CN105111290A - Antibacterial polypeptide of bacillus amyloliquefaciens NCPSJ7 and method for preparing antibacterial polypeptide - Google Patents
Antibacterial polypeptide of bacillus amyloliquefaciens NCPSJ7 and method for preparing antibacterial polypeptide Download PDFInfo
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Abstract
The invention relates to an antibacterial polypeptide of bacillus amyloliquefaciens NCPSJ7 and a method for preparing the antibacterial polypeptide, and belongs to the field of technologies for preparing antibacterial polypeptides. The method includes purification means of precipitating ammonium sulfate; desalinating the ammonium sulfate; exchanging anions; carrying out exclusion chromatography; carrying out hydrophobic chromatography and the like to obtain the antibacterial polypeptide of the bacillus amyloliquefaciens NCPSJ7 by means of purification. The antibacterial polypeptide of the bacillus amyloliquefaciens NCPSJ7 and the method have the advantages that the molecular weight of the antibacterial polypeptide is 3.0-5.8KDa, and the lowermost inhibition concentration for oxysporum of watermelons is 0.031mg/mL; the antibacterial polypeptide in an apparent form is light yellow powder; the application temperature range of the antibacterial polypeptide is 4-80 DEG C; the application pH (potential of hydrogen) range of the antibacterial polypeptide is 1-11; the antibacterial polypeptide is high in heat stability, is acid-resistant and alkali-resistant and has potential application value.
Description
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide and preparation method thereof, belong to antimicrobial polypeptide preparing technical field.
Background technology
Plant diseases main reason is invading pathogens plant, causes huge financial loss thus.Chemical pesticide, elimination pathogenic bacteria, avoids Plant diseases, and the aspect such as to reduce the loss has played Main Function.But long-term a large amount of chemical pesticide that uses also exists potential harm, and it causes environmental pollution, causes agricultural byproducts Pesticide Residues to increase, and cause food safety question day by day serious, direct harm humans is healthy; Chemical pesticide has has also killed and wounded other beneficial microorganisms, insect and livestock and poultry while suppression pathogenic bacteria, destroys the eubiosis; Further, pathogenic fungi and resistance are constantly deepened and are expanded, the amount of application of increasing agricultural chemicals of having to, thus can cause the vicious cycle that chemical pesticide is applied.
Along with the enhancing of people's environmental consciousness, and understanding in depth chemical pesticide negative impact, how efficiently to kill crop pathogens, maintaining ecological balance simultaneously, obtain high-quality, safe agricultural-food become the problem of people's growing interest, impel people to go to seek control of plant disease approach safely and effectively.Biological control (i.e. microbial metabolites (agricultural antibiotic) or microorganism live bacteria preparation) controlling plant diseases is utilized to not only avoid the series of problems that chemical pesticide brings, and safely, effectively and lastingly, oneself is paid close attention to widely through developing into a kind of effective measure of controlling plant diseases.But be limited to the biological control research of microbial cells at present, the study on prevention of the secondary metabolite secreted by antagonistic microbe also only rests on laboratory stage.Therefore, from antagonistic microbe, be separated the antagonistic substance of stability and high efficiency more, create conditions for popularization antimicrobial substance carries out mass control Plant diseases, for the biotechnology exploitation of controlling plant diseases lays the foundation.
Bacillus amyloliquefaciens
bacillusamyloliquefaciensnCPSJ7 is separated and obtains in Shandong Province's Laiwu City ginger field soil, be deposited in China typical culture collection center, deposit number is CCTCCNO:M2013098(number of patent application: CN201310112580.9, denomination of invention: broad-spectrum antimicrobial Bacillus amyloliquefaciens strain and application thereof), the thalline of invention bacterial strain and fermented liquid have antagonism peach head mold canker, cucumber fusarium axysporum, graw mold of tomato, jujube anthrax, Pear black spot, pears penicilliosis, apple brown rot, alternaria leaf spot of apple, the Plant diseases that the plant pathogenic fungis such as watermelon blight cause and postharvest disease of fruits and vegetables and antagonism streptococcus aureus, Salmonella paratyphi A, Vibrio parahemolyticus, the effect of the food-borne pathogens such as yeast and spoilage organism.A kind of bacillus amyloliquefaciens NCPSJ7 antibacterial protein product and preparation method thereof, Chinese patent 201410198531.6, disclose a kind of antibacterial protein with broad spectrum antibacterial performance, its molecular weight is 20-40kDa, and its main component is: 24kDa and 36kDa two kinds of antibacterial proteins; Its morphological appearance is brownish-yellow powder.Experiment proves, it is 0.086mg/mL to withered germ of water-melon minimum inhibitory concentration.
Summary of the invention
The object of the present invention is to provide a kind of can the antibacterial polypeptide significantly suppressing the activity of withered germ of water-melon and preparation method thereof.
Technical scheme of the present invention
A kind of bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide, its molecular weight is 3.0-5.8KDa, is 0.031mg/mL to withered germ of water-melon minimum inhibitory concentration.
Bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide of the present invention, its mode of appearance is buff powder; Its Applicable temperature scope 4-80oC; Its applicable pH range 1-11.
Above-mentioned bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide, preferably, its molecular weight is 3.0-4.5KDa; The bacteriostatic activity of the polypeptide in this molecular weight ranges is better.
A preparation method for above-mentioned bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide, comprises the following steps:
(1) bacillus amyloliquefaciens fermented liquid centrifugal after in supernatant liquor, add the saturation ratio of ammonium sulfate to 80%, centrifugal after 4oC leaves standstill 24h, precipitate and redissolve with B, obtain redissolution liquid;
(2) liquid that will redissolve take B as damping fluid, carries out desalination, obtain concentrated solution with dialysis tubing;
(3) concentrated solution DEAE anion-exchange chromatography post is separated: balance chromatography column with B, and the B being 0.4mol/L with NaCl content is elution, collects elution peak, obtains active ingredient 1;
(4) active ingredient 1 Superdex75 exclusion chromatography post is separated: elutriant is B, collects second elution peak, obtains active ingredient 2;
(5) the damping fluid NaCl content in active ingredient 2 is that rear octyl sepharose-4 hydrophobic chromatography post of B replacement of 1.5mol/L carries out purifying: first carry out the linear wash-out of 0-100%B, then carry out B-C-ddH
2the gradient elution of O, collects elution peak during gradient elution, obtains product;
Described B is pH is 7.0, concentration is the PBS of 50mmol/L;
The PBS of described C to be concentration be 20mmol/L;
Molecular weight cut-off MwCO≤3000 of described dialysis tubing.
MwCO is different, and the molecular weight distribution of the polypeptide of concentrated solution is different, and the present invention adopts the object of the dialysis tubing of MwCO≤3000 to be to obtain polypeptide of the present invention.
The saturation ratio of ammonium sulfate is different, and the polypeptide in the precipitation obtained is different; The object of the saturation ratio of 80% is adopted to be to obtain polypeptide of the present invention.
DEAE anion-exchange chromatography post, refers to the diethylamino ethyl anion-exchange chromatography post of post footpath 26mm, filler height 100mm.
Superdex75 exclusion chromatography post, post footpath 16mm, packing layer 900mm.
Octyl sepharose-4 hydrophobic chromatography post, post footpath 16mm, packing layer 100mm.
Bacillus amyloliquefaciens fermented liquid in the present invention is by bacillus amyloliquefaciens
bacillusamyloliquefaciensnCPSJ7 carries out cultivating, fermenting forms.
Beneficial effect
The present invention adopts the means of purification such as ammonium sulfate precipitation, desalination, anionresin, exclusion chromatography, hydrophobic chromatography, and purifying obtains a kind of bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide.Prepared antimicrobial polypeptide, obviously can suppress the activity of withered germ of water-melon (be 0.031mg/mL to withered germ of water-melon minimum inhibitory concentration), and have stronger thermostability, acid and alkali-resistance, have potential using value.
Accompanying drawing explanation
Fig. 1 is exclusion chromatography collection of illustrative plates;
Fig. 2 is Octyl sepharose hydrophobic chromatography, 50mmol/LPBS-20mmol/LPBS-ddH
2o gradient elution collection of illustrative plates;
Fig. 3, after Octyl sepharose hydrophobic chromatography, elution peak fungistatic effect figure;
Fig. 4, after Octyl sepharose hydrophobic chromatography, the electrophoretogram of active elution peak 1; 15.5%Tricine-SDS-PAGE:A, the electrophoretic band after hydrophobic chromatography; B, low molecular weight protein (LMWP) marker, main band molecular weight is respectively 22.0,14.4,7.8,5.8,3.3 from top to bottom;
Fig. 5, the antibacterial result figure of comparative example different concns antimicrobial polypeptide of the present invention;
Fig. 6, antimicrobial polypeptide temperature stability experimental result picture of the present invention;
Fig. 7, antimicrobial polypeptide potential of hydrogen stability experiment result figure of the present invention;
Fig. 8, the antibacterial result figure of comparative example different concns antibacterial protein.
Embodiment
The present invention's DEAE anion-exchange chromatography used post, Superdex75 exclusion chromatography post, octyl sepharose-4 hydrophobic chromatography post, be all purchased from GE medical treatment China, product type is respectively 17-0709-01,17-1044-01,17-0946-02.
embodiment 1the preparation of antimicrobial polypeptide crude product concentrated solution
Bacillus amyloliquefaciens
bacillusamyloliquefaciensthe Culture and fermentation conditions of NCPSJ7 is shown in the embodiment 2 of " broad-spectrum antimicrobial Bacillus amyloliquefaciens strain and application thereof ", Chinese patent 2013101125809.
4L fermented supernatant fluid can be replaced by other parameters through this parameter of noncentricity of the centrifugal 10min(of 10000r/min, on result without impact), retain supernatant liquor; The saturation ratio of ammonium sulfate to 80% is added in supernatant liquor, then leave standstill 24 hours in 4oC, can replace by other parameters through this parameter of noncentricity of the centrifugal 20min(of 11000r/min, on result without impact), the precipitation PBS that about 50mL concentration is 50mmol/L, pH7.0 redissolves, and obtains redissolution liquid.
Redissolution liquid is put into dialysis tubing (molecular weight cut-off MwCO≤3000), dialysis tubing being put into the concentration that 1000mL is housed is that the PBS(of 50mmol/L, pH7.0 is as buffered soln) beaker, beaker is placed on magnetic stirring apparatus and stirs, buffered soln is replaced every 2h, after 24h, changing liquid interval time is reclaim solution in dialysis tubing after 6-8h, 48h, is crude product concentrated solution.
embodiment 2dEAE anion-exchange chromatography column chromatography
Concentrated solution post footpath 26mm, the DEAE anion-exchange chromatography post of filler height 100mm carries out being separated (wherein, post footpath, filler is high is conventional selection, select other post footpaths, filler high on result without impact): normal pressure loading, then working concentration is 50mmol/L, the PBS of pH7.0 balances 2 column volumes, again with the NaCl content concentration that is 0.4mol/L for 50mmol/L, the PBS of pH7.0 is that elutriant carries out wash-out, arrange this flow velocity of flow velocity 1mL/min(to arrange for conventional, its change does not affect result), the absorbing wavelength arranging Ultraviolet Detector is that this wavelength of 280nm(is arranged for conventional), collect elution peak, active ingredient 1(to contain NaCl content be the concentration of 0.4mol/L is 50mmol/L, the PBS of pH7.0).
embodiment 3exclusion chromatography
Active ingredient 1 post footpath 16mm, the Superdex75 exclusion chromatography post of packing layer 900mm is separated (wherein, post footpath, filler is high is conventional selection, select other post footpaths, filler high on result without impact): take concentration as 50mmol/L, the PBS of pH7.0 is elutriant, arrange this flow velocity of flow velocity 0.8mL/min(to arrange for conventional, its change does not affect result), the absorbing wavelength arranging Ultraviolet Detector is that this wavelength of 280nm(is arranged for conventional, Fig. 1 is shown in by wash-out collection of illustrative plates) collect second elution peak, active ingredient 2(to contain concentration be 50mmol/L, the PBS of pH7.0).
embodiment 4octyl sepharose-4 hydrophobic chromatography
Octyl sepharose-4 hydrophobic chromatography post of active ingredient 2 post footpath 16mm, packing layer 100mm carries out chromatography (wherein, post footpath, the high routine that is of filler are selected, select other post footpaths, filler high on result without impact): the damping fluid in active ingredient 2 to be replaced with NaCl content be the concentration of 1.5mol/L is the PBS of 50mmol/L, pH7.0, adopt normal pressure loading, elution program: 0-100%B linear elution washes foreign protein off, then according to 50mmol/LPBS-20mmol/LPBS-ddH2O gradient elution; The absorbing wavelength arranging Ultraviolet Detector is that this wavelength of 280nm(is arranged for conventional) collect elution peak (Fig. 2 is shown in by wash-out collection of illustrative plates) when gradient elution, obtain the bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide activeconstituents that concentration is 0.08-0.24mg/mL.
embodiment 5the Tricine-SDS-PAGE electrophoresis of bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide
Adopt the bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide activeconstituents band of 15.5%Tricine-SDS-PAGE electrophoresis detection embodiment 4, separation gel: squeegee: concentrated glue=3.5:1.2:1, sample concentration 0.25mg/mL, applied sample amount 10 μ L, voltage 30V, when band enters separation gel, voltage is transferred to 100V, terminate to electrophoresis, coomassie brilliant blue R250 dyeing 2h, decolouring.Electrophoresis result is shown in Fig. 4, and bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide molecular weight concentrates between 3.0-5.8KDa, is polypeptide class antimicrobial substance.
embodiment 6bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide is to the bacteriostatic experiment of withered germ of water-melon
Pathogenic bacteria (withered germ of water-melon) bacterium block is produced with the punch tool of diameter 5mm, be inoculated in PDA flat board, 28oC produces bacterium block with 5mm punch tool from pathogenic bacteria mycelia edge after cultivating 3d, be suspended in the sterilized water of 400 μ L with after aseptic cutter chopping, get 50 μ L bacteria suspensions and coat on PDA flat board.Flat board is placed 2-4 Oxford cup, and spacing is not less than 4cm, adds 200 μ L samples, cultivate 2-3d under 28oC condition in the cup of each Oxford, suppresses withered germ of water-melon growth result to see Fig. 3.In Fig. 3, the sample in the cup of Oxford contains the paraxin of the 2.5mg/mL of 100 μ L bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptides (concentration is 0.12mg/mL) and 100 μ L.
embodiment 7bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide is to the mensuration of test fungal minimum inhibitory concentration
Damping fluid in embodiment 4 bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide activeconstituents is removed, obtains the pure bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide of light yellow powder solid.
Be the PBS of 50mmol/L, pH7.0 by pure bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide concentration, be diluted to respectively concentration be 1,0.5,0.25,0.125,0.063,0.031mg/mL; Then add paraxin according to the volume ratio of 1:1, final concentration be respectively 0.5,0.25,0.125,0.063,0.031,0.016mg/mL.Bacteriostatic experiment is carried out to the bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide after dilution; Experimental result shows, its minimum effective Mlc is 0.031mg/mL(Fig. 5).
embodiment 8the stability study of bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide
Dissolved by the PBS of pure bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide 50mmol/L, pH7.0, concentration is 1mg/mL.Get 500 μ L respectively at 4,25,40,60,80, process 30min under 100oC condition, then carry out the bacteriostatic experiment (each treatment condition are in triplicate) to withered germ of water-melon.Experimental result (as shown in Figure 6) shows, and through the antimicrobial polypeptide for the treatment of of different temperature, through 4-80oC process, does not significantly change the inhibition of withered germ of water-melon, shows that its Applicable temperature scope is 4-80oC.
Dissolved by the PBS of pure bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide 50mmol/L, pH7.0, concentration is 1mg/mL.Get 1.5mL sample, regulate pH to 1,2,3,5,7(CK with 2M hydrochloric acid or 2MNaOH, do not regulate), 9,10,11,12,13, under 4oC condition, place 4h, recall to initial pH(7.16), the multiple hole of 2, each sample, Odontothrips loti measures inhibition zone size, with the sample do not processed for contrast.Experimental result (as shown in Figure 7) shows, and this antimicrobial polypeptide can keep its bacteriostatic activity within the scope of pH1-11, and under basic conditions (pH >=12), lose bacteriostatic activity, show that its applicable soda acid scope is pH1-11, within the scope of this soda acid, fungistatic effect does not have considerable change.
comparative example
Chinese patent 201410198531.6, discloses a kind of antibacterial protein with spectrum antibacterial performance, and its molecular weight is 20-40kDa, and its main component is: 24kDa and 36kDa.Be dissolved in the PBS of 50mmol/L, pH7.0, according to the method gradient dilution of embodiment 7, measured the minimum inhibitory concentration that it effectively can suppress withered germ of water-melon.Experimental result shows, and its minimum effective Mlc is shown in 0.086mg/mL(Fig. 8).
Claims (7)
1. a bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide, is characterized in that, its molecular weight is 3.0-5.8KDa, is 0.031mg/mL to withered germ of water-melon minimum inhibitory concentration.
2. bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide according to claim 1, is characterized in that, its mode of appearance is buff powder; Its Applicable temperature scope 4-80oC; Its applicable pH range 1-11.
3. bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide according to claim 1 and 2, is characterized in that, its molecular weight is 3.0-4.5KDa.
4. a preparation method for the bacillus amyloliquefaciens NCPSJ7 antimicrobial polypeptide described in claim 1,2 or 3, is characterized in that, comprise the following steps:
(1) bacillus amyloliquefaciens fermented liquid centrifugal after in supernatant liquor, add the saturation ratio of ammonium sulfate to 80%, centrifugal after 4oC leaves standstill 24h, precipitate and redissolve with B, obtain redissolution liquid;
(2) liquid that will redissolve take B as damping fluid, carries out desalination, obtain concentrated solution with dialysis tubing;
(3) concentrated solution DEAE anion-exchange chromatography post is separated: balance chromatography column with B, and the B being 0.4mol/L with NaCl content is elution, collects elution peak, obtains active ingredient 1;
(4) active ingredient 1 Superdex75 exclusion chromatography post is separated: elutriant is B, collects second elution peak, obtains active ingredient 2;
(5) the damping fluid NaCl content in active ingredient 2 is that rear octyl sepharose-4 hydrophobic chromatography post of B replacement of 1.5mol/L carries out purifying: first carry out the linear wash-out of 0-100%B, then carry out B-C-ddH
2the gradient elution of O, collects elution peak during gradient elution, obtains product;
Described B is pH is 7.0, concentration is the PBS of 50mmol/L;
The PBS of described C to be concentration be 20mmol/L;
Molecular weight cut-off MwCO≤3000 of described dialysis tubing.
5. preparation method according to claim 4, is characterized in that, DEAE anion-exchange chromatography post, refers to the diethylamino ethyl anion-exchange chromatography post of post footpath 26mm, filler height 100mm.
6. preparation method according to claim 4, is characterized in that, Superdex75 exclusion chromatography post, post footpath 16mm, packing layer 900mm.
7. preparation method according to claim 4, is characterized in that, octyl sepharose-4 hydrophobic chromatography post, post footpath 16mm, packing layer 100mm.
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Citations (3)
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| CN1611511A (en) * | 2004-03-27 | 2005-05-04 | 中国科学院等离子体物理研究所 | Bacillus subtilis antibacterial peptide separating and purifying method |
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| CN103951736A (en) * | 2014-05-09 | 2014-07-30 | 山东省农业科学院农产品研究所 | Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product |
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Patent Citations (3)
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| CN1611511A (en) * | 2004-03-27 | 2005-05-04 | 中国科学院等离子体物理研究所 | Bacillus subtilis antibacterial peptide separating and purifying method |
| CN103173397A (en) * | 2013-04-02 | 2013-06-26 | 山东省农业科学院农产品研究所 | Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof |
| CN103951736A (en) * | 2014-05-09 | 2014-07-30 | 山东省农业科学院农产品研究所 | Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product |
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| 王英国等: "解淀粉芽孢杆菌抗菌活性物质的分离纯化及抑菌活性研究", 《中国生物工程杂志》 * |
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