CN102851339A - Production method of Sublancin antibacterial peptide - Google Patents
Production method of Sublancin antibacterial peptide Download PDFInfo
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- CN102851339A CN102851339A CN 201210076365 CN201210076365A CN102851339A CN 102851339 A CN102851339 A CN 102851339A CN 201210076365 CN201210076365 CN 201210076365 CN 201210076365 A CN201210076365 A CN 201210076365A CN 102851339 A CN102851339 A CN 102851339A
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Abstract
The invention provides a preparation method of Sublancin antibacterial peptide, which comprises the following steps: (1) inoculating bacillus subtilis into a medium for fermentation, filtering the fermentation liquor; (2) orderly performing centrifugation, cation exchange chromatography, hydrophobic chromatography, size exclusion chromatography, and anion exchange chromatography of the fermentation liquor in step (1), finally performing ultrafiltration concentration and drying to obtain the Sublancin antibacterial peptide. The method of the invention is low in cost, increased in production efficiency, and high in product purity; the prepared Sublancin antibacterial peptide has purity of up to more than 99%; the process is simple, and easy to popularize and apply, and can realize large-scale production.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to a kind of production method of Sublancin antibacterial peptide.
Background technology
The Sublancin antibacterial peptide is to produce a kind of endogenous polypeptide with anti-microbial activity by subtilis (Bacillus subtilis).By Ribosome biogenesis, carry out post transcriptional modificaiton and transhipment by leader sequence.Its physical properties is colourless crystallization or white crystalline powder, and is soluble in water.Sublancin contains disulfide linkage, so good stability.
The Sublancin antibacterial peptide has strong the inhibition and killing action to gram-positive microorganism, can be applicable to food fresh keeping, anti-infective and inhibition environment disinfected.At present, at home and abroad there is no the Sublancin antibacterial peptide production method that can realize suitability for industrialized production.Report about Sublancin antibacterial peptide preparation method is also fewer.(the Sun H.Paik in 1998 such as Paik, Anu Chakicherla, and J.Norman Hansen.1998.Identification and Characterization of the Structural and Transporter Genes for, and the Chemical and Biological Properties of, Sublancin 168, a Novel Lantibiotic Produced by Bacillus subtilis 168.THE JOURNAL OF BIOLOGICAL CHEMISTRY.273:23134-23142.) reported a kind of method for preparing the Sublancin antibacterial peptide in the laboratory.Bacterial strain is B.subtilis 168, and the fermentation scale is 1L.Every 1L substratum contains sucrose 20g, citric acid 11.7g, Na
2SO
44g, (NH
4)
2HPO
44.2g, yeast extract 5g, KCl 0.762g, MgCl
26H
2O 0.418g, MnCl
24H
2O 0.0543g, FeCl
36H
2O 0.049g, ZnCl
20.0208g, use NH
4OH transfers pH 6.8～6.9.After fermentation condition is 37 ℃ of aerlbic culture 16h, 37 ℃ of cultivations of 200rpm shaking table 28h.Purification process is: readjust the distribution ferment nutrient solution pH2.5 with phosphoric acid; The centrifugal cell of removing; Cross 25ml volume hydrophobic chromatography post; Freeze-drying; With the centrifugal precipitation of removing after 0.1% the trifluoroacetic acid dissolving; After the separation and purification through 2 anti-phase C-18 analytical columns of HPLC, through freeze-drying, in-20 ℃ of preservations.The method fermentation small scale yields poorly and unstable.Purge process has adopted highly sophisticated device high performance liquid chromatography (HPLC), and is all higher to chemical reagent and operator's requirement, and this just causes adopting the refining Sublancin antibacterial peptide cost of this method high, the characteristics that yield poorly.Therefore, aforesaid method is not suitable for scale operation, can not satisfy the needs of suitability for industrialized production.
Summary of the invention
The object of the present invention is to provide a kind of fermentative production of high purity Sublancin antibacterial peptide and the method for purifying, to overcome the deficiency that does not have suitability for industrialized production high purity Sublancin antibacterial peptide in the prior art.
The present invention is by large quantity research, the series of process data are explored, by technical renovation, fermentating formula and technique have been optimized, enlarge the fermentation scale, improve the output of Sublancin antibacterial peptide, reduced production cost, found out the fermentation process that a cover can scale operation Sublancin antibacterial peptide; And design, the combination of purge process have been carried out; solved purifying to the dependence of highly sophisticated device and the problem that yields poorly; make the purifying of Sublancin antibacterial peptide satisfy the requirement of large-scale production; realize the large-scale purification of Sublancin antibacterial peptide by a series of chromatography technology, finally realize the preparation of industrialization of high purity Sublancin antibacterial peptide.
The purification flow process of Sublancin antibacterial peptide pure substance is seen Fig. 1.
The invention provides a kind of preparation method of Sublancin antibacterial peptide, described production method comprises:
(1) the subtilis inoculation medium ferments, filtering fermentating liquid;
(2) fermented liquid that step (1) is obtained is centrifugal, and by cation-exchange chromatography, hydrophobic chromatography, size exclusion chromatography, anion-exchange chromatography, last ultrafiltration and concentration, drying namely get the Ssublancin antibacterial peptide.
Wherein, employed bacterial strain is B.subtilis CVCC20076 in the step (1), and fermented liquid is by ceramic membrane filter, and described ceramic membrane aperture is 20nm.
Substratum described in the step (1) consists of every 1L fermention medium and contains: Semen Maydis powder 10-50g, dregs of beans 10-50g, peptone 5-20g, glucose (or sucrose) 2-20g, yeast powder 0-10g, ammonium sulfate 0-10g, ammonium nitrate 0-10g, sal epsom 0-1.5g, manganous sulfate 0-1.5g, phosphoric acid salt 0-10g.Hydro-oxidation sodium is regulated pH7.0-8.0, and the fermentation scale is 10-20m
3
In addition, the inoculum size of subtilis is 1 * 10 in the step (1)
6-10
7CFU/mL, leavening temperature are 30-37 ℃, and fermentation time is 24-32h, air flow 600-1000m
3/ h, rotating speed 120-200r/min, pH 6-7.
Wherein the described centrifugal condition of step (2) is the centrifugal 20-40min of 10000-15000g.
The present invention finds out the washing, the elutriant that are fit to chromatography by attempting different ionic concn and pH value.
The preparation method of Sublancin antibacterial peptide provided by the invention, wherein the cation-exchange chromatography step is with buffer A counterion displacement chromatography post, with ion exchange column on the fermented liquid, the volume ratio of applied sample amount and cation-exchange chromatography column packing is 20: 1, the loading flow velocity is 50mL/min, reach baseline values with buffer A flushing chromatography column to uv-absorbing afterwards, carry out isocratic elution with buffer A and buffer B by 3: 1 volume ratio again, collect elutriant; Described buffer A is 20mM NaAc solution, pH4.0; Described buffer B is the solution that contains 20mM NaAc and 1M NaCl, pH4.0.
This step is utilized Sublancin antibacterial peptide and other foreign protein and the electrically charged interactional principle of chromatography column weighting material, and the Sublancin antibacterial peptide is separated with other foreign protein.Because the iso-electric point of Sublancin antibacterial peptide is different from the iso-electric point of other foreign protein, the intensity from chromatography column weighting material (cationite) combination under suitable salt concn and pH condition is different, thereby the purpose peptide is separated with foreign protein.This step can be removed the foreign protein such as most of material proteins and tropina in the fermented liquid, and the Sublancin antibacterial peptide purity that obtains is more than 70%.
In the method for the present invention, step 2) described hydrophobic chromatography step is to use buffer A ' balance hydrophobic chromatography post, the elutriant that ion exchange chromatography is collected carries out equal-volume with 2M NaCl and mixes loading hydrophobic chromatography post, the volume ratio of applied sample amount and hydrophobic chromatography column packing is that 2.5: 1 loading flow velocitys are 30mL/min, use buffer A ' the washing chromatographic column, until uv-absorbing to baseline values, is used buffer B again ' carry out linear gradient elution, collect elutriant; Described buffer A ' for containing the solution of 20mM NaAc and 1M NaCl, pH4.0, described buffer B ' be 20mM NaAc, pH4.0.
Wherein, step 2) described size exclusion chromatography is with the elutriant loading of hydrophobic chromatography collection step, and each sampling volume is 5% of column volume, and flow velocity is 2mL/min, collects the antibacterial peptide elution peak.
Wherein, step 2) size exclusion chromatography step is with the SEC buffer solution for cleaning and is full of the size exclusion chromatography post, behind baseline values, and loading again.Described SEC damping fluid is 20mM Na
2HPO
4Mixing solutions with 150mM NaCl.
This step can be separated according to each component molecular size in the sample.In the process of chromatography column of flowing through, high molecular weight protein is subjected to the obstruction of filler little, and preferentially by chromatography column, small molecular protein is subjected to the obstruction of filler long by the time of chromatography column greatly.Can further remove the impurity albumen larger than Sublancin molecular weight through size exclusion chromatography.The Sublancin antibacterial peptide purity of this moment is high to more than 99%.
With anion-exchange chromatography post on the elutriant of size exclusion chromatography collection step, flow velocity 2ml/min collects the liquid that flows out.
The anion-exchange chromatography Main Function is to remove the impurity substances such as the residual DNA that brings from fermented liquid and endogenous toxic material, guarantees the security of Sublancin antibacterial peptide end product.
The Sublancin antibacterial peptide product that the present invention also provides aforementioned production method to prepare.
To namely get Sublancin antibacterial peptide pure substance after the Sublancin antibacterial peptide solution ultrafiltration and concentration that obtain, the drying at last.
According to the resulting high purity Sublancin antibacterial peptide of production method provided by the invention, its purity＞99%, molecular weight is 3876.48Da, has excellent antibacterial effect.Beneficial effect of the present invention:
(1) the present invention has replaced other raw material of chemical reagent-grade in the laboratory take cereal products such as corn and soyabean processing product as main raw material, has reduced production cost;
(2) the present invention has brought up to the suitability for industrialized production rank with the fermentation scale, has realized the suitability for industrialized production of Sublancin antibacterial peptide.
(3) the present invention has improved the production efficiency of Sublancin antibacterial peptide, and the content of Sublancin antibacterial peptide can reach 80-100 μ g/mL in the fermented liquid.
(4) the present invention adopts industrialization Chinese style chromatography column, for the large scale purification of realizing the Sublancin antibacterial peptide has been paved road, and is easy to apply.
(5) the present invention's Sublancin antibacterial peptide purity made from extra care reaches more than 99%, can be used for the production of the products such as medicine, foodstuff additive and environment disinfected agent.
Method of the present invention can realize large scale fermentation, and output is high, and cost is low, and product purity is high, and has realized quality stability.Purge process requires low to operator, technique simply is easy to apply, can accomplish scale production.
Description of drawings
Fig. 1 is the method for preparing purified schema of Sublancin antibacterial peptide of the present invention.
Fig. 2 is the Sublancin antibacterial peptide pure substance HPLC color atlas that the present invention prepares.
Fig. 3 is the Sublancin antibacterial peptide pure substance molecular weight mass spectroscopy figure that the present invention prepares.
Fig. 4 is Sublancin antibacterial peptide mass spectrum fragment HPLC color atlas.
Fig. 5 is Sublancin antibacterial peptide fragment 1, fragment 3 mass spectrums.
Fig. 6 is Sublancin antibacterial peptide fragment 2 mass spectrums.
Fig. 7 is the second order ms figure of Sublancin antibacterial peptide fragment 2.
Fig. 8 is the content detection color atlas of Sublancin antibacterial peptide in the fermented liquid.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, used chemical reagent is conventional commercial reagent among the embodiment, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Embodiment 1
Fermentation scale: 10m
3The 1L fermentation culture contains: Semen Maydis powder 20g, dregs of beans 20g, peptone 5g, glucose 5g, ammonium sulfate 5g, sal epsom 0.8g.Transfer pH7.5 with sodium hydroxide.Nutrient solution is through 121 ℃ of temperature, tank pressure 0.08Mpa, the 30min that holds time sterilization.Subtilis B.subtilis CVCC20076 is available from Chinese industrial microbial strains preservation administrative center.The bacterial classification inoculation amount is 1 * 10
6CFU/mL.The fermentation culture temperature: 34 ℃, air flow: 600m
3/ h, stirring velocity: 120r/min, pH7.0.Fermented incubation time: 32h.The membrane pore size 20nm of ceramic membrane filter obtains the fermented liquid of Sublacin antibacterial peptide.
Embodiment 2
Fermentation scale: 20m
3The 1L fermentation culture contains: Semen Maydis powder 50g, dregs of beans 50g, peptone 20g, glucose 20g, ammonium sulfate 5g, yeast powder 5g, dipotassium hydrogen phosphate 5g.Transfer pH8.0 with sodium hydroxide.Nutrient solution is through 121 ℃ of temperature, and tank pressure 0.15Mpa holds time and sterilized in 30 minutes.Subtilis B.subtilis CVCC20076 bacterial classification inoculation amount is 1 * 10
7CFU/mL.The fermentation culture temperature: 30 ℃, air flow: 1000m
3/ h, stirring velocity: 160r/min, pH:6.5.Fermented incubation time: 24h.The membrane pore size 10nm of ceramic membrane filter obtains the fermented liquid of Sublacin antibacterial peptide.
Embodiment 3
Fermentation scale: 10m
3The 1L fermentation culture contains: Semen Maydis powder 10g, dregs of beans 10g, peptone 5g, sucrose 2g, ammonium sulfate 5g.Transfer pH7.0 with sodium hydroxide.Nutrient solution is through 121 ℃ of temperature, and tank pressure 0.10Mpa holds time and sterilized in 30 minutes.Subtilis B.subtilis CVCC20076 bacterial classification inoculation amount is 1 * 10
6CFU/mL.The fermentation culture temperature: 37 ℃, air flow: 720m
3/ h, stirring velocity: 200r/min, pH:6.0.Fermented incubation time: 28h.The membrane pore size 30nm of ceramic membrane filter obtains the fermented liquid of Sublacin antibacterial peptide.
The purification process of embodiment 4 Sublancin antibacterial peptides
Contain by fermentation antibacterial peptide in the secondary fermentation liquid through behind the ceramic membrane filter, collect supernatant liquor through the centrifugal 30min of 12000g for subsequent use.Fermented liquid after pretreatment finally obtains the pure substance of Sublancin antibacterial peptide through a series of solid phase extraction techniques, technique is as follows:
1, ion exchange chromatography
At ambient temperature, the ion exchange column packing volume is 1L.With ion-exchange buffer A (20mM sodium-acetate NaAc, pH4.0) the counterion displacement chromatography post of 7L, flow velocity is 50ml/min.Get the fermented liquid that embodiment 3 makes and carry out the chromatography column loading, applied sample amount is about 20L, and flow velocity is 50ml/min.After the loading, with ion-exchange buffer A flushing chromatography column, until uv-absorbing is near baseline values.Use buffer A: the ratio of buffer B (20mMNaAc+1MNaCl, pH4.0)=3: 1 is carried out isocratic elution, and elution peak namely contains antibacterial peptide, collects the elutriant that contains the sublancin antibacterial peptide.Collect complete after, with 5L buffer B flushing chromatography column, and clean with 3L 0.1M NaOH, at last with 20% ethanol preservation chromatography column.The purity that this step obtains antibacterial peptide is about 70%.
2, hydrophobic chromatography
At ambient temperature, the hydrophobic chromatography filler is 0.2L.The thick product of sublancin antibacterial peptide and the 2M NaCl that first ion exchange chromatography are collected carry out the equal-volume mixing for standby use.With buffer A (20mM NaAc+1M NaCl, pH4.0) balance hydrophobic chromatography post, flow velocity is 30ml/min.After the chromatography column balance is good, keep flow velocity, the whole loadings of the sample that ion-exchange is collected, applied sample amount compares at 2.5: 1 with the chromatography column filler.After the loading, wash chromatographic column with buffer A, until uv-absorbing is to baseline.Use buffer B (20mM NaAc, pH4.0) to carry out linear gradient elution, buffer B from 0 to 100% in the 30min.In when buffer B proportion during to 80%-90%, the Sublancin antibacterial peptide is eluted, the collection elutriant.After wash-out is complete, uses 0.6L 0.1M NaOH to clean chromatography column, and preserve chromatography column with 20% ethanol.The purity that this step obtains antibacterial peptide is about 95%.
3, size exclusion chromatography
At ambient temperature, the size exclusion chromatography filler is 0.4L.The fermented liquid that contains the Sublancin antibacterial peptide through ion-exchange, hydrophobic chromatography after, also need could obtain highly purified desired substance through size exclusion chromatography.Use first SEC damping fluid (20mM Na
2HPO
4+ 150mM NaCl) clean and be full of purification system, connect size exclusion chromatography post (Superdex G50 filler) equilibrium system, flow velocity is 2ml/min.Sample introduction behind baseline values, size exclusion chromatography post on the elutriant that the hydrophobic chromatography step is obtained, each sampling volume is 15ml, antibacterial peptide goes out the peak about 70min, collect the antibacterial peptide elution peak.At last, preserve chromatography column with 20% ethanol.This step obtains the purity of antibacterial peptide greater than 95%.
4, anion-exchange chromatography
The purpose of this step is to remove remaining host DNA.At ambient temperature, anion exchange filler is 1ml.Use first 20ml PBS (20mM Na
2HPO
4+ 150mM NaCl) balance anion displacement chromatography post.With the sample loading that size exclusion chromatography is collected, flow velocity is 2ml/min (that is: all samples stream is worn, remaining host DNA is adsorbed on the chromatography column in the sample, and antibacterial peptide does not adsorb).The liquid that collect to flow out namely gets not contain and participates in host DNA, purity greater than 99% antibacterial peptide.Cleaning chromatography column with damping fluid (20mM Tris+1MNaCl, pH8.0) also preserves with 20% ethanol.
The antibacterial peptide that the anion-exchange chromatography step is obtained is ultrafiltration and concentration, drying according to a conventional method, and the freeze-drying sealing is preserved.
The HPLC analysing and detecting method of embodiment 5 Sublancin antibacterial peptides
The Sublancin antibacterial peptide that embodiment 4 obtains is analyzed with HPLC.Instrument has: Agilent 1260 Infinity binary liquid chromatographic systems (available from Agilent company); Agilent ZORBAX 300StableBond (300SB) C8 HPLC chromatographic column (available from Agilent company); Mobile phase A: 0.1% trifluoroacetic acid aqueous solution; Mobile phase B: 80% acetonitrile solution; Column temperature: 25 ℃, detect wavelength: 280nm; Peak pressure: 200bar, flow velocity: 1ml/min, analyze gradient and see Table 1:
Table 1 HPLC analyzes the gradient eluent gradient
Detected result is seen Fig. 2.As shown in Figure 2, carrying analysis software with Agilent1260 calculates going out peak area per-cent.
Table 2 goes out calculated by peak area
The result shows: the purity of the Sublancin antibacterial peptide that embodiment 4 obtains is 99.42%.
Draw the Sublancin antibacterial peptide purity that the present invention prepares＞99% by the analysis to the HPLC color atlas.
The fermented liquid that makes with embodiment 1,2 adopts embodiment 4 described methods to carry out purifying, the Sublancin antibacterial peptide refined solution that obtains is through the HPLC stratographic analysis, find Sublancin antibacterial peptide purity all＞99%, illustrate with substratum provided by the invention and production method and purification process to obtain highly purified Sublancin antibacterial peptide.
Embodiment 6 Sublancin antibacterial peptide molecular weight mass spectroscopies
Sample preparation: the highly purified sublancin antibacterial peptide freeze-dried products that above-described embodiment 4 is obtained dissolves by 1: 100 (w/v) with ultrapure water, gets 40 μ L loadings.
Plant and instrument: Agilent 1290HPLC liquid chromatograph, Thermo LTQ mass spectrograph.
Chromatographic column: Agilent Eclipse Plus C18 RRHD 1.8 μ m, 2.1mm * 150mm
Column temperature: 60 ℃; Detect wavelength: 214nm; Sample size: 5 μ L; Mobile phase A: 0.1% aqueous formic acid; Mobile phase B: 0.085% formic acid+20% water+80% acetonitrile; Eluent gradient table 3:
Table 3 mass spectrum eluent gradient table
Mass spectrometric detection condition: sheath gas flow (arb): 15; Assist gas flow (arb): 5; Thermospray voltage (kV): 4.7; Capillary temperature (℃): 275; Capillary voltage (V): 49; Sleeve lens offset voltage (V): 120.
Adopt molecular weight mass spectroscopy such as Fig. 3 of the Sublancin antibacterial peptide pure substance of the inventive method preparation, get the trial-product molecular weight according to collection of illustrative plates and be: 3876.48Da.The fermented liquid that makes with embodiment 1,2 adopts embodiment 4 described methods to carry out purifying, the Sublancin antibacterial peptide refined solution that obtains is through the molecular weight mass spectroscopy, obtaining Sublancin antibacterial peptide molecular weight is 3876.48Da, illustrates that the material with substratum provided by the invention and production method and purification process acquisition is the Sublancin antibacterial peptide.
Embodiment 7 Sublancin antibacterial peptide sequence mass spectroscopies
Sample preparation: the pancreatin enzyme is cut the liquid dialysis, adds trypsinase-TPCK solution by 1: 50 (mg/mg), and enzyme was cut 18 hours in 37 ℃.Getting supernatant liquor after centrifugal is used for analyzing.
Plant and instrument: Agilent 1290 HPLC liquid chromatographs, Thermo LTQ mass spectrograph
Chromatographic column: Agilent Eclipse Plus C18 RRHD 1.8 μ m 2.1mm * 150mm
Column temperature: 60 ℃; Detect wavelength: 214nm; Sample size: 5 μ L; Mobile phase A: 0.1% aqueous formic acid; Mobile phase B: 0.085% formic acid+20% water+80% acetonitrile; Eluent gradient table 4:
Table 4 sequence mass spectrum eluent gradient table
Mass spectrometric detection condition: sheath gas flow (arb): 15; Assist gas flow (arb): 5; Thermospray voltage (kV): 4.7; Capillary temperature (℃): 275; Capillary voltage (V): 49; Sleeve lens offset voltage (V): 120.
Adopt the mass spectroscopy of the Sublancin antibacterial peptide sequence of the present invention's preparation, the results are shown in Figure 4-Fig. 7.
Table 5
The Sublancin antibacterial peptide forms 3 fragments after cutting through enzyme, and the molecular weight of each fragment is consistent with theoretical sequence.The Sublancin antibacterial peptide sequence of prompting the present invention preparation is: GLGKAQCAALWLQCASGGTIGCGGGAVACQNYRQFCR, wherein glucose of the upper connection of Cys22.Consistent with theoretical sequence.
The test of embodiment 8 fungistatic effects
Measure the Sublancin antibacterial peptide to the MIC of CMCCB26003 and CVCC1882 two strain streptococcus aureuses according to the micro-dilution method that standard committee of U.S. clinical labororatory (Clinical and Laboratory Standards Institute, CLSI) is recommended.Concrete operations are as follows: get aseptic 96 hole Microdilution plates, and add 100 μ L MH liquid nutrient mediums in each hole; In the first hole of every row, add the 100 μ L Sublancin antibacterial peptides that obtain after the anion-exchange chromatography step of embodiment 4; Carry out serial dilution by 2 coubling dilutions, last hole discards 100 μ L liquid; In 15min, with 100 μ L 1 * 10
6CFU/mL bacterium liquid adds in each hole, hatches 16～18h for 37 ℃.Do simultaneously blank (not adding bacterium) and negative control group (only add bacterium liquid and do not add antibacterial peptide).The result judges: when the negative control group bacterium obviously grows, the blank group is during without bacterial growth, with in the experimental group hole fully bacteria growing inhibiting (be in the hole solution the same with the blank group clarify bright) lowest concentration of drug as the MIC of bacterium to medicine.When single jumping hole occurring, the highest drug level of record bacteria growing inhibiting is jumped the hole phenomenon as repeatedly occurring, and does not then record the result, again test.
Result's demonstration, the Sublancin antibacterial peptide that embodiment 4 makes is respectively the MIC of CMCCB26003 and CVCC1882 two strain streptococcus aureuses: 1.3 μ g/mL (0.335 μ M) and 4.69 μ g/mL (1.21 μ M).
The fermented liquid that embodiment 2,3 is made is behind embodiment 4 described method purifying, the refined solution of gained carries out bacteriostatic experiment by above-mentioned same procedure, found that, embodiment 2,3 fermented liquid make the Sublancin antibacterial peptide behind the inventive method purifying all have significant bacteriostatic action to CMCCB26003 and CVCC1882 two strain streptococcus aureuses.
The content detection of Sublancin antibacterial peptide in embodiment 9 fermented liquids
Adopt the detection method of embodiment 5, get the fermented liquid that embodiment 1 obtains, applied sample amount 5 μ L.Typical curve equation in 40 μ g/mL～5000 μ g/mL linearity range is y=48.03x+0.614, R
2=1.000.The fermented liquid color atlas is seen Fig. 8.Carry software through Agilent 1260 HPLC instrument, the content of Sublancin antibacterial peptide is 92.72 μ g/mL in the regression Calculation fermented liquid.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a method for preparing the Sublancin antibacterial peptide is characterized in that comprising the steps:
1) the subtilis inoculation medium ferments, filtering fermentating liquid;
2) with step 1) the gained fermented liquid successively carries out centrifugal, cation-exchange chromatography, hydrophobic chromatography, size exclusion chromatography, anion-exchange chromatography, and last ultrafiltration and concentration, drying namely get the Sublancin antibacterial peptide.
2. antibacterial peptide preparation method as claimed in claim 1 is characterized in that step 1) middle fermented liquid is by ceramic membrane filter, and described ceramic membrane aperture is 20nm.
3. antibacterial peptide preparation method as claimed in claim 1 or 2, it is characterized in that step 1) described in substratum consist of every 1L fermention medium and contain: Semen Maydis powder 10-50g, dregs of beans 10-50g, peptone 5-20g, glucose (or sucrose) 2-20g, yeast powder 0-10g, ammonium sulfate 0-10g, ammonium nitrate 0-10g, sal epsom 0-1.5g, manganous sulfate 0-1.5g, phosphoric acid salt 0-10g, pH 7.0-8.0.
4. antibacterial peptide preparation method as claimed in claim 1 or 2 is characterized in that step 1) in the inoculum size of subtilis be 1 * 10
6-10
7CFU/mL, leavening temperature are 30-37 ℃, and fermentation time is 24-32h, air flow 600-1000m
3/ h, rotating speed 120-200r/min, pH 6.0-7.0.
5. antibacterial peptide preparation method as claimed in claim 1 or 2 is characterized in that step 2) described centrifugal condition is the centrifugal 20-40min of 10000-15000g.
6. antibacterial peptide preparation method as claimed in claim 1 or 2, it is characterized in that, described cation-exchange chromatography is with buffer A counterion displacement chromatography post, with the fermented liquid loading, the volume ratio of applied sample amount and cation-exchange chromatography column packing is 20: 1, the loading flow velocity is 50ml/min, reach baseline values with buffer A flushing chromatography column to uv-absorbing afterwards, carry out isocratic elution with buffer A and buffer B by 3: 1 volume ratio again, collect elutriant, described buffer A is 20mM NaAc solution, pH4.0, described buffer B is the solution that contains 20mMNaAc and 1M NaCl, pH4.0.
7. antibacterial peptide preparation method as claimed in claim 1 or 2, it is characterized in that, step 2) described hydrophobic chromatography step is to use buffer A ' balance hydrophobic chromatography post, the elutriant that ion exchange chromatography is collected carries out equal-volume with 2M NaCl and mixes the loading chromatography column, the volume ratio of applied sample amount and hydrophobic chromatography column packing is 2.5: 1, the loading flow velocity is 30ml/min, use buffer A ' the washing chromatographic column, until uv-absorbing is to baseline values, use again buffer B ' carry out linear gradient elution, collect elutriant; Described buffer A ' for containing the solution of 20mM NaAc and 1M NaCl, pH4.0, described buffer B ' be 20mM NaAc, pH4.0.
8. antibacterial peptide preparation method as claimed in claim 1 or 2, it is characterized in that step 2) described size exclusion chromatography is the elutriant loading with the hydrophobic chromatography collection step, each sampling volume is 5% of column volume, flow velocity is 2ml/min, collects the antibacterial peptide elution peak.
9. high purity antibacterial peptide production method as claimed in claim 1 or 2 is characterized in that, with anion-exchange chromatography post on the elutriant of size exclusion chromatography collection step, flow velocity 2ml/min collects the liquid that flows out.
10. the antibacterial peptide product that obtains such as the described production method of claim 1～9 any one.
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| CN103194411A (en) * | 2013-04-08 | 2013-07-10 | 中国农业大学 | Bacillus subtilis and application of bacillus subtilis to production of antimicrobial peptide |
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