JP3991043B2 - Sugar compounds - Google Patents

Description

  The present invention relates to a sugar compound useful in the field of carbohydrate research, a glycolytic enzyme useful for producing the sugar compound, and a bacterium belonging to the genus Fucoidanobacter useful for producing the sugar compound.

  Fucoidan is a sulfated polysaccharide contained in brown algae and has been reported to have various biological activities such as anticoagulant action, fat serum clearing action, antitumor action, cancer metastasis inhibiting action, anti-AIDS virus infection action, It is a useful substance.

  However, fucoidan is a sulfated polysaccharide with an extremely large molecular weight, and there are problems such as antigenicity, homogeneity, and anticoagulant activity in order to use it as a pharmaceutical product as it is. Therefore, fucoidan must be decomposed to some extent. Many.

  Therefore, it was desired to elucidate the structure of fucoidan and elucidate the relationship with biological activity, but fucoidan is a highly branched polymer, has many types of sugars, and sulfate groups bind to various positions. Therefore, the structural analysis is very difficult. There is a method for analyzing the structure of oligosaccharides by using an enzyme that degrades polysaccharides to analyze the structure of polysaccharides. Among the currently reported fucoidan-degrading enzymes, the sugar chain structure of the degradation products There are no commercially available products that have been identified or are standard products of fucoidan oligosaccharides.

  For the reasons described above, there has been a demand for a sugar compound whose structure has been clarified, a glycolytic enzyme useful for the production of the sugar compound, and a microorganism useful for the production of the sugar compound.

  An object of the present invention is a novel useful for research on fucoidan, such as structural analysis of fucoidan, identification of enzymatic degradation products of fucoidan, and sugar compounds that can be used to search for biological activity, partial decomposition of fucoidan, and production of fucoidan oligosaccharides. It is to provide a novel microorganism useful for the production of a novel endo-type fucoidan-degrading enzyme and a sugar compound.

〔式中、ＸはＨ又は下記式（３）：

で表される基を示し、ＹはＨ又は下記式（４）、若しくは（５）：

で表される基を示すが、ＸとＹが共にＨであることはない。また、ＺはＨ又は下記式（６）：

で表される基を示す〕で表される化合物の少なくとも一つのアルコール性水酸基が硫酸エステル化している糖化合物又はその塩に関する。
前記一般式（１）又は（２）で表される化合物の例としては、下記の式（７）〜式（１５）で表される化合物が挙げられる。

If the present invention is outlined, the first invention of the present invention is represented by the following general formula (1) or (2):

[Wherein X is H or the following formula (3):

Y represents H or the following formula (4) or (5):

In which X and Y are not both H. Z is H or the following formula (6):

A saccharide compound or a salt thereof in which at least one alcoholic hydroxyl group of the compound represented by
Examples of the compound represented by the general formula (1) or (2) include compounds represented by the following formulas (7) to (15).

The second invention of the present invention relates to an endo-type fucoidan degrading enzyme characterized by having the following physicochemical properties.
(I) Action: Acts on fucoidan to liberate at least the compounds represented by the formulas (7) and (8).
(II) Optimal pH: The optimal pH of the enzyme is 6 to 10.
(III) Optimal temperature: The optimal temperature of this enzyme is 30 to 40 ° C.

  The third invention of the present invention is a novel microorganism useful for the production of the sugar compound of the first invention of the present invention, which has a menaquinone in the electron transport chain and has a GC content of about 60%. It relates to bacteria belonging to the genus Novacter.

  In the formulas (1), (2), (4) to (15), and (17) to (25) described in the present specification, “˜” represents both α anomer and β anomer in mannose or galactose. Means that an anomer exists.

  When the inventors act on the fucoidan with the enzyme according to the second invention of the present invention or the bacterial cell extract or the culture supernatant of the third invention of the present invention, the sugar compound of the present invention is produced. As a result, the present invention was completed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing an elution pattern when a pyridyl- (2) -aminated sugar compound (PA-a) of a sugar compound (a) is separated by an L-column.
FIG. 2 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-b) of the sugar compound (b) is separated by an L-column.
FIG. 3 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-c) of the sugar compound (c) is separated by an L-column.
FIG. 4 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-d) of the sugar compound (d) is separated by an L-column.
FIG. 5 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-e) of the sugar compound (e) is separated by an L-column.
FIG. 6 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-f) of the sugar compound (f) is separated by an L-column.
FIG. 7 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-g) of the sugar compound (g) is separated by an L-column.
FIG. 8 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-h) of the sugar compound (h) is separated by an L-column.
FIG. 9 is a diagram showing an elution pattern when the pyridyl- (2) -aminated sugar compound (PA-i) of the sugar compound (i) is separated by an L-column.
FIG. 10 is a diagram showing the results obtained by mass analysis (negative measurement) of the sugar compound (a).
FIG. 11 is a view showing the results obtained by mass analysis (negative measurement) of the sugar compound (b).
FIG. 12 is a diagram showing the results obtained by mass analysis (negative measurement) of the sugar compound (c).
FIG. 13 is a diagram showing the results obtained by mass analysis (negative measurement) of the sugar compound (d).
FIG. 14 is a diagram showing the results obtained by mass analysis (negative measurement) of the sugar compound (e).
FIG. 15 is a diagram showing the results obtained by mass analysis (negative measurement) of the sugar compound (f).
FIG. 16 shows the results obtained by mass analysis (negative measurement) of the sugar compound (g).
FIG. 17 is a diagram showing the results obtained by mass analysis (negative measurement) of the sugar compound (h).
FIG. 18 is a diagram showing the results obtained by mass analysis (negative measurement) of the sugar compound (i).
FIG. 19 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (a).
FIG. 20 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (b).
FIG. 21 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (c).
FIG. 22 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (d).
FIG. 23 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (e).
FIG. 24 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (f).
FIG. 25 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (g).
FIG. 26 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (h).
FIG. 27 is a diagram showing the results obtained by mass-mass analysis (negative measurement) of the sugar compound (i).
FIG. 28 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (a).
FIG. 29 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (b).
FIG. 30 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (c).
FIG. 31 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (d).
FIG. 32 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (e).
FIG. 33 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (f).
FIG. 34 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (g).
FIG. 35 is a diagram showing a ¹ H-NMR spectrum of a sugar compound (h).
FIG. 36 is a diagram showing a ¹ H-NMR spectrum of sugar compound (i).
FIG. 37 is a graph showing the relationship between the pH and relative activity (%) of the enzyme according to the second invention of the present invention.
FIG. 38 is a graph showing the relationship between the reaction temperature (° C.) and the relative activity (%) of the enzyme according to the second invention of the present invention.
FIG. 39 is a graph showing the relationship between the pH after treatment with the enzyme according to the second invention of the present invention and the residual activity (%).
FIG. 40 is a graph showing the relationship between the treatment temperature (° C.) and the residual activity (%) of the enzyme according to the second invention of the present invention.
FIG. 41 is a view showing an elution pattern when sugar compounds (a) to (i) are separated by DEAE-Sepharose FF.
FIG. 42 is a diagram showing an elution pattern when sugar compounds (h) and (i) are separated by DEAE-Sepharose FF.

Hereinafter, the present invention will be described in detail.
The strain used in the second invention of the present invention may be any strain as long as it belongs to the genus Flavobacterium and has the ability to produce the endo-type fucoidan degrading enzyme of the present invention. Specific examples of the strain having the ability to produce endo-type fucoidan degrading enzyme include, for example, Flavobacterium sp. SA-0082 strain is mentioned. If the endo-type fucoidan-degrading enzyme derived from the strain is allowed to act on fucoidan, the sugar compound according to the first invention of the present invention can be obtained.

This strain is a strain newly obtained by the present inventors from seawater in Aomori Prefecture, and its mycological properties are as follows.
1. Flavobacterium sp. SA-0082 strain a. Morphological properties (1) The bacterium is a short bacterium.
Width 0.8-1.0μm
Length 1.0-1.2μm
(2) Presence or absence of spores None (3) Gram staining negative b. Physiological properties (1) Growth temperature range Can grow at 37 ° C or less. A suitable growth temperature is 15 to 28 ° C.
(2) Attitude toward oxygen Aerobic (3) Catalase positive (4) Oxidase positive (5) Urease weak positive (6) Acid production D-glucose positive Lactose positive Maltose positive D-mannitol positive Sucrose negative Trehalose negative (7) Hydrolysis Degradation Starch Negative Gelatin Positive Casein Negative Esculin Positive (8) Nitrate Reduction Negative (9) Indole Formation Negative (10) Hydrogen Sulfide Generation Negative (11) Milk Coagulation Negative (12) Sodium Requirement Positive (13) Salts Requirement Growth in 0% saline medium Negative Growth in 1% saline medium Negative Growth in seawater medium Positive (14) Quinones Menaquinone 6
(15) GC content of intracellular DNA 32%
(16) OF-test O
(17) Color of the village Yellowish (18) No mobility (19) No sliding

  This strain is known as Bergey's Manual of Systemic Bacteriology, Volume 1 (1984), and Birge's Manual of Detergent Bacteriology (Bergey's Manual of Bacterial Minofol of Biobiology). (1994) is considered to be a related bacterium of Flavobacterium aquatile and Flavobacterium meningosepticum, but does not assimilate sucrose and form acid. Inability to decompose casein That can degrade, that it can liquefy gelatin, except urease is positive, that can not be decomposed casein is the latter, different points slow growth at 37 ° C.. Therefore, this strain was identified as a bacterium belonging to Flavobacterium, and Flavobacterium sp. It was named SA-0082.

The above strain is Flavobacterium sp. SA-0082 is displayed, and FERM BP-5402 (date of original deposit;) is placed at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry [Address: 1-3-3 Higashi 1-3, Tsukuba, Ibaraki, Japan (postal code 305)]. The deposit was made on March 29, 1995, the date of request for transfer to the international deposit date; February 15, 1996).
The nutrient source to be added to the culture medium of the strain used in the second invention of the present invention is not limited as long as it is used by the strain used and produces the endo-type fucoidan degrading enzyme according to the second invention of the present invention. For example, fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, glycerol, saccharose, maltose, lactose, starch and the like can be used as the nitrogen source, yeast extract, peptone, casamino acid, corn steep liquor, meat extract, Defatting soybeans, ammonium sulfate, ammonium chloride and the like are suitable. In addition, minerals such as sodium salt, phosphate, potassium salt, magnesium salt, zinc salt, and metal salts may be added.
This strain grows very well in seawater or artificial seawater containing the above nutrients.

When cultivating the endo-fucoidan-degrading enzyme-producing bacterium according to the second invention of the present invention, the production amount varies depending on the culture conditions. Generally, the culture temperature is 15 ° C. to 30 ° C., and the pH of the medium is 5 to 5. 9 is good, and the production amount of the endo-type fucoidan-degrading enzyme of the present invention reaches the maximum after 5 to 72 hours of aeration stirring culture. It is a matter of course that the culture conditions are set so that the production amount of the endo-type fucoidan degrading enzyme of the present invention is maximized according to the strain used, the composition of the medium and the like.
The endo-type fucoidan degrading enzyme according to the second invention of the present invention is present in the cells and the culture supernatant.

  Flavobacterium sp. The SA-0082 strain is cultured in an appropriate medium, the cells are collected, and the cells are disrupted by a commonly used cell disruption means such as ultrasonic treatment to obtain a cell-free extract.

  Next, a purified enzyme preparation can be obtained from this extract by a commonly used purification means. For example, purification is performed by salting out, ion exchange column chromatography, hydrophobic binding column chromatography, gel filtration, etc., and the purified end-type fucoidan-degrading enzyme according to the second invention of the present invention which does not contain other fucoidan-degrading enzymes is obtained. be able to. Further, since a large amount of the present enzyme (extracellular enzyme) is also present in the culture supernatant obtained by removing the bacterial cells from the above-mentioned culture broth, it can be purified by the same purification means as the intracellular enzymes.

The chemical and physicochemical properties of the endo-type fucoidan degrading enzyme according to the second invention of the present invention are as follows, and the intracellular enzyme and extracellular enzyme show the same properties except for the molecular weight.
(I) Action: Acts on fucoidan to liberate at least the sugar compounds represented by the above formulas (7) and (8).
(II) Optimal pH: The optimal pH of the enzyme is 6 to 10 (FIG. 37).
That is, FIG. 37 is a graph showing the relationship between the pH and relative activity of the enzyme, where the vertical axis indicates relative activity (%) and the horizontal axis indicates pH.
(III) Optimal temperature: The optimal temperature of this enzyme is 30 to 40 ° C. (FIG. 38).
That is, FIG. 38 is a graph showing the relationship between the temperature and relative activity of this enzyme, where the vertical axis indicates relative activity (%) and the horizontal axis indicates temperature (° C.).
(IV) pH stability: The enzyme is stable between pH 6 and 11.5 (FIG. 39).
That is, FIG. 39 is a graph showing the relationship between the pH of the enzyme treated and the residual activity, where the vertical axis indicates the residual activity (%) and the horizontal axis indicates the pH.
(V) Temperature stability: This enzyme is stable at about 30 ° C. or less (FIG. 40).
That is, FIG. 40 is a graph showing the relationship between the temperature at which this enzyme is treated and the residual activity, where the vertical axis shows the residual activity (%) and the horizontal axis shows the treatment temperature (° C.).
(VI) Molecular weight: The molecular weight of the enzyme was determined by gel filtration using Sephacryl S-200 (Pharmacia), and Flavobacterium sp. In the case of the extracellular enzyme of SA-0082 strain, it was about 70,000, and in the case of the intracellular enzyme, it was about 460,000.
(VII) Method for measuring enzyme activity:

The enzyme activity of the endo-type fucoidan degrading enzyme according to the second invention of the present invention was measured as follows.
That is, 50 μl of a fucoidan solution derived from 2.5% gagome kelp, 10 μl of the end-type fucoidan degrading enzyme according to the second invention of the present invention, and 83 mM phosphate buffer pH 7.5 containing 60 μl of 667 mM sodium chloride After mixing and reacting at 37 ° C. for 3 hours, 105 μl of the reaction solution and 2 ml of water were mixed and stirred, and the absorbance (AT) at 230 nm was measured. As a control, instead of the endo-type fucoidan degrading enzyme according to the second invention of the present invention, the reaction was carried out under the same conditions using only the above-mentioned buffer solution in which the enzyme of the present invention was dissolved, and instead of the fucoidan solution. The reaction was performed using only water, and the absorbance was measured in the same manner (AB1 and AB2).

One unit of enzyme is the amount of enzyme that releasably cleaves a glycosidic bond between 1 μmol of mannose and uronic acid per minute in the above reaction system. Quantification of the cleaved bond was performed by calculating the molar extinction coefficient of unsaturated uronic acid generated during the elimination reaction as 5.5. The enzyme activity was determined by the following formula.
(AT-AB1-AB2) × 2.105 × 120 / 5.5 × 105 × 0.01 × 180 = U / ml
2.105: Volume of sample for measuring absorbance (ml)
120: Volume of enzyme reaction solution (μl)
5.5: Molar molecular extinction coefficient of unsaturated uronic acid at 230 nm (/ mM) 105: Volume of reaction solution used for dilution (μl)
0.01: Amount of enzyme solution (ml)
180: Reaction time (minutes)

  The protein was quantified by measuring the absorbance at 280 nm of the enzyme solution. At that time, the absorbance of a 1 mg / ml protein solution was calculated as 1.0.

As described below, the present inventors determined the mechanism of action of the endo-type fucoidan degrading enzyme according to the second invention of the present invention.
(1) Preparation of gagome kelp fucoidan Dry gagome kelp was pulverized with a free crusher M-2 type (manufactured by Nara Machinery Co., Ltd.), treated in 10% 85% methanol at 70 ° C for 2 hours, filtered, and residue Was treated in 10 volumes of methanol at 70 ° C. for 2 hours and filtered. A 20-fold amount of water was added to the residue, treated at 100 ° C. for 3 hours, and an extract was obtained by filtration. After making the salt concentration of the extract the same as that of a 400 mM sodium chloride solution, cetylpyridinium chloride was added until no further precipitation occurred, followed by centrifugation. The precipitate is thoroughly washed with ethanol, and when cetylpyridinium chloride is completely removed, desalting and low molecular weight removal are performed by an ultrafilter (exclusion molecular weight of filter membrane: 100,000) (Amicon). The resulting precipitate was removed by centrifugation. This supernatant was freeze-dried to obtain purified gagome kelp fucoidan. The yield was about 4% based on the dry gagome kelp powder weight.
(2) Decomposition of fucoidan by endo-type fucoidan-degrading enzyme and purification of degradation product Large-scale preparation of the degradation product was performed by allowing the end-type fucoidan-degrading enzyme of the second invention of the present invention to act on purified gagome kelp-derived fucoidan. .

  That is, 600 ml of 5% gagome kelp-derived fucoidan solution, 750 ml of 100 mM phosphate buffer (pH 8.0), 150 ml of 4M sodium chloride, and 3.43 ml of the end-type fucoidan-degrading enzyme solution of the present invention of 1750 mU / ml. The mixture was mixed and reacted at 25 ° C. for 144 hours.

  The reaction solution was dialyzed using a dialysis membrane having a pore size of 3500, and fractions having a molecular weight of 3500 or less were collected. This fraction was desalted with a microacylizer G3 (manufactured by Asahi Kasei Co., Ltd.) and then subjected to nine fractions (a), (b), (c), (d), (e), (f), DEAE-Sepharose FF. Fractionated into (g), (h), and (i). The elution pattern is shown in FIG. The horizontal axis indicates the fraction number, the left vertical axis and the black circle in the figure indicate the sugar content by phenol sulfuric acid method at an absorbance of 480 nm, the right vertical axis and the white circle in the figure indicate the content of unsaturated glucuronic acid. The absorbance is 235 nm, the rightmost vertical axis and the dotted line in the figure indicate the ammonium acetate concentration (M) in the eluate. The fraction numbers of the fractions are (a): 42 to 43, (b): 84 to 91, (c): 51 to 52, (d): 79, (e): 102 to 103, (f), respectively. : 62-63, (g): 45, (h): 75, and (i): 77.

  For fractions (h) and (i), the above fraction numbers 64-78 were collected and repurified with DEAE-Sepharose FF. The elution pattern is shown in FIG. The horizontal axis indicates the fraction number, the left vertical axis and the black circle in the figure indicate the sugar content by phenol sulfuric acid method at an absorbance of 480 nm, the right vertical axis and the white circle in the figure indicate the content of unsaturated glucuronic acid. The absorbance is 235 nm, the rightmost vertical axis and the dotted line in the figure indicate the ammonium acetate concentration (M) in the eluate. The fraction numbers of the fractions were (h): 92 to 96 and (i): 99 to 103, respectively.

(3) Structural analysis of enzyme reaction product (a) Confirmation of uniformity of each fraction Nine fractions (a), (b), (c), (d), (e), (f) , (G), (h), and (i) were each partially PA-reduced by 2-aminopyridylation of the reducing end using GlycoTAG and GlycoTAG Reagent Kit (both manufactured by Takara Shuzo). Sugars (PA-a), (PA-b), (PA-c), (PA-d), (PA-e), (PA-f), (PA-g), (PA-h), and (PA-i) was obtained. (PA-a), (PA-b), (PA-c), (PA-d), (PA-e), (PA-f), (PA-g), (PA-h), and ( PA-i) was found to be homogeneous by HPLC analysis.

The HPLC conditions were as follows.
Apparatus: L-6200 type (manufactured by Hitachi, Ltd.)
Column: L-column (4.6 × 250 mm) {Chemicals Inspection Association}
Eluent: 50 mM acetic acid-triethylamine (pH 5.) for substances of the above formulas (7), (8), and (9) [(PA-a), (PA-b), and (PA-c)]. 5)
For substances of formula (10), (12), (13) and (15) [(PA-d), (PA-f), (PA-g) and (PA-i)] 100 mM acetic acid-triethylamine (pH 5)
For the substance of formula (11) [(PA-e)], 0 to 10 minutes is 200 mM acetic acid-triethylamine (pH 3.8) 10 to 60 minutes is 200 mM acetic acid-triethylamine (pH 3.8) and 0 The ratio of 200 mM acetic acid-triethylamine (pH 3.8) containing 0.5% tetrahydrofuran was linearly changed from 100: 0 to 20:80.
200 mM acetic acid-triethylamine (pH 3.8) containing 200 mM acetic acid-triethylamine (pH 3.8) and 0.5% tetrahydrofuran from 0 to 60 minutes for the substance of formula (14) [(PA-h)] The ratio was linearly changed from 80:20 to 50:50.
Detection: Detected with a fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 nm.
Flow rate: 1 ml / min Column temperature: 40 ° C

(A) Analysis of reducing end sugar and neutral sugar composition of enzyme reaction product Each PA sugar (PA-a), (PA-b), (PA-c), (PA-d), (PA -E), (PA-f), (PA-g), (PA-h), and (PA-i) were hydrolyzed by treatment with 4N hydrochloric acid at 100 ° C. for 3 hours, and the reducing end sugar was analyzed by HPLC. I investigated.
Further, the hydrolyzate was converted to a reducing end using a glyco tag (manufactured by Takara Shuzo) and a glyco tag regent kit (manufactured by Takara Shuzo), and the neutral sugar composition was examined by HPLC. The HPLC conditions were as follows.
Apparatus: L-6200 type (manufactured by Hitachi, Ltd.)
Column: Palpack type A (4.6 mm × 150 mm) (manufactured by Takara Shuzo) Eluent: 700 mM borate buffer (pH 9.0): acetonitrile = 9: 1 Detection: Fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) Detection was performed at an excitation wavelength of 310 nm and a fluorescence wavelength of 380 nm.
Flow rate: 0.3 ml / min Column temperature: 65 ° C

As a result, the reducing terminal sugars of (PA-a), (PA-b), (PA-c), (PA-d), (PA-e), (PA-f), and (PA-i) Were all mannose. The neutral sugar composition is (PA-a), (PA-b), (PA-c), (PA-e), (PA-f), and (PA-i) are equimolar amounts of mannose and fucose. And (PA-d) had a mannose to fucose ratio of 2: 1.
The reducing terminal sugars of (PA-g) and (PA-h) are both galactose, and the neutral sugar composition of (PA-g) is that the ratio of galactose to fucose is 1: 2, (PA- h) had a ratio of galactose to fucose of 2: 1.
In addition, in order to investigate the configuration of mannose, one of the constituent sugars, a reaction system that can measure only D-mannose according to the instructions using F-kit glucose / fructose and phosphomannose isomerase (both manufactured by Boehringer Mannheim Yamauchi). Separately, 100 μg each of (a), (b), (c), (d), (e), (f), and (i) was neutralized after hydrolysis with 2N hydrochloric acid at 100 ° C. for 3 hours. Things were measured in this reaction system. As a result, since D-mannose was detected from all of (a), (b), (c), (d), (e), (f), and (i), (a), (b), Mannose of the constituent sugars of (c), (d), (e), (f), and (i) was all found to be D-type.

  F-kit lactose / galactose (manufactured by Boehringer Mannheim Yamanouchi) that can detect only D-type galactose was used to examine the configuration of galactose, which is one of the constituent sugars of (g) and (h). That is, (g) and (h) 100 μg of each was hydrolyzed with 2N hydrochloric acid at 100 ° C. for 3 hours and then neutralized and then measured using this kit. As a result, since galactose was detected from (g) and (h), all of the constituent sugars galactose of (g) and (h) were found to be D-type.

  In order to investigate the configuration of fucose, which is another constituent sugar, according to the method described in Clinical Chemistry Vol. 36, pp. 474-476 (1990), the above (a), (b) , (C), (d), (e), (f), (g), (h) and (i) 100 μg of each was hydrolyzed with 2N hydrochloric acid at 100 ° C. for 3 hours and neutralized after this reaction. Measured with system. In this reaction system, D-fucose is not detected and only L-fucose can be detected. As a result, L-fucose was detected from (a), (b), (c), (d), (e), (f), (g), (h) and (i).

(C) Analysis of molecular weight of enzyme reaction product Next, (a), (b), (c), (d), (e), (f), (g), (h) and (i) When subjected to mass spectrometry using an API-III mass spectrometer (PerkinElmer-Sciex), their molecular weights were 564, 724, 1128, 1062, 1448, 644, 632, 1358, and 1288, respectively. . In (b) and (c), a divalent anion formed the main signal. In (d), monovalent ions, monovalent ions with sodium and divalent ions were detected. In (e), divalent ions with four sodium ions, trivalent ions with three sodium ions, and tetravalent ions with one sodium ion were detected. In (f), monovalent ions with two sodium ions were detected. In (g), monovalent ions and divalent ions in which monovalent ions and sodium were bonded were detected. In (h), monovalent, divalent, trivalent, and tetravalent ions having 4, 3, 2, and 1 sodium ions were detected, respectively. In (i), a monovalent ion with two sulfate groups off and one sodium and a divalent ion with two sulfate groups removed were detected.

  In addition, according to mass-mass (MS / MS) analysis in negative mode, (a) is a monovalent sulfate ion (molecular weight 97), a monovalent ion (molecular weight 157) obtained by removing water and hydrogen ions from unsaturated hexuronic acid, unsaturated A monovalent ion (molecular weight 175) from which hydrogen ions are removed from hexuronic acid, a monovalent ion (molecular weight 225) from which water molecules and hydrogen ions are removed from a combination of fucose and sulfuric acid, and a hydrogen ion from which fucose and sulfuric acid are combined Monovalent ion with a molecular weight of 243, monovalent ion with a molecular weight of 319 from which an unsaturated hexuronic acid and mannose are combined (molecular weight 319), hydrogen ion from a combination of fucose, sulfuric acid and mannose A monovalent ion (molecular weight 405) was detected.

  Similarly, by negative mode MS / MS analysis, (b) is a monovalent sulfate ion (molecular weight 97), a monovalent ion (molecular weight 175) obtained by removing hydrogen ions from unsaturated hexuronic acid, and a combination of fucose and sulfuric acid. Monovalent ions (molecular weight 243) from which hydrogen ions have been removed, divalent ions (molecular weight 321) from which two hydrogen ions have been removed from a combination of unsaturated hexuronic acid, fucose, mannose and sulfuric acid, fucose, sulfuric acid and mannose A monovalent ion (molecular weight 405) from which hydrogen ions were removed from those bonded to each other, and a monovalent ion (molecular weight 417) from which hydrogen ions were removed from those obtained by combining unsaturated hexuronic acid, mannose and sulfuric acid were detected.

  In addition, by negative mode MS / MS analysis, (c) is a monovalent sulfate ion (molecular weight 97), a monovalent ion obtained by removing hydrogen ions from unsaturated hexuronic acid (molecular weight 175), and a combination of fucose and sulfuric acid. Monovalent ions (molecular weight 225) from which water molecules and hydrogen ions were removed, monovalent ions (molecular weight 243) from which hydrogen ions were removed from the combination of fucose and sulfuric acid, mannose, hexuronic acid, mannose, fucose and sulfuric acid were combined Divalent ions (molecular weight 371) from which two hydrogen ions were removed, monovalent ions (molecular weight 405) from which hydrogen ions were removed from a combination of fucose, sulfuric acid and mannose, fucose, unsaturated hexuronic acid, mannose and hexuron A monovalent ion (molecular weight 721) in which water and hydrogen ions were removed from a combination of acid and sulfuric acid was detected.

  Also, negative mode MS / MS analysis revealed that (d) divalent ions were converted into monovalent sulfate ions (molecular weight 97), monovalent ions (molecular weight 175) from which hydrogen ions were removed from unsaturated hexuronic acid, fucose and sulfuric acid. Monovalent ions (molecular weight 225) from which water molecules and hydrogen ions are removed from those bonded to each other, monovalent ions (molecular weight 243) from which hydrogen ions are removed from those from which fucose and sulfuric acid are combined, fucose, mannose and sulfate groups are combined. Monovalent ions (molecular weight 405) from which hydrogen ions have been removed, divalent ions (molecular weight 450) from which two sulfate groups have been removed from (d), two sulfate groups have been obtained from (d), and sulfate groups 1 from (d). A divalent ion (molecular weight 490) from which two hydrogen ions were removed was detected.

  In addition, by negative mode MS / MS analysis, (e) is a monovalent sulfate ion (molecular weight 97), a monovalent ion (molecular weight 225) in which water molecules and hydrogen ions are removed from a combination of fucose and sulfuric acid, fucose A monovalent ion (molecular weight 243) from which hydrogen ions have been removed from a combination of sulfuric acid, a monovalent ion (molecular weight 345) from which two hydrogen ions have been removed from a combination of fucose, two molecules of sulfate groups and sodium ions, ( e) 2 sulfate groups, 3 sodium ions attached, 6 hydrogen ions removed trivalent ion (molecular weight 450), (e) 1 sulfate groups and 6 hydrogen ions Trivalent ions (molecular weight 476) with three sodium ions, monovalent ions with hydrogen ions removed from unsaturated hexuronic acid, mannose, fucose and sulfuric acid combined (Molecular weight 563), and an unsaturated hexuronic acid and mannose-fucose and 3 molecules of monovalent ions sulfate caught water molecule and a hydrogen ion from the union (molecular weight 705) was detected.

  Further, by negative mode MS / MS analysis, (f) is a monovalent sulfate ion (molecular weight 97), a monovalent ion (molecular weight 243) obtained by removing hydrogen ions from a combination of fucose and sulfuric acid, unsaturated hexuronic acid Monovalent ions (molecular weight 421) from which water and hydrogen ions were removed from a combination of mannose, sulfuric acid and sodium were detected.

  In addition, by negative mode MS / MS analysis, (g) is a monovalent ion (molecular weight 405) obtained by removing hydrogen ions from a combination of fucose, sulfuric acid and galactose, and two fucose molecules, one galactose molecule and one sulfate group. Monovalent ions (molecular weight 551) were detected in which hydrogen ions were removed from the molecules bound.

  In addition, from the negative mode MS / MS analysis, 3 molecules of sodium were bound to (h) and 5 molecular hydrogen ions were taken, monovalent sulfate ion (molecular weight 97), fucose and sulfuric acid bound Detects a monovalent ion (molecular weight 225) from which water molecules and hydrogen ions have been removed, and a monovalent ion (molecular weight 1197) from which one molecule of hydrogen ions has been removed from a combination of two fucose molecules, four galactose molecules and three sulfate groups. It was done.

  In addition, by negative mode MS / MS analysis, from divalent ions from which two sulfate groups were removed from (i) to monovalent sulfate ions (molecular weight 97), fucose, two molecules of sulfuric acid and sodium bound to hydrogen Monovalent ions (molecular weight: 345) from which ions were removed were detected.

(D) Analysis of sugar composition of enzyme reaction product From the results of mass spectrometry, (a), (b), (c), (d), (f), and (i) are unsaturated hexuronic acids in the molecule. It was suggested that there is a high possibility that In order to prove the presence of hexuronic acid having an unsaturated bond in the molecule of the enzyme reaction product, the following experiment was conducted. It is known that there is strong absorption at 230 to 240 nm when unsaturated bonds exist in the molecule. Therefore, 230 to 240 nm of purified aqueous solutions of the oligosaccharides (a), (b), (c), (d), (e), (f), (g), (h), and (i). (A), (b), (c), (d), (e), (f), and (i) have strong absorption, and there are unsaturated bonds in the molecule. It has been suggested. Moreover, since it was also confirmed that the absorbance at 230 to 240 nm increases with the progress of the decomposition reaction of fucoidan by this enzyme, this enzyme is a glycosidic bond between mannose and hexuronic acid or between galactose and hexuronic acid in fucoidan. It was strongly suggested that cleaved by elimination reaction.

  Since most of the enzymatic reaction products have unsaturated hexuronic acid at the non-reducing end and the reducing end is mannose, it is suggested that there are molecular species in which fucoidan prepared by alternation of hexuronic acid and mannose exists. It was.

Since most of the constituent sugars of fucoidan are fucose, fucoidan is more easily decomposed by acid than general polysaccharides. On the other hand, hexuronic acid and mannose bonds are known to be relatively strong against acids. In order to clarify the kind of hexuronic acid in the molecular species in which hexuronic acid and mannose existing alternately in the molecule of fucoidan in Gagome kelp are used, the present inventors have found that Carbohydrate Research 125th. Vol. 283-290, with reference to the method of 1984, first, fucoidan was dissolved in 0.3 M oxalic acid and treated at 100 ° C. for 3 hours, and then molecular weight fractionation was performed. The adsorbed matter was further collected with an anion exchange resin. This material was freeze-dried and then hydrolyzed with 4N hydrochloric acid, adjusted to pH 8, converted to PA, and uronic acid was analyzed by HPLC. The HPLC conditions were as follows.
Apparatus: L-6200 type (manufactured by Hitachi, Ltd.)
Column: Palpack type N (4.6 mm x 250 mm) (Takara Shuzo)
Eluent: 200 mM acetic acid-triethylamine buffer (pH 7.3): acetonitrile = 25: 75
Detection: Detected with a fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 nm.
Flow rate: 0.8 ml / min Column temperature: 40 ° C

The standard substance of PA hexuronic acid is glucuronic acid manufactured by Sigma, galacturonic acid manufactured by Wako Pure Chemicals, and iduronic acid hydrolyzed 4-methylumbelliferyl α-L-iduronide manufactured by Sigma. Mannuronic acid and guluronic acid were separated from an alginic acid manufactured by Wako Pure Chemical with an anion exchange resin according to the method described in Acta Chemica Scandinavica, Vol. 15, pages 1397 to 1398, 1961. This was obtained by converting to PA.
As a result, it was found that glucuronic acid was the only hexuronic acid contained in the molecular species of fucoidan.

  Furthermore, glucuronic acid in the hydrolyzate of the above molecular species was separated from D-mannose with an anion exchange resin, and after lyophilization, the specific rotation was measured. The glucuronic acid was D-glucuronic acid. There was found.

  Further, the fucoidan derived from Gagome kelp was previously treated with the endo-type fucoidan degrading enzyme according to the second invention of the present invention, but was also acid-hydrolyzed with oxalic acid in the same manner as above, but D-glucuronic acid and D-mannose were used. A polymer in which is alternately bound was not detected. From this, it was found that the skeleton structure of the fucoidan molecular species cleaved by the elimination reaction of the enzyme of the present invention has a structure in which D-glucuronic acid and D-mannose are alternately bonded.

  Furthermore, in order to investigate each bond position of D-glucuronic acid and D-mannose and the anomeric configuration of the glycosidic bond, the polymer obtained by oxalic acid decomposition was subjected to NMR analysis.

Ｄ−グルクロン酸の１位の立体配置はそのビシナル結合定数が７．６ＨｚであることからβーＤ−グルクロン酸であると決定した。
また、マンノースの１位の立体配置はその化学シフト値が５．２５ｐｐｍであることからαーＤ−マンノースであると決定した。
構成糖の結合様式は^１Ｈ検出異種核検出法であるＨＭＢＣ法を用いて行った。^１Ｈ−ＮＭＲの帰属にはＤＱＦ−ＣＯＳＹ法及びＨＯＨＡＨＡ法を、^１３Ｃ−ＮＭＲの帰属にはＨＳＱＣ法を用いた。
ＨＭＢＣスペクトルにより１ーＨと４’−Ｃの間及び４’−Ｈと１ーＣの間、１’−Ｈと２−Ｃの間及び２ーＨと１’−Ｃの間にそれぞれクロスピークが認められた。このことからＤ−グルクロン酸はβ結合でＤ−マンノースの２位に、Ｄ−マンノースはα結合でＤ−グルクロン酸の４位にそれぞれ結合していることが明らかとなった。 The NMR measurement results of the polymer are shown below. However, the chemical shift value in ¹ H-NMR was expressed with the chemical shift value of the methyl group of triethylamine as 1.13 ppm, and the chemical shift value of the methyl group of triethylamine as 9.32 ppm in 13C-NMR.
¹ H-NMR (D ₂ O)
δ 5.25 (1H, br-s, 1-H), 4.32 (1H, d, J = 7.6 Hz, 1′-H), 4.00 (1H, br-s, 2-H), 3.71 (1H, m, 5'-H), 3.69 (1H, m, 5-CH H), 3.68 (1H, m, 3-H), 3.63 (1H, m, 5-CH H), 3.63 (1H, m, 4'-H), 3.57 (1H, m, 4-H), 3.54 (1H, m, 3'-H); 53 (1H, m, 5-H), 3.25 (1H, t, J = 8.5 Hz, 2'-H)
¹³ C-NMR (D ₂ O)
δ 175.3 (C of 5′-COOH), 102.5 (1′-C), 99.6 (1-C), 78.5 (2-C), 77.9 (4′-C), 77.0 (3'-C), 76.7 (5'-C), 73.9 (5-C), 73.7 (2'-C), 70.6 (3-C), 67. 4 (4-C), 61.0 (5-CH ₂ OH C)
The peak assignment numbers are as shown in the following formula (16):

The configuration at the 1-position of D-glucuronic acid was determined to be β-D-glucuronic acid because its vicinal binding constant was 7.6 Hz.
The steric configuration at the 1-position of mannose was determined to be α-D-mannose because the chemical shift value was 5.25 ppm.
The binding mode of the constituent sugars was performed using the HMBC method which is a ¹ H detection heterogeneous nuclear detection method. ^The DQF-COSY method and the HOHAHA method were used for ¹ H-NMR assignment, and the HSQC method was used for ¹³ C-NMR assignment.
Cross peaks between 1-H and 4'-C, between 4'-H and 1-C, between 1'-H and 2-C and between 2-H and 1'-C by HMBC spectrum, respectively. Was recognized. From this, it was revealed that D-glucuronic acid was bonded to the 2-position of D-mannose by β bond, and D-mannose was bonded to the 4-position of D-glucuronic acid by α-bond.

(E) Analysis of the sugar bonding mode and sulfate group binding position of the enzyme reaction product In order to investigate the binding mode of the constituent sugar and sulfate group, the enzyme reaction product was subjected to a 500 MHz nuclear magnetic resonance apparatus JNM-α500 (manufactured by JEOL Ltd.). ) For NMR spectrum analysis. From this analysis result, (a) is the above formula (7), (b) is the above formula (8), (c) is the above formula (9), (d) is the above formula (10), (e) is the above formula. (11) and (f) are represented by the above formula (12), (g) is represented by the above formula (13), (h) is represented by the above formula (14), and (i) is represented by the above formula (15). (A) 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, and (b) A structure in which unsaturated D-glucuronic acid and L-fucose bonded to two sulfate groups are bonded to D-mannose which is a reducing terminal residue bonded to a sulfate group; D-glucuronic acid and L-fucose having a sulfate group bonded to D-mannose, which is a group, bind to D-glucuronic acid. (D) has a structure in which mannose is bonded and L-fucose bonded with unsaturated D-glucuronic acid and a sulfate group is bonded to the D-mannose; Group, D-glucuronic acid and L-fucose with two sulfate groups bonded to each other, D-mannose to D-glucuronic acid, and unsaturated D-glucuronic acid to D-mannose (E) shows that sulfate group, D-glucuronic acid, and L-fucose having two sulfate groups bonded to D-mannose, which is the reducing terminal residue, bind to the D-glucuronic acid. (D) having a structure in which D-mannose to which a sulfate group is bonded is bonded, and further L-fucose and unsaturated D-glucuronic acid are bonded to the D-mannose. (G) shows that D-mannose, which is a reducing terminal residue, has a structure in which unsaturated D-glucuronic acid and L-fucose having a sulfuric acid group are bonded to D-mannose, which is a reducing terminal residue to which an acid group is bonded. -L-fucose having a sulfate group bonded to galactose, and having a structure in which L-fucose having a sulfate group is bonded to the L-fucose, (h) is a reducing end having a sulfate group bonded thereto. L-fucose with a sulfate group bound to D-galactose, and one sugar chain has a structure branched into two starting from D-galactose as a residue. L-fucose to which is bound is bound, and the other sugar chain has a structure in which D-galactose to which a sulfate group is bound is bound to D-galactose to which a sulfate group is bound, (i) D-mannose as a terminal residue A sulfate group, D-glucuronic acid, and L-fucose to which a sulfate group was bonded were bound to D-glucuronic acid, D-mannose was bound to D-mannose, and two sulfate groups were bound to the D-mannose. It was found to have a structure in which L-fucose and unsaturated D-glucuronic acid were combined.

  A compound contained in the sugar compound according to the first invention of the present invention was obtained by allowing the endo-type fucoidan degrading enzyme according to the second invention of the present invention to act on fucoidan. Below, formula (7), formula (8), formula (9), formula (10), formula (11), formula (12), formula (13) are examples of the sugar compound of the first invention of the present invention. ), Formula (14), and compounds represented by formula (15), that is, (a), (b), (c), (d), (e), (f), (g), (h) And the physical properties of (i).

  1, 2, 3, 4, 5, 6, 7, 8, and 9, each pyridyl (-2-) aminated sugar compound (PA-a), (PA-b), (PA-c), The (PA-d), (PA-e), (PA-f), (PA-g), (PA-h), and (PA-i) HPLC elution patterns are shown. Relative fluorescence intensity, horizontal axis indicates retention time (minutes). Further, FIG. 10 (a), FIG. 11 (b), FIG. 12 (c), FIG. 13 (d), FIG. 14 (e), FIG. 16 shows the mass spectrum of (g), FIG. 17 shows the mass spectrum of (h), FIG. 18 shows the mass spectrum of (i), FIG. 19 shows the mass spectrum of (a), FIG. 20 shows the mass spectrum of (b), and FIG. 22 (d), FIG. 23 (e), FIG. 24 (f), FIG. 25 (g), FIG. 26 (h), FIG. 27 (i) In each figure, the vertical axis represents relative intensity (%), and the horizontal axis represents m / z value.

FIG. 28 is a diagram of (a), FIG. 29 is a diagram of (b), FIG. 30 is a diagram of (c), FIG. 31 is a diagram of (d), FIG. 32 is a diagram of (e), and FIG. 34 shows the ¹ H-NMR spectrum of (g), FIG. 35 shows (h), and FIG. 36 shows (i). In each figure, the vertical axis represents the signal intensity, and the horizontal axis represents the chemical shift value (ppm). Show.
The chemical shift value in ¹ H-NMR was expressed assuming that the chemical shift value of HOD was 4.65 ppm.

Physical property molecular weight 564 of (a)
MS m / z 563 [M−H ⁺ ] ⁻
MS / MS m / z 97 [HSO4] ⁻ , 157 [unsaturated D-glucuronic acid-H ₂ O—H ⁺ ] ⁻ , 175 [unsaturated D-glucuronic acid -H ⁺ ] ⁻ , 225 [L-fucose sulfate] _{^{-H 2 O-H +] -}} , 243 [L- fucose _{^{-H +] -, 319 [-H}} 2 O-H +] that the unsaturated D- glucuronic acid D- mannose bound -, 405 [ M-unsaturated D-glucuronic acid-H ⁺ ] ⁻ , 483 [M-SO ₃ —H ⁺ ] ^−
1 H-NMR (D ₂ O)
δ 5.78 (1H, d, J = 3.7 Hz, 4 ″ -H), 5.26 (1H, d, J = 1.2 Hz, 1-H), 5.12 (1H, d, J = 4) .0Hz, 1'-H), 5.03 (1H, d, J = 6.1Hz, 1 "-H), 4.47 (1H, dd, J = 3.4, 10.4Hz, 3 '-H), 4.21 (1H, br-s, 2-H), 4.12 (1H, m, 5'-H), 4.10 (1H, dd, J = 3.7, 5.8 Hz, 3 ″ -H), 4.03 ((1H, d, J = 3.4 Hz, 4′-H), 3.86 (1H, m, 3-H), 3.83 (1H, d-d, J = 4.0, 10.4 Hz, 2'-H), 3.72 (1H, m, 4-H), 3.72 (1H, m, 5-H), 3.70 ( 2H, m, 5-CH ₂ of _{H 2), 3.65 (1H,} d-d, J = 5.8,6. Hz, 2 "-H), 1.08 (3H, d, J = 6.7Hz, H 3 of the 5'-CH ₃₎
Sugar composition L-fucose: unsaturated D-glucuronic acid: D-mannose = 1: 1: 1 (one molecule each)
1 molecule of sulfate group (3rd position of L-fucose)
In addition, the numbers of the peaks in ¹ H-NMR are as shown in the following formula (17).

Physical property molecular weight of (b) 724
MS m / z 723 [M−H ⁺ ] ⁻ , 361 [M−2H ⁺ ] ²⁻
MS / MS m / z 97 [HSO ₄ ] ⁻ , 175 [unsaturated D-glucuronic acid -H ⁺ ] ⁻ , 243 [L-fucose sulfate-H ⁺ ] ⁻ , 321 [M-SO ₃ −2H ⁺ ] ^{2 -,} 405 [M- unsaturated D- glucuronic acid -2SO ₃ -H ^{+] -,} 417 [M-L-fucose -2SO ₃ -H ^{+] -
1} H-NMR (D ₂ O)
δ 5.66 (1H, d, J = 3.4 Hz, 4 ″ −H), 5.27 (1H, d, J = 7.3 Hz, 1 ″ −H), 5.25 (1H, d, J = 1.8 Hz, 1-H), 5.21 (1H, d, J = 3.7 Hz, 1'-H), 4.50 (1H, d, J = 3.1 Hz, 4'-H), 4 .32 (1H, q, J = 6.7 Hz, 5′-H), 4.27 (1H, dd, J = 3.7, 10.4 Hz, 2′-H), 4.21 (1H , Dd, J = 3.4, 6.7 Hz, 3 ″ -H), 4.18 ((1H, dd, J = 1.8, 11.0 Hz, 5-CH H), 4 .15 (1H, br-s, 2-H), 4.10 (1H, dd, J = 5.8, 11.0 Hz, 5-CH H), 3.99 (1H, dd) , J = 3.1, 10.4 Hz, 3′-H), 3.90 (1H, m, 5- ), 3.82 (1H, m, 3-H), 3.82 (1H, m, 4-H), 3.54 (1H, br-t, J = 7.3 Hz, 2 ″ -H), 1.11 (3H, d, J = 6.7 Hz, 5′-CH ₃ H ₃ )
Sugar composition L-fucose: unsaturated D-glucuronic acid: D-mannose = 1: 1: 1 (one molecule each)
3 molecules of sulfate group (2nd and 4th positions of L-fucose and 6th position of D-mannose)
In addition, the numbers of the peaks in ¹ H-NMR are as shown in the following formula (18).

Physical property molecular weight of (c) 1128
MS m / z 1127 [M−H ⁺ ] ⁻
MS / MS m / z 97 [HSO ₄ ] ⁻ , 175 [unsaturated D-hexuronic acid-H ⁺ ] ⁻ , 225 [L-fucose sulfate-H ₂ O—H ⁺ ] ⁻ , 243 [L-fucose sulfate − H ⁺ ] ⁻ , 371 [M-unsaturated D-glucuronic acid-L-fucose-SO ₃ -2H ⁺ ] ²⁻ , 405 [sulfate L-fucose and D-mannose bonded to each other −H ⁺ ] ⁻ 721 [M-D-mannose -L--fucose _-SO 3 _-H 2 ^{O-H +] -
1} H-NMR (D ₂ O)
δ 5.69 (1H, d, J = 3.7 Hz, (4) ″-H), 5.34 (1H, s, (1) -H), 5.16 (1H, s, 1-H), 5.10 (1H, d, J = 4.0 Hz, (1) ′-H), 5.05 (1H, d, J = 3.7 Hz, 1′-H), 4.93 (1H, d, J = 6.4 Hz, (1) ″-H), 4.50 (1H, dd, J = 3.4, 10.7 Hz, 3′-H), 4.47 (1H, dd, J = 3.4, 10.4 Hz, (3) ′ − H), 4.39 (1H, d, J = 7.9 Hz, 1 ″ −H), 4.33 (1H, br−s, (2 ) -H), 4.14 (1H, m, 2-H), 4.12 (1H, m, (3) ″-H), 4.12 (1H, m, 5′-H), 4. 12 (1H, m, (5) '-H), 4.04 (1H, m, 4'-H), 4.03 (1H m, (4) '-H), 3.85 (1H, m, 2'-H), 3.85 (1H, m, (2)'-H), 3.82 (1H, m, 3- H), 3.82 (1H, m, (3) -H), 3.73 (1H, m, 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) H2 of -CH2), 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, 2 ″ -H), 1.09 (3H, d, J = 6.7Hz, (5) ' - H 3 of _{CH 3), 1.07 (1H,} d, J = 6.7Hz, 5 _H 3 of -CH ₃₎
Sugar composition L-fucose: unsaturated D-glucuronic acid: D-glucuronic acid: D-mannose = 2: 1: 1: 2 (two molecules each of L-fucose and D-mannose, unsaturated D-glucuronic acid and D- 1 molecule for each glucuronic acid)
2 molecules of sulfate group (3rd position of each L-fucose)
In addition, the number of the peak assignment in ¹ H-NMR is as shown in the following formula (19).

Physical property molecular weight of (d) 1062
MS m / z 1061 [M−H ⁺ ] ⁻
MS / MS m / z 97 [HSO ₄ ] ⁻ , 175 [unsaturated hexuronic acid-H +] ⁻ , 225 [L-fucose sulfate-H ₂ O—H ⁺ ] ⁻ , 243 [L-fucose sulfate −H ⁺ ] ^-, 405 [L-ones fucose D- mannose and sulfate groups attached _{^{-H +] -, 450 [M}} -2SO 3 -2H +] 2-, 490 [M-SO 3 -2H +] 2-
¹ H-NMR (D ₂ O)
δ 5.67 (1H, d, J = 3.7 Hz, (4) ″-H), 5.32 (1H, br-s, (1) -H), 5.17 (1H, d, J = 3) .5 Hz, 1′-H), 5.17 (1 H, br-s, 1-H), 4.93 (1 H, d, J = 6.4 Hz, (1) ″-H), 4.71 ( 1H, m, 1 ″ -H), 4.53 (1H, d, 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, 2′-H), 4.18 (1H, m, 5-CH ₂ H), 4.13 (1H, m, (2) -H), 4.12 (1H, m, (3) ″-H), 4.08 (1H, m, 5-CH ₂ H), 4.07 (1H, m, 2-H) ), 3.98 (1H, dd, J = 3.4, 11.0, 3′-H), 3.88 (1 H, m, 5-H), 3.82 (1H, m, 4 ″ -H), 3.78 (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 = 7.9 Hz, 2 ″ -H), 1.10 (3H, d, J = 6.7 Hz, 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, one D-glucuronic acid molecule and D -Mannose 2 molecule)
3 molecules of sulfate group (2nd and 4th positions of L-fucose and 6th position of D-mannose on the reducing end side)
In addition, the numbers of the peaks in ¹ H-NMR are as shown in the following formula (20).

Physical property molecular weight of (e) 1448
MS m / z 767 [M + 4Na-6H ⁺ ] ^2-
503.7 [M + 3Na-6H ⁺ ] ^3-
366.5 [M + Na-5H ⁺ ] ⁴⁻
MS / MS m / z 97 [HSO ₄ ] ⁻ , 225 [L-fucose sulfate-H ₂ O—H ⁺ ] ⁻ , 243 [L-fucose sulfate −H ⁺ ] ⁻ , 345 [L-fucose disulfate + Na-2H _{^{+] -, 450 [M +}} 3Na-2SO 3 -6H +] 3-, 477 [M + 3Na-SO 3 -6H +] 3-, 563 [ unsaturated D- glucuronic acid and D- mannose L- fucose and sulfate binding -H ⁺ ] ⁻ , 705 [unsaturated D-glucuronic acid, D-mannose sulfate and L-fucose disulfate bonded together —H ₂ O—H ⁺ ] ^−
1 H-NMR (D ₂ O)
δ5.58 (1H, d, J = 3.4 Hz, (4) ″-H), 5.35 (1H, br-s, (1) -H), 5.22 (1H, d, J = 6) .7 Hz, (1) ″-H), 5.19 (1H, d, J = 3.7 Hz, 1′-H), 5.19 (1H, d, J = 3.7 Hz, (1) ′ − H), 5.16 (1H, d, J = 1.8 Hz, 1-H), 4.62 (1H, d, J = 7.6 Hz, 1 ″ -H), 4.50 (1H, m, 4'-H), 4.50 (1H, m, (4) '-H), 4.30 (1H, m, 5'-H), 4.30 (1H, m, (5)'-H ), 4.30 (1H, m, (5) -CH ₂ H), 4.25 (1H, m, 2'-H), 4.25 (1H, m, (2) -H), 4 .25 (1H, m, (2) ′-H), 4.20 (1H, m, (5) —CH ₂ H), 4. 18 (1H, m, 5-CH ₂ H), 4.16 (1H, m, (3) ″-H), 4.08 (1H, m, 5-CH ₂ H), 4.07 ( 1H, m, 2-H), 4.02 (1H, m, 3'-H), 4.02 (1H, m, (3) '-H), 3.85 (1H, m, 5-H) ), 3.85 (1H, m, (5) -H), 3.78 (1H, m, 3-H), 3.78 (1H, m, (3) -H), 3.76 (1H) , M, 4 "-H), 3.76 (1H, m, 5" -H), 3.75 (1H, m, 4-H), 3.75 (1H, m, (4) -H) 3.58 (1H, m, 3 ″ -H), 3.55 (1H, m, (2) ″-H), 3.18 (1H, t, J = 8.2 Hz, 2 ″ -H) , 1.10 (3H, d, J = 6.7Hz, (5) '- CH 3 of _{H 3), 1.09 (3H,} d, J 6.7 Hz, _H 3 of the 5'-CH ₃₎
Sugar composition L-fucose: unsaturated D-glucuronic acid: D-glucuronic acid: D-mannose = 2: 1: 1: 2 (two molecules each of L-fucose and D-mannose, unsaturated D-glucuronic acid and D- 1 molecule for each glucuronic acid)
6 molecules of sulfate group (2nd and 4th positions of each L-fucose and 6th position of each D-mannose)
In addition, the numbers of the peaks in ¹ H-NMR are as shown in the following formula (21).

Physical property molecular weight of (f) 644
MS m / z 687 [M + 2Na-3H ⁺ ] ⁻
MS / MS m / z 97 [HSO ₄ ] ⁻ , 243 [L-fucose sulfate-H ⁺ ] ⁻ , 421 [unsaturated D-glucuronic acid and D-mannose sulfate + Na—H ₂ O-2H ⁺ ] ^−
1 H-NMR (D ₂ 0)
δ 5.60 (1H, d, J = 3.4 Hz, 4 ″ -H), 5.24 (1H, br-s, 1-H), 5.08 (1H, d, J = 4.0 Hz, 1 '-H), 4.94 (1H, d, J = 6.7 Hz, 1 "-H), 4.45 (1H, dd, J = 3.1, 10.4 Hz, 3'-H) , 4.20 (1H, br-s , 2H), 4.14 (2H, m, 5-CH 2 of _{H 2), 4.14 (1H,} m, 5'-H), 4.09 , (1H, m, 3 ″ -H), 4.01 (1H, d, J = 3.1 Hz, 4′-H), 3.91 (1H, m, 5-H), 3.85 (1H , M, 3-H), 3.85 (1H, m, 2′-H), 3.75 (1H, t, J = 9.8 Hz, 4-H), 3.59 (1H, t, J = 6.7 Hz, 2 "-H), 1.06 (3H, d, J = 6.4 Hz, 5'-C) ₃ of _H 3)
Sugar composition L-fucose: unsaturated D-glucuronic acid: D-mannose
1: 1: 1 (1 molecule each of L-fucose, D-mannose and unsaturated D-glucuronic acid)
2 molecules of sulfate group (3rd position of L-fucose and 6th position of D-mannose)
In addition, the numbers of the peaks in ¹ H-NMR are as shown in the following formula (22).

Physical property molecular weight of (g) 632
MS m / z 631 [M−H ⁺ ] ⁻
MS / MS m / z 405 [D-galactose combined with L-fucose sulfate-H ⁺ ] ⁻ , 551 [L-fucose sulfate combined with L-fucose and D-galactose—H ⁺ ] ^−
1 H-NMR (D ₂ 0)
δ 5.15 (1H, d, J = 4.3 Hz, F ₁ 1-H), 4.93 (1H, d, J = 3.7 Hz, F ₂ 1-H), 4.53 (1H, d− d, J = 2.4, 10.4 Hz, F ₁ 3-H), 4.49 (1H, d, J = 7.6 Hz G ₁ 1-H), 4.46 (1H, dd, J = 3.1, 10.7 Hz, F ₂ 3-H), 4.36 (1H, q, J = 6.7 Hz F ₂ 5-H), 4.14 (1H, q, J = 6.7 Hz F) ₁ 5-H), 4.09 (1H, d, J = 2.4 Hz F ₁ 4-H), 4.03 (1H, d, J = 3.1 Hz F2 4-H), 3.97 (1H , Dd, J = 4.3, 10.4 Hz, F ₁ 2-H), 3.90 (1H, br-s, G ₁ 4-H), 3.81 (1H, dd, J _{= 3.7,10.7Hz, F 2 2-H} ), 3.59 ( _{H, m, G 1 3-} H), 3.59 (1H, m, G 1 5-H), 3.59 (2H, m, H 2 of _{G 1 5-CH 2),} 3.56 (1H _{, m, G 1 2-H} ), 1.19 (3H, d, J = 6.7Hz, F 1 5-CH 3 of _{H 3), 1.14 (3H,} d, J = 6.7Hz, F _{2 H} 3 of 5-CH ₃₎
Sugar composition L-fucose: D-galactose = 2: 1
(2 L-fucose molecules and 1 D-galactose molecule)
2 molecules of sulfate group (3rd position of each L-fucose)
In addition, the numbers of the peaks in ¹ H-NMR are as shown in the following formula (23).

(H) Physical property molecular weight 1358
MS m / z 1445 [M + 4Na-5H ⁺ ] ⁻
MS / MS m / z 97 [HSO ₄ ] ⁻ , 225 [L-fucose sulfate-H ₂ O—H ⁺ ] ⁻ , 1197 [M-2SO ₃ —H ⁺ ] ^−
1 H-NMR (D ₂ 0)
δ 5.19 (1H, d, J = 4.3 Hz, F ₁ 1-H), 4.93 (1H, d, J = 3.7 Hz, F ₂ 1-H), 4.62 (1H, m, G ₂ 1-H), 4.59 (1H, m, G ₁ 1-H), 4.54 (1H, dd, J = 2.7, 10.6 Hz, F ₁ 3-H), 4 .46 (1H, m, F ₂ 3-H), 4.46 (1H, d, J = 7.6 Hz G ₃ 1-H), 4.41 (1H, br-s, G ₁ 4-H) 4.41 (1H, d, J = 7.6 Hz, G ₄ 1-H), 4.37 (1H, q, J = 6.4 Hz 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 = 6.7 Hz, F ₁ 5-H), 4.09 (1H, d, J = 2.7 Hz, F ₁ 4-H), 4.04 (1H, d, J = 2. 8 Hz, F ₂ 4-H), 3.98 (H of 1H, m, G ₁ 5-CH ₂ ), 3.96 (1H, dd, J = 4.3, 10.6 Hz, F ₁ 2 _{-H), 3.88 (1H, br} -s, G 2 4-H), 3.93 (1H, m, G 3 of _{5-CH 2 H), 3.86} (1H, m, G 1 5 -H), 3.81 (1H, m, F ₂ 2-H), 3.81 (1H, m, G ₁ 5-CH ₂ H), 3.80 (1H, m, G ₃ 5-H ), 3.80 (H of 1H, m, G ₃ 5-CH ₂ ), 3.66 (1H, m, G ₂ 3-H), 3.65 (1H, m, G ₁ 2-H), 3.64 _{(1H, m,} of _{G 2 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 H), 3.56 (1H, m, G ₄ 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, H 3 of _{F 1 5-CH 3),} 1.14 ( _{3H, d, J = 6.4Hz,} H 3 of the F 2 5-CH ₃₎
Sugar composition L-fucose: D-galactose = 1: 2
(2 L-fucose molecules and 4 D-galactose molecules)
5 molecules of sulfate group (3-position of each L-fucose, 3-position of D-galactose at the reducing end, and 3-position of D-galactose bound to 6-position of D-galactose at the reducing end, and D-galactose thereof 3rd position of D-galactose bound to 6th position)
In addition, the assignment number of the peak in ¹ H-NMR is as the following formula (24).

Physical property molecular weight of (i) 1288
MS m / z 1149 [M + Na-2SO ₃ -2H ⁺ ] ⁻
MS / MS m / z 97 [HSO ₄ ] ⁻ , 345 [L-fucose disulfate + Na-2H ⁺ ] ⁻ ,
¹ H-NMR (D ₂ 0)
δ 5.68 (1H, d, J = 3.4 Hz, (4) ″-H), 5.34 (1H, br-s, (1) -H), 5.19 (1H, m, 1-H) ), 5.19 (1H, m, (1) '-H), 4.94 (1H, m, 1'-H), 4.94 (1H, d, J = 6.4 Hz, (1) " -H), 4.72 (1H, d, J = 7.9 Hz, 1 "-H), 4.54 (1H, m, (4) '-H), 4.48 (1H, dd, J = 3.3, 10.6 Hz, 3′-H), 4.38 (1H, q, J = 6.4 Hz, 5′-H), 4.34 (1H, q, J = 6.7 Hz, (5) ′-H), 4.29 (1H, m, (2) ′-H), 4.20 (1H, m, 5-CH ₂ H), 4.14 (1H, m, (2 ) -H), 4.13 (1H, m, (3) ″-H), 4.09 (1H, m, 5-CH ₂ H), 4.08 (1H, m, 2-H), 4.05 (1H, d, J = 3.3 Hz, 4'-H), 3.99 (1H, m, (3) '-H ), 3.89 (1H, m, 5-H), 3.83 (1H, m, 4 "-H), 3.81 (1H, m, 2'-H), 3.80 (1H, m) , 4-H), 3.78 (1H, m, 3-H), 3.78 (1H, m, (3) -H), 3.68 (2H, m, (5) -CH ₂ H ₂ ), 3.62 (1H, m, (2) ″-H), 3.60 (1H, m, 3 ″ -H), 3.60 (1H, m, 5 ″ -H), 3.58 (1H, m, (5) -H), 3.56 (1H, m, (4) -H), 3.17 (1H, t, J = 7.9 Hz, 2 ″ -H), 1.15 (3H, d, J = 6.4Hz , H 3 of the _{5'-CH 3), 1.11 (} 3H, d, J = 6.7Hz, 5) '- _H 3 of CH ₃₎
Sugar composition L-fucose: unsaturated D-glucuronic acid: D-glucuronic acid: D-mannose = 2: 1: 1: 2 (two molecules each of L-fucose and D-mannose, unsaturated D-glucuronic acid and D- 1 molecule for each glucuronic acid)
4 molecules of sulfate group (3-position of L-fucose bonded to D-mannose at the reducing end, 2-position and 4-position of the other L-fucose, and 6-position of D-mannose at the reducing end)
In addition, the numbers of the peaks in ¹ H-NMR are as shown in the following formula (25).

In addition, the sugar compound according to the first invention of the present invention caused a cell extract or culture supernatant obtained from a bacterium belonging to the genus Fucoidanobacter according to the third invention of the present invention to act on fucoidan. It can also be manufactured in some cases.
The strain according to the third invention of the present invention may be any strain as long as it belongs to the genus Fucoidanobacter, and specific examples of the strain include, for example, Fucoidanobacter marinus) SI-0098 strain.

This strain is a strain newly obtained by the present inventors from seawater in Aomori Prefecture, and its mycological properties are as follows.
2. Fucoidanobacter Marinas SI-0098
a. Morphological properties (1) The bacterium is a sphere to short gonococcus and sometimes takes the form of a diuretic bacterium.
Width 0.5-0.7μm
Length 0.5-0.7μm
(2) Presence or absence of spores None (3) Gram staining negative b. Physiological properties (1) Temperature range for growth Can grow at 37 ° C. A suitable growth temperature is 15 to 28 ° C.
(2) Attitude toward oxygen Aerobic (3) Catalase positive (4) Oxidase negative (5) Urease negative (6) Hydrolysis Starch positive Gelatin negative Casein negative Esculin positive (7) Nitrate reduction negative (8) Indole production negative (9) Hydrogen sulfide production Positive (10) Milk coagulation Negative (11) Sodium requirement Positive (12) Salt requirement Growth in 0% saline medium Negative Growth in 1% saline medium Negative Growth in seawater medium Positive (13) Quinones Menaquinone 7
(14) 61% GC content of intracellular DNA
(15) Cell wall diaminopimelic acid negative (16) Glycolyl test negative (17) Hydroxy fatty acid presence positive (18) OF-test O
(19) Colors of villages Not producing characteristic colony pigments (20) With mobility (21) Without gliding (22) Flagella Polar hair

  This strain is classified into group 4 (Gram-negative aerobic bacilli and cocci) according to the basic classification described in Virgie's Manual of Determinative Bacteriology, Volume 9 (1994). However, this strain has menaquinone 7 in the electron transport chain and is greatly different from bacteria belonging to Group 4 in that the GC content is 61%. Basically, gram-negative bacteria have ubiquinone in the electron transport chain, and gram-positive bacteria have menaquinone.

  The Gram-negative bacteria Flavobacterium and Cytophaga have exceptionally menaquinone in the electron transport chain, but the GC content of the bacteria belonging to these genera is the soil bacterium Cytophaga avenchicola (Cytophaga). arvensicola) 43-46%, marine bacteria Cytophaga diffluens, Cytophaga fermentans, Cytophaga marina (Cytophaga marina), and 42% The nature of is completely different. Furthermore, when the homology between the 16S rDNA sequence of this strain and the already identified strain was compared, the homology was also 76.6% in the already identified strain, Verrucomicrobium spinosum. . When the homology of the 16S rDNA sequence is 90% or less, it is generally known that the genus of both bacteria is different. Therefore, the present inventors are bacteria of a new genus that does not belong to the existing genus. Therefore, this strain was named Fucoidanobacter marinas SI-0098.

  The strain is indicated as Fucoidanobacter marinus SI-0098 and is registered with FERM BP at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry [Address: 1-3-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan] -5403 (original deposit date; March 29, 1995, request date for transfer to international deposit date; February 15, 1996).

  The saccharide compound of the first invention of the present invention is released by culturing the Fucoidanobacter genus bacteria according to the third invention of the present invention and allowing the cell extract or culture supernatant to act on fucoidan. Can be made. As a strain to be cultured, it belongs to the genus Fucoidanobacter, and if the bacterial cell extract or culture supernatant is allowed to act on fucoidan, the strain can obtain the sugar compound of the first invention of the present invention. Any strain may be used, and specific examples of the strain include Fucoidanobacter marinus SI-0098 strain.

  The nutrient source added to the medium of the Fucoidanobacter genus bacterial strain is utilized by the strain, and the bacterial extract is a fungal extract and a culture supernatant capable of producing the sugar compound of the first invention of the present invention from fucoidan. Any carbon source may be used, for example, fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, glycerol, saccharose, maltose, lactose, starch, etc., and nitrogen sources include yeast extract, peptone, Casamino acid, corn steep liquor, meat extract, defatted soybean, ammonium sulfate, ammonium chloride and the like are suitable. In addition, minerals such as sodium salt, phosphate, potassium salt, magnesium salt, zinc salt, and metal salts may be added.

  This strain grows very well in seawater or artificial seawater containing the above nutrients.

  In culturing Fucoidanobacter bacterium according to the third invention of the present invention, the culture temperature is generally 15 ° C. to 30 ° C., the pH of the medium is preferably 5 to 9, and the aerated stirring culture for 5 to 72 hours. Thus, the endo-type fucoidan-degrading enzyme activity of the bacterial cell extract and the culture supernatant reaches the maximum. It is a matter of course that the culture conditions are set so as to maximize the endo-fucoidan degradation activity of the intracellular extract and culture supernatant produced by the strain according to the strain to be used and the medium composition.

  Fucoidanobacter sp. SI-0098 strain is cultured in an appropriate medium, and after completion of the culture, the cells and the culture supernatant are obtained by centrifugation. Further, the obtained microbial cells can be obtained by centrifuging the cells after centrifuging the cells normally used, for example, by ultrasonic disruption.

  The culture supernatant or the bacterial cell extract concentrated by ultrafiltration is mixed with a phosphate buffer containing sodium chloride, and fucoidan is added thereto to react.

  After completion of the reaction, the reaction solution is fractionated using a column for molecular weight fractionation, whereby the sugar compound according to the first invention of the present invention can be obtained.

  The sugar compound according to the first invention of the present invention is useful as a reagent for sugar chain engineering. When a PA product is prepared by the method described in Japanese Patent Publication No. 5-65108 and a PA product is prepared, the sugar compound is used as a reagent for sugar chain engineering. A very useful material is provided. Furthermore, the sugar compound of the present invention is expected to be applied for physiological activity as an anticancer agent, a cancer metastasis inhibitor, and an antiviral agent.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for producing an example of a sugar compound according to the first invention of the present invention will be described by way of examples, but the present invention is limited only to the scope of the following examples. is not.

Example 1
Flavobacterium sp. SA-0082 (FERM BP-5402) was dispensed into 600 ml of a medium consisting of artificial seawater (made by Jamarin Laboratory) pH 7.5 containing 0.1% glucose, 1.0% peptone and 0.05% yeast extract. Sterilized (120 ° C., 20 minutes) into a 2-liter Erlenmeyer flask and cultured at 24 ° C. for 20 hours to form a seed culture solution. Artificial seawater (jamarin laboratory) pH7 containing fucoidan 0.3% derived from gagome kelp, 0.5% peptone, 0.01% yeast extract, and 0.01% antifoam (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) 20 liters of the medium consisting of 5 was placed in a 30 liter jar fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the above seed culture solution was inoculated and cultured at 24 ° C. for 20 hours under conditions of an aeration rate of 10 liters per minute and a stirring speed of 125 revolutions per minute. After completion of the culture, the culture solution was centrifuged to obtain bacterial cells and a culture solution supernatant.

  The culture supernatant was concentrated by ultrafiltration (exclusion molecular weight of filtration membrane 10,000) (manufactured by Amicon), and the endo-type fucoidan-degrading enzyme of the present invention was measured. As a result, an activity of 6 milliunit per 1 ml of the culture was detected. It was done.

  On the other hand, the cells obtained in the main culture were suspended in a 20 mM acetate-phosphate buffer solution (pH 7.5) containing 200 mM sodium chloride, subjected to ultrasonic disruption, and centrifuged to obtain a cell extract. . When the activity of the endo-type fucoidan-degrading enzyme of the present invention in this bacterial cell extract was measured, an activity of 20 mU was detected in 1 ml of the medium.

  Ammonium sulfate is added to the concentrated solution of the above culture supernatant so that the final concentration becomes 90% saturation, and after stirring and centrifuging, the precipitate is suspended in the same buffer as the above bacterial cell extract, and 50 mM Dialyzed thoroughly with 20 mM acetate-phosphate buffer (pH 7.5) containing sodium chloride. After removing the precipitate produced by dialysis by centrifugation, the precipitate is adsorbed on a column of DEAE-Sepharose FF equilibrated in advance with 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride, and the adsorbate is adsorbed on the same buffer. After sufficiently washing with the solution, the active fraction was collected by eluting with a sodium chloride gradient of 50 mM to 600 mM. Next, sodium chloride is added to the active fraction to a final concentration of 4M, and the active fraction is adsorbed to a column of phenyl sepharose CL-4B equilibrated in advance with 20 mM phosphate buffer (pH 8.0) containing 4M sodium chloride. The adsorbate was thoroughly washed with the same buffer and eluted with a 4M to 1M sodium chloride gradient to collect the active fraction. Next, this active fraction was concentrated with an ultrafilter (Amicon), and then gel filtered with Sephacryl S-200 equilibrated with a 10 mM phosphate buffer containing 50 mM sodium chloride to collect the active fraction. Next, sodium chloride was added to this active fraction to a final concentration of 3.5 M and adsorbed to a column of phenyl sepharose HP equilibrated in advance with 10 mM phosphate buffer (pH 8) containing 3.5 M sodium chloride. The adsorbed material was washed with the same buffer solution and eluted with a gradient of 3.5 M to 1.5 M sodium chloride, and the active fraction was collected to obtain a purified enzyme. When the molecular weight of this enzyme was determined from the retention time of Sephacryl S-200, it was about 70,000. The above purification steps are shown in Table 1.

Example 2
Flavobacterium sp. SA-0082 (FERM BP-5402) was dispensed with 600 ml of an artificial seawater (made by Jamarin Laboratory) pH 7.5 containing 0.25% glucose, 1.0% peptone and 0.05% yeast extract. Sterilized (120 ° C., 20 minutes) was inoculated into a 2-liter Erlenmeyer flask and cultured at 24 ° C. for 24 hours to obtain a seed culture solution. A medium consisting of artificial seawater (manufactured by Jamarin Laboratory) pH 7.5 containing 0.25% glucose, 1.0% peptone, 0.05% yeast extract, and 0.01% antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.) 20 liters was placed in a 30 liter jar fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the above seed culture solution was inoculated and cultured at 24 ° C. for 24 hours under conditions of an aeration rate of 10 liters per minute and a stirring rate of 125 revolutions per minute. After completion of the culture, the culture solution was centrifuged to obtain bacterial cells and a culture solution supernatant.

  The culture supernatant was concentrated by ultrafiltration (exclusion molecular weight of filtration membrane 10,000) (Amicon), and the endo-type fucoidan-degrading enzyme of the present invention was measured. As a result, 1 mU of activity per 1 ml of the culture was detected. .

  On the other hand, the cells obtained in the main culture were suspended in a 20 mM acetate-phosphate buffer solution (pH 7.5) containing 200 mM sodium chloride, subjected to ultrasonic disruption, and centrifuged to obtain a cell extract. . When the activity of the endo-type fucoidan-degrading enzyme of the present invention in this bacterial cell extract was measured, an activity of 5 mU was detected in 1 ml of the medium.

  To this extract, ammonium sulfate is added so that the final concentration becomes 90% saturation, and after stirring, the mixture is centrifuged. The precipitate is suspended in the same buffer as the above-mentioned cell extract, and 20 mM acetic acid containing 50 mM sodium chloride is added. -Fully dialyzed against phosphate buffer (pH 7.5). After removing the precipitate produced by dialysis by centrifugation, the precipitate is adsorbed on a column of DEAE-Sepharose FF equilibrated in advance with 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM sodium chloride, and the adsorbate is adsorbed on the same buffer. After sufficiently washing with the solution, the active fraction was collected by eluting with a sodium chloride gradient of 50 mM to 600 mM. Next, sodium chloride is added to the active fraction to a final concentration of 4M, and the active fraction is adsorbed to a column of phenyl sepharose CL-4B equilibrated in advance with 20 mM phosphate buffer (pH 8.0) containing 4M sodium chloride. The adsorbate was thoroughly washed with the same buffer and eluted with a 4M to 1M sodium chloride gradient to collect the active fraction. Next, this active fraction was concentrated with an ultrafilter and then subjected to gel filtration with Sephacryl S-300 equilibrated in advance with 10 mM phosphate buffer containing 50 mM sodium chloride to collect the active fraction. When the molecular weight of this enzyme was determined from the retention time of Sephacryl S-300, it was about 460,000. Next, this active fraction was dialyzed against 10 mM phosphate buffer (pH 7) containing 25 mM sodium chloride. This enzyme solution was adsorbed on a column of Mono Q HR 5/5 previously equilibrated with 10 mM phosphate buffer (pH 7) containing 250 mM sodium chloride, and the adsorbate was sufficiently washed with the same buffer, and then 250 mM. And eluted with a gradient of 450 mM sodium chloride, and the active fractions were collected to obtain a purified enzyme. The above purification steps are shown in Table 2.

Example 3
The endo-type fucoidan degrading enzyme (intracellular enzyme) obtained in Example 1 of the present invention was allowed to act on the purified gagome kelp-derived fucoidan to prepare a degradation product.
Specifically, 16 ml of a 2.5% fucoidan solution, 12 ml of 50 mM phosphate buffer (pH 7.5), 4 ml of 4M sodium chloride, and 8 ml of the endo-type fucoidan degrading enzyme solution of the present invention of 32 mU / ml were mixed. The reaction was carried out at 0 ° C. for 48 hours.
The reaction solution was subjected to molecular weight fractionation using a column of Cellulofine GCL-300 (manufactured by Seikagaku Corporation), and fractions having a molecular weight of 2000 or less were collected. This fraction was desalted with a microacylator G3 (manufactured by Asahi Kasei Co., Ltd.) and then separated into three fractions with DEAE-Sepharose FF. As a result, 41 mg, 69 mg, and 9.6 mg of the substances (a), (b), and (c) described above were obtained, respectively.

Example 4
The end product fucoidan-degrading enzyme (extracellular enzyme) obtained in Example 2 of the present invention was allowed to act on the purified gagome kelp-derived fucoidan to prepare a degradation product.

  Specifically, 16 ml of a 2.5% fucoidan solution, 12 ml of 50 mM phosphate buffer (pH 7.5), 4 ml of 4M sodium chloride, and 8 ml of the endo-type fucoidan degrading enzyme solution of the present invention of 32 mU / ml were mixed. The reaction was carried out at 0 ° C. for 48 hours.

  The reaction solution was subjected to molecular weight fractionation using a column of Cellulofine GCL-300 (manufactured by Seikagaku Corporation), and fractions having a molecular weight of 2000 or less were collected. This fraction was desalted with a microacylator G3 (manufactured by Asahi Kasei Co., Ltd.), and then the three fractions were separated with DEAE-Sepharose FF and freeze-dried. As a result, 40 mg, 65 mg, and 9.2 mg of the substances (a), (b), and (c) described above were obtained, respectively.

Example 5
Fucoidanobacter marinas SI-0098 strain (FERM BP-5403) was added to 0.3% fucoidan derived from Gagome kelp, 0.5% peptone, 0.05% yeast extract, and anti-foaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.). ) Artificial seawater containing 0.01% (manufactured by Jamarin Laboratory) 600 ml of medium consisting of pH 7.5 was placed in a 2 liter Erlenmeyer flask and inoculated into a medium sterilized at 120 ° C. for 20 minutes, and then at 25 ° C. for 48 hours. Incubation was carried out at a stirring speed of 120 minutes per minute. After completion of the culture, the culture solution was centrifuged to obtain bacterial cells and a culture solution supernatant.

  The culture supernatant was concentrated by ultrafiltration (excluded molecular weight of filtration membrane 10,000) (manufactured by Amicon), and the endo-type fucoidan-degrading enzyme of the present invention was measured. As a result, 0.2 mU activity per 1 ml of the culture was detected. It was.

  On the other hand, the cells obtained in the main culture were suspended in a 20 mM acetate-phosphate buffer solution (pH 7.5) containing 200 mM sodium chloride, subjected to ultrasonic disruption, and centrifuged to obtain a cell extract. . When the activity of the endo-type fucoidan-degrading enzyme of the present invention in this bacterial cell extract was measured, an activity of 20 mU was detected in 1 ml of the medium.

Example 6
A degradation product was prepared by reacting the purified fucoidan derived from Gagome kelp with the intracellular enzyme of Fucoidobacter marinas SI-0098 obtained in Example 5 of the present invention.

  That is, 16 ml of 2.5% fucoidan solution, 20 ml of 100 mM phosphate buffer (pH 8.0) containing 800 mM sodium chloride, and 20 mU / ml of Fucoid Novacter marinas obtained in Example 5 of the present invention. 4 ml of intracellular enzyme solution of SI-0098 strain was mixed and reacted at 25 ° C. for 48 hours.

  The reaction solution was subjected to molecular weight fractionation using a cellulofine GCL-300 column, and fractions having a molecular weight of 2000 or less were collected. This fraction was desalted with a microacylator G3, and then three fractions were separated with DEAE-Sepharose FF and freeze-dried. As a result, 38 mg, 60 mg, and 8.2 mg of the aforementioned substances (a), (b), and (c) were obtained, respectively.

  According to the present invention, a novel end useful for research on fucoidan, such as structural analysis of fucoidan, identification of enzymatic degradation product of fucoidan, and sugar compounds that can be used for bioactivity search, partial decomposition of fucoidan and production of fucoidan oligosaccharide. Novel microorganisms useful for the production of types of fucoidan degrading enzymes and sugar compounds have been provided.

It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-a) of a sugar compound (a) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-b) of a saccharide | sugar compound (b) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-c) of a saccharide | sugar compound (c) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-d) of a saccharide | sugar compound (d) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-e) of a saccharide | sugar compound (e) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-f) of a saccharide | sugar compound (f) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-g) of a saccharide | sugar compound (g) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-h) of sugar compound (h) is isolate | separated with an L-column. It is a figure which shows the elution pattern when the pyridyl- (2) -aminated sugar compound (PA-i) of sugar compound (i) is isolate | separated with an L-column. It is a figure which shows the result obtained by the mass analysis (negative measurement) of the sugar compound (a). It is a figure which shows the result obtained by the mass analysis (negative measurement) of a sugar compound (b). It is a figure which shows the result obtained by the mass analysis (negative measurement) of a sugar compound (c). It is a figure which shows the result obtained by the mass analysis (negative measurement) of a sugar compound (d). It is a figure which shows the result obtained by the mass analysis (negative measurement) of the sugar compound (e). It is a figure which shows the result obtained by the mass analysis (negative measurement) of a sugar compound (f). It is a figure which shows the result obtained by the mass analysis (negative measurement) of a sugar compound (g). It is a figure which shows the result obtained by the mass analysis (negative measurement) of a sugar compound (h). It is a figure which shows the result obtained by the mass analysis (negative measurement) of sugar compound (i). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (a). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (b). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (c). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (d). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (e). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (f). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (g). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of a sugar compound (h). It is a figure which shows the result obtained by the mass-mass analysis (negative measurement) of sugar compound (i). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (a). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (b). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (c). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (d). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (e). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (e). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (g). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (h). It is a diagram showing ¹ H-NMR spectrum of the sugar compound (i). It is a figure showing the relationship of pH and relative activity (%) of the enzyme which is 2nd invention of this invention. It is a figure showing the relationship between the reaction temperature (degreeC) and relative activity (%) of the enzyme which is 2nd invention of this invention. It is a figure showing the relationship between pH which processed the enzyme which is 2nd invention of this invention, and residual activity (%). It is a figure showing the relationship between the processing temperature (degreeC) and residual activity (%) of the enzyme which is 2nd invention of this invention. It is a figure which shows the elution pattern when sugar compound (a)-(i) is isolate | separated by DEAE-Sepharose FF. It is a figure which shows the elution pattern when sugar compounds (h) and (i) are isolate | separated by DEAE-Sepharose FF.

Claims (3)

The following general formula (1):

[Wherein X is H or the following formula (2):

Wherein Y represents an alcoholic hydroxyl group or a sulfated alcoholic hydroxyl group] or a salt thereof. A sugar compound represented by the following formula (13) or a salt thereof.

A sugar compound represented by the following formula (14) or a salt thereof.

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