JP2010252680A - New microorganism having polysaccharide-producing ability and polysaccharide produced thereby - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new microorganism having polysaccharide-producing ability and a polysaccharide produced by the microorganism, especially a new species belonging to the genus Rhizobium and the polysaccharide produced by the new species. <P>SOLUTION: The new microorganism having the polysaccharide-producing ability is Rhizobium JW (Accession number: NITE P-736). The polysaccharide is produced by the microorganism. As a result, a polysaccharide having a β-(1→3) bonded glucan and a β-(1→4) bonded glucan can be produced industrially. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a novel microorganism having the ability to produce polysaccharides, polysaccharides produced thereby, in particular, new strains belonging to the genus Rhizobium and polysaccharides produced thereby.

  Polysaccharides are sugars in which monosaccharide molecules are polymerized by glycosidic bonds, and are an important group of substances used in a wide range of foods, fibers, papermaking, cosmetics, dentifrices, adhesives, medical materials, etc. is there. Generally, it has hydrophilicity, but is insoluble and soluble in water, and all are produced by living organisms, and exist as, for example, cell walls, exoskeletons, energy storage substances, or secretory substances.

  Among polysaccharides, in particular, β-glucan is known to have an action of activating macrophages, NK cells, T cells, or killer T cells to enhance immunity and resistance. By enhancing the body's immunity and resistance, it is possible to increase the ability to eliminate bacteria and foreign substances that have invaded the body, and to suppress the onset of disease, to suppress allergic reactions, and to suppress tumors such as cancer it can. Furthermore, since various effects such as a decrease in blood glucose level, diuretic action, blood pressure adjustment, and a decrease in blood cholesterol and triglyceride levels can be expected, various clinical trials have been conducted.

  Accordingly, attempts have been made to industrially produce various polysaccharides including β-glucan. In other words, research and development has been conducted on methods for screening new microorganisms that produce polysaccharides and for producing these polysaccharides to produce polysaccharides, and improving culture conditions for microorganisms that produce polysaccharides to improve polysaccharide production efficiency. ing. For example, as a method for causing a novel microorganism to produce a polysaccharide, Japanese Patent Application Laid-Open No. 2004-049013 discloses a novel strain of the genus Aureobasidium that secretes and produces β-glucan outside the cell (Patent Document 1). . As a method for improving the production efficiency of polysaccharides by improving the culture conditions of microorganisms that produce polysaccharides, Japanese Patent Application Laid-Open No. 2008-043308 discloses an improved method for producing macrohomopsis gum using macrohomopsis. (Patent Document 2).

JP 2004-049013 A JP 2008-043308 A

  Thus, in order to industrially produce various polysaccharides including β-glucan, further research and development are desired.

  The present invention has been made to solve such problems, and includes a novel microorganism capable of producing a polysaccharide and a polysaccharide produced therefrom, in particular, a new strain belonging to the genus Rhizobium and a polysaccharide produced thereby. The purpose is to provide.

  The present inventors have found that a new strain belonging to the genus Rhizobium produces a polysaccharide, and completed the following invention.

(1) A novel microorganism capable of producing a polysaccharide, which is Rhizobium JW (reception number: NITE AP-736).

(2) A polysaccharide produced from Rhizobium JW (reception number: NITE AP-736), which is a novel microorganism.

(3) The polysaccharide according to (2), wherein the polysaccharide is a polysaccharide having a β- (1 → 3) -linked glucan and / or a β- (1 → 4) -linked glucan.

(4) The polysaccharide according to (2) or (3), wherein the polysaccharide is glucan.

(5) The polysaccharide according to (4), wherein the glucan is β-glucan.

  According to the novel microorganism capable of producing a polysaccharide and the polysaccharide produced thereby according to the present invention, a polysaccharide having a β- (1 → 3) -linked glucan and / or a β- (1 → 4) -linked glucan is industrially produced. Can be produced.

It is a figure which shows the alignment of the base sequence of Rhizobium JW and Rhizobium radiobacter found by the homology search. In the figure, asterisks indicate locations where these sequences match, and portions enclosed by squares indicate locations where the sequences are different. It is a figure which shows the difference in the growth number of Rhizobi m JW by the difference in the carbon source in a culture medium. It is a figure which shows the difference in the polysaccharide production amount of Rhizobium JW by the difference in the carbon source in a culture medium. It is a figure which shows the difference in the growth number of Rhizobium JW by the difference in the sucrose density | concentration in a culture medium. It is a figure which shows the difference in the growth number of Rhizobium JW by the difference in the nitrogen source in a culture medium. It is a figure which shows the difference in the polysaccharide production amount of Rhizobium JW by the difference in the nitrogen source in a culture medium. It is a figure which shows the difference in the growth number of Rhizobium JW by the difference in the pH of a culture medium. It is a figure which shows the difference in the polysaccharide production amount of Rhizobium JW by the difference in pH of a culture medium. It is a figure which shows the pH change of the culture medium in the growth process of Rhizobium JW. It is a figure which shows the result of having developed the hydrolyzate of the polysaccharide by trifluoroacetic acid by thin layer chromatography. It is a figure which shows the result of having developed the hydrolyzate of the polysaccharide by alpha-amylase by thin layer chromatography. It is a figure which shows the result of having developed the hydrolyzate of the polysaccharide by a cellulase or a dextranase by thin layer chromatography. It is a figure which shows the result of having developed the hydrolyzate of the polysaccharide by laminarinase by thin layer chromatography. It is a figure which shows the result of having developed the hydrolyzate of the polysaccharide by a cellulase and a laminarinase by the thin layer chromatography.

  Hereinafter, the novel microorganisms having the ability to produce polysaccharides according to the present invention and the polysaccharides produced thereby will be described in detail. A novel microorganism having the ability to produce a polysaccharide according to the present invention is Rhizobium JW (reception number: NITE AP-736).

  The “microorganism” used in the present invention is used interchangeably with “fungus”, “strain”, “microorganism strain” or “bacteria”. Accordingly, the “new microorganism” can also be expressed as, for example, a “new strain”. As shown in Examples described later, the inventors have discovered a novel microorganism that has the ability to produce a polysaccharide and is not included in a known species belonging to the genus Rhizobium. The new microorganism was named Rhizobium JW, deposited on April 20, 2009 at the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation, and given the receipt number NITE AP-736. It can be obtained more.

  The novel microorganism according to the present invention has been isolated from the rhizosphere soil of burdock seedlings growing naturally on the farm of the National University Corporation Hokkaido University. Moreover, in this application, the base sequence of the 16S rRNA gene of the novel microorganisms which have the polysaccharide production ability based on this invention is disclosed (sequence number 1). Therefore, most simply, a person skilled in the art will synthesize a probe or primer targeting the 16S rRNA gene of the novel microorganism according to the present invention based on this sequence information and attach it to the roots of burdock or other plants. It is also possible to detect the presence of the target fungus in the soil and isolate it. In particular, the 16S rRNA gene can be specifically amplified by PCR and quantified.

  Furthermore, for example, by separating a nucleic acid mixture obtained by amplifying a highly conserved rRNA region by denaturing gradient gel electrophoresis (DGGE method), determining the sequence of the separated nucleic acid, and measuring the concentration It is also possible to know the microflora structure showing the abundance ratio of various microorganisms in the same or similar microbial groups present in the same sample. That is, in the present invention, when referring to “detection targeting 16S rRNA gene”, in addition to detection using a probe of 16S rRNA gene, in order to know the presence of the gene such as PCR using 16S rRNA gene as a template. All detection methods are included.

  In addition to Rhizobium JW, the novel microorganisms according to the present invention include all mutants and progeny obtained by mutating naturally or by artificial means, all having Rhizobium JW as microorganisms according to the present invention. Is included.

  The culture of the novel microorganism according to the present invention is not particularly limited, and a medium or buffer that can be appropriately selected by those skilled in the art can be used. Such media include, for example, MEM (Eagle's Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), IMDM (Iscove's Modified 40A, RP , LPM, Ham's F10, Ham'sF12, DCCM1, DCCM2, BGJ, BME (Basal Medium Eagle), GMEM (Glassow's Modified Eagle's Medium), L-15 (LeibovMc, 15A) Fisher, Schneider, MW (Modified Wingradsky's nitrogen-fr e mineral medium), and the like MW agar plate medium. In addition, if necessary, known medium components, known balanced salt components such as Earl balanced salt solution, Hanks balanced salt solution, Dulbecco PBS, spinner salt solution, various cell growth factors, antibiotics, vitamins , Hormones, pH adjusters, serum, other biological components, and the like can be added. Examples of the buffer include 4- (2-hydroxyethyl) -piperazin-1-ethanesulfonic acid (HEPES) buffer, fetal bovine serum (FBS), gelatin aqueous solution, phosphate buffered saline (PBS), and the like. be able to.

  In addition, the optimal conditions for the growth of the novel microorganisms and the production of polysaccharides according to the present invention can be studied by appropriately changing the composition of the medium used in the present invention. For example, these optimal conditions can be examined by appropriately changing the carbon source and nitrogen source contained in the medium. Examples of such carbon sources include sucrose, glucose, lactose, dextran, maltose, Fructose can be exemplified, and examples of the nitrogen source include yeast extract, tryptone, urea, sodium nitrate and the like.

  In addition, the pH of the medium used in the present invention is preferably pH 3 to 11, more preferably pH 4 to 9, and further preferably pH 5 or pH 8.

  Next, the polysaccharide according to the present invention is produced from Rhizobium JW (reception number: NITE AP-736), which is a novel microorganism.

  That is, the polysaccharide according to the present invention is characterized in that it is produced by a novel microorganism, Rhizobium JW. This polysaccharide is a β- (1 → 3) -linked glucan and / or β- (1 → 4). Since it has a bound glucan and this polysaccharide itself may be a glucan, or even a β-glucan, it may be a novel polysaccharide.

  The structure analysis of the polysaccharide according to the present invention is not particularly limited, and methods, reagents, devices and the like that can be appropriately selected by those skilled in the art can be used. For example, various acids such as hydrochloric acid, sulfuric acid, fluoroacetic acid, Using various hydrolases such as α-amylase, β-amylase, glucoamylase, dextranase, amylopullulanase, β-glucanase, cellulase (endoglucanase, exoglucanase), hemicellulase, arabanase, pectinase, phytase, xylanase Hydrolysis, and the resulting hydrolyzate is subjected to various chromatographies such as thin-layer chromatography (TLC) and gas chromatograph mass spectrometry (Gas Chromatography Mass Spectrometry). The constituent monosaccharides, oligosaccharides or their binding can be analyzed using various mass spectrometry methods such as try; GC-MS). In addition, the structure of this polysaccharide can be analyzed using nuclear magnetic resonance spectroscopy (NMR) or the like.

  The measurement of the molecular weight of the polysaccharide according to the present invention is not particularly limited, and a method, a reagent, an apparatus, or the like that can be appropriately selected by those skilled in the art can be used. For example, the measurement can be performed using gel filtration chromatography. Can do. The measurement by gel filtration chromatography may be carried out according to a conventional method, and is not particularly limited. C. Moore, J.M. Polym. Sci. 1964, Vol. 2, pp. 835-843, and the like.

  The measurement of the viscosity of the polysaccharide according to the present invention is not particularly limited, and methods, reagents, devices, and the like that can be appropriately selected by those skilled in the art can be used. For example, the measurement can be performed using a B-type viscometer. it can. As a measurement using a B-type viscometer, for example, an appropriate amount of the polysaccharide aqueous solution according to the present invention is filled in a sleep adjusted to 25 ° C., left to stand for 5 minutes, and then measured for 20 seconds at an appropriate rotational speed. Thus, the viscosity can be measured.

  The use of the polysaccharide according to the present invention can be appropriately selected. For example, it can be applied to uses such as pharmaceutical compositions, food additives, and skin external preparations.

  When used as a pharmaceutical composition, known methods can be used for its formulation. In addition, the dosage form can be appropriately selected. As such an administration form, for example, when it is prepared as an oral administration preparation, tablets, granules, powders, capsules, coating agents, liquid agents, suspensions, etc. In the case of a preparation for parenteral administration, forms such as injections, drops, suppositories and the like can be mentioned. In addition, the dosage can be appropriately set depending on the formulation form of the pharmaceutical composition, the administration method, the purpose of use, and the age, weight and symptom of the administration subject applied thereto.

  When used as a food additive, the form thereof can be appropriately selected. For example, the polysaccharide according to the present invention is prepared as it is as a food, added to other foods, or capsules, tablets, etc. The arbitrary form normally used for health food can be mentioned. In addition, when ingested or administered by blending in food, appropriately mixed with excipients, extenders, binders, thickeners, emulsifiers, colorants, fragrances, food additives, seasonings, etc. Depending on the application, it can be formed into a powder, granule, tablet or the like. Furthermore, it can be ingested by preparing a food by mixing it into a food material and commercializing it as a functional food.

  When used as a skin external preparation, the dosage form can be appropriately selected. For example, a solution system, a solubilization system, an emulsification system, a powder dispersion system, a water-oil two-layer system, and a water-oil-powder three-layer system. In addition to gels, mists, sprays, mousses, roll-ons, sticks, and the like, preparations impregnated or coated on sheets such as non-woven fabrics can be used. Further, the polysaccharide according to the present invention includes, for example, fats and oils such as vegetable oils, waxes such as lanolin and beeswax, hydrocarbons, fatty acids, higher alcohols, esters, various surfactants, pigments, fragrances, vitamins, A skin external preparation can be produced by appropriately blending ingredients used as raw materials for normal skin external preparations such as plant and animal extract components, ultraviolet absorbers, antioxidants, preservatives, and bactericides. . Further, it can be used together with licorice extract components such as glycyrrhetinic acid, which is a raw material for external preparations for anti-inflammatory skin, diphenhydramine hydrochloride, azulene, dl-α-tocopherol and its derivatives, vitamin B2, vitamin B6 and the like.

  Hereinafter, the novel microorganisms having the ability to produce polysaccharides according to the present invention and the polysaccharides produced thereby will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

  Inorganic salt solution used in this example, and Modified Winogradsky's nitrogen-free mineral medium (MW medium) and Modified Winogradsky's nitrogen-free mineral medium agar plate medium (MW) It is as follows.

(Composition of inorganic salt solution; in 1 L of aqueous solution) pH 7.2
50g potassium dihydrogen phosphate
Magnesium sulfate heptahydrate 25g
Sodium chloride 25g
Iron sulfate heptahydrate 1g
1g of molybdenum tetraoxide disodium dihydrate
Manganese sulfate tetrahydrate 1g
The pH of this inorganic salt solution was adjusted to 7.2 with 1 mol / L sodium hydroxide. In addition, the inorganic salt solution can be stored at room temperature, but the inorganic salt is very difficult to dissolve, so it was mixed again at the time of use.

(Composition of MW medium; in 1005 mL of medium) pH 6.2
Inorganic salt solution 5mL
Calcium carbonate 100mg
Yeast extract 50mg
Sucrose 10g
The prepared inorganic salt solution was added to ion-exchanged water and subjected to ultrasonic treatment to dissolve the salt, and then calcium carbonate, sucrose and yeast extract were added and dissolved. Next, filtration was performed with a 0.45 μm PTFE membrane (OMNIPORE; Millipore). The filtered medium was adjusted to pH 6.2 with 2 mol / L sulfuric acid and then sterilized by autoclaving at 121 ° C. for 15 minutes.

(Composition of MW agar plate medium; in 1 L of medium) pH 7
MW medium 1L
Agar 15g
Agar was added to the MW medium before being autoclaved, and sterilized by autoclaving at 121 ° C. for 15 minutes. Thereafter, it was poured into a petri dish and cooled to prepare a MW agar plate medium.

<Example 1> Identification of a new strain The present inventors isolated and identified a new strain from the soil attached to the roots of burdock growing on a farm of National University Corporation Hokkaido University by the following method.

(1) Isolation of strains

  The soil adhering to the roots of burdock seedlings growing on the farm of the National University Corporation Hokkaido University is collected as a sample. The mixture was stirred for 10 minutes and then allowed to stand for 10 minutes. Subsequently, 100 μL of the supernatant obtained by this operation was seeded on a MW agar plate medium (pH 7), applied with a conage bar, and then cultured at 25 ° C. for 4 days. Among the appearing colonies, a strain with good polymer formation in which the production of an adhesive substance was confirmed was visually selected and picked up with a platinum ear. This was streaked on a nutrient agar medium (Oxoid) and cultured at 25 ° C. for 2 days until a single colony was obtained to isolate the strain. The isolated strain was inoculated into 5 mL of MW medium, shake-cultured under conditions of 25 ° C. and 100 rpm for 24 hours, and then the obtained strain was L-dried.

(2) Identification of Rhizobium JW The strain L-dried in this Example (1) was streaked on a nutrient agar medium (Oxoid) at Techno Suruga Lab Co., Ltd. and cultured at 30 ° C. for 24 hours. The obtained strain was subjected to 16S rRNA gene nucleotide sequence analysis and bacteriological property test to estimate the attribution taxon.

[2-1] Nucleotide sequence analysis of 16S rRNA gene First, genomic DNA of the strain was extracted using InstaGene Matrix (BIO-RAD) according to the attached instruction. Using this extracted genomic DNA as a template, PCR amplification of 16S rRNA gene was performed using PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) according to the attached instruction manual.

  Next, the base sequence of the amplified gene was determined using an ABI 3130xl genetic analyzer (Applied Biosystems) and BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the attached instructions for use. The obtained sequence data was subjected to nucleotide sequence determination using ChromaPro 1.4 (Technology Pty Ltd.). The base sequence is shown in SEQ ID NO: 1.

  Next, with respect to the base sequence of this Example (2) [2-1] shown in SEQ ID NO: 1, the bacterial reference strain database (Apollon DB-BA Ver4. 0; Technosurga Laboratories) and known genes on international base sequence databases (GenBank, DDBJ and EMBL), and homology search was performed (SF Altschul et al., Gapped BLAST and PSI-BLAST; a new generation of protein database search programs, Nucleic Acids Res., 1997, 25, 3389).

  Furthermore, for the base sequence of this Example (2) [2-1] shown in SEQ ID NO: 1, the top 15 homology search strains in the bacterial reference strain database (Apollon DB-BA Ver4.0; Technosuruga Lab.) The 16S rRNA gene was subjected to a simple molecular phylogenetic analysis using Apollon 2.0 (Techno Suruga Lab.) (N. SAITOU et al., The newborn-joining method; a new method for phytogenetics biotechnology, Molecule. Evol., 1987, No. 4, page 406).

  As a result of homology search in the bacterial reference strain database (Apollon DB-BA Ver4.0; Technosuruga Laboratories), the nucleotide sequence of SEQ ID NO: 1 is the most homologous nucleotide sequence, and 16S rRNA of Rhizobium radiobacter ATCC19358 strain It showed 99.4% homology with that of the gene. On the other hand, as a result of homology search in the international nucleotide sequence databases (GenBank, DDBJ and EMBL), the nucleotide sequence of SEQ ID NO: 1 is 100% homologous to that of the 16S rRNA gene of Agrobacterium tumefaciens as the most homologous nucleotide sequence. Showed sex. Since Agrobacterium tumefaciens has now been transferred to Rhizobium radiobacter, the results of homology search in the bacterial reference strain database (Apollon DB-BA Ver4.0; Technosuruga Laboratories) and the international nucleotide sequence database (GenBank, DDBJ) And EMBL) all suggest that this strain is closely related to Rhizobium radiobacter.

  Furthermore, as a result of simple molecular phylogenetic analysis using the 16S rRNA gene, this strain forms a cluster with the 16S rRNA gene of the Rhizobium radiobacter in the strain group formed of the 16S rRNA gene of the Rhizobium genus. It was shown to be closely related on the base sequence.

[2-2] Bacteriological property test Next, this isolated strain was subjected to a mycological property test at Techno Suruga Lab. The results are as follows.
1) Morphological properties 30 ° C. on a nutrient agar medium (Oxoid)
1. Cell morphology Sponge 2. Cell size 0.6-0.7 × 1.2-1.5 μm
3. 3. There is mobility. Gram staining negative 5. Colony form 5-1. Medium Nutrient agar medium 5-2. Culturing time 24 hours 5-3. Diameter 1.0-2.0mm
5-4. Color tone Light yellow 5-5. Shape Round 5-6. Raised state Lenticular shape 5-7. Perimeter edge Full edge 5-8. Surface shape, etc. Smooth 5-9. Transparency Opaque 5-10. Viscosity buttery 2) Physiological properties Growth temperature test 1-1.37 ° C +
1-2.45 ° C. −
2. Catalase +
3. Oxidase +
4). Acid production from glucose −
5). Gas production from glucose −
6). O / F test (oxidation) +
7). O / F test (fermentation)-
8). Reduction of nitrate +
9. Indole generation +
10. Glucose acidification −
11. Arginine dihydrolase −
12 Urease +
13. Esculin hydrolysis ＋
14 Gelatin hydrolysis-
15. β-galactosidase +
16. Growth in the presence of 2% NaCl +
17. Growth on MacConkey agar +
18. Acid production from xylose +
19. Acid production from mannitol +
20. Acid production from lactose + (weak reaction)
21. Utilization test 21-1. Glucose +
21-2. L-arabinose +
21-3. D-Mannose +
21-4. D-mannitol +
21-5. N-acetyl-D-glucosamine +
21-6. Maltose +
21-7. Glycerol +
21-8. Potassium gluconate +
21-9. n-Capric acid-
21-10. Adipic acid −
21-11. d1-malic acid +
21-12. Sodium citrate −
21-13. Phenyl acetate −

  The bacteriological properties of this strain (2) [2-2] for this strain almost matched the bacteriological properties of Rhizobium radiobacter (translated by Toshikazu Sakazaki, other than the enteric bacteria found in clinical materials) Identification of Gram-negative, aerobic and facultative anaerobes, Modern Publishing, 1993, J. M. Young et al., A revision of Rhizobium Frank 1889, with an endored description of the genf es gen sf es gen s, and the gen sf Conn 1942 and Allorhizobium undecola de Lajudie et al. 1998 as new combinations; Rhizobium radioba ter, R.rhizogenes, R. rubi, R.undicola and R.vitis, Int.J. Syst. Evol.Microbiol., 2001 year, No. 51, pp. 89). From this, it was shown that this strain is closely related to Rhizobium radiobacter on mycological characteristics.

(3) Taxonomic consideration From the above, the 16S rRNA gene base sequence of this strain showed high homology with the base sequence of the Rhizobium radiobacter 16S rRNA gene, and the bacteriological properties of this strain were Rhizobium radiobacter. Since this mycobacterial property was almost the same, this strain was confirmed to be closely related to Rhizobium radiobacter.

  However, the base sequences of the 16S rRNA gene of this strain and Rhizobium radiobacter had 9 base differences as shown in FIG. 1, and all were clearly different. From this, it can be said that although this strain is closely related to Rhizobium radiobacter, it is different as a species.

  Based on the above results, this strain is a novel microorganism, which was named Rhizobium JW and deposited on April 20, 2009 at the National Institute of Technology and Evaluation Microbiology Deposit Number as NITE AP-736. did.

Example 2 Optimal Conditions for Rhizobium JW Culture and Polysaccharide Production Rhizobium JW isolated and identified in Example 1 was cultured to produce a polysaccharide. At this time, by changing the pH of the carbon source, nitrogen source and medium to be added to the medium and attempting to culture Rhizobium JW and produce polysaccharides, the amount of cell growth and polysaccharides due to differences in carbon source, nitrogen source and pH The amount produced was compared. Moreover, the pH change of the culture medium in the growth process of a microbial cell was confirmed.

(1) Examination of optimal carbon source By changing the carbon source contained in the culture medium, the optimal carbon source for cell growth and polysaccharide production was examined.

[1-1] Confirmation of the amount of bacterial cell growth First, the soil attached to the roots of burdock seedlings growing on the farm of the National University Corporation Hokkaido University was collected as a sample, and this was put into 10 mL of sterile water together with the roots. The mixture was stirred for 1 minute with a vortex mixer (Scientific Industries) at room temperature and then allowed to stand for 10 minutes. Subsequently, 100 μL of the supernatant obtained by this operation was seeded on a MW agar plate medium (pH 7), applied with a conage bar, and then cultured at 25 ° C. for 4 days. Among the emerged colonies, the strains were isolated by visually selecting strains with good polymer formation in which the production of sticky substances was confirmed in the surroundings and picking them up with platinum ears. The isolated strain was inoculated into 5 mL of MW medium and cultured with shaking at 25 ° C. and 100 rpm for 24 hours. 3 mL of 5 mL of this culture solution was added to 100 mL of a medium prepared by changing sucrose in the MW medium to another carbon source, and cultured under shaking at 25 ° C. and 100 rpm for 10 days. As the carbon source, sucrose, glucose, lactose, dextran, maltose and fructose were used, respectively. The growth amount of the bacterial cells was confirmed by measuring the turbidity (absorbance 600 nm) of each culture medium every day. The result is shown in FIG.

  As shown in FIG. 2, it was confirmed that the amount of cell growth when sucrose was used as a carbon source was the largest as compared with the case where glucose, lactose, dextran, maltose, and fructose were each used as a carbon source. It was.

[1-2] Measurement of Dry Weight of Extracted Polysaccharide Next, the cells contained in each culture solution cultured for 10 days by the same method as in Example (1) [1-1] The polysaccharide produced and secreted into the culture solution was extracted and its dry weight was measured. The extraction method will be described in detail below.

  It sterilized by putting 100 mL of the adhesive culture solution obtained in this example (1) [1-1] at 70 ° C. for 15 minutes. Thereafter, centrifugation was performed at 4 ° C. and 15000 × g for 20 minutes, and a precipitate containing cells and 100 mL of a supernatant containing polysaccharides were collected.

  Next, about 10 mL of sterilized water is added to the precipitate containing cells and mixed, and then centrifuged at 4 ° C., 15000 × g for 20 minutes to obtain a precipitate containing cells and a supernatant containing polysaccharides. Washing was done by dividing. After performing this precipitate washing operation twice, the precipitate containing cells was removed to obtain about 20 mL of the supernatant.

  The obtained supernatants were combined to make about 120 mL, and 3 times the amount of cooled isopropanol was added thereto, mixed and allowed to stand at 4 ° C. for 24 hours to precipitate polysaccharides contained in the supernatant. Thereafter, the mixture was centrifuged at 4 ° C., 15000 × g, 20 minutes. After removing the supernatant and recovering the polysaccharide, it was dissolved in 50 mL of distilled water. By performing this operation twice, a polysaccharide dissolved in 50 mL of distilled water was obtained.

  Thereafter, an equal amount of 1 mol / L NaOH was added thereto and mixed, followed by centrifugation under conditions of 4 ° C., 10,000 × g, 20 minutes. After removing the precipitate and recovering the supernatant, 3 mol / L of acetic acid was added thereto until it became neutral to precipitate the polysaccharide, and an equal amount of methanol was added and mixed. After centrifugation at 4 ° C. and 10,000 × g for 20 minutes, the supernatant was removed to recover the polysaccharide, and distilled water was added and dissolved to a final concentration of 5%. Further, in order to purify the recovered polysaccharide, an equal amount of methanol was added to precipitate the polysaccharide, followed by centrifugation under conditions of 4 ° C. and 10,000 × g for 20 minutes. The supernatant was removed again to recover the polysaccharide, and 30 mL of distilled water was added and dissolved.

  Further, an equal amount of a solution obtained by mixing chloroform and 1-butanol in a volume ratio of 4: 1 (Sevag solution) was added and stirred for 10 minutes with a vortex mixer (Scientific Industries), then 30 Left at room temperature for 5 minutes. After removing the separated Sevag solution by centrifuging at 4 ° C. and 10,000 × g for 20 minutes (Staub, AM Removal of Proteins: Sevag Method. In: R L. Whistler (ed.), Methods in Carbohydrate Chemistry, Vol.5, pp.5-6.New York: Academic Press, Inc., 1965.) By exchanging distilled water three times a day Dialysis was performed for 2 days to purify the polysaccharide. The purified polysaccharide was lyophilized and stored at -20 ° C. FIG. 3 shows the dry weights of the polysaccharides obtained by culturing using sucrose, glucose, lactose, dextran, maltose and fructose as carbon sources, extracted as described above.

  As shown in FIG. 3, it is confirmed that the dry weight of the polysaccharide obtained by culturing sucrose as a carbon source is the largest compared with the case where glucose, lactose, dextran, maltose and fructose are used as carbon sources, respectively. It was done.

  From the results of this Example (1) [1-1] and [1-2], it was confirmed that sucrose was suitable as a carbon source for cultivation and polysaccharide production of Rhizobium JW.

(2) Examination of optimal sucrose concentration Next, the optimal sucrose concentration for growth of a microbial cell was examined by changing the density | concentration of the sucrose contained in a culture medium.

  First, 3 mL of a culture solution obtained by the same method as in Example (1) [1-1] was used, and the sucrose concentration of the MW medium was 0.2 w / v%, 0.5 w / v%, 1. Each medium was changed to 0 w / v%, 2.0 w / v%, and 3.0 w / v%, and then cultured with shaking at 25 ° C. and 100 rpm for 5 days. By measuring the turbidity (absorbance 600 nm) of each culture medium every day, the amount of bacterial cell growth was confirmed. The result is shown in FIG.

  As shown in FIG. 4, the growth amount of the cells when cultured in an MW medium containing 1.0% sucrose is 0.2%, 0.5%, 1.0%, 2.0% by weight. MW containing 3.0% and 3.0% sucrose (the most sucrose was confirmed as compared with the case of culturing in a medium. From the above results, the preferred sucrose concentration in the culture of Rhizobium JW is 1. It was confirmed to be 0%.

(3) Examination of optimal nitrogen source Next, the optimal nitrogen source for proliferation of a microbial cell and polysaccharide production | generation was examined by changing the nitrogen source contained in a culture medium.

[3-1] Confirmation of growth amount of bacterial cells First, 3 mL of the culture solution obtained by the same method as in Example (1) [1-1] is used, with the yeast extract of the MW medium replaced with another nitrogen source. The medium was added to 100 mL of the medium prepared at a weight ratio of 0.01%, and cultured with shaking at 25 ° C. and 100 rpm for 10 days. As the nitrogen source, yeast extract, tryptone, urea and sodium nitrate were used, respectively. The growth amount of the bacterial cells was confirmed by measuring the turbidity (absorbance 600 nm) of each culture medium every day. The result is shown in FIG.

  As shown in FIG. 5, it was confirmed that the amount of cell growth when urea was used as a nitrogen source was the largest as compared with the case where tryptone, yeast extract, and sodium nitrate were used as nitrogen sources.

[3-2] Measurement of dry weight of extracted polysaccharide This example (1) can be cultured using yeast extract, tryptone, urea and sodium nitrate, respectively, as a nitrogen source, extracted in the same manner as [1-2]. The dry weight of the obtained polysaccharide is shown in FIG.

  As shown in FIG. 6, it was confirmed that the dry weight of the polysaccharide obtained by culturing yeast extract as a nitrogen source was the largest as compared with the case where tryptone, urea and sodium nitrate were each used as the nitrogen source.

  From the results of this Example (1) [3-1] and [3-2], it was confirmed that urea is suitable as the nitrogen source in the culture of Rhizobium JW, and the nitrogen source in the production of Rhizobium JW polysaccharide. As a result, it was confirmed that a yeast extract is suitable.

(4) Examination of optimum pH Next, the optimum pH for cell growth and polysaccharide production was examined by changing the pH of the medium.

[4-1] Confirmation of growth amount of bacterial cells First, 3 mL of a culture solution obtained by the same method as in Example (1) [1-1] was added to 1 mol / L sodium hydroxide or 2 mol in an MW medium. / L sulfuric acid was added to 100 mL of the medium adjusted to pH 4, 5, 6, 7 and 8, respectively, and cultured with shaking at 25 ° C. and 100 rpm for 10 days. By measuring the turbidity (absorbance 600 nm) of each culture medium every day, the amount of bacterial cell growth was confirmed. The result is shown in FIG.

  As shown in FIG. 7, it was confirmed that the amount of bacterial cell growth when the pH of the medium was 5 or 8 was larger than that when the pH of the medium was 4, 6, and 7.

[4-2] Measurement of Dry Weight of Extracted Polysaccharide Extracted by the same method as in Example (1) [1-2], and can be cultured with pH of 4, 5, 6, 7 and 8, respectively. The dry weight of the polysaccharide is shown in FIG.

  As shown in FIG. 8, it was confirmed that the dry weight of the polysaccharide obtained by culturing with the medium pH set to 8 was the largest compared with the case where the culture was cultivated with the medium pH set to 4, 5, 6 and 7. It was.

  From the results of this Example (1) [4-1] and [4-2], it was confirmed that pH 8 was suitable as the culture medium for Rhizobium JW culture and polysaccharide production.

(5) Confirmation of pH change of medium during growth process of Rhizobium JW Next, the pH change of the medium during the growth process of Rhizobium JW was confirmed over time.

  First, 3 mL of the culture solution obtained by the same method as in Example (1) [1-1] was added to 100 mL of MW medium and cultured with shaking for 10 days under the conditions of 25 ° C. and 100 rpm. By measuring the pH of the culture medium every day, changes in the pH of the medium during the growth of the cells were confirmed. The result is shown in FIG.

  As shown in FIG. 9, it was confirmed that pH 6.2, which is the pH at the time of preparing the MW medium, became about pH 5.6 after 2 days of culture and decreased slightly thereafter, but was almost constant.

<Example 3> Examination of properties of polysaccharides produced by Rhizobium JW (1) Examination of solubility of polysaccharides produced by Rhizobium JW Various concentrations of polysaccharides produced by Rhizobium JW are 1 mg / 1 mL. In a different solvent, the solubility was examined. Examples of the solvent include distilled water, 0.1 mol / L sodium hydroxide solution, 0.2 mol / L sodium hydroxide solution, 0.4 mol / L sodium hydroxide solution, 0.6 mol / L sodium hydroxide solution,. 8 mol / L sodium hydroxide solution, 1.0 mol / L sodium hydroxide solution, methanol, acetone, dimethyl sulfoxide, dimethylformamide, chloroform, tetrahydrofuran, ether, toluene and ethyl acetate. The results are shown in Table 1.

  As shown in Table 1, the polysaccharide produced by Rhizobium JW dissolves very well in 1.0 mol / L sodium hydroxide solution and into 0.6 mol / L sodium hydroxide solution and 0.8 mol / L sodium hydroxide solution. It was confirmed that it melted well. Moreover, although it is inferior to 0.6 mol / L sodium hydroxide solution, 0.8 mol / L sodium hydroxide solution and 1.0 mol / L sodium hydroxide solution, it is soluble in distilled water and 0.4 mol / L sodium hydroxide solution. It was confirmed. Furthermore, it was confirmed that it was insoluble in 0.1 mol / L sodium hydroxide solution, 0.2 mol / L sodium hydroxide solution, methanol, acetone, dimethyl sulfoxide, dimethylformamide, chloroform, tetrahydrofuran, ether, toluene and ethyl acetate. It was.

(2) Examination of monosaccharide constituting the polysaccharide produced by Rhizobium JW The polysaccharide produced by Rhizobium JW was hydrolyzed with trifluoroacetic acid, and the components constituting the polysaccharide were examined.

  Hydrolysis was carried out by adding 1 mL of 2 mol / L trifluoroacetic acid to 3.5 mg of polysaccharide produced by Rhizobium JW and reacting at 100 ° C. for 30 minutes, 1 hour to 2 hours, respectively. Thereafter, the trifluoroacetic acid contained in the hydrolyzate was volatilized at 80 ° C., and the process of adding a few drops of ethanol was repeated twice to dry the hydrolyzate. The dried hydrolyzate was dissolved in 0.01 mL of MilliQ water (Millipore), and the components contained in the hydrolyzate were confirmed using thin layer chromatography. The developing solvent was 85% acetonitrile and multiple development was performed twice. As the indicator, an ethanol solution containing 0.03% of naphthol and 5% of sulfuric acid was used as a spray reagent. The result is shown in FIG.

  As shown in FIG. 10, the polysaccharide produced | generated by Rhizobium JW was hydrolyzed with trifluoroacetic acid, and all the components contained in the obtained hydrolyzate were glucose. From this result, it was confirmed that the component which comprises the polysaccharide produced | generated by Rhizobium JW is only glucose, ie, the polysaccharide produced | generated by Rhizobium JW is a glucan.

(3) Examination of the binding form of glucose constituting the polysaccharide produced by Rhizobium JW The polysaccharide produced by Rhizobium JW was hydrolyzed by various enzymes, and the binding form of glucose possessed by the polysaccharide was examined.

[3-1] Hydrolysis with α-amylase 2 mg of polysaccharide produced by Rhizobium JW was dissolved in 1 mL of distilled water, and an equal amount of 50 mmol / L sodium acetate buffer (pH 5) containing 1820 U of α-amylase was added. It was. The reaction solution was reacted at 20 ° C. for 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours and 24 hours, and then allowed to stand at 100 ° C. for 10 minutes. The reaction was stopped. The components contained in the hydrolyzate were confirmed using thin layer chromatography. The developing solvent was 85% acetonitrile and multiple development was performed twice. As the indicator, an ethanol solution containing 0.03% of naphthol and 5% of sulfuric acid was used as a spray reagent. The result is shown in FIG.

  As shown in FIG. 11, the component hydrolyzed by α-amylase was not detected, and the polysaccharide remaining without being separated at the position of the origin was detected deeply. A thin signal was detected at the position of sucrose, which is considered to be detected due to insufficient purification of sucrose derived from the medium. From this, it was shown that the polysaccharide produced | generated by Rhizobium JW does not receive the hydrolysis by (alpha) -amylase, and it was confirmed that it does not have an (alpha)-(1-> 4) coupling | bonding.

[3-2] Hydrolysis by cellulase 2 mg of polysaccharide produced by Rhizobium JW was dissolved in 1 mL of distilled water, and an equal volume of 50 mmol / L sodium acetate solution (pH 5) containing 138 U of cellulase was added. The reaction solution was reacted at 37 ° C. for 10 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours, and then allowed to stand at 100 ° C. for 10 minutes to stop the reaction. The components contained in the hydrolyzate were confirmed using thin layer chromatography. The developing solvent was 85% acetonitrile and multiple development was performed twice. As the indicator, an ethanol solution containing 0.03% of naphthol and 5% of sulfuric acid was used as a spray reagent. The result is shown in FIG.

  As shown in FIG. 12, the component hydrolyzed by cellulase was detected as a dark signal at a position indicating glucose. In addition, many thin signals were detected between the position slightly below maltose and the position where the hydrolyzate was spotted. These were oligosaccharides produced as a result of degradation of the polysaccharide produced by Rhizobium JW with cellulase. Was shown. Moreover, the signal of the hydrolyzate which remained without developing was detected deeply at the position where the hydrolyzate was spotted. From this, it was shown that the polysaccharide produced | generated by Rhizobium JW receives the hydrolysis by a cellulase and produces | generates glucose and an oligosaccharide. That is, it was confirmed that the polysaccharide produced by Rhizobium JW has a β- (1 → 4) linked glucan. Furthermore, since the polysaccharide remains at the position where the hydrolyzate is spotted, it is suggested that the polysaccharide produced by Rhizobium JW has a binding form other than β- (1 → 4) bond that is degraded by cellulase. It was done.

[3-3] Hydrolysis by dextranase 2 mg of polysaccharide produced by Rhizobium JW was dissolved in 1 mL of distilled water, and an equal volume of 50 mmol / L sodium acetate solution (pH 6) containing 258 U of dextranase was added. It was. The reaction solution was reacted at 37 ° C. for 10 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours, and then allowed to stand at 100 ° C. for 10 minutes to stop the reaction. The components contained in the hydrolyzate were confirmed using thin layer chromatography. The developing solvent was 85% acetonitrile and multiple development was performed twice. As the indicator, an ethanol solution containing 0.03% of naphthol and 5% of sulfuric acid was used as a spray reagent. The result is shown in FIG.

  As shown in FIG. 12, a component hydrolyzed by dextranase was not detected, and a signal of the hydrolyzate remaining without development was detected deeply at the position where the hydrolyzate was spotted. This indicates that the polysaccharide produced by Rhizobium JW is not subject to hydrolysis by dextranase and has no α- (1 → 6) bond.

[3-4] Hydrolysis by laminarinase 2 mg of polysaccharide produced by Rhizobium JW was dissolved in 1 mL of distilled water, and an equal volume of 50 mmol / L citrate buffer (pH 6.8) solution containing 3.38 U of laminarinase was added. added. The reaction solution was reacted at 50 ° C. for 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 18 hours and 24 hours, and then allowed to stand at 100 ° C. for 10 minutes to stop the reaction. The components contained in the hydrolyzate were confirmed using thin layer chromatography. The developing solvent was 85% acetonitrile and multiple development was performed twice. As the indicator, an ethanol solution containing 0.03% of naphthol and 5% of sulfuric acid was used as a spray reagent. The result is shown in FIG.

  As shown in FIG. 13, the component hydrolyzed by laminarinase was detected as a dark signal at a position indicating glucose. In addition, a thin signal was detected slightly below the position indicating maltose, which indicated oligosaccharides produced as a result of degradation of the polysaccharide produced by Rhizobium JW with laminarinase. Moreover, the signal of the hydrolyzate which remained without developing was detected deeply at the position where the hydrolyzate was spotted. From this, it was shown that the polysaccharide produced | generated by Rhizobium JW receives the hydrolysis by laminarinase and produces | generates glucose and an oligosaccharide. That is, it was confirmed that the polysaccharide produced by Rhizobium JW has a β- (1 → 3) linked glucan. Furthermore, since the polysaccharide remains at the position where the hydrolyzate is spotted, it is suggested that the polysaccharide produced by Rhizobium JW has a binding form other than β- (1 → 3) bond that is degraded by laminarinase. It was done.

[3-5] Hydrolysis with cellulase and laminarinase 2 mg of polysaccharide produced by Rhizobium JW was dissolved in 1 mL of distilled water, and an equal volume of 50 mmol / L sodium acetate solution (pH 5) containing 138 U of cellulase; An equal volume of 50 mmol / L citrate buffer (pH 6.8) solution containing 38 U laminarinase was added. The reaction solution was allowed to react at 37 ° C. for 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 18 hours, and 24 hours, and then left at 100 ° C. for 10 minutes to stop the reaction. The components contained in the hydrolyzate were confirmed using thin layer chromatography. The developing solvent was 85% acetonitrile and multiple development was performed twice. As the indicator, an ethanol solution containing 0.03% of naphthol and 5% of sulfuric acid was used as a spray reagent. The result is shown in FIG.

  As shown in FIG. 14, the components hydrolyzed by cellulase and laminarinase were detected as a dark signal at the position indicating glucose. Moreover, the thin signal which shows an oligosaccharide was detected between the position which spotted the hydrolyzate from the position slightly lower than the position which shows maltose. Furthermore, the signal of the hydrolyzate remaining without spreading was detected deeply at the position where the hydrolyzate was spotted. This indicates that the polysaccharide produced by Rhizobium JW undergoes hydrolysis by cellulase and laminarinase to produce glucose and oligosaccharides. That is, it was confirmed that the polysaccharide produced by Rhizobium JW has β- (1 → 3) -linked glucan and / or β- (1 → 4) -linked glucan.

  In addition, since the polysaccharide remains at the position where the hydrolyzate is spotted, the polysaccharide produced by Rhizobium JW is β- (1 → 4) bond that is degraded by cellulase and / or β that is degraded by laminarinase. -It was suggested that it has a binding form (a branched structure is expected) other than a (1 → 3) bond and / or a bond that is degraded by the joint action of cellulase and laminarinase.

  According to the present Example as described above, a polysaccharide having β- (1 → 3) -linked glucan and / or β- (1 → 4) -linked glucan can be industrially produced.

  In addition, the novel microorganisms which have the polysaccharide production | generation ability based on this invention, and the polysaccharide produced | generated by this are not limited to embodiment mentioned above, It can change suitably.

Claims (5)

  A novel microorganism capable of producing a polysaccharide, which is Rhizobium JW (reception number: NITE AP-736).   A polysaccharide produced from Rhizobium JW (reception number: NITE AP-736), a novel microorganism.   The polysaccharide according to claim 2, wherein the polysaccharide is a polysaccharide having β- (1 → 3) -linked glucan and / or β- (1 → 4) -linked glucan.   The polysaccharide according to claim 2 or 3, wherein the polysaccharide is glucan.   The polysaccharide according to claim 4, wherein the glucan is β-glucan.