CN109679875A - A kind of Dell's Ford bacterium and application - Google Patents
A kind of Dell's Ford bacterium and application Download PDFInfo
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- CN109679875A CN109679875A CN201910045759.4A CN201910045759A CN109679875A CN 109679875 A CN109679875 A CN 109679875A CN 201910045759 A CN201910045759 A CN 201910045759A CN 109679875 A CN109679875 A CN 109679875A
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- phoxim
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- ford bacterium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- 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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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
Abstract
The catabolic enzyme preparation and preparation method thereof of phoxim, specially a kind of Dell's Ford bacterium and application are remained in the technical field utilized the present invention relates to microorganism more particularly to a kind of degradation storage grain and on processing of farm products byproduct.The Dell Ford bacterium, by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number are as follows: CGMCC NO.16844.It is prepared for a kind of enzyme preparation using the bacterial strain, which is used for degrading phoxim for the first time, and Determination of Phoxim Residues can degrade 90%~99% or more.
Description
Technical field
The technical field utilized the present invention relates to microorganism more particularly to a kind of degradation store in grain and processing of farm products
The catabolic enzyme preparation and preparation method thereof of phoxim, specially a kind of Dell's Ford bacterium and application are remained on byproduct.
Background technique
With the disabling of high-toxic pesticide, low-toxin farm chemicals gradually capture market.2015~2016 is new in the examination & approval of Ministry of Industry and Information's pesticide
The phoxim medicine enterprise to issue licence has 455.Pesticide net report organophosphorus insecticide year sales volume in 2015 accounts for insecticide at 19.4 ten thousand tons
Total dosage 64%, and wherein phoxim sales volume first.Phoxim is a kind of less toxic organophosphorus insecticide, in agricultural, fishery and herding
There is use in industry.2015 in 12 kinds of staple crops such as vegetables, wheat phoxim dosage rank the first, be widely used in
Lepidopterous larvae, subterranean pest-insect, sanitary insect pest, stored grain insects etc..Largely, Use out of range cause in recent years Determination of Phoxim Residues it is exceeded repeatly
See not fresh, processing of farm products, water and soil, fruits and vegetables etc. are frequently detected excess.
The toxicity of phoxim is taken seriously gradually, although phoxim ambient stable, is easy to photodissociation higher than 120 DEG C, its light
Solution product generates the thio Mortopl for having very big toxicity to mammal, to honeybee and fish high poison.It is reported that phoxim can make to grow up
The reproductive system of male mouse is abnormal completely.Long Term Contact phoxim can endanger the organs such as the heart of people, liver and system genitale
System.Therefore, Determination of Phoxim Residues excessive problem can not be ignored.National standard (GB 14869-1994) clear stipulaties in China: phoxim
Maximum residue limit per kilogram must not exceed 0 .05mg in grain (raw grain) and fruits and vegetables.
The organophosphorus insecticides such as phoxim are long in silo, soil and root crop ratio residence time on leaf vegetables.In grain
In food processing, the organophosphorus insecticides such as phoxim residual mainly in the appearance skin portion of crops, accordingly act as feed wheat bran,
There is phoxim severe overweight in the agricultural and sideline products such as rice bran.
Summary of the invention
Object of the present invention is to being largely excessively used for current phoxim causes frequently to be detected exceeded status, provides
A kind of speed of growth is fast, cultural method is simple, energy is efficient, stablizes Dell's Ford bacteria strain of safe disposal phoxim, the bacterial strain
For the Determination of Phoxim Residues in degrade silo grain or processing of farm products byproduct, it is prepared for a kind of enzyme preparation using the bacterial strain,
The enzyme preparation is used for degrading phoxim for the first time, and Determination of Phoxim Residues can degrade 90%~99% or more.
The object of the invention is achieved by the following technical programs.
A kind of Dell's Ford bacterium, by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preservation
Number are as follows: CGMCC NO. 16844.
The Dell Ford bacterium is by the separated acquisition of Shandong Scientific Research Academy ecological Studies and to save, in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center preservation, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode:
100101, deposit number is: the classification naming of CGMCC NO. 16844, strain are: Dell Ford bacteriumDelftiaSp., join
According to microorganism (strain): D39, the deposit date is on November 30th, 2018.
Another object of the present invention is to provide the applications that Dell Ford bacterium of the invention is used for degrading phoxim, further
It is application of the Dell Ford bacterium for phoxim in degrade silo grain or processing of farm products byproduct.
Dell Ford bacterium of the invention is used for the application of degrading phoxim, the enzyme system that will be prepared using Dell's Ford bacterium
Agent is added in system to be processed in a manner of spraying or stirring, make 0.05 g/kg of enzyme preparation concentration in system~
0.30 g/kg reacts 1~24 h under conditions of temperature is 20~37 DEG C.
Dell Ford bacterium of the invention is used for the application of degrading phoxim, and the preparation method of the enzyme preparation is specific to walk
It is rapid as follows:
(1) Dell's Ford bacterium D39 bacterial strain is taken to line on LB solid medium, 25~30 DEG C of inversion 1~2d of activation culture are made
Bacterial strain after activation；
(2) strain inoculated after taking step (1) to activate in LB liquid medium, temperature be 25~30 DEG C, revolving speed be 150~
Under conditions of 200rpm, seed liquor is made in 6~8h of shaking table culture；
(3) seed liquor made from step (2) is taken, is inoculated in LB liquid medium by 5~10% percent by volume, in temperature
Under conditions of being 150~200rpm for 25~30 DEG C, revolving speed, expands 3~5d of culture, Dell's Ford bacterium D39 bacterium solution is made；
(4) Dell's Ford bacterium D39 bacterium solution made from step (3) is taken, under conditions of 5000~10000rpm, centrifugation 5~
10min collects thallus, is suspended in the phosphate buffer of the .0 of pH6 .0~9 of 10~30 times of volumes, and it is thin to carry out ultrasonic wave
Born of the same parents are broken, under conditions of 4~25 DEG C, 5000~8000rpm, are centrifuged 5~8min, discard cell fragment, collect supernatant, system
Obtain efficient degradation enzyme preparation.
According to the present invention preferably, the LB solid medium in the step (1), every liter of component are as follows: peptone 10g,
Yeast extract 5g, sodium chloride 10g, agar 15g, water are settled to 1L, and pH is natural.
According to the present invention preferably, the LB liquid medium in the step (2) and step (3), every liter of component are as follows:
Peptone 10g, yeast extract 5g, sodium chloride 10g, water are settled to 1L, and pH is natural.
According to the present invention preferably, the ultrasonic cell-break in the step (4), condition are as follows: broken time/gap
Time is 20sec/10sec, total time 15min, power 170w.
The invention has the benefit that
The present invention screen to obtain a kind of speed of growth is fast, cultural method is simple, can efficiently, stablize the Dell of degradable phoxim
Ford bacteria strain, and develop the enzyme preparation of degrading phoxim using Dell's Ford bacterium for the first time, the enzyme preparation to the grain storage of silo and
Determination of Phoxim Residues in the byproduct of processing of farm products can degrade 90%~99% or more.In addition, Dell Ford bacterium D39 of the invention
Enzyme preparation simple process is easy to be mass produced.
Detailed description of the invention
Fig. 1 is the hydrolysis that D39 is formed after degrading phoxim on SMM plate.
Fig. 2 is that D39 shakes training for 24 hours to the degradation of phoxim.
Fig. 3 is that HPLC detects bacterial strain D39 of the present invention to the degradation rate of phoxim.
Specific embodiment
For a better understanding of the present invention, below with specific example come the technical solution that the present invention will be described in detail, but this
Invention is not limited thereto.
The Dell's Ford bacteria strain characteristic of the invention of embodiment 1
Dell's Ford bacteria strain of the invention, bacterium colony is rounded, milky, there is protrusion, and flush edge, form is full, smooth wet
Profit, cell are in rod-short, and size is (0.6~0.8) μ m (1.0~1.5) μm, no gemma.The physiology of bacterial strain D39 is raw
Change feature are as follows: contact enzyme reaction is the positive, V~P reaction, clark and Lubsreaction, nitrate reduction reaction, indole test, lemon
Hydrochlorate is feminine gender, the aobvious feminine gender of Gram's staining using test.
Culture, separation, screening step and the 16S rDNA identification of the Dell's Ford bacteria strain of the invention of embodiment 2
D39 of the invention Dell Ford Pseudomonas (DelftiaSp.) bacterial strain is obtained by following culture, separation, screening
It arrives:
20 samples are taken using five point sampling from Heze Juye Hou Tuncun different wheat field and cotton field within 2 months (1) 2016 year.
Each sample weighs 10 g of soil in laboratory, is respectively placed in the SMM culture medium of 90mL, the formula of SMM culture medium are as follows:
NH4NO31.0 g/L, KH2PO40.5 g/L, K2HPO41.5 g/L, MgSO40.2 g/L, NaCl 0.5g/L adjust pH=7.0
Afterwards, phoxim, in 30 DEG C, 150 rpm shaking table cultures are added by 100 mg/L；
(2) above-mentioned culture solution shifts weekly inoculation once, is accessed in fresh SMM culture medium with 10% inoculum concentration, and gradually mention
The concentration of high (100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L) phoxim is up to 500 mg/L.Shaking flask knot
Shu Hou takes the pregnant solution of 0.1 mL to be coated in the basal medium SMM plate containing 500 mg/L phoxim stostes, places constant temperature
It cultivates that the picking speed of growth is fast, bacterium colony is regular after 3 d in incubator and has degradation bacteria 39 of transparent circle, transferred respectively in containing
It is further isolated and purified in the SMM plate of 500 mg/L phoxim stostes.By activated single colonie microscope inspection, guarantee pure
Single colonie.
(3) screening of degradation bacteria strains: primary dcreening operation is first big according to the size picking colony of plate hydrolysis circle and hydrolysis circle is also big
Degradation bacteria has selected 2,4,7,8,9,22,23,26,30,34,37,39 totally 12 plants of phoxim degradation bacterias.Secondary screening using HPLC into
The measurement of row degradation property.It takes 1 mL of culture solution to be placed in the scale test tube of tool plug with 2 d of bacterium amount shaking flask is met, 5 mL dichloromethanes is added
Alkane, acutely stratification after concussion, discards upper strata aqueous phase, and organic phase takes 1 mL in microcentrifugation after the dehydration of anhydrous slufuric acid ammonium
1 mL methanol dissolution (chromatographically pure) is added, HPLC determination condition are as follows: mobile phase methanol in Guan Zhong after being dried with nitrogen: water=80:20, stream
1 mL.min of speed-1, the operation wavelength of 10 uL of sample volume, variable-wavelenght detector are 280 nm, using quantified by external standard method.By
Measurement, screening obtain the highest bacterial strain 39 of degradation rate.
(4) the 16S rDNA identification of bacterial strain
The genomic DNA of bacterial strain 39 is extracted as template, carries out the amplification of 16S rRNA gene.Pair of primers is respectively as follows: just
To primer: 5 '~AGAGTTTGATCCTGGCTCAG~3 ', reverse primer: 5 '~TACGGCTACCTTGTTACGACTT~3 '；
25 μ L systems are as follows: 1 μ L of template, each 1 μ L, Mix(dNTP+Taq+ amplification buffer of primer (1mmol L)) 12.5 μ L,
9.5 μ L of ultrapure water.Polymerase chain reaction condition: 94 DEG C, 5 min；94 ℃, 30 sec ;52.3 ℃, 45 sec
;72 ℃, 1.5 min ;Circulation 30 times, 72 DEG C of 10 min of extension.16S rRNA is recycled using PCR QIAquick Gel Extraction Kit
Genetic fragment, agarose gel electrophoresis detect amplified production size (1.5 kb or so) after, by its TA clone it is laggard
Row sequencing (being completed by BIOAISA company).Sequencing result passes through on-line analysis (http: www .ncbi .nlm.nih
.gov), in GenBank 16S rRNA gene order carry out similarity system design, find the sequence withDelftiasp's
16S rRNA sequence homology is up to 99%.Determine the bacterial strain be Dell's Ford Pseudomonas (DelftiaSp.), it is named as D39.
The toxicological assessment of 3 Dell's Ford bacterium D39 enzyme preparation of embodiment
(1) acute toxicity test method: selecting maximum tolerated dose method, and SPF grades KM mouse 20, half male and half female, if 3.0 g/
Mono- dosage group of kgBW, is prepared with distilled water, and stirring and evenly mixing rear concentration of test liquid is 13.3%.Gastric infusion (hold by stomach-filling
Amount is 20 mL/kgBW), stomach-filling 3 times, it is spaced 4 h.Seek the oral maximum tolerated dose of male and female chmice acute (MTD).
The acute toxicity tests: Dell's Ford bacterium D39 enzyme preparation gives mouse with 3.0 g/kgBW dosage stomach-fillings,
Experimental animal diet and activity are normal, after having no any poisoning symptom and death assay, carry out gross anatomy, meat to animal
Eye observes each internal organs no abnormality seen and shows the female male mouse oral maximum tolerated dose (MTD) of Dell's Ford bacterium D39 enzyme preparation powder
It is all larger than 3.0 g/kgBW.
(2) 90 d feeding trials
Animal single cage is fed, ad lib and drinking-water, the activity of daily observation experiment animal and growing state.Feeding contains sample respectively
Product 2.5,5.0,10.0% and feed not with sample, continuous 90 d.Claim a weight respectively within 2nd, 5,13 week, records to appetite
With surplus appetite.Calculate influence of the enzyme preparation to rat body weight.It does not send out after experiment making gross examination to each dosage group animal
Now obvious lesion only carries out the dissection of maximum dose level group and control animals main organs.
During experiment, flexibly, reaction is quick, and coat is clean and tidy for groups of animals action, and eye muzzle is without secretion, and feed drinking-water is just
Often, growth and development is good, behavior without exception and poisoning symptom, no death.By feeding in 13 weeks, practical food ration weekly is calculated
And average weight weekly.Through analyzing, each dosage group male and female mouse starting weight, weight gain, compared with the control group, there was no significant difference.Each group
Between difference be not present dose-response relationship, it is not statistically significant, illustrate the sample to Growth in Rats hair the region between the heart and the diaphragm have no significant effect.
Embodiment 4 is used in the Determination of Phoxim Residues in degradation silo grain
(1) Dell's Ford bacterium D39 bacterial strain is taken to line on LB solid medium, 25 DEG C of inversion activation culture 1d, after activation is made
Bacterial strain；
(2) strain inoculated after taking step (1) to activate is in LB liquid medium, temperature is 25 DEG C, revolving speed is 150rpm's
Under the conditions of, seed liquor is made in shaking table culture 8h；
(3) seed liquor made from step (2) is taken, is inoculated in LB liquid medium by 10 percent by volume, is 25 in temperature
DEG C, revolving speed be 150rpm under conditions of, expand culture 3d, be made Dell's Ford bacterium D39 bacterium solution；
(4) Dell's Ford bacterium D39 bacterium solution made from step (3) is taken, under conditions of 8000rpm, 10min is centrifuged, collects bacterium
Body is suspended in the phosphate buffer of the pH7.0 of 30 times of volumes, carries out ultrasonic cell-break, in 10 DEG C, 8000rpm
Under the conditions of, it is centrifuged 8min, discards cell fragment, collects supernatant, efficient degradation enzyme preparation is made.
The ultrasonic cell-break, condition are as follows:
Broken time/off time is 20sec/10sec, total time 15min, power 170w.
The applying step of above-mentioned enzyme preparation phoxim in degradation silo grain is as follows:
Enzyme preparation obtained above is taken, is added in system to be processed in a manner of spraying or stirring, makes enzyme system in system
Agent concentration is 0.05~0.30g/kg, under conditions of temperature is 25 DEG C, reacts 12h.The system to be processed refers to,
Silo grain or processing of farm products byproduct to be degraded.
It takes after above-mentioned processing with enzyme preparation 10 grams of wheat to be put into triangular flask using five point sampling plus water is settled to 100mL,
Shaking table 150rpm shakes 30min, takes 1mL wheat solution that 5 mL methylene chloride are added and extracts 3 times, organic phase is dehydrated through anhydrous slufuric acid ammonium
Afterwards, it takes 1 mL in microcentrifugal tube, 1 mL methanol dissolution (chromatographically pure) is added after being dried with nitrogen, HPLC detects the drop of phoxim
Solution rate, the degradation rate of phoxim enzyme preparation is up to 93.3%.
Embodiment 5 is for remaining phoxim in food processing byproduct wheat bran of degrading
(1) Dell's Ford bacterium D39 bacterial strain is taken to line on LB solid medium, 25 DEG C of inversion activation culture 1d, after activation is made
Bacterial strain；
(2) strain inoculated after taking step (1) to activate is in LB liquid medium, temperature is 25 DEG C, revolving speed is 150rpm's
Under the conditions of, seed liquor is made in shaking table culture 8h；
(3) seed liquor made from step (2) is taken, is inoculated in LB liquid medium by 5 percent by volume, is 25 in temperature
DEG C, revolving speed be 150rpm under conditions of, expand culture 3d, be made Dell's Ford bacterium D39 bacterium solution；
(4) Dell's Ford bacterium D39 bacterium solution made from step (3) is taken, under conditions of 8000rpm, 10min is centrifuged, collects bacterium
Body is suspended in the phosphate buffer of the pH7.0 of 30 times of volumes, carries out ultrasonic cell-break, in 10 DEG C, 8000rpm
Under the conditions of, it is centrifuged 8min, discards cell fragment, collects supernatant, efficient degradation enzyme preparation is made.
The ultrasonic cell-break, condition are as follows:
Broken time/off time is 20sec/10sec, total time 15min, power 170w.
The applying step of above-mentioned enzyme preparation phoxim in degradation silo grain is as follows:
Enzyme preparation obtained above is taken, enzyme preparation, which is added, in spraying or stirring makes enzyme preparation concentration 0.10g/kg in system,
Under conditions of temperature is 25 DEG C, 10h is reacted, detects the phoxim degradation rate on wheat bran.The sampling of wheat bran and the HPLC of phoxim
Detection method is with above-described embodiment 4, through HPLC detection phoxim enzyme preparation to the degradation rate of phoxim on wheat bran up to 96.8%.
Embodiment 6
Bacterial strain D39 monoclonal method of scoring switching of the present invention is in the SMM plate of the stoste of phoxim containing 500mg/L, in 25 DEG C of constant temperature
The local SMM culture medium of strain growth becomes transparent after culture 48h in incubator, illustrates that phoxim is degraded.Without bacterial strain
The place of growth, the phoxim in culture medium remain as muddy milky, are specifically shown in attached drawing 1.
Embodiment 7
Percent by volume of the invention bacterial strain D39 on aseptic operating platform by 10 is inoculated in the liquid of the stoste of phoxim containing 500mg/L
In SMM culture medium (being CK control not connect bacterium), shaken under conditions of temperature is 25 DEG C, revolving speed is 180rpm after training 2d to pungent
Attached drawing 2 is shown in the degradation of sulphur phosphorus, as can be seen from Figure 2 the right the triangular flask containing D39 in phoxim be degraded after culture solution
Become clear.And it compares phoxim in CK triangular flask culture solution and remains as muddy milky.
Embodiment 8
The culture solution of shaking flask 2d in above-described embodiment 7 is taken to carry out the detection of phoxim degradation rate using HPLC.The specific steps are, point
The D39 and control culture solution for not taking 1mL are placed in the scale test tube of tool plug, and 5 mL methylene chloride are added, acutely stand after concussion
Layering, discards upper strata aqueous phase, and organic phase takes 1mL in microcentrifugal tube, be added after being dried with nitrogen after the dehydration of anhydrous slufuric acid ammonium
1 mL methanol dissolves (chromatographically pure), HPLC determination condition are as follows: mobile phase methanol: water=80:20,1 mL.min of flow velocity-1, sample volume
The operation wavelength of 10uL, variable-wavelenght detector are 280nm, using quantified by external standard method.As can be seen from Figure 3 phoxim is compareed
Appearance time be 4.361min, and in the culture solution containing D39 at the same time between appearance before and after section without substance peak, D39
It has been completely degraded.
Sequence table of the present invention is as follows
Sequence table
<110>Shandong Scientific Research Academy ecological Studies institute
<120>a kind of Dell's Ford bacterium and application
<130> 18-1-1497
<141>
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1439
<212> DNA
<213>
<400> 1
accgtaccgg ctgccttacc atgcagtcga acggtaacag gtcttcggac gctgacgagt 60
ggcgaacggg tgagtaatac atcggaacgt gcccagtcgt gggggataac tactcgaaag 120
agtagctaat accgcatacg atctgaggat gaaagcgggg gaccttcggg cctcgcgcga 180
ttggagcggc cgatggcaga ttaggtagtt ggtgggataa aagcttacca agccgacgat 240
ctgtagctgg tctgagagga cgaccagcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtgggga attttggaca atgggcgaaa gcctgatcca gcaatgccgc 360
gtgcaggatg aaggccttcg ggttgtaaac tgcttttgta cggaacgaaa aagctccttc 420
taatacaggg ggcccatgac ggtaccgtaa gaataagcac cggctaacta cgtgccagca 480
gccgcggtaa tacgtagggt gcgagcgtta atcggaatta ctgggcgtaa agcgtgcgca 540
ggcggttatg taagacagat gtgaaatccc cgggctcaac ctgggaactg catttgtgac 600
tgcatggcta gagtacggta gagggggatg gaattccgcg tgtagcagtg aaatgcgtag 660
atatgcggag gaacaccgat ggcgaaggca atcccctgga cctgtactga cgctcatgca 720
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccct aaacgatgtc 780
aactggttgt tgggaattag ttttctcagt aacgaagcta acgcgtgaag ttgaccgcct 840
ggggagtacg gccgcaaggt tgaaactcaa aggaattgac ggggacccgc acaagcggtg 900
gatgatgtgg tttaattcga tgcaacgcga aaaaccttac ccacctttga catggcagga 960
agtttccaga gatggattcg tgctcgaaag agaacctgca cacaggtgct gcatggctgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgtcatt 1080
agttgctaca ttcagttgag cactctaatg agactgccgg tgacaaaccg gaggaaggtg 1140
gggatgacgt caagtcctca tggcccttat aggtggggct acacacgtca tacaatggct 1200
ggtacagagg gttgccaacc cgcgaggggg agctaatccc ataaaaccag tcgtagtccg 1260
gatcgcagtc tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc ggatcagcat 1320
gccgcggtga atacgttccc gggtcttgta cacaccgccc gtcacaccat gggagcgggt 1380
ctcgccagaa gtaggtagcc taaccgcaag gagggcgcta ccacgacggt cgcggcgcc 1439
Sequence table
<110>Shandong Scientific Research Academy ecological Studies institute
<120>a kind of Dell's Ford bacterium and application
<130> 18-1-1497
<141>
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1439
<212> DNA
<213>
<400> 1
accgtaccgg ctgccttacc atgcagtcga acggtaacag gtcttcggac gctgacgagt 60
ggcgaacggg tgagtaatac atcggaacgt gcccagtcgt gggggataac tactcgaaag 120
agtagctaat accgcatacg atctgaggat gaaagcgggg gaccttcggg cctcgcgcga 180
ttggagcggc cgatggcaga ttaggtagtt ggtgggataa aagcttacca agccgacgat 240
ctgtagctgg tctgagagga cgaccagcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtgggga attttggaca atgggcgaaa gcctgatcca gcaatgccgc 360
gtgcaggatg aaggccttcg ggttgtaaac tgcttttgta cggaacgaaa aagctccttc 420
taatacaggg ggcccatgac ggtaccgtaa gaataagcac cggctaacta cgtgccagca 480
gccgcggtaa tacgtagggt gcgagcgtta atcggaatta ctgggcgtaa agcgtgcgca 540
ggcggttatg taagacagat gtgaaatccc cgggctcaac ctgggaactg catttgtgac 600
tgcatggcta gagtacggta gagggggatg gaattccgcg tgtagcagtg aaatgcgtag 660
atatgcggag gaacaccgat ggcgaaggca atcccctgga cctgtactga cgctcatgca 720
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccct aaacgatgtc 780
aactggttgt tgggaattag ttttctcagt aacgaagcta acgcgtgaag ttgaccgcct 840
ggggagtacg gccgcaaggt tgaaactcaa aggaattgac ggggacccgc acaagcggtg 900
gatgatgtgg tttaattcga tgcaacgcga aaaaccttac ccacctttga catggcagga 960
agtttccaga gatggattcg tgctcgaaag agaacctgca cacaggtgct gcatggctgt 1020
cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgtcatt 1080
agttgctaca ttcagttgag cactctaatg agactgccgg tgacaaaccg gaggaaggtg 1140
gggatgacgt caagtcctca tggcccttat aggtggggct acacacgtca tacaatggct 1200
ggtacagagg gttgccaacc cgcgaggggg agctaatccc ataaaaccag tcgtagtccg 1260
gatcgcagtc tgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc ggatcagcat 1320
gccgcggtga atacgttccc gggtcttgta cacaccgccc gtcacaccat gggagcgggt 1380
ctcgccagaa gtaggtagcc taaccgcaag gagggcgcta ccacgacggt cgcggcgcc 1439
Claims (8)
1. a kind of Dell's Ford bacterium, by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preservation is compiled
Number are as follows: CGMCC NO. 16844.
2. the application that Dell's Ford bacterium as described in claim 1 is used for degrading phoxim.
The phoxim in silo grain or processing of farm products byproduct 3. Dell's Ford bacterium as claimed in claim 2 is used to degrade
Using.
4. the application that Dell's Ford bacterium as claimed in claim 2 is used for degrading phoxim, which is characterized in that Dell's good fortune will be used
The enzyme preparation that special bacterium is prepared is added in system to be processed in a manner of spraying or stirring, makes enzyme preparation in system
Concentration is 0.05~0.30g/kg, under conditions of temperature is 20~37 DEG C, reaction 1~for 24 hours.
5. the application that Dell's Ford bacterium as claimed in claim 4 is used for degrading phoxim, which is characterized in that the enzyme preparation
Preparation method, the specific steps are as follows:
(1) Dell's Ford bacterium D39 bacterial strain is taken to line on LB solid medium, 25~30 DEG C of inversion 1~2d of activation culture are made
Bacterial strain after activation；
(2) strain inoculated after taking step (1) to activate in LB liquid medium, temperature be 25~30 DEG C, revolving speed be 150~
Under conditions of 200rpm, seed liquor is made in 6~8h of shaking table culture；
(3) seed liquor made from step (2) is taken, is inoculated in LB liquid medium by 5~10% percent by volume, in temperature
Under conditions of being 150~200rpm for 25~30 DEG C, revolving speed, expands 3~5d of culture, Dell's Ford bacterium D39 bacterium solution is made；
(4) Dell's Ford bacterium D39 bacterium solution made from step (3) is taken, under conditions of 5000~10000rpm, centrifugation 5~
10min collects thallus, is suspended in the phosphate buffer of the .0 of pH6 .0~9 of 10~30 times of volumes, and it is thin to carry out ultrasonic wave
Born of the same parents are broken, under conditions of 4~25 DEG C, 5000~8000rpm, are centrifuged 5~8min, discard cell fragment, collect supernatant, system
Obtain efficient degradation enzyme preparation.
6. the application that Dell's Ford bacterium as claimed in claim 5 is used for degrading phoxim, which is characterized in that the step (1)
In LB solid medium, every liter of component is as follows: peptone 10g, yeast extract 5g, sodium chloride 10g, agar 15g, water constant volume
To 1L, pH is natural.
7. the application that Dell's Ford bacterium as claimed in claim 5 is used for degrading phoxim, which is characterized in that the step (2)
With the LB liquid medium in step (3), every liter of component is as follows: peptone 10g, yeast extract 5g, sodium chloride 10g, water are fixed
Hold to 1L, pH is natural.
8. the application that Dell's Ford bacterium as claimed in claim 5 is used for degrading phoxim, which is characterized in that the step (4)
In ultrasonic cell-break, condition is as follows: broken time/off time is 20sec/10sec, and total time 15min, power is
170w。
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Cited By (3)
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| CN112877243A (en) * | 2021-02-08 | 2021-06-01 | 常州大学 | Darfeld lakeda bacterium for degrading acephate and application thereof |
| CN115404191A (en) * | 2022-11-03 | 2022-11-29 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Soil remediation composite microbial agent containing Delftia D39 and Junpian M1031 and preparation method thereof |
| CN116790415A (en) * | 2023-04-21 | 2023-09-22 | 中国科学院地理科学与资源研究所 | Delford bacteria and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112877243A (en) * | 2021-02-08 | 2021-06-01 | 常州大学 | Darfeld lakeda bacterium for degrading acephate and application thereof |
| CN115404191A (en) * | 2022-11-03 | 2022-11-29 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Soil remediation composite microbial agent containing Delftia D39 and Junpian M1031 and preparation method thereof |
| CN115404191B (en) * | 2022-11-03 | 2023-01-20 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Soil remediation composite microbial agent containing Delftia D39 and Junpian M1031 and preparation method thereof |
| CN116790415A (en) * | 2023-04-21 | 2023-09-22 | 中国科学院地理科学与资源研究所 | Delford bacteria and application thereof |
| CN116790415B (en) * | 2023-04-21 | 2023-12-08 | 中国科学院地理科学与资源研究所 | Delford bacteria and application thereof |
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| CN109679875B (en) | 2020-06-23 |
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