CN108949872A - A kind of method of a small amount of fermenting and producing Lantibiotics Subtilomycin - Google Patents

Abstract

The invention belongs to fermenting and producing fields, integrated condition more particularly to the method for a small amount of fermenting and producing Lantibiotics Subtilomycin a kind of, during the fermented and cultured are as follows: initial pH value 8.25, revolving speed 169r/min, 30.24 DEG C of temperature, liquid amount 37.9% and inoculum concentration 1.46%；Nutrition proportion are as follows: glucose 11.4g/L, yeast powder 5g/L, zinc sulfate 0.00056g/L and calcium chloride 0.0595g/L, incubation time are for 24 hours that the fermenting microbe used is bacillus subtilis (Bacillus subtilis) SX3411.In bacillus subtilis SX3411 fermentation liquid when crude separation Subtilomycin, the antibacterial activity of Subtilomycin is can be detected in 20%, 30% and 40% ammonium sulfate precipitation fermentation liquid, wherein 30% ammonium sulfate precipitation can get the maximum amount of Subtilomycin.The phosphate buffer of 30% ammonium sulfate precipitation pH7.4 of fermentation liquid, concentration 50mmol/L are suspended, then by the ultra-filtration centrifuge tube of 3kD, the Subtilomycin of preliminary purification can be obtained.

Description

A kind of method of a small amount of fermenting and producing Lantibiotics Subtilomycin

Technical field

The present invention relates to a kind of methods from a small amount of fermenting and producing Lantibiotics Subtilomycin, belong to fermentation life Production field.

Background technique

Lantibiotics (Lantibiotics) are a kind of to be generated in bacterium, is encoded by gene, on ribosomes Synthesis, post translational processing modification, the antibacterial peptide of a kind of multi-ring containing thioether ring.Sent out in a variety of gram-positive bacteriums Existing Lantibiotics, most of it is inhibited to gram-positive bacterium, lactein Nisin therein is sheep The representative of hair sulfur bacteria element, there is application in food antiseptic.Sheep is formed according to catalysis in Lantibiotics biosynthesis mechanism The enzyme characteristic of hair methyllanthionine, Lantibiotics can be divided into four classes: the modification of I class Lantibiotics is mainly complete by two kinds of enzymes At that is, a kind of dehydratase (dehydratase) LanB and a kind of cyclase (cyclase) LanC；II class Lantibiotics, Synthetic reaction only has single lanthiopeptin synzyme (lanthipeptide synthetase) LanM catalysis, and the end enzyme N- is Dehydratase domain, but it is not homologous with LanB, and the end C- is LanC class (LanC-like) cyclase domain；Group III Lantibiotics and IV The synthesis of class Lantibiotics is acted on by three functional enzyme LanKC and LanL respectively.LanKC and LanL contains the end N- lyases (lyase) domain and center kinase domain, but have the different ends C-；Contain zinc-binding motif in the end the C- domain of LanC and LanM and LanL (Cys-Cys-His/Cys), and the end the C- cyclase domain of LanKC does not have zinc-binding motif.

Lantibiotics have specificity, including many Antibiotic Resistances to the effect of gram-positive bacteria (antibiotic resistant) pathogen, such as vancomycin resistance staphylococcus aureus (vancomycin-resistant Staphylococcus aureus, VRSA), vancomycin-resistant enterococcus (vancomycin-resistant Enterococci, VRE) and methicillin-resistant staphylococcus aureus (methicillin-resistant Staphylococcus aureus, MRSA) etc..Researches show that lanthiopeptin bacteriocins can be with the precursor substance lipid of cell wall II is combined, so that the normal of cell wall be interfered to synthesize the destruction for causing cell.In addition to this antifungal mechanism, Lantibiotics with Lipid forms polymer, and cell membrane is made to form duct, thus bacterial cell contents is allowed to be lost and cause death.Because of wool There are two types of completely different antifungal mechanism, pathogenetic bacteria is not easy to be evolved simultaneously for two kinds of antibacterial machines sulfur bacteria element tool very much The drug resistance of reason, this allows pathogenetic bacteria not allow to be also easy to produce the drug resistance for Lantibiotics.Only have compared to conventional antibiotic Single antifungal mechanism and the generation for leading to drug resistance, the antifungal mechanism of Lantibiotics obviously occupy advantage, and wool sulphur Bacterium is known as preferable physicochemical property.Thus, Lantibiotics have fabulous in related fieldss such as food, medicine and feeds Utilization prospects.

Subtilomycin is the I class Lantibiotics found in bacillus subtilis MMA7 for 2013.To regard for oneself Your blue Robert W.Phelan et al. obtains bacillus subtilis from the sponge (Haliclona simulans) in ocean Bacterial strain MMA7, the bacterial strain secrete a kind of Novel wool sulfur bacteria element Subtilomycin.Subtilomycin gene cluster includes Structural gene SubA, two modification gene SubB (LanB-like dehydratase) and SubC (LanC-like Cyclase), extracellular serine proteinase SubP (cutting for being responsible for leader peptide), abc transport Protein S ubT and immune protein SubI (own growth can be made not influenced by Subtilomycin).Subtilomycin to clinically in food pathogenicity remove from office Lan Shi positive bacteria shows bacteriostatic activity, including monocyte Listeria (Listeria monocytogenes), production gemma shuttle Shape bacillus (Clostridium sporogenes), the drug resistant staphylococcus aureus of MRSA and heterogeneous vancomycin intermediary (heterogeneous vancomycin-intermediate-level-resistant Staphylococcus aureus, The gram-positive bacterias such as hVISA), while also there is bacteriostatic activity to some Gram-negative bacterias, such as: pseudomonas aeruginosa (Pseudomonas aeruginosa), Vibrio anguillarum (Vibrio anguillarum), Aeromonas hydrophila (Aeromonas ) and monospore Pseudomonas (Alteromonas sp) etc. hydrophila.But needing greater concentrations of Subtilomycin just has pair The bacteriostasis of Gram-negative bacteria.We are also separated to one plant near one pig farm of the Nanchang City, Jiangxi Province town Ma Qiu in early period Bacillus subtilis (Bacillus subtilis) SX3411 bacterial strain of antibacterial peptide is produced, which can produce Lantibiotics Subtilomycin.Culture presevation is in China General Microbiological culture presevation administrative center, culture presevation CGMCC NO.14396.Have many characteristics, such as antibacterial activity, thermal stability, resistance to acid and alkali and the water solubility of wide spectrum in view of Subtilomycin, So that Subtilomycin has good medical value, and is very suitable for carrying out the industrial production of scale, make it have good The good processing and application prospect in related fieldss such as food, medicine and feeds.

Summary of the invention

The technical problem to be solved by the present invention is to the sides of a small amount of fermenting and producing Lantibiotics Subtilomycin a kind of Method, this method are as follows:

(1) fermenting microbe: zymophyte is bacillus subtilis (Bacillus subtilis) SX3411, the culture presevation In China General Microbiological culture presevation administrative center, culture presevation CGMCC NO.14396；

Bacillus subtilis (Bacillus subtilis) the SX3411 bacterial strain by molecular biology method and Immunobloting hybridization analysis, it was demonstrated which create Lantibiotics Subtilomycin.

The bacillus subtilis SX3411 is sampled under 37 DEG C of cultivation temperature in different time, and is tuned into identical bacterium Liquid concentration, i.e. OD₆₀₀Value is 1.0.The Immunoblotting analysis detection in the case where cellular biomass is essentially identical The expression quantity variation of Subtilomycin precursor protein SubA in the cell: bacillus subtilis SX3411 starts intracellular in 5h SubA albumen is expressed, the expression quantity highest in 12h, expression quantity gradually decreases over time, until all disappearing when 25h It loses, again without the expression of SubA after 25h.

The bacillus subtilis SX3411 is using 27 DEG C, 30 DEG C, 33 DEG C, 37 DEG C, 40 DEG C, 43 DEG C of temperature culture 12h After sample, and be tuned into identical bacterial concentration, i.e. OD₆₀₀Value is 1.0.In the case where cellular biomass is essentially identical The expression quantity variation of Immunoblotting analysis detection Subtilomycin precursor protein SubA in the cell: 6 kinds of culture temperature There is the expression of SubA under degree, i.e. temperature can express SubA between 27 DEG C to 43 DEG C, wherein expressing at 33 DEG C or so Highest is measured, expression quantity is minimum at 43 DEG C or so.

(2) fermentation time: the fermentation time that fermented and cultured bacillus subtilis SX3411 generates Subtilomycin is 24- 30 hours；Further it is advisable within 24 hours with fermentation；

(3) fermentation comprehensive condition of culture are as follows: initial pH value 8.25, revolving speed 169r/min, 30.24 DEG C of temperature, liquid amount 37.9%, inoculum concentration 1.46%；

(4) component of fermentation medium are as follows: glucose 11.4g/L, yeast powder 5g/L, zinc sulfate 0.00056g/L, chlorination Calcium 0.0595g/L.

In preliminary purification fermentation liquid the step of Subtilomycin are as follows:

(1) fermentation liquid is precipitated using the ammonium sulfate of 20%-40%, obtains fermentation liquid ammonium sulfate precipitation；

(2) phosphate buffer of fermentation liquid ammonium sulfate precipitation pH7.4, concentration 50m mol/L are suspended and is precipitated, then passed through The Subtilomycin of preliminary purification can be obtained in the ultra-filtration centrifuge tube of 3kD.

Preferably, in preliminary purification fermentation liquid the step of Subtilomycin are as follows:

(1) fermentation liquid is precipitated using 30% ammonium sulfate, obtains fermentation liquid ammonium sulfate precipitation；

(2) phosphate buffer of fermentation liquid ammonium sulfate precipitation pH7.4, concentration 50m mol/L are suspended and is precipitated, then passed through The Subtilomycin of preliminary purification can be obtained in the ultra-filtration centrifuge tube of 3kD.

The utility model has the advantages that by a small amount of fermenting and producing Lantibiotics Subtilomycin of bacillus subtilis SX3411, A certain amount of Subtilomycin can be quickly obtained；Production can also be amplified on this basis.

Detailed description of the invention

Fig. 1 structural gene SubA PCR products electrophoresis map (M:DNA maker DL2000；Swimming lane 1:SubA)

Fig. 2 .SubB, SubC and SubP PCR products electrophoresis map (M:DNA maker DL-5000 swimming lane 1:SubC；Swimming lane 2:SubP；Swimming lane 3:SubB)

Fig. 3 .SubP to SubB sections, SubB to SubC sections, SubP-SubB-SubC sections of gene PCR product electrophoretogram (M:DNA maker DL-5000；Swimming lane 1:SubP-SubB；Swimming lane 2-3:SubB-SubC；Swimming lane 4-5:SubP-SubB-SubC)

Fig. 4 .3 × SubA gene colony PCR product electrophoretogram (M:DNA marker DL-1000；Swimming lane 1:3 × SubA)

Fig. 5 recombinant plasmid pET-41a (+) -3 × SubA double enzyme digestion product electrophoretogram (M:DNA marker DL-10000； Swimming lane 1:pET-41a (+) -3 × SubA double digestion)

The SDS-PAGE of Fig. 63 × SubA of recombinant protein expression detects (M:BlueProtein Marker (14kDa-100kDa)；Swimming lane 1: the e. coli bl21 bacterial protein without induction；Swimming lane 2: the recombinant bacterium without induction Body total protein；3:37 DEG C of swimming lane, 0.5mmol/L IPTG induces the recombination bacterial protein of 6h；4:37 DEG C of swimming lane, 0.5mmol/L The recombinant bacterium ultrasonic disruption supernatant of IPTG induction 6h；5:37 DEG C of swimming lane, 0.5mmol/L IPTG induces the recombinant bacterium of 6h super Sonication precipitating)

The SDS-PAGE of 3 × SubA of Fig. 7 recombinant protein after purification detects (M:BlueProtein Marker (14kDa-100kDa)；Swimming lane 1: the dissolution supernatant after inclusion body purification；Swimming lane 2: the total egg of recombination thallus induced through IPTG It is white；Swimming lane 3: the recombinant bacterium ultrasonic disruption supernatant induced through IPTG)

The specific detection immunoblotting analysis chart (M:Blue of Fig. 8 antibody anti-3 × SubA Protein Marker(14kDa-100kDa)；1:37 DEG C of swimming lane and 0.5mmol/L IPTG induce 6 hours recombinant bacterium BL21- PET41a-3 × SubA thallus ultrasonic disruption precipitating；Swimming lane 2: recombinant bacterium BL21-pET41a-3 × SubA bacterium without induction Body ultrasonic disruption precipitating)

In Fig. 9 different time bacillus subtilis SX3411 SubA protein expression immunoblotting analysis chart (M: Blue Protein Marker(14kDa-100kDa)；The bacillus subtilis SX3411 of 1-20:37 DEG C of swimming lane culture Thallus ultrasonic disruption supernatant, incubation time be respectively 0h, 5h, 12h, 15h, 20h, 21h, 22h, 23h, for 24 hours, 25h, 26h, 27h, 28h, 29h, 30h, 32h, 36h, 40h, 44h and 48h)

Under Figure 10 different temperatures in SX3411 SubA protein expression immunoblotting analysis chart (M:Blue Protein Marker(14kDa-100kDa)；Swimming lane 1-6: the bacillus subtilis SX3411 thallus that incubation time is 12h is super Sonication supernatant, cultivation temperature are respectively 27 DEG C, 30 DEG C, 33 DEG C, 37 DEG C, 40 DEG C, 43 DEG C)

(A: different pH value ferment to bacterial strain SX3411 for influence of Figure 11 .SX3411 fermentation condition single factor test to bacteriostatic activity Liquid bacteriostatic activity influences；B: different seed liquor inoculum concentrations influence bacterial strain SX3411 fermentation liquid bacteriostatic activity；C: different liquid amounts Bacterial strain SX3411 fermentation liquid bacteriostatic activity is influenced；D: different temperatures influences bacterial strain SX3411 fermentation liquid bacteriostatic activity；E: no Bacterial strain SX3411 fermentation liquid bacteriostatic activity is influenced with revolving speed；IZD:diameter of inhibition zone)

Figure 12 different carbon sources influences [A: separate sources carbon source (1:CK to SX3411 fermentation liquid bacteriostatic activity；2: grape Sugar；3: fructose；4: sucrose；5: mannitol；6: soluble starch)；B: glucose]

Figure 13 nitrogen source influences [A: nitrogen source types (1:CK to bacterial strain SX3411 fermentation liquid bacteriostatic activity；2: peptone；3: pancreas Peptone；4: soy peptone；5: beef extract；6: yeast powder)；B: dusty yeast concentration]

Figure 14 inorganic salts influence [A: inorganic salts type (1:CK to bacterial strain SX3411 fermentation liquid bacteriostatic activity；2:NaCl；3: K2HPO4；4:CaCl；5:FeSO4·7H2O；6:ZnSO4)；B:Zn²⁺Concentration]

Initial pH value and revolving speed are to the reciprocal effect for producing antibacterial peptide in Figure 15 condition of culture

Initial pH value and temperature are to the reciprocal effect for producing antibacterial peptide in Figure 16 condition of culture

Initial pH value and liquid amount are to the reciprocal effect for producing antibacterial peptide in Figure 17 condition of culture

Initial pH value and inoculum concentration are to the reciprocal effect for producing antibacterial peptide in Figure 18 condition of culture

Revolving speed and temperature are to the reciprocal effect for producing antibacterial peptide in Figure 19 condition of culture

Revolving speed and liquid amount are to the reciprocal effect for producing antibacterial peptide in Figure 20 condition of culture

Revolving speed and inoculum concentration are to the reciprocal effect for producing antibacterial peptide in Figure 21 condition of culture

Temperature and liquid amount are to the reciprocal effect for producing antibacterial peptide in Figure 22 condition of culture

Temperature and inoculum concentration are to the reciprocal effect for producing antibacterial peptide in Figure 23 condition of culture

Liquid amount and inoculum concentration are to the reciprocal effect for producing antibacterial peptide in Figure 24 condition of culture

Glucose and dusty yeast concentration are to the reciprocal effect for producing antibacterial peptide in Figure 25 culture medium

Glucose and zinc ion concentration are to the reciprocal effect for producing antibacterial peptide in Figure 26 culture medium

Glucose and calcium ion concentration are to the reciprocal effect for producing antibacterial peptide in Figure 27 culture medium

Yeast powder and zinc ion concentration are to the reciprocal effect for producing antibacterial peptide in Figure 28 culture medium

Yeast powder and calcium ion concentration are to the reciprocal effect for producing antibacterial peptide in Figure 29 culture medium

Zinc ion and calcium ion concentration are to the reciprocal effect for producing antibacterial peptide in Figure 30 culture medium

Figure 31 bacillus subtilis SX3411 tunning ammonium sulfate precipitation fungistatic effect

The SDS-PAGE electrophoretic effects and inhibition zone of Figure 32 bacillus subtilis SX3411 tunning ammonium sulfate precipitation object The correspondence result figure of size

Specific embodiment

Embodiment 1

The identification of subtilomycin gene cluster:

The extraction of step 1. bacillus subtilis strain SX3411 complete genome DNA

Take out the bacillus subtilis strain SX3411 that laboratory is stored in -60 DEG C of low temperature refrigerators, picking after being recovered It grows normal bacterium colony and connects bacterium and LB liquid medium expansion culture.Using OMEGA bacterium complete genome DNA extracts kit (Omega Bio-tek, Inc product) extracts bacillus subtilis strain SX3411 complete genome DNA.

Step 2. design of primers

According to Subtilomycin gene in the bacillus subtilis B.subtilis Strain MMA7 provided on NCBI Structural gene SubA (1038bp-1208bp), dehydrase gene SubB in cluster (GenBank sequence number: JX912247.1) (2271bp-5381bp) and cyclase gene SubC (5406bp-6728bp), serine protease gene SubP (1296bp- DNA sequence dna 2270bp).SubA, SubB, SubC, SubP gene are separately designed using Primer5.0 and Oligo6.0 software Primer.In addition, according to Subtilomycin gene cluster in B.subtilis Strain MMA7 on NCBI (GenBank sequence number: JX912247.1 putting in order for all DNA sequence) and its gene cluster, uses Primer5.0 and Oligo6.0 software to distinguish Design SubP to SubB sections of primer (1296bp-5381bp), SubB to SubC sections of primer (2271bp-6728bp), SubP extremely SubB to SubC sections of primer (1296bp-6728bp).For details, see the appendix one for correlated series.Primer is sent to Nanjing Jin Sirui biotechnology Co., Ltd's synthesis.Design of primers is shown in Table 1.

The primer used in 1 PCR of table detection

Step 3.PCR response procedures

Reaction condition are as follows: 94 DEG C initial denaturation 5 minutes, then 94 DEG C be denaturalized 1 minute, 50 DEG C annealing 1 minute (according to piece segment length Degree is appropriate to be changed), 72 DEG C of extension, 30 seconds (suitably being changed according to fragment length), totally 30 circulations, then 72 DEG C extension 10 minutes, instead Answering system is 50 μ L systems.After amplification, 10 μ L PCR products is taken to observe clip size in 1.5% agarose gel electrophoresis.

The identification of step 4.Sub A structural gene sequence

Using bacterial strain SX3411 genomic DNA as template, PCR amplification is carried out by primer S1 and S2, obtains the DNA of Sub A Sequence, band at 170bp (Fig. 1).After obtained PCR product is sequenced and BLAST is compared, in ncbi database Sub A structural gene sequence is completely the same (GenBank sequence number: JX912247.1), illustrates bacillus subtilis SX3411 base Because of the structural gene SubA in group containing Subtilomycin.

Step 5.SubB, the detection respectively of SubC and SubP sequence

Pass through the dehydrase gene SubB, cyclase gene SubC and serine egg of the Subtilomycin provided on NCBI White enzyme gene SubP basic sequence designs three pairs of primers (table 1), then carries out PCR amplification.As a result as shown in Figure 2, PCR amplification goes out Segment by agargel electrophoresis detect, size is respectively SubB 3100bp or so (theoretical 3111bp), SubC 1300bp Left and right (theoretical 1323bp) and SubP 1000bp or so (theoretical 975bp).The sequence of acquisition is sequenced, as a result and Related gene sequence is consistent (GenBank sequence number: JX912247.1) in the Subtilomycin gene cluster of publicity.

Step 5.SubB, the joint-detection of SubC and SubP sequence

Pass through SubP to SubB sections, SubB to SubC sections, the SubP-SubB-SubC sections of gene order institutes provided on NCBI Three pairs of primers (table 1) of design, carry out PCR amplification, the DNA fragmentation size expanded is that: SubP-SubB is respectively respectively 4100bp or so (theoretical 4086bp), SubB-SubC are that 4500bp or so (4458bp) and SubP-SubB-SubC is 5400 left Right (5433bp) (Fig. 2-4).The sequence of acquisition is sequenced, as a result with phase in the Subtilomycin gene cluster of publicity Correlation gene sequence is consistent (GenBank sequence number: JX912247.1).

It can determine in bacillus subtilis SX3411 genome really by pcr amplification product electrophoretogram and sequencing comparison result The real structural gene SubA containing Subtilomycin, dehydrase gene SubB and cyclase gene SubC, serine protease Gene SubP.And modification puts in order with processed gene as in SubP-SubB-SubC, with bacillus subtilis MMA7 The related gene of Subtilomycin biological synthesis gene cluster puts in order unanimously.

Embodiment 2

The immunobloting detection that Subtilomycin structural gene SubA is expressed in SX3411 bacterial strain

Step 1. sequence and design of primers

According to Subtilomycin gene cluster (GenBank sequence in the bacillus subtilis strain MMA7 provided on NCBI Number: the sequence of the structural gene SubA in JX912247.1), design three times copy 3 × SubA of concatermer gene order is (see sequence Table).It is needed according to the correlation properties and expression of the gene order of 3 × SubA of concatermer and expression vector pET-41a (+), choosing Suitable restriction enzyme site Kpn I and BamH I is selected, is separately designed out using Primer5.0 and Oligo6.0 software and is drawn accordingly Object, such as table 2.3 × SubA of concatermer sequence and primer are all synthesized by Nanjing Jin Sirui biotechnology Co., Ltd.

The primer used in 2 PCR amplification of table

Note: whereinGGTACCFor Kpn I restriction enzyme site,GGATCCFor BamH I restriction enzyme site

The expression vector establishment of 3 × SubA of step 2. concatermer

By the PCR product of 3 × SubA gene, after recycling and expression vector pET-41a (+) carries out double digestion processing.3× SubA and plasmid pET-41a (+) is simultaneously using Kpn I and BamH I restriction enzymes double zyme cutting processing (37 DEG C, 3h).By enzyme It cuts product and uses Tiangeng DNA small fragment Purification Kit respectively.3 × SubA and plasmid pET-41a (+) after purification is used T4DNA Ligase law temperature joining is stayed overnight, and is formed recombinant plasmid pET-41a (+) -3 × SubA, is then converted bacillus coli DH 5 alpha, PCR amplification detects positive colony (Fig. 4).It extracts recombinant plasmid and carries out double digestion processing simultaneously with Kpn I and BamH I.Recombination There is the segment almost the same with aim sequence size after double digestion in plasmid pET-41a (+) -3 × SubA, is 570bp left Right (Fig. 5).Recombinant plasmid send to Nanjing Genscript Biotechnology Co., Ltd. and carries out sequencing identification, and sequencing result passes through DNAMAN Molecular biology software compares correctly, entirely defines 3 × SubA gene and has been properly inserted on expression vector pET-41a (+).

The SDS-PAGE of 3 × SubA of step 3. prokaryotic expression recombinant protein is detected

- 3 × SubA of successful recombinant plasmid pET-41a (+) will be sequenced and be transformed into competent cell e. coli bl21 (DE3) in.Recombinant bacterium in different IPTG concentration, different cultivation temperature induction after, carry out SDS- after collecting thallus alignment processing The detection of PAGE protein electrophoresis.Recombinant bacterium BL21-pET41a (+) -3 × SubA is 37 DEG C in condition, 0.5mmol/L IPTG induction The expression of lower albumen is preferably (Fig. 6).According to electrophoresis result it is found that IPTG induces recombination bacterial protein cracking in 6 hours Liquid and the recombination bacterial protein lysate comparison without IPTG induction and the empty carrier e. coli bl21 bacterium without induction Body total protein (Fig. 6, swimming lane 1,2,3) compare, there is apparent target stripe (size is about 54kDa) (Fig. 6, swimming lane 3).Through (5) Fig. 3-6, swimming lane 4 compare, it is known that purpose egg for recombinant bacterium ultrasonic disruption supernatant and precipitating after induction in IPTG 6 hours It is white to be mainly collected in ultrasonic wave precipitating (Fig. 6, swimming lane 5), show recombinant plasmid pET-41a (+) -3 × SubA in cell Expression albumen mainly exist with inclusion bodies.

Step 5.3 × SubA recombinant protein inclusion body preliminary purification

Existence form of the 3 × SubA of recombinant expression protein in e. coli bl21 is inclusion body.In order to which preliminary purification should Albumen obtains more pure 3 × SubA of destination protein, takes the method to inclusion body washing dissolution to purify, incites somebody to action Through 37 DEG C, 0.5mmol/L IPTG induces 6 hours recombinant bacterium bacterial sediments through ultrasonic disruption, and precipitating is collected after centrifugation (i.e. Inclusion body), it will be precipitated and dissolved in brokenly among bacterium buffer, then use inclusion body washing buffer I (1mol/L Tris-HCl (PH8.0) mother liquor 25mL, Triton X-100 5mL, NaCl 1.426g are settled to 500mL, 4 DEG C of preservations with distilled water) and packet Contain body washing buffer II (1mol/L Tris-HCl (PH8.0) mother liquor 25mL, urea 60.06g, NaCl 14.62g, with double steamings Water is settled to 500mL, 4 DEG C of preservations) it is respectively washed inclusion body, the precipitating after centrifugation uses solubilization of inclusion bodies buffer (Tris again 0.2422g, NaCl 2.92g, imidazoles 0.034g, guanidine hydrochloride 57.198g, beta -mercaptoethanol 1mmol/L, are settled to distilled water 100mL, adjusting pH value is 8.0,4 DEG C of preservations) 4 DEG C of dissolutions overnight, centrifuging and taking supernatant, supernatant is recombinant protein after purification 3×SubA。

It takes equally through 37 DEG C, 0.5mmol/L IPTG induces 6 hours recombinant bacterium bacterial protein lysates and ultrasounds Wave is crushed supernatant, and the loading after processing together with protein solution after purification, by SDS-PAGE electrophoresis, purification effect is shown in Fig. 7.As seen from the figure, protein solution foreign protein after purification has lacked very much, and the concentration of destination protein is obviously got higher, recombinant protein 3 × SubA has obtained preliminary purifying.

The preparation of step 6. antibody

3 × SubA recombinant protein inclusion body purification sample of prokaryotic expression carries out SDS-PAGE electrophoresis.It is used after electrophoresis The 0.25M KCl solution of pre-cooling cuts destination protein band in being dyed on horizontal shaker, with clean blade, soaks in distilled water 5-15min is steeped, wherein albumen can be used as antigen use.

The adhesive tape with 3 × SubA recombinant protein cut is mixed with Freund's complete adjuvant, and 2 New Zealand great Bai are immunized Rabbit.Period has carried out 8 injecting immunes, in 103 days immune acquisition antiserums, purifies, obtains through ProteinA affinity column chromatography The SubA antibody protein that must be purified.

The SubA antibody protein ELISA detection that step 7. purifies

The concentration for being detected antibody is 3.42mg/mL.The potency of elisa assay anti-SubA, the results showed that dilution At 50000 times, microplate reader detects the absorbance value of ELISA considerably beyond the value of blank control (rabbit anteserum before immune) (table 3) illustrates that the antibody anti-3 × SubA obtained has higher potency.

The potency of table 3ELISA analysis anti-3 × SubA

Step 8.Immunoblotting detects the expression of 3 × SubA albumen to determine that antibody can detect SubA

37 DEG C, 0.5mmol/L IPTG induce 6 hours recombinant bacterium BL21-pET-41a (+) -3 × SubA thallus through ultrasound After wave is broken, SDS-PAGE electric current, then Immunoblotting detects the amalgamation and expression of 3 × SubA, it is seen that the weight after induction About there is an apparent hybrid belt at 54kDa in group bacterium, and the hybridization of 54kDa size does not occur in the recombinant bacterium not induced Band (Fig. 8), shows that antibody anti-3 × SubA can detect SubA.

Step 9.Immunoblotting detects the expression of the SubA albumen of different time bacillus subtilis SX3411

In order to which the precursor protein SubA for detecting Subtilomycin in bacillus subtilis SX3411 is special in expression intracellular Property, the thallus that bacillus subtilis SX3411 is collected under 37 DEG C of cultivation temperature is designed, thallus is handled through ultrasonic disruption After take supernatant and anti-3 × SubA antibody to carry out Immunoblotting analysis.

As shown in Figure 9, there is hybrid belt at the position about 6kDa, theoretical molecular weight (6.16kDa) base with SubA This is consistent.Bacillus subtilis SX3411 starts intracellular expression SubA albumen in 5h, the expression quantity highest in 12h, with The passage expression quantity of time gradually decreases, until all disappearing when 25h, again without the expression of SubA after 25h.

Step 10.Immunoblotting detects the expression of SubA albumen in bacillus subtilis SX3411 under different temperatures

For the precursor protein for detecting the Subtilomycin in the bacillus subtilis SX3411 under different cultivation temperatures SubA intracellular expression characteristic, this experiment cultivate withered grass at a temperature of 27 DEG C, 30 DEG C, 33 DEG C, 37 DEG C, 40 DEG C, 43 DEG C this 6 kinds respectively Bacillus SX3411, while being sampled at the 12nd hour, thallus taken after ultrasonic disruption is handled supernatant anti-3 × SubA antibody carries out hybridization check.

As shown in Figure 10, occurs the specific hybrid band of about 6kDa size under 6 kinds of cultivation temperatures, with the liter of temperature Height, the expression quantity of SubA albumen first increases to be reduced again, the expression quantity highest at 33 DEG C or so, and expression quantity is minimum at 43 DEG C or so. Through speculating, cultivation temperature is 33 DEG C or so and is relatively conducive to bacillus subtilis SX3411 synthesis SubA albumen；And cultivation temperature is 43 DEG C or it is higher when, be unfavorable for bacillus subtilis SX3411 growth synthesis SubA albumen.

The single factor analysis of 3 fermentation condition of embodiment

The detection of step 1. bacteriostatic activity

Each culture dish topples over 30mL or so LB solid medium, and 100 μ L indicator bacteria staphylococcus aureus suspensions are added (OD₆₀₀=1, then dilute 10^-5), bacterium solution can not be excessive, to plate even spread, 37 DEG C of culture 10min.Then by 2 layers of filter paper It is gently sticked on the antibacterial plate prepared, the fermentation liquid of 25 μ L SX3411 bacterial strains is added on filter paper.Using sterile new Fresh LB liquid medium is as control.After adding sample, setting 4 DEG C of refrigerator 4h or more expands fermentation liquid sufficiently on agarose plate It dissipates, is then placed within 37 DEG C of constant incubator culture 12h or so, periodically checks the case where inhibition zone occurs, make a record and survey Measure antibacterial circle diameter.

The single factor analysis of step 2. fermentation condition

Bacillus subtilis SX3411 is seeded to basal fermentation medium (i.e. LB culture medium, peptone 10.0g, yeast Extract 5.0g, NaCl 5.0g, pH 7.2 adds water constant volume 1L, 121 DEG C of sterilizing 20min), culture to OD₆₀₀To get kind when=1 Sub- culture medium.Inoculate to basal fermentation medium carry out it is each under the conditions of fermented and cultured for 24 hours, utilize the antibacterial work of fermentation liquid Property detect each fermentation condition to bacillus subtilis SX3411 produce antibacterial material influence.The single factor test of selection has culture medium 5 initial pH value, seed liquor inoculum concentration, shaking flask liquid amount (ventilatory capacity), cultivation temperature and revolving speed factors.

Step 3. initial pH value of medium produces the influence of antibacterial material to bacillus subtilis SX3411

The initial pH value of basal fermentation medium is respectively set 5,6,7,8,9,10, is sterilized spare.It is withered by what is prepared The seed liquor of careless bacillus SX3411 according to 2% inoculum concentration to different initial pH values basal fermentation medium, 30 DEG C, 200r/min is cultivated for 24 hours.

Step 4. inoculum concentration produces the influence of antibacterial material to bacillus subtilis SX3411

By the seed liquor of the bacillus subtilis SX3411 prepared respectively according to 0.5%, 2%, 4%, 6%, 8%, 10% inoculum concentration is seeded to different basal fermentation mediums, and 30 DEG C, 200r/min is cultivated for 24 hours.

Step 5. shaking flask liquid amount (ventilatory capacity) produces the influence of antibacterial material to bacillus subtilis SX3411

By the 250mL shaking flask liquid amount of basal fermentation medium be respectively set to 50mL, 75mL, 100mL, 125mL, 150mL, 175mL (corresponding ventilatory capacity is respectively 20%, 30%, 40%, 50%, 60%, 70%) sterilize spare.It will preparation The seed liquor of good bacillus subtilis SX3411 according to 2% inoculum concentration to different initial pH values basal fermentation medium, 30 DEG C, 200r/min shaking table constant temperature incubation is for 24 hours.

Step 6. temperature produces the influence of antibacterial material to bacillus subtilis SX3411

The seed liquor of the bacillus subtilis SX3411 prepared is seeded to different bases according to 2% inoculum concentration respectively Plinth fermentation medium sets 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, the training of 200r/min shaking table constant temperature for shaking table parameter respectively It supports for 24 hours.Revolving speed produces the influence of antibacterial material to bacillus subtilis SX3411: by the bacillus subtilis SX3411's prepared Seed liquor is seeded to different basal fermentation mediums according to 2% inoculum concentration respectively, and shaking table parameter is set as 30 DEG C respectively, 140r/min, 160r/min, 180r/min, 200r/min, 220r/min shaking table constant temperature incubation are for 24 hours.

Each single factor test bacteriostatic activity analysis of step 7.

Above-mentioned bacillus subtilis SX3411 is taken to cultivate fermentation liquid for 24 hours respectively, 10 000r/min are centrifuged 15min, take Supernatant to get arrive antibacterial material crude extract.Using staphylococcus aureus as indicator bacteria, measure under the conditions of each single factor test to withered grass The influence of the bacteriostatic activity of bacillus SX3411 fermentation liquid.The result shows that in initial pH single-factor influence, with initial pH 8 When, preferably, produced fermentation liquid has best bacteriostatic activity (Figure 11 A) for bacterial strain SX3411 growth；When the seed liquor of bacterial strain SX3411 is dense Degree is OD₆₀₀Be inoculated with when=1, when inoculum concentration 2%~4% produced fermentation liquid bacteriostatic activity preferably (Figure 11 B)；Bacterial strain SX3411 is grown preferably in shaking flask liquid amount 40%~50%, and the bacteriostatic activity of produced fermentation liquid is preferably (Figure 11 C)；Difference training The influence that temperature produces antibacterial material to bacterial strain SX3411 is supported, the bacteriostatic activity of produced fermentation liquid is best when with 25 DEG C~30 DEG C (Figure 11 D)；Preferably, the bacteriostatic activity of produced fermentation liquid is most for culture growth when revolving speed is 160~180r/min by bacterial strain SX3411 Good (Figure 11 E)；

Single factor analysis of the 4 fermentation medium nutritional ingredient of embodiment to bacteriostatic activity

The setting of step 1. analysis condition

Bacillus subtilis SX3411 is seeded to seed liquid culture medium, culture to OD₆₀₀When=1, inoculate to different carbon The fermentation medium in source, nitrogen source and inorganic ion carries out fermented and cultured for 24 hours, detects each hair using the bacteriostatic activity of fermentation liquid Ferment condition produces the influence of antibacterial material to bacillus subtilis SX3411.The single factor test of selection have carbon source, nitrogen source and inorganic salts from Sub 3 factors.

Step 2. prepares different carbon source culture medium

Glucose, fructose, sucrose, mannitol, soluble starch are added by carbon source basal medium with 1% amount respectively (peptone 10g/L, yeast extract 5g/L, NaCl 5g/L, pH value are adjusted in 7), are control with carbon source basal medium.It will Bacillus subtilis SX3411 is seeded in the shaking flask of 50% liquid amount according to 2% inoculum concentration, is put in 30 DEG C of constant-temperature table 180r/ Min, culture is for 24 hours.

Step 3. prepares different nitrogen sources culture medium

Peptone, tryptone, soy peptone, beef extract and yeast powder are added by nitrogen source basis with 1% amount respectively (glucose 10g/L, NaCl 5g/L, pH value are adjusted in 7) culture medium, are control with nitrogen source basal medium.By bacillus subtilis Bacterium SX3411 is seeded in the shaking flask of 50% liquid amount according to the inoculum concentration of 2% seed liquor, is put in 30 DEG C of constant-temperature table 180r/ Min, culture is for 24 hours.

Step 4. inorganic ion produces the influence of antibacterial material to bacillus subtilis SX3411

The most suitable carbon source and nitrogen source obtained using above-mentioned experiment prepares different inorganic ion trainings as inorganic salts basal medium Base is supported, it is different according to utilization of the microorganism to inorganic ion, respectively with 0.5%NaCl, 0.5%K₂HPO₄, 0.05% CaCl, 0.0 005%FeSO₄·7H₂0,0.0 005%ZnSO₄Amount be added inorganic salts basal medium in, with inorganic alkali Basal culture medium is control.Bacillus subtilis SX3411 is seeded in the shaking flask of 50% liquid amount according to 2% inoculum concentration, is put In 30 DEG C of constant-temperature table 180r/min, culture is for 24 hours.

The analysis of step 5. bacteriostatic activity

Bacterial strain SX3411 is taken to cultivate fermentation liquid for 24 hours, 10 000r/min are centrifuged 15min, abandon thallus and take supernatant, i.e., Antibacterial material crude extract is obtained, with the ammonium sulfate precipitation Subtilomycin of 30% concentration, every 40mL culture solution gained precipitating is used 2mL concentration is the suspension of 50mmo1/L PBS (pH 7.4) buffer, is slightly mentioned Subtilomycin.With staphylococcus aureus For indicator bacteria, influence of the different inorganic ions to the bacteriostatic activity of bacillus subtilis SX3411 fermentation liquid, 3 weights are measured It is multiple.

Step 6. carbon source produces the influence result and analysis of antibacterial material to bacillus subtilis SX3411

Different carbon source is not much different to the bacterial strain SX3411 influence for producing antibacterial material, and detection fermentation liquid thick leach protein can produce Raw preferable fungistatic effect, can effectively accumulate Subtilomycin.Wherein the fungistatic effect of sweet dew alcohol and glucose is best, Antibacterial circle diameter respectively reaches 23.18mm and 23.21mm；Followed by sucrose and soluble starch, antibacterial circle diameter are respectively 21.97mm and 21.76mm；Fructose and carbon source control inhibition zone effect be not much different, antibacterial circle diameter be respectively 21.19mm and 21.43mm (Figure 12 A).The price and acquisition channel of comprehensive mannitol and glucose sugar, therefore select glucose sugar for optimum carbon source.No Influence with concentration of glucose to bacterial strain SX3411 fermentation secretion Subtilomycin is unobvious, and comprehensive microorganism utilizes carbon source Efficiency and economic factor, the concentration of final choice glucose are 1% (Figure 12 B).

Step 7. nitrogen source produces the influence result and analysis of antibacterial material to bacillus subtilis SX3411

Different nitrogen sources, which have the bacterial strain SX3411 activity for producing antibacterial material, to be significantly affected.When yeast powder is nitrogen source, slightly mention The inhibition zone of Subtilomycin detection reaches 22.66mm；When followed by soy peptone is nitrogen source, antibacterial circle diameter is 22.18mm；When beef extract and peptone are respectively nitrogen source, the antibacterial circle diameter of the two is 20.47mm and 20.67mm respectively；Nothing The culture medium slow growth of nitrogen source, does not detect bacteriostatic activity, illustrates that nitrogen source secretes bacillus subtilis SX3411 Subtilomycin has important influence (Figure 13 A).According to comparison soy peptone and yeast powder price between the two, choosing It is optimum nitrogen source with yeast powder.

According to Figure 13 B it is found that when dusty yeast concentration is 1%, bacterial strain SX3411 production antibacterial material bacteriostatic activity is best, antibacterial Loop diameter is 23.62mm, most beneficial for the accumulation of antibacterial material；When dusty yeast concentration is 0.5%, antibacterial circle diameter is 22.82mm bacteriostasis is preferable；The yeast powder of 0.1% concentration is substantially not detectable bacteriostatic activity, illustrates the nitrogen source of low concentration not Conducive to the growth of bacterial strain SX3411；Nitrogen source be 1.5% and 2% concentration yeast powder when, bacteriostatic diameter be respectively 21.13mm and 18.40mm.Therefore select the yeast powder of 1% concentration as nitrogen source.

Step 8. inorganic ion produces the influence result and analysis of antibacterial material to bacillus subtilis SX3411

In the inorganic salts basal medium containing only 1% glucose and 1% yeast powder, it is separately added into each inorganic ion, Fermented and cultured bacillus subtilis SX3411 is heavy obtained by every 40mL culture solution using 30% ammonium sulfate precipitation fermentation liquid crude protein Forming sediment with 2mL concentration is that 50mmo1/L PBS (pH 7.4) buffer suspends, and is slightly mentioned Subtilomycin.Detection slightly mentions The bacteriostatic activity of Subtilomycin, experimental result are as shown in Figure 14 A: compared with the control that inorganic salts are not added, having added different nothings There is rising or falling for bacteriostatic activity in the fermentation liquid of machine salt.The antibacterial circle diameter of control is 23.15mm, and in inorganic salts ZnSO₄, CaCl have facilitation to its bacteriostatic activity, antibacterial circle diameter is respectively 26.00mm, 25.25mm；As addition K₂HPO₄ When, the antibacterial circle diameter for slightly being mentioned Subtilomycin is 15.70mm, bacteriostatic activity decline；NaCl,FeSO₄·7H₂O is to it Influence unobvious, antibacterial circle diameter is respectively 24.53mm, 24.30mm.It follows that ZnSO₄Most beneficial for bacterial strain SX3411 points Antibacterial material is secreted, CaCl takes second place.

The ZnSO of various concentration is added into the culture medium without inorganic salts₄, fermented and cultured is carried out, detects fermentation liquid respectively Bacteriostatic activity, with without minimal medium be control, identify different Zn²⁺The influence that concentration ferments to bacterial strain SX3411, knot Fruit is as shown in Figure 14B.The Zn of high concentration²⁺The growth that can inhibit bacterial strain SX3411 is unfavorable for the secretion and product of Subtilomycin It is tired；The Zn of 0.01~0.1mmol/L²⁺Bacillus subtilis SX3411 can be promoted to secrete and accumulate Subtilomycin, wherein 0.05mmol/L is the most advantageous.

5 response phase method optimum culture condition of embodiment

Step 1. experimental design and obtained regression equation

According to experiment of single factor obtain as a result, selection to bacillus subtilis SX3411 secretion Subtilomycin have shadow Loud factor: initial pH value, shaking speed, cultivation temperature, liquid amount and inoculum concentration carry out response surface experiment, and incubation time is For 24 hours, using antibacterial circle diameter as response.Table 4 is to carry out experimental design using Box-Benhnken to obtain actual value and recurrence side The predicted value of journey.Regression analysis is carried out to antibacterial circle diameter, the quadratic regression equation of fitting is as follows:

Y=24.10-1.27A-0.61B+0.421C-2.25D-0.32E-1.56AB-3.31AC-2.2 2AD-0.23AE- 0.80BC+1.44BD+2.84BE+1.28CD-3.09CE+2.00DE-1.77A²-2.81B²-3.22C²-1.23D²-0.32E²

Wherein Y indicates that antibacterial circle diameter, A indicate initial pH value of medium, and B indicates shaking speed, and C indicates cultivation temperature, D Indicate that liquid amount, E indicate inoculum concentration.

The design of 4 condition of culture response surface experiments of table and actual value and predicted value

The significance analysis of step 2. experimental result

Table 5 is the variance point that the quadratic regression equation (ANOVA) obtained after regression equation analysis is carried out according to experimental result Analysis.Significance analysis is carried out to each factor, wherein A is initial pH value, B is shaking speed, C is cultivation temperature, D is dress liquid Amount, E are inoculum concentration, AB, AC, AD, BC, BD, BE, CD, CE, DE, A²、B²、C²、D²Bacillus subtilis SX3411 is produced Subtilomycin has conspicuousness, wherein producing Subtilomycin influence degree to bacillus subtilis SX3411 is liquid amount > pH value > revolving speed > temperature > inoculum concentration (table 5).

The variance analysis of 5 culture medium regression model of table

Note: * * * highly significant, * are significant

The regression equation that step 3. fits carries out error statistics analysis

Statistical has been carried out to precision, confidence level, accuracy, multiple correlation coefficient using Design-Expert software Analysis, is shown in Table 6.Wherein R²=0.981639, show the good relationship of the regression equation；Adj R-Squared value and Pred R- Squared value is high and difference is lower than 0.2, illustrates that the regression model can explain fermentation process；C.V. it is worth less than 10%, illustrates reality The accuracy tested and with a high credibility；Accuracy (Adeq Precision) value is greater than 4, illustrates rationally.The regression equation can be used In the analysis prediction to fermentation condition.

The analysis of 6 regression equation error statistics of table

Two interactive response surface analysis of factor in step 4. condition of culture

According to test result, the interactive response surface design of two factors and contour map have been obtained after analysis processing, point Not as shown in Figure 15 to 24.From figure it is upper can be simple and clear observe 5 factors to bacillus subtilis SX3411 secretion The influence of Subtilomycin.Contour illustrates two interactive conspicuousnesses of factor, and ellipse indicates there is conspicuousness, Circle indicates that reciprocation is not significant.

From figure 15, it can be known that the significant interaction of initial pH value and revolving speed, under certain conditions, with initial pH value Increase, antibacterial circle diameter is in downward trend after rising, and with the increase of revolving speed, antibacterial circle diameter is in first to rise becoming of declining afterwards Gesture.Antibacterial circle diameter has maximum value under suitable initial pH value and revolving speed, and it is 8.25 which, which appears in initial pH value, ~8.5, revolving speed is in the range of 160~175rpm.

As can be seen from Figure 16, the significant interaction of initial pH value and temperature, under certain conditions, with initial pH value Increase, antibacterial circle diameter is in the trend risen, and as the temperature increases, antibacterial circle diameter is in downward trend after first rising.Suppression Bacterium loop diameter suitable pH value and at a temperature of have maximum value, the maximum value appear in initial pH value be 7.75~8.25, temperature Degree is in 30~31 DEG C of range.

As can be seen from Figure 17, the significant interaction of initial pH value and liquid amount, under certain conditions, with initial pH value Increase, antibacterial circle diameter in first rise after downward trend, with the increase of liquid amount, antibacterial circle diameter in decline become Gesture.Antibacterial circle diameter has maximum value under suitable pH value and liquid amount, and it is 7.5~8 which, which appears in initial pH value, In the range that liquid amount is 30%~40%.

As can be seen from Figure 18, the reciprocation of initial pH value and inoculum concentration is not significant, under certain conditions, with initial pH The increase of value, antibacterial circle diameter is in downward trend after first rising, and with the increase of inoculum concentration, antibacterial circle diameter becomes in decline Gesture.Antibacterial circle diameter under suitable pH value and inoculum concentration have maximum value, the maximum value appear in initial pH value be 7.75~ 8.5, inoculum concentration is in 1%~1.5% range.

As can be seen from Figure 19, the significant interaction of revolving speed and temperature, under certain conditions, with the increase of revolving speed, suppression Bacterium loop diameter is in downward trend after first rising, and as the temperature increases, antibacterial circle diameter is in downward trend after first rising.Suppression Bacterium loop diameter suitable revolving speed and at a temperature of have maximum value, the maximum value appear in revolving speed be 170~175r/min, temperature For in 30~31 DEG C of ranges.

As can be seen from Figure 20, the significant interaction of revolving speed and liquid amount, under certain conditions, with the increase of revolving speed, Antibacterial circle diameter is in downward trend after first rising, and with the increase of liquid amount, antibacterial circle diameter is in downward trend.Inhibition zone Diameter has maximum value under suitable revolving speed and liquid amount, and it is 170~178r/min, liquid amount which, which appears in revolving speed, For in 25%~35% range.

As can be seen from Figure 21, the significant interaction of revolving speed and inoculum concentration, under certain conditions, with the increase of revolving speed, Antibacterial circle diameter is in downward trend, and with the increase of inoculum concentration, antibacterial circle diameter is in downward trend.Antibacterial circle diameter is closing Under suitable revolving speed and liquid amount have maximum value, the maximum value appear in revolving speed be 170~175r/min, inoculum concentration be 1%~ In 1.5% range.

As can be seen from Figure 22, the significant interaction of temperature and liquid amount, under certain conditions, as the temperature increases, Antibacterial circle diameter is in downward trend after first rising, and with the increase of liquid amount, antibacterial circle diameter is in downward trend.Inhibition zone Diameter has maximum value at suitable temperature and liquid amount, and it is 29~31 DEG C which, which appears in temperature, and liquid amount is In 25%~30% range.

As can be seen from Figure 23, the significant interaction of temperature and inoculum concentration, under certain conditions, as the temperature increases, Antibacterial circle diameter is in downward trend after first rising, and with the increase of inoculum concentration, antibacterial circle diameter is in downward trend.Inhibition zone Diameter has maximum value at suitable temperature and inoculum concentration, and it is 29.5~31.5 DEG C which, which appears in temperature, inoculum concentration For in 1%~1.75% range.

As can be seen from Figure 24, the significant interaction of liquid amount and inoculum concentration, under certain conditions, with the increasing of liquid amount Add, antibacterial circle diameter is in downward trend, and with the increase of inoculum concentration, antibacterial circle diameter is in downward trend.Antibacterial circle diameter There is maximum value under suitable liquid amount and inoculum concentration, it is 25%~35% which, which appears in liquid amount, and inoculum concentration is In 1%~1.75% range.

The best Synthetical cultivation condition of step 5.

It is optimized using Design-Expert software, fermenting B. subtilis SX3411 secretes Subtilomycin Best Synthetical cultivation condition are as follows: initial pH value 8.25, revolving speed 169r/min, 30.24 DEG C of temperature, liquid amount 37.9%, inoculation Amount 1.46%, inhibition zone predicted value are 30.04mm.According to practical operation, initial pH value 8.25, revolving speed 170r/min, temperature are selected The condition of culture of 30 DEG C of degree, liquid amount 38%, inoculum concentration 1.5% carries out verification test, and the antibacterial circle diameter of measuring is 29.83mm meeting predicted value.

6 response phase method optimization culture based component of embodiment

Step 1. experimental design and obtained regression equation

According to experiment of single factor obtain as a result, selection to bacillus subtilis SX3411 secretion Subtilomycin have shadow Loud factor: glucose, yeast powder, zinc sulfate and calcium chloride carry out response surface experiment, incubation time be for 24 hours, it is straight with inhibition zone Diameter is response.Table 7 is to carry out experimental design using Box-Benhnken to obtain the predicted value of actual value and regression equation.To suppression Bacterium loop diameter carries out regression analysis, and the quadratic regression equation of fitting is as follows:

Y=25.65+2.23A-3.68B-0.37C-0.53D+1.33AB+1.64AC-0.20AD-0.7 5BC-1.77BD- 0.21CD-3.74A²-1.70B²-2.34C²-3.09D²。

Wherein Y indicates that antibacterial circle diameter, A indicate that concentration of glucose, B indicate dusty yeast concentration, and C indicates sulfuric acid zinc concentration, D Indicate calcium chloride concentration.

The design of 7 culture medium response surface experiments of table and actual value and predicted value

The significance analysis of step 2. experimental result

Table 8 is the variance point that the quadratic regression equation (ANOVA) obtained after regression equation analysis is carried out according to experimental result Analysis.Significance analysis is carried out to each factor.Analysis is it is found that A glucose, B yeast powder, D calcium chloride, AB, AC, BD, A²、B²、 C²、D²Producing antibacterial peptide to bacillus subtilis SX3411 has conspicuousness, and wherein C zinc sulfate does not have conspicuousness, is likely present it The influence of his factor.Wherein to bacillus subtilis SX3411 produce antibacterial peptide influence degree be yeast powder > glucose > calcium ion > Zinc ion.

The variance analysis of 8 culture medium regression model of table

Note: * * * highly significant, * are significant

The regression equation that step 3. fits carries out error statistics analysis

Statistical has been carried out to precision, confidence level, accuracy, multiple correlation coefficient using Design-Expert software Analysis, is shown in Table 9.Wherein R²=0.98192, show the good relationship of the regression equation；Adj R-Squared value and Pred R- Squared value is high and difference is lower than 0.2, illustrates that the regression model can explain fermentation process, but there are still the shadows of other factors It rings；C.V. it is worth the accuracy and with a high credibility for illustrating experiment less than 10%；Accuracy (Adeq Precision) value is greater than 4, says It is bright reasonable.The regression equation can be used for the prediction of the analysis to fermentation medium.

The analysis of 9 regression equation error statistics of table

Two interactive response surface analysis of factor in step 4. medium component

According to test result, the interactive response surface design of two factors and contour map have been obtained after analysis processing, point Not as shown in Fig. 4-19 to Fig. 4-24.From figure on can be simple and clear observe four factors to bacillus subtilis SX3411 Secrete the influence of Subtilomycin.Contour illustrates the interactive conspicuousness of two factors, and ellipse indicates to have significant Property, circle indicates that reciprocation is not significant.

As can be seen from Figure 25, the significant interaction of glucose and yeast powder, with the increase of concentration of glucose, inhibition zone is straight Diameter is in the trend declined afterwards is first risen, and with the increase of dusty yeast concentration, antibacterial circle diameter is in first gentle rear downward trend. Antibacterial circle diameter has maximum value under suitable glucose and dusty yeast concentration, and it is 10 which, which appears in concentration of glucose, ~12.5g/L, dusty yeast concentration are in the range of 5~7.5g/L.

As can be seen from Figure 26, the significant interaction of glucose and zinc sulfate, with the increase of concentration of glucose, inhibition zone is straight Diameter is in the trend declined afterwards is first risen, and with the increase of sulfuric acid zinc concentration, antibacterial circle diameter is in first to rise again gentle rear decline Trend.Antibacterial circle diameter has maximum value under suitable glucose and sulfuric acid zinc concentration, which appears in glucose Concentration is 10~12.5g/L, and sulfuric acid zinc concentration is in the range of 0.00025~0.00075g/L.

As can be seen from Figure 27, the reciprocation of glucose and calcium chloride is not significant, with the increase of concentration of glucose, inhibition zone Diameter is in the trend declined afterwards is first risen, and with the increase of calcium chloride concentration, antibacterial circle diameter variation is not significant.Work as glucose Concentration is 10~12.5g/L, and when calcium chloride concentration is 0.05~0.075g/L range, antibacterial circle diameter reaches maximum value.

As can be seen from Figure 28, the reciprocation of yeast powder and zinc sulfate is not significant, with the increase of dusty yeast concentration, inhibition zone Diameter is in the first gentle rear trend declined, and with the increase of sulfuric acid zinc concentration, antibacterial circle diameter variation is not significant.Inhibition zone is straight Diameter has maximum value under suitable yeast powder and sulfuric acid zinc concentration, and it is 5~7.5g/L which, which appears in dusty yeast concentration, Sulfuric acid zinc concentration is in the range of 0.00025~0.00075g/L.

As can be seen from Figure 29, the significant interaction of yeast powder and calcium chloride, with the increase of dusty yeast concentration, inhibition zone is straight Diameter is in the first gentle rear trend declined, and with the increase of calcium chloride concentration, antibacterial circle diameter variation is not significant.Antibacterial circle diameter There is maximum value under suitable yeast powder and calcium chloride concentration, it is 5~7.5g/L, chlorine which, which appears in dusty yeast concentration, Change in the range that calcium concentration is 0.05~0.075g/L.

As can be seen from Figure 30, the reciprocation of zinc sulfate and calcium chloride is not significant, with the increase of sulfuric acid zinc concentration, inhibition zone Diameter change is not significant, and with the increase of calcium chloride concentration, antibacterial circle diameter variation is not significant.Antibacterial circle diameter is in suitable sulphur There is maximum value, it is 0.00025~0.00075g/L, chlorine which, which appears in sulfuric acid zinc concentration, under sour zinc and calcium chloride concentration Change in the range that calcium concentration is 0.05~0.075g/L.

The best comprehensive nutrient proportioning components of step 5.

It is optimized using Design-Expert software, fermenting B. subtilis SX3411 secretes Subtilomycin Optimal medium component are as follows: glucose 11.4g/L, yeast powder 5g/L, 0.000 56g/L of zinc sulfate, calcium chloride 0.059 5g/L, inhibition zone predicted value are 27.83mm.To verify related nutritional proportioning components, glucose 11.4g/L, yeast powder 5g/ are selected The nutrient media components of L, 0.000 6g/L zinc sulfate, 0.06g/L calcium chloride carry out verification test, the antibacterial circle diameter of measuring For 27.22mm, meet predicted value.

The preliminary purification of embodiment 7Subtilomycin

The acquisition of step 1. bacterial strain SX3411 fermentation liquid

For 24 hours by aforementioned prioritization scheme fermented and cultured by bacillus subtilis SX3411,10 000r/min of culture solution is centrifuged 20min abandons thallus and takes supernatant to get bacillus subtilis SX3411 fermentation liquid is arrived.

The ammonium sulfate precipitation of Subtilomycin in step 2. bacterial strain SX3411 fermentation liquid

The coarse extraction of Subtilomycin uses salting out method, in fermentation liquid reinforcing body ammonium sulfate respectively to 20%, 30%, 40%, 50%, 60%, 70% saturation degree is set at 4 DEG C overnight.High speed freezing centrifuge is centrifuged at 10 000r/min, 4 DEG C 20min.Precipitating obtained by every 40mL culture solution is that 50mmo1/L PBS (pH 7.4) buffer suspends with 2mL concentration.With golden yellow Staphylococcus is indicator bacteria, carries out bacteriostatic experiment, observes antibacterial situation and antibacterial circle diameter size, and more each saturation degree precipitates egg White bacteriostatic activity.

According to Figure 31 it is found that can effectively be precipitated in bacillus subtilis SX3411 fermentation liquid at 20%~40% Subtilomycin, fungistatic effect are fine；And the polypeptide fungistatic effect that 50%~70% saturation degree obtains is poor；Wherein 30% The Subtilomycin fungistatic effect that ammonium sulfate saturation degree obtains is best.

Step 3. ultrafiltration slightly mentions the Subtilomycin in bacterial strain SX3411 tunning

The ultra-filtration centrifuge tube of 3kD is placed in 0.2mo1/L NaOH solution, impregnates 30min or more, is taken out, is soaked in 20% It is saved in alcohol.Wash with distilled water after super filter tube, the Subtilomycin for taking 500 μ L ammonium sulfate precipitations slightly to mention is in 3kD ultrafiltration Guan Zhong, 10 000r/min are centrifuged 10min, and trapped fluid volume is about 100 μ L in super filter tube, discard the filtered solution in collecting pipe.It is past 400 μ L 50mmo1/L PBS (pH 7.4) buffers are added in super filter tube, 10 000r/min are centrifuged 10min, cut in super filter tube Liquid stay product is about 100 μ L, discards the filtered solution in collecting pipe.Repeat the step 2 time.It is cut what 3kD super filter tube ultrafiltration obtained It stays liquid to be stored in 1.5mL centrifuge tube, using staphylococcus aureus as indicator bacteria, respectively takes 25 μ L progress disc diffusion method to do antibacterial Activity determination.

Step 4.SDS-PAGE electrophoresis detection ultrafiltration slightly mentions Subtilomycin

The Subtilomycin sample for the bacillus subtilis SX3411 for taking ultrafiltration to obtain, with 15%SDS-PAGE gel into Row protein electrophorese utilizes the stripe size of coomassie brilliant blue staining observation Subtilomycin.The result shows that 30% ammonium sulfate It precipitates obtained band to become apparent from, size is in 4kD or so；And it is also big containing 4kD or so in 20% and 40% ammonium sulfate precipitation object Small band, but clarity reduces.Inhibition zone size after analysis SDS-PAGE in the clarity and step 1 of band is just right Should get up (Figure 32), wherein, suppression most (see electrophoretogram in Figure 32) in the Subtilomycin amount that 30% ammonium sulfate precipitation obtains Bacterium circle is secondly the Subtilomycin that 20% ammonium sulfate precipitation obtains also maximum (see inhibition zone size column diagram in Figure 32), 40% ammonium sulfate precipitation obtains relatively minimal.

Claims (3)

1. a kind of method of a small amount of fermenting and producing Lantibiotics Subtilomycin, it is characterised in that:

(1) fermenting microbe: zymophyte is bacillus subtilis (Bacillus subtilis) SX3411, and the culture presevation is in State's General Microbiological Culture preservation administrative center, culture presevation CGMCC NO.14396；

(2) fermentation time: the fermentation time that fermented and cultured bacillus subtilis SX3411 generates Subtilomycin is that 24-30 is small When；

(3) fermentation comprehensive condition of culture are as follows: initial pH value 8.25, revolving speed 169r/min, 30.24 DEG C of temperature, liquid amount 37.9%, Inoculum concentration 1.46%；

(4) component of fermentation medium are as follows: glucose 11.4g/L, yeast powder 5g/L, zinc sulfate 0.00056g/L, calcium chloride 0.0595g/L。

2. a kind of method of a small amount of fermenting and producing Lantibiotics Subtilomycin according to claim 1, feature The step of being Subtilomycin in preliminary purification fermentation liquid are as follows:

(1) fermentation liquid is precipitated using the ammonium sulfate of 20%-40%, obtains fermentation liquid ammonium sulfate precipitation；

(2) phosphate buffer of fermentation liquid ammonium sulfate precipitation pH7.4, concentration 50m mol/L are suspended and is precipitated, then pass through 3kD Ultra-filtration centrifuge tube, the Subtilomycin of preliminary purification can be obtained.

3. a kind of method of a small amount of fermenting and producing Lantibiotics Subtilomycin according to claim 2, feature The step of being Subtilomycin in preliminary purification fermentation liquid are as follows:

(1) fermentation liquid is precipitated using 30% ammonium sulfate, obtains fermentation liquid ammonium sulfate precipitation；

(2) phosphate buffer of fermentation liquid ammonium sulfate precipitation pH7.4, concentration 50m mol/L are suspended and is precipitated, then pass through 3kD Ultra-filtration centrifuge tube, the Subtilomycin of preliminary purification can be obtained.