CN102249952A - Method for separating and purifying water-soluble antibiotic Zwittermicin A in Bacillus thuringiensis - Google Patents
Method for separating and purifying water-soluble antibiotic Zwittermicin A in Bacillus thuringiensis Download PDFInfo
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- CN102249952A CN102249952A CN2011101176426A CN201110117642A CN102249952A CN 102249952 A CN102249952 A CN 102249952A CN 2011101176426 A CN2011101176426 A CN 2011101176426A CN 201110117642 A CN201110117642 A CN 201110117642A CN 102249952 A CN102249952 A CN 102249952A
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Abstract
The invention discloses a method for separating and purifying water-soluble antibiotic Zwittermicin A in Bacillus thuringiensis through an isoelectrofocusing electrophoresis method, which comprises the following steps: preliminarily purifying the supernate of a Bacillus thuringiensis fermentation liquid with anhydrous alcohol and petroleum ether, and sampling; preparing the supernate sample, an amphoteric electrolyte liquid, bromphenol blue, distilled water and agarose into a gel liquid, heating to 75-85 DEG C until the materials are sufficiently dissolved, and pouring the liquid into a medical rubber tube with a diameter of about 0.3cm while keeping the temperature; cooling the liquid for two hours, and introducing into an electrophoresis cell; and respectively adding electrolyte into negative and positive poles as required, and immersing both ends of the rubber tube into the electrode liquid to carry out isoelectrofocusing electrophoresis, thus separating and purifying the water-soluble antibiotic Zwittermicin A. As the experiment agents and samples are isolated from the air in the invention, the oxidation is avoided, the result is more accurate, the method is simple to operate, and the antibiotic Zwittermicin A can effectively separated out.
Description
Technical field
The invention belongs to vegetable chemistry, agrochemistry, biologic pharmacological science, plant protection, food chemistry, analytical chemistry, organic chemistry, biochemical field, the method for the water soluble antibiotics in particularly a kind of separation and purification bacillus thuringiensis-Zwittermicin A.
Background technology
At present, widely used in the world chemical insecticide when killing and wounding insect, has has also killed and wounded pest natural enemy in a large number, has caused the severe contamination of ecological balance damage and environment, has brought grave danger for human life security.In addition, insect progressively develops immunity to drugs to the chemical insecticide of life-time service, forms the vicious cycle that chemical insecticide is used.And biotic pesticide can partly overcome above shortcoming.Therefore the insect on the crops such as active development and applying biological agricultural chemicals substituting chemical pesticide control fruit and vegetables has become human common recognition.
The toxalbumin that bacillus thuringiensis (Bacillus thuringiensis subsp. is called for short Bt) produces is natural sterilant, and it is a kind of harmless boilogical agricultural chemicals that the prospect utilized is arranged most of generally acknowledging both at home and abroad at present.Bt desinsection toxalbumin has not been given play to due effect so far as yet in pollution-free food productions such as pollution-free vegetable, on using, push away for a long time and wideless, one of its reason mainly is the cubage output with toxalbumin, and Bt liquid fermenting products production cost is higher both at home and abroad, and the scope of application is little; It two is that to be subjected to uviolizing be to degrade to toxalbumin, drenching with rain in addition and spraying is the loss that causes soluble in water, use time-sensitive, only be suitable for, beet armyworm, the insecticidal effect and the cost that bore moth property primary pest are less competitive than chemical pesticide the killing of responsive insect.
Zwittermicin A is the water soluble antibiotics that bacillus thuringiensis produces during the fermentation, insecticidal activity is extremely low, but the insecticidal toxicity to the insecticide active substance toxalbumin of Tribactur has synergism, it not only makes, and the insecticidal crystal protein relevant with desinsection (as Cry1A, Cry1B, Cry1C and CryZ to Cry6 class) is active to increase several times even thousands of times, and can reduce the resistance of insect.Therefore this antibiotic existence can reduce the fermentation costs of Bt effectively, for the application of Bt engineering bacteria is laid a good foundation.Along with the needs of people to biotic pesticide, the research of separation and purification Zwittermicin A method becomes focus.
1994, Stabb provided two kinds of methods that detect Zwittermicin A that bacillus thuringiensis produced: to phage-sensitive with grass is given birth to the growth fungistatic effect of Erwinia.1996, Sandra etc. provided other three kinds: bunch analysis of fatty acid methyl ester, the RAPD of 10 bases reaches the Auele Specific Primer PCR reaction at zmaR (resistant gene of Zwittermicin A).And in above five kinds of methods, the test of inhibition zone is relatively accurate and simple.Shao Tie plums in 2005 etc. have carried out perfect to the inhibition zone measuring method of Bt bacterial strain.
Present only separation purification method has: (1) ion-exchange-(Manker etc. 1994 for high-voltage paper phase electrophoretic method (Silo-Souh etc. 1994) (2) ion-exchange-high performance liquid chromatography; Silo-Souh etc. 1998; Shen Xihui, king open plum 2004; Chen Shouwen, what advances 2007) etc. method.Above-mentioned purification process step is more, and test is complicated, and the high performance liquid chromatograph device is relatively more expensive, needs the professional and technical personnel simultaneously, and does not also have the standard substance of Zwittermicin A so far.And the method test of the water soluble antibiotics-Zwittermicin A in isoelectric focusing electrophoresis method separation and purification bacillus thuringiensis operation is simple relatively, and has measured the iso-electric point of Zwittermicin A, for the industrialization of Zwittermicin A lays the foundation.
Summary of the invention
The method that the purpose of this invention is to provide the water soluble antibiotics-ZwittermicinA in a kind of separation and purification bacillus thuringiensis.
Concrete steps are:
The fermented liquid that with bacillus thuringiensis is Bacillus thuringiensis subsp is through the centrifugal 10min of 10000r/min, fermented liquid supernatant liquid, at 50 ℃ with supernatant liquor water vacuum concentration to 50 times; According to volume ratio, in concentrated solution, add the dehydrated alcohol of 1/2 volume, through the centrifugal 10min of 10000r/min, abandon precipitated phase then, keep water; Aqueous phase adds isopyknic sherwood oil, shakes up, and leaves standstill, and water phase separated and sherwood oil abandon the sherwood oil phase mutually, keeps water; Both must collect standby through the supernatant samples of dehydrated alcohol and sherwood oil preliminary purification; With pH 3.5～9.5 carrier electrolytic liquid 1.5-2.5mL, supernatant samples 0.5-1.5mL, one of tetrabromophenol sulfonphthalein, distilled water 6-8mL, agarose 0.05-1.5g mixes, mixed glue solution is warming up to 75～85 ℃ of dissolvings fully, insulation is poured into even thickness with glue, diameter is about in the medical sebific duct of 0.3cm, cool off and insert tiselius apparatus after two hours, anode adds 60～80mL0.5mol/L acetum, negative electrode adds 60～80mL0.5mol/L sodium hydroxide solution, the two ends of sebific duct all need be immersed in the electrode solution, use the JY5000 electrophoresis apparatus, adjust electric current, make power output remain on 1～2W, when the tetrabromophenol sulfonphthalein color in the pipe is concentrated into when a bit, stopping electrophoresis, is the center with sebific duct with the tetrabromophenol sulfonphthalein point, is cut to the segment of 1cm respectively, then as can be known, near the water soluble antibiotics-Zwittermicin A that goes out with the electrophoretic method separation and purification exactly in that section pipe of tetrabromophenol sulfonphthalein through the bacteriostatic test analysis.
Isoelectric focusing electrophoresis is to utilize amphotericeledrolyte to make a good pH gradient in gel under optimized conditions, amphipathic molecule will be moved to the iso-electric point (pI) of positively charged not or negative electricity during electrophoresis, and microbiotic-Zwittermicin A is also gathered a narrow zone, thereby obtains separation and purification.In conjunction with the bacteria inhibition assay result, calculating antibiotic iso-electric point is 3.59～3.81.
Isoelectric focusing electrophoresis method of the present invention, be that experiment reagent and sample and air are isolated, avoid oxidation, make its result more accurate, and this method is simple to operate, microbiotic-Zwittermicin A can be isolated, and measure its iso-electric point, for a large amount of separation and purification preparations of microbiotic-Zwittermicin A are later on laid a good foundation.And this method also can apply to other amphotericeledrolyte in the separation determination.
Description of drawings:
Accompanying drawing is microbiotic of the present invention-Zwittermicin A molecular structure.
Zwittermicin A molecular structure has 2 imino-s, 3 ketone groups, 4 amino and 5 hydroxyls as can be seen, and hydroxyl is staggered in the carbon backbone chain both sides, and molecular weight is the linear amino polyol of 396Da.Owing to be rich in amino and hydroxyl, have positive and negative electrolysis from, infer thus and can separate this microbiotic and measure its iso-electric point pI by the isoelectric focusing electrophoresis test.
Embodiment
Embodiment:
The fermented liquid that with bacillus thuringiensis is Bacillus thuringiensis subsp is through the centrifugal 10min of 10000r/min, fermented liquid supernatant liquid, at 50 ℃ with supernatant liquor water vacuum concentration to 50 times; According to volume ratio, in concentrated solution, add the dehydrated alcohol of 1/2 volume, through the centrifugal 10min of 10000r/min, abandon precipitated phase then, keep water; Aqueous phase adds isopyknic sherwood oil, shakes up, and leaves standstill, and water phase separated and sherwood oil abandon the sherwood oil phase mutually, keeps water; Both must collect standby through the supernatant samples of dehydrated alcohol and sherwood oil preliminary purification; With pH 6.5 carrier electrolytic liquid 2mL, supernatant samples 1mL, one of tetrabromophenol sulfonphthalein, distilled water 7mL, agarose 0.1g mixes, mixed glue solution is warming up to 80 ℃ of dissolvings fully, insulation is poured into even thickness with glue, diameter is about in the medical sebific duct of 0.3cm, cool off and insert tiselius apparatus after two hours, anode adds the 70mL0.5mol/L acetum, negative electrode adds the 70mL0.5mol/L sodium hydroxide solution, the two ends of sebific duct all need be immersed in the electrode solution, use the JY5000 electrophoresis apparatus, adjust electric current, make power output remain on 1.5W, when the tetrabromophenol sulfonphthalein color in the pipe is concentrated into when a bit, stop electrophoresis, be the center with sebific duct with the tetrabromophenol sulfonphthalein point, be cut to the segment of 1cm respectively, then through the bacteriostatic test analysis as can be known, near being exactly the water soluble antibiotics-Zwittermicin A that goes out with the electrophoretic method separation and purification in that section pipe of tetrabromophenol sulfonphthalein, described reagent is analytical pure.
Claims (1)
1. the method for the water soluble antibiotics-Zwittermicin A in the separation and purification bacillus thuringiensis, it is characterized in that concrete steps are: the fermented liquid that with bacillus thuringiensis is Bacillus thuringiensis subsp is through the centrifugal 10min of 10000r/min, fermented liquid supernatant liquid, at 50 ℃ with supernatant liquor water vacuum concentration to 50 times; According to volume ratio, in concentrated solution, add the dehydrated alcohol of 1/2 volume, through the centrifugal 10min of 10000r/min, abandon precipitated phase then, keep water; Aqueous phase adds isopyknic sherwood oil, shakes up, and leaves standstill, and water phase separated and sherwood oil abandon the sherwood oil phase mutually, keeps water; Both must collect standby through the supernatant samples of dehydrated alcohol and sherwood oil preliminary purification; With pH 3.5～9.5 carrier electrolytic liquid 1.5-2.5mL, supernatant samples 0.5-1.5mL, one of tetrabromophenol sulfonphthalein, distilled water 6-8mL, agarose 0.05-1.5g mixes, mixed glue solution is warming up to 75～85 ℃ of dissolvings fully, insulation is poured into even thickness with glue, diameter is about in the medical sebific duct of 0.3cm, cool off and insert tiselius apparatus after two hours, anode adds 60～80mL0.5mol/L acetum, negative electrode adds 60～80mL0.5mol/L sodium hydroxide solution, the two ends of sebific duct all need be immersed in the electrode solution, use the JY5000 electrophoresis apparatus, adjust electric current, make power output remain on 1～2W, when the tetrabromophenol sulfonphthalein color in the pipe is concentrated into when a bit, stopping electrophoresis, is the center with sebific duct with the tetrabromophenol sulfonphthalein point, is cut to the segment of 1cm respectively, then as can be known, near the water soluble antibiotics-Zwittermicin A that goes out with the electrophoretic method separation and purification exactly in that section pipe of tetrabromophenol sulfonphthalein through the bacteriostatic test analysis; Described reagent is analytical pure.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645111A (en) * | 2017-03-01 | 2017-05-10 | 桂林理工大学 | Method for rapidly detecting concentration of antibiotic Zwittermicin A through electrochemical luminescence |
| CN108191713A (en) * | 2017-12-17 | 2018-06-22 | 桂林理工大学 | A kind of method that zwiitermicin A is isolated and purified in the zymotic fluid from Bt |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5736382A (en) * | 1995-06-06 | 1998-04-07 | Wisconsin Alumni Research Foundation | Bacillus cereus strain DGA34 |
| CN101508656A (en) * | 2009-03-20 | 2009-08-19 | 武汉科诺生物科技股份有限公司 | Ion exchange recovery method of synergistic substance in Bt fermentation |
-
2011
- 2011-05-06 CN CN2011101176426A patent/CN102249952A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5736382A (en) * | 1995-06-06 | 1998-04-07 | Wisconsin Alumni Research Foundation | Bacillus cereus strain DGA34 |
| CN101508656A (en) * | 2009-03-20 | 2009-08-19 | 武汉科诺生物科技股份有限公司 | Ion exchange recovery method of synergistic substance in Bt fermentation |
Non-Patent Citations (5)
| Title |
|---|
| HAIYIN HE等: "Zwittermicin A, an Antifungal and Plant Protection Agent from Bacillus cereus", 《TETRAHEDRON LETTERS》, vol. 35, no. 16, 31 December 1994 (1994-12-31), pages 2499 * |
| LAURA A. SILO-SUH等: "Target Range of Zwittermicin A, an Aminopolyol Antibiotic from Bacillus cereus", 《CURRENT MICROBIOLOGY》, vol. 37, 31 December 1998 (1998-12-31), pages 6 - 11 * |
| NICHOLE A. BRODERICK等: "Synergy Between Zwittermicin A and Bacillus thuringiensis subsp. kurstaki Against Gypsy Moth (Lepidoptera: Lymantriidae)", 《BIOLOGICAL CONTROL》, vol. 29, no. 1, 31 December 2000 (2000-12-31) * |
| 刘中信等: "Zwitterm ic in A的分离纯化及其稳定性的初步研究", 《微生物学通报》, vol. 34, no. 2, 31 December 2007 (2007-12-31), pages 212 - 215 * |
| 徐素平等: "抗生素分离纯化技术", 《安徽农学通报》, vol. 15, no. 7, 31 December 2009 (2009-12-31), pages 79 - 1 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645111A (en) * | 2017-03-01 | 2017-05-10 | 桂林理工大学 | Method for rapidly detecting concentration of antibiotic Zwittermicin A through electrochemical luminescence |
| CN108191713A (en) * | 2017-12-17 | 2018-06-22 | 桂林理工大学 | A kind of method that zwiitermicin A is isolated and purified in the zymotic fluid from Bt |
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Application publication date: 20111123 |