CN103396957B - A kind of marine microorganism polysaccharide deriving from carp streptomycete and preparation method thereof - Google Patents
A kind of marine microorganism polysaccharide deriving from carp streptomycete and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of marine microorganism polysaccharide deriving from carp streptomycete and preparation method thereof, the present invention adopts the carp streptomycete Streptomyces carpaticus OUCMDZ-726 fermentation of marine source to get everything ready the marine microorganism polysaccharide of novel chemical structure, it is the linear homopolymer that 3-deoxidation-D-glycerine-D-gala ketononose connects with α-2,8.The structure of marine microorganism polysaccharide of the present invention is unique, and derive from marine microorganism, can be used for precursor or the intermediate of PRODUCTION TRAITS newtype drug, can be fully used, its function and activity can be developed widely.
Description
Technical field
The invention belongs to marine microorganism field, be specifically related to a kind of marine microorganism polysaccharide deriving from carp streptomycete and preparation method thereof.
Background technology
Abundant Microbial resources are contained in ocean, due to special living environment, cause the building-up process of marine microorganism different from land microorganism, produce the active polysaccharide that many novel structure effects are special.About the research of marine microorganism exocellular polysaccharide receives publicity day by day, from general environment marine microorganism, marine organisms symbiotic and epiphyte microorganism and ocean particular surroundings microorganism, many novel polysaccharide compounds are isolated to.Research shows, this kind of polysaccharide mostly by multiple monose according to a certain percentage and the mixed polysaccharide that forms of mode of connection.Monose forms the pectinose, ribose, the wood sugar that relate in five-carbon sugar, glucose in hexose, seminose, semi-lactosi, allose, rhamnosyl and Fucose, glucosamine in aminosugar, GalN, the glucuronic acid in acid sugar, galacturonic acid.Wherein glucose, semi-lactosi and seminose are the most common.Sugarless components such as acetic acid, succsinic acid, pyruvic acid, phosphoric acid and sulfuric acid also can be substituted on polysaccharide with the form of acyl group, hemiacetal and ether.In recent years, the mannosans of novel structure is also separated with galactan sulfate and obtains from marine microorganism fermented liquid.But the report that there is not yet so far about the linear homopolymer being component units with 3-deoxidation-D-glycerine-D-gala ketononose.
Summary of the invention
The object of this invention is to provide a kind of marine microorganism polysaccharide deriving from carp streptomycete and preparation method thereof, described marine microorganism polysaccharide is the linear homopolymer that 3-deoxidation-D-glycerine-D-gala ketononose connects with α-2,8, and it is by the carp streptomycete of marine source
streptomyces carpaticusoUCMDZ-726 fermentation is standby and obtain.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
Carp streptomycete OUCMDZ-726, its Classification And Nomenclature is carp streptomycete
streptomyces carpaticus, its deposit number is: CGMCC 7461.
Derive from the marine microorganism polysaccharide of described carp streptomycete, it is the linear homopolymer that 3-deoxidation-D-glycerine-D-gala ketononose connects with α-2,8, and its structural formula is such as formula shown in I: wherein, n=25;
Formula I.
Present invention also offers the preparation method of described marine microorganism polysaccharide, it comprises the following steps:
Carp streptomycete OUCMDZ-726 described in fermentation culture, obtain the fermented product containing described marine microorganism polysaccharide, fermented liquid is isolated again from this fermented product, gained fermented liquid is concentrated, desalination, concentrate except the polysaccharide soln after desalting, be dissolved in water after drying, make the weight percent concentration of polysaccharide in solution be 0.1% ~ 10%; Then be separated by ion-exchange chromatography separation, gel chromatographic columns, gained polysaccharide soln concentrated, desalination, the solution after desalination is concentrated, dry, prepare polysaccharide described in formula I.
Further, described fermentation culture adopts Gause I synthetic medium, cultivates in 28 ~ 30 DEG C of constant incubators.
Further, described desalination adopts the method for dialysis.
Further, described simmer down to concentrating under reduced pressure.
Further, described ion-exchange chromatography is separated into chromatographic column Q Sepharose Fast Flow, and elutriant used respectively is distilled water, 0.3 mol/L sodium chloride aqueous solution and 0.6 mol/L sodium chloride aqueous solution.
Further, described gel chromatographic columns is separated into chromatographic column Sephadex G-100, and elutriant used is distilled water.
Further, described drying is lyophilize; Or make polysaccharide precipitation with 95% ethanol adding 3 ~ 6 times of volumes, gained to be deposited in 40 ~ 80 DEG C of baking ovens dry 0.01 ~ 8 hour.
Compared with prior art, advantage of the present invention and technique effect are: the present invention is first from marine microorganism carp streptomycete
streptomyces carpaticusthe linear homopolymer connected with α-2,8 for component units with 3-deoxidation-D-glycerine-D-gala ketononose is separated in OUCMDZ-726 fermented liquid.The structure of formula I polysaccharide of the present invention is unique, and derive from marine microorganism, can be used for precursor or the intermediate of PRODUCTION TRAITS newtype drug, can be fully used, its function and activity can be developed widely.
After reading the specific embodiment of the present invention by reference to the accompanying drawings, the other features and advantages of the invention will become clearly.
Accompanying drawing explanation
Figure 1A is the High Performance Gel Permeation chromatogram of described microbial polysaccharide (the linear homopolymer that 3-deoxidation-D-glycerine-D-gala ketononose connects with α-2,8).
Figure 1B is the typical curve drawn retention time (High Performance Gel Permeation Chromatography mensuration) of the molecular weight logarithm of polysaccharide standard substance and regression equation thereof.
Fig. 2 is the infrared spectra of described microbial polysaccharide.
Fig. 3 oligosaccharides that to be described microbial polysaccharide obtain through degrading is at the separation and purification figure of gel chromatographic columns Bio-Gel P 4.
Fig. 4 is the ESI-MS spectrum of described microbial polysaccharide through the oligosaccharide compositions F2 obtained that degrades.
Fig. 5 is the ESI-MS spectrum of described microbial polysaccharide through the oligosaccharide compositions F3 obtained that degrades.
Fig. 6 is the ESI-MS spectrum of described microbial polysaccharide through the oligosaccharide compositions F4 obtained that degrades.
Fig. 7 is the ESI-MS spectrum of described microbial polysaccharide through the oligosaccharide compositions F5 obtained that degrades.
Fig. 8 is the ESI-MS spectrum of described microbial polysaccharide through the oligosaccharide compositions F6 obtained that degrades.
Fig. 9 A is the ESI-CID-MS/MS spectrum of described microbial polysaccharide through the fragmention m/z 1267 of the oligosaccharide compositions F6 obtained that degrades.
Fig. 9 B is the mass spectrometry fragmentation pattern diagram of described microbial polysaccharide through the oligosaccharide compositions F6 obtained that degrades.
Figure 10 A is the ESI-CID-MS/MS spectrum of described microbial polysaccharide through the fragmention m/z 1017 of the oligosaccharide compositions F5 obtained that degrades.
Figure 10 B is the mass spectrometry fragmentation pattern diagram of described microbial polysaccharide through the oligosaccharide compositions F5 obtained that degrades.
Figure 11 A is the ESI-CID-MS/MS spectrum of described microbial polysaccharide through the fragmention m/z 767 of the oligosaccharide compositions F4 obtained that degrades.
Figure 11 B is the mass spectrometry fragmentation pattern diagram of described microbial polysaccharide through the oligosaccharide compositions F4 obtained that degrades.
Figure 12 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
13c NMR composes.
Figure 13 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
1h NMR composes.
Figure 14 is the DEPT spectrum of described microbial polysaccharide through the oligosaccharide compositions F2 obtained that degrades.
Figure 15 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
1h-
13c HMQC composes.
Figure 16 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
1h-
13c HMQC part amplifies spectrum.
Figure 17 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
1h-
1h COSY composes.
Figure 18 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
1h-
1h COSY part amplifies spectrum.
Figure 19 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
1h-
13c HMBC composes.
Figure 20 oligosaccharide compositions F2's that to be described microbial polysaccharide obtain through degrading
1h-
13c HMBC part amplifies spectrum.
Figure 21 oligosaccharide compositions F3's that to be described microbial polysaccharide obtain through degrading
1h-
1h COSY composes.
Figure 22 oligosaccharide compositions F3's that to be described microbial polysaccharide obtain through degrading
1h-
1h COSY part amplifies spectrum.
Figure 23 oligosaccharide compositions F3's that to be described microbial polysaccharide obtain through degrading
1h-
13c HMBC composes.
Figure 24 oligosaccharide compositions F3's that to be described microbial polysaccharide obtain through degrading
1h NMR composes.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail.
Embodiment 1
One, the preservation of carp streptomycete
Carp streptomycete of the present invention obtains by being separated in Zhanjiang mud sample, and it is a kind of marine microorganism, gram positive bacterium.Gause I synthetic medium is cultivated its base silk gray, gas silk gray.
The 16S rDNA gene order numbering of this bacterial strain is KC832293.1(gene numbering is GI:483124984), described bacterial strain is carp streptomycete through morphological specificity and 16S rDNA Sequence Identification
streptomyces carpaticusoUCMDZ-726.This bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on April 11st, 2013; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on April 11st, 2013; Carp streptomycete
streptomyces carpaticusdeposit number: CGMCC No. 7461.
Two, the concrete preparation method of described microbial polysaccharide is as follows:
1, fermentative production
The fermentation culture of microorganism: by the ordinary method of culturing micro-organisms, gets described carp streptomycete
streptomyces carpaticusoUCMDZ-726 is appropriate, is inoculated on Gause I agar solid slant culture base, cultivates 7 days in 28 DEG C of incubators.
Get the slant culture carp streptomycete of 7 days
streptomyces carpaticusoUCMDZ-726 is appropriate, and (substratum forms: Zulkovsky starch 20 g, KNO containing 100 milliliters of seed culture fluids to be inoculated into one
31 g, K
2hPO
43H
2o 0.5 g, MgSO
47H
2o 0.5 g, FeSO
40.01 g, NaCl 0.5 g, agar 20 g, antiseptic sea water 1 liter, pH 7.5) Erlenmeyer flask in, under 28 DEG C, 120 revs/min conditions, shaking table cultivates 48 hours, obtains seed culture medium.
Get this seed culture fluid, be inoculated in the Erlenmeyer flask of 100 in-built 100 milliliters of productive culture liquid (substratum composition is with seed culture fluid substratum) respectively by 5% inoculum size, be loaded into the production fermentation of 28 DEG C, 120 revs/min 7 days enterprising behavior phases of shaking table, obtain the fermented product containing formula I polysaccharide.The same terms bottom fermentation twice, obtains fermented product 60 liters altogether.
2, containing the preparation of the fermentation broth coarse extract of described microbial polysaccharide
By described carp streptomycete
streptomyces carpaticusfermented product (the about 60 liters) suction filtration of OUCMDZ-726, leaching liquid portion, with activated carbon decolorizing, namely slowly joins 1.5% gac in fermented liquid and is incubated 5 ~ 10 minutes in 100 DEG C of water-baths, be cooled to after room temperature until solution, with the Büchner funnel suction filtration containing diatomite filter cake to dry.Solution after decolouring is concentrated into certain volume.The ethanol of 95% of precooling 2 hours is slowly joined (volume ratio 5:1) in concentrated solution, and limit edged stirs, and has a large amount of white flock precipitate to separate out, the solution after alcohol precipitation is put into refrigerator left undisturbed overnight.Incline and supernatant liquid, sediment fraction Büchner funnel three metafiltration paper suction filtration, and in 40 DEG C of oven dry after dewatering for several times with dehydrated alcohol successively, it is 3500 daltonian dialysis tubing dialysis 2 days that the supernatant liquor be dissolved in after appropriate distilled water is placed in molecular weight cut-off, after conductivitimeter test dialysis, outer liquid stops dialysis to during steady state value, by dialyzate concentrate drying, must containing the crude product (26.5g) of formula I polysaccharide.
The separation and purification of microbial polysaccharide I described in 3
It is every milliliter of solution containing the Crude polysaccharides of 60 mg that the polysaccharide crude got containing formula I polysaccharide is made into concentration, be separated by anion-exchange column Q Sepharose Fast Flow, use distilled water, 0.3 mol/L sodium chloride aqueous solution, 0.6 mol/L sodium chloride aqueous solution wash-out, 2 column volumes successively respectively, collect the elutriant of 0.6 mol/L sodium chloride aqueous solution, by gained polysaccharide soln concentrating under reduced pressure, then carry out dialysis and remove salt, by except the polysaccharide soln concentrating under reduced pressure after desalting, after dry, adding distil water dissolves, and makes the weight percent concentration of polysaccharide in solution be 5%.
Be separated further by gel chromatographic columns Sephadex G-100 again, use distilled water wash-out, sulfuric acid-phynol method detects, collect the elutriant of polysaccharide elution peak, by gained polysaccharide soln concentrating under reduced pressure, then carry out dialysis and remove salt, by except the polysaccharide soln concentrating under reduced pressure after desalting, drying, (1.968 g) to obtain described microbial polysaccharide I sterling.
Drying in described step 2 and 3 is lyophilize or makes polysaccharide precipitation with 95% ethanol adding 3 ~ 6 times of volumes, gained to be deposited in 40 ~ 80 DEG C of baking ovens dry 0.01 ~ 8 hour.
Three, the structural analysis of described microbial polysaccharide I
Purity and the molecular weight of described microbial polysaccharide I are measured by High Performance Gel Permeation Chromatography, and as shown in Figure 1A, from Figure 1A, microbial polysaccharide I presents single symmetrical peak to result, illustrates that the purity of microbial polysaccharide I is high; Under same analysis condition, draw various polysaccharide standard substance (molecular weight: 84.4,47.1,25.5,12.5,9.6,5.7 and 2.5 kilodaltons) molecular weight logarithm to the typical curve of retention time, and obtain the regression equation (Figure 1B) of typical curve, by the retention time of microbial polysaccharide I is substituted into typical curve regression equation, the molecular-weight average calculating microbial polysaccharide I is about 6.2 kilodaltons.
The infrared spectra (IR) of described microbial polysaccharide I as shown in Figure 2.Wave number 3402 cm
– 1strong absorption peak be the stretching vibration of O-H, 2938 cm
– 1absorption peak is the stretching vibration of C-H, 1732 cm
– 1absorption peak is C=O stretching vibration in-COOH, 1615 cm
– 1absorption peak is the asymmetric stretching vibration of C=O, 1401 cm
– 1absorption peak is C=O symmetrical stretching vibration, 1213 cm
– 1absorption peak is OH angle vibration absorption peak in-COOH, 1064 cm
– 1absorption peak is the C-O stretching vibration of C-O-H ring inner ether and the angle vibration of O – H.
For studying the structure of described microbial polysaccharide I further, adopting acid hydrolyzation to degrade to polysaccharide, selecting gel column Bio-Gel P4 to carry out separation and purification to degraded product.Adopt electrospray ionization mass spectrometry (ESI-MS) to measure the molecular weight of oligose fragment to gained oligose fragment, by electron spray ionisation-collision induced dissociation second order ms (ESI-CID-MS/MS) and proton nmr spectra (
1h NMR), carbon-13 nmr spectra (
13c NMR), undistorted polarization transfer strengthen spectrum (DEPT), with nuclear chemistry displacement Correlated Spectroscopy (
1h-
1h COSY), heteronuclear Multiple-quantum chemical shift correlated spectroscopy (
1h-
13c HMQC) and heteronuclear multiple-bond chemical shift correlated spectroscopy (
1h-
13c HMBC) etc. technology combine the structure of oligosaccharide compositions studied, finally determine the structure of polysaccharide I.
The degraded of described microbial polysaccharide I: described microbial polysaccharide I is made into the solution that weight percent concentration is 1%, adding appropriate hydrochloric acid to reaction system concentration of hydrochloric acid is 0.01 mol/L, at 30 DEG C, 40 DEG C, 50 DEG C and 60 DEG C time respectively react 1 ~ 6 hour com-parison and analysis different condition to the impact of degraded, adopt High Performance Thin Layer Chromatography to be analyzed degraded product and detect polysaccharide degraded situation.Final selected 0.01 mol/L hydrochloric acid prepares the oligosaccharides of described microbial polysaccharide I for 3 hours as degradation condition 60 DEG C of degradeds.
The preparation of oligosaccharides: adopt gel column Bio-Gel P4(100 × 1.6 cm) degraded product of chromatography column to polysaccharide I be separated, and elutriant is 0.2 mol/L bicarbonate of ammonia, adopts Composition distribution on-line checkingi, collects main peak part, concentrated, lyophilize.Gained oligosaccharides carries out secondarily purified under the same conditions, and collect main peak part, after desalination, namely freeze-drying obtains oligosaccharides.Gained oligosaccharides is called after F2, F3, F4, F5 and F6 successively.Polysaccharide I degrades the separation and purification at gel Bio-Gel P4 chromatography column of gained product oligosaccharides, as shown in Figure 3.
The determination of oligosaccharide molecular amount: by the molecular weight (Fig. 4 ~ Fig. 8) of each oligosaccharide compositions of ESI-MS spectrum analysis of negative-ion mode, result shows that the molecular weight of F2, F3, F4, F5, F6 is respectively 268,518,768,1018 and 1268 dalton.
The sequential analysis of oligosaccharides: application electrospray ionization mass spectrometry also utilizes the oligosaccharides of shock dispersion inductive technology to the homogeneous fragment of gained to carry out sequential analysis.First, carry out sequential analysis to oligosaccharide compositions F6, its ESI-CID-MS/MS composes as shown in Figure 9 A.Several comparatively significant mass spectra peak as we can see from the figure: mass-to-charge ratio (m/z) 249,267,499,517,749,767,999 and 1017 represents the interannular scission fragments ion B produced from the fracture of glycosidic link respectively
1/ Z
1, C
1/ Y
1, B
2/ Z
2, C
2/ Y
2, B
3/ Z
3, C
3/ Y
3, and B
4/ Z
4, C
4/ Y
4, these fragments are derive from the reducing end of pentasaccharides-F6 component and the characteristic ion fragment of non-reducing end respectively, and prove this oligosaccharides further.In addition, have also appeared two peculiar fragment peaks near the m/z 499 and m/z 749:
m/z455 Hes
m/z705.They all have the difference of 44 with each other, therefore infer that above-mentioned two fragment peaks are that the phenomenon that decarboxylation occurs in high energy field for carboxyl that trisaccharide and disaccharides exist caused.The mass spectrometry fragmentation pattern diagram of oligosaccharide compositions F6 as shown in Figure 9 B.
ESI-CID-MS/MS analysis being carried out to oligosaccharide compositions F5, compared with F6 fragment, except having lacked larger molecular weight fragments 1267 in the ESI-CID-MS/MS spectrum of F5, as shown in Figure 10 A, in collection of illustrative plates, having occurred the identical fragment peak with F6, as
m/z249,267,499,517,749 and 767, namely represent the fragmention B of the interannular fracture produced from the fracture of glycosidic link respectively
1/ Z
1, C
1/ Y
1, B
2/ Z
2, C
2/ Y
2and B
3/ Z
3.The mass spectrometry fragmentation pattern diagram of oligosaccharide compositions F5 as shown in Figure 10 B.
Find kindred circumstances by ESI-CID-MS/MS spectrum (Figure 11 A) observing oligosaccharide compositions F4, occur the fragmention B of the interannular fracture that the identical fracture from glycosidic link produces
1/ Z
1, C
1/ Y
1, B
2/ Z
2's
m/z249,267,499 and 517 peaks.The mass spectrometry fragmentation pattern diagram of oligosaccharide compositions F4 as shown in Figure 11 B.
Thus the repeating unit of deducibility polysaccharide I to be saccharide residue molecular weight be 250 compound, i.e. 3-deoxidation-D-glycerine-D-gala ketononose.Molecular weight between this and each oligosaccharide compositions F2 ~ F6 recorded previously by first mass spectrometric all has 250 daltonian differences consistent, oligosaccharide compositions F2(268 dalton) be that this difference adds a part water (18 dalton) just, wherein 535 is dimeric molecular ion peak, so suppose F2(268 dalton) be the repeating unit of polysaccharide I, and oligosaccharide compositions F3(518 dalton) be that two such repeating units deduct a part water, in like manner reason deducibility oligosaccharide compositions F4(768 dalton) be that three such repeating units deduct two molecular waters; Oligosaccharide compositions F5(1018 dalton) be that four such repeating units deduct trihyarol; Oligosaccharide compositions F6(1268 dalton) be that five such repeating units deduct four molecular waters.For determining that continuation adopts by its structure further
1h NMR,
13c NMR, DEPT,
1h-
1h COSY,
1h-
13c HMQC and
1h-
13c HMBC spectrum is resolved the chemical structure of oligosaccharide compositions F2, finally to determine its structure.
Oligosaccharide compositions F2's
13c NMR composes (Figure 12) and has 9 carbon signals, and is C1 ~ C9 by its order number consecutively from low field to High-Field.The characteristic signal peak of carboxyl carbon is had at chemical shift 176.8 ppm place.
1h NMR composes in (Figure 13) except reactive hydrogen, has nine hydrogen signals, and have 7 even oxygen hydrogen signals at 3.40 ~ 3.80 ppm, there are two sp in high field district
3hydridization hydrogen 2.01 ppm and 1.62 ppm.
13c NMR and DEPT composes containing 1 carboxyl (176.9 ppm) in (Figure 14) prompting structure, 1 hemiacetal quaternary carbon (96.5 ppm), the sp of 5 company's oxygen
3the tertiary carbon (68.3,69.2,70.4,70.7,71.6 ppm) of hydridization, the secondary carbon (63.4 ppm) of the sp3 hydridization of 1 company's oxygen, also has the sp of 1 high field in addition
3the secondary carbon (39.2 ppm) of hydridization.According to it
1h NMR and
13the data that C NMR composes, infer in structure and should contain sugared ring.
Continue the two dimensional NMR wave spectrum of resolving oligosaccharide compositions F2 data (
1h-
13c HMQC,
1h-
13c HMBC,
1h-
1h COSY).According to
1h-
13c HMQC composes (Figure 15,16) and belongs to the hydrogen atom on each carbon atom.?
1h-
1h COSY composes in (Figure 17,18), can infer the signal between H3 to H9.Observe again
1h-
13c HMBC composes (Figure 19,20), and calm strong according to H9a, H9b(a, b is uprightly good for) and C1 and C2 between coherent signal, and C1 is that a carboxyl can only be connected in end, can infer that C2 and C9 is connected, and C1 can only be connected on C2.Because C2 connects oxygen carbon, and chemical shift and field on the low side thereof, deducibility C2 is carboxylic ketone structure, connects two " O ".Last according to signal correction between H3 and C2, so C3 should be connected every an oxygen with C2, be namely connected by 1 oxygen between C2 and C3, form a hexa-atomic sugared ring structure, final deterministic compound is the acidity nine carbon sugar compounds with carboxyl, and the structural formula of F2 is as follows:
Molecular weight 268 dalton that its molecular weight and ESI-MS determine is consistent, can accurately prove from marine microorganism further
streptomyces carpaticusthe repeated structural unit of the exocellular polysaccharide extracted in OUCMDZ-726 is 3-deoxidation-D-glycerine-D-gala ketononose.
In order to illustrate the mode of connection of polysaccharide I, resolve the two dimensional NMR wave spectrum of oligosaccharide compositions F3 further.?
1h-
1with hydrocarbon coupling, 1.54 ppm of the middle high field region of H COSY spectrum (Figure 21,22) and 2.53 ppm illustrate that these two hydrogen are two hydrogen on C3, because its chemical displacement value is the non-hydrogen connected on oxygen carbon, also deducibility can only be the hydrogen on C3; Hydrogen signals all for this compound is belonged to simultaneously.
1h-
13c HMBC composes the signal that (Figure 23) provides, and can find C2 and H8 signal correction, proves that the mode of connection between this oligosaccharide compositions F3 and disaccharides is 2, the 8 3-deoxidation-D-glycerine-D-gala ketononoses connected.Simultaneously binding oligosaccharide compositions F3
1h NMR composes (Figure 24) can prove that the structural unit of oligosaccharides is the linear chain structure that the 3-deoxidation-D-glycerine-D-gala ketononose connected by α-2,8 forms further.
The NMR (Nuclear Magnetic Resonance) spectrum of comprehensive oligosaccharide compositions F2 and F3, the sign of each quasi-molecular ions of Negative electrospray ionization of oligosaccharides and the sequential analysis of electron spray ionisation-collision induced dissociation second order ms, and according to molecular weight 6.2 kilodalton of polysaccharide, can determine that polysaccharide I is α-2, the polymkeric substance of 8 25 the 3-deoxidation-D-glycerine-D-gala ketononose unit compositions connected, the structural formula of marine microorganism polysaccharide is as follows:
Formula I
Wherein n=25.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (4)
1. derive from a preparation method for the marine microorganism polysaccharide of carp streptomycete, it is characterized in that: carp streptomycete OUCMDZ-726, its Classification And Nomenclature is carp streptomycete
streptomyces carpaticus, its deposit number is: CGMCC No. 7461; Described polysaccharide is the linear homopolymer that 3-deoxidation-D-glycerine-D-gala ketononose connects with α-2,8, and its structural formula is such as formula shown in I: wherein, n=25;
Formula I;
It comprises the following steps:
Carp streptomycete OUCMDZ-726 described in fermentation culture, obtain the fermented product containing described marine microorganism polysaccharide, fermented liquid is isolated again from this fermented product, gained fermented liquid is concentrated, desalination, concentrate except the polysaccharide soln after desalting, be dissolved in water after drying, make the weight percent concentration of polysaccharide in solution be 0.1%-10%; Then be separated by ion-exchange chromatography separation, gel chromatographic columns, gained polysaccharide soln concentrated, desalination, the solution after desalination is concentrated, dry, prepare polysaccharide described in formula I; Described ion-exchange chromatography is separated into chromatographic column Q Sepharose Fast Flow, and elutriant used respectively is distilled water, 0.3 mol/L sodium chloride aqueous solution and 0.6 mol/L sodium chloride aqueous solution; Described gel chromatographic columns is separated into chromatographic column Sephadex G-100, and elutriant used is distilled water; Described drying is lyophilize; Described drying or make polysaccharide precipitation with 95% ethanol adding 3-6 times of volume, is deposited in dry 0.01-8 hour in 40-80 DEG C of baking oven by gained.
2. the preparation method of marine microorganism polysaccharide according to claim 1, is characterized in that described fermentation culture adopts Gause I synthetic medium, cultivates in 28 ~ 30 DEG C of constant incubators.
3. the preparation method of marine microorganism polysaccharide according to claim 1, is characterized in that described desalination adopts the method for dialysis.
4. the preparation method of marine microorganism polysaccharide according to claim 1, is characterized in that described simmer down to concentrating under reduced pressure.
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| CN112094357B (en) * | 2020-09-15 | 2022-11-25 | 淄博职业学院 | Purification method of crude polysaccharide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1184108A (en) * | 1990-04-24 | 1998-06-10 | 比奥塔科学管理有限公司 | Anti-viral compounds |
| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1184108A (en) * | 1990-04-24 | 1998-06-10 | 比奥塔科学管理有限公司 | Anti-viral compounds |
| CN103114064A (en) * | 2013-03-08 | 2013-05-22 | 浙江省柑桔研究所 | Marine actinomycete with antibacterial activity to multiple plant pathogens |
Non-Patent Citations (3)
| Title |
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| Calcium Ion Binding of Three Different Types of Oligo/Polysialic Acids As Studied by Equilibrium Dialysis and Circular Dichroic Methods;Yasushi Shimoda et al.;《Biochemistry》;19941231;第33卷;第1202-1208页 * |
| Characterization of Marine Streptomyces carpaticus and Optimization of Conditions for Production of Extracellular Protease;R.Haritha et al.;《Microbiology Journal》;20111231;第1页摘要、第2页最后一段 * |
| 三株海洋微生物中胞外多糖的分离、结构和抗氧化活性研究;孙海红;《中国博士学位论文全文数据库 医药卫生科技辑》;20091015(第10期);第E079-2页 * |
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