CN101139564B - Duganella bacterium and uses thereof - Google Patents
Duganella bacterium and uses thereof Download PDFInfo
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- CN101139564B CN101139564B CN2007101200749A CN200710120074A CN101139564B CN 101139564 B CN101139564 B CN 101139564B CN 2007101200749 A CN2007101200749 A CN 2007101200749A CN 200710120074 A CN200710120074 A CN 200710120074A CN 101139564 B CN101139564 B CN 101139564B
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Abstract
The invention discloses a Duganella bacterium and its application. The Duganella bacterium is sp. B2 CGMCC No:2056. Culturing Duganella sp. B2 CGMCC No:2056 will get purple bacteriocin. The fermentingcondition for the bacterial strain is: culturing 24-60 h under 4-28 DEG C. The fermenting culture medium contains in each liter: starch 10-20 g, ferrisulphas 0.01-0.05 g, potassium nitrate 0.8-1.2 g,dipotassium hydrogen phosphate 0.5-1 g, and magnesium sulfate 0.5-1 g. The pH value of the fermenting culture medium is 6-9. The purple bacteriocin from the bacterial strain is a natural product, andfree from any hidden harm due to artificial pigment. Tests show that the purple bacteriocin is of good biologic activity, will generate no side effect on animal bodies, and can be widely used in suchfields as medicine and industries, etc.
Description
Technical field
The present invention relates to a Duganella bacterium and application thereof.
Background technology
Pigment generally is divided into synthetic food color and natural pigment, and both all have been widely used in aspects such as food colorant, daily cosmetics and fodder additives.Synthetic food color is meant with chemical synthesis process prepared organic pigment, mainly is to extract from coal tar or is that raw material is synthetic with arene compounds such as benzene, toluene, naphthalene, aniline.After synthetic food color occurs, accepted by the user rapidly with advantages such as its lovely luster, strong coloring force, good stability, color matching convenience, low prices, but along with subject development such as modern medicine, food hygienes, it is found that synthetic colour exists toxicological issues, some is harmful to HUMAN HEALTH, even brings out cancer etc.Therefore, more and more countries has been stopped using the bigger kind of some toxicity, country that has even the use of having forbidden synthetic colour.Because the natural pigment major part is the edible portion from plant, microorganism, animal, extracting the course of processing is pure physical process, can not change on the structure.Therefore, natural pigment is safe and have the extensive attention that certain nutrient value causes people mostly because of it, and its Application and Development research becomes the focus in vegetable chemistry and the Food science field.
Natural pigment comprises plant pigments, animal pigment, microorganism pigment.Realize industrialization easily because of microbial fermentation, from microorganism, extract pigment, not limited by resource, environment and spatial, have plant pigments and animal pigment incomparable superiority.
Violacein is a kind of indole derivatives, is that tryptophane forms through oxidation in microbe, and molecular structure is suc as formula (I), and molecular weight is 343.Since finding for 19 end of the centurys, people have carried out a large amount of explorations to its biological function, find that it has a lot of biological functions, paid close attention to by people.Evidence, violacein has very strong biological activity: (1) has broad-spectrum antibacterial activity, as anti-staploylococcousaureus, Bacilluss sp, streptococcus sp., mycobacterium, Neisserig, pseudomonas (Sanchez et al., Reevaluation of the Violacein BiosyntheticPathway and its Relationship to Indolocarbazole Biosynthesis.Journal 2006.7,1231-1240); (2) anti-oxidant (Konzen et al., Antioxidant properties of violacein:possible relation on its biological function.Journal 2006.14,8307-8313); (3) antitumor cell (de Carvalho et al., Cytotoxic activity of violacein in humancolon cancer cells.Journal 2006.); (4) antiviral property or the like, can also process various material cloth (Akira SHIRATA*1 as dyestuff, Isolation of Bacteria ProducingBluish-Purple Pigment and Use for Dyeing.Japan Agricultural ResearchQuarterly.2000.34), in a word, violacein has very high medical value and prospects for commercial application.
Now the product violacein bacterium of open report has Chromobacterium violaceum, Chromobacterium fluviatile, Janthinobacterium lividum, Alteromonasluteoviolacea, wherein studying main is Chromobacterium violaceum, but it is an opportunistic pathogen, and output lower (0.43g/L) (Armando et al., Factorial design and responsesurface optimization of crude violacein for Chromobacterium violaceumproduction.Journal 2001.V23,1963-1969).
It is (Proposal to reclassify Zoogloearamigera IAM 12670 (P.R.Dugan 115) as Duganella zoogloeoides gen.nov. such as Hiraishi that Du rolls Bordetella (Duganella), sp.nov.Int J Syst Bacteriol 1997,47,1249-1252.) a newer genus proposing in 1997, comprise two kinds at present: 1) D.zoogloeoides, bacterium colony is slightly yellow, and 19th century separated from sewage; 2) D.violaceinigra (Wen-Jun Li, Yu-Qin Zhang, Dong-Jin Park, Chang-Tian Li, Li-Hua Xu, Chang-Jin Kim and Cheng-Lin Jiang, Duganellaviolaceinigra sp.nov., a novel mesophilic acterium isolated from forestsoil.Int J Syst Evol Microbiol, 2004,54,1811-1814), bacterium colony is purple darkly, separates from the forest soil of Yunnan in 2004.At present, it is limited that known Du rolls the kind number of Bordetella, and its applied research seldom.
Summary of the invention
The purpose of this invention is to provide a kind of new bacterial strain that can secrete, produce violacein.
The bacterial strain B2 of generation violacein provided by the present invention separates from glacier, Tianshan Mountains, Xinjiang, be that Du rolls the novel species of Bordetella (Duganella), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 18th, 2007 and (be called for short CGMCC, the address is: the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City), preservation registration number is CGMCC № 2056.
This bacterial strain is rod-short, no brood cell, pod membrane, Gram-negative.On Gao Shi substratum (starch 20g/L, ferrous sulfate 0.01g/L, saltpetre 1g/L, dipotassium hydrogen phosphate 0.5g/L, sal epsom 0.5g/L) plate, the bacterium colony avy blue, rounded, surface wettability is smooth, neat in edge.Support on base (peptone 5g/L, beef extract 3g/L) plate the bacterium colony lavender at nutrient agar medium.Pigment produces follows thalli growth, and final colony diameter can reach 2～3mm, and blueness is more shallow around the later stage bacterium colony.The oxydase of this bacterium, the catalase reaction positive, the urase reaction negative, hydrolyzable polychrom, gelatin, utilize cellobiose, maltose, sucrose, trehalose, starch, glycogen, hold together ox sugar,, D-rock sugar is only carbon source, do not utilize glycerine, erythrose, D-pectinose, ribose, D-wood sugar, L-wood sugar, ribitol, beta-methyl D-xyloside, semi-lactosi, glucose, fructose, seminose, sorbose, rhamnosyl, melampyrin, inositol, N.F,USP MANNITOL, sorbyl alcohol; G+C content is 63.68%, and 16srDNA sequence and Du roll Salmonella (Duganella zoogloeoides, ATCC 25935
T) similarity is 97%, lipid acid moiety and Du roll the Salmonella basically identical.This bacterium all can grow under the scope of 4 ℃-28 ℃ culture temperature and pH 6-9.
Second purpose of the present invention provides the method that Du rolls Bordetella (Duganella sp.) B2CGMCC № 2056 production violaceins of using.
The method of production violacein provided by the present invention is that fermentation Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 and obtains violacein.
The fermentation condition that described Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 can be 4 ℃-28 ℃ cultivations 24-60 hour.
The fermentation condition that described Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 is preferably 25 ℃ of cultivations 50 hours.
Described Du rolls in every liter of the fermention medium of Bordetella (Duganella sp.) B2 CGMCC № 2056 and can contain: starch 10-20g, ferrous sulfate 0.01-0.05g, saltpetre 0.8-1.2g, dipotassium hydrogen phosphate 0.5-1g, sal epsom 0.5-1g; The pH of described fermention medium is 6-9.
In order to improve the output of violacein, described Du rolls in every liter of the fermention medium of Bordetella (Duganella sp.) B2 CGMCC № 2056 contains the 0.3-0.5g tryptophane.
Described Du rolls the fermention medium of Bordetella (Duganella sp.) B2 CGMCC № 2056 and specifically can prepare as follows: starch 15g, ferrous sulfate 0.03g, saltpetre 1g, dipotassium hydrogen phosphate 0.7g, sal epsom 0.5g, tryptophane 0.5g adds water to 1000ml; The pH of described fermention medium is 7.0.
Also comprise the step of rolling purifying violacein Bordetella (Duganella sp.) B2 CGMCC № 2056 thalline from described Du in the aforementioned production method.
Described purification process can be with can dissolve purple the organic solvent solution of bacillin roll the broken thing that Bordetella (Duganella sp.) B2 CGMCC № 2056 somatic cells or described Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 somatic cells from described Du and extract violacein.
Described organic solvent solution is the ethanol of 5-100%, the Virahol of 5-100% or the tetrahydrofuran (THF) of 5-100%; Described organic solvent solution is preferably the ethanol of 5-100%; Described percentage composition is volumn concentration.
In order to make the better effects if of fermentation, before carrying out fermentation culture, frozen bacterial classification also activates through dull and stereotyped the cultivation, and plate culture medium commonly used contains following substances (every liter of amount): peptone 5g/L, and beef extract 3g/L, agar 20g, pH are 7.0.
In process of production, the described thalline that obtains generally can adopt centrifugation, as obtaining thalline in that 7000 * g is centrifugal.Also can utilize Plate Filtration method commonly used on the common fermentation industry to obtain cell.Described somatic cells is that adding concentration is the ethanol of 5-100%, the Virahol of 5-100% or the tetrahydrofuran (THF) of 5-100% in thalline through the process that fragmentation obtains violacein, obtains the ethanolic soln of cyanine behind the sonic oscillation, obtains cyanine after the drying.Solvent is the ethanol of 5-100% preferably, with the extraction using alcohol violacein time, amount of alcohol added can be controlled in every 100ml fermented liquid usually and adds 20-300ml, the ethanolic soln drying mode commonly used that has pigment can adopt vacuum-drying, promptly can obtain the thick product of violacein after the drying.Because pigment is positioned on the cytolemma, also can directly utilize described organic solvent extraction cell, obtains the pigment crude product.
The productive rate of rolling Bordetella (Duganella sp.) B2 CGMCC № 2056 production violaceins with Du of the present invention is higher, reach 0.45-1.7g violacein/L fermented liquid, the violacein that obtains belongs to all-natural product, there is not the harm of artificial color potential, Du of the present invention rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 not to be had pathogenic, fast growth all can produce pigment at 4 ℃-28 ℃ condition bottom fermentations, is convenient to suitability for industrialized production.Experiment showed, that chromobacterium have good biological activity, can not produce any side effect, in each field such as medical science, industry, good application prospects is arranged animal body.
Description of drawings
Fig. 1 rolls the HPLC separating spectrum of the main component of the pigment that Bordetella (Duganella sp.) B2 CGMCC № 2056 produces for Du
Fig. 2 rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 pigment main component mass spectrum results for LC-MS Du
Fig. 3 rolls the nucleus magnetic hydrogen spectrum spectrogram of Bordetella (Duganella sp.) B2 CGMCC № 2056 pigment principal constituents for Du
Fig. 4 is for 16S rDNA sequence being the tree-shaped graph of a relation of phylogeny that Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 and the genus of being correlated with is planted of fundamental construction
Embodiment
The isolation identification that embodiment 1, Du roll Bordetella (Duganella sp.) B2 CGMCC № 2056
1, sample collecting
Pedotheque is gathered in No. 1 glacier from Xinjiang.
2, the separation of bacterial strain and screening
Preparation separating plate substratum: peptone 5g, beef extract 3g, agar 20g adds water 1000ml, and pH 7.0.With above-mentioned substratum with 1.05kg/cm
2, 121 ℃, high pressure steam sterilization 20 minutes.
Pedotheque is made the suspension of 2% (2g/100ml), increase progressively by 10 times and be diluted to 2 * 10
8Get each extent of dilution sample liquid 0.2mL and evenly coat respectively on the above-mentioned isolation medium flat board, cultivated 7 days for 25 ℃, observe and find to have blue single bacterium colony, select and get this bacterium colony, the separation of ruling, the single bacterium colony of the picking bacterium separation of ruling again repeats step after the cultivation, until (detecting with microscope for pure bacterial strain, cellular form is consistent to be pure bacterial strain), with this bacterial strain called after B2, just Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056.
3, the evaluation of bacterial strain
With Duganella zoogloeoides IAM 12670
T(buying the using microbe institute in Tokyo Univ Japan) and Duganella violaceinigra YIM 31327T (buying in German microbial strains preservation center) are type strain, according to document (eastern elegant pearl etc. common bacteria identification handbook .2001.) in the method described, Du is rolled Bordetella (Duganella sp.) B2 CGMCC № 2056 carries out morphological specificity observation and oxydase, hydrogen peroxide enzymatic determination; All Physiology and biochemistry experiments are all tested under 25 ℃ of conditions, and all experiments as various enzyme biopsy surveys, utilization of carbon source etc. are all with API20NE and API50CH test kit (BioMerieux) test.
According to document (Owen, R.J. ﹠amp; Pitcher, D..Current methods for estimating DNAbase composition and levels of DNA-DNA hybridization.In Chemical Methodsin Bacterial Systematics, (1985) pp.67-93.Edited by M.Goodfellow ﹠amp; D.E.Minnikin.London:Academic Press) method of describing in is rolled Bordetella (Duganellasp.) B2 CGMCC № 2056 to Du and is carried out the G+C assay; According to document (Lane, D.J..16S/23S rRNAsequencing.In Nucleic Acid Techniques in Bacterial Systemat ics, (1991) pp.115-175.Edited by E.Stackebrandt ﹠amp; M.Goodfellow.Chichester:Wiley.) method of describing in, Du is rolled Bordetella (Duganella sp.) B2 CGMCC № 2056 carry out 16s rDNA sequencing,, utilize the 16S rDNA sequence of the Related Bacteria bacterial strain that the Blast search software accesses earlier from databases such as GenBank and EMBL according to sequencing result, use Clustal W 1.8[Thompson subsequently, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F.﹠amp; Higgins, D.G..The CLUSTAL_X windowsinterface:flexible strategies for multiple sequence alignment aided byquality analysis tools.Nucleic Acids Res (1997) 25,4876-4882] carry out multisequencing comparison, with MEGA 3 (Kumar, S., Tamura, K.﹠amp; Nei, M..MEGA3:integrated softwarefor molecular evolutionary genetics analysis and sequence alignment.BriefBioinform (2004) 5,150-163.) carry out data analysis, repeat number 1000 times, and adopt minimum evolution method (Minimum Evolution) method to carry out the structure of systematic evolution tree.The full-automatic identification systems analysis of U.S. MIDI of lipid acid component, according to document (Sasser, M..Identification of bacteria by gaschromatography of cellular fatty acids.USFCC Newsl (1990) 20,16., Ka ¨ mpfer, P.﹠amp; Kroppenstedt, R.M..Numerical analysis of fatty acid patterns ofcoryneform bacteria and related taxa.CanJ Microbiol (1996) 42,989-1005) the middle method of describing is carried out.
The result shows: 1) Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 and is rod-short, no brood cell, pod membrane, Gram-negative.On Gao Shi substratum (starch 20g/L, ferrous sulfate 0.01g/L, saltpetre 1g/L, dipotassium hydrogen phosphate 0.5g/L, sal epsom 0.5g/L) plate, the bacterium colony avy blue, rounded, surface wettability is smooth, neat in edge.Support on base (peptone 5g/L, beef extract 3g/L) plate the bacterium colony lavender at nutrient agar medium.Pigment produces follows thalli growth, and final colony diameter can reach 2～3mm, and blueness is more shallow around the later stage bacterium colony.
2) oxydase, the catalase reaction positive of shutting out and rolling Bordetella (Duganella sp.) B2 CGMCC № 2056, the urase reaction negative, hydrolyzable polychrom, gelatin, utilize cellobiose, maltose, sucrose, trehalose, starch, glycogen, hold together ox sugar, D-rock sugar is sole carbon source, does not utilize glycerine, erythrose, D-pectinose, ribose, D-wood sugar, L-wood sugar, ribitol, beta-methyl D-xyloside, semi-lactosi, glucose, fructose, seminose, sorbose, rhamnosyl, melampyrin, inositol, N.F,USP MANNITOL, sorbyl alcohol.
3) Du's G+C content (mol%) of rolling Bordetella (Duganella sp.) B2 CGMCC № 2056 is 63.68%, being positioned at G+C content (mol%) scope (63-64%) that Du rolls Bordetella.
4) Du roll Bordetella (Duganella sp.) B2 CGMCC № 2056 16s rDNA sequence shown in the sequence in the sequence table 1, total 1406bp, (ATCC 25935 with Duganella zoogloeoides
T) similarity is 97%.Carry out the multisequencing homology analysis, and constructing system growth tree (Fig. 4), the result shows that the B2 bacterium should be Du and rolls Bordetella.Among Fig. 4, the numerical value on the branch is 1000 times result of bootstrapping; Line segment 0.01 is represented 1/100 evolutionary distance unit.
5) Du's main lipid acid of rolling Bordetella (Duganella sp.) B2 CGMCC № 2056 consists of C16:0 (24.61%), C14:0 (0.88%), C12:0 (6.26%), 3-OH C10:0 (4.25%), 3-OH C12:0 (4%), C18:1 ω 7c (4.27%), through the full-automatic identification systems analysis of U.S. MIDI, (ATCC 25935 with Duganellazoogloeoides
T) similarity reaches 0.682.
According to the result of study to morphology, cultural characteristic, Physiology and biochemistry and the 16S rDNA of bacterial strain B2, bacterial strain B2 has the morphological specificity that typical Du rolls Bordetella, and the composition of lipid acid and genomic dna G+Cmol% content are supported The above results.But because its 16SrDNA sequence is with the Duganella zoogloeoides lAM12670 of this genus
T, D.violaceinigra YIM 31327
TSimilarity only is respectively 97%, 96%, and bacterial strain B2 has obvious difference (as table 1) with two kinds that Du rolls Bordetella, therefore bacterial strain B2 is decided to be Du and rolls the novel species (DuganeHa sp.) of Bordetella.In the table 1, nutrient agar (agar 20g adds water to 1000ml for peptone 5g, beef extract 3g, regulates pH 7.0) growing state is measured as follows: streak inoculation was cultivated 7 days for 28 ℃ in the nutrient agar medium plate, observed to have or not bacterium colony to produce.
Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 and all can grow under the scope of 4 ℃-28 ℃ culture temperature and pH6-9.
Table 1 bacterial strain B2 and Du roll the existing type strain physiological property difference of Bordetella
Annotate: "+" positive reaction, "-" negative reaction
1, strain culturing
The preparation plate culture medium: peptone 5g, beef extract 3g, agar 20g adds water to 1000ml, regulates pH 7.0.At 121 ℃ of 15min that sterilize down, dull and stereotyped, the access of cooling back is shut out and is rolled Bordetella (Duganella sp.) B2 CGMCC № 2056, cultivates 72 hours down at 25 ℃ with plate culture medium.
Preparation seed liquid nutrient medium (every liter of content): peptone 5g, beef extract 3g regulates pH7.0.Single Du of picking rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 colony inoculations in aseptic liquid nutrient medium from flat board, cultivates under 25 ℃ of temperature, and rotating speed is 200rpm (rotation radius is 32mm).Cultivate that thalline is in logarithmic phase after 24 hours.
The preparation fermentation broth: starch 15g, ferrous sulfate 0.03g, saltpetre 1g, dipotassium hydrogen phosphate 0.7g, sal epsom 0.5g, tryptophane 0.5g adds water to 1000ml, and pH is 7.0.The 100ml substratum packed into sterilize in the 500ml culturing bottle, the seed 10mL of logarithmic phase is inserted in the fermention medium (10% inoculum size) cultivate down at 25 ℃, mixing speed is 200rpm (rotation radius is 32mm).Cultivate that thalline is in logarithmic growth after 10 hours, when thalline enters lag phase after 50 hours and pigment produces and also is in lag phase, cultivated 50 hours, stop to ferment.
2, the extraction of cyanine
With fermented liquid centrifugal 10min under 7000 * g, abandon supernatant liquor, in throw out, add dehydrated alcohol (every 100ml fermented liquid adds 50ml ethanol), with eddy mixer with its mixing, vibrated 1 hour in the 200W ultrasonic cleaner then, the centrifugal 5min of liquid 9000 * g that will vibrate obtains the ethanolic soln of pigment.If still have pigment to remain in the cell residue, can repeat the aforesaid operations step, till no longer pigment can being extracted.With resulting ethanolic soln vacuum-drying, promptly obtain the crude extract of cyanine.
3, the physics-chem characteristic of cyanine detects
1) light stability
The pigment ethanolic soln of undried in the 2ml step 2 is added to light transmission preferably in the 10ml glass test tube, in case the ethanol volatilization, placed outdoor natural light under irradiation respectively 0,12 and 24 hour, handle repetition 3 times for every kind with sealing the sealing of film and paraffin.The rate of loss of pigment is as shown in table 2 behind the illumination certain hour.
Table 2. Du rolls the rate of loss of Bordetella (Duganella sp.) B2 CGMCC № 2056 pigments under natural light irradiation
By the data in the table as can be seen, the light stability of pigment in ethanolic soln is relatively poor, and after 24 hours, pigment is with a toll of 64.2%, and color also changes, and becomes light blue by original mazarine.
2) thermostability
With 1ml concentration is that pigment ethanol (alcohol concn is 100%) solution and the concentration of 0.5g/L is that the 1.0g/L aqueous solution places the 1.5ml polypropylene tube to carry out Temperature Treatment respectively, qualitatively judges the thermostability of pigment according to the rate of loss of pigment.Take three kinds of Temperature Treatment modes: airbath is heated 4h respectively for 60 ℃, 100 ℃, 121 ℃ of heating of high pressure steam pot 15min.Handle for every kind and repeat 3 times.Thermostability result is as shown in table 3.The result shows, when with ethanol during as solvent, the pigment absorbance is loss not.When with water during as solvent, the pigment loss when operative temperature is 100 ℃ is greater than 60 ℃.
The rate of loss of table 3 pigment under differing temps
The pigment extract of undried in the step 2 is preserved under 4 ℃ in refrigerator with the masking foil shading, and in 30 days, the range estimation pigmentary colours do not change substantially at least.
3) ph stability of pigment
The pigment ethanolic soln of undried in 5ml step 2 (pigment concentration is 0.438g/L) adds HCl, and when HCl concentration was 2.4mol/L, solution colour became green by original mazarine; Add NaOH, when NaOH concentration was 0.24mol/L, solution colour became yellow by original mazarine.
4) solvability and the dissolving color of pigment in different solvents
Gained cyanine shown color in different solvents has a great difference.With water, sherwood oil is solvent, is shown as dusty blue; As solvent, be shown as blueness with methyl alcohol, ethanol, Virahol, 50% methyl alcohol, 50% ethanol, 50% Virahol, 50% acetone, 50% tetrahydrofuran (THF); As solvent, be shown as purple with acetone, ethyl acetate, tetrahydrofuran (THF).
Can observe directly a certain amount of pigment crude extract by range estimation can be dissolved in methyl alcohol very soon, and solvability is best; Tetrahydrofuran (THF) and ethanol take second place; Water and sherwood oil are the poorest to the solvability of pigment crude extract.
4, cyanine purifying and structure elucidation
1) separation and purification of cyanine
Adopt high performance liquid chromatography (HPLC) (U.S. Waters company, Waters 2695), chromatographic column is InertsilODS-35um 250 * 4.6mm (purchasing the company in DIKMA), column temperature is a room temperature, PDA diode-array detector (Waters 2996), the detection wavelength is 575nm, moving phase is used 70% methanol-water system (volume ratio of methyl alcohol and water is 70: 30), each sample size 0.2mL, flow velocity is 1mL/min, collects concentrated main peaks (retention time is 4 minutes a elution peak) and has obtained the pigment main ingredient.
2) liquid chromatograph mass spectrography
By the isolated pigment main component of high performance liquid chromatography, by LC-MS separation and purification again, (mass spectrograph is: LCQ Deca XP (U.S. power ﹠ light company); Mass spectral condition is: ion source: the ESI source; Shell gas: 35arb; Auxiliary gas: 0arb; Spray voltage: 3.5kV; Capillary temperature: 300 ℃; Flow velocity: 1mL/min; Splitting ratio 9: 1; Mass spectrum speed: 0.1mL/min.Secondary mass spectrum fragmentation energies: (1) 35% (2) 38%), mass spectrum has provided certain structure information (as Fig. 2) simultaneously.The molecular weight that shows the pigment main ingredient is 344.2, and this molecular weight with the violacein of report is consistent.
3) nucleus magnetic resonance
Carry out nuclear magnetic resonance spectroscopy by the isolated pigment main component of high performance liquid chromatography according to the method for describing in the following document, with JNM ECA-600 (JEOL company) nuclear magnetic resonance spectrometer, magneticstrength is 14.096T,
1The H resonant frequency is 600.17MHz, and solvent is DMSO, at room temperature carries out data gathering.90 degree pulse widths are 11.3us, and sampling number is 16384, and scanning times is 16 times, and deuterated reagent is d-DMSO, has obtained nuclear magnetic spectrogram result as shown in Figure 3.Spectrogram 3 has provided the chemical shift of the H (formula (II)) of each position of pigment main component more clearly, by with document (T.Hoshino, T.Kondo, T.Uchiyama, N.Ogasawara, Biosynthesis of violacein:a novel rearrangement in tryptophan metabolismwith a 1,2-shift of the indole, Agric.Biol.Chem.51 1987) 965-968.) in the chemical displacement value comparison (table 4) of violacein, obtain corresponding structure information.
The comparison of table 4 Du's Gan Shi B2 bacterium pigment principal constituent nucleus magnetic hydrogen spectrum chemical displacement value and data in literature
Formula (II)
Because method is the same with reference, by the contrast of H chemical shift figure, as can be known Du to roll Bordetella (Duganella sp.) B2 CGMCC № 2056 main component compounds are violaceins, its structure is suc as formula (III):
ESI-MSm/z:344.24 (posotive) Mw:343.24
Formula (III)
The result shows that the output of rolling the violacein of Bordetella (Duganella sp.) B2 CGMCC № 2056 under above-mentioned fermentation condition of shutting out is the 1.7g/L fermented liquid.
1, the cultivation of bacterial strain
The preparation plate culture medium: peptone 5g, beef extract 3g, agar 20g adds water to 1000ml, regulates pH7.0.At 121 ℃ of 15min that sterilize down, dull and stereotyped, the access of cooling back is shut out and is rolled Bordetella (Duganella sp.) B2 CGMCC № 2056, cultivates 72 hours down at 25 ℃ with plate culture medium.
Preparation seed liquid nutrient medium (every liter of content): peptone 5g, beef extract 3g regulates pH7.0.Single Du of picking rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 colony inoculations in aseptic liquid nutrient medium from flat board, cultivates under 25 ℃ of temperature, and mixing speed is 200rpm (rotation radius is 32mm).Cultivate that thalline is in logarithmic phase after 24 hours.
The preparation fermentation broth: starch 10g, ferrous sulfate 0.01g, saltpetre 0.8g, dipotassium hydrogen phosphate 0.7g, sal epsom 1g, tryptophane 0.5g adds water to 1000ml, and pH is 7.0.The 100ml substratum packed into sterilize in the 500ml culturing bottle, the seed 10mL of logarithmic phase is inserted in the fermention medium (10% inoculum size) cultivate down at 25 ℃, mixing speed is 200rpm (rotation radius is 32mm).Cultivated 24 hours, and stopped fermentation.
2, the extraction of cyanine
With fermented liquid centrifugal 10min under 7000 * g, abandon supernatant liquor, in throw out, add ethanol (every 100ml fermented liquid adds 50ml ethanol), with eddy mixer with its mixing, vibrated 1 hour in the 200W ultrasonic cleaner then, the centrifugal 5min of liquid 9000 * g that will vibrate obtains the ethanolic soln of pigment.If still have pigment to remain in the cell residue, can repeat the aforesaid operations step, till no longer pigment can being extracted.With resulting ethanolic soln vacuum-drying, promptly obtain the crude extract of cyanine.
Method according to step 4 among the embodiment 2 is carried out cyanine purifying and structure elucidation, and the result shows that the output of rolling the violacein of Bordetella (Duganella sp.) B2 CGMCC № 2056 under above-mentioned fermentation condition of shutting out is the 0.56g/L fermented liquid.
1, the cultivation of bacterial strain
The preparation plate culture medium: peptone 5g, beef extract 3g, agar 20g adds water to 1000ml, regulates pH 7.0.At 121 ℃ of 15min that sterilize down, dull and stereotyped, the access of cooling back is shut out and is rolled Bordetella (Duganella sp.) B2 CGMCC № 2056, cultivates 72 hours down at 25 ℃ with plate culture medium.
Preparation seed liquid nutrient medium (every liter of content): peptone 5g, beef extract 3g regulates pH7.0.Single Du of picking rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 colony inoculations in aseptic liquid nutrient medium from flat board, cultivates under 25 ℃ of temperature, and mixing speed is 200rpm (rotation radius is 32mm).Cultivate that thalline is in logarithmic phase after 24 hours.
The preparation fermentation broth: starch 20g, ferrous sulfate 0.05g, saltpetre 1.2g, dipotassium hydrogen phosphate 0.7g, sal epsom 0.8g adds water to 1000ml, and pH is 8.0.The 100ml substratum packed into sterilize in the 500ml culturing bottle, the seed 10mL of logarithmic phase is inserted in the fermention medium (10% inoculum size) cultivate down at 25 ℃, mixing speed is 200rpm (rotation radius is 32mm).Cultivated 50 hours, and stopped fermentation.
2, the extraction of cyanine
With fermented liquid centrifugal 10min under 7000 * g, abandon supernatant liquor, in throw out, add ethanol (every 100ml fermented liquid adds 50ml ethanol), with eddy mixer with its mixing, vibrated 1 hour in the 200W ultrasonic cleaner then, the centrifugal 5min of liquid 9000 * g that will vibrate obtains the ethanolic soln of pigment.If still have pigment to remain in the cell residue, can repeat the aforesaid operations step, till no longer pigment can being extracted.With resulting ethanolic soln vacuum-drying, promptly obtain the crude extract of cyanine.
Method according to step 4 among the embodiment 2 is carried out cyanine purifying and structure elucidation, and the result shows that the output of rolling the violacein of Bordetella (Duganella sp.) B2 CGMCC № 2056 under above-mentioned fermentation condition of shutting out is the 0.45g/L fermented liquid.
Sequence table
<160>1
<210>1
<211>1406
<212>DNA
＜213〉Du rolls Bordetella (Duganella sp.)
<400>1
ccttaccatg?caagtcgaac?ggcagcgcgg?ggcaacctgg?cggcgagtgg?cgaacgggtg 60
agtaatatat?cggaacgtac?cctggagtgg?gggataacgt?agcgaaagtt?acgctaatac 120
cgcatacgat?ctaaggatga?aagtgggggc?ttcgcaagaa?cctcatgctc?ctggagcggc 180
cgatatctga?ttagctagtt?ggtggggtaa?aggcctacca?aggcatcgat?cagtagctgg 240
tctgagagga?cgaccagcca?cactggaact?gagacacggt?ccagactcct?acgggaggca 300
gcagtgggga?attttggaca?atgggcgaaa?gcctgatcca?gcaatgccgc?gtgagtgaag 360
aaggccttcg?ggttgtaaag?ctcttttgtc?agggaagaaa?cggtgagagc?taatatctct 420
tgctaatgac?ggtacctgaa?gaataagcac?cggctaacta?cgtgccagca?gccgcggtaa 480
tacgtagggt?gcaagcgtta?atcggaatta?ctgggcgtaa?agcgtgcgca?ggcggttttg 540
taagtctgtc?gtgaaatccc?cgggctcaac?ctgggaattg?cgatggagac?tgcaaggcta 600
gaatctggca?gaggggggta?gaattccacg?tgtagcagtg?aaatgcgtag?agatgtggag 660
gaacaccgat?ggcgaaggca?gccccctggg?tcaagattga?cgctcatgca?cgaaagcgtg 720
gggagcaaac?aggattagat?accctggtag?tccacgccct?aaacgatgtc?tactagttgt 780
cgggttttaa?ttaacttggt?aacgcagcta?acgcgtgaag?tagaccgcct?ggggagtacg 840
gtcgcaagat?taaaactcaa?aggaattgac?ggggacccgc?acaagcggtg?gatgatgtgg 900
attaattcga?tgcaacgcga?aaaaccttac?ctacccttga?catgtacgga?agccacgaga 960
gatcgaggtg?tgctcgaaag?agaaccgtaa?cacaggtgct?gcatggctgt?cgtcagctcg 1020
tgtcgtgaga?tgttgggtta?agtcccgcaa?cgagcgcaac?ccttgtcatt?agttgctacg l080
aaagagcact?ctaatgagac?tgccggtgac?aaaccggagg?aaggtgggga?tgacgtcaag 1140
tcctcatggc?ccttatgggt?agggcttcac?acgtcataca?atggtacata?cagagggccg 1200
ccaacccgcg?agggggagct?aatcccagaa?agtgtatcgt?agtccggatt?gtagtctgca 1260
actcgactac?atgaagttgg?aatcgctagt?aatcgcggat?cagcatgtcg?cggtgaatac 1320
gttcccgggt?cttgtacaca?ccgcccgtca?caccatggga?gcgggtttta?ccagaagtag 1380
gtagcttaac?cgcaaggggg?gcgcta 1406
Claims (10)
1. Du rolls Bordetella (Duganella sp.) B2 bacterial strain, and its preserving number is CGMCC № 2056.
2. method of producing violacein is that fermentation culture Du rolls Bordetella (Duganella sp.) B2 bacterial strain CGMCC № 2056 and obtains violacein.
3. method according to claim 2 is characterized in that: the fermentation condition that described Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 is 4 ℃-28 ℃ cultivations 24-60 hour.
4. method according to claim 3 is characterized in that: the fermentation condition that described Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 is 25 ℃ of cultivations 50 hours.
5. method according to claim 2, it is characterized in that: described Du rolls in every liter of the fermention medium of Bordetella (Duganella sp.) B2 CGMCC № 2056 to be contained: starch 10-20g, ferrous sulfate 0.01-0.05g, saltpetre 0.8-1.2g, dipotassium hydrogen phosphate 0.5-1g, sal epsom 0.5-1g; The pH of described fermention medium is 6-9.
6. method according to claim 5 is characterized in that: described Du rolls in every liter of the fermention medium of Bordetella (Duganella sp.) B2 CGMCC № 2056 contains the 0.3-0.5g tryptophane.
7. method according to claim 6, it is characterized in that: described Du rolls the fermention medium of Bordetella (Duganella sp.) B2 CGMCC № 2056 and prepares as follows: starch 15g, ferrous sulfate 0.03g, saltpetre 1g, dipotassium hydrogen phosphate 0.7g, sal epsom 0.5g, tryptophane 0.5g adds water to 1000ml; The pH of described fermention medium is 7.0.
8. according to arbitrary described method in the claim 2 to 7, it is characterized in that: also comprise the step of rolling purifying violacein Bordetella (Duganella sp.) B2 CGMCC № 2056 thalline from described Du in the described method.
9. method according to claim 8 is characterized in that: described purification process extracts violacein for rolling the broken thing that Bordetella (Duganella sp.) B2 CGMCC № 2056 somatic cells or described Du rolls Bordetella (Duganella sp.) B2 CGMCC № 2056 somatic cells from described Du with the organic solvent of energy dissolve purple bacillin;
Described organic solvent solution is the ethanol of 5-100%, the Virahol of 5-100% or the tetrahydrofuran (THF) of 5-100%; Described percentage composition is volumn concentration.
10. method according to claim 9 is characterized in that: described organic solvent solution is the ethanol of 5-100%; Described percentage composition is volumn concentration.
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| GB2473401B (en) * | 2008-07-11 | 2012-10-17 | Univ Tsinghua | Recombinant bacteria for producing deoxyviolacein and uses thereof |
| CN101319219B (en) * | 2008-07-11 | 2010-12-01 | 清华大学 | Method for preparing deoxidized violacein and special recombinant bacterium |
| CN102153633A (en) * | 2010-12-27 | 2011-08-17 | 江苏九阳生物制药有限公司 | Method for extracting and separating bacitracin |
| CN102703367B (en) * | 2011-03-28 | 2014-04-16 | 清华大学 | Method for preparing violacein and special bacterium thereof |
| CN104774786B (en) * | 2015-03-23 | 2017-08-25 | 四川理工学院 | One Duganella bacterium and its application |
| TWI570191B (en) * | 2015-12-10 | 2017-02-11 | The use of natural pigments as nail polish |
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| 温露等.南海海洋细菌Pseudomonas sp. 产生的一种抗肿瘤蓝色素.中山大学学报（自然科学版）44 4.2005,44(4),第63-65，69页. |
| 温露等.南海海洋细菌Pseudomonas sp. 产生的一种抗肿瘤蓝色素.中山大学学报(自然科学版)44 4.2005,44(4),第63-65,69页. * |
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