CN101724578A - Method for producing novel exopolysaccharide from Phyllobacterium sp. nov. 921F - Google Patents
Method for producing novel exopolysaccharide from Phyllobacterium sp. nov. 921F Download PDFInfo
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- CN101724578A CN101724578A CN200810170766A CN200810170766A CN101724578A CN 101724578 A CN101724578 A CN 101724578A CN 200810170766 A CN200810170766 A CN 200810170766A CN 200810170766 A CN200810170766 A CN 200810170766A CN 101724578 A CN101724578 A CN 101724578A
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Abstract
The invention relates to bacteria which can produce exopolysaccharide and a method for producing exopolysaccharide from the bacteria. The bacteria have simple requirement for the nutrition and are particularly easy to culture in semisynthetic culture medium to produce high yield of exopolysaccharide. The bacteria can be cultured in the liquid culture medium of gravy which contains 3 percent of sucrose for 72h to produce 1.5g/100ml of exopolysaccharide. The exopolysaccharide is whiter powder, can easily dissolve in water and has the weight average molecular weight of 736kD and the 1.0 percent solution apparent viscosity of 655mps at room temperature. The study on the nature of the exopolysaccharide shows that the exopolysaccharide is formed by the repeating units of glucose, galactose and pyruvate which are at the molar ratio of 1:1:1. Preliminarily judged by the mass spectrum and the nuclear magnetism, the exopolysaccharide produced from the Phyllobacterium sp. nov. 921F is the polysaccharide which is formed by the repeating units of 4)-Belta-D-Glcp (1->3)-Alpha-D-Galp (4, 6-S-Pyr)-(1->. The exopolysaccharide is different from the polysaccharide which is produced from any industrial bacteria. The Phyllobacterium sp. nov. 921F used for producing the exopolysaccharide has the characteristics of high quality, good stability, short production period and low cost, thereby being capable of realizing the industrial application and production.
Description
Technical field
Biotechnology
Background technology
The bacterial strain of having reported at present that can produce exocellular polysaccharide has following a few class: the acidic polysaccharose that the D-glucose that Pseudomonas aeruginosa (P.aeruginosa) produces, D-semi-lactosi, D-seminose, L-rhamnosyl and D-glucuronic acid are formed, the exocellular polysaccharide that the glucose that C.ulvaensis produces, wood sugar constitute, and in conjunction with the D-seminose of bacillus (M.tuberculosis) generation, the polysaccharide that pectinose is formed.Suis S.equi and S.zooepidernicus can produce the hyaluronic acid of being made up of glucuronic acid and glucosamine.Thermophilus streptococcus (S.Thermophilus) produces the polysaccharide that is made of repeating unit D-semi-lactosi, D-glucose and N-acetyl semi-lactosi.The yellow sporangium of bird rape (X.Campestris) can produce xanthan gum, and structure is made up of D-glucose, D-seminose, D-glucuronic acid.Also there is the kind of minority can produce the homogeneity glycan, for example the outer dextran of the born of the same parents of leuconos toc (Leuconostoc sp.) and Pediococcus (Pediococcus sp.) generation; The outer bacteria cellulose of the born of the same parents that bacillus aceticus (A.xylinum) produces etc.
Phyllobacterium sp.nov.921F can produce exocellular polysaccharide, according to the inventor by known to the inspection information, literature search, at present not about leaf bacillus Phyllobacterium produce exocellular polysaccharide the research report.
Summary of the invention
The purpose of this invention is to provide a kind of bacterial strain Phyllobacterium sp.nov.921F, and utilize this bacterial strain to produce the method for bacterium exocellular polysaccharide.
Separation and purification of the present invention goes out the bacterial strain Phyllobacterium sp.nov.921F that a strain derives from the fungus sporophore surface, has the characteristic that produces exocellular polysaccharide.Depositary institution: Chinese typical culture collection center.Address: Chinese Wuhan, Wuhan University.Preservation date is on May 16th, 2005, and preserving number is M205043.Characteristics of the present invention are:
Isolate a strain first and have the Phyllobacterium sp.nov.921F bacterial strain that produces the bacterium exocellular polysaccharide.This bacterial strain Gram-negative, direct rod shape, 0.4-0.8 μ m * 0.8-2.0 μ m.Adnation flagellar movement with 1 to several utmost point hairs or long wave shape.Aerobic, be the strict aerobic respiration metabolic type of terminal electron acceptor with the molecular oxygen.Optimum temperuture is 28～34 ℃.The catalase positive.Chemoheterotrophic bacteria utilizes various sugar, as: glucose, lactose, sucrose, maltose, pectinose and organic acid (malonate, gluconate) are as carbon source.Can utilize ammonium salt, nitrate and most amino acids as nitrogenous source.Not hydrolyzed starch, pectin and Mierocrystalline cellulose.According to uncle Jie Shi handbook the 9th edition, the form of bacterial strain of the present invention is consistent with Phyllobacterium with physiological and biochemical property, be defined as Phyllobacterium, compare different (the feature contrast of this bacterial strain and other bacterial classifications of Phyllobacterium sees Table 1) but its form belongs to known bacterium with physiological and biochemical property and this.This bacterial classification is carried out 16S rRNA gene sequencing, and the sequence of the Phyllobacterium similarity of comparing is the highest among its result and the Genebank, and its similarity is 98.7%.It is simple that this bacterial classification has a nutritional requirement, easily cultivates the characteristics that exopolysaccharides is high.Be made up of the repeating unit that glucose, semi-lactosi and pyruvic acid form through basic this polysaccharide of chemical determination, the mole ratio of components is 1:1:1.Only mass spectrum, nuclear-magnetism tentatively judge the exocellular polysaccharide structure that Phyllobacterium sp.nov.921F produces may be → 4)-β-D-Glcp (1 → 3)-α-D-Galp (4,6-S-Pyr)-(1 → repeat the saccharan that segment constitutes.The polysaccharide that this polysaccharide any industrial producing strain different from the past is produced is a kind of novel bacteria exocellular polysaccharide that can realize industrial application.
Description of drawings
Fig. 1: standard sugar gas chromatographic analysis result.
Fig. 2: Phyllobacterium sp.nov.921F bacterial strain is produced exocellular polysaccharide acidolysis end product gas chromatographic analysis result.
Fig. 3: molecular weight is the second order ms of 805 components.
Fig. 4: molecular weight is the second order ms of 1200 components.
Fig. 5: molecular weight is 805 components
1H-NMR.
Fig. 6: molecular weight is 805 components
13C-NMR.
Fig. 7: molecular weight is 805 components
13C-NMR.
Embodiment
1. the separation and purification of bacterial strain:
The acquisition of Phyllobacterium sp.nov.921F bacterial strain of the present invention: gather the fungus sporophore sample from Changbai Mountain Yu Mai, join 25ml enrichment medium (sucrose 2g, NaNO are housed
31g, K
2HPO
40.5g, MgSO
40.5g, NH
4Cl0.5g, FeSO
40.01g, H
2O1000ml, in triangular flask pH7.2), 30 ℃, 160r/mim shake-flask culture 3d.After then enrichment culture liquid suitably being diluted with stroke-physiological saline solution, get 0.2ml coating primary dcreening operation isolation medium (sucrose 10g, NaNO
31g, K
2HPO
40.5g, MgSO
40.5g, NH
4Cl0.5g, FeSO
40.01g, agar 15g, H
2O1000ml, pH7.5) flat board.Be inverted for 30 ℃ and cultivate 48h, obtaining to choose has the milky bacterium colony of thickness on the flat board, obtain the bacterium that a strain can be produced exocellular polysaccharide.
2. the evaluation of bacterial strain:
It is that bacterial strain is identified by form and Physiology and biochemistry that " uncle Jie Shi bacteriology identification handbook " the 9th version carries out bacterial strain that separation is obtained, its feature is consistent with Phyllobacterium, determine that it is Phyllobacterium, this genus has 2 kinds of report at present, physiological and biochemical property in contrast belongs to belongs to known bacterium with this and compares different (the feature contrast of Phyllobacterium sp.nov.921F and other bacterial classification of Phyllobacterium sees Table 1), again this bacterial classification is carried out 16S rRNA gene sequencing, the result is as follows:
CATGCgAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCATCTCTACGGAATAACTCAGGGAAACTTGTGCTAATACCGTATACGCCCTTTTGGGGAAAGATTTATCGGAGATGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAgACTccTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGACTATTAAGTCAGAGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTTGATACTGGCAGTCTTGAGTTCGAGAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGGCGAAGGCGGCTCACTGGCTCGATACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACTATGAGAG
The sequence of the Phyllobacterium similarity of comparing is the highest among its result and the Genebank, and similarity is 98.7%.According to above result this kind is designated as Phyllobacterium sp.nov.921F.
3. produce exocellular polysaccharide with Phyllobacterium sp.nov.921F bacterial strain:
The pre-culture of bacterial classification is inoculated in 100ml fermention medium (sucrose 30g, peptone 7g, K are housed
2HPO
42g, MgSO
40.25g, FeSO
40.01g, H
2O1000ml in 250ml triangular flask pH8), behind 30 ℃ of shake-flask culture 48h, crosses film with nutrient solution and removes thalline.Then, 95% ethanol that adds 2 times of volumes is in fermented liquid, glass stick stirs to flocks occurring, after preserving 12h under 4 ℃, centrifugal removal supernatant liquor is after precipitation is redissolved with distilled water, Crude polysaccharides removes albumen through the Sevag method, be further purified the product polysaccharide with ethanol sedimentation again, lyophilize obtains the polysaccharide finished product.
4.Phyllobacterium sp.nov.921F analysis of polysaccharide:
At first utilize alditol acetate derivative vapor-phase chromatography (GC) that monose is formed and determine, promptly get the 25mg sample and add 3ml2mol/L trifluoroacetic acid (TFA), 100 ℃ of hydrolysis 6h, decompression eliminates TFA, adds 50mgNaBH again
4With 5mg internal standard substance inositol, it is carried out derivatize after, with gas chromatographic column on the sample (DB225 capillary column (30cm * 25mm)).Determine the monose composition and keep the content that peak area is determined each monose relatively according to relative retention time.Fig. 1 is the standard sugar analytical results, and Fig. 2 is this polysaccharide acidolysis end product, and through comparison as can be known, this polysaccharide is made up of glucose, semi-lactosi, and its mol ratio is 1:1.
Product with degraded carries out classification through Bio-Gel P4 and obtains 3 components again, determines that through the one-level mass spectrum molecular weight is respectively 806,1200 and 1594Da.It is carried out second order ms (Fig. 3, Fig. 4) respectively, and the result shows that this polysaccharide is made up of the repeating unit that glucose, semi-lactosi and pyruvic acid substituting group form, and a mole ratio of components is 1:1:1.Through nuclear-magnetism
1H-NMR,
13C-NMR interpretation of result (Fig. 5, Fig. 6), the repeating unit of polysaccharide is → 4)-β-D-Glcp (1 → 3)-α-D-Galp (4,6-S-Pyr)-(1 →.
Discriminating between table 1 Phyllobacterium (Phyllobacterium) is planted
Claims (3)
1. a leaf bacillus (Phyllobacterium sp.nov.921F), this bacterial classification carries out preservation by China typical culture collection center, and depositary institution is called for short CCTCC, and preserving number is M205043.
2. a leaf bacillus (Phyllobacterium sp.nov.921F) produces its 16SrDNA nucleotide sequence of sugared bacterium: SEQ ID NO.1
3. the bacterium exocellular polysaccharide of a leaf bacillus (Phyllobacterium sp.nov.921F) bacterial strain generation is characterized in that being made up of the repeating unit that glucose, semi-lactosi and pyruvic acid form, and a mole ratio of components is 1:1:1.The repeating unit of polysaccharide is → 4)-β-D-Glcp (1 → 3)-α-D-Galp (4,6-S-Pyr)-(1 →.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2460780C1 (en) * | 2011-04-11 | 2012-09-10 | Учреждение Российской академии наук Институт биологии Уфимского научного центра РАН (ИБ УНЦ РАН) | Exopolysaccharide producer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2460780C1 (en) * | 2011-04-11 | 2012-09-10 | Учреждение Российской академии наук Институт биологии Уфимского научного центра РАН (ИБ УНЦ РАН) | Exopolysaccharide producer |
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