CN1502688A - Bacterial strain with broad-spectrum degradation ability to organic phosphorus pesticide and screening method thereof - Google Patents
Bacterial strain with broad-spectrum degradation ability to organic phosphorus pesticide and screening method thereof Download PDFInfo
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- CN1502688A CN1502688A CNA021488835A CN02148883A CN1502688A CN 1502688 A CN1502688 A CN 1502688A CN A021488835 A CNA021488835 A CN A021488835A CN 02148883 A CN02148883 A CN 02148883A CN 1502688 A CN1502688 A CN 1502688A
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Abstract
The present invention relates to a method for screening strain with broad-spectrum degradation capability for degrading organophosphorus pesticide and its screened strain. Said method includes the following steps: respectively inoculating the strain in LB perfect medium, activating, centrifuging the activated bacteria and collecting thallus, using Burk culture medium to suspend thallus, then culturing in liquid Burk inorganic salt culture medium in which methyl thiophos 0.5 mg/ml is added, diluting bacteria solution and coating it on the Burk inorganic salt solid culture medium in which 0.2 mg/ml vapona and 0.2 mg/ml methyl thiophos are added, culturing in culture box, selecting single colony, respectively inoculating separated single colony strain on several Burk inorganic salt solid culture medium plates containing organophosphorous pesticide, observing counteracting ring and selecting strain with large counteracting ring.
Description
Technical field
The present invention relates to a kind of bacterial strain, particularly a kind of bacterial strain that organophosphorus pesticide is had broad-spectrum degradation capability also relates to the screening method of this bacterial strain.
Background technology
Organophosphorus pesticide is the primary categories in the agricultural chemicals, account for 60% (Zhang Liyan of agricultural chemicals total amount, organophosphorus pesticide, Beijing: chemistry of fuel industry press, 1973), be that agriculture production is requisite, the pesticide producing output of China in 2000 is 60.7 ten thousand tons of (National Bureau of Statistics of the People's Republic of China, 2001 China Statistical Yearbooks, the China Statistics Press).Be hypertoxic more than 80% in organophosphorus pesticide, as acephatemet, parathion-methyl, thiophos, monocrotophos, SD-1750 etc., only other large-tonnage organophosphorus pesticide also had Rogor, omethoate and Volaton etc. to the usage quantity of acephatemet just up to 6.5 ten thousand tons in 1 year.But along with the development of agricultural chemicals, it has also caused people's great attention to the pollution problem that environment caused.
Because organophosphorus pesticide has and the similar structure of vagusstoff, so they all have the function that suppresses human acetylcholinesterase, to the toxicity of people's various degrees.Acute poisoning can cause that people's muscle spasm, pupil contraction, expiratory dyspnea are until death.The chronic poisoning of low dosage can be induced polyneuropathy, apoplexy etc.Therefore develop new product, effectively degrading pesticide residues has become the problem that presses for solution.
Organophosphorus contains three phosphide keys, so often be called as phosphotriester.General organophosphorus can be divided into two types, and the one, phosphorus passes through two keys and combines (P=O) with oxygen, as acephatemet, omethoate, SD-1750 etc.; Another kind is that phosphorus passes through two keys and combines (P=S) with sulphur, as thiophos, parathion-methyl, Volaton, isocarbophos, Chlorpyrifos 94 (Le Siben) etc.There are some researches show will reduce its toxicity greatly if a phosphoric acid ester bond in the organophosphorus is hydrolyzed, is example with the thiophos, will make its toxicity reduce by 100 times of (Serdar, C.M., and D.T.Gibson.1985.Bio/Technology3:567-571; Serdar, C.M.December 1996.U.S.patent 5,589,386).Therefore, the phosphatide key of destruction organophosphorus is to reduce the toxic efficient ways of organophosphorus pesticide.
Discover in a large number that in recent years some soil microorganismss (bacterium, fungi) can degrading organic phosphor pesticides, this mainly is because it can secrete a kind of phosphoesterase degrading organic phosphor, product after the degraded can be used as carbon, nitrogen, the phosphorus source of microorganism growth simultaneously, for its growth provides nutrition (Sethunathan, N., and T.Yoshida.1973.Can.J.Microbiol.19:873-875; Chaudhry, G.R., et al., 1988.Appl.Environ.Microbiol.54:288-293; Li Shubin, all benevolence is superfine, the microbiology circular, 1999,26, No.1:27-30).But the microorganism majority of the degradable organophosphorus of report can only be degraded a kind of or an organophosphorus at present.
Summary of the invention
The object of the present invention is to provide a kind of bacterial strain of the multiple organophosphorus pesticide of effectively degrading, and the culture condition and the degradation characteristic of this bacterial strain are studied.Purpose is in order further to study this bacterial strain of good degradation characteristic to be arranged, and utilizes engineered means to clone its organic phosphorous chemicals decomposing enzyme gene, and it is efficiently expressed, the cheap organic phosphorous chemicals decomposing enzyme of producing.
The invention provides the bacterial strain of the multiple organophosphorus pesticide of a kind of degradable, it is the radial pattern edaphic bacillus (Agrobacterium radiobacter, CGMCC bacterium numbering 1150) that belongs to Agrobacterium, and we are with its called after OP150.This bacterial strain is a strain gramstaining reaction negative bacterium, and thalline is rod-short, and size is 0.8 * 1.5-3.0 micron, and 1-2 root flagellum is generally arranged, no gemma, and oxydase and catalase are determined as the positive, and bacterium colony generally is slick.This bacterium can grow being added with on the minimal medium of organophosphorus pesticide, and clearing up agricultural chemicals provides carbon source for its growth.
Bacterium OP150 adopts the agricultural chemicals enriching method to separate to obtain among the present invention.Key step is as follows:
1. the anthology laboratory is preserved and is screened available from the 11 strain bacterium (the bacterium list of file names is as follows) at Chinese microorganism strain preservation center.
The used bacterial classification one hurdle table of table 1 screening
This experiment of originating of bacterium name
Numbering
F85 (aspergillus niger) preserves F85 in this laboratory
92-1 (yeast) preserves 92-1 in this laboratory
90-2 (yeast) preserves 90-2 in this laboratory
66-1 (bacterium) preserves 66-1 in this laboratory
83-4 (bacterium) preserves 83-4 in this laboratory
89-1 (bacterium) preserves 89-1 in this laboratory
Agrobacterium tumefaciens CGMCC, 1.1415 1415
Radial pattern edaphic bacillus CGMCC, 1.150 150
Pseudomonas diminuta CGMCC, 1.220 220
Flavobacterium?sp. ATCC，27551 27551
Arthrobacter CGMCC, 1.8 8
Annotate: CGMCC: Chinese common micro-organisms culture presevation administrative center
ATCC:The Global Bioresource Center (global Biological resources center)
2. bacterium is connected to respectively 5mL LB perfect medium (peptone 1%, yeast powder 0.5%, NaCl1%, pH7.0) in activation, get the good centrifugal collection thalline of bacterium of activation, discard the substratum supernatant, with Burk substratum (K
2HPO
40.02%, KH
2PO
40.08%, MgSO
4.7H
2O 0.02%, CaSO
4.H
2O 0.01%, NaMoO
4.2H
2O 0.0003%, FeSO
4.7H
2O 0.0005%, (NH
4)
2SO
40.1%, yeast powder 0.05%, pH7.2) hanged thalline after, be connected in the 50mL liquid B urk minimal medium by 1%, be added with parathion-methyl 0.5mg/mL (pure product) in the substratum, 32 ℃ of concussions were cultivated 36 hours.
Bacterium liquid with the sterilized water gradient dilution after separate application on the Burk inorganic salt solid medium that is added with 0.2mg/mL SD-1750 (pure product) and 0.2mg/mL parathion-methyl (pure product), place 32 ℃ of incubators to cultivate.Picking list bacterium colony is through the plate streaking purifying.
3. single bacterium colony bacterial strain that will be separated to is inoculated in respectively on the Burk inorganic salt solid medium flat board that contains organophosphorus pesticide parathion-methyl (0.05%), acephatemet (0.05%), thiophos (0.05%), observes and clears up circle.
4. be chosen in and clear up all bigger bacterial strain of circle on several agricultural chemicals flat boards, numbering is stored on the inclined-plane.Wherein, being numbered 150 bacterium, to clear up circle on three kinds of agricultural chemicals flat boards all maximum, illustrates that its organic phosphorus pesticide degradation characteristic is the best.
Bacterial strain OP150 of the present invention compares its advantage with other pesticide degradation bacterias of having reported at present and is: 1) degradation rate height, can 100% degraded for parathion-methyl, for its degradation rate of other organophosphorus pesticides also up to 60-80%; 2) has broad spectrum, can act on multiple organophosphorus pesticide, no matter substrate is the organophosphorus (acephatemet, omethoate, SD-1750 etc.) of the two keys of phosphorus oxygen or the organophosphorus (as thiophos, parathion-methyl, Volaton, isocarbophos, Chlorpyrifos 94 (Le Siben) etc.) of the two keys of phosphorus sulphur, all has good effect.3) organic phosphorous chemicals decomposing enzyme of expressing is active high, therefore can be used for the new organic phosphorous chemicals decomposing enzyme gene of separating clone, producer gene engineering strain, thereby the extensive cheap organic phosphorous chemicals decomposing enzyme of producing.
Embodiment
Embodiment 1
Be inoculated in the liquid minimal medium that contains the 0.5mg/mL parathion-methyl and activate being stored in bacterial strain OP150 on the inclined-plane, bacterium liquid after the activation is chosen a ring and is inoculated in respectively on the solid Burk minimal medium flat board that contains 0.05% parathion-methyl, thiophos, acephatemet, omethoate, SD-1750, Volaton, Chlorpyrifos 94 respectively, place 32 ℃ of incubators to cultivate, after cultivating 24 hours, 48 hours and 72 hours, observe the growing state of clearing up circle and bacterium on the agricultural chemicals flat board respectively.After 24 hours, promptly have to clear up on parathion-methyl, acephatemet, SD-1750, omethoate, thiophos flat board and iris out now, bacterium also has a small amount of growth; After 48 hours, all have on each agricultural chemicals flat board to clear up and iris out now; Clear up circle after 72 hours and further enlarge, bacterium is further growth also.Fig. 1 is 72 hours rear oxidation Rogor flat boards, can obviously find out the circle of clearing up of central authorities.What deserves to be mentioned is that the substratum in the dull and stereotyped central authorities of parathion-methyl and thiophos presents yellow, this is because parathion-methyl and thiophos hydrolyzed product are p-nitrophenols is due to the yellow.Simultaneously as can be seen, clear up circle big than on the thiophos flat board on the parathion-methyl flat board, this organic phosphorous chemicals decomposing enzyme that illustrates that this bacterium produces can better act on parathion-methyl.
Can find out from present embodiment, bacterial strain degradable organophosphorus pesticide of the present invention, and on minimal medium, grow, illustrate that this bacterium can utilize degraded product to provide required carbon source for its growth.
Embodiment 2
The OP150 inoculation that is stored on the inclined-plane is activated in the liquid minimal medium that contains the 0.5mg/mL parathion-methyl, the bacterium that activation is good is transferred and cultivates in a large number in 50mL LB substratum, with the centrifugal collection thalline of the bacterium that grows to logarithmic phase, remove substratum, and bacterium is washed twice with LB perfect medium sticking on the thorough removal bacterium surface with the Burk minimal medium.With 50mL Burk minimal medium thalline is hanged, getting 2% is inoculated in the 5mL Burk liquid minimal medium, in this substratum, be added with parathion-methyl, thiophos, acephatemet, omethoate, SD-1750 and Chlorpyrifos 94 respectively, do contrast simultaneously, organophosphorus pesticide amount in the contrast is identical with experiment tube, but do not inoculate bacterium liquid, but replace with the Burk substratum.After concussion is cultivated 36 hours on 32 ℃ of shaking tables, use the chloroform extraction nutrient solution respectively, the content of various agricultural chemicals in the gas Chromatographic Determination substratum, the results are shown in Table 2.
From showing listed result as can be seen, this bacterium for parathion-methyl to clear up effect best, cleared up fully through cultivating the back parathion-methyl, from nutrient solution, do not detect.
Different agricultural chemicals of table 2 and bacterium OP150 cultivate the pesticide residue after 36 hours
Cultivate back substratum middle peasant before pesticide name is cultivated in the substratum and clear up rate
The residual content (μ g) of the pesticide volume that コ goes into (μ g) medicine (%)
Parathion-methyl 421.7 0 100
Thiophos 457 169.5 62.9
Acephatemet 489.8 97.2 80.2
Omethoate 489.1 101.2 79.3
SD-1750 469.5 113.4 75.8
Chlorpyrifos 94 445.5 155.3 65.1
Annotate: the preceding pesticide volume of residual content/cultivation of clearing up the preceding pesticide volume of rate (%)=cultivation-cultivation back agricultural chemicals
Embodiment 3
Our purpose is to utilize engineered means clone organic phosphorous chemicals decomposing enzyme gene, and it is efficiently expressed the cheap organic phosphorous chemicals decomposing enzyme of producing.Therefore measuring the enzymic activity of agricultural chemicals zymolase in the former bacterial strain, is the strong method of the degradation of pesticide characteristic of this bacterial strain of preliminary evaluation.Because parathion-methyl and thiophos produce p-nitrophenol after the hydrolysis of agricultural chemicals zymolase, present yellow, therefore we can measure p-nitrophenol content by the method for colorimetric, mensuration is with reference to the method (Serdar of Serdar etc., C.M.et al., 1982, Appl.Environ.Microbiol. 44:246) carries out.
The bacterium OP150 that activation is good with 2% amount transfer in 5mL Burk minimal medium, (be added with 0.05% acephatemet) and cultivate 36 hours after, centrifugal collection thalline, bacterium is suspended in the 50mmol/L Tris-HCl damping fluid (pH7.5) again, centrifugal removal cell debris, supernatant liquor are used for carrying out enzyme activity determination.
The measuring method of enzymic activity: get 0.9mL Tris-HCl damping fluid (50mmol/L, pH7.5) thiophos and the 0.1mL enzyme liquid to be measured of adding 0.005mL 10mg/mL, shaking all, the back is incubated 10 minutes at 37 ℃, add the colour developing of 1mL1% sodium bicarbonate, the content of colorimetric estimation hydrolysate p-nitrophenol under the 415nm wavelength.Regulation under these conditions, enzyme unit (u) that lives is defined as the per minute required enzyme amount of 1 μ mol thiophos of degrading.
Measuring the organic phosphorous chemicals decomposing enzyme enzymic activity that OP150 produces according to above method is the 9.36u/mL nutrient solution.
Claims (6)
1, a kind of screening has the method for the bacterial strain of broad-spectrum degradation capability to organophosphorus pesticide, it is characterized in that it may further comprise the steps:
A) choose the bacterial strain of some amount, they are connected to respectively in the LB perfect medium activate, get the good centrifugal collection thalline of bacterium of activation, discard the substratum supernatant, after having hanged thalline with the Burk substratum, be connected in the 50ml liquid B urk minimal medium by 1%, be added with parathion-methyl 0.5mg/ml in the substratum, certain hour is cultivated in concussion at a certain temperature;
B) bacterium liquid with the sterilized water gradient dilution after separate application on the Burk inorganic salt solid medium that is added with 0.2mg/mL SD-1750 and 0.2mg/mL parathion-methyl, place the incubator of certain temperature to cultivate, picking list bacterium colony is through the plate streaking purifying;
Single bacterium colony bacterial strain that c) will be separated to is inoculated in respectively on several Burk inorganic salt solid medium flat boards that contain organophosphorus pesticide, observes and clears up circle;
D) be chosen in and clear up all bigger bacterial strain of circle on several agricultural chemicals flat boards.
2, according to claims 1 described method, it is characterized in that, in step a), shake cultivation 36 hours down at 32 ℃.
According to claims 1 described method, it is characterized in that 3, in step b), the temperature of incubator is 32 ℃.
According to claims 1 described method, it is characterized in that 4, in step c), organophosphorus pesticide comprises parathion-methyl (0.05%), acephatemet (0.05%) and thiophos (0.05%).
5, a kind of radial pattern edaphic bacillus (Agrobacterium radiobacter, the CGMCC bacterium numbering 1150) bacterial strain that requires each described method to filter out according to aforesaid right.
6, the organic phosphorous chemicals decomposing enzyme that bacterial strain according to claim 5 produced.
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| CNA021488835A CN1502688A (en) | 2002-11-22 | 2002-11-22 | Bacterial strain with broad-spectrum degradation ability to organic phosphorus pesticide and screening method thereof |
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| CNA021488835A CN1502688A (en) | 2002-11-22 | 2002-11-22 | Bacterial strain with broad-spectrum degradation ability to organic phosphorus pesticide and screening method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306026C (en) * | 2005-05-25 | 2007-03-21 | 厦门大学 | Bacillus alcaigenes and its application for degrading methamidophos |
| CN1306023C (en) * | 2004-09-30 | 2007-03-21 | 中国科学院动物研究所 | Degradation bacteria for highly effective degrading organophosphorus pesticide and its use |
| CN103881929A (en) * | 2014-03-13 | 2014-06-25 | 湛江德玥生物工程有限公司 | Purification compound bacterium and purification compound probiotic preparation as well as preparation method of compound probiotic preparation |
| CN110699293A (en) * | 2019-11-06 | 2020-01-17 | 广东省生物资源应用研究所 | Method for screening bacteria with phosphate solubilizing function |
| CN111621459A (en) * | 2020-06-11 | 2020-09-04 | 青岛大学威海创新研究院 | Broad-spectrum pesticide degradation strain and preparation method and application thereof |
-
2002
- 2002-11-22 CN CNA021488835A patent/CN1502688A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306023C (en) * | 2004-09-30 | 2007-03-21 | 中国科学院动物研究所 | Degradation bacteria for highly effective degrading organophosphorus pesticide and its use |
| CN1306026C (en) * | 2005-05-25 | 2007-03-21 | 厦门大学 | Bacillus alcaigenes and its application for degrading methamidophos |
| CN103881929A (en) * | 2014-03-13 | 2014-06-25 | 湛江德玥生物工程有限公司 | Purification compound bacterium and purification compound probiotic preparation as well as preparation method of compound probiotic preparation |
| CN103881929B (en) * | 2014-03-13 | 2017-03-01 | 湛江德玥生物工程有限公司 | Purification compound bacteria and purification composite probiotics preparations and preparation method |
| CN110699293A (en) * | 2019-11-06 | 2020-01-17 | 广东省生物资源应用研究所 | Method for screening bacteria with phosphate solubilizing function |
| CN111621459A (en) * | 2020-06-11 | 2020-09-04 | 青岛大学威海创新研究院 | Broad-spectrum pesticide degradation strain and preparation method and application thereof |
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