CN1611511A - Bacillus subtilis antibacterial peptide separating and purifying method - Google Patents
Bacillus subtilis antibacterial peptide separating and purifying method Download PDFInfo
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- CN1611511A CN1611511A CNA2004100144837A CN200410014483A CN1611511A CN 1611511 A CN1611511 A CN 1611511A CN A2004100144837 A CNA2004100144837 A CN A2004100144837A CN 200410014483 A CN200410014483 A CN 200410014483A CN 1611511 A CN1611511 A CN 1611511A
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Abstract
The invention relates to a kind of separation and purification method of hay bacillus antibiotic peptide. The method contains the steps as follows: preparation of virgin extract, separation of anion exchange column DEAE-Sepharose FF, separation of hydrophobic column SOURCE 15 PHE, separation of molecular sieve column Sephacryl S-200HR, and separation and purification of anti-phase C18 column, and finally, it can get 3 categories main antibiotic peptide. The antibiotic peptide mentioned above has high thermal stability and ability of resisting protease hydrolysis, and also microorganism can generate the drug tolerance to it difficultly. The separation and purification technology of the invention has the features such as quick process, high efficiency, and high recovery ratio, and according to the identification of mass spectrum, the product purity can achieve up to more than 98%.
Description
Technical field
The present invention relates to a kind of separating and purifying method of antisepsis peptide of wilted hay bacilli.
Background technology
Antibacterial peptide be in the organism a kind of micromolecule polypeptide with anti-microbial activity through inducing generation (relative molecular mass is usually 1 * 10
4D is following), extensively be present among bacterium, fungi, batrachians, insect, higher plant, Mammals and even the mankind.Have sterilization, the bacteriostatic activity of wide spectrum, not only act on gram positive bacterium, and act on fungi, insect and some virus and tumour.Owing to antibacterial peptide has a series of advantage such as the difficult resistant mutation that produces of target bacterial strain that common microbiotic does not have, contain the Degradation that the amino acid whose cyclic peptide of D-type can be resisted the protein in body enzyme, therefore become a focus of modern science and applied research; Simultaneously owing to it plays the new source that crucial effect becomes new drug in the biophylaxis system.
Subtilis also has at present as microbial pesticide uses, and its composition is the crude extract of fermentation of bacillus subtilis liquid, because impurity is more, has limited its use range.
Summary of the invention
The present invention relates to the method for antibacterial peptide in a kind of rapidly and efficiently separating and purifying fermentation of bacillus subtilis liquid, its preparation technology is rationally simple, the rate of recovery is high, operational condition is gentle, and has determined that the molecule of the antibacterial peptide of extraction constitutes.
Subtilis is that a kind of can the secretion is harmful to the soil microorganisms that pathogenic fungi has the antibacterial peptide that suppresses lethal effect to multiple kinds of crops.This class antibacterial peptide has the thermostability height, the ability of tolerance protease hydrolysis, and microorganism is difficult for it is produced resistance.Because this type of antibacterial peptide is present in the fermented liquid of subtilis jointly with the form of multiple analog usually, and ring texture and lipid side chain are arranged.According to above characteristics, preferred separation process for separating and purifying of the present invention is:
1, the preparation of the crude extract of fermentation of bacillus subtilis liquid is to utilize the device of ultra-filtration that fermented liquid is concentrated and purifying, the material of molecular weight cut-off more than 1000D.
More precisely be that fermented liquid (molecular weight cut-off is 1000D) 6000r/min centrifuging and taking supernatant liquor behind the cross-flow ultrafiltration system ultrafiltration and concentration of Millipore company with subtilis JA obtains.
2, DEAE-Sepharose FF column chromatography: crude extract is fully dialysed with A damping fluid (20mmol/L pH6.8 phosphate buffered saline buffer), the centrifugal collection supernatant liquor of 8000r/min, cross A damping fluid equilibrated DEAE-Sepharose FF anion-exchange column, (contain 1.0mol/L (NH with the B damping fluid
4)
2SO
4The A damping fluid, this concentration also is the purpose that fully takes into account separation and purification: both guaranteed all antibacterial peptides wash-out from the post all, avoid introducing too much foreign protein again)) carry out linear gradient elution with A damping fluid on-line mixing, collection all has the active peak of bacteriostatic action to aspergillus niger, gibberella saubinetii and Rhizoctonia solani Kuhn, and the active eluant of collecting is concentrated (molecular weight cut-off is 3000D) through the Stirred of Millipore company Cells 8050 ultrafiltration cups.
3, SOURCE 15PHE column chromatography: with the SOURCE 15PHE post of B damping fluid pre-equilibration on the active constituent of anion column collection, the drip washing of B damping fluid is to baseline, carry out drip washing with the A damping fluid again, obtain active peak with the distilled water wash-out at last, collect the merging back and concentrate.
4, Sephacryl S-200HR column chromatography: the sample after concentrating is crossed and is contained 0.5mol/L (NH
4)
2SO
4((NH herein
4)
2SO
4Concentration between 0.15-0.5mol/L, get final product, selecting 0.5 concentration for use is in order to avoid the non-special interaction between the separating medium in antibacterial peptide and the molecular sieve column as far as possible) the molecular sieve Sephacryl S-200HR post of A damping fluid pre-equilibration, then with same buffer solution elution, obtain two elution peaks, collect active peak sample concentration, again through the Milli-Q water postlyophilization of fully dialysing.
5, reversed-phase HPLC separates: the cryodesiccated sample of previous step separates with 30% acetonitrile (containing 0.1%TFA) dissolving and through 30% acetonitrile (containing 0.1%TFA) balance C18 post, carry out linear gradient elution with 30%～80% acetonitrile (containing 0.1%TFA), collect the polypeptide of some molecular weight about 1000D to 1500D.When acetonitrile concentration reached 50% left and right sides, three kinds of target products that anti-microbial activity is arranged were successively by wash-out.
6, the molecular weight of three kinds of antibacterial peptides of mass spectroscopy is followed successively by AFP1:1462.645D, AFP2:1476.390D, AFP3:1490.530D; Amino acid composition analysis shows AFP1 (1462.645D) by 1 Ala, 1 Ile, and 1 Pro, 1 Thr, 3 Glx (Glu or Gln), 2 Tyr and 1 Orn form; AFP2 (1476.390D) is by 1 Ala, 1 Ile, and 1 Pro, 1 Thr, 3 Glx (Glu or Gln), 2 Tyr and 1 Orn form; AFP3 (1490.530D) then is by 1 Val, 1 Ile, and 1 Pro, 1 Thr, 3 Glx (Glu or Gln), 2 Tyr and 1 Orn form.Three kinds of antibacterial peptides all have very strong inhibition ability to fusarium graminearum and Rhizoctonia solani Kuhn.
Involved in the present invention to various buffer formulation laboratory manuals in all can find.
Three kinds of antibacterial peptide purity height of the present invention have bacteriostatic activity efficiently, can be used as biological control and use, and the bacteriostatic experiment of human disease fungi shows also has restraining effect to beriberic pathogenic fungi.
Preparation technology of the present invention rapidly and efficiently, target product purity height, degree of purity of production is identified and can be reached more than 98% through mass spectrum.Three kinds of antibacterial peptide called after AFP1, AFP2 and AFP3.
The control that above-mentioned AFP1, AFP2 through purifying and AFP3 can be used for diseases and pests of agronomic crop (as rice sheath blight disease, wheat scab, watermelon blight etc.); Can be used for treating people and animals' the tetter that causes by fungi (as beriberi etc.) simultaneously, wherein obvious to trichophyton, Trichophyton mentagrophytes effect; The fodder additives that also can be used as domestic animal is with the feed mold that prevents fungies such as aspergillus niger, flavus to cause; In addition normal mammalian cell is had no adverse effects, but to JEG-3, part virus then has obvious lethal effect.This indication antibacterial peptide has a good application prospect in treatment and preventing cancer and anti-virus aspect.
Embodiment
The step of the separating and purifying method of antisepsis peptide of wilted hay bacilli of the present invention is as follows:
1, the preparation of the crude extract of fermentation of bacillus subtilis liquid is that fermented liquid (molecular weight cut-off is 1000D) 6000r/min centrifuging and taking supernatant liquor behind the cross-flow ultrafiltration system ultrafiltration and concentration of Millipore company with subtilis JA obtains.
2, DEAE-Sepharose FF column chromatography: crude extract is fully dialysed with A damping fluid (20mmol/L pH6.8 phosphate buffered saline buffer), the centrifugal collection supernatant liquor of 8000r/min, cross A damping fluid equilibrated DEAE-Sepharose FF anion-exchange column, (contain 1.0mol/L (NH with the B damping fluid
4)
2SO
4The A damping fluid) with A damping fluid on-line mixing carry out linear gradient elution, collection all has the active peak of bacteriostatic action to aspergillus niger, gibberella saubinetii and Rhizoctonia solani Kuhn, and the active eluant of collecting is concentrated (molecular weight cut-off is 3000D) through the Stirred of Millipore company Cells 8050 ultrafiltration cups.
3, SOURCE 15PHE column chromatography: with the SOURCE 15PHE post of B damping fluid pre-equilibration on the active constituent of anion column collection, the drip washing of B damping fluid is to baseline, carry out drip washing with the A damping fluid again, obtain active peak with the distilled water wash-out at last, collect the merging back and concentrate.
4, Sephacryl S-200HR column chromatography: the sample after concentrating is crossed and is contained 0.5mol/L (NH
4)
2SO
4The molecular sieve Sephacryl S-200HR post of A damping fluid pre-equilibration, then with same buffer solution elution, obtain two elution peaks, collect active peak sample concentration, again through the Milli-Q water postlyophilization of fully dialysing.
5, reversed-phase HPLC separates: the cryodesiccated sample of previous step separates with 30% acetonitrile (containing 0.1%TFA) dissolving and through 30% acetonitrile (containing 0.1%TFA) balance C18 post, carry out linear gradient elution with 30%～80% acetonitrile (containing 0.1%TFA), collect the polypeptide of some molecular weight about 1000D to 1500D.When acetonitrile concentration reached 50% left and right sides, three kinds of target products that anti-microbial activity is arranged were successively by wash-out.
The molecular weight of three kinds of antibacterial peptides of mass spectroscopy is followed successively by AFP1:1462.645D, AFP2:1476.390D, AFP3:1490.530D; Amino acid composition analysis shows AFP1 (1462.645D) by 1 Ala, 1 Ile, and 1 Pro, 1 Thr, 3 Glx (Glu or Gln), 2 Tyr and 1 Orn form; AFP2 (1476.390D) is by 1 Ala, 1 Ile, and 1 Pro, 1 Thr, 3 Glx (Glu or Gln), 2 Tyr and 1 Orn form; AFP3 (1490.530D) then is by 1 Val, 1 Ile, and 1 Pro, 1 Thr, 3 Glx (Glu or Gln), 2 Tyr and 1 Orn form.
Claims (5)
1, a kind of separating and purifying method of antisepsis peptide of wilted hay bacilli, be crude extract, after the separation and purification of the separation of the separation of the separation of anion-exchange column DEAE-Sepharose FF, drainage column SOURCE15 PHE, molecular sieve column Sephacryl S-200HR and anti-phase C18 post, obtain 3 kinds of main antibacterial peptides fermentation of bacillus subtilis liquid.
2, the separating and purifying method of antisepsis peptide of wilted hay bacilli as claimed in claim 1, the crude extract that it is characterized in that fermentation of bacillus subtilis liquid are to utilize the device of ultra-filtration that fermented liquid is concentrated and purifying, the material of molecular weight cut-off more than 1000D.
3, the separating and purifying method of antisepsis peptide of wilted hay bacilli as claimed in claim 1, the crude extract that it is characterized in that fermentation of bacillus subtilis liquid is that fermented liquid with subtilis JA is behind the cross-flow ultrafiltration system ultrafiltration and concentration of Millipore company, the material of molecular weight cut-off more than 1000D, and the supernatant liquor obtained of the speed high speed centrifugation more than 6000r/min.
4, the separating and purifying method of antisepsis peptide of wilted hay bacilli as claimed in claim 1, it is characterized in that: (1), through the separation of anion-exchange column DEAE-Sepharose FF: with crude extract with after the A damping fluid-20mmol/L pH6.8 phosphate buffered saline buffer is fully dialysed, centrifugal collection supernatant liquor, cross anion-exchange column, with B damping fluid-contain 1.0mol/L (NH through A damping fluid equilibrated DEAE-Sepharose FF
4)
2SO
4A damping fluid and A damping fluid on-line mixing, make (the NH in the damping fluid
4)
2SO
4Concentration increases with elution of bound heterogeneity on anion-exchange column by linear gradient, therefrom collect the active peak that aspergillus niger, gibberella saubinetii and Rhizoctonia solani Kuhn is all had bacteriostatic action, and be that 3000D ultrafiltration cup concentrates through molecular weight cut-off the active eluant of collecting; (2), the separation of drainage column SOURCE 15 PHE: anion column separated that the back is collected and on the active constituent of ultrafiltration and concentration through the SOURCE of B damping fluid pre-equilibration 15 PHE posts, elder generation's B damping fluid will be not and SOURCE 15 PHE post bonded impurity flush awaies, carry out drip washing with the A damping fluid again and further remove impurity, obtain active peak with the distilled water wash-out at last, collect the merging back and concentrate; (3), the separation of molecular sieve column Sephacryl S-200HR: the sample after concentrating is crossed and is contained 0.5mol/L (NH
4)
2SO
4The molecular sieve Sephacryl S-200HR post of A damping fluid pre-equilibration, then with same buffer solution elution, obtain two elution peaks, collect active peak sample concentration, again through the Milli-Q water postlyophilization of fully dialysing; (4), the separation of anti-phase C18 post: the cryodesiccated sample of previous step separates with 30% acetonitrile (containing 0.1%TFA) dissolving and through 30% acetonitrile (containing 0.1%TFA) balance C18 post, the acetonitrile (30%～80% that increases progressively by linear gradient with concentration, contain 0.1%TFA) wash-out, collect the polypeptide of some molecular weight about 1000D to 1500D, when acetonitrile concentration reached 50% left and right sides, three kinds of target products that anti-microbial activity is arranged were successively by wash-out.
5, the separating and purifying method of antisepsis peptide of wilted hay bacilli as claimed in claim 1, it is characterized in that: (1), through the separation of anion-exchange column DEAE-Sepharose FF: crude extract is fully dialysed with A damping fluid-20mmol/L pH6.8 phosphate buffered saline buffer, the centrifugal collection supernatant liquor of 8000r/min, cross A damping fluid equilibrated DEAE-Sepharose FF anion-exchange column, with B damping fluid-contain 1.0mol/L (NH
4)
2SO
4A damping fluid and A damping fluid on-line mixing carry out linear gradient elution, collect the active peak that aspergillus niger, gibberella saubinetii and Rhizoctonia solani Kuhn is all had bacteriostatic action, and be that 3000D ultrafiltration cup concentrates through molecular weight cut-off the active eluant of collecting; (2), the separation of drainage column SOURCE 15 PHE: with the SOURCE 15 PHE posts of B damping fluid pre-equilibration on the active constituent of anion column collection, the drip washing of B damping fluid is to baseline, carry out drip washing with the A damping fluid again, obtain active peak with the distilled water wash-out at last, collect the merging back and concentrate; (3), the separation of molecular sieve column Sephacryl S-200HR: the sample after concentrating is crossed and is contained 0.5mol/L (NH
4)
2SO
4The molecular sieve Sephacryl S-200HR post of A damping fluid pre-equilibration, then with same buffer solution elution, obtain two elution peaks, collect active peak sample concentration, again through the Milli-Q water postlyophilization of fully dialysing; (4), the separation of anti-phase C18 post: the cryodesiccated sample of previous step separates with 30% acetonitrile (containing 0.1%TFA) dissolving and through 30% acetonitrile (containing 0.1%TFA) balance C18 post, carry out linear gradient elution with 30%～80% acetonitrile (containing 0.1%TFA), collect the polypeptide of some molecular weight about 1000D to 1500D, when acetonitrile concentration reached 50% left and right sides, three kinds of target products that anti-microbial activity is arranged were successively by wash-out.
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|---|---|---|---|
| CNB2004100144837A CN1271084C (en) | 2004-03-27 | 2004-03-27 | Bacillus subtilis antibacterial peptide separating and purifying method |
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|---|---|---|---|
| CNB2004100144837A CN1271084C (en) | 2004-03-27 | 2004-03-27 | Bacillus subtilis antibacterial peptide separating and purifying method |
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| CN1271084C CN1271084C (en) | 2006-08-23 |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100379874C (en) * | 2006-01-20 | 2008-04-09 | 南京农业大学 | Method for producing antibiotic peptide by using Bacillus amyloliquefaciens and product thereof |
| CN100463919C (en) * | 2006-11-15 | 2009-02-25 | 江苏省农业科学院 | Extracellular antiseptic protein for Bacillus subtilis and its separation and purification method |
| CN101974586A (en) * | 2010-08-18 | 2011-02-16 | 南京农业大学 | Production method of bacillus subtilis antimicrobial lipopeptide and application thereof in feeds |
| CN102010884A (en) * | 2010-06-13 | 2011-04-13 | 浙江大学 | Antibacterial active matter as well as preparation method and application thereof |
| CN102102110A (en) * | 2010-12-01 | 2011-06-22 | 青岛科技大学 | Method for preparing antibacterial extracellular product of bacillus subtilis |
| CN102191194A (en) * | 2011-03-17 | 2011-09-21 | 四川师范大学 | Bacillus subtilis and antibacterial protein thereof |
| CN101062204B (en) * | 2006-04-30 | 2012-04-04 | 北京赛而生物药业有限公司 | Method for preparing capsule made from asarum, scutellaria and so on and the capsule prepared by using said method |
| CN105111290A (en) * | 2015-09-24 | 2015-12-02 | 山东省农业科学院农产品研究所 | Antibacterial polypeptide of bacillus amyloliquefaciens NCPSJ7 and method for preparing antibacterial polypeptide |
| CN106146608A (en) * | 2015-03-16 | 2016-11-23 | 江西科诺生物科技有限公司 | A kind of extraction preparation method of apidaecin |
| CN109096379A (en) * | 2018-08-14 | 2018-12-28 | 黑龙江八农垦大学 | A kind of identification of the new function and its antibacterial peptide of bacillus subtilis AMEP412 albumen |
-
2004
- 2004-03-27 CN CNB2004100144837A patent/CN1271084C/en not_active Expired - Fee Related
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100379874C (en) * | 2006-01-20 | 2008-04-09 | 南京农业大学 | Method for producing antibiotic peptide by using Bacillus amyloliquefaciens and product thereof |
| CN101062204B (en) * | 2006-04-30 | 2012-04-04 | 北京赛而生物药业有限公司 | Method for preparing capsule made from asarum, scutellaria and so on and the capsule prepared by using said method |
| CN100463919C (en) * | 2006-11-15 | 2009-02-25 | 江苏省农业科学院 | Extracellular antiseptic protein for Bacillus subtilis and its separation and purification method |
| CN102010884A (en) * | 2010-06-13 | 2011-04-13 | 浙江大学 | Antibacterial active matter as well as preparation method and application thereof |
| CN102010884B (en) * | 2010-06-13 | 2012-09-19 | 浙江大学 | Antibacterial active matter as well as preparation method and application thereof |
| CN101974586A (en) * | 2010-08-18 | 2011-02-16 | 南京农业大学 | Production method of bacillus subtilis antimicrobial lipopeptide and application thereof in feeds |
| CN102102110A (en) * | 2010-12-01 | 2011-06-22 | 青岛科技大学 | Method for preparing antibacterial extracellular product of bacillus subtilis |
| CN102191194A (en) * | 2011-03-17 | 2011-09-21 | 四川师范大学 | Bacillus subtilis and antibacterial protein thereof |
| CN102191194B (en) * | 2011-03-17 | 2013-06-05 | 四川师范大学 | Bacillus subtilis and antibacterial protein thereof |
| CN106146608A (en) * | 2015-03-16 | 2016-11-23 | 江西科诺生物科技有限公司 | A kind of extraction preparation method of apidaecin |
| CN105111290A (en) * | 2015-09-24 | 2015-12-02 | 山东省农业科学院农产品研究所 | Antibacterial polypeptide of bacillus amyloliquefaciens NCPSJ7 and method for preparing antibacterial polypeptide |
| CN105111290B (en) * | 2015-09-24 | 2018-07-03 | 山东省农业科学院农产品研究所 | Bacillus amyloliquefaciens NCPSJ7 antibacterial polypeptides and preparation method thereof |
| CN109096379A (en) * | 2018-08-14 | 2018-12-28 | 黑龙江八农垦大学 | A kind of identification of the new function and its antibacterial peptide of bacillus subtilis AMEP412 albumen |
| CN109096379B (en) * | 2018-08-14 | 2021-04-02 | 黑龙江八一农垦大学 | Novel function of bacillus subtilis AMEP412 protein and identification of antibacterial peptide thereof |
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|---|---|
| CN1271084C (en) | 2006-08-23 |
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