CN104694601A - High-efficiency preparation method of Iturin A and homologue of Iturin A - Google Patents
High-efficiency preparation method of Iturin A and homologue of Iturin A Download PDFInfo
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Abstract
The invention belongs to the technical field of microbes and particularly relates to a high-efficiency preparation method of Iturin A and a homologue of Iturin A. The method comprises the following steps: culturing a Bacillus strain and a genetically modified bacterium thereof in a primary fluid medium, and then inoculating a secondary fluid medium with the primary fluid medium as a seed solution, wherein the strain can generate Iturin A and the homologue of Iturin A; after inoculating the secondary fluid medium with the seed solution, performing fermentation culture and the feed-batch culture of amino acid and the like; and collecting Iturin A and the homologue thereof from a fermentation culture solution after fermentation is over. The method provided by the invention has the characteristics of high production efficiency, easy industrialization and the like.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to the method that one efficiently prepares iraq subtilis actinomycin A (Iturin A) and homologue thereof.
Background technology
Iraq subtilis actinomycin A (Iturin A) and homologue thereof, be mainly derived from the secondary metabolite of bacillus (Bacillus) bacterial strain such as subtilis (Bacillus.subtilis), the advantage such as there is broad spectrum resistance and not easily develop immunity to drugs, be considered to a kind of natural, there is the efficient antimicrobial substance suppressing plant and animal pathogenic bacteria.Especially Iturin A has strong antifungal property, also suppresses the activity of part bacterium.
Usual bacillus (Bacillus) bacterial strain such as subtilis (B.subtilis) produces two class secondary metabolites, one class is the surfactin (Surfactin) with bio-surfactant function, and another kind of is iraq subtilis actinomycin A (Iturin A) and the homologue thereof of anti-mycotic activity.
Iturin class material is the mixture of multiple homologue composition, and wherein main component is Iturin A, and basic structure (see accompanying drawing 1) is a cyclic peptide be made up of 7 a-amino acids, is beta-amino lipid acid at N-end.The size differences of the fat chain group on its side chain, constitutes its homologue.
The structure and energy of Iturin A is closely related, and the fat chain group on its side chain is larger (n value is larger), and activity is stronger; Its anti-mycotic activity is also relevant with the cytolemma of target cell, and it improves membrane passage by forming ionic channel on cytolemma thus act on target site.Medically also in the dermatophytid infection of some humans and animals, do clinical experiment with natural Iturin A.Due to its broad-spectrum antifungal and low toxicity and low-metamorphic reactive, it is made to get a good chance of becoming a kind of antifungal drug of high value.
But, up to now, all there is no the production method of efficient iraq subtilis actinomycin A and homologue thereof both at home and abroad.Especially the technology of bacillus (Bacillus) bacterial strain such as subtilis (B.subtilis) fermentative production iraq subtilis actinomycin A and homologue thereof is utilized not break through, it yields poorly, cost is high, it is comparatively large that zymotechnique controls difficulty, governs its suitability for industrialized production and commercial applications.
From report both domestic and external in recent decades, the Study on Fermentation course of iraq subtilis actinomycin A and homologue thereof mainly divides three phases, first stage (before nineteen ninety-five) is traditional liquid submerged fermentation (submerged fermentation, SMF), exploration fermentation condition is mainly concentrated on the impact of Biomass and yield.The people such as Sandrin (1990) carry out shake-flask culture with subtilis S499 and produce iraq subtilis actinomycin A and surfactin, when its production peak is respectively 280mg/liter(cultivation 72h) and 760mg/liter.The people such as Shoda (1991,1993) carry out small canister liquid fermenting with subtilis NB22, control the output of iturin, when production peak reaches 620mg/liter(cultivation 72h by changing temperature and air flow in fermenting process).The people such as Hbid (1996) carry out 20L ferment tank with Bacillus subtilus S499, and controlled the output of iturin by the control changing oxygen transmission in fermenting process, production peak reaches 1388mg/liter.The difficulty mainly faced in the research work of this one-phase is the problem of foam overflow, and the use of chemical defoamer is restricted, because chemical defoamer affects the transfer rate of oxygen and affects the physiological property of microorganism.
Subordinate phase (1992-2007) is solid fermentation (solid stated fermentation, SSF), and utilization of resources research department of Tokyo technical college is the significant contribution person in this stage.The people such as Akihiro (1993,1995) carry out the production of iturin as substrate with Testa Tritici, its output comparatively submerged fermentation improves 5-6 doubly, and their trial is afterwards with bean curd tankage as fermentation substrate, and its output comparatively submerged fermentation improves 6-8 doubly.The people such as Mizumoto (2006,2007) solid fermentation produces iturin, and its output reaches wet solid medium and 5591 μ g/g of 3300mg/kg respectively and to wet solid medium.Solid fermentation is that fermenting process is simple compared with the advantage of submerged fermentation, and thalline and production concentration are larger, and solvent when extracting is less.But because solid state fermentation production unit is subject to the restriction of factors, the problems such as easy pollution microbes, make this technology be applied to large scale fermentation production and are subject to serious restriction.
In order to the output of liquid state fermentation can be improved, research worker gets on to find new outlets from the novel method of fermentation, so the research of iturin zymotechnique phase III (2004-) mainly concentrates in the innovation of fermentation process, the maximum contribution person of this stage research or the researchist of utilization of resources research department of Tokyo technical college.2006, the people such as this research department Mohammad were again sprouted by the spore in hot activation induction batch fermentation late period and carry out Secondary Fermentation and produce iturin, and its output comparatively one time fermentation improves 1.0g/L, reaches 4.0g/L.Secondary Fermentation is the huge innovation on fermentation process, has very large application potential.Also just after a year, 2007, or the people such as Mohammad produces iraq subtilis actinomycin A with recombinant bacterial strain B.sutbtilis168 quiescent culture on microbial film, and the submerged fermentation that its output is more traditional improves 2 times, reaches 0.8g/L.But on microbial film, the quick growth of thalline causes nutrition supply not enough, and result causes producing more spore, affects ferment effect.Japanese Showa Denko K. K in 2004, Yoneda, Tadashi (meter Tian Zheng etc.) (WO2004/029273 in the patent of application; CN1685056A in 2005) describe the preparation method of Iturin A and homologue thereof.Be specially and use bacterial strain B.subtilis SD142 and mutant strain 1,2 thereof, in the synthetic medium containing 2% or more concentration soyflours, produce the Iturin A of 1.5g/L or more output and the method for homologue thereof.It has carried out 5L tank fermentation research, result, and in the fermented liquid of wild strain SD142, the output of Iturin A and homologue thereof reaches 3.8g/L; In fermented liquid with mutant strain 1,2, the output of Iturin A and homologue thereof reaches 6.7g/L; But the method production cost is still higher, and there is the increase along with nitrogenous source, it is serious that fermentation produces foam, increases the disadvantages such as zymotechnique difficulty, be difficult to realize suitability for industrialized production.2009, patent ZL200910058559.9 reports, adopt the genetic improvement bacterial strain Bacillus subtilis ZK-H6(CGMCC No.2897 of subtilis) or Bacillus subtilis ZK-3(CGMCC No.0924), add adsorption of immobilization cell material and sticky dose, after inoculation seed liquor, the fed-batch fermentation carrying out fermentation culture and flow feeding liquid is cultivated, and Iturin A and homologue output thereof can reach 8.0g/L.
The secondary metabolite research work of the present inventor's long campaigns subtilis (Bacillus subtilis).Find after carrying out genetic improvement to a plants endogenetic subtilis (B subtilis), mutant strain Bacillus subtilis ZK-H28 has high yield Iturin A and homologue thereof, produces hardly or the micro-characteristic producing Surfactin.
Subsequently, the present inventor is studied for the processing parameter of Iturin A and homologue thereof and technological method utilizing subtilis (B subtilis) fermentation, obtain the technological method improving strain cell density in fermentation system, effectively control the foam of fermentation generation, supplement the high yield Iturin A such as the amino acid precursor of Iturin A and homologue synthesis thereof and homologue thereof, therefore complete the present invention.
Summary of the invention
Object of the present invention:
An object of the present invention, is to provide one " amino acid and liquid nutrient medium fed batch fermentation Technology ", fermentative production Iturin A and homologue thereof;
Object two of the present invention, is to provide a kind of new strains for the production of Iturin A and homologue thereof;
Object three of the present invention, is to provide the substratum for the production of Iturin A and homologue thereof.
Object four of the present invention, the stream be to provide for the production of Iturin A and homologue thereof adds the method for amino acid precursor.
More particularly, the invention provides a kind of method preparing Iturin A and homologue thereof, comprise following steps:
The bacterium that can produce Iturin A and homologue thereof is cultivated, as seed liquor in level liquid substratum (such as, culture medium A hereinafter described); Described bacterium is mainly selected from the genetic improvement bacterial strain of the subtilis (Bacillus subtilis) of bacillus, bacillus amyloliquefaciens (Bacillus.amyloliquefaciens), Bacillus licheniformis (Bacillus licheniformis), bacillus firmus (Bacillus firmus) bacterial strain and above-mentioned bacterial strains.
To cultured seed liquor be inoculated in second stage liquid nutrient medium (such as, substratum B) hereinafter described and cultivate in above-mentioned first step liquid nutrient medium; Cultivate one suitable period after second stage liquid nutrient medium inoculation first step seed liquor, the fed-batch fermentation starting to carry out flow feeding liquid (such as, amino acid feed supplement liquid hereinafter described and feed supplement liquid C) is cultivated.
After fermentation ends, from above-mentioned fermentation culture, collect Iturin A and homologue thereof.
Specific embodiment of the invention scheme is, adopt known generation Iturin A and the Bacillus sp of homologue thereof, as the genetic improvement bacterium of subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus.amyloliquefaciens), Bacillus licheniformis (Bacillus licheniformis), bacillus firmus (Bacillus firmus) bacterial strain and above-mentioned bacterial strains; Wherein subtilis, as: wild-type B. subtilis and genetic improvement bacterium thereof, subtilis S499, NB22, genetic improvement bacterium Bacillus sutbtilis ZK-H6, ZK-3, ZK-H28 etc., carry out second order fermentation cultivation in liquid medium within, select different substratum at every grade of fermentation stage.In the fermentation of the second stage, to be suitable for the liquid nutrient medium of second stage fermentation, such as substratum B hereinafter described, as fermention medium.After inoculation first step seed liquor, cultivate for some time at a suitable temperature, such as, after 3-30 hours, select suitable feed supplement liquid 26 DEG C of-37 DEG C of fermentation culture, such as amino acid feed supplement liquid hereinafter described and feed supplement liquid C, carry out fed batch fermentation cultivation.
The mode of second stage liquid culture flow feeding (such as amino acid feed supplement liquid and feed supplement liquid C) can adopt continuously (at the uniform velocity or at the uniform velocity non-), and stream adds and/or intermittent type fed-batch mode, and Continuous Flow add mode is preferred.
Described continuous (at the uniform velocity or at the uniform velocity non-) fed-batch mode, feed supplement liquid (such as amino acid feed supplement liquid and the feed supplement liquid C) Continuous Flow be applicable to is added in the fermentor tank of the second stage, until stop first about 10 hours of fermentation (lower tank) with certain stream rate of acceleration (at the uniform velocity or at the uniform velocity non-).
Described intermittent type fed-batch mode is the mode adding a defective material with certain interval of time, and feed supplement liquid (such as amino acid feed supplement liquid and the feed supplement liquid C) stream be applicable to adds in the fermentor tank of the second stage by intermittent type.Intermittent time is preferably every feed supplement in 1-10 hour 1-20 time, is more preferably about interval feed supplement in 1 hour 1-5 time.Those skilled in the art also as required, can adopt other applicable timed interval feed supplement.
Fermentation condition: temperature 26 DEG C-37 DEG C, PH:6-8
Fermentation time: 3-6 days
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
Adopt present invention process, when fermenting in the second stage, stream adds amino acid feed supplement liquid and feed supplement liquid C carries out liquid culture, can supplement the amino acid precursor of Iturin A and homologue synthesis thereof, maintain the good carbon-nitrogen ratio of zymotechnique demand; Improve the concentration of bacterial cell in fermentation system, realize the fermentation of higher density, greatly reduce the foam that fermenting process produces, be conducive to controlling the technical parameter such as dissolved oxygen concentration, temperature, thus improve the output of Iturin A and homologue thereof.
In preferred specific embodiments, the present invention also adopts the technical schemes such as such as new strains to improve the output of Iturin A and homologue thereof further.
In preferred the inventive method, the present invention provides a strain for the production of the genetic improvement bacterial strain Bacillus subtilis ZK-H28 of the subtilis of Iturin A and homologue thereof especially; It is preserved in No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on March 15th, 2013, Institute of Microorganism, Academia Sinica's (postcode 100101) China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number is CGMCC No.7321.This bacterial strain produces hardly or trace produces Surfactin.
The culture medium A that second order fermentation of the present invention adopts, B, and amino acid feed supplement liquid, feed supplement liquid C, it is specifically composed as follows:
Culture medium A (g/mL): Zulkovsky starch 0.2%-6.0% glycerine 0.3%-2.0%
Glucose 0.1%-10.0% peptone 0.1%-3.0%
Yeast extract paste 0.1%-2.0% magnesium sulfate 0.01%-1.0%
Potassium primary phosphate 0.2%-1.0% sodium-chlor 0.01%-1.0%
Substratum B(g/mL):
| Composition | General range | Preferable range |
| Semen Maydis powder | 0.1%-6.0% | 0.3%-4.0% |
| Maltose | 0.1%-4.0% | 0.3%-2.0% |
[0041]
| Sucrose/molasses | 0.1%-5.0% | 0.5%-3.0% |
| Glycerine | 0.1%-2.0% | 0.5%-1.5% |
| Glucose | 0.1%-10.0% | 1.5%-5.0% |
| Peptone | 0.3%-6.0% | 0.7%-4.0% |
| Analysis for soybean powder or analysis for soybean powder hydrolyzed solution | 0.1%-12.0% | 1.5%-8.0% |
| Yeast extract paste | 0.1%-5.0% | 0.5%-2.0% |
| Fishbone powder | 0.1%-5.0% | 0.6%-3.0% |
| Potassium primary phosphate | 0.01%-5.0% | 0.05%-2.0% |
| Ammonium nitrate | 0.01%-5.0% | 0.05%-2.0% |
| Magnesium sulfate | 0.01%-5.0% | 0.05%-2.0% |
| Sodium-chlor | 0.01%-5.0% | 0.05%-2.0% |
Feed supplement liquid C(g/mL):
| Composition | General range | Preferable range |
| Maltose | 0.1%-5.0% | 0.5%-3.0% |
| Yeast extract paste | 0.1%-3.0% | 0.5%-2.0% |
| Sucrose/molasses | 0.1%-8.0% | 1.5%-5.0% |
| Semen Maydis powder | 0.1%-3.0% | 0.3%-2.0% |
| Glucose | 0.3%-12.0% | 0.5%-9.0% |
| Analysis for soybean powder or analysis for soybean powder hydrolyzed solution | 0.1%-12.0% | 0.5%-6.0% |
| Ammonium nitrate | 0.1%-5.0% | 0.5%-3.0% |
| Fishbone powder | 0.1%-5.0% | 0.5%-3.0% |
Usually, adding 18 kinds of D types in fermenting process or/and L-type amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg, Pro, Trp), is all useful to the fermentation yield of raising Iturin A and homologue thereof.In preferred scheme, the amino acid classes added and content thereof are as following table.
Amino acid (D type and/or L-type) feed supplement liquid (g/mL):
| Composition | General range | Preferable range |
| Lys | 1.0x10 -6-9.0x10 -4 | 5.0x10 -5-3.0x10 -4 |
| Ser | 1.0x10 -6-2.0x10 -3 | 3.0x10 -5-5.0x10 -4 |
| Gln | 1.0x10 -6-8.0x10 -4 | 5.0x10 -5-4.0x10 -4 |
| Asp | 1.0x10 -6-9.0x10 -3 | 6.0x10 -5-9.0x10 -4 |
| Asn | 1.0x10 -6-8.0x10 -3 | 6.0x10 -5-8.0x10 -4 |
| Glu | 1.0x10 -6-5.0x10 -3 | 5.0x10 -5-5.0x10 -4 |
| Pro | 1.0x10 -6-8.0x10 -3 | 5.0x10 -5-5.0x10 -4 |
| Tyr | 1.0x10 -6-6.0x10 -4 | 3.0x10 -5-1.0x10 -4 |
| Thr | 1.0x10 -6-5.0x10 -4 | 5.0x10 -5-3.0x10 -4 |
The zymotechnique whole process of the preferred embodiment of the invention is:
Bacillus sp after activation is inoculated in culture medium A, solid culture or be loaded on 26 DEG C of-37 DEG C of shake-flask culture in triangular flask and, after 10-30 hours, be inoculated in the fermentor tank being added with sterilising medium B with the inoculum size of 5%--30% and carry out fermentative production.Strain inoculation is in 26 DEG C-37 DEG C after the fermentor tank of the second stage, and preferably 28 DEG C of-33 DEG C of fermentation culture are after 3-30 hours, starts the flow feeding of amino acid feed supplement liquid and feed supplement liquid C.
The flow feeding mode of amino acid feed supplement liquid and feed supplement liquid C has two kinds, and one is continuously that (at the uniform velocity or at the uniform velocity non-) stream adds, and one is that intermittent type stream adds, and Continuous Flow add mode is preferred.
When adopting Continuous Flow add mode, with the speed of 0.001-10.0L/h continuously (at the uniform velocity or at the uniform velocity non-) stream add amino acid feed supplement liquid and/or feed supplement liquid C, until stop fermentation (lower tank) precontract 10 hours.
When adopting intermittent type stream to add amino acid feed supplement liquid and/or feed supplement liquid C, intermittent time can be every feed supplement in 1-10 hour 1-20 time, be preferably about interval feed supplement in 5 hours 3-10 time, each feed supplement amount is 0.001%-0.6% of fermented liquid cumulative volume, preferably 0.01%-0.2%.
Fermentation condition: temperature 26 DEG C-37 DEG C, PH:6-8
Fermentation time: 3-6 days
Accompanying drawing 2 is shown in full technical process of the present invention.
Adopt present invention process, bacterial strain produces Iturin A and homologue thereof with higher substrate conversion efficiency and Product formation speed, and the output of Iturin A and homologue thereof can reach more than 9.0g/L.
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
In preferred the inventive method, provide a strain for the production of the genetic improvement bacterial strain Bacillus subtilis ZK-H28 of the subtilis of Iturin A and homologue thereof especially; It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on March 15th, 2013, and preserving number is CGMCC No.7321.This bacterial strain produces hardly or trace produces Surfactin.
Subtilis (Bacillus subtilis) ZK-H28 has following feature on gene level:
Subtilis (Bacillus subtilis) Iturin A synthetic gene is bunch primarily of 4 genomic constitutions: ituD, ituA, ituB, ituC, as shown in Figure 3.
Existing document discloses the Iturin A synthetic gene bunch sequence (Tsuge of subtilis (Bacillus subtilis) RH14, K., et al. (2001). " Cloning, sequencing, and characterization of the iturin A operon. "
j Bacteriol183 (21): 6265-6273.).Through gene order comparison, the ituD gene of the Iturin A synthetic gene of subtilis (B.subtilis) ZK-H28 bunch, ituD gene with the Iturin A synthetic gene bunch of B.subtilis RH14 bacterial strain, nucleotide sequence and aminoacid sequence there are differences.Its difference characteristic is:
There is the base difference in 40 sites in the ituD gene order of ZK-H28 bacterial strain and RH14 bacterial strain, wherein the base difference (87,116,137,204,248 in 15 sites, 304,305,310,318,346,350,363,367,368,392 sites), cause the change of 15 amino acid sites after translating into amino acid, these amino acid sites are respectively F in RH14 bacterial strain, E, Q, M, K, P, P, T, H, T, P, K, D, V, A; In ZK-H28 bacterial strain, these amino acid sites are respectively L, Q, H, L, E, H, L, A, R, N, A, E, N, E, D.(the ituD gene order of ZK-H28 bacterial strain and RH14 bacterial strain and aminoacid sequence attached)
The present invention has the following advantages:
One. adopt amino acid feed supplement liquid and liquid nutrient medium fed batch fermentation, the good carbon-nitrogen ratio of zymotechnique demand can be maintained, supplement the amino acid precursor of Iturin A and homologue synthesis thereof, significantly can improve the output of Iturin A and homologue thereof.
Two. this process advan is in the concentration improving bacterial cell in fermentation system, realize the fermentation of higher density, greatly reduce the foam that fermenting process produces, be conducive to controlling the technical parameter such as dissolved oxygen concentration, temperature, thus improve the productive rate of Iturin A and homologue thereof.
Three. extraction process flow process is simple, and the rate of recovery is high, easy to operate
Accompanying drawing explanation
Accompanying drawing 1 is Iturin A structure.
Accompanying drawing 2 is the schema of Iturin A and homologue fermentative production thereof.
By clearly finding out in figure that first producing bacterial classification with Iturin A and homologue thereof carries out second order fermentation cultivation according to required condition, in the fermentation culture process of the second stage, carry out flow feeding, then the process that aftertreatment obtains Iturin A and homologue thereof is carried out to tunning.
Accompanying drawing 3 is subtilis (Bacillus subtilis) Iturin A synthetic genes bunch.
Embodiment
Embodiment 1
With 1000mL triangular flask 12, every bottled 300mL culture medium A (Zulkovsky starch 1.0% glycerine 0.6%, glucose 5.0%, peptone 1.0%, yeast extract paste 0.5%, magnesium sulfate 0.5%, potassium primary phosphate 0.5%, sodium-chlor 0.3%), in 120 DEG C of sterilizings 30 minutes, subtilis (Bacillus subtilis) ZK-H28 bacterium liquid after cooling after inoculation activation, under being placed in 30 DEG C of temperature, shake-flask culture 18 hours.Cultured strain liquid is inoculated in by the inoculum size of 5%-10% in the 100L fermentor tank of in-built 50L sterilising medium B, (substratum B: Semen Maydis powder 2.0%, maltose 1.0%, sucrose 0.5%, glycerine 0.5%, glucose 1.0%, peptone 1.0%, analysis for soybean powder hydrolyzed solution 5.0%, yeast extract paste 0.5%, fishbone powder 1.0%, potassium primary phosphate 0.5%, ammonium nitrate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.3%), at 28 DEG C of-30 DEG C of temperature, aeration-agitation fermentation is after 5-15 hours, amino acid feed supplement liquid (g/mL) (Lys1.0x10 is added with the speed Continuous Flow of 0.01-1.0L/h
-5, Ser1.0x10
-4, Gln2.0x10
-4, Asp5.0x10
-4, Asn1.0x10
-4, Glu2.0x10
-4, Pro2.0x10
-4, Tyr3.0x10
-5, Thr1.0x10
-5), add culture medium C (culture medium C: maltose 1.0%, yeast extract paste 0.5%, sucrose 2.0% with the speed Continuous Flow of 0.02-2.0L/h, Semen Maydis powder 1.0%, glucose 2.0%, analysis for soybean powder hydrolyzed solution 3.0%, ammonium nitrate 1.0%, fishbone powder 1.0%), until stop fermentation (lower tank) precontract 12 hours.Fermentation PH6-8, fermentation period 5 days.
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
After testing, through fermentation in 5 days, Iturin A and homologue output thereof can reach 10.5g/L fermented liquid, and product recovery rate reaches more than 85%.
Embodiment 2
With 1000mL triangular flask 10, every bottled 300mL culture medium A (Zulkovsky starch 2.0%, glycerine 0.5%, glucose 5.0%, peptone 3.0%, yeast extract paste 0.3%, magnesium sulfate 0.6%, potassium primary phosphate 0.6%, sodium-chlor 0.4%), in 120 DEG C of sterilizings 30 minutes, subtilis (Bacillus subtilis) ZK-H6 (CGMCC No.2897) the bacterium liquid after cooling after inoculation activation, under being placed in 30 DEG C of temperature, shake-flask culture 18 hours.Cultured strain liquid is inoculated in (substratum B: Semen Maydis powder 2.0% in the 100L fermentor tank of in-built 50L sterilising medium B by the inoculum size of 5%-10%, maltose 1.0%, sucrose 0.5%, glycerine 0.5%, glucose 1.0%, peptone 1.0%, analysis for soybean powder hydrolyzed solution 5.0%, yeast extract paste 0.5%, fishbone powder 1.0%, potassium primary phosphate 0.5%, ammonium nitrate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.3%), at 28 DEG C of-30 DEG C of temperature, aeration-agitation fermentation is after 5-15 hours, amino acid feed supplement liquid (g/mL) (Lys 1.0x10 is added with the speed Continuous Flow of 0.01-1.0L/h
-5, Ser 1.0x10
-4, Gln 2.0x10
-4, Asp 5.0x10
-4, Asn 1.0x10
-4, Glu 2.0x10
-4, Pro 2.0x10
-4, Tyr 3.0x10
-5, Thr1.0x10
-5), add culture medium C (culture medium C: maltose 1.0%, yeast extract paste 0.5%, sucrose 2.0% with the speed Continuous Flow of 0.02-2.0L/h, Semen Maydis powder 1.0%, glucose 2.0%, analysis for soybean powder hydrolyzed solution 3.0%, ammonium nitrate 1.0%, fishbone powder 1.0%), until stop fermentation (lower tank) precontract 12 hours.Fermentation PH6-8, fermentation period 5 days.
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
After testing, through fermentation in 5 days, Iturin A and homologue output thereof can reach 9.5g/L fermented liquid, and product recovery rate reaches more than 80%.
Embodiment 3
With 1000mL triangular flask 12, every bottled 300mL culture medium A (Zulkovsky starch 4.0%, glycerine 1.0%, glucose 3.0%, peptone 1.0%, yeast extract paste 0.5%, magnesium sulfate 0.5%, potassium primary phosphate 0.5%, sodium-chlor 0.3%), in 120 DEG C of sterilizings 30 minutes, subtilis (Bacillus subtilis) the ZK-H28 bacterium liquid after cooling after inoculation activation, under being placed in 30 DEG C of temperature, shake-flask culture 18 hours.Cultured strain liquid is inoculated in by the inoculum size of 5%-10% in the 100L fermentor tank of in-built 50L sterilising medium B, (substratum B: Semen Maydis powder 3.0%, maltose 1.0%, sucrose 1.5%, glucose 1.0%, peptone 2.0%, analysis for soybean powder hydrolyzed solution 4.0%, yeast extract paste 0.5%, fishbone powder 1.0%, potassium primary phosphate 0.5%, ammonium nitrate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.3%), at 28 DEG C of-30 DEG C of temperature, aeration-agitation fermentation, after 3-15 hours, adds amino acid feed supplement liquid (g/mL) (Lys 0.5x10 with intermittent type stream
-5, Ser 1.5x10
-4, Gln5.0x10
-5, Asp 5.0x10
-4, Asn 2.0x10
-4, Glu 7.0x10
-5, Pro 6.0x10
-4, Tyr 3.0x10
-5, Thr 1.0x10
-5), the intermittent time is about interval feed supplement in 1 hour 2 times, and each feed supplement amount is the 0.01%-0.1% of fermented liquid cumulative volume; With intermittent type fed-batch medium C(culture medium C: maltose 1.0%, yeast extract paste 0.5%, sucrose 2.0%, Semen Maydis powder 1.0%, glucose 2.0%, analysis for soybean powder hydrolyzed solution 3.0%, ammonium nitrate 1.0%, fishbone powder 1.0%), the intermittent time is about interval feed supplement in 2 hours 3 times, each feed supplement amount is the 0.01%-0.2% of fermented liquid cumulative volume, until stop fermentation (lower tank) precontract 12 hours.Fermentation PH6-8, fermentation period 5 days.
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
After testing, through fermentation in 5 days, Iturin A and homologue output thereof can reach 10.0g/L fermented liquid, and product recovery rate reaches more than 85%.
Embodiment 4
With 1000mL triangular flask 15, every bottled 300mL culture medium A (Zulkovsky starch 4.0%, glycerine 1.0%, glucose 3.0%, peptone 1.0%, yeast extract paste 0.5%, magnesium sulfate 0.5%, potassium primary phosphate 0.5%, sodium-chlor 0.3%), in 120 DEG C of sterilizings 30 minutes, subtilis (Bacillus subtilis) ZK-H6 (CGMCC No.2897) the bacterium liquid after cooling after inoculation activation, under being placed in 30 DEG C of temperature, shake-flask culture 18 hours.Cultured strain liquid is inoculated in by the inoculum size of 5%-20% in the 100L fermentor tank of in-built 50L sterilising medium B, (substratum B: Semen Maydis powder 2.0%, maltose 1.0%, sucrose 0.5%, glycerine 0.5%, glucose 1.0%, peptone 1.0%, analysis for soybean powder hydrolyzed solution 5.0%, yeast extract paste 0.5, fishbone powder 1.0%, potassium primary phosphate 0.5%, ammonium nitrate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.3%), at 28 DEG C of-30 DEG C of temperature, aeration-agitation fermentation, after 5-15 hours, adds amino acid feed supplement liquid (g/mL) (Lys 5.0x10 with intermittent type stream
-5, Ser 7.0x10
-5, Gln 8.0x10
-5, Asp 3.0x10
-4, Asn 5.0x10
-4, Glu 2.0x10
-4, Pro8.0x10
-5tyr 1.0x10
-5, Thr 1.0x10
-5), the intermittent time is about interval feed supplement in 1 hour 3 times, and each feed supplement amount is the 0.01%-0.1% of fermented liquid cumulative volume; With intermittent type fed-batch medium C(culture medium C: maltose 1.5%, yeast extract paste 0.6%, sucrose 2.0%, Semen Maydis powder 3.0%, glucose 2.5%, analysis for soybean powder hydrolyzed solution 5.0%, ammonium nitrate 1.0%, fishbone powder 1.5%), the intermittent time is about interval feed supplement in 1 hour 2 times, each feed supplement amount is the 0.01%-0.2% of fermented liquid cumulative volume, until stop fermentation (lower tank) precontract 12 hours.Fermentation PH6-8, fermentation period 5 days.
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
After testing, through fermentation in 5 days, Iturin A and homologue output thereof can reach 8.0g/L fermented liquid, and product recovery rate reaches more than 85%.
Embodiment 5
With 1000mL triangular flask 12, every bottled 300mL culture medium A (Zulkovsky starch 3.0%, glycerine 1.5%, glucose 3.0%, peptone 1.0%, yeast extract paste 0.5%, magnesium sulfate 0.5%, potassium primary phosphate 0.5%, sodium-chlor 0.3%), in 120 DEG C of sterilizings 30 minutes, bacillus firmus (Bacillus firmus) BF-I (preservation of this laboratory) the bacterium liquid after cooling after inoculation activation, under being placed in 30 DEG C of temperature, shake-flask culture 18 hours.Cultured strain liquid is inoculated in by the inoculum size of 5%-20% in the 100L fermentor tank of in-built 50L sterilising medium B, (substratum B: Semen Maydis powder 2.0%, maltose 1.0%, sucrose 0.5%, glycerine 0.5%, glucose 2.0%, peptone 2.0%, analysis for soybean powder hydrolyzed solution 4.0%, yeast extract paste 0.5%, fishbone powder 1.0%, potassium primary phosphate 0.5%, ammonium nitrate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.3%), at 28 DEG C of-30 DEG C of temperature, aeration-agitation fermentation is after 6-15 hours, amino acid feed supplement liquid (g/mL) (Lys1.0x10 is added with the speed Continuous Flow of 0.01-1.0L/h
-5, Ser2.0x10
-4, Gln3.0x10
-4, Asp5.0x10
-4, Asn2.0x10
-4, Glu2.0x10
-4, Pro3.0x10
-4, Tyr2.0x10
-5, Thr1.0x10
-5), add culture medium C (culture medium C: maltose 2.0%, yeast extract paste 0.5%, sucrose 1.0% with the speed Continuous Flow of 0.01-2.0L/h, Semen Maydis powder 2.0%, glucose 3.0%, analysis for soybean powder hydrolyzed solution 3.0%, ammonium nitrate 1.0%, fishbone powder 3.0%), until stop fermentation (lower tank) precontract 12 hours.Fermentation PH6-8, fermentation period 5 days.
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
After testing, through fermentation in 5 days, Iturin A and homologue output thereof can reach 7.5g/L fermented liquid, and product recovery rate reaches more than 85%.
Embodiment 6
With 1000mL triangular flask 15, every bottled 300mL culture medium A (Zulkovsky starch 3.0%, glycerine 1.0%, glucose 5.0%, peptone 1.5%, yeast extract paste 0.5%, magnesium sulfate 0.5%, potassium primary phosphate 0.5%, sodium-chlor 0.3%), in 120 DEG C of sterilizings 30 minutes, subtilis (Bacillus subtilis) ZK-3 (CGMCC No.0924) after cooling after inoculation activation) bacterium liquid, under being placed in 30 DEG C of temperature, shake-flask culture 18 hours.Cultured strain liquid is inoculated in by the inoculum size of 5%-20% in the 100L fermentor tank of in-built 50L sterilising medium B, (substratum B: Semen Maydis powder 3.0%, maltose 1.0%, sucrose 0.5%, glycerine 0.5%, glucose 3.0%, peptone 2.0%, analysis for soybean powder hydrolyzed solution 5.0%, yeast extract paste 0.5%, fishbone powder 1.0%, potassium primary phosphate 0.5%, ammonium nitrate 0.5%, magnesium sulfate 0.2%, sodium-chlor 0.3%), at 28 DEG C of-30 DEG C of temperature, aeration-agitation fermentation is after 4-15 hours, amino acid feed supplement liquid (g/mL) (Lys1.0x10 is added with the speed Continuous Flow of 0.01-1.0L/h
-5, Ser3.0x10
-4, Gln4.0x10
-4, Asp5.0x10
-4, Asn8.0x10
-5, Glu1.0x10
-4, Pro4.0x10
-4, Tyr2.0x10
-5, Thr1.0x10
-5), culture medium C (culture medium C: maltose 2.0%, yeast extract paste 0.5%, sucrose 1.0% is added with the speed Continuous Flow of 0.02-2.0L/h, Semen Maydis powder 1.0%, glucose 2.0%, analysis for soybean powder hydrolyzed solution 3.0%, ammonium nitrate 1.0%, fishbone powder 2.0%), until stop fermentation (lower tank) precontract 12 hours.Fermentation PH6-8, fermentation period 5 days.
After fermentation ends, fermented liquid removing thalline and other solid substance, adjust the pH value of fermented liquid to 2-5 with sulfuric acid or hydrochloric acid, or saltout with the ammonium sulfate of saturation ratio at 40%-70%.Collecting precipitation, carries out silica gel column chromatography, using ethanol, methyl alcohol and trichloromethane as eluting solvent, detects (developing agent is for propyl carbinol: Glacial acetic acid: water=100:5:20) with silica gel thin-layer chromatography method.Collect the elutriant containing iraq subtilis actinomycin A and homologue thereof, leaving standstill with under stirring state after concentrated, cool gradually, iraq subtilis actinomycin A and homologue thereof are separated out in solid form, through collecting, and washing, dry, obtain iraq subtilis actinomycin A and homologue product thereof.
High performance liquid chromatography is adopted to detect Iturin A and homologue thereof, condition used: chromatographic column C1810.7 φ × 250mm; Moving phase is 45% acetonitrile: water: 10mM ammonium acetate; Flow velocity 1mL/min; Determined wavelength 280nm.
After testing, through fermentation in 5 days, Iturin A and homologue output thereof can reach 6.5g/L fermented liquid, and product recovery rate reaches more than 85%.
Attached: the ituD gene order of ZK-H28 bacterial strain and RH14 bacterial strain and aminoacid sequence
The ituD gene order of 1.ZK-H28 bacterial strain and RH14 bacterial strain
Note: green difference site box indicating one group of codon representing RH14 and ZK-H28, the amino acid of its correspondence is inconsistent in RH14 and ZK-H28
Claims (6)
1. the high efficiency preparation method of an iraq subtilis actinomycin A and homologue thereof, comprise bacterial classification, preparation technology, it is characterized in that: bacterial classification used is the Bacillus strain that can produce iraq subtilis actinomycin A and homologue thereof, as: subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus.amyloliquefaciens), Bacillus licheniformis (Bacillus licheniformis), the genetic improvement bacterium of bacillus firmus (Bacillus firmus) bacterial strain and above-mentioned bacterial strains, preparation technology is divided into two-stage, the first step is that seed liquor is cultivated, used medium is liquid nutrient medium A, the second stage is fermentation culture, used medium is liquid nutrient medium B, after cultivating for some time under the suitable conditions, the fed batch fermentation carrying out amino acid feed supplement liquid and feed supplement liquid C is cultivated, after fermentation ends, from above-mentioned fermentation culture, collect iraq subtilis actinomycin A and homologue thereof.
2. the preparation method of iraq subtilis actinomycin A according to claim 1 and homologue thereof, is characterized in that: described bacterial classification is the genetic improvement bacterial strain Bacillus subtilis ZK-H28 of subtilis, CGMCCNo.7321.
3. the preparation method of iraq subtilis actinomycin A and homologue thereof according to claim 1, is characterized in that: by g/mL, substratum and feed supplement liquid specifically composed as follows:
Culture medium A:
Zulkovsky starch 0.2%-6.0% glycerine 0.3%-2.0% glucose 0.1%-10.0% peptone 0.1%-3.0% yeast extract paste 0.1%-2.0% magnesium sulfate 0.01%-1.0% potassium primary phosphate 0.2%-1.0% sodium-chlor 0.01%-1.0%
Substratum B:
Semen Maydis powder 0.3%-4.0%, maltose 0.3%-2.0%, sucrose/molasses 0.5%-3.0%, glycerine 0.5%-1.5%, glucose 1.5%-5.0%, peptone 0.7%-4.0%, analysis for soybean powder or analysis for soybean powder hydrolyzed solution 1.5%-8.0%, yeast extract paste 0.5%-2.0%, fishbone powder 0.6%-3.0%, potassium primary phosphate 0.05%-2.0%, ammonium nitrate 0.05%-2.0%, magnesium sulfate 0.05%-2.0%, sodium-chlor 0.05%-2.0%
Feed supplement liquid C:
Maltose 0.5%-3.0%, yeast extract paste 0.5%-2.0%, sucrose/molasses 1.5%-5.0%, Semen Maydis powder 0.3%-2.0%, glucose 0.5%-9.0%, fishbone powder 0.5%-3.0% analysis for soybean powder or analysis for soybean powder hydrolyzed solution 0.5%-6.0%, ammonium nitrate 0.5%-3.0%
Amino acid (D type and/or L-type) feed supplement liquid:
Lys 5.0x10
-5-3.0x10
-4，Ser 3.0x10
-5-5.0x10
-4，Gln 5.0x10
-5-4.0x10
-4，Asp 6.0x10
-5-9.0x10
-4，Asn 6.0x10
-5-8.0x10
-4，Glu 5.0x10
-5-5.0x10
-4，Pro 5.0x10
-5-5.0x10
-4，Tyr 3.0x10
-5-1.0x10
-4，Thr5.0x10
-5-3.0x10
-4。
4. the preparation method of iraq subtilis actinomycin A and homologue thereof according to claim 1, is characterized in that: the mode that stream adds amino acid feed supplement liquid and feed supplement liquid C is Continuous Flow add mode and/or intermittent type fed-batch mode.
5. the preparation method of iraq subtilis actinomycin A and homologue thereof according to claim 4, it is characterized in that: Continuous Flow add mode is at the uniform velocity or non-at the uniform velocity Continuous Flow add mode, amino acid feed supplement liquid and/or feed supplement liquid C is added, until stop fermentation precontract 10 hours with the speed Continuous Flow of 0.001-10.0L/h.
6. the preparation method of iraq subtilis actinomycin A and homologue thereof according to claim 4, it is characterized in that: the intermittent time that intermittent type stream adds amino acid feed supplement liquid and/or feed supplement liquid C can be every feed supplement in 1-10 hour 1-20 time, be preferably about interval feed supplement in 5 hours 3-10 time, each feed supplement amount is 0.001%-0.6% of fermented liquid cumulative volume, preferably 0.01%-0.2%.
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