enzyme preparation, it is characterised in that：The active component of the enzyme preparation is to occupy larva Providence
(Providencia vermicola) bacterial strain or the biodegradable enzyme by the bacterial strain biosynthesis, the deposit number of the bacterial strain
For CGMCC No.13520.
Dropped 9. one kind occupies larva Providence (Providencia vermicola) bacterial strain as claimed in claim 1
Solve the application in residues of pesticides.
A kind of 10. application of the biodegradable enzyme preparation in degrading pesticide residues as claimed in claim 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710759539.9A CN107446856B (en) | 2017-08-30 | 2017-08-30 | Providencia juveniles strain, enzyme preparation and application thereof in degrading pesticide residue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710759539.9A CN107446856B (en) | 2017-08-30 | 2017-08-30 | Providencia juveniles strain, enzyme preparation and application thereof in degrading pesticide residue |
Publications (2)
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994488A (en) * | 2011-12-20 | 2013-03-27 | 辽宁中科生物工程有限公司 | Pesticide degradation enzyme preparation |
| CN104531582A (en) * | 2014-12-29 | 2015-04-22 | 北京市农林科学院 | Strain for degrading pendimethalin pesticide and fungicide and application of strain |
| CN104894000A (en) * | 2015-02-09 | 2015-09-09 | 暨南大学 | Application of Providencia sp. 2D in remediation of dibutyl phthalate contaminated soil |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994488A (en) * | 2011-12-20 | 2013-03-27 | 辽宁中科生物工程有限公司 | Pesticide degradation enzyme preparation |
| CN104531582A (en) * | 2014-12-29 | 2015-04-22 | 北京市农林科学院 | Strain for degrading pendimethalin pesticide and fungicide and application of strain |
| CN104894000A (en) * | 2015-02-09 | 2015-09-09 | 暨南大学 | Application of Providencia sp. 2D in remediation of dibutyl phthalate contaminated soil |
Non-Patent Citations (4)
| Title |
|---|
| ANASTASIA EM CHIRNSIDE ET AL: "Isolation of a selected microbial consortium from a pesticide-contaminated mix-load site soil capable of degrading the herbicides atrazine and alachlor", 《SOIL BIOLOGY & BIOCHEMISTRY》 * |
| E EI-BESTAWY ET AL: "Isolation, identification and acclimatization of Atrazine-resistant soil bacteria", 《ANNALS OF AGRICULTURAL SCIENCE》 * |
| M SUREKHA RANI ET AL: "Isolation and characterization of a chlorpyrifos-degrading bacterium from agricultural soil and its growth response", 《AFRICAN JOURNAL OF MICROBIOLOGY RESEARCH》 * |
| 李增智 等: "《食品安全的理论与实践》", 31 December 2005 * |
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