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    • 专利摘要:
    The present invention provides new endo-type fucoidan hydrolase and novel microorganisms useful for the production of the sugar compound. A sugar compound refers to a compound represented by formula (1) wherein at least one alcohol hydroxyl group is sulfated and Y is hydrogen or a group of formula (2), and salts thereof. Formula 1 (2)
    公开号:KR19990008099A
    申请号:KR1019970707623
    申请日:1996-04-22
    公开日:1999-01-25
    发明作者:사카이다케시；기무라히토미；고지마가오루；이카이가츠시게；아키요시스미코；나카니시요시쿠니；가토이쿠노신
    申请人:오미야히사시；다카라슈조 주식회사；후쿠시가즈에；리서치인스티튜트포글리코테크놀로지；
    IPC主号:
    专利说明:

    Sugar compound
    It has been reported that fucoidan, a sulfated polysaccharide contained in Phaeophyta, has various biological activities including anticoagulation, lipidemia-eradication, anti-cancer, cancer metastasis-inhibiting and anti-AIDS virus infection effects . That is, the fucoidan is very useful as a medicine.
    When the fucoidan itself is used as a drug, however, the fucoidan is a sulfated polysaccharide having an extremely large molecular weight, which causes problems such as antigenicity, uniformity and anticoagulant activity. Therefore, it is often necessary to decompose the fucosanide to a certain extent.
    Therefore, it has been eager to clarify the structure of fucoidan and clarify its relation with its biological activity. However, the fucosanic acid is a polymer compound having a plurality of side chains and a sugar forming the various constituents. Moreover, its sulfate group is bonded at various positions. This feature makes it very difficult to analyze the structure of fucoidan. In order to analyze the structure of the polysaccharide, it is known to treat the polysaccharide with an enzyme capable of degrading the polysaccharide and to analyze the structure of the thus formed oligosaccharide. However, none of the other fucoidan degrading enzymes that produce known glycoconjugate products or any of the so far reported to serve as a standard for fucoidan oligosaccharides are commercially available.
    For this reason, there has been a demand for a sugar compound having an identified structure, a polysaccharide degrading enzyme useful for production of such a sugar compound, and a microorganism useful for the production of the sugar compound.
    It is an object of the present invention to provide a novel endo-fucosane useful for fucosidation studies such as the structure analysis of fucosanic acid, the identification of an enzymatic degradation product of fucosidase, and the detection of biological activity thereof, such as the production of a sugar compound, a fucoidan oligosaccharide - < / RTI > novel microorganisms useful for the production of lyases and sugar compounds.
    Briefly, a first object of the present invention relates to a sugar compound represented by the formula (1) or (2) wherein at least one alcohol hydroxyl group is sulfated, or a salt thereof.

    In this formula,
    X is hydrogen or a group of formula (3)
    Y is hydrogen or a group of formula 4 or 5 with the proviso that X and Y can not be simultaneously hydrogen,
    Z is hydrogen or a group of formula (6).
    Examples of the compound represented by the formula (1) or (2) are those represented by the formulas (7) to (15)


    The second invention of the present invention relates to an endo-fucoidan-lyase characterized by having the following physicochemical properties.
    (I) function: acting on the fucosic anhydride and thereby releasing at least the compound represented by formula (7) or (8).
    (II) Optimum pH value: pH 6-10.
    (III) Optimum temperature: 30 to 40 占 폚.
    A third invention of the present invention relates to a bacterium belonging to the genus Fucoidanobacter, which is a microorganism having menaquinone in the electron transport chain and having a GC of 60%, which is useful for the production of the sugar compound of the first invention of the present invention.
    In the formulas (1), (2), (4) to (15) and (17) to (25), ~ denotes that mannose or galactose is produced both in the α- and β-anomers.
    It has been found by the present invention that the sugar compound of the present invention can be obtained by treating the fucoidan with the cell extract or the culture supernatant of the bacterium of the second invention or the third invention of the present invention, Is completed.
    The present invention relates to a sugar compound useful in the field of carbohydrate research, a polysaccharide lyase useful in the production of such a sugar compound, and a microorganism belonging to the genus Fucoidanobacter useful in the production of the sugar compound.
    Figure 1 also shows the dissolution pattern of pyridyl- (2) -aminated (PA-a) compound (a) eluted from the L column.
    Figure 2 is an elution pattern of the pyridyl- (2) -aminated (PA-b) compound (b) eluted from the L column.
    Figure 3 also shows the elution pattern of pyridyl- (2) -amidated (PA-c) per molecule compound (c) eluting from the L column.
    Figure 4 also shows the elution pattern of pyridyl- (2) -aminated (PA-d) per compound (d) eluting from the L column.
    Figure 5 also shows the dissolution pattern of pyridyl- (2) -aminated (PA-e) compound (e) eluted from the L column.
    Figure 6 is an elution pattern of the pyridyl- (2) -aminated (PA-f) per compound (f) eluted from the L column.
    Figure 7 also shows the elution pattern of the compound (g) per pyridyl- (2) -aminated (PA-g) eluted from the L column.
    Figure 8 is an elution pattern of the compound (h) per pyridyl- (2) -aminated (PA-h) column eluted from the L column.
    Figure 9 also shows the dissolution pattern of pyridyl- (2) -aminated (PA-i) compound (i) eluted from the L column.
    10 is a mass spectrogram (negative measurement) of the sugar compound (a).
    11 is a mass spectrogram (negative measurement) of the sugar compound (b).
    12 is a mass spectrogram (negative measurement) of the sugar compound (c).
    13 is a mass spectrogram (negative measurement) of the sugar compound (d).
    14 is a mass spectrogram (negative measurement) of the sugar compound (e).
    15 is a mass spectrogram (negative measurement) of the sugar compound (f).
    16 is a mass spectrogram (negative measurement) of the sugar compound (g).
    17 is a mass spectrogram (negative measurement) of the sugar compound (h).
    18 is a mass spectrogram (negative measurement) of the sugar compound (i).
    19 is a mass-mass spectrogram (negative measurement) of the sugar compound (a).
    20 is a mass-mass spectrogram (negative measurement) of the sugar compound (b).
    21 is a mass-mass spectrogram (negative measurement) of the sugar compound (c).
    22 is a mass-mass spectrogram (negative measurement) of the sugar compound (d).
    23 is a mass-mass spectrogram (negative measurement) of the sugar compound (e).
    24 is a mass-mass spectrogram (negative measurement) of the sugar compound (f).
    25 is a mass-mass spectrogram (negative measurement) of the sugar compound (g).
    26 is a mass-mass spectrogram (negative measurement) of the sugar compound (h).
    27 is a mass-mass spectrogram (negative measurement) of the sugar compound (i).
    28 is a 1 H-NMR spectrum diagram of the sugar compound (a).
    29 is a 1 H-NMR spectrum of the sugar compound (b).
    30 is a 1 H-NMR spectrum diagram of the sugar compound (c).
    31 is a 1 H-NMR spectrum diagram of the sugar compound (d).
    32 is a 1 H-NMR spectrum of the sugar compound (e).
    33 is a 1 H-NMR spectrum of the sugar compound (f);
    34 is a 1 H-NMR spectrum diagram of the sugar compound (g).
    35 is a 1 H-NMR spectrum diagram of the compound (h).
    36 is a 1 H-NMR spectrum diagram of the sugar compound (i);
    37 is an exemplary graph showing the relationship between the relative activity (%) of the enzyme according to the second invention of the present invention and the pH value.
    38 is an exemplary graph showing the relationship between relative activity (%) and temperature (占 폚) of the enzyme according to the second invention of the present invention;
    39 is an exemplary graph showing the relationship between the residual activity (%) of the enzyme and the pH value according to the second invention of the present invention at the time of treatment.
    40 is an exemplary graph showing the relationship between the residual activity (%) and the temperature (占 폚) of the enzyme according to the second invention of the present invention at the time of treatment;
    Fig. 41 is an elution pattern of the sugar compounds (a) to (i) separated by DEAE-Sepharose FF.
    FIG. 42 is an elution pattern of the sugar compounds (h) and (i) separated by DEAE-Sepharose FF.
    Hereinafter, the present invention will be described in more detail.
    The strain used in the second invention of the present invention may be any one as long as it belongs to the genus Flabobacterium and can produce the endo-fucoidan-lyase of the present invention. As a specific example of the strain capable of producing endo-fucoidan-lyase, Flavobacterium sp. SA-0082 strain can be mentioned. The sugar compound of the first invention of the present invention can be obtained by treating the fucoidan with an endo-fucoidan-lyase of this strain origin.
    The strain first discovered by the present inventor from seawater of Aomori, Japan, has the following mycological characteristics.
    1. Flavobacterium sp. SA-0082 strain
    a. Morphological characteristics
    (1) simple phase;
    Width: 0.8 to 1.0 μm
    Length: 1.0 to 1.2 μm
    (2) Spores: None
    (3) Gram stain: -
    b. Physiological characteristic
    (1) Growth temperature range: Growth below 37 ℃, optimum growth temperature 15-28 ℃
    (2) Behavior for oxygen: aerobic
    (3) Catalase: +
    (4) Oxidase: +
    (5) urease: about +
    (6) Acid formation D-glucose: +
    Lactose: +
    Maltose: +
    D-mannitol: +
    Scooter: -
    Trehalose: -
    (7) Hydrolyzed starch: -
    Gelatin: +
    Casein: -
    Esculin: +
    (8) Reduction of nitrate: -
    (9) Indole formation: -
    ) 10) Hydrogen sulfide formation: -
    (11) Solidification of milk: -
    (12) Sodium Requirement: +
    (13) Composition of salt
    Growth in NaCl-free medium: -
    Growth in 1% NaCl medium: -
    Growth in sea water medium: +
    (14) Quinone: Menaquinone 6
    (15) GC content of intracellular DNA: 32%
    (16) OF-test: O
    (17) Colony color: yellow
    (18) Mobility: None
    (19) Slippery: None
    This strain is described in Bergeys ' s Manual of Systematic Bacteriology, Vol. 1 (1984) and Bergey ' s Manual of Determinative Bacteriology, Vol. (F. aquatile) and F. meningosepticum, which are described in US Pat. However, this strain is different from the former in that it can not form any acid via sucrose metabolism, can not decompose casein, can degrade esculin, liquefy gelatin, and is urease-positive, It differs from the latter in that it can not decompose casein and grows slowly at 37 ° C. Therefore, this strain was identified as a bacterium belonging to the genus Flavobacterium and named as Flavobacterium sp. SA-0082.
    The strain was named Flavobacterium sp. SA-0082 and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (Ibaraki, Japan, 305, Higashi 1-chome, Deposited with Accession No. FERM BP-5402 to the National Institute of Bioscience and Human-Technology as deposited as FERM P-14872 on May 29 (as of February 15, 1996) )
    The nutrients to be added to the culture medium for culture to be used in the second invention of the present invention are not limited as long as the strains used can produce the endo-fucoidan-lyase of the second invention of the present invention using such nutrients. Suitable examples of a nitrogen source include yeast extract, peptone, casamino acid, lactic acid, lactose and starch, while suitable examples of carbon sources include fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, glycerol, saccharose, maltose, Corn steep liquor, meat extract, defatted soybean, ammonium sulfate and ammonium chloride. The medium may further contain inorganic substances and metal salts such as sodium salts, phosphates, potassium salts, magnesium salts and zinc salts.
    The strain also grows well in seawater or in artificial seawater containing the aforementioned nutrients.
    In culturing the strain producing the endo-fucoid epidermal-lyase of the second invention of the present invention, the yield of the enzyme varies depending on the culture conditions. In general, the culture temperature is in the range of 15 to 30 DEG C and the pH of the culture medium is preferably in the range of 5 to 9. [ The yield of endo-fucoidan-lyase is maximized by incubating the strain for 5 to 72 hours under aerobic conditions and agitation. As a matter of course, the culture conditions are appropriately selected according to the strain, the medium composition and the like used so as to achieve the maximum yield.
    The endo-fucoidan-lyase of the second invention of the present invention is contained in both cell and cell supernatant.
    The above-mentioned Flavobacterium sp. SA-0082 is cultured in an appropriate medium, and the cells are collected and disintegrated by means commonly used for cell destruction such as ultrasonication. Thereby, cell-free extracts can be obtained.
    Subsequently, the extract is purified by a purification means conventionally used in the art, whereby a purified enzyme preparation is produced. For example, the purification can be carried out by salting out, ion exchange chromatography, hydrophobic binding column chromatography, gel filtration and the like, whereby the purified endo-fucose of the second invention of the present invention which does not contain any fucoidan- A two-terminal-lyase is obtained.
    The culture supernatant obtained by removing the cells from the above-mentioned culture medium also contains a large amount of such enzymes (extracellular enzymes), which can be purified by the same means used for the purification of intracellular enzymes.
    The endo-fucoid epoxidase of the second invention of the present invention has the following chemical and physical properties. The extracellular enzymes are the same as the intracellular enzymes in their properties except molecular weight.
    (I) inducing the release of the sugar compound represented by at least formulas (7) and (8) by acting on the fucoident.
    (II) Optimum pH value: pH 6 to 10 (FIG. 37).
    37 is an exemplary graph showing the relationship between the relative activity of the enzyme and the pH value, wherein the ordinate represents the relative activity (%) and the abscissa represents the pH value.
    (III) Optimum temperature: 30 to 40 占 폚 (FIG. 38).
    38 is an exemplary graph showing the relationship between the relative activity of the enzyme and the temperature, wherein the ordinate represents the relative activity (%) and the abscissa represents the temperature.
    (IV) pH stability: stable within the pH range of 6 to 11.5 (FIG. 39).
    39 is an example graph showing the relationship between the residual activity of the enzyme and the pH value at the time of treatment, the ordinate indicates the residual activity (%) and the abscissa indicates the pH value.
    (V) Temperature stability: stable at a temperature of about 30 캜 or less (Fig. 40).
    40 is an exemplary graph showing the relationship between the residual activity of the enzyme and the temperature at the time of treatment, wherein the ordinate indicates the residual activity (%) and the abscissa indicates the temperature.
    (VI) Molecular weight: The molecular weight of the enzyme measured by gel filtration using Sephacryl S-200 (manufactured by Pharmacia) is about 70,000 for the outer cell enzyme of the strain Flavobacterium sp. SA-0082 or the inner cell of the strain And about 460,000 for the enzyme.
    (VII) Method for measuring enzyme activity
    The activity of the endo-fucoid epoxidase of the second invention of the present invention is measured as follows.
    50 μl of a 2.5% solution of a fucoidan originating from Kjellmaniella crassifolia, 10 μl of the endo-fucoidan-lyase of the second invention of the present invention and 60 μl of 66 mM mM sodium chloride-containing 83 mM phosphate buffer (pH 7.5) Mixed and reacted at 37 캜 for 3 hours. Then, 105 μl of the reaction mixture is mixed with 2 ml of water under stirring and the absorbance (AT) is measured at 230 nm. As a control, the reaction mixture and the fucoidan solution prepared by the same method, except that the endo-fucoidan-lyase of the second invention of the present invention was replaced with the above-mentioned buffer used alone to dissolve the enzyme, Absorbance (AB1 and AB2) of these is measured using the other reaction mixture prepared by the same method except for the substitution.
    The amount of enzyme that 1 pmol of the glycoside bond between mannose and uronic acid can be cleaved exclusively in 1 minute is referred to as 1U. The cleaved bond is measured by taking the mmol molecular extinction coefficient of the unsaturated uronic acid formed in the elimination reaction as 5.5. The activity of the enzyme is determined according to the following formula:
    Activity (U / ml) =
    (AT-AB1-AB2) x 2.105 x 120 / (5.5 x 105 x 0.01 x 180);
    2.105: Volume (ml) of the sample for which the absorbance is to be measured.
    120: volume (l) of enzyme reaction mixture,
    5.5: mmol molecular extinction coefficient (/ mM) of the unsaturated uronic acid at 230 nm,
    105: volume (μl) of the reaction mixture used for dilution,
    0.01: volume of enzyme (ml), and
    180: reaction time (minutes).
    The protein is calculated by measuring the absorbance of the enzyme solution at 280 nm and taking the absorbance of the 1 mg / ml protein solution as 1.0.
    The inventors measured the action mechanism of the endo-fucoidan-lyase of the second invention of the present invention as follows:
    (1) Preparation of gel maniella crassifolia fucoidan
    The dried gel mania crasifolia is pulverized with Frees Mill Model M-2 (manufactured by Nara Kikai Seisakusho) and treated with 10 times 85% methanol at 70 캜 for 2 hours. It is then filtered and the residue is treated with an additional 10-fold portion of methanol at 70 < 0 > C for 2 hours. After filtration, 20 times the amount of water is added to the residue. The mixture is then treated at 100 < 0 > C for 3 hours and filtered to give an extract. The salt concentration of the extract is adjusted to the same level as that of the 400 mM solution of sodium chloride. Cetylpyridinium chloride is then added until the precipitate no longer forms. After centrifugation, the precipitate is thoroughly washed with ethanol to completely remove cetylpyridinium chloride. Subsequently, it is desalted and the low molecular weight material is removed by using an ultrafilter (exclusion molecular weight of ultrafiltration membrane: 100,000, manufactured by Amicon). The precipitate thus formed is removed by centrifugation. The supernatant is lyophilized to give a purified gel. Mania crasifolia fucoidan. The yield of the product is about 4% based on the weight of the dry gel mania crasifolia powder.
    (2) Decomposition and decomposition of fucoidan by endo-fucoidan-lyase Purification of product
    The purified fucoidan of the genus Maniella crassifolia is treated with the endo-fucoidan-lyase of the second invention of the present invention to obtain a large amount of the decomposition product.
    That is, 600 ml of a 5% solution of fuco-ethane in the origin of gel mania crasifolia, 750 ml of 100 mM phosphate buffer (pH 8.0), 150 ml of 4M sodium chloride and 3.43 ml of a 1750 mU / ml solution of the present invention endo-fucoidan- The mixture is reacted at 25 DEG C for 144 hours.
    Then, the reaction mixture is dialyzed using a dialysis membrane having a pore size of 3500 and a fraction having a molecular weight of 3500 or less is taken. (A), (b), (c), (d) and (e) with DEAE-Sepharose FF after desalting with Micro Acilyzer G3 (manufactured by Asahi Chemical Industry Co., Ltd.) , (f), (g), (h) and (i). Figure 41 shows its elution pattern, where the abscissa represents the fraction number, the left ordinate and the closed circles represent the sugar content of the sample as measured by the phenol-sulfuric acid method and expressed as absorbance at 480 nm, Represents the unsaturated glucuronic acid content of the sample expressed in absorbance at 235 nm, and the rightmost ordinate and dotted line represent the ammonium acetate concentration (M) in the eluate. The fraction numbers of the fractions are as follows: (a): 42 to 43, (b): 84 to 91, (c): 51 to 52, (d): 79, (e) ): 62 to 63, (g): 45, (h): 75, and (i): 77.
    With respect to fractions (h) and (i), fractions 64 to 78 described above are combined and redefined as DEAE-Sepharose FF. Figure 42 shows its elution pattern, where the abscissa represents the fraction number, the left ordinate and the closed circles represent the sugar content of the sample as measured by the phenol-sulfuric acid method and expressed as absorbance at 480 nm, Represents the unsaturated glucuronic acid content of the sample expressed in absorbance at 235 nm, and the rightmost ordinate and dotted line represent the ammonium acetate concentration (M) in the eluate. The fraction numbers of the fractions are respectively (h): 92 to 96, and (i): 99 to 103.
    (3) Structural analysis of enzyme reaction product
    Identify the uniformity of each fraction
    Each portion of the nine fractions (a), (b), (c), (d), (e), (f), (g), (h) and (i) described above was incubated with GlycoTAG and GlycoTAG reagent kit (PA-a), (PA-b), (PA-c), and (PA-c) by pyridyl- (2) (PA-d), (PA-e), (PA-f), (PA-g), (PA-h) and (PA-i) which are then analyzed by HPLC. (PA-a), (PA-b), (PA-c), (PA-d) (PA-i) are each a uniform material.
    HPLC is carried out under the following conditions.
    Equipment: Model L-6200 (manufactured by Hitachi, Ltd.).
    Column: L-column (4.6 x 250 mm) [Kagaku Yakuhin Kensa Kyokai (Foundation)]
    Solvent: 50 mM acetic acid-triethylamine (pH 5.5) was added to the substances of the formulas (7), (8) and (9) (i.e., (PA-a), (PA- ;
    (PA-d), (PA-f), (PA-g) and (PA-i)] of the formulas (10), (12), (13) Triethylamine (pH 5);
    200 mM acetic acid-triethylamine (pH 3.8) was added to the substance of the above formula (11) (i.e., (PA-e)) for 0 to 10 minutes and 200 mM acetic acid-triethylamine ) And 200 mM acetic acid-triethylamine ((pH 3.8) (former: changed to a linear ratio of 100: 0 to 20:80) containing 0.5% tetrahydrofuran and the substance of formula (14) 200 mM acetic acid-triethylamine (pH 3.8) containing 80% acetic acid-triethylamine (pH 3.8) and 0.5% tetrahydrofuran (electron: the latter is 80: 20 to 50: 50 linear ratio).
    Detection: Fluorescence measurement detector F-1150 (manufactured by Hitachi, Ltd.), absorption wavelength: 320 nm, fluorescence wavelength: 400 nm.
    Flow rate: 1 ml / min
    Column temperature: 40 占 폚.
    Analysis of Reductive and Neutral Sugar Compositions of Enzyme Reaction Products
    (PA-a), (PA-b), (PA-c), (PA-d) And (PA-i) were hydrolyzed by treatment with 4N hydrochloric acid at 100 ° C for 3 hours, and the reducing end sugars were examined by HPLC.
    Subsequently, the reducing ends of these hydrolysates were pyridyl- (2) -aminated (PA) using GlycoTAG and GlycoTAG reagent kits (each manufactured by Takara Shuzo Co., Ltd.) and the neutral sugar composition was analyzed by HPLC . HPLC is carried out under the following conditions.
    Equipment: Model L-6200 (manufactured by Hitachi, Ltd.).
    Column: PALPAK Type A (4.6 mm x 150 mm, manufactured by Takara Shuzo, Co., Ltd.).
    Eluent: 700 mM borate buffer (pH 9.0); Acetonitrile = 9: 1.
    Detection: Fluorescence measurement detector F-1150 (manufactured by Hitachi, Ltd.), absorption wavelength: 310 nm, fluorescence wavelength: 380 nm.
    Flow rate: 0.3 ml / min, and
    Column temperature: 65 ° C.
    (PA-a), (PA-b), (PA-c), (PA-d) And (PA-i) all carry mannose as a reducing end. With respect to the neutral sugar composition, (PA-a), (PA-b), (PA-c), (PA-d) contains mannose and fucose in a 2: 1 ratio.
    (PA-g) and (PA-h) each carry galactose as a reducing end. Regarding the neutral sugar composition, (PA-g) contains 1: 2 galactose and fucose, while (PA-h) contains 2: 1 galactose and fucose.
    Furthermore, the form of mannose, which is one of constituent sugars, is inspected in the following manner. The reaction system in which only D-mannose can be measured by using F-kit glucose / fructose and phosphomonous isomerase (each manufactured by Boehringer Mannheim-Yamanouchi) is manufactured according to the manufacturer's description. Separately, 100 占 퐇 portions of the present compounds (a), (b), (c), (d), (e), (f) and (i) were each hydrolyzed with 2N hydrochloric acid at 100 占 폚 for 3 hours, And then measured in this reaction system. As a result, D-mannose is detected from both the sugar compounds (a), (b), (c), (d), (e), (f) and (i). Thus, it has been found that both of the present compounds (a), (b), (c), (d), (e), (f) and (i) contain D-mannose as a constituent sugar.
    Also, the form of galactose, which is one of the constituent sugars of (g) and (h), is examined using F-kit lactose / galactose (manufactured by Boehringer Mannheim-Yamanouchi), where only D-galactose can be detected. That is, 100 μg of each of (g) and (h) is hydrolyzed with 2N hydrochloric acid at 100 ° C. for 3 hours, and neutralization is carried out in this reaction system. As a result, galactose is detected from (g) and (h). Therefore, it is proved that both (g) and (h) contain D-galactose as a constitutional sugar.
    Furthermore, the shape of the fucose, which is another constituent, is inspected in the following manner. (A), (b), (c), (d), (e), (f) and (c) described above, according to the method described in Clinical Chemistry, 36, 474-476 (g), (h) and (i) were hydrolyzed at 2O < 0 > C for 3 hours with 2N hydrochloric acid, neutralized, and then subjected to the present reaction in which only L-fucose Perform measurements within the system. As a result, L-fucose is detected from the sugar compounds (a), (b), (c), (d), (e), (f), (g), (h) and (i).
    Molecular weight analysis of enzyme reaction product
    Then, the API-III mass spectroscopy (Perkin-Elmer) was performed using the compounds (a), (b), (c), (d) Mass spectrometric analysis is carried out using a spectrophotometer. Accordingly, these materials were found to have molecular weights of 564, 724, 1128, 1062, 1448, 644, 632, 1358 and 1288, respectively. (b) and (c), the divalent anion forms a main signal. Monovalent ions and sodium-bonded monovalent ions and divalent ions are detected from (d). A divalent ion to which a sodium ion is bonded, a trivalent ion to which three sodium ions are bonded, and a tetravalent ion to which a sodium ion is bonded are detected in (e). A monovalent ion to which two sodium ions are bonded is detected from (f). Monovalent ions, sodium monovalent ions, and divalent ions are detected from (g). Mono-, di-, tri-, and tetravalent ions having four, three, two, and one sodium ion, respectively, are detected in (h). The two sulfate groups are removed and the monovalent ions to which the sodium ions are bonded and the two sulfate groups to which the two sulfate groups have been removed are detected in (i).
    (Molecular weight of 97), water molecules and monovalent ions in which hydrogen ions are removed from unsaturated hexuronic acid (molecular weight of 157), and hydrogen ions are unsaturated in water by mass / mass (MS / MS) Monovalent ions (molecular weight 175) removed from hexuronic acid, monovalent ions (molecular weight 225) in which water molecules and hydrogen ions are removed from sulfated fucose, monovalent ions in which hydrogen ions are removed from sulfated fucose (molecular weight 243 ), A monovalent ion (molecular weight: 319) in which water molecules and hydrogen ions are removed from unsaturated hexuronic acid combined with mannose, and a monovalent ion (molecular weight: 405) removed from sulfated fucose in which the hydrogen ion is combined with mannose a).
    (Molecular weight: 97), monovalent ions (molecular weight: 175) in which hydrogen ions are removed from unsaturated hexuronic acid, monovalent ions in which hydrogen ions are removed from sulfated fucose, Ion (molecular weight 243), unsaturated hexuronic acid in which two hydrogen ions are bound to sulfated mannose and divalent ion (molecular weight 321) removed from sulfated fucose, sulfated fucose in which hydrogen ion is combined with mannose, or sulfated (Molecular weight: 405) removed from fucose coupled with mannose, and monovalent ions (molecular weight 417) removed from unsaturated hexuronic acid in which hydrogen ions are bound to sulfated mannose are detected (b) similarly to the above do.
    (Molecular weight 97), monovalent ions (molecular weight 175) in which hydrogen ions are removed from unsaturated hexuronic acid, water molecules and hydrogen ions are removed from the sulfated fucose by a negative MS / MS spectrometry Sulfated fucose having a monovalent ion (molecular weight: 225), a monovalent ion (molecular weight: 243) in which a hydrogen ion is removed from sulfated fucose, and a mannose bonded to hexuronic acid in which two hydrogen ions are bound to mannose (Molecular weight: 405) removed from the sulfated fucose in which the hydrogen ion is bound to mannose, and water and hydrogen ions bonded to the mannose bound to the sulfated fucose and hexuronic acid The monovalent ions (molecular weight 721) removed from the unsaturated hexuronic acid are detected in (c).
    (Molecular weight 97), monovalent ions (molecular weight 175) in which hydrogen ions are removed from unsaturated hexuronic acid, water molecules and hydrogen ions are removed from the sulfated fucose by a negative MS / MS spectrometry (Molecular weight: 405) removed from sulfated fucose in which the hydrogen ion is bound to mannose, and 2 < RTI ID = 0.0 > (Molecular weight: 450), and the divalent ion (molecular weight: 490) in which the sulfate group and the two hydrogen ions are removed from the (d) Divalent ions.
    (Molecular weight: 97), water molecules and hydrogen ions were removed from the sulfated fucose by a negative MS / MS spectrophotometry, hydrogen ions were separated from the sulfated fucose (Molecular weight 243), two hydrogen ions are removed from the disulfated fucose, the two sulfate groups are removed from the monovalent ions (molecular weight 345), (e) in which the sodium ions are bonded, A trivalent ion (molecular weight 450) in which three sodium ions are bonded and six hydrogen ions are removed, a sulfate group and six hydrogen ions are removed from (e) and three sodium ions are bonded thereto 476), unsaturated hexuronic acid with hydrogen ion conjugated with mannose and monovalent ion (molecular weight 563) removed from sulfated fucose, and unsaturated hexuronic acid with water molecule and hydrogen ion bonded with sulfated mannose and 1 removed from the sulfated-fucose-ion (molecular weight 705) is detected in (e).
    (Molecular weight: 97), hydrogen ions were removed from the sulfated fucose (molecular weight: 243), and unsaturated hexuronic acid combined with sulfated mannose were dissolved in water And a monovalent ion (molecular weight 421) in which two hydrogen ions are removed and the sodium ion is bonded thereto is detected in (f).
    (Molecular weight: 405) removed from sulfated fucose in which hydrogen ions are bound to galactose, and sulfated fucose or galactose bonded to fucose in which hydrogen ions are bound to galactose by galactose- (Molecular weight 551) removed from the fucose bound to the sulfated fucose bound to the enzyme is detected in (g).
    From the material in which three sodium ions are bonded to (h) and five hydrogen ions are removed therefrom, monosulfate ions (molecular weight 97), water molecules and hydrogen ions are removed from the material by negative MS / MS spectroscopy Monovalent ions (molecular weight of 225) removed from sulfated fucose and monovalent ions (molecular weight of 1197) in which hydrogen ions and two sulfate groups are removed from (h) are detected.
    From the divalent ions removed from (i) by two sulfate groups and two hydrogen ions by a negative MS / MS spectroscopic analysis, it is possible to obtain a mixture of a monovalent sulfate ion (molecular weight 97) and two disulfurized fucose The hydrogen ion is removed and a monovalent ion (molecular weight: 345) in which a sodium ion is bonded is detected.
    Analysis of sugar composition of enzyme reaction product
    As the result of mass spectrometry indicates, the sugar components (a), (b), (c), (d), (e), (f) and (i) contain unsaturated hexuronic acid It is likely.
    Thus, the following experiments were conducted to demonstrate that each of these enzymatic reaction products contained hexuronic acid, which contained unsaturated bonds in its own molecule. Strong absorption at 230 to 240 nm is known to be due to unsaturated bonds in the molecule. Therefore, the absorbance of the aqueous solution of the purified oligosaccharides (a), (b), (c), (d), (e), (f), (g), (h) nm. Consequently, aqueous solutions of each of (a), (b), (c), (d), (e), (f) and (i) show strong absorption, suggesting the presence of unsaturated bonds in the molecule do. The absorbance at 230 to 240 nm increases as the enzymatic degradation of the glucoheteride progresses. This strongly suggests that the enzyme cleaves the glycosidic bond between fucoidan mannose and hexuronic acid or galactose and hexuronic acid through the elimination reaction.
    Most enzyme reaction products contain unsaturated hexuronic acid at the non-reducing end and mannose at the reducing end, suggesting that the produced fucoidan involves a molecular species made up of alternating hexachronic acids and mannos.
    Because it contains fucose as the major constituent saccharide, the fucoidan is more susceptible to degradation by acids than conventional polysaccharides. On the other hand, the bond between hexuronic acid and mannose is known to be relatively resistant to acid. The inventors of the present invention have found that, with reference to the method described in Carbohydrate Research, 125, 283-290 (1984), the inventors of the present invention have found that, in the following manner, It was attempted to identify the hexuronic acid in the sugar chain contained in the fucoidan. First, the fucodane is dissolved in 0.3M oxalic acid and treated at 100 DEG C for 3 hours. Subsequently, molecular weight fractionation is carried out and fractions having a molecular weight of 3,000 or more are combined. It is then further treated with an anion exchange resin and adsorbent material is collected. The thus obtained material is lyophilized and hydrolyzed with 4N hydrochloric acid. After adjusting the pH value to 8, the pyridyl- (2) -amidation is performed and the uronic acid is analyzed by HPLC. HPLC is carried out under the following conditions.
    Equipment: Model L-6200 (manufactured by Hitachi, Ltd.)
    Column: PALPAK Type N (4.6 mm x 250 mm, manufactured by Takara Shuzo Co., Ltd.)
    Eluent: 200 mM acetic acid-triethylamine buffer (pH 7.3): acetonitrile = 25: 75;
    Detection: Fluorescence measurement detector F-1150 (manufactured by Hitachi, Ltd.) Excitation wavelength: 320 nm, Fluorescence wavelength: 400 nm,
    Flow rate: 0.8 ml / min
    Column temperature: 40 占 폚.
    As a standard for PA hexuronic acid, glucuronic acid manufactured by Sigma Chemical Co., galacturonic acid manufactured by Wako Pure Chemical Industries, Ltd., 4-methylumbelliferyl- mannuronic acid and ferulonic acid (obtained from Wako Pure Chemical Industries, Ltd.) obtained by hydrolysis of aluconic acid and duronic acid obtained by hydrolyzing -L-iduronide, and Acta Chemica Scandinavicaj, 15 , 1397-1398 (1961), followed by separation into an anion exchange resin.
    As a result, it has been found that only glucuronic acid is contained as the aforementioned intramolecular hexuronic acid of fucoidan.
    Moreover, the glucuronic acid in the hydrolyzate of the molecular species is separated from D-mannose using an anion exchange resin and lyophilized. Then, its specific rotation is measured. As a result, it becomes apparent that the glucuronic acid is the preferential type, that is, D-glucuronic acid.
    In addition, the fucoidan of the genus Maniella crassifolia is hydrolyzed with the endo-fucoidan hydrolase of the second invention of the present invention followed by oxalic acid analogously as described above. However, no polymer was found in which D-glucuronic acid and D-mannose were intertwined alternately. Based on these results, it becomes apparent that the enzyme of the present invention cleaves a molecular species having a skeletal structure formed by mutual alternation of D-glucuronic acid and D-mannose through elimination reaction.
    Further, the polymers obtained by hydrolysis with oxalic acid are subjected to NMR spectroscopy to examine the binding sites of D-glucuronic acid and D-mannos and the anomer arrangement of glycosidic linkages.
    The obtained NMR spectrum of the polymer is as follows. The chemical shifts in the 1 H-NMR spectrum are represented by 1.13 ppm by taking a chemical shift of the methyl groups in triethylamine, while those in the 13 C-NMR spectrum take chemical shifts of the methyl group in triethylamine Expressed as 9.32 ppm.
    1 H-NMR (D 2 O )
    (1H, br-s, 2-H), 3.71 (1H, d, J = M, 3-H), 3.63 (1H, m, 5-CH), 3.63 M, 5H), 3.55 (1H, m, 4-H), 3.57 , 2'-H)
    13 C-NMR (D 2 O )
    77.5 (4'-C), 77.0 (3'-C), 78.5 (2-C) 76.7 (5'-C), 73.9 (5-C), 73.7 (2'-C), 70.6 (3-C), 67.4 (4-C), (C of 5-CH 2 OH) 61.0
    The peaks can be respectively assigned to positions indicated by numerical values in the chemical formula (16).
    With respect to the arrangement at position 1 of D-glucuronic acid, this is identified as -D-glucuronic acid due to the close-coupling constant of 7.6 Hz.
    With respect to the arrangement at position 1 of D-mannose, this is identified as a-D-mannose with a chemical shift of 5.25 ppm.
    The binding scheme per constituent is analyzed by the HMBC method, i.e., 1 H-detected multiple-coupled binuclear multiple quantum interference spectra.
    The DQF-COZY and HOHAHA methods are used for assignment in the 1 H-NMR spectrum, while the HSQC method is used for assignment in the 13 C-NMR spectrum.
    In the HMBC spectrum, the cross-peaks are shown between 1-H and 4'-C together with 4'-H and 1-C and between 1'-H and 2'-C with 2-H and 1'- Lt; / RTI > This fact indicates that D-glucosonic acid is bound to the 2-position of D-mannos via the Bond while D-mannose is bound to the 4-position of D-glucuronic acid through the
    Analysis of binding site and sugar binding method of sulfate group in enzyme reaction product
    In order to examine the binding manner of the constituent sugar and the sulfate group, the enzyme reaction product was analyzed by NMR spectroscopy using a nuclear magnetic resonance spectrometer model JNM-a500 (500 Mz; manufactured by JEOL Ltd.). The analytical data thus obtained show that the compounds (a), (b), (c), (d), (e), (f), (g) , (8), (9), (10), (11), (12), (13), (14) and (15). In other words, the following facts are clear. The present compound (a) has a structure in which an unsaturated d-glucuronic acid to which a sulfate group is bound and a 1-fucose to D-mannose which is a reducing terminal residue are bonded. The present compound (b) has a structure in which unsaturated D-glucuronic acid having two sulfate groups bonded thereto and D-mannose being a reducing terminal residue having L-fucose bonded to a sulfate group are bonded. The present compound (c) is prepared by reacting D-glucuronic acid and D-fucose with a sulfate group and D-mannose with a reducing terminal residue, wherein D-mannose is bound to D-glucuronic acid and unsaturated D- Fucose to which D-mannose is further bound, and sulfate group-bonded L-fucose is further bound to D-mannose. The present compound (d) is prepared by combining a sulfate group, D-glucuronic acid, and L-fucose conjugated with two sulfate groups to D-mannose, which is a reducing terminal residue, and D- mannosine is added to D-glucuronic acid And an unsaturated D-glucuronic acid is also bound to D-mannose. The present compound (e) is prepared by combining a sulfate group, D-glucuronic acid and L-fucose conjugated with two sulfate groups to D-mannose, which is a reducing terminal residue, and D- Fucose having two sulfate groups bonded thereto, and an unsaturated D-glucuronic acid is bonded to D-mannose. The present compound (f) has a structure in which unsaturated D-glucuronic acid and L-fucose to which a sulfate group is bonded are bonded to D-mannose which is a reducing terminal residue bonded with a sulfate group. The compound (g) has a structure in which L-fucose to which a sulfate group is bonded is bonded to D-galactose which is a reducing terminal residue, and L-fucose with a sulfate group is further bonded to L-fucose. In one of the sugar chains of the sugar chain (h), L-fucose having a sulfate group bonded to D-galactose and L-fucose having a sulfate group are further bonded to L-fucose, Galactose, which is a reducing end group bonded with a sulfate group, in which D-galactose to which a sulfate group is bonded is bonded to D-galactose to which a sulfate group is bonded is composed of two side chains. The present compound (i) binds to D-mannose in which L-fucose linked with a sulfate group, D-glucuronic acid and sulfate group is a reducing terminal residue, D-mannos is further bound to D- glucuronic acid, Fucose and unsaturated D-glucuronic acid combined with two sulfate groups are further bonded to D-mannose.
    The compound included in the first invention of the present invention is obtained by treating the fucoidan with the endo-fucoidan-lyase of the second invention of the present invention.
    Subsequently, the compound represented by the formula (7), (8), (9), (10), (11), (12), (13), (14) (B), (c), (d), (e), (f), (g), (h) and (i) are illustrated.
    1, 2, 3, 4, 5, 6, 7, 8 and 9 are pyridyl- (2) -amidated sugar compounds (PA-a) -d), (PA-e), (PA-f), (PA-g), (PA-h) and In each figure, the ordinate indicates relative fluorescence intensity while the abscissa indicates retention time (min). (A), (b), (c), (d), (e), (f), (g), 19, 20, 21, 22, 23, 24, 25, 26 and 27 show the mass spectra of the sugar compounds (a), (b), (c), (d) ), (e), (f), (g), (h) and (i). In each figure, the ordinate represents relative strength (%), while the abscissa represents m / z.
    (A), (b), (c), (d), (e), (f), (g ), (h), and (i) represents each of 1 H-NMR spectrum. In each figure, the ordinate represents the signal intensity and the abscissa represents the chemical shift (ppm).
    The chemical shift of the 1 H-NMR spectrum is expressed by taking the chemical shift of the HOD as 4.65 ppm.
    Physical properties of compound (a):
    Molecular weight: 564
    MS m / z: 563 [MH < + & gt ; ] - .
    MS / MS m / z: 97 (HSO 4] -, 157 [ unsaturated D- glucuronic -H 2 OH +] -, 175 [ unsaturated D- glucuronic -H +] -, 225 [sulfated L- Fucose-H 2 OH + ] - , 243 [sulfated L-fucose-H + ] - , 319 [unsaturated D-glucuronic acid bound to D-mannose-H 2 OH + ] - , 405 [M- D- glucuronic acid -H +] -, 483 [M -SO 3 -H +] -.
    1 H-NMR (D 2 O )
    (1H, d, J = 4.0 Hz, 1'-H), 5.25 (1H, d, J = , 5.03 (1H, d, J = 6.1 Hz, 1-H), 4.47 (1H, dd, J = 3.4, 10.4 Hz, 3'- (1H, m, 5'-H), 4.10 (1H, dd, J = 3.7,5.8Hz, 3H), 4.03 m, 3-H), 3.83 (1H, dd, J = 4.0, 10.4 Hz, 2'-H), 3.72 2H, m, 5-CH 2 of H 2), 3.65 (1H, dd, J = 5.8, 6.1Hz, 2H), 1.08 (3H, d, J = 6.7, the 5'-CH 3 H 3)
    Sugar composition:
    L-fucose: Unsaturated D-glucuronic acid: D-mannose = 1: 1: 1 (one molecule each).
    Sulfate group:
    1 molecule (at position 3 of L-fucose).
    In the < 1 > H-NMR spectrum, peaks can be designated at positions indicated by the numerical values of the following chemical formulas:
    Physical properties of compound (b):
    Molecular weight: 724
    MS m / z: 723 [MH < + & gt ; ] - , 361 [M-2H + ] 2- .
    MS / MS m / z: 97 [HSO 4 ] - , 175 [Unsaturated D-glucuronic acid-H + ] - , 321 [M-SO 3 -2H + ] 2- , 405 [M-unsaturated D- acid -2SO 3 -H +] -, 417 (ML- fucose -2SO 3 -H +] -.
    1 H-NMR (D 2 O )
    (1H, d, J = 1.8 Hz, 1-H), 5.25 (1H, d, J = 7.3 Hz, 4.51 (1H, d, J = 3.1 Hz, 4'-H), 4.32 (1H, q, J = 6.7 Hz, 5'- , 4.27 (1H, dd, J = 3.7, 10.4 Hz, 2'-H), 4.21 (1H, dd, J = 3.4, 6.7 Hz, , H of 5-CH), 4.15 (1H, br-s, 2-H), 4.10 (1H, dd, J = 5.8, 11.0 Hz, H of 5-CH) M, 3-H), 3.90 (1H, m, 5-H), 3.82 , J = 7.3Hz, 2-H ), 1.11 (3H, d, J = 6.7Hz, of the 5'-CH 3 H 3)
    Sugar composition:
    L-fucose: Unsaturated D-glucuronic acid: D-mannose = 1: 1: 1 (one molecule each).
    Sulfate group:
    3 molecule (at the 2- and 4-positions of L-fucose and 6-position of D-mannose).
    In the < 1 > H-NMR spectrum, peaks can be respectively assigned to the positions represented by the numerical values of the following chemical formula (18).
    Physical properties of compound (c):
    Molecular Weight: 1128.
    MS m / z: 1127 [MH < + & gt ; ] - .
    MS / MS m / z: 97 [HSO 4 ] - , 175 [Unsaturated D-hexuronic acid-H + ] - , 225 [sulfated L-fucose-H 2 OH + ] - , 243 [sulfated L- course -H +] -, 371 [M- unsaturated D- glucuronic acid -L- fucose -SO 3 -2H +] 2-, 405 ( L- sulfated fucose bonded to mannose, D- -H +] - , 721 [MD-mannose-L-fucose-SO 3 -H 2 OH + ] - .
    1 H-NMR (D 2 O )
    (1H, d, J = 3.7 Hz, (4) -H), 5.34 (1H, s, J = 4.0 Hz, (1) -H), 5.05 (1H, d, J = 3.7 Hz, 1'-H), 4.93 (1H, d, J = 6.4 Hz, D, J = 3.4, 10.7 Hz, 3'-H), 4.47 (1H, dd, J = 3.4, 10.4 Hz, (1H, m, 5H), 4.13 (1H, m, 3H), 4.33 (1H, m, 4'H), 4.12 (1H, m, (5) '- H), 4.04 M, (3) -H), 3.73 (lH, m, 3-H), 3.85 , 4-H), 3.73 ( 1H, m, 5-H), 3.73 (1H, m, (4) -H), 3.70 (2H, m, 5-CH 2 of H 2), 3.70 (2H, m , 5 of the -CH 2 H 2), 3.67 ( 1H, m, 5-H), 3.62 (1H, m, 4-H), 3.62 (1H, m, (2) -H), 3.62 (1H , m, (5) -H), 3.51 (1H, t, J = 8.9 Hz, 3-H), 3.28 (1H, t, J = 7.9 Hz, 6.7Hz, (5) '- CH 3 a H 3), 1.07 (1H, d, J = 6.7Hz, of the 5'-CH 3 H 3)
    Sugar composition:
    L-fucose: D-glucuronic acid: D-glucuronic acid: D-mannose = 2: 1: 1: 2 (L-fucose and D-mannos: two molecules each, unsaturated D- D-glucuronic acid: one molecule each).
    Sulfate group:
    2 molecule (at the 3-position of L-fucose).
    In the < 1 > H-NMR spectrum, peaks can be respectively assigned to positions indicated by numerical values of the following chemical formula 19.
    Physical properties of compound (d):
    Molecular weight: 1062.
    MS m / z: 1061 [MH < + & gt ; ] - .
    MS / MS m / z: 97 < RTI ID = 0.0 > [HSO4]-, 175 [unsaturated hexuronic acid-H+]-, 225 [sulfated L-fucose-H2O-H+]-, 243 [sulfated L-fucose-H+]-, 405 [D-mannose-H+Gt; L-fucose < / RTI > coupled to <-Or [sulfated D-mannose-H+≪ RTI ID = 0.0 > L-fucose &-, 450 [M-2SO3-2H+]2-, 490 [M-SO3-2H+]2-.
    1 H-NMR (D 2 O )
    (1H, d, J = 3.7 Hz, (4) -H), 5.32 (1H, br-s, ), 5.17 (1H, br-s, 1-H), 4.93 (1H, d, J = 6.4 Hz, J = 3.4 Hz, 4'-H), 4.33 (1H, q, J = 6.7 Hz, 5'-H), 4.28 (1H, dd, J = 3.5, 11.0 Hz, , m, 5-CH 2 of H), 4.13 (1H, m , (2) -H), 4.12 (1H, m, (3) -H), 4.08 (1H, m, H of 5-CH 2) , 4.07 (1H, m, 2H), 3.98 (1H, dd, J = 3.4,11.0,3'-H), 3.88 ), 3.78 for (1H, m, 3-H ), 3.78 (1H, m, 4-H), 3.78 (1H, m, (3) -H), 3.67 (2H, m, (5) -CH 2 H 2), 3.63 (1H, m, (2) -H), 3.60 (1H, m, 3-H), 3.59 (1H, m, 5-H), 3.57 (1H, m, (4) -H ), 3.57 (1H, m, (5) -H), 3.16 (1H, t, J = 7.9Hz, 2-H), 1.10 ( in 3H, d, J = 6.7Hz, 5'-CH 3 H 3 )
    Sugar composition:
    L-fucose: Unsaturated D-glucuronic acid: D-glucuronic acid: D-mannose = 1: 1: 1: 2 (L-fucose, unsaturated D-glucuronic acid and D- Molecule, D-mannose: two molecules).
    Sulfate group:
    3 molecule (at the 2- and 4-positions of L-fucose and the 6-position of the reducing end of D-mannose).
    In the 1 H-NMR spectrum, peaks can be respectively assigned to positions indicated by the numerical values of the following chemical formula (20).
    Physical properties of compound (e):
    Molecular Weight: 1448.
    MS m / z: 767 [M + 4Na + -6H +] 2-, 503.7 [M + 3Na + -6H +] 3- and 366.5 [M + Na + -5H + ] 4-.
    MS / MS m / z: 97 < RTI ID = 0.0 > [HSO4]-, 225 [sulfated L-fucose-H2O-H+]-, 243 [sulfated L-fucose-H+]-, 345 [disulfated L-fucose + Na+-2H+]-, 450 [M + 3Na+-2SO3-6H+]3-, 477 [M + 3Na+-SO3-6H+]3-, 563 [D-mannose-H+Unsaturated D-glucuronic acid and sulfated L-fucose coupled to < RTI ID = 0.0 >-Or [sulfated D-mannose-H+Unsaturated D-glucuronic acid and L-fucose coupled to < RTI ID = 0.0 >-, 705 [sulfated D-mannose-H2O-H+Unsaturated D-glucuronic acid and disulfated L-fucose coupled to < RTI ID = 0.0 >-.
    1 H-NMR (D 2 O )
    d, J = 6.7 Hz, (1) - (1H), 5.55 (1H, d, J = 3.4 Hz, D), 5.19 (1H, d, J = 3.7 Hz, 1 H-), 5.16 (1H, m, 4H), 4.62 (1H, d, J = 7.6Hz, 1 H), 4.50 , m, 5'-H), 4.30 (1H, m, (5) '- H), 4.30 (1H, m, (5) H a -CH 2), 4.25 (1H, m, 2'-H) , 4.25 (1H, m, ( 2) -H), 4.25 (1H, m, (2) '- H), 4.20 (1H, m, (5) H a -CH 2), 4.18 (1H, m, 5-CH of the 2 H), 4.16 (1H, m, (3) -H), 4.08 (1H, m, H of 5-CH 2), 4.07 ( 1H, m, 2-H), 4.02 (1H, m, 3'-H), 4.02 (1H, m, (3) -H), 3.85 m, 3-H), 3.78 (1H, m, (3) -H), 3.76 M), 3.18 (1H, t, J = 8.2Hz), 3.75 (1H, m, (4) , 2-H), 1.10 ( 3H, d, J = 6.7Hz, (5) '- CH 3 a H 3), 1.09 (3H, d, J = 6.7Hz, of the 5'-CH 3 H 3)
    Sugar composition:
    L-fucose: D-glucuronic acid: D-glucuronic acid: D-mannose = 2: 1: 1: 2 (L-fucose and D-mannosic acid: two molecules each, unsaturated D- And D-glucuronic acid: one molecule each).
    Sulfate group:
    6 molecules (at the 2- and 4-positions of each L-fucose and the 6-position of each D-mannose).
    In the 1 H-NMR spectrum, peaks can be respectively assigned to positions indicated by the numerical values of the following chemical formula (21).
    Physical properties of compound (f):
    Molecular weight: 644.
    MS m / z: 687 [M + 2 Na + 3 H + ] - .
    MS / MS m / z: 97 [HSO 4] -, 243 [ sulfated L- fucose -H +] -, 421 [sulfated D- mannose + Na + -H 2 O-2H + combines the unsaturated D- Glucuronic acid] - .
    1 H-NMR (D 2 O )
    (1H, d, J = 4.0 Hz, 1'-H), 4.94 (1H, (1H, d, J = 6.7 Hz, 1 H), 4.45 (1H, dd, J = 3.1, 10.4 Hz, 3'- m, 5-CH 2 H 2 ), 4.14 (1H, m, 5'-H), 4.09 , 3.91 (1H, m, 5H), 3.85 (1H, m, 3-H) , 3.59 (1H, t, J = 6.7Hz, 2-H), 1.06 (3H, d, J = 6.4Hz, of the 5'-CH 3 H 3)
    Sugar composition:
    L-fucose: unsaturated D-glucuronic acid: D-mannose = 1: 1: 1 (one molecule each of L-fucose, D-mannose and unsaturated D-glucuronic acid).
    Sulfate group:
    2 molecule (at the 3-position of L-fucose and the 6-position of D-mannose).
    In the < 1 > H-NMR spectrum, peaks can be respectively assigned to positions represented by numerical values of the following chemical formula (22).
    Physical properties of compound (g):
    Molecular Weight: 632.
    MS m / z: 631 [MH < + & gt ; ] - .
    MS / MS m / z: 405 [D- galactose -H + L- sulfated fucose bonded to] -, 551 [L- fucose bonded to sulfated D- galactose -H + L- fucose bound to the - Or [sulfated L-fucose bound to L-fucose bound to D-galactose-H + ] - or [M-SO 3 -H + ] - .
    1 H-NMR (D 2 O )
    δ5.15 (1H, d, J = 4.3Hz, F 1 1H), 4.93 (1H, d, J = 3.7Hz, F 2 1H), 4.53 (1H, dd, J = 2.4, 10.4Hz , F 1 3-H), 4.49 (1H, d, J = 7.6Hz, G 1 1H), 4.46 (1H, dd, J = 3.1, 10.7Hz, F 2 3-H), 4.36 (1H, q, J = 6.7Hz F 2 5 -H), 4.14 (1H, q, J = 6.7Hz F 1 5-H), 4.09 (1H, d, J = 2.4Hz F 1 4-H), 4.03 (1H , d, J = 3.1Hz F 2 4-H), 3.97 (1H, dd, J = 4.3, 10.4Hz, F 1 2-H), 3.90 (1H, br-s, G 1 4-H), 3.81 (1H, dd, J = 3.7 , 10.7Hz, F 2 2H), 3.59 (1H, m, G 1 3-H), 3.59 (1H, m, G 1 5-H), 3.59 (2H, m , G 1 5-CH 2 of H 2), 3.56 (1H, m, G 1 2-H), 1.19 (3H, d, J = 6.7Hz, F 1 5-CH 3 a H 3), 1.14 (3H , d, J = 6.7Hz, F 2 5-CH 3 a H 3)
    Sugar composition:
    L-fucose: D-galactose = 2: 1 (two L-fucose molecules and one D-galactose molecule).
    Sulfate group:
    2 molecules (at the 3-position of each L-fucose).
    In the < 1 > H-NMR spectrum, peaks can be respectively assigned to positions indicated by numerical values of the following chemical formula (23).
    Physical properties of compound (h):
    Molecular Weight: 1358.
    MS m / z: 1445 [M + 4 Na + 5 H + ] - .
    MS / MS m / z: 97 [HSO 4 ] - , 225 [sulfated L-fucose-H 2 OH + ] - , 1197 [M-2 SO 3 -H + ] - .
    1 H-NMR (D 2 O )
    δ5.19 (1H, d, J = 4.3Hz, F 1 1H), 4.93 (1H, d, J = 3.7Hz, F 2 1H), 4.62 (1H, m, G 2 1H) , 4.59 (1H, m, G 1 1H), 4.54 (1H, dd, J = 2.7, 10.6Hz, F 1 3-H), 4.46 (1H, m, F 2 3-H), 4.46 (1H , d, J = 7.6Hz G 3 1H), 4.41 (1H, br-s, G 1 4-H), 4.41 (1H, d, J = 7.6Hz G 4 1H), 4.37 (1H, q, J = 6.4Hz F 2 5 -H), 4.27 (1H, m, G 1 3-H), 4.24 (1H, br-s, G 3 4-H), 4.21 (1H, m, G 3 3 -H), 4.19 (1H, m , G 4 3-H), 4.15 (1H, br-s, G 4 4-H), 4.13 (1H, q, J = 6.7Hz, F 1 5-H), 4.09 (1H, d, J = 2.7Hz, F 1 4-H), 4.04 (1H, d, J = 2.8Hz, F 2 4-H), 3.98 (1H, m, G 1 5-CH 2 of H ), 3.96 (in 1H, dd, J = 4.3, 10.6Hz, F 1 2-H), 3.88 (1H, br-s, G 2 4-H), 3.93 (1H, m, G 3 5-CH 2 H), 3.86 (1H, m , G 1 5-H), 3.81 (1H, m, F 2 2-H), 3.81 (1H, m, H of G 1 5-CH 2), 3.80 (1H, m , G 3 5-H), 3.80 (1H, m, G 3 5-CH 2 of H), 3.66 (1H, m , G 2 3-H), 3.65 (1H, m, G 1 2-H), 3.64 (1H, m, G 2 of the 5-CH 2 H), 3.64 (1H, m, G 4 H of 5-CH 2), 3.61 ( 1H, m, G 4 5-H), 3.58 (1H, m , G 2 2-H), 3.56 (1H, m, G 2 5-CH 2 of H), 3.56 (1H, m , G 4 of the 5-CH 2 H), 3.55 (1H, m, G 4 2- H), 3.54 (1H, m , G 2 5-H), 3.54 (1H, m, G 3 2-H), 1.20 (3H, d, J = 6.7Hz, F 1 5-CH 3 a H 3), 1.14 (3H, d, J = 6.4Hz, F 2 5- of CH 3 H 3)
    Sugar composition:
    L-fucose: D-galactose = 1: 2 (two L-fucose molecules and four D-galactose molecules).
    Sulfate group:
    5-molecule (the 3-position of each L-fucose, the 3-position of the D-galactose at the reducing end and the 3-position of the D-galactose at the 6-position of the reducing terminal D- Position of D-galactose bound to the 6-position of D-galactose).
    In the < 1 > H-NMR spectrum, peaks can be respectively assigned to positions indicated by the numerical values of the following chemical formula (24).
    Physical properties of compound (i):
    Molecular Weight: 1288.
    MS m / z: 1149 [M + Na + -2SO 3 -2H +] -.
    MS / MS m / z: 97 [HSO 4 ] - , 345 [desulfated L-fucose + Na + -2H + ] - .
    1 H-NMR (D 2 O )
    (1H, m, 1H), 5.19 (1H, d, J = (1H, d, J = 7.9 Hz, 1H), 4.94 (1H, (1H, d, J = 3.3, 10.6 Hz, 3'-H), 4.38 (1H, q, J = 6.4 Hz, of H), 4.20 (1H, m , 5-CH 2 - 5'-H), 4.34 (1H, q, J = 6.7Hz, (5) '- H), 4.29 (1H, m, (2)' H), 4.14 (1H, m , (2) -H), 4.13 (1H, m, (3) -H), 4.09 (1H, m, H of 5-CH 2), 4.08 ( 1H, m, 2 (1H, m, 5H), 3.83 (1H, d, J = m, 4-H), 3.81 (1H, m, 2'-H), 3.80 -H), 3.68 (2H, m , (5) H 2) of -CH 2, 3.62 (1H, m , (2) -H), 3.60 (1H, m, 3-H), 3.60 (1H, m (5H), 3.58 (1H, m, (5) -H), 3.56 3H, d, J = 6.4Hz, 5'-CH 3 a H 3), 1.11 (3H, d, J = 6.7Hz, (5) '- of CH 3 H 3)
    Sugar composition:
    L-fucose: D-glucuronic acid: D-glucuronic acid: D-mannose = 2: 1: 1: 2 (L-fucose and D-mannose: two molecules each, unsaturated D- D-glucuronic acid: one molecule each).
    Sulfate group:
    4 molecule (at the 3-position of L-fucose bonded to D-mannose at the reducing end, at the 2- and 4-positions of the other L-fucose and 6-position of D-mannose at the reducing end).
    In the 1 H-NMR spectrum, peaks can be respectively assigned to positions indicated by the numerical values of the following chemical formula (25).
    The sugar compound of the first invention of the present invention can be produced by treating the fucoidan with the cell extract or the culture supernatant of the third invention of the present invention belonging to the genus Fucoidanobacter.
    The strain of the third invention of the present invention may be any strains of the present invention belonging to the genus Fukao danobacter. As a specific example thereof, F. marinus strain SI-0098 can be mentioned.
    This strain Fucoidanobacter marinus SI-0098, which was first discovered by the present inventor from seawater of Aomori, has the following mycological characteristics.
    2. Fucoidanobacter mariners strain SI-0098
    a. Morphological characteristic
    (1) single phase (often pancytopically);
    Width: 0.5 - 0.7 탆
    Length: 0.5 - 0.7 ㎛
    (2) Spores: None
    (3) Gram-staining: -
    b. Physiological properties
    (1) Growth temperature range: Growth possible at 37 ℃, optimum growth temperature 15 ~ 28 ℃.
    (2) Behavior for oxygen: aerobic
    (3) Catalase: +
    (4) Oxidase: -
    (5) Urease: -
    (6) Hydrolyzed starch: +
    Gelatin: -
    Casein: -
    Esculin: +
    (7) Reduction of nitrate: -
    (8) Indole formation: -
    (9) Hydrogen sulfide formation: +
    (10) Solidification of milk: -
    (11) Sodium Requirement: +
    (12) Composition of salt
    Growth in NaCl-free medium: -
    Growth in 1% NaCl medium: -
    Growth in sea water medium: +
    (13) Quinone: Menaquinone 7
    (14) GC content of intracellular DNA: 61%
    (15) Diaminopimel acid in the cell wall: -
    (16) Glycolyl test: -
    (17) presence of hydroxy fatty acid: +
    (18) OF-test: O
    (19) Colony color: Does not form characteristic color.
    (20) Mobility: Yes
    (21) Runtime: None
    (22) Single hair: polar short hair
    According to the classification described in Bergey's Manual of Determinative Bacteriology, 9 (1994), this strain is classified as Group 4 (gram-negative aerobic bacilli and streptococci). However, this strain differs from group 4 bacteria, which generally have menaquinone in the electron transport chain and contain 61% GC. Basically, Gram-negative bacteria have ubiquinone in the electron transport chain and Gram-positive bacteria have menaquinone.
    Gram-negative bacteria belonging to the genus Flavobacterium and Cytophaga have exceptionally menaquinone in the electron transport chain, but they are generally different from the above-mentioned strains in the GC content, so that the soil bacteria, C. arvensicola contains 43 to 46% of GC and the marine bacteria such as C. diffluens, C. fermentans, C. parasitica, C. marina and C. uliginosa each contain 42% of GC. When comparing this strain with the strains identified for homology of the 16S rDNA sequence, even the most homologous (Verrucomicrobium spinosum) exhibits 76.6% homology thereto. It is well known that two bacteria having 90% or less homology are mutually different. Therefore, the inventor determined that this strain was a new bacteria not belonging to any genus in which this strain existed, and named this Fucoidanobacter mariners SI-0098.
    The above strain was named Fukoi Danbaut Mariners SI-0098 and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (Ibaraki, Japan, Higashi 1-chome, BP-5403 (the original deposit was issued on 29 March 1995 and was transferred to the International Deposit on February 15, 1996).
    The microorganism of the third invention of the present invention belonging to the genus Fucoidanobacter can be cultured in a medium and the fucoidan can be treated with a cell extract or a culture supernatant to thereby obtain the saccharide compound of the first invention of the present invention. When the microorganism belonging to the fucoidanobacter and capable of producing the sugar compound of the first invention of the present invention when treating the fucoidan with the cell extract or its culture supernatant may be any microorganism to be used. As a specific example of the strain, mention may be made of a strain of Fucoidanobacter marinus SI-0098.
    If the strains used can be used to produce cell extracts or culture supernatants capable of producing the sugar compound of the first invention, the nutrients to be added to the medium of the bacterial strain belonging to the genus Fukao danobacter may be any . Suitable examples of a carbon source include fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, glycerol, saccharose, maltose, lactose and starch. Suitable examples of the nitrogen source include yeast extract, peptone, , Meat extract, defatted soybean, ammonium sulfate, and ammonium chloride. The culture medium may also contain inorganic substances and metal salts such as sodium salts, phosphates, potassium salts, magnesium salts and zinc salts.
    These strains also grow well in seawater or artificial seawater containing the aforementioned nutrient sources.
    In order to cultivate bacteria belonging to the genus Fucoidanobacter according to the third invention of the present invention, the culture temperature is generally in the range of 15 to 30 DEG C and the pH value of the culture medium is preferably in the range of 5 to 9. [ Endopucoidan-lyase activity in cell extracts and culture supernatants reaches their maximum by culturing the strains under aeration and agitation for 5 to 72 hours. As a matter of course, the culture conditions are appropriately selected in accordance with the strain, medium composition and the like used for achieving the maximum activity of the endo-fucoidan-lyase of the intracellular extract and the culture supernatant.
    After culturing the Fukoianobacter mariners SI-0098 strain in a suitable medium, the culture medium is centrifuged to produce a culture supernatant with the cells. Also, the harvested cells are pulverized by a method commonly used for pulverizing cells such as ultrasonication. By centrifuging the pulverized cells, cell extracts can be obtained.
    Subsequently, the cell supernatant or cell extract is concentrated by ultrafiltration and mixed with a phosphate buffer containing sodium chloride. The fucoidan is then added thereto and reacted together.
    After the reaction is completed, the reaction mixture is fractionated by using a column for molecular weight fractionation. Therefore, the sugar compound of the first invention of the present invention can be obtained.
    The sugar compound of the first invention of the present invention is useful as a reagent for sugar chain technology. Pyridyl- (2) -aminated (PA) according to the method described in Japanese Patent Publication No. 65108/1993 These compounds make it possible to produce highly useful PA compounds as reagents for sugar chain technology. In addition, it is expected that the physiological activity of the sugar compound of the present invention can be applied to anticancer, cancer metastasis inhibiting and antiviral drugs.
    Best Mode for Carrying Out the Invention:
    The following examples will be provided to illustrate examples of methods for producing the sugar compound of the first invention of the present invention, but it should be understood that the present invention is not limited thereto.
    Example 1
    Flavobacterium sp. SA-0082 (FERM BP-5402) was pipetted into a 2-1 Erlenmeyer flask and sterilized at 120 ° C for 20 minutes containing 0.1% glucose, 1.0% peptone and 0.05% yeast extract And cultured in 600 ml of medium containing artificial seawater (pH 7.5, manufactured by Jamarin Laboratary). The strain is then incubated at 24 DEG C for 20 hours, thereby producing a seed culture. 30-1 jar fermenter containing 0.3% of fucoidan, 0.5% of peptone, 0.01% of yeast extract and 0.01% of defoamer (manufactured by Shin-Etsu Chemical Co., Ltd.) produced in gel mania crassifolia 20 liters of medium containing artificial seawater is supplied and sterilized at 120 DEG C for 20 minutes. After cooling, the medium is inoculated with 600 ml of the above-mentioned seed culture, followed by aeration at a rate of 10 1 / min and incubation at 24 캜 for 20 hours with stirring at 125 rpm. After completion of the culture, the culture medium is centrifuged to obtain a culture supernatant with the cells.
    The culture supernatant is concentrated by ultrafiltration (membrane exclusion molecular weight: 10,000, manufactured by Amicon) to analyze the endo-fucoidan-lyase of the present invention. Thus, 6 mU / ml activity of the culture medium is detected.
    Alternatively, the cells obtained by the main culture are suspended in 20 mM acetate-phosphate buffer (pH 7.5) containing 200 mM sodium chloride, pulverized by ultrasonication and centrifuged to thereby produce a cell extract. Upon analysis of the endo-fucoidan-lyase of the present invention of this cell extract, 20 mU / ml activity of the culture medium is detected.
    Ammonium sulfate is added to the above-mentioned concentrate of the culture supernatant to finally achieve 90% saturation. After dissolving by stirring, the mixture is centrifuged and the precipitate is suspended in the same buffer as mentioned above in which the cells are suspended. The suspension is then fully dialyzed against 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride. The precipitate formed by dialysis is removed by centrifugation and adsorbed on a DEAE-Sepharose FF column equilibrated with 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride. The adsorbed material is washed well with the same buffer and allowed to evolve by linear gradient elution with 50 mM to 600 mM sodium chloride. The active fractions are combined and sodium chloride is added thereto to produce a final concentration of 4M. And then adsorbed on a Phenyl Sepharose CL-4B column equilibrated with 20 mM phosphate buffer (pH 8.0) containing 4M sodium chloride. The adsorbed material is then washed well with the same buffer and allowed to evolve by linear gradient elution with 4M to 1M sodium chloride. The active fractions are combined and concentrated with an ultrafilter (Amicon). Then, gel filtration is carried out using a Sephacryl S-200 gel equilibrated with 10 mM phosphate buffer containing 50 mM sodium chloride. The active fractions are combined and sodium chloride is added thereto to produce a final concentration of 3.5 M. It is then adsorbed onto a Phenyl Sepharose HP column equilibrated with 10 mM phosphate buffer (pH 8) containing 3.5 M sodium chloride. The adsorbed material is then washed with the same buffer and developed by linear gradient elution with 3.5 M to 1.5 M sodium chloride. The active fractions are combined to produce the purified enzyme. The molecular weight of the enzyme measured from the retention time of Sephacryl S-200 is about 70,000. Table 1 summarizes the purification steps mentioned above.
    stepTotal protein (mg)Total activity (mU)Specific activity (mU / mg)yield(%) Cell extract980114,000116100 Ammonium sulfate - Salts473108,00022894.7 DEAE-Sepharos FF21686,40040075.8 Phenyl Sepharose CL-4B21.957,3002,62050.3 Sephacryl S-2003.7046,20012,50040.5 Phenyl Sepharose HP1.5341,20027,00036.1
    Example 2
    Flavobacterium sp. SA-0082 (FERM BP-5402) was pipetted in a 2-1 Erlenmeyer flask and sterilized at 120 ° C for 20 minutes containing 0.25% glucose, 1.0% peptone and 0.05% yeast extract And cultured in 600 ml of medium containing artificial seawater (pH 7.5, manufactured by Jamarin Laboratary). The strain is then incubated at 24 DEG C for 24 hours, thereby producing a seed culture. 30-1 jar fermenter containing 0.25% glucose, 1.0% peptone, 0.05% yeast extract and 0.01% defoamer (KM 70 manufactured by Shin-Etsu Chemical Co., Ltd.) and sterilized at 120 ° C for 20 minutes 20 L of medium containing artificial seawater (pH 7.5, manufactured by Jamarin Laboratory) is supplied. After cooling, the medium is inoculated with 600 ml of the above-mentioned seed culture, followed by aeration at a rate of 10 1 / min and cultivation at 24 캜 for 24 hours with stirring at 125 rpm. After completion of the cultivation, the culture medium is centrifuged to thereby produce a cell and culture suspension.
    The culture supernatant is concentrated by ultrafiltration (membrane exclusion molecular weight: 10,000, manufactured by Amicon) to analyze the endo-fucoidan-lyase of the present invention. Thus, 1 mU / ml of the activity of the culture medium is detected.
    Alternatively, the cells obtained by the main culture are suspended in 20 mM acetate-phosphate buffer (pH 7.5) containing 200 mM sodium chloride, pulverized by ultrasonication and centrifuged to thereby produce a cell extract. In the endo-fucoidan-lyase assay of this cell extract, 5 mU / ml activity of the culture medium is detected.
    Ammonium sulfate is added to this extract to achieve a final 90% saturation. After dissolution by stirring, the mixture is centrifuged and the precipitate is suspended in the same buffer as mentioned above in which the cells are suspended. The suspension is then fully dialyzed against 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride. The precipitate formed by dialysis is removed by centrifugation and adsorbed on a DEAE-Sepharose FF column equilibrated with 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride. The adsorbed material is then washed well with the same buffer and allowed to proceed by linear gradient elution with 50 mM to 600 mM sodium chloride. The active fractions are combined and sodium chloride is added thereto to produce a final concentration of 4M. And then adsorbed on a Phenyl Sepharose CL-4B column equilibrated with 20 mM phosphate buffer (pH 8.0) containing 4M sodium chloride. The adsorbed material is then washed well with the same buffer and allowed to evolve by linear gradient elution with 4M to 1M sodium chloride. The active fractions are combined and concentrated by ultrafiltration. Then, gel filtration is performed using Sepakril S-300 equilibrated with 10 mM phosphate buffer containing 50 mM sodium chloride. The active fractions are combined. The molecular weight of the enzyme measured from the retention time of Sephacryl S-300 is about 460,000. The active fraction is then dialyzed against 10 mM phosphate buffer (pH 7) containing 250 mM sodium chloride. The enzyme solution is adsorbed onto a Mono Q HR5 / 5 column equilibrated with 10 mM phosphate buffer (pH 7) containing 250 mM sodium chloride. The adsorbed material is washed well with the same buffer and allowed to evolve by linear gradient elution with 250 mM to 450 mM sodium chloride. The active fractions are combined to produce the purified enzyme. Table 2 summarizes the purification steps mentioned above.
    stepTotal protein (mg)Total activity (mU)Specific activity (mU / mg)yield(%) Cell extract61,900101,0001.63100 Ammonium sulfate - Salts33,80088,6002.6287.7 DEAE-Sepharos FF2,19040,40018.440.0 Phenyl Sepharose CL-4B48.229,00060128.7 Sephacryl S-3007.2419,6002,71019.4 Mono Q0.82415,00018,20014.9
    Example 3
    The purified fucoidan generated in gel mania crassifolia is treated with the endo-fucoidan-lyase (intracellular enzyme) of the present invention obtained in Example 1, thereby producing its decomposition product.
    Namely, 16 ml of a 2.5% bupocecidane solution, 12 ml of 50 mM phosphate buffer (pH 7.5), 4 ml of a 4 M sodium chloride solution and 8 ml of the 32 mU / ml endo-fucoidan-lyase solution of the present invention were mixed together And reacted at 25 DEG C for 48 hours.
    Subsequently, the reaction mixture is subjected to molecular weight fractionation using a Cellulofin GCL-300 column (manufactured by Seikagaku Kogyo), and fractions having a molecular weight of 2,000 or less are collected. After desalting with Micro Acilyzer G3 (manufactured by Asahi Chemical Industry Co., Ltd.), this fraction was separated into three fractions by DEAE-Sepharose FF, whereby the above-mentioned compounds (a), (b), and (c) are produced at 41 mg, 69 mg and 9.6 mg, respectively.
    Example 4
    The purified fucoidan produced in gel mania crassifolia is treated with the endo-fucoidan-lyase (extracellular enzyme) of the present invention obtained in Example 2, thereby producing its decomposition product.
    That is, 16 ml of a 2.5% bupocecidane solution, 12 ml of 50 mM phosphate buffer (pH 7.5), 4 ml of a 4 M sodium chloride solution and 8 ml of a 32 mU / ml endo-fucoidan-lyase solution of the present invention were mixed together And reacted at 25 DEG C for 48 hours.
    Subsequently, the reaction mixture is subjected to molecular weight fractionation using a cellulphin GCL-300 column (manufactured by Seikagaku Kogyo), and fractions having a molecular weight of 2,000 or less are collected. After desalting with Micro Acilyzer G3 (Asahi Chemical Industry Co., Ltd.), this fraction was separated into three fractions by DEAE-Sepharose FF and freeze-dried to give the above-mentioned compounds (a), ), And (c), respectively, of 40 mg, 65 mg and 9.2 mg, respectively.
    Example 5
    (FERM BP-5403) was cultured in the presence of 0.3% fucoidan, 0.5% peptone, 0.05% yeast extract and 0.01% defoamer (KM70, Shin-Etsu) produced in gel mania crassifolia (Manufactured by Jamarin Laboratory Co., Ltd.) containing artificial seawater (pH 7.5, manufactured by Chemical Co., Ltd.) and sterilized (120 DEG C, 20 minutes). The strain is pipetted into a 2-1 Erlenmeyer flask and sterilized at 120 ° C for 20 minutes. The strain is then incubated at 25 DEG C for 48 hours with stirring at 120 rpm. After the cultivation is complete, the culture medium is centrifuged to produce a culture supernatant with the cells.
    The culture supernatant is concentrated by ultrafiltration (membrane exclusion molecular weight: 10,000, manufactured by Amicon) to analyze the endo-fucoidan-lyase of the present invention. Thus, 0.2 mU / ml activity of the culture medium is detected.
    Alternatively, the cells obtained by the main culture are suspended in 20 mM acetate-phosphate buffer (pH 7.5) containing 200 mM sodium chloride, pulverized by ultrasonication and centrifuged to thereby produce a cell extract. In the endo-fucoidan-lyase assay of the present invention of this cell extract, 20 mU / ml activity of the culture medium is detected.
    Example 6
    The purified fucoidan produced in gel mania crassifolia is treated with the intracellular enzyme of the fucoidanobacter mariners SI-0098 strain of the present invention obtained in Example 5, thereby producing the decomposition product thereof.
    That is, 20 ml of 100 mM phosphate buffer (pH 8.0) containing 16 ml of a 2.5% buco-dacellated solution, 800 mM of sodium chloride, and 4 ml of the inventive solution of 20 mU / ml of fucoidan The enzyme solution in the bacterial mariners SI-0098 strain is mixed together and reacted at 25 DEG C for 48 hours.
    The reaction mixture is then subjected to molecular weight fractionation using a cellulphin GCL-300 column and fractions of molecular weight less than 2,000 are collected. After desalting with Micro Acilyzer G3, the fractions were separated into three fractions by DEAE-Sepharose FF and freeze-dried to give 38 mg (a), (b) and (c) , 60 mg and 8.2 mg.
    Accordingly, the present invention provides a method for the analysis of the structure of a fucosanic anhydride, the identification of an enzymatic degradation product of a fucosanthan and the detection of the biological activity of a fucosanthan, such as the production of a sugar compound, partial decomposition of a fucose endo, and production of a fucosan oligosaccharide. Lt; / RTI > endo-fucosidase-lyase useful in the study of < RTI ID = 0.0 >
    权利要求:
    Claims (12)
    [1" claim-type="Currently amended] A sugar compound of formula (I) or a salt thereof, wherein at least one alcohol hydroxyl group is sulfated.
    Formula 1

    (2)

    (3)

    Formula 4

    Formula 5

    6

    In this formula,
    X is a hydrogen atom or a group of the formula (3)
    Y is hydrogen or a group of formula 4 or 5, provided that X and Y can not be simultaneously hydrogen,
    Z is hydrogen or a group of formula (6).
    [2" claim-type="Currently amended] A sugar compound of formula (7) or a salt thereof.
    Formula 7

    [3" claim-type="Currently amended] A sugar compound of the formula (8) or a salt thereof.
    8

    [4" claim-type="Currently amended] A sugar compound of the formula (9) or a salt thereof.
    Formula 9

    [5" claim-type="Currently amended] A sugar compound of the formula (10) or a salt thereof.
    10

    [6" claim-type="Currently amended] A sugar compound of the formula (11) or a salt thereof.
    Formula 11

    [7" claim-type="Currently amended] A sugar compound of formula (12) or a salt thereof.
    Formula 12

    [8" claim-type="Currently amended] A sugar compound of the formula (13) or a salt thereof.
    Formula 13

    [9" claim-type="Currently amended] A sugar compound of formula (14) or a salt thereof.
    Formula 14

    [10" claim-type="Currently amended] A sugar compound of the formula (15) or a salt thereof.
    Formula 15

    [11" claim-type="Currently amended] Endo-fucoidan-lyase having the following physiochemical properties:
    (1) Function: a sugar compound glass of at least formulas (7) and (8) acting on fucoidan;
    (2) optimum pH value: in the range of pH 6 to 10;
    (3) Optimum temperature: in the range of 30 to 40 占 폚.
    [12" claim-type="Currently amended] A bacterium belonging to the genus Fucoidanobacter having menaquinone in the electron transport chain and containing about 60% GC.
    类似技术:
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    JP3605414B2|2004-12-22|
    AU2466497A|1998-12-03|
    US6379947B2|2002-04-30|
    EP0870771A1|1998-10-14|
    US20010046696A1|2001-11-29|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1995-04-28|Priority to JP127453/95
    1995-04-28|Priority to JP12745395
    1996-04-22|Application filed by 오미야히사시, 다카라슈조 주식회사, 후쿠시가즈에, 리서치인스티튜트포글리코테크놀로지
    1999-01-25|Publication of KR19990008099A
    2004-07-23|Application granted
    2004-07-23|Publication of KR100414607B1
    优先权:
    申请号 | 申请日 | 专利标题
    JP127453/95|1995-04-28|
    JP12745395|1995-04-28|
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