TWI243853B - Glucuronofucan sulfate - Google Patents

Abstract

Three enzymes degrading sulfated glucuronofucan; processes for producing these enzymes; activators for these enzymes; glucuronofucan sulfate and degradation products thereof; and a novel microorganism.

Description

1243853 A7 B7 V. Description of the invention (1) Technical scope The present invention relates to a novel microorganism that can be used to produce enzymes capable of decomposing various sulfated polysaccharides contained in brown algae, and can be used in the field of sugar chain engineering. An enzyme that decomposes sulfated glucuronidated fucoidan, and a method for manufacturing the enzyme, which activates various factors of the enzyme, can be used as a deacetylation and deglucosylation of sugar chain engineering reagents Sulfated glucuronidated fucoidan and sulfated glucuronidated fucoidan, and methods for their production. Background Art Brown algae contains various sulfated polysaccharides. For example, ① sulfated fucose formed only from fucose and sulfate groups, and ② sulfated glucuronide fucose containing glucuronic acid, mannose, fucose, and sulfate groups are known Glycans, for example, polysaccharide-U containing fucose sulfuric acid described in the W097 / 26896 gazette (the constituent sugars and their molar ratios are fucose, mannose, galactose: glucuronic acid: sulfate group = about 1 0 · _ 7: 4: 5 ·· 2 0, hereinafter referred to as U-fucoidan), ③ sulfated fucoidan formed from fucose and galactose, such as PCT / JP00 / 00965 The sulfated fucose galactan (the constituent sugar and its molar ratio is fucose · galactose = 1: 1 ~ 6, hereinafter referred to as G-fucoid Sugar) and other polysaccharides containing sulfated fucose. These polysaccharides are collectively referred to as fucoidan or fucoidan, and their structure varies depending on the seaweed from which they are derived. For example, it is known that sulfated polysaccharides extracted separately from females of Haiba Mata, Kumbu, Okinawa algae, algae or Wakame have different structures. Moreover, these polysaccharides containing sulfated fucose, -4- This paper size applies to the Chinese National Standard (CNS) A4 specifications (210X 297 mm) A7 B7 1243853 V. Description of the invention (3) The identified isolates produce sulfated polysaccharide-degrading enzymes, or produce several sulfated polysaccharide-degrading enzymes from one strain. Furthermore, in a recent study on the sulfated polysaccharide of Okinawa algae, it was reported that the polysaccharide can inhibit the fixation of the bacterium Helicobacter pylori causing gastric ulcer in the gastric mucosa. The complex of polysaccharide and fibroblast proliferation factor can be used as Fibroblast proliferation promoter and oral administration of the polysaccharide can prevent infections caused by bacteria and viruses. Therefore, the physiological activity of Okinawa algae and the chemical structure of sulfated polysaccharides derived from algae of Okinawa are under study. For example, there are two reports of this polysaccharide, one is the sulfated glucose while acidifying fucans described in Glycoconjugate Journal, Vol. 16, pages 19-26 (1999), where fucose: glucose is acidic: Molar ratio of sulfate group: ethyl group is 6.1: 1.0: 2.9: 1, molecular weight is about 56,000; the other is sulfated glucose described in Applied Glycan Science Vol. 43, 143-148 (1996) Alkylated fucose, in which the molar ratio of fucose: glucuronic acid: xylose: sulfate group: acetamyl group is 3 ~ 4: 0.8 ~ 1.2: 0 · 1 ~ 0.3: 0.8 ~ 1.2 ... 0.5 ~ 1, molecular weight is about 500,000 ~ 600,000. However, with today's physical and chemical analysis, the average structure is judged. Furthermore, for the development of pharmaceuticals, oligosaccharides that can maintain a uniform structure must be obtained. It is very difficult for polysaccharides to obtain oligosaccharides with uniform structure. Because of the above, there is an urgent need for microorganisms that can decompose sulfated polysaccharides from various brown algae, and specifically decompose enzymes of sulfated polysaccharides (that is, sulfated glucuronidated fucose) from marine algae in Okinawa, And Enzyme-6- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Explanation of the invention Glucuronidated Fucoidan and sulfated glucuronidated Fucoid The oligosaccharide is hydrolyzed to dissociate the acetamyl group to make the acetic acid free. The optimal p Η is in the range of about 6 to 9.1, and the optimal temperature is in the range of about 2 3 to 4 5. The second invention of the present invention relates to a "_d-glucose acidase", which is characterized by the following physical and chemical properties: the enzyme acts on deacetylated sulfated gluconofucoid and deacetylated Sulfated glucuronidation of fucoxanthin 'and the hydrolysis of α-D-glucosaccharic acid-based bond to dissociate d-glucoside acid', its optimal pH is about 5 · 8 ~ 7 The range of 8 and the optimal temperature is in the range of about 14 to 2 9 ° C. The third invention of the present invention relates to an endo_q_L_fucosylase, which is characterized by having the following physicochemical properties. The enzyme acts on deacetylated sulfated gluconate and then acidified fucoidan and deacetylated sulfated glucuronidated fucoidan to decompose α-L-fucosyl hydrolyzed inwardly. An oligosaccharide having L-fucose at the reducing end is produced, and the optimum pH is in the range of about 4.5 to 7.5, and the optimum temperature is in the range of about 23 to 4 2 t. In the invention, fucoidan deacetylase, aD-glucuronidase, and endo-α-L-fucosidase can be cultivated by culturing Then, the enzyme is obtained from the culture and produced. The fourth invention of the present invention relates to a method for producing deacetylated sulfated glucuronidated fucoidan, which is characterized by making the rock of the first invention of the present invention Fucoidan deacetylase acts on sulfated glucuronidated fucoidan to obtain a deacetylated sulfated glucuronidated fuctan that removes at least one molecule of ethyl amidinyl group. In the fourth invention, deacetylation can be performed on sodium chloride, calcium salts, and / -8-This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm)

Binding

1243853 A7 B7 5. Description of the invention (6) or coexistence of protein. The fifth invention of the present invention relates to a deacetylated sulfated glucuronidated fucoidan or a salt thereof, which is obtained by the method of the fourth invention of the present invention. The sixth invention of the present invention relates to a method of A method for producing deacetylated deglucouronated sulfated glucuronidated fucoidan is characterized in that a-D-glucuronidase of the second invention of the present invention acts on deacetylated sulfated glucose Uric acid and rock fucoidan are obtained to remove at least one molecule of glucuronic acid residues, and deacetylated and deglucourated sulfated glucuronidated fucose. In the sixth aspect of the present invention, the deglucuronidation can be performed in the coexistence of sodium chloride, calcium salt, and / or protein. The seventh invention of the present invention relates to a deacetylated deglucouronated sulfonated glucuronidated fucoidan or a salt thereof, which is obtained by the method of the sixth invention of the present invention. The eighth invention of the present invention relates to a method for producing a sulfated glucuronidated fucoidan oligosaccharide, which is characterized in that fucoidan deacetylase, pan-D-glucuronidase, and Endo-a-L-fucose enzyme acts on sulfated glucuronidated fucose to obtain sulfated glucuronidated fucoidan. In this case, fucoidan deacetylase, π-D-glucosaminidase and endo-fucosinase can act simultaneously, and fucoidan deacetylase, a -D-Glucosidase and endo-a-L-fucosylase act in sequence. The ninth invention of the present invention relates to a method for producing a sulfated glucuronidated fuco-oligosaccharide, which is characterized in that π-D-glucuronidase and endo-α--9- China National Standard (CNS) Α4 specification (210X297 mm)

Pretend

1243853 A7 _____ B7_______ 5. Description of the invention (7) L-fucosidase acts on alkali-treated deacetylated sulfated glucuronidated fucoidan to obtain sulfated glucuronidated fucoidan . In this case, 'a-D-glucuronidase and endo-a-L-fucosinase can be simultaneously acted on'. Alternatively, α-D-glucuronidase can be used, and then -Take -L-Fucose enzyme. The eighth and ninth inventions of the present invention can be carried out in the coexistence of sodium chloride, calcium salt and / or protein. The tenth invention of the present invention relates to a sulfated glucuronidated rock drug oligosaccharide or a salt thereof, which is obtained by the method of the eighth to ninth inventions of the present invention. The 11th invention of the present invention relates to a sugar compound having a chemical structure selected from the following general formulae (I) to (111) ...

Binding

-10- This paper size applies to China National Standard (CNS) A4 specifications (210X297 male f) 1243853 A7 B7 V. Description of the invention (8)

This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 1243853 A7 B7 V. Description of the invention (

〔I 2

X

OH.H

〇 ° X 0H

2 This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm).

This paper size applies to China National Standard (CNS) A4 (210x 297 mm) 1243853 A7 B7 V. Description of the invention (11) (where R is Η, S03H or CH3CO; and η is an integer greater than 0). The twelfth invention of the present invention relates to a sulfated glucuronidated fucoidan or a salt thereof, which is characterized by the following physical and chemical properties: The sulfated polysaccharide contains fucose and glucoaldehyde as constituent sugars For acid, its molar ratio is 3 5 ~ 44: 10. For example, the sulfated sugar represented by the following general formula (VIII) is used as an essential component of sugar: -14- This paper size applies Chinese National Standard (CNS) Α4 specifications (210X297 mm) 1243853 A7 B7 V. Description of the invention (12

This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1243853 A7 B7 V. Description of the invention (14) Figure 7 shows the sulfated glucuronidated fucoidan i_ obtained by the present invention i _ ( 1) 1 H_NMR spectrum. FIG. 8 shows a 13C-NMR spectrum chart of the sulfated glucosyl acid-fucose oligosaccharide i_ (1) obtained by the present invention. FIG. 9 shows a mass analysis spectrum (mass spectrogram) of the sulfated glucuronidated fuctooligosaccharide i_ (1) obtained by the present invention. FIG. 10 shows a mass analysis spectrum (mass spectrum) chart of the sulfated glucuronidated fuctooligosaccharide _ (2) obtained by the present invention. FIG. 11 shows a 1 H-NMR spectrum chart of the sulfated glucuronidated fuco-oligosaccharide iO) obtained by the present invention. Fig. 12 shows an I3c-NMR spectrum chart of the sulfated glucuronidated fuco-oligosaccharide 1- (3) obtained by the present invention. Fig. 13 shows a mass spectrometry (mass spectrum) chart of the sulfated glucose obtained by the present invention while acidifying the fucoidan oligosaccharide 1-(3). Fig. 14 shows a mass spectrometry (mass spectrum) chart of the sulfated glucuronidated fuctooligosaccharide 1- (4) obtained by the present invention. Fig. 15 shows a 1H-NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 1- (5) obtained by the present invention. FIG. 16 shows a 13 NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 1- (5) obtained by the present invention. FIG. 17 shows a mass analysis spectrum (mass spectrum) of the sulfated glucuronidated fuco-oligosaccharide h (5) obtained by the present invention. FIG. 18 shows a mass analysis spectrum (mass spectrum) of the sulfated glucuronidated fuco-oligosaccharide 1_ (6) obtained by the present invention. -17- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1243853 A7 B7 V. Description of the invention (15) Figure 19 shows the sulfated glucuronidated fucoid oligomer obtained by the present invention 1 H-NMR spectrum of sugar 1- (7). Fig. 20 shows a 13C_NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 1- (7) obtained by the present invention. FIG. 21 shows a mass analysis spectrum (mass spectrum) of the sulfated glucuronidated fuco-oligosaccharide ^ (7) obtained by the present invention. Fig. 22 shows a mass analysis spectrum (mass spectrum) of the sulfated glucuronidated fuco-oligosaccharide ^ (8) obtained by the present invention. Fig. 23 shows the 1H-NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 2- (3) obtained by the present invention. Fig. 24 shows a 13C-NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 2- (3) obtained by the present invention. Fig. 25 shows a mass analysis spectrum (mass spectrum) of the sulfated glucuronidated fuctooligosaccharide 2- (3) obtained by the present invention. Fig. 26 shows a 1H-NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 2- (5) obtained by the present invention. Fig. 27 shows a 13C-NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 2- (5) obtained by the present invention. Fig. 28 shows a mass analysis spectrum (mass spectrogram) of the sulfated glucuronidated fucoidan low molecular compound obtained by the present invention, namely, oligosaccharide 2- (5). Fig. 29 shows the 1H-NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide 3- (3) obtained by the present invention. FIG. 30 shows a C-NMR spectrum chart of the sulfated glucuronidated fuctooligosaccharide J-(J) obtained by the present invention. -18- The paper size is suitable for the National Suzuka Standard (CNS) A4 specification (; 297 public love)

Binding

1243853

AT

The bacteria listed below have the bacteriological properties such as a. Morphological properties The bacteria are the presence or absence of cocci spores with a diameter of 1.2 ~ 1.6 # m. · No Gram staining: negative b · Physical properties (1 ) Reproduction temperature ·· Reproduction at 25 ° C (2) Preference for oxygen: aerobic (3) catalase: positive (4) oxidase: negative (5) salt requirement: reproduction in 0% salt medium : Reproduction negative in 1% salt medium: Reproduction negative in seawater medium: positive (6) g Kun line: methyl hydrazine 7 (7) GC content of DNA in the bacteria 52% (8) 〇F Test ·· No acid generation (9) Tone of the community: Does not produce characteristic community pigments (10) Mobility: Negative '(1 1) Slip-out: Negative (12) Flagella: None Handbook of Bacteriology (Bergey, Manuai 〇f

Determinatlve Bacteri.丨 〇gy) The basic taxonomy described in Volume 9 Π " 4) can be classified as Category 4 (Grann-negative aerobic bacteria and bacteria). However -21-This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) 1243853 A7 B7

This paper size applies to Chinese National Standard (CNS) A4 (210 x 297 mm) 1243853 A7 B7 V. Description of the invention (22) (where R is Η, S03H or CH3CO). Furthermore, the molecular weight, sugar composition, and sulfate group content of the sulfated glucuronidated fucoidan, along with the harvest time of the sulfated glucuronidated fucoidan raw material, the drying method of the material, and the raw material The preservation method, the heating conditions during the extraction of the sulfated glucuronidated fucoidan, or the pΗ conditions are different. For example, there is a case where sulfated glucuronidated fucoidan is hydrolyzed by acid. Therefore, the molecular weight, molecular weight distribution, sugar composition, or sulfate group content of the sulfated glucuronidated fucoidan described in this specification are just examples, and the sulfated glucuronidated fucoidan is described as an example. The extraction treatment conditions can easily change its molecular weight, molecular weight distribution, sugar composition, or sulfuric acid group content. For example, if extracted from Okinawa algae by an extraction method at pH 6.0, 95 ° C, and 2 hours, a sulfated glucuronidated fucoidan having the above molecular weight and sugar composition can be obtained. That is, by adjusting the conditions at the time of preparation, a sulfated glucuronidated fucose having an arbitrary molecular weight, molecular weight distribution, sugar composition, or sulfate group content can be prepared. For example, the main constituent sugars of the above-mentioned sulfated glucuronidated fucose, although the average fucose contains about 2 sulfate residues, but in general, the sulfate-based chemistry of ester binding on sugars Sexual instability, easily cut off by acid, alkali or heat. For example, if heat treatment is performed under acidic or alkaline conditions, the sulfuric acid content or acetamidine content will decrease. That is, it is possible to desulfurize or deacetylate the sulfated glucuronidated fucoidan. In addition, when desulfurizing or deacetylating, adjusting the types and concentrations of acids and bases, and the temperature and time during heat treatment, the amount of sulfuric acid groups and acetic acid groups can be adjusted. Although there is no special limitation, for example, with about 0.5 ~ 1 N sodium hydroxide-25- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention ( 23) The deacetylated sulfated glucuronidated fucoidan obtained by treating the solution at 25 ° C for 24 hours is suitable as the α-D-glucosidase or endo-a-L- The substrate of fucose enzyme. Therefore, the sulfated glucuronidated fucans in this specification include the sulfated glucuronidated fucans having the aforementioned characteristics, or they are decomposed by the sulfated glucuronidated fucans of the present invention. Enzymes and low-molecular sulfated glucuronidated fucans are all derived from brown algae. Although its source is not particularly limited, for example, brown algae of the genus Pinnaceae, such as Okinawa sea algae, stone sea algae, rough sea algae, rough sea algae, etc., due to the large content of sulfated glucuronidated fucoidan Therefore, it is particularly suitable as a raw material. For example, sulfated polysaccharides from algae in Okinawa, because their main chain is formed by L-fucose, which is weaker in acid resistance than ordinary sugars, so it is considered to be extracted with hydrochloric acid such as pΗ 3.0 and 0.2N. Will cause low molecular weight of sulfated polysaccharides. That is, the sulfated polysaccharide derived from the seaweed of Okinawa, like other polysaccharides mainly composed of sulfated L-fucose, can be easily lowered by heating or acid treatment. The sulfated glucuronidated fucoidan of the present invention has a sulfate group and / or a carboxyl group in the molecule, and the group can react with various bases to form a salt. The state of the salt formed by the sulfonated glucuronidated fucoidan of the present invention is stable, and it is usually provided in the form of a salt such as sodium and / or potassium and / or calcium. The salts of these substances can be introduced into the free sulfated glucuronidated fucoidan of the present invention using a cation exchange resin such as Dowex 50W. Furthermore, they can perform a known salt exchange method as necessary to convert them into various desired salts. The salt of sulfated glucuronidated fucoidan of the present invention can be used in medicine. 26- This paper size is applicable to Chinese National Standard (CNS) A4 specifications (210X297 mm) 1243853 A7 B7 V. Description of the invention (24) Permissible salts, such as metals such as sodium and potassium, salts such as bow, magnesium and zinc, and ammonium. When producing the sulfated glucuronidated fucoidan used in the present invention, a water-soluble soluble extract of brown algae is first obtained. At this time, in order to prevent the low-molecularization of the sulfated gluconic acid fucoidan, it is preferable to use a water-soluble solvent extract solution obtained at a pH of 4 to 9 and a temperature of 100 ° C or lower. Furthermore, amino acids, low-molecular-weight pigments, and the like in the above-mentioned extracts can be effectively removed by the ultra-fluoridation method. Furthermore, activated carbon treatment and the like are effective in removing the hydrophobic substance. In this way, a sulfated polysaccharide solution from brown algae can be obtained. Further, if this solvent is separated by an anion exchange column, a sulfated glucuronidated fucoidan having a higher purity can be obtained. The sulfated glucuronidated fucoidan obtained by the above method showed a hair-growing effect. Therefore, for example, it can be used as a component of a hair growth agent. Furthermore, the sulfated polysaccharide solution obtained by the above method or the sulfated glucuronidated fucoidan refined through an anion exchange column can be used as the α-D-glucosaccharinase and Endo-a-L-fucose fenase substrate. Furthermore, the fucoidan deacetylase, α-D-glucosidase, and endo-a-L-fucosinase of the present invention can be used as a substrate for measuring activity during purification, Alternatively, it can also be used to manufacture deacetylated sulfated glucuronidated fucoidan, deacetylated deglucuronated sulfated glucuronidated fucoidan, and sulfated glucuronidated fucoidan. Raw materials for oligosaccharides. The sulfated glucuronidated fucoidan in this specification means an oligosaccharide generated from the sulfurized glucuronidated fucoidan. Although it basically contains sulfate group, glucuronyl group, fucosyl group, and acetamyl group, there are also only -27- This paper size is applicable to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (25) The situation formed by sulfate group and fucose group; from the case of sulfated glucuronidated fucose, it is called sulfated glucuronidated fucoidan. The fucoidan deacetylating enzyme in the present invention refers to the action of sulfated glucuronidated fucoidan and sulfated glucuronidated fuctooligosaccharide to hydrolyze the acetamyl group. An enzyme that releases acetic acid. The physical and chemical properties of the fucoidan deacetylase of the present invention are as follows. (I) Action: It acts on sulfated glucono-acid fucoidan and sulfated gluco-acid fucoidan, etc., to hydrolyze the acetamyl group and free acetic acid. (II) Optimum pH: The optimum pH of this enzyme is in the range of about 6 to 9.1 (Figure 1). That is, Fig. 1 is a graph showing the relationship between pΗ 値 and relative activity during the reaction of the present enzyme, in which the vertical axis represents relative activity (0 / 〇) and the horizontal axis represents pH 値. (III) Optimum temperature: The optimal temperature of this enzyme is in the range of about 2 3 ~ 4 5 t (Figure 2). That is, Fig. 2 is a graph showing the relationship between temperature and relative activity during the reaction of the present enzyme, wherein the vertical axis represents relative activity (%) and the horizontal axis represents temperature (rc). (IV) Molecular weight: When measured by gel filtration, it is about 30,000 to 50,000. The confirmation of fucoidan deacetylase of the present invention was performed by measuring the degradation activity of a sulfonated glucuronidated fuctooligosaccharide having an acetamyl group and its reducing terminal group with a fluorescently labeled compound. get on. By using these fluorescently labeled oligosaccharides, a trace and high-sensitivity activity measurement system can be constructed. In addition, the fumaran deacetylase was allowed to act on sulfated glucosamine acidified fucoid -28-

1243853 V. Description of the invention (26 After the oligosaccharide, the sugar product acidified rock of the reaction product is used to separate the oligosaccharide and the amount of sulfated glucose, or the reaction is performed to generate the amount of acetic acid and the Bacterial enzyme activity. The present invention === can confirm the rock drug .. ,, heart stone / lysin deacetylase can be used to produce cell-free extracts of bacteria or by various column chromatography The purified enzyme solution is measured. ::: In the application state, the deethylated enzyme of the present invention can be used on gasified steel and / or eggs and played outside right ^ ^ _ _ early alone or hunting by a combination of the two to sleep First of all, the explanation of gasification sodium, the gasification sodium of the test reagent, food two, mother 7, artificial seawater, etc. can be used regardless of any substance as long as it contains sodium chloride. Add to the deacetylation enzyme reaction solution of the present invention The gasification sodium concentration is preferably in the range of ^ • ^ he rhyme, and the range of sac ~ cafu is more preferable. Then, the protein is explained to activate the fucoidan deacetylase of the present invention. For the purpose of adding the protein to the reaction solution, as long as it does not decompose the fucoidan deacetylase of the present invention, Any protein can be used without hindering its reaction. Among them, bovine serum white and white are preferred. Furthermore, by way of example, fucoidan can be degraded from the rock of the present invention by fucoidan SI- Protein extracted from the bacterial cell of strain 1234. The concentration of protein added to the fucoxan deacetylase reaction solution of the present invention is preferably in the range of 0.001 to 10 mg / ml, and 0.001 to 丨The range of mg / ml is more preferable. In the present invention, -D • glucuronidase refers to deacetylated sulfated glucuronide and fucoidan and deacetylated sulfated glucanase. Uronic acid-29 This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) Binding 1243853 A7 ______ B7 V. Invention 27—) '' 'Chemical fucoidan, etc. The α-D-glucuronic acid bond between the acid and fucose is hydrolyzed to make the enzyme free of D-glucuronic acid. The physicochemical properties of the a-D-glucosaminidase of the present invention are as follows. (I) Action ·· It acts on deacetylated sulfated glucuronidated fucoidan and deethylated sulfated glucuronidated fuctooligosaccharide, etc., while -glucose is bonded to the acid group It is hydrolyzed to make D-glucuronic acid free. (II) Optimum pH: The optimal pH of this enzyme is in the range of about 5.8 to 7.8 (Figure 3). That is, Fig. 3 is a graph showing the relationship between ρ η and relative activity when the present enzyme reacts, wherein the vertical axis represents relative activity (%), and the horizontal axis represents ρ Η 値. (III) Optimum temperature: The optimal temperature of this enzyme is in the range of about 14 ~ 29 ° C (Figure 4). That is, FIG. 4 is a graph showing the relationship between temperature and relative activity during the reaction of the present enzyme, wherein the vertical axis represents relative activity (%), and the horizontal axis represents temperature (° C). (IV) Molecular weight: by gel During filtration measurement, it was about 120,000 to 180,000. Confirmation of the a-D-glucosaccharinase of the present invention can be carried out by measuring the degradation activity of the deacetylated sulfated glucuronide fucose. Furthermore, a compound labeled with sulfated glucuronidated fuco-oligosaccharide and its reducing end group by 2-aminopyridine fluorescence can also be used as the substrate of this enzyme, and by using these fluorescently labeled oligosaccharides Glycan can build a trace and high sensitivity activity measurement system. After sulfated glucosyl acid and fuco-oligosaccharides react with pan-D_glucuronidase, the glucuronic acid and sulfated glucuronidated fucoidan contained in the reaction product are separated and separated by Determination of total sugar content and total sugar content «30- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1243853 A7 ______B7 V. Description of the invention (28 一) " ~-Acid content, and by The mass analysis of the reaction product can also confirm the activity of "D_ 匍 glucosidase. The activity of the glucuronidase of the present invention can also be obtained by the cell-free extract of the producing bacteria or by various column layers. The enzyme solution after purification is measured. In one embodiment, the slave-D-glucosidase of the present invention can be activated in the presence of sodium gas, calcium ion and / or protein. These factors are independent of each other. Or it can be effective by combining two or three kinds. First, the sodium gasification will be explained. Sodium gasification, salt, seawater, artificial seawater, etc. of the test reagent can be used regardless of any substance as long as it contains sodium gasification. In the present invention, D-glucose: y: the opposite of acidase The concentration of sodium vaporized solution in the liquid is preferably in the range of 0 mM to iM, and more preferably in the range of i mM to 600 mM. Next, as for the calcium ion, as long as calcium ion can be generated, regardless of any substance Both can be used. Preferred soluble calcium salts include, for example, calcium chloride and calcium acetate. Furthermore, even poorly soluble calcium salts, such as calcium carbonate, calcium phosphate, calcium citrate, and calcium sulfate, are used in sulfated glucose. Sulfated polysaccharides such as alkyd fucoidan will slowly generate calcium ions in a dissolved state, so they can be used for the activation of a-D glucoglucosidase in the present invention. Furthermore, in sulfated glucose In the case where the ions of the sulfonic acid group and the carboxyl group of uronic acid contain calcium salts, since the matrix becomes a source of calcium ions, the calcium ion source added for activation can be reduced. Depending on the situation, even if not added The calcium salt can also act. The calcium ion concentration added to the π_D-glucosaminidase reaction solution of the present invention is preferably in the range of 0 i mM to 200 mM, and is in the range of 1 mM to 100. The range of mM is more preferable. The protein is described in order to make the present invention general_D_ Portuguese -31-This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1243853

DESCRIPTION OF THE INVENTION (29) For the purpose of activating a sugar a enzyme, adding "· D-glucosaminidase protein ..." to the reaction solution does not decompose the invention. Any protein may be used. Among them, bovine serum = its reaction, Another example is 1. It is expected that the Shiming-D-glucopurinase reaction solution from the 1234 strain of the present invention is extracted from the bacterial cell of the present invention: 2. Added to this hairpin ancient / ancient 1, It is better to be in the range of 0.001 ~ 10 gram / stroke &lt; from Bei / Chen degree, and more preferably in the range. Normalization in the range of 0.01 ~ g / ml:!: Within the month ... L- Fucose enzymes, which can act on deacetylated glutamate. Acidified sulfated glucuronidated fucose and sulfated glucosamine fossil bath oligomers Shoutang, while -fucosyl group The bond is hydrolyzed in an inward manner so that the reducing end has [• fucose oligosaccharide-producing enzyme: within the present invention ... fucose I enzyme, a natural sulfated glucoside acidified diatom I Glycosides function, but for sulfated glucuronidated fucans previously treated with fucoidan deacetylase and pan-D-glucosidase, or The sulfated glucuronidated fucoidan with co-existence of enan deacetylase and "_D.glucosidase has a good effect. However, even if it is a natural sulfated glucuronidated fucoidan, if you borrow Removal of acetamyl and glucose by acid groups from any reason such as enzymes from other marine bacteria or physical or chemical factors is suitable as a substrate for the endo-fucosidase of the present invention. Within the present invention-to- The physical and chemical properties of L-Langangsufenfen enzyme are as follows: 0 (I) Action: It acts on deacetylated deglucuronidated sulfated glucuronic acid-32- This paper size applies to Chinese national standards (CNS ) A4 specification (210X 297 mm) 1243853 A7 ___B7 V. Description of the invention (3Q &quot; &quot;) ^ &quot; Fossil algal and gadolinated glucuronidated fuctooligosaccharides will act as -L- Fucosyl bond is hydrolyzed inward to generate oligosaccharides with L-fucose at the reducing end. (II) Optimum pH 値: The optimal pH of this enzyme is about 4 5 ~ 7 4 (Figure 5). That is, Figure 5 is a graph showing the relationship between pH 値 and relative activity during the reaction of this enzyme. The vertical axis indicates relative activity (%), and the horizontal axis indicates pH 値. (III) Optimum temperature: The optimal temperature of the enzyme is in the range of about 23 to 42Ό (Figure 6). That is, Figure 6 shows the The relationship between temperature and relative activity during enzyme reaction, where the vertical axis represents relative activity (%) and the horizontal axis represents temperature (...). (Iv) Molecular weight: When measured by gel filtration, it is about 150,000 ~ 200,000. The confirmation of the -L-fucosinase in the present invention can be carried out by measuring the degradation activity of deacetylation, deglucouration, sulfated glucuronidation, and fucoidan. Furthermore, the sulfated glucuronidated fucoid oligosaccharide is treated with -D · fucosylase in the present invention, and its reducing end is labeled with 2-aminopyridine. The compounds can also be used as a substrate for this enzyme, and trace and high-sensitivity activity measurement systems can be constructed by using these fluorescently labeled oligosaccharides. For example, after using 8Fuc-4S-PA (described below) for a substrate and reacting it, if the reaction product is analyzed by HPLC, the activity of endo-L · fucosidase can be measured. In the present invention, the activity of the enzyme can be measured with -L_fucose, and the cell-free extract of the producing bacteria or the enzyme solution purified by various column chromatography can be used for measurement. -33- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 ____B7 V. Description of the invention (31) In an embodiment, the present invention is within the scope of the present invention. L-Fucoidan Enzymes can be activated in the presence of sodium chloride, calcium ions and / or proteins. These factors have activation effects either alone or by a combination of two or three. First of all, for the explanation of sodium sulfide, sodium hydride, common salt, seawater and artificial seawater of the test drug must contain sodium fumes, and any substance can be used. The concentration of sodium chloride added in the -L-fucosidase reaction solution of the present invention is preferably within a range of 0 mM to 1 M <, and within a range of 1 mM to 600 mM. For the better. Next, as for calcium ions, any substance can be used as long as calcium ions can be generated. Preferred soluble calcium salts are, for example, calcium carbonate and calcium acetate. Furthermore, even in the case of poorly soluble calcium salts such as calcium carbonate, calcium phosphate, calcium citrate, and calcium sulfate, calcium is slowly produced when sulfated polysaccharides such as sulfated glucuronidated fucoidan are dissolved. Therefore, it can be used in the present invention. "·-L-fucosinase activation. Furthermore, the sulfate ion and the carboxyl group of sulfated glucuronide contain calcium salt when the counter ion contains calcium salt. Since the matrix becomes the source of calcium ions, the calcium ion source added for activation can be reduced. Depending on the situation, the enzyme can also be used without adding about salt. Added in the present invention-"-B-rock The calcium ion concentration in the fucose enzyme reaction solution is preferably in the range of 0-1 to 200, and more preferably in the range of 1 mM to 100 mM. Next, the protein will be described, and the protein added to the reaction solution for the purpose of activating the α-fucosinase in the present invention, as long as it does not distinguish ~ within the present invention-not-L-rock desert sugar answer Enzymes do not inhibit their reactions' Any protein is fine. Among them, the use of bovine serum albumin as the car six -34- This paper size applies the Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 1243853 A7 _B7__ 5. The invention description (32) is good. Furthermore, for example, a protein extracted from the fucoidan strain SI-1234 strain decomposed by the fucoidan of the present invention can also be used. The protein concentration of the -a-L-fucosidase reaction solution added in the present invention is preferably in the range of 0.001 to 10 mg / ml, and in the range of 0.01 to 1 mg / ml. Better. Microorganisms used in the present invention for the production of fucoidan deacetylase, π-D-glucosidase, and endo-α-L · fucosidase, as long as they are capable of producing the above sulfated polysaccharides There are no particular restrictions on the amount of the dissolved or sulfated glucuronidated fucoidan enzyme, and for example, it is suitable to use a fucobacterium. The above-mentioned fucobacteria bacteria include all microorganisms that are classified as homologous from the bacteriological nature or are classified as homologous based on the same sequence as the base sequence of 16S rDNA. Invented Fucoidan Deacetylase, -D-Glucosidase and Endo-a-L-yandose #enzyme, the same enzyme that can decompose sulfated glucose while acidifying fucoidan Of all microorganisms. It is suitable for culturing and producing microorganisms of fucoidan deacetylase, π-D-glucosidase and endo-π-L-fucosidase of the present invention, and the nutrient source added to the culture medium, as long as it can It can be used by the used microorganisms, and the enzyme can be produced in the presence of it. The carbon source can be seaweeds such as sulfated glucuronide, fucoidan, Okinawa algae, alginic acid, kumbu polysaccharide, fucoidan Sugar, glucose, mannitol, glycerol, sucrose, maltose, and starch; suitable as nitrogen source, such as yeast extract, protein gland, enzyme protein amino acid, corn extract, meat extract, defatted soybean, sulfuric acid Ammonium, ammonium chloride, urea and uric acid. Other gases such as sodium, potassium, thorium, and # 5 can also be added -35- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (33 compounds, phosphates Or sulphate, etc. Moreover, the culture medium of the microorganism may be seawater or a commercially available artificial seawater culture medium. Furthermore, the culture conditions and the composition of the medium should of course be set according to the microorganisms used to enable the fucoid of the present invention Glycan deacetylase, _ D _ glucuronidase and endo-from-L-fucosidase produced the largest amount. For example, at a culture temperature of 15 ~ 301, the culture medium { ^ Is 5 to 9 and aeration and agitation culture is preferably performed for 5 to 72 hours. Under the aforementioned conditions, the present invention <fucoidan deacetylase, a_D-glucosidase, and internal _l_ rock The production amount of glycosaminoglycan can reach the maximum. After the end of the culture, the bacterial cells are separated from the culture supernatant by centrifugation γ, and the rock bath glycosaminoglycan deethyl brewing enzyme U-glucuronide of the present invention can be obtained from them respectively. Enzyme and endo_yase_fucosidase. Although not particularly limited, but by way of example, The fucoidan of the invention decomposes Fucoidophila SI 1234 and cultivated in an appropriate culture medium. Then collect the bacterial cells' and then use conventional cell disruption methods (such as ultrasonic treatment) to break the bacterial cells to obtain cell-free cells. Extraction solution. Then, by the conventional refining method: a refined enzyme sample can be obtained from this extraction solution. It can be extracted by salting out, ion exchange &amp; column chromatography, hydrophobic column chromatography, and gel filtration. Refined 'can obtain a purification that does not substantially contain other sulfated fucose-containing degrading enzymes of the present invention. The fucoidan deacetylase, ^ D · glucanase, and endogenous medicinal enzymes can be obtained. Furthermore,' if used, the Sulfuric acid while acidifying fucoidan-immobilized resin can easily separate the D-glucose pure enzyme from the a.L. rock surface of the present invention. Furthermore, in the above culture supernatant Since there are also fucoids of the present invention-36-1243853 A7 _____B7 V. Description of the invention (35) Deacetylation degluconization desulfurization and sulfation of various glucuronic acid ratios from 4: 1 to 4: 0 Glucuronidated fucoidan. In order to obtain the deacetylated deglucose of the present invention Alkylated sulfated glucuronide fucose, a-D-glucuronidase acts on deacetylated sulfated glucose while acidifying fucose, and then free D-glucuronide It can be removed by, for example, ultrathorium method, gel filtration, anion exchange column treatment, etc. If necessary, treatments such as desalting and freeze-drying can be performed. In the case of deglucose acidification treatment, if sulfated glucoaldehyde is used in advance, Acidified fucoidan treatment with fucoidan deacetylation enzyme and alkaline treatment to remove acetamidine can efficiently deglucose acidification. For example, sulfation of algae from Okinawa Deacetylated sulfated glucuronidated fucose obtained by deacetylation of glucuronidated fucoidan 'about 1 molecule of D-glucose per 4 molecules of fucose while acid, but borrowed By removing D-glucuronic acid with -D -glucose: y: acidase, it can be adjusted to deacetylated degluconated sulfated glucuronidated fucoid with various glucose content and acid content. sugar. By adjusting conditions such as the amount of enzymes and base mass, reaction time, reaction temperature, and P Η 値 used in the de-glucuronic acid reaction, for example (but not limited to), appropriate amounts of sodium gasification, bovine serum albumin, and 500 The α-D-glucuronidase of the present invention in mlJ is mixed into 1 g of deacetylated sulfated glucuronidated fucose and allowed to react at 20 ° C and about ρΗ7 for various times. Production of deacetylated degluconated degluconated sulfated glucuronidated fucose with various glucosinic acid content of fucose and glucosinolate from 4: 1 to 4: 0 . Deacetylated, de-glucuronidated, sulfated, glucuronidated, fucoidan -38 · This paper size applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (36) It is used as a sugar chain engineering reagent as it is, but in addition to being used as the substrate of the endo-pan-L_fucosidase of the present invention and used for the activity measurement, the sulfuric acid of the present invention as a reaction product thereof Glucuronidated fuco-oligosaccharide can also be used as a test reagent for sugar chain engineering. When the deacetylated degluconated sulfated glucuronidated fucose of the present invention is prepared, the deacetylated sulfated glucuronidated fuctan or deacetylated sulfated glucan The dissolution of the acidified fucoidan can be carried out by a common method, and the deacetylated sulfated glucuronidated fucoidan or the deacetylated sulfated glucuronidated fucoidan in the solution is dissolved. Although the concentration of the sugar-containing substance can be its highest dissolved concentration, it is usually better to select it in consideration of its operability and enzyme activity. As the dissolving solution of the deacetylated sulfated glucuronidated fucoidan, it can be selected from water, a buffer solution, and the like according to the purpose. The pH of the dissolving solution is usually around neutral, and the enzyme reaction is usually carried out around 20 ° C. By adjusting the amount of enzyme, the composition of the reaction solution, and the reaction time, the degree of de-glucuronic acid can be adjusted. Deacetylated, de-glucuronidated, sulfated-glucuronidated fucoidan can be further purified by ion exchange resin treatment and ultrafiltration if necessary, and desalted, aseptically treated, and frozen if necessary Drying. The deacetylated de-glucuronidated sulfated glucuronidated fucoidan of the present invention has a sulfate group and / or a carboxyl group in the molecule, and the group reacts with various bases to form a salt. The state of the salt formed by the deacetylation, deglucouration, sulfated glucuronidation, and fucoidan of the present invention is usually provided in the form of salts such as sodium and / or potassium and / or calcium. The salts of these substances can be introduced by using cation exchange resins such as Dowex 50W to introduce the free deacetation and deglucose of the present invention-39- This paper size applies to China National Standard (CNS) A4 (210X297) (%) 1243853 A7 B7 V. Description of the invention (37) Uronic acid sulfated glucuronidated fucose. Furthermore, they can carry out well-known salt exchange methods as needed to transform into various desired salts. In the present invention, the salts of deacetylated deglucouronated sulfated glucuronidated fucoidan can use pharmaceutically acceptable salts, such as alkali metals such as sodium and potassium, alkaline earth metals such as calcium, magnesium, and zinc, and Ammonium and other salts. In this specification, the sulfated glucuronidated fucoidan refers to the action of the fucoidan deacetylase of the present invention on the sulfated glucuronidated fucoidan, or an alkali treatment to After deacetylation, the reducing terminal sugar obtained by the action of a-D-glucosidase and endo-a-L-fucosidase is an oligosaccharide of L-fucose. For example, a sugar compound having a chemical structure selected from the following general formulae (I) to (111) is not limited thereto. -40- This paper size applies to Chinese National Standard (CNS) Α4 size (210 X 297 mm) 1243853 Α7 Β7 V. Description of invention (38

(In the formula, R is Η, S03H or CH3CO; η is an integer of 0 or more). -41-

This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of invention (39

〇 X X

J (where R is Η, S03H or CH3CO; η is an integer of 0 or more) 〇 42

This paper size applies to China National Standard (CNS) Α4 size (210 X 297 mm) 1243853

V. Description of the invention (4Q (\ HH »—ί

X

〇 X π: (where R is Η, S03H or CH3CO; η is an integer of 0 or more) -43-

This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1243853 A7 B7 V. Description of the invention (41) The sulfated glucuronide fuco-oligosaccharide of the present invention can be used to make the rock of the present invention Fucoidan deacetylase, π-D-glucosidase and endo-aL-fucosinase act on sulfated glucuronidated fucoidan or sulfated glucuronidated fucoidan The sugar-containing substance can be efficiently prepared, and the π-D · glucosaminidase and endo-α-L-fucosinase of the present invention can also be used to remove chemical substances which have undergone chemical treatment such as alkali treatment in advance. Acetate is prepared by sulfated glucuronidation of fucoidan. As the sulfated glucuronide fucoidan-containing substance, for example, a partially refined product of sulfated glucuronidated fucose, a fucose-containing polysaccharide component derived from brown algae, and brown algae are suitably used. Aqueous solvent extracts or brown algae algae. Furthermore, when the π-L-fucosidase in the present invention is applied to the deacetylation, deglucouration, sulfated glucuronidation, and fucoidan of the present invention, of course, the sulfuric acid of the present invention can also be obtained. Glucuronidated Fucoidan. For example, but not limited to, by deacetylating the sulfated glucuronidated fucoidan from Okinawa algae, and then using the a_D-glucuronidase and endo-α-L of the present invention -Fucosidase acts to obtain a sugar compound or a salt thereof having a chemical structure selected from the above general formulae (I) to (III). When preparing the sulfated glucuronidated fuctooligosaccharide of the present invention, the dissolution of the sulfated glucuronidated fucoidan or the sulfated glucuronidated fucoidan can be performed by a common method. Although the concentration of the sulfated glucuronidated fucoidan of the present invention or the content of the sulfated glucuronidated fucoidan in the dissolving solution can be its highest dissolved concentration, it is generally considered in consideration of its operability and cost. The amount of fucoidan deacetylase, α-D-glucosaccharinase, and endo-me-L-rock dextrose enzymes in the invention is preferably selected. As the sulfated glucose road-44- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (42 Acidified lycoglycan solution, depending on the purpose Choose from water, buffer solution, etc. The pH of the dissolving solution is usually around neutral, and the enzyme reaction is usually carried out around 25 ° C. By adjusting the fucoidan deacetylase, α of the present invention used in the reaction, -D · Glucosidase and Endo-Pan-l · Fucosylase The mixing ratio and usage amount, the composition of the reaction solution and the reaction time, etc. Molecular weight and the content of glucuronic acid and acetamyl group. Sulfated glucuronidated fucoidan can be obtained by 3 kinds of enzymes, namely fucoidan deacetylase, a_D_glucoside of the present invention. Acidase and endo-pan-L-fucosidase are decomposed, however, the reaction of these three enzymes can be performed simultaneously or separately. That is, the sulfated glucuronidation fucoidan is prepared in advance by the present The fucoidan deacetylating enzyme and ___ glucuronidase were invented to deacetylate and deglucose After the fucoidan deacetylase and α-D.glucose acidase of the present invention are deactivated by heat treatment, acid or test treatment, etc., then the fuccosidase of the present invention acts, Then the molecular weight distribution of the sulfated glucuronidated fuctooligosaccharide of the present invention and the adjustment of the glucose and acid and acetamyl content will become easy. The sulfated glucuronide of the present invention obtained in the above manner will be easily acidified. Fucoidan, through molecular weight division or anion exchange column division, can further adjust the molecular weight division or uniform charge distribution of the present invention sulfated glucuronidated oligosaccharide. The molecular weight division is usually applied by The method used can be, for example, gel filtration or molecular weight division membrane. The low-molecular-weight compound can be further subjected to purification operations such as ion exchange resin treatment and activated carbon treatment if necessary, and can be further subjected to desalination treatment, aseptic treatment, and freezing if necessary. Drying, etc. By this method, a uniform structure can be obtained (determined by NMR analysis -45- This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm)

Binding

1243853 A7 B7 V. Description of the invention (made by (43)) The sulfated glucuronide fuco-oligosaccharide of the present invention. The sulfated glucuronidated fucoidan oligosaccharide of the present invention has a sulfuric acid group and a carboxymethyl group in the molecule, and the group reacts with various bases to form a salt. The salt state formed by the sulfated glucuronidation of fuco-oligosaccharides of the present invention is stable, and is usually provided in the form of salts such as sodium and / or potassium and / or calcium. The salts of these substances can be introduced into the free sulfonated glucuronidated fucoidan of the present invention using a cation exchange resin such as Dowex 50W. Furthermore, they can perform known salt exchange methods as needed to transform into various desired salts. As the salt of the sulfated glucuronidated fuco-oligosaccharide of the present invention, pharmaceutically acceptable salts such as alkali metals such as sodium and potassium, alkaline earth metals such as calcium, magnesium, and zinc, and ammonium salts can be used. Furthermore, the sulfated glucuronidated fucoidigosaccharides of the present invention can be used as a test reagent for sugar chain engineering. For example, if 2-aminopyridination (PA) is performed by the method described in Japanese Patent Publication No. 5-65108 and the PA-form of the oligosaccharide is adjusted, it can be used as the fucoidan of the present invention. A substrate for measuring the activity of glycan deacetylase, -D-glucosidase, and endo-me-L-fucosidase. In this way, the sulfated glucuronidated fuco-oligosaccharide of the present invention is extremely useful as a reagent for sugar chain engineering. The fucoidan deacetylase, α-D-glucosaccharase and endo-pan-L-fucosidase of the present invention can be used for sulfated glucuronidation of fucoidan. Structural analysis of sulfated glucuronidated fucoidan used for low molecularization. Furthermore, since the fucoidan deacetylase, α-D-glucosaminidase and endo-α-L-fucose fenase of the present invention have vaporized sodium, protein and Coexistence of calcium ions can improve stability and response speed. Therefore, -46-This paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 1243853 A7 B7. ΛΛ, V. Description of the invention ( ） The coexistence of these sulfated glucuronidated fucoidan decomposing enzyme activation factors can efficiently perform the above-mentioned low-molecularization. Furthermore, the sulfated glucuronidated fucoid oligosaccharide, deacetylated sulfated glucuronidated fucoidan, and deacetylated deglucuronidated sulfated glucuronidated fucoidan of the present invention Glycan can be used as a sugar chain engineering reagent, and can also be used as the fucoidan deacetylase, π-D-glucuronidase and endo-a-L-fucoid A substrate for measuring the activity of glycosidase. Examples Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the scope of the following examples. '' Reference Example 1 (1) Preparation of Crude Sulfated Glucuronidated Fucoidan Fraction 625 g of salted marine algae from Okinawa, suspended in 4375 ml of 30 mM sodium phosphate buffer (ρΗ6.0 ·) After 5 minutes of treatment at 8000 revolutions / minute with a homogenizer, the mixture was treated at 95 ° C for 1 hour, and the supernatant was obtained by centrifugation. 10 g of activated carbon was added to the obtained supernatant, and the mixture was stirred for 30 minutes, and the supernatant was obtained by centrifugation. The obtained supernatant was concentrated to 2 liters by means of an external filter equipped with a hollow fiber exclusion molecular weight of 100,000, and then the solvent was replaced with 20 mM sodium vaporization, and then freeze-dried to obtain a crude of 10.9 grams. Sulfate glucuronidation of dried fucoidan. (2) Method for determination of sulfated glucuronidation fucoidan decomposition activity The fucoidan deacetylase, α-D-glucose: y: acidase and endo-a-L-rock of the present invention Although fucosinase alone acts on sulfated glucuronidated fucoidan, although it cannot produce oligosaccharides efficiently, it can combine sulfur-47 by combining this paper. This paper standard is applicable to China National Standard (CNS) A4 specifications. (210 X 297 mm) 1243853 A7 B7 1 45 ~ V. Description of the invention () Acidified glucuronide fucose decomposes, making it low molecular efficiently. The activity of degrading sulfated glucuronidated fucans in the presence of the three enzymes mentioned above is called "sulfated glucuronidation fucoidan decomposition activity", which can be counted by the following activity measurement methods: Into. That is, 10 microliters of a 1% crude sulfated glucuronidated fucoidan solution, 52.5 microliters of 50 mM imidazole hydrochloride buffer (ρΗ6 · 6), and 5.5 microliters of 4M gas Sodium chloride, 2 microliters of 1M calcium carbonate and 10 microliters of 5 mg / ml bovine serum albumin and 20 microliters coexist with fucoidan deacetylase, "-d-glucose: y : The enzyme solution of acidase and endo-α-L-fucose enzyme was mixed and reacted at 30 ° C for 3 hours, and then the reaction solution was treated at 100 ° C for 10 minutes. HPLC analysis to determine the degree of low-molecularization. As a control, the fucoidan deacetylase, a-D-glucosidase and endo-a-L-rock of the present invention were coexisted. The enzyme solution of trehalase was replaced by a buffer solution to dissolve the enzyme solution, and the crude sulfated glucuronidated fucoidan was reacted by using water instead. The HPLC analysis was performed in the same way. The so-called 1 unit Sulfated glucuronide fucoidan decomposition activity refers to the fucosyl bond of 1 micromolar sulfated glucuronide rock and fucose in 1 minute in the above reaction system The amount of broken enzyme. The amount of fucosyl bond cut can be obtained by the following formula. Mathematical formula 1 {(ΙΟχΙΟΟΟχ l / 100) / MG} x {(MG / M) -l} x {1 /(180x0.02)} = U / ml 1 0 x 1000 x 1/100 ·· Crude sulfated glucuronidated fucoidan added in the reaction system (micrograms) MG: Matrix crude sulfated glucose The average molecular weight of uronic acid fucoidan-48- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1243853 A7 B7 V. Description of the invention (46) M: The average molecular weight of the reaction product ( MG / M) -1: 1 molecule of sulfated glucuronidated fucoidan is cut by enzymes 180 ··· reaction time (minutes) 0.02: amount of enzyme solution (ml) Furthermore, the conditions of HPLC are as follows Device: Model L-6200 (manufactured by Yori Manufacturing Co., Ltd.) Column: OHpak SB-806HQ (8 X 300 mm, manufactured by Showa Denko Corporation) Eluent: 50 mM sodium chloride containing 5 mM sodium azide J: Parallax tortuosity detector (Shodex RI-71, manufactured by Showa Denko Corporation)

Flow rate: 1 ml / min. Column temperature: 25 ° C. In order to measure the average molecular weight of the reaction product, a commercially available pullulan (Pullulan) (STANDARD P-82, manufactured by Showa Denko Corporation) with a known molecular weight was used. In the same condition analysis of the above HPLC analysis, the relationship between the molecular weight and the retention time of the budding spore sugar was expressed as a curve, and it was used as a standard curve for measuring the molecular weight of the reaction product. The protein was quantified by measuring the absorbance at 280 nm of the enzyme solution. In this case, the absorbance of the protein solution at 1 mg / ml was taken as 1.0. Example 1 Fucoidan-degrading Fucoidophile SI-1234 strain was inoculated on a culture medium containing acidified crude sulfated glucuronide from algae from Okinawa, prepared in accordance with the method of Reference Example 1 (1). Fucoidan part 0.2% and protein gland-49- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1243853 A7 B7 47 V. Description of the invention (1% artificial seawater (ρΗ8 · 0 ) (Manufactured by Gamarinly Labradure), 50 ml of the culture medium was prepared at 120 ° C by a 20-minute autoclave. After inoculation, it was cultured at 24 ° C for 7 2 hours as the culture broth. Contains 0.2% of crude sulfated glucuronidated fucoidan from algae in Okinawa, prepared by the method of Reference Example 丨 (^), 1% of protein glands, and an antifoaming agent (KM70, Shin-Etsu Chemical Industry Co., Ltd. 600 ml of pH 8.0 culture medium made of artificial seawater was added to a 2-liter Erlenmeyer flask at 115. (: 10-minute autoclave treatment to obtain the culture medium, 7 bottles were prepared in this way, Inoculate 5 ml per Erlenmeyer flask, at a rotation speed of 90 revolutions per minute, Incubate at 24t for 72 hours. After the end of the culture, the culture solution is centrifuged to obtain the bacterial cells and the culture supernatant. Binding The obtained bacterial cells are suspended in 250 ml of 100 mM sodium vaporized and 10 mM calcium vaporized In 10 mM imidazole-hydrochloric acid buffer solution (pH 70), the solution was centrifuged to obtain a supernatant after ultrasonic disruption. The obtained supernatant was fully dialyzed with the same buffer solution, centrifuged, and the supernatant was processed. It is a crude enzyme solution. The obtained crude enzyme solution is fed into a 300 ml DEAE-cellulose A_800 column equilibrated with the same buffer solution, washed with the same buffer solution, and then washed with 100 mM to 400 mM. Gaseous sodium lysate was used for gradient elution, and the active dissolution fraction was collected. In this way, a part of the purified enzyme solution was obtained. Furthermore, the "sulfated glucuronidation" contained in the above culture supernatant and crude enzyme solution was measured. The fucoidan activity j was confirmed to be active in any of them, and the activity of 0.4 mlJ was detected per 1 ml of the culture medium in the bacterial cell extract. Example 2 (1) The crude value described in Example 1 The enzyme solution acts on the crude -50- 1243853 A7 described in Reference Example 丨 (丨) _ B7 V. Description of the invention (48 ~ Γ 'Sulfated glucuronidated fucoidan to prepare the sulfated glucuronidated fuctooligosaccharide of the present invention. That is, 5 g of crude sulfuric acid The glucuronidated fucoidan was partially dissolved in 1 liter of 10 mM imidazole-hydrochloric acid buffer (pH 6.6) containing 25 ° C sodium chloride and 20 mM calcium carbonate, and then added 35 mU of Example 1 The crude enzyme solution described was allowed to react at 30t for 6 days. The reaction solution was centrifuged, and the resulting supernatant was treated with an external machine equipped with an exclusion limit equipped with a medium molecular weight of 10,000, and the molecular weight was recovered. The oligosaccharide part below 10,000 is used as the sulfated glucuronidated fucolytic enzyme digestion part1. (2) Example 2 (1) The obtained sulfated glucuronidated fucoidan enzyme digestion part 1 was desalted by a desalination apparatus (Micro-Asilaza-G3, manufactured by Asahi Kasei Corporation). In desalted sulfated glucuronide fucoidanase digestion Shao 1, imidazole was added to a concentration of 5 μM and sodium gasification was added to a concentration of 20 mM. The mixture was fed into Equilibrate 1 liter of DEAE-Cellulose A-8 0 0 column with 5 mM imidazole-hydrochloric acid buffer solution (pH 7.0) containing 20 mM sodium vaporization, wash with the same buffer, and borrow Gradient dissolution from 20 mM to 600 mM sodium vaporization. The total sugar content of the eluted portion was measured by the g-sulfuric acid method, and the total sugar content was measured by the Weijun-sulfuric acid method. As a result, at least 8 of the eluted fractions had obvious peaks, and the peaks were collected as oligosaccharides i_ (丨) ~ (8), which were respectively concentrated to 40 ml by an evaporator. After that, it was fed into a celluloid GCL-25 column preliminarily equilibrated with 10% ethanol, and then eluted and desalted with 10% ethanol. Thereby, the sulfated glucuronidated fucoidan oligosaccharides 1- (丨) ~ (8) of the present invention can be obtained, that is, oligosaccharides 1- (1) ~ (8). -51-Standards for this paper: Standards for Financial Standards (CNS) A4 (2 脳 297 public love) 1243853 A7 _____ B7 V. Description of the invention (49) (3) For the oligosaccharide obtained from Example 2 (2) ( 1) ~ (8), analysis of reducing terminal sugar and sugar composition by fluorescent labeling method using 2-aminotitanium, and fucose using UFC measurement reagent Takara (manufactured by Takara Shuzo) When determining the absolute configuration, it was found that the reducing terminal sugars of oligosaccharides 1_ (1) to (8) were L-fucose. Regarding sugar composition, oligosaccharides I · (丨) are formed only from fucose, and oligosaccharides 1- (2) to (8) are formed from fucose and glucuronic acid. Then, the sulfuric acid content (by the turbidimetric method using barium chloride) and the gluconic acid content (by the carbazole-sulfuric acid method) were measured, and the mass analysis device (API-III, Parkin Emma-Sec Company's) analysis quality. Furthermore, nmR analysis was performed using a JNM_α500 nuclear magnetic resonance apparatus (manufactured by Japan Electronics Corporation). The analysis sample was replaced with heavy water by a fixed method, and then the structure was analyzed. The method of combining sugars is performed using the Μ BM B C method (a 1 Η-detection of heteronuclear detection method). 1H-NMR is assigned using the dqf-COSY method and ΗΟΗΑΗΑ method, and 13C-NMR is assigned using the HSQC method. The physical properties of oligosaccharides 1- (1) to (8) are shown below. (a) The physical property quality analysis and NMR analysis results of oligosaccharide 1- (1) are shown below. The NMR spectrum of the sulfated glucose of the present invention while acidifying fucoidan 1- (1) is shown in FIG. The '13 C-NMR spectrum shown in 7 is shown in FIG. 8, and the mass spectrum is shown in FIG. 9. In Figs. 7 and 8, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In Fig. 9, the vertical axis represents the relative intensity (0 / 〇), and the horizontal axis represents m / z 値. Molecular weight · 762 MS m / z 380.2 [M-2H +] 2 · The results of analysis by 1NMR-NMR and 13C_NMR are shown in the table! As shown. -52 · This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7

V. Description of the invention (5Q Table 1 Chemical shift 値 (ppm) 13c-nmr 'HNMR Chemical shift 値, multiplicity, binding constant F1-1 97.0 4.48, d, 7.6 F1-2 70.8 3.44, dd, 7.6, 10.1 F1- 3 78.4 3.59, dd, 3.7, 10.1 F1-4 68.6 3.86, d, 3.7 F1-5 71.4 3.66, q, 6.8 F1-6 16.5 1.13, d, 6.8 F2-1 96.5 4.98, d, 4.0 F2-2 67.5 3.85 , dd, 4.0, 10.4 F2-3 77.5 3.98, dd, 3.1, 10.4 F2-4 80.5 4.65, d, 3.1 F2-5 67.2 4.30, q, 6.8 F2-6 16.5 1.13, d, 6.8 F3-1 99.9 4.99, d, 4.0 F3-2 68.0 3.72, dd, 4.0, 10.6 F3-3 76.1 3.90, dd, 3.0, 10.6 F3-4 80.3 4.65, d, 3.0 F3-5 67.4 4.34, q, 6.7 F3-6 16.5 1.11, d , 6.7 F4-1 98.4 4.97, d, 4.0 F4-2 69.3 3.59, dd, 4.0, 10.4 F4-3 70.8 3.76, dd, 3.4, 10.4 F4-4 72.8 3.68, d? 3.4 F4-5 68.0 4.21, q, 6.7 F4-6 16.0 1.09, d, 6.7 The sugar composition is only L-fucose (4 molecules) -53- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) 1243853 51 is 1 X Ο Ο

X 〔IV〕 A7 B7 V. Description of the invention (sulfate group 2 molecules In addition, the numbers of the peak assignments in iH-NMR and 13C-NMR are shown by the following formula (IV). 〇-=

Sr

-54- This paper size applies to Chinese National Standard (CNS) A4 (210 x 297 mm)

1243853 A7 B7 V. Description of the invention (52) (b) The results of the physical quality analysis of oligosaccharides 1- (2) are shown below. The sulfated glucuronide-fucose oligosaccharides 1- (2) The mass spectrum of) is shown in Fig. 10. In Fig. 10, the vertical axis indicates the relative intensity (%), and the horizontal axis indicates m / z 値. Molecular weight: 1456 MS m / z 484.6 [M-3H +] 3 'sugar composition L-fucose: D-glucuronic acid = 7: 1 sulfate group 3 molecules (c) oligosaccharide 1-(3) physical properties The results of mass analysis and NMR analysis are shown below. The sulfated glucuronidated fuctooligosaccharide 1-(3) 1 Η-NMR spectrum of the present invention is shown in Figure 11 and 13 C-NMR spectrum As shown in Figure 12, the mass spectrum is shown in Figure 13. In FIGS. 11 and 12, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In FIG. 13, the vertical axis represents relative strength (%), and the horizontal axis represents m / z 値 0 Molecular weight: 1682 MS m / z 873.4 [M + 3Na + -5H +] 2 ·, 862.4 [M + 2Na + -4H +] 2 , 574.4 [M + 2Na + -5H +] 3, 567.2 [M + Na + -4H +] 3-, 425.2 [M + Na + -5H +] 4-, 419.8 [M-4H +] 4 ·, by 1H-NMR and 13C-NMR analysis results are shown in Table 2. -55- This paper size is in accordance with Chinese National Standard (CNS) A4 (210x 297 mm) 1243853 A7 B7 V. Description of the invention (Table 2 Chemical shift 値 (ppm) 13c_nmr lH-NMR Chemical shift 値, multiplicity, binding constant F1-1 97.1 4.47, d, 7.9 F1-2 70.9 3.44, dd, 7.9, 10.1 F1-3 78.6 3.57, dd, 3.2, 10.1 F1-4 68.7 3.85, d, 3.2 F1-5 71.5 3.65, q, 6.7 F1 -6 16.6 1.13, d, 6.7 F2-1 96.6 4.98, d, 4.0 F2-2 67.6 3.86, dd, 4.0, 8.6 F2-3 77.6 3.97, dd, 2.9, 8.6 F2-4 80.6 4.65, d, 2.9 F2- 5 67.4 4.29, q, 6.7 F2-6 16.7 1.11, d, 6.7 F3-1 100.1 4.99, d, 4.0 F3-2 68.1 3.74, dd, 4.0, 10.4 F3-3 77.4 3.89, dd, 2.8, 10.4 F3-4 80.6 4.62, d, 2.8 F3-5 67.4 4.33 q, 6.7 F3-6 16.6 1.12, d, 6.7 F4-1 99.6 4.95, d, 4.0 F4-2 67.7 3.72, dd, 4.0, 10.4 F4-3 74.8 3.86, dd , 3.1, 10.4 F4-4 69.1 3.95, d, 3.1 F4-5 68.1 4.22, q, 6.8 F4-6 16.2 1.09, d, 6.8 F5-1 95.1 5.00, d, 4.0 -56- This paper size applies to Chinese national standards (CNS) A4 size (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (Table 2 (continued) Chemical shift 値(ppm) 13c-nmr ^ -NMR Chemical shift 値, multiplicity, binding constant F5-2 71.4 4.04, dd, 4.0, 10.4 F5-3 73.7 4.19, dd, 2.8, 10.4 F5-4 68.1 4.00, d, 2.8 F5 -5 67.4 4.20 q, 6.7 F5-6 16.2 1.11, d, 6.7 F6-1 94.3 5.08, d, 4.0 F6-2 67.4 3.89, dd, 4.0, 10.4 F6-3 76.5 3.99, dd ,, 10.4 F6-4 80.3 4.58, d, 2.4 F6-5 67.8 4.37, q, 6.7 F6-6 16.7 1.12, d, 6.7 F7-1 99.1 4.99, d, 4.0 F7-2 68.1 3.74, dd, 4.0, 10.4 F7-3 76.4 3.91, dd , 3.0, 10.4 F7-4 80.2 4.64, d, 3.0 F7_5 67.4 4.37, q, 6.7 F7-6 16.7 1.12, d, 6.7 F8-1 98.7 4.96, d, 4.0 F8-2 69.4 3.57, dd, 4.0, 10.1 F8 -3 70.9 3.76, dd, 3.0, 10.1 F8-4 73.0 3.66, d, 3.0 F8-5 68.1 4.23, q, 6.7 F8-6 16.1 1.07, d, 6.7 GA-1 100.4 5.16, d, 4.0 GA-2 72.1 3.48, dd, 4.0, 9.8 -57- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (55 Table 2 (continued) Chemical shift 値 (ppm) 13c -nmr ^ -NMR Chemical shift 値, multiplicity, binding constant GA-3 74.0 3.59, t, 9.8 GA-4 72.7 3.36, t, 9.8 GA-5 73.7 3 .81, d, 9.8 GA-6 177.3 Sugar composition L-fucose ·· D-glucuronic acid = 8 ·· 1 sulfate group 4 molecules Furthermore, the numbers of the peak assignments in iH-NMR and 13C-NMR are as follows Equation (V) is shown. -58- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (56 〔V] GA 1

Η c F 7 F6 F 5 Η

S even 1 U *

OH -59- This paper size applies Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1243853 A7 B7 V. Description of the invention () (d) Oligosaccharide 1- (4) The results of physical property quality analysis are as follows As shown below, the mass spectrum of the sulfated glucuronidated fucoid: oligosaccharide 1- (4) of the present invention is shown in FIG. 14. In Fig. 14, the vertical axis represents the relative intensity (%), and the horizontal axis represents m / z 値. Molecular weight: 2376 MS m / z 598.8 [M + Na + -5H +] 4-, 474.6 [M-5H +] 5 · Sugar composition L · Fucose: D_glucuronic acid = 11: 2 sulfate group 5 molecules (e) The results of the physical property quality analysis of oligosaccharides 1-(5) are shown below. The 1H-NMR spectrum of the sulfated glucuronidated fucoidan 1- (5) of the present invention is shown in Fig. 15 The 13C-NMR spectrum is shown in FIG. 16 and the mass spectrum is shown in FIG. 17. In Fig. 15 and Fig. 16, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In Fig. 17, the vertical axis represents the relative intensity (%), and the horizontal axis represents m / z 値. Molecular weight: 2602 MS m / z 666.4 [M + 3Na + -7H + wide, 661.0 [M + 2Na + -6H +] 4 &quot;, 524.0 [M + Na + -6H +] 5-, 433 · 0 [Μ-6Η +] 6-, The analysis results by 1H-NMR and 13C-NMR are shown in Table 3. -60- This paper scale is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (58) Table 3 Chemical shift 値 (ppm) 13c-nmr iH-NMR chemical shift 値, Multiplicity, binding constant F1-1 96.8 4.48, d, 8.0 F1-2 70.6 3.45, dd, 8.0, 9.5 F1-3 78.3 3.58, m F1-4 68.3 3.85, m F1-5 71.2 3.66, q, 6.7 F1- 6 16.2 1.14, d, 6.7 F2-1 96.3 4.99, d, 4.0 F2-2 67.4 3.86, m F2-3 77.1 3.98, m F2-4 80.2 4.65, m F2-5 67.0 4.30, q, 6.7 F2-6 16.3 1.10, d, 6.7 F3-1 99.7 5.00, d, 4.0 F3-2 67.8 3.75, m F3-3 77.0 3.90, m F3-4 80.2 4.63, m F3-5 67.4 4.34, q, 6.7 F3-6 16.3 1.13, d, 6.7 F4_l 99.2 4.96, d, 4.0 F4-2 67.4 3.73, m F4-3 74.5 3.86, m F4-4 68.7 3.96, m F4-5 67.8 4.22, q, 6.7 F4-6 15.8 1.10, d, 6.7 F5 -1 94.7 5.01, d, 4.0 -61-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (Table 3 (continued) Chemical shift 値 (ppm) 13c -nmr b-NMR Chemical shift 値, multiplicity, binding constant F5-2 71.1 4.05, dd, 4.0, 10.0 F5-3 73. 4 4.20, m F5-4 67.8 4.01, m F5-5 67.0 4.21, q, 6.7 F5-6 15.8 1.12, d, 6.7 F6-1 94.0 5.09, d, 4.0 F6-2 67.0 3.90, m F6-3 76.2 3.99 , m F6-4 79.9 4.60, m F6-5 67.4 4.38, q, 6.7 F6-6 16.4 1.13, d, 6.7 F7-1 98.7 5.00, d, 4.0 F7-2 67.8 3.75, m F7-3 76.0 3.91, m F7-4 79.8 4.65, m F7-5 67.4 4.38, q, 6.7 F7-6 16.4 1.13, d, 6.7 F8-1 99.2 4.96, d, 4.0 F8-2 67.4 3.73, m F8-3 74.5 3.86, m F8- 4 68.7 3.96, m F8-5 67.8 4.22, q, 6.7 F8-6 15.8 1.10, d, 6.7 F9-1 94.7 5.01, d? 4.0 F9-2 71.1 4.05, dd, 4.0, 10.0 -62- This paper size applies China National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (Table 3 (continued) Chemical shift 値 (ppm) 13c_nmr ^ -NMR Chemical shift 値, multiplicity, binding constant F9-3 73.4 4.20, m F9-4 67.8 4.01, m F9-5 67.0 4.21, q, 6.7 F9-6 15.8 1.12, d, 6.7 F10-1 94.0 5.09, d, 4.0 F10-2 67.0 3.90, m F10-3 76.2 3.99 , m F10-4 79.9 4.60, m F10-5 67.4 4.38, q, 6.7 F10-6 16.4 1.13, d, 6.7 F11-1 98.7 5.00, d, 4.0 F11-2 67.8 3.75, m F11-3 76 .0 3.91, m F11-4 79.8 4.65, m F11-5 67.4 4.38, q, 6.7 F11-6 16.4 1.13, d, 6.7 F12-1 98.3 4.96, d, 4.0 F12-2 69.1 3.58, m F12-3 70.5 3.77, m F12-4 72.6 3.67, d, 4.0 F12-5 67.8 4.23, q, 6.7 F12-6 15.8 1.08, d, 6.7 GA1-1 100.0 5.16, d, 4.0 GA1-2 71.8 3.49, dd, 4.0, 10.0 GA1-3 73.6 3.60, t, 10.0 binding

-63- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1243853 A7 B7 V. Description of the invention (Table 3 (continued) Chemical shift 値 (ppm) 13c-nmr ^ -NMR chemical shift 値, Multiplicity, binding constant GA1-4 72.4 3.37, t, 10.0 GA1-5 73.2 3.82, d, 10.0 GA1-6 176.9 GA2-1 100.0 5.16, d, 4.0 GA2-2 71.8 3.49, dd, 4.0, 10.0 GA2-3 73.6 3.60, t, 10.0 GA2-4 72.4 3.37, t, 10.0 GA2-5 73.2 3.82, d, 10.0 GA2-6 176.9 Sugar composition L-fucose: D-glucuronic acid = 12: 2 sulfate group 6 molecules In addition, the numbers of the peak assignments in iH-NMR and 13C-NMR are shown in the following formula (XI).

1243853 A7 B7 V. Description of the invention (63) Η 1F 8

F 1 W F 7 F 1 1

F 1 0 F 9 In addition, this substance is hereinafter referred to as 12Fuc-6S-2GlcUA 〇 (f) oligosaccharide 1- (6) -66- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm)

1243853 A7 B7 V. Description of the invention () The results of the mass analysis are shown below. The mass spectrum of the sulfated glucuronidated fuctooligosaccharide 1-(6) of the present invention is shown in FIG. 18. In Fig. 18, the vertical axis represents relative intensity (%), and the horizontal axis represents Πΐ / ζ 値. Molecular weight: 3296 MS m / z 840.0 [M + 3Na + _7H +] 4, 672.0 [M + 3Na + -8H +] 5, 556.0 [M + 2Na + -8H +] 6-, 473.2 [M + Na + -8H +] 7 · Sugar composition L_fucose: D-glucuronic acid = 15: 3 sulfate group 7 molecules (g) oligosaccharide 1- (7) The physical property quality analysis and NMR analysis result are shown below. The present invention The 1 H-NMR spectrum of the sulfated glucuronidated fuctooligosaccharide 1- (7) is shown in FIG. 19, the 13 C-NMR spectrum is shown in FIG. 20, and the mass spectrum is shown in FIG. 21. In Figs. 19 and 20, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In addition, in FIG. 21, the vertical axis represents relative strength (%), and the horizontal axis represents m / z 値 0. Molecular weight: 3522 MS m / z 896.6 [M + 3Na + -7H +] 4, 712.6 [M + 2Na + -7H +] 5 * 、 597.2 [M + 3Na + -9H 十] 6 ·, 505.4 [M + Na 十 -8H +] 7-, 439.6 [M-8H +] 8 · The analysis results by 1Η-NMR and 13C-NMR are shown in Table 4. Show. -67- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

Binding

1243853 A7 B7 V. Description of the invention (65) Table 4 Chemical shift 値 (ppm) 13c-nmr ^ -NMR Chemical shift 値, multiplicity, binding constant F1-1 96.8 4.46, d? 8.0 F1-2 70.6 3.43, dd, 8.0, 9.5 F1-3 78.3 3.57, m F1-4 68.3 3.83, m F1-5 71.2 3.65, q, 6.7 F1-6 16.2 1.12, d, 6.7 F2-1 96.3 4.98, d, 4.0 F2-2 67.3 3.84, m F2-3 77.1 3.96, m F2-4 79.9 4.65, m F2-5 67.1 4.29, q, 6.7 F2-6 16.3 1.09, d, 6.7 F3-1 99.7 4.98, d, 4.0 F3-2 67.8 3.73, m F3 -3 77.0 3.88, m F3-4 80.2 4.61, m F3-5 67.1 4.33, q, 6.7 F3-6 16.3 1.11, d, 6.7 F4-1 99.3 4.94, d, 4.0 F4-2 67.3 3.71, m F4-3 74.5 3.84, m F4-4 68.7 3.93, m F4-5 67.8 4.20, q, 6.7 F4-6 15.9 1.07, d, 6.7 F5-1 94.7 4.99, d, 4.0 -68- This paper size applies to the Chinese National Standard (CNS ) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (Table 4 (continued) Chemical shift 値 (ppm) 13c-nmr bNMR Chemical shift 値, multiplicity, binding constant F5-2 71.2 4.03, m F5 -3 73.4 4.18, m F5-4 67.8 3.99, m F5-5 67.1 4.19, q, 6.7 F5-6 15.9 1.09, d, 6.7 F6-1 94.0 5.07, d , 4.0 F6-2 67.1 3.88, m F6-3 76.2 3.98, m F6-4 79.8 4.58, m F6-5 67.4 4.36, q, 6.7 F6-6 16.4 1.11, d, 6.7 F7-1 98.7 4.98, d, 4.0 F7-2 67.8 3.73, m F7-3 76.0 3.90, m F7-4 79.8 4.64, m F7-5 67.1 4.36, q, 6.7 F7-6 16.4 1.11, d, 6.7 F8-1 99.3 4.94, d, 4.0 F8- 2 67.3 3.71, m F8-3 74.5 3.84, m F8-4 68.7 3.93, m F8-5 67.8 4.20, q, 6.7 F8-6 15.9 1.07, d, 6.7 F9-1 94.7 4.99, d, 4.0 F9-2 71.2 4.03, m -69- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (67 Table 4 (continued) Chemical shift 値 (ppm) 13c-nmr ^- NMR chemical shift 値, multiplicity, binding constant F9-3 73.4 4.18, m F9-4 67.8 3.99, m F9-5 67.1 4.19, q, 6.7 F9-6 15.9 1.09, d, 6.7 F10-1 94.0 5.07, d, 4.0 F10-2 67.1 3.88, m F10-3 76.2 3.98, m F10-4 79.8 4.58, m F10-5 67.4 4.36, q, 6.7 F10-6 16.4 1.11, d, 6.7 F11-1 98.7 4.98, d, 4.0 F11 -2 67.8 3.73, m F11-3 76.0 3.90, m F11-4 79.8 4.64, m F11-5 67.1 4.36, q, 6.7 F11-6 16.4 1.11, d, 6.7 F12-1 99.3 4.94, d, 4.0 F12-2 67.3 3.71, m F12-3 74.5 3.84, m F12-4 68.7 3.93, m F12-5 67.8 4.20, q, 6.7 F12-6 15.9 1.07, d, 6.7 F13-1 94.7 4.99, d, 4.0 F13- 2 71.2 4.03, m F13-3 73.4 4.18, m -70- This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) 1243853 A7 B7 V. Description of the invention (68) Table 4 (continued) Chemical shift 値(ppm) 13c-nmr ^ -NMR Chemical shift 値, multiplicity, binding constant F13-4 67.8 3.99, m F13-5 67.1 4.19, q, 6.7 F13-6 15.9 1.09, d, 6.7 F14-1 94.0 5.07, d , 4.0 F14-2 67.1 3.88, m F14-3 76.2 3.98, m F14-4 79.8 4.58, m F14-5 67.4 4.36, q, 6.7 F14-6 16.4 1.11, d, 6.7 F15-1 98.7 4.98, d, 4.0 F15-2 67.8 3.73, m F15-3 76.0 3.90, m F15-4 79.8 4.64, m F15-5 67.1 4.36, q, 6.7 F15-6 16.4 1.11, d, 6.7 F16-1 98.3 4.97, d, 4.0 F16- 2 69.1 3.57, m F16-3 70.6 3.75, m F16-4 72.6 3.66, m F16-5 67.8 4.23, q, 6.7 F16-6 15.8 1.06, d, 6.7 GA1-1 100.1 5.15, d, 4.0 GA1-2 71.8 3.47, dd, 4.0, 10.0 GA1-3 73.6 3.58, t, 10.0 GA1-4 72.4 3.36, t, 10.0 -71-This paper size applies to China Standard (CNS) A4 (210X 297 mm) 1243853 A7 B7 V. Description of the invention (69) Table 4 (continued) Chemical shift 値 (ppm) 13c_nmr ^ -NMR chemical shift 値, multiplicity, binding constant GA1-5 73.2 3.81, d, 10.0 GA1-6 177.0 GA2-1 100.1 5.15, d, 4.0 GA2-2 71.8 3.47, dd, 4.0, 10.0 GA2-3 73.6 3.58, t, 10.0 GA2-4 72.4 3.36, t, 10.0 GA2- 5 73.2 3.81, d, 10.0 GA2-6 177.0 GA3-1 100.1 5.15, d, 4.0 GA3-2 71.8 3.47, dd, 4.0, 10.0 GA3-3 73.6 3.58, t, 10.0 GA3-4 72.4 3.36, t, 10.0 GA3 -5 73.2 3.81, d, 10.0 GA3-6 177.0 Sugar composition L-fucose ·· D-glucuronic acid = 1 6 ·· 3 sulfate group 8 molecules, 1 再-NMR and 13 C-NMR The number of the spike assignment is shown in the following formula (X11). -72- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 1243853 A7 B7

1243853 A7 B7 V. Description of the invention (71

F 1 4: F 1 3 -74-

This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) 1243853 V. Description of the invention (72 A7 B7

The substance is hereinafter referred to as 16Fuc_8S-3GlcUA. (h) Physical properties of oligosaccharide 1- (8) The results of the mass analysis are shown below. 'The quality of the sulfated glucosamine acidified fucoidigosaccharide 1- (8) of the present invention is shown in FIG. 22. In Fig. 22, the vertical axis represents relative strength (%), and the horizontal axis represents m / z 値. Molecular weight: 4216 MS m / z 1092.0 [M + 7Na + -llH +] 4_, 869.0 [M + 6Na + _llH +] 5, 713.0 [M + 3Na + -9H +] 6 ', 611.0 [M + 3Na + -10H +] 7' &gt; 529 2 [M + Na, 9H +] 8 ·, 470.2 [M + Na, 10H +] 9-sugar composition L-fucose: D-glucose while acid = 19: 4 sulfate 9 molecular example 3 (1) A part of the crude enzyme solution described in Example 1 was applied to the crude sulfated gluconate acidified fucoidan portion described in Reference Example 1 (i) to prepare the sulfated glucuronidated fucoidan of the present invention. . That is, 3 grams of crude sulfated gluconate was partially dissolved in 1 liter of 250 mM sodium chloride, 20 mM vaporized dextrose, and 1 gram of bovine serum albumin at 10 mM. Also, after adding hydrochloric acid buffer solution (ρΗ6.6) to 29 mU, a part of the purified enzyme described in Example 1 was added and allowed to react at 30 ° C for 3 days. The reaction solution was centrifuged, and the resulting supernatant was equipped with an outer limit filter for removing hollow fiber with a molecular weight of 10,000, and an oligosaccharide part with a molecular weight of less than 10,000 was recovered as sulfated glucuronidated fucoid Glycanase digestion part 2. (2) The sulfated glucuronide fucoidanase digested part 2 obtained in Example 3 (1) was passed through a desalination device (micro-Asilaza-G3, Asahi Kasei Industries-75-) Applicable to China National Standard (CNS) A4 (210X 297mm)

Binding

Line 1243853 A7 B7 V. Description of Invention (73) Company) Desalination. In the desalted sulfated glucuronide fucoidan digest part 2, add imidazole to a concentration of 5 μM, and add sodium chloride to a concentration of 10 mM. A 1 liter DEAE-cellulose A-800 column equilibrated with 5 mM imidazole-hydrochloric acid buffer (pH 7.0) containing 10 mM steel chloride, washed with the same buffer, and passed 10 mM to 600 mM sodium chloride gradient elution. The total sugar content of the eluted portion was measured by the phenol-sulfuric acid method, and the total uronic acid content was measured by the carbazole-sulfuric acid method. As a result, at least 5 of the eluted portions had obvious peaks, and the peaks were collected as oligosaccharides 2- (1) to (5), which were respectively concentrated by an evaporator to 40. After milliliters, it was fed into a celluloid GCL-25 column preliminarily equilibrated with 10% ethanol, and eluted and desalted with 10% ethanol. Thus, oligosaccharides 2- (1) to (5) can be obtained. (3) Structural analysis of oligosaccharides For the desalted products of oligosaccharides 2-(1) to (5) obtained in Example 3 (2), the terminal sugar was reduced by the fluorescent labeling method of 2-aminopyridine. When analyzing the sugar composition, it was found that all the reducing terminal sugars of the oligosaccharide were L-fucose. In addition, as for the sugar composition, oligosaccharide 2- (1) is formed only from fucose, while oligosaccharides 2- (2) to (5) are formed from fucose and glucose while the acid is present. Then, the sulfuric acid content (by the turbidimetric method using barium vaporization) and the uronic acid content (by the Cajun-sulfuric acid method) were measured. (Manufactured by Max Cox) Analytical quality. Furthermore, NMR analysis was performed using a JNM-Pan 500 type nuclear magnetic resonance device (manufactured by Japan Electronics Co., Ltd.). The analysis sample was replaced with heavy water by a fixed method, and the structure was analyzed. The method of combining sugar is based on the use of HMBC method (for a 1 Η-detection of heterogeneous nuclear detection method) -76- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 ____B7__ 5 The invention description (74) line. 1H-NMR is assigned using the DQF-COSY method and HOHAHA method, and 13C-NMR is assigned using the HSQC method. The physical properties of oligosaccharides 2- (1) to (5) are shown below. (a) Physical properties of oligosaccharide 2- (1) As a result of the above analysis, the substance was determined to be the same substance as oligosaccharide 1- (1). (b) Physical properties of oligosaccharide 2- (2) As a result of the above analysis, it was determined that this substance was the same substance as oligosaccharide 1- (3). (c) The physical property quality analysis and NMR analysis results of oligosaccharide 2- (3) are shown below. The 1 H-NMR of the sulfated glucuronidated fucoidan 2- (3) of the present invention is shown below. The spectrum is shown in Fig. 23, the 13C-NMR spectrum is shown in Fig. 24, and the mass spectrum is shown in Fig. 25. In Figs. 23 and 24, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In addition, the vertical axis in FIG. 25 indicates the relative strength (%), and the horizontal extraction indicates m / z 値 0. Molecular weight: 1536 MS m / z 800.2 [M + 3Na + -5H +] 2_, 789.2 [M + 2Na + -4H +] 2- , 526.0 [M + 2Na + -5H +] 3-, 518.6 [M + Na + -4H +] 3 ·, 388.8 [M + Na + -5H +] 4, 383.2 [M-4H +] 4 · By 1H-NMR and 13C- The results of the NMR analysis are shown in Table 5. • 77- This paper size applies to the Chinese National Standard (CNS) A4 (210 x 297 mm) 1243853 A7 B7 V. Description of the invention (Table 5 Chemical shift 値 (ppm) 13c_nmr ^ -NMR chemical shift 値, multiplicity, Bonding constant F1 -1 97.1 4.47, d, 7.9 F1-2 70.9 3.44, dd, 7.9, 9.8 F1-3 78.6 3.58, dd, 2.8, 9.8 F1-4 68.7 3.85, d, 2.8 F1-5 71.5 3.66, q, 6.8 F1-6 16.6 1.13, d, 6.8 F2-1 96.6 4.99, d, 4.0 F2-2 67.7 3.85, dd, 4.0, 8.6 F2-3 77.6 3.98, dd, 3.1, 8.6 F2-4 80.6 4.65, d, 3.1 F2-5 67.3 4.30, q, 6.8 F2-6 16.6 1.13, d, 6.8 F3-1 100.1 4.99, d, 4.0 F3-2 68.2 3.75, dd, 4.0, 11.3 F3-3 77.4 3.90, dd, 2.7, 11.3 F3 -4 80.6 4.62, d, 2.7 F3-5 67.4 4.34, q, 6.8 F3-6 16.7 1.12, d, 6.8 F4-1 99.6 4.96, d, 4.0 F4-2 67.8 3.73, dd, 4.0, 10.4 F4-3 74.9 3.86, dd, 3.1, 10.4 F4-4 69.1 3.95, d, 3.1 F4-5 68.1 4.21, q, 6.8 F4-6 16.2 1.09, d, 6.8 F5-1 95.1 5.01, d, 4.0 -78- This paper size applies China National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of invention (Table 5 (continued) Chemical position Shift (ppm) 13c-nmr ^ -NMR Chemical shift 値, multiplicity, binding constant F5-2 71.4 4.05, dd, 4.0, 10.4 F5-3 73.7 4.18, dd, 3.1, 10.4 F5-4 68.1 4.00, d, 3.1 F5-5 67.3 4.19, q, 6.4 F5-6 16.2 1.13, d, 6.4 F6-1 94.2 5.08, d, 4.0 F6-2 67.6 3.85, m F6-3 75.8 4.00, m F6-4 80.0 4.59, d, 2.4 F6-5 67.4 4.37, q, 6.8 F6-6 16.8 1.14, d, 6.8 F7-1 98.3 5.01, d, 4.0 F7-2 69.5 3.66, dd, 4.0, 10.7 F7-3 70.0 3.87, dd, 2.5, 10.7 F7-4 81.6 4.48, d, 2.5 F7-5 67.4 4.37, q, 6.8 F7-6 16.8 1.11, d, 6.8 GA1-1 100.3 5.16, d, 4.0 GA1-2 72.1 3.47, dd, 4.0, 9.8 GA1-3 74.0 3.59, t, 9.8 GA1-4 72.7 3.37, dd, 9.8, 10.4 GA1-5 73.6 3.81, d, 10.4 GA1-6 177.3--79- This paper size applies to China National Standard (CNS) A4 (210X 297 male) 1243853 A7 B7 V. Description of the invention (77) Sugar composition L-fucose · D-glucuronic acid = 7 · 1 1 molecule of sulfate The numbers are shown in the following formula (VI). GA1

3 2 H F 7 X H H F 6 F 5 ^

Η Η H -oyo / H% 〔VI〕

H

啕 3 F 2 even 1 -80-

This paper size applies Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1243853 A7 B7 V. Description of the invention (78) (d) Properties of oligosaccharide 2- (4) The results of the above analysis determine the substance It is the same substance as oligosaccharides 1-(5). (e) The results of the physical quality analysis of oligosaccharide 2- (5) are shown below. The 1H-NMR spectrum of the sulfated glucuronidated fuco-oligosaccharide 2- (5) of the present invention is shown in Fig. 26 The 13C-NMR spectrum is shown in Figure 27 and the mass spectrum is shown in Figure 28. In Figs. 26 and 27, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In Fig. 28, the vertical axis represents relative strength (%), and the horizontal axis represents m / z 値. Molecular weight 2456 MS m / z 854.8 [M + 5Na + _8H +] 3, 847.2 [M + 4Na + -7H +] 3, 840.4 [M + 3Na + -6H +] 3-, 63 5.4 [M + 4Na + -8H + 广, 630.0 [M + 3Na + -7H +] 4 ·, 624.4 [M + 2Na + _6H +] 4 ·, 503.6 [M + 3Na + -8H +] 5 ·, 499.4 [M + 2Na + -7H +] 5 ·, 495.0 [M + Na + -6H +] 6 ', 416.0 [M + 2Na +-8H +] 6, 412.4 [M + Na + -7H +] 6-, 408 · 8 [Μ · 6Η +] 6 · Results of analysis by 1H-NMR and 13C_NMR As shown in Table 6. -81-This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (79 Table 6 Chemical shift 値 (ppm) 13c-nmr ^ -NMR Chemical shift 値, more Severity, binding constant F1-1 97.1 4.48, d, 8.0 F1-2 70.9 3.45, dd, 8.0, 9.5 F1-3 78.6 3.59, m F1-4 68.7 3.87, m F1-5 71.5 3.67, q, 6.7 F1-6 16.6 1.15, d, 6.7 F2-1 96.6 4.99, d, 4.0 F2-2 67.6 3.87, m F2-3 77.5 3.99, m F2-4 80.6 4.66, m F2-5 67.4 4.30, q, 6.7 F2-6 16.6 1.10 , d, 6.7 F3-1 100.1 5.00, d, 4.0 F3-2 68.1 3.78, m F3-3 77.5 3.88, m F3-4 80.6 4.63, m F3-5 67.6 4.35, q, 6.7 F3-6 16.7 1.13, d , 6.7 F4-1 99.6 4.97, d, 4.0 F4-2 67.8 3.73, m F4-3 74.8 3.87, m F4-4 69.1 3.96, m F4-5 68.1 4.22, q, 6.7 F4-6 16.2 1.10, d, 6.7 F5-1 95.1 5.02, d, 4.0 -82- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (8Q) Table 6 (continued) Chemical shift 値(ppm) 13c-nmr ^ -NMR Chemical shift 値, multiplicity, binding constant F5-2 71.4 4.06, dd, 4.0, 10.0 F5-3 7 3.7 4.20, m F5-4 68.1 4.01, m F5-5 67.4 4.21, q, 6.7 F5-6 16.2 1.12, d, 6.7 F6-1 94.3 5.09, d, 4.0 F6-2 67.4 3.88, m F6-3 75.8 4.00 , m F6-4 80.0 4.60, m F6-5 67.6 4.38, q, 6.7 F6-6 16.8 1.13, d, 6.7 F7-1 99.1 5.02, d, 4.0 F7-2 68.2 3.76, m F7-3 76.4 4.00, m F7-4 80.5 4.66, m F7-5 67.6 4.38, q, 6.7 F7-6 16.7 1.13, d, 6.7 F8_l 99.6 4.97, d, 4.0 F8-2 67.8 3.73, m F8-3 74.8 3.87, m F8-4 69.1 3.96, m F8-5 68.1 4.22, q, 6.7 F8-6 16.2 1.10, d, 6.7 F9-1 95.0 5.02, d, 4.0 F9-2 71 · 4 4.06, dd, 4.0, 10.0 -83- Applicable for this paper size China National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (81 Table 6 (continued) Chemical shift 値 (ppm) 13C-NMR ^ -NMR chemical shift 値, multiplicity, binding constant F9-3 73.7 4.20, m F9-4 68.1 4.01, m F9-5 67.4 4.21, q, 6.7 F9-6 16.2 1.12, d5 6.7 F10-1 94.2 5.09, d? 4.0 F10-2 67.4 3.88, m F10-3 75.8 4.00, m F10-4 80.0 4.60, m F10-5 67.6 4.38, q, 6.7 F10-6 16.8 1.13, d, 6.7 F11-1 98.3 5.02, d, 4.0 F11-2 69.5 3.67, m F11-3 70 .0 3.88, m F11-4 81.6 4.49, m F11-5 67.6 4.38, q, 6.7 F11-6 16.7 1.12, d, 6.7 GA1-L 100.4 5.17, d, 4.0 GA1-2 72.1 3.49, dd, 4.0, 10.0 GA1-3 74.0 3.60, t, 10.0 GA1-4 72.7 3.37, t, 10.0 GA1-5 73.6 3.82, d, 10.0 GA1-6 177.3 GA2-1 100.3 5.17, d, 4.0 GA2-2 72.1 3.49, dd, 4.0, 10.0 GA2-3 74.0 3.60, t, 10.0 -84- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 1243853 A7 B7 V. Description of the invention (82) Table 6 (continued) Chemical shift 値 ( ppm) 13c-nmr! h-nmr Chemical shift 値, multiplicity, binding constant GA2-4 72.7 3.37, t, 10.0 GA2-5 73.6 3.82, d, 10.0 GA2-6 177.3 Sugar composition L-fucose · D -Glucuronic acid = 1 1: 2 sulfate group 6 molecules Furthermore, the number of the peak assignment in iH-NMR and 13C-NMR is shown by the following formula (XIII). -85- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 Α7 Β7 V. Description of the invention (83 GA 1

F4 F3 F2 Fl ο π:

X -86- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm)

^ § X 1243853 A7 B7 V. Description of the invention (84) 〇 H J——〇-Ϊ &amp; X οχGA2

Η *

Furthermore, this substance is hereinafter referred to as 11Fuc-6S-2 (Hcl; A), which is seen from the enzyme reaction products obtained in the above Examples 2 and 3. For example, the respective oligosaccharides 1- (1), 1 The mass difference between 5) and 1- (7) is equivalent to the mass of 4 molecules of rock lotus, 2 molecules of sulfate group and 1 molecule of glucuronic acid. Mass difference between oligosaccharides 1- (2), 1_ (4), 1- (6) and 1- (8), oligosaccharides 2- (1), 2- (2) and 2- (4) The mass difference between the two and the mass difference between the oligosaccharides 2- (3) and 2- (5) are also the same. Therefore, it is considered that the crude enzyme solution obtained in Example 1 and the partially purified enzyme solution include the repeating unit {that is, 4 molecules of fucose, 2 molecules of sulfate group and 1 unit of glucosinolic acid ((-3F-3 (4S) Fl-3 (4S) Fl-3 (GUl-2) Fl-), where pan-L-fucose is abbreviated as F, sulfate group is simple S, π-D-glucuronic acid is abbreviated as GU)}. However, there is no fucoidan-87 in the reaction product. This paper is in accordance with Chinese National Standards (CNS) A4 specifications (210 x 297). 1243853 A7 B7 V. Description of the invention (85) Sugar 4 molecules, sulfate groups 2 The oligosaccharide formed by the molecule and one glucuronic acid molecule is similar to the structure of the oligosaccharide, which is formed by four fucose molecules and two sulfate group molecules. That is, "'vaguely shows that the crude enzyme solution and part of the purified enzyme solution obtained in Example 1 (1) are at least a mixture of glucosidase and fucosidase. Example 4 (1) The preparation of sulfated glucuronidated fucoidan celluloid is prepared as an affinity resin for the enzyme contained in the partially purified enzyme solution obtained in Example 1 (1). The crude sulfated gluconate-acidified fucoidan described in i (丨) is partially fixed to aminocellulose (manufactured by Biochemical Industry). The method of immobilization is based on the instructions of the biochemical industry. That is, 1.5 g of crude sulfated glucose was dissolved in $ 00 ml of water while the acidified rock glycan was partially dissolved in hydrochloric acid to make pH 4.5, and 50 ml of aminocellulose and 3 g were added. 1-ethyl · 3- (dimethylaminopropyl) carbodiimide hydrochloride, stirred at 4 C for 20 hours, filtered, and thoroughly washed with water to obtain sulfated glucuronidated fucoidan Sugar-Celluloid. (2) Separation of α-D-glucosidase and endo-a-L-fucosinase by sulfated glucuronidation fucoidan · Celluloid Obtained from Example 1 (1) Part of the purified enzyme solution was thoroughly dialyzed with 10 mM imidazole-hydrochloric acid buffer solution (pH 75) containing 50% of sodium gas and 10 mM of calcium gas, and then 50 ml of sulfuric acid equilibrated with the same buffer solution was used. The glucuronidated fucoidan celluloid was washed with the same buffer solution, and then eluted with a gradient of 50 mM to 1M sodium gaseous sodium. If the "sulfated glucuronidated fucoidan decomposition activity" of the dissolution part is measured, the recovery rate of the activity is about 2%. Renxifang only has a slight "sulphated glucuronidated fucoidan decomposition" Live-88-

1243853 A7 B7 V. "Illustrative Invention" Residual dissolution content and sulfated glucuronide fucoidan mixed with unabsorbed part of the mixture, then "sulfated glucuronidation fucoidan decomposition activity Becomes approximately equal to the activity added to the column. Because the above non-adsorbed part is used alone, it will not reduce the molecular weight of sulfated glucuronidated fucoidan, and it has only a little "dissolved activity of sulfated glucuronidated fucoidan" When used alone, it will not reduce the molecular weight of sulfated glucuronidated fucoidan at all. It can be determined that the unadsorbed and dissolved parts of the sulfated glucuronidated fucoidan are decomposed by different actions. And, if the dissolving part is not caused to act in advance on the sulfated gluconate acidified fucoidan, the unabsorbed part will not act. This suggests that the dissolution part is to cut off the sulfate. The side chain of glucuronidated fucoidan (that is, a-D -glucuronyl bond or sulfate ester bond, etc.) The part of the fucoidan whose molecular weight does not change much), and the non-adsorbed part is to cut off its main chain (that is, a-L -fucosyl bond). Example 5 (1) Identification of oligosaccharides by 2-aminopyridine fluorescent labeling (ρ Αβ) method was performed to confirm the function mechanism of the non-adsorbed portion and dissolved portion contained in Example 4 (2) I. The following experiments. Take 50 mol of each of the dried 1- (1) ~ (8) and 2- (3) and 2- (5) sulfated glucuronidated fucoidan, using a glycosyl marker and a glycosyl group The labeling reagent kits (both manufactured by Takara Shuzo Co., Ltd.) are labeled (PA) with a reducing amino group using 2-aminopyridine to prepare a PA compound of the oligosaccharide. (2) The effects of the non-adsorbed portion and the eluted portion described in Example 4 (2) on the 10 types of oligosaccharide PA compounds described in Example 5 (1) were investigated by the following reactions. -89 This paper size applies to Chinese Standard (CNS) A4 (210 X 297 mm)

Binding

Line 1243853 A7 B7 V. Description of the invention (87 reaction system 50 microliters 50 mM imidazole hydrochloride buffer (ρίί6.6) 2 3 microliters of water 5 microliters 4M sodium gasification 2 microliters 1M calcium gasification 5 microliters 5¾ g / ¾ liters of bovine serum albumin 10 microliters 2 picomoles / microliters of oligosaccharide palate 5 microliters of water or the non-adsorbed portion described in Example 4 (2) or the dissolved portion described in Example 4 (2) After all the above components were mixed, they were reacted at 3crc for 3 hours, and then centrifuged at 10000 for 10 minutes, and the supernatant was subjected to HpLC * analysis under the following conditions, and then the effect on each oligosaccharide PA compound was confirmed. Device: L-6200 (manufactured by Hitachi, Ltd.) Column: OHpak SB-803 (8x300 mm, manufactured by Showa Denko Corporation) Eluent: 0.2mM sodium chloride containing 5mM sodium azide and 10% methylsulfoxide Detection: Fluorescence Detector F-1150 (manufactured by Yuri Seisakusho), excitation wavelength 320 nm 'fluorescence wavelength 400 nm.

Flow rate · 1ml / min column temperature: 50 ° C k The results of the above analysis are determined. The unabsorbed portion and dissolved portion described in Example 4 (2) are used for various sulfated glucuronidated fucoidan. The presence or absence of p A compound action, and the retention time in the SB803 column before and after the action, are shown in Table 7. -90-

1243853 A7 B7 V. Description of the invention (88) Table 7 Whether the PA compound of the oligosaccharide acts on the dissolution part of the non-adsorbed part so that the retention time before and after the action of the dissolution part K1) None 9.74 9.72 1-有 None 9.09 9.22 H3) None 8.94 9.06 H4) None 8.76 8.91 H5) None 8.56 8.65 H6) None 8.50 8.60 1_⑺ None 8.40 8.48 H8) None 8.31 8.40 2-⑶ None 8.92 9.04 2-⑶ No 8.56 8.68 As shown in Table 7, the unadsorbed portion of Example 4 (2) had no effect on the PA compound of the oligosaccharide. On the other hand, although the dissolution portion of Example 4 (2) had no effect on sulfated glucuronidated fuctooligosaccharides 1-(1), it had effects on other sulfated glucuronidated fuctooligosaccharides. . From this result, it is strongly shown that the dissolution part of 4 (2) is an enzyme that cuts off the glucuronic acid of the sulfated glucuronidated fuctooligosaccharide. For example, if one fucose molecule is Chess from oligosaccharide 1- (3), oligosaccharide 2-(3) can be generated. The change in the holding time at this time was considered to have changed from 8.94 minutes to 8.92 minutes. However, in the reaction of the dissolution part of Example 4 (2) to the oligosaccharide 1- (3), the holding time was changed from 8.94 minutes to 9.06 minutes. Therefore, it can be concluded that this reaction is not for cutting fucose-91-This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1243853 A7 _______B7 V. Description of the invention Γ89 ~~) ^ &quot; ~ of reaction. Similarly, by comparing the oligosaccharides 2_ (5) and 1- (4) and 2- (3) and 1- (2) ', it can be concluded that the reaction of the dissolution part of Example 4 (2) is not to cut off the sulfate. reaction. In addition, by comparing the oligosaccharides and qi ”, and bu㈠), 1 · (7) and 1- (6) and 1- (3) and 1- (2), it is suggested that the dissolution part of Example 4 (2) The reaction is not a reaction to cut off the sulfated fucose. (3) In order to confirm that the elution portion described in Example 4 (2) has a -0-glucosidase activity, the elution portion described in Example 4 (2) The reaction was performed on the stone wild acidified gluconate oligosaccharides 1-(3) described in Example 2, and the mass change was analyzed. First, the following reaction system was constructed. The reaction system was 32.1 ml of 50 mM imidazole hydrochloric acid buffer solution (ρΗ6 · 6) 2.0 ml of 4M sodium gasification, 0.8 ml of 1M calcium gasification, 4.0 ml of 5 mg / ml of bovine serum albumin, 10 mg of sulfated glucuronide fuco-oligosaccharide b (3) as described in Example 2 The dissolution part described in 4 (2) After mixing all the above components, it was reacted at 30 ° C for 5 days. For desalting, a celluloid GCL-25 column equilibrated with 10% ethanol (4 X 90 cm), aliquot each 9.1 ml of the solution, and measure the total sugar content (-sulfuric acid method) and the total sugar content (Ye Jun-sulfuric acid) law As a result, the color development by the phenol-sulfuric acid method was strongly positive, and the color development by the katu-sulfuric acid method was negative. That is, it was strongly shown that sulfated glucuronide containing glucuronic acid Glucuronic acid cleaving in acidified fucoidan oligosaccharide 1- (3). Furthermore, in order to confirm the cleaving of glucuronic acid, the phenol-sulfuric acid method-92- Chinese paper standard applies to this paper size (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 5. The quality of the part of the invention description (9Q) which is strongly positive is analyzed by mass. Results The quality of these parts is 1506, which means that it is present and the content of sulfuric acid The mass (1506) caused by the removal of glucuronic acid in the modified glucuronidated fuco-oligosaccharide 1- (3). Furthermore, the oligomerization with sulfated glucuronidated fucoid cannot be detected because The sugars 1-(3) were consistent in mass (1682), and it was found that the degluconation reaction proceeded completely. Based on the above results, it was determined that the elution part described in Example 4 (2) had -D-glucose Glycosidase is present. It is the same substance as the mass after excision of glucuronic acid from sulfated glucuronidated fuco-oligosaccharide 1- (3) (1506). Having the structure of formula (VII) of. This paper -93- applies China National Standard Scale (CNS) A4 size (210 X 297 mm) 1243853 A7 B7 V. invention is described in (91 [VII]

Sentence 8 F7 F6 P5

F 3 F 2 F 1 -94- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 ----------_. V. Description of the invention (92) This substance is hereinafter referred to as 8Fuc-4S 0, thereby detecting the present invention "__D-Glucosidase For sulfated glucuronidated fuctooligosaccharide b (3) obtained in Example 5 (丨) The best reaction conditions of p A compound. In addition, when determining the 々-D-glucosidase activity of the present invention, the activity was measured by the following reaction system. 50 microliters of the reaction system contains 1〇. 50 mM sodium gasified 50 imidazole-hydrochloric acid buffer (PH7.0) 2 1 microliter of water 4 microliters 1 micrometer of calcium vaporized 10 microliters 3 mg / ml bovine serum albumin 10 microliters 4 picomolars / micro 5 microliters of α-D-glucosidase solution after mixing all the above components with 5 μl of α-D-glucuronidase solution. The reaction was performed at 2 ° C for 3 hours at 100 ° C. (Centrifuge after 10 minutes of treatment. The supernatant was subjected to Hplc analysis under the following conditions. 0 Device: L_ 6200 (manufactured by Hitachi). Column: L · Column (4.6x250 mm, corporate foundation Product inspection association) Eluate: 50 mM acetic acid-triethylamine buffer (ρΗ5.0 · 0) containing 0.3% butanol Detection: Fluorescence detector F-1150 (manufactured by Hitachi), excitation wavelength 320 nm, fluorescence The light wavelength is 400 nm. -95- This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 _____B7 Five invention instructions (93 ^) a '&quot;

Flow rate: 1 ml / min column temperature 1: 4 (TC 1 unit of the present invention α_D-glucose: y: acidase, which refers to the reaction system in which 1 micromolar of Example 5 (1 ) The amount of the enzyme that cleaves the glucuronic acid group of the sulfated glucuronidated glutamate 1- (3) PA compound. The amount of the glucuronide group that is cut off is borrowed It is calculated from the following formula: Mathematical formula 2 DGA / 18 × 0.05 = U / ml DGA: Example 5 (1) which was cut off Sulfated glucose obtained from acidified fuctooligosaccharide 1- ( 3) Amount of PA compound (micromolar) 180: reaction time (minutes) 0.005: amount of enzyme solution (ml) (4) 8FUC-4S described in Example 5 (3) is identified by 2-aminopyridine (PA conversion) Take 50 nanomolar of 8FUC-4S as described in Example 5 (3), and use a glycosyl labeling agent and a glycosyl labeling reagent kit (both manufactured by Takara Shuzo Co., Ltd.). Emei is fluorescently labeled (PA-based) to modulate the PA compound of the oligosaccharide. This substance is hereinafter referred to as 8Fuc-4S-PA 〇 (5) As described in Example 4 (2) using 8Fuc-4S-PA Review of the role of the absorbing and dissolving parts Example 4 (2) The effects of the non-adsorbed portion and the eluted portion on 8Fuc-4S-PA described in Example 5 (4) were investigated by the following reaction system: 50 microliters of 50 mM imidazole · hydrochloric acid buffer (PH6.6) -96 -

This paper size applies to China National Standard (CNS) Α4 size (210 X 297 public love) 1243853

2 3 microliters of water 5 microliters of 4M sodium gasification 2 microliters of 1M gasification_ 5 microliters 5 mg / ml bovine serum albumin

10 microliters 2 picomoles / microliters 8Fuc-4S_pA 5 microliters of water or the unadsorbed portion described in Example 4 (2) or the dissolved portion described in Example 4 (2). 3〇. The reaction was performed for 3 hours, and the mixture was centrifuged at 10 ° C for 10 minutes. The supernatant was subjected to HPLC analysis under the conditions described in 5 (3). As a result of the above analysis, the non-adsorbed portion described in Example 4 (2) may be 8Fm_ 4S-PA was solved, and the dissolution part described in Example 4 (2) could not dissolve 盱 4_PA. Furthermore, aw 4S- The decomposed product obtained by PA decomposition is dissolved at the same position as the sulfated glucuronidated fucoidan obtained in Example 5 (1) at the same position. In this case, a large number of compounds are dissolved in Example 2 and Example 3. There is no contradiction in obtaining the same substance as sulfated glucuronidated fuctooligosaccharide 1- (1). That is, the non-adsorbed portion described in the example can be judged to be deacetylated degluconated. Within the action of sulfated glucuronidated fucoidan and sulfated glucuronidated fuctooligosaccharide-α-L-fucosinase. Based on this result, the sulfuric acid prepared in Example 4 can be determined. Glucuronidated fucosides are useful resins for separating the glucuronidase of the present invention and the -L-fucosidase from within the present invention. (6) Who 'review by the present invention - no -l · A enzyme for fucose 8Fuc_4S_ -97-

1243853 A7 B7 V. Invention description (95) Optimum reaction conditions of PA. In addition, when the endo-me-L-fucosidase activity of the present invention is determined, the activity is measured by the following reaction system. 50 microliters of 50 mM acetic acid buffer solution (ρΗ5 · 5) containing 40 mM sodium vaporization 23 microliters of water 2 microliters 1 M calcium vaporization 10 microliters 3 mg / ml bovine serum albumin 10 microliters 4 pico Moore / microliter 8Fuc-4S-PA 5 microliters of endo-a-L-fucosidase was reacted at 30 ° C for 3 hours, and the reaction solution was analyzed in the same manner as above. One unit of the in-α-L-fucosinase of the present invention refers to the amount of enzyme that cuts 1 micromole of 8Fuc-4S-PA in one minute in the above reaction system. The amount of the fucosyl bond that was cut was obtained by the following formula. Mathematical formula 3 DPA / 180x0.005 = U / ml DPA: the amount of 8Fuc-4S-PA cut off (micromolar) 180: reaction time (minutes) 0.005 ·· enzyme volume (ml) Example 6 Example The non-adsorbed portion and the eluted portion described in 4 (2) were prepared by pre-treatment with a 10 mM imidazole-hydrochloric acid buffer solution containing 100 mM sodium vaporization, 10 mM vaporization # 5, and 5 μM sodium azide. (ΡΗ7.5) Equilibrium gel filtration with Sephacryl S-200 column (4.4 X 100 cm), divided into 13.5 ml per solution -98- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Binding

Line 1243853 A7 B7 V. Description of the invention (96) Take the solvent. For the eluted fraction, the activity of the π_D-glucosaccharinase of the present invention and the -L-fucosylase within the present invention was measured separately, and the unadsorbed portion described in Example 4 (2) In the method described in Example 5 (2), the substrate was a sulfated glucuronidated fucoidan i 3), and the elution portion described in Example 4 (2) was used in Example 5 (2). 5) The method described in the measurement. The a-D-glucosidase and α-L_fucosidase activities of the present invention were measured. The molecular weight of the -D-glucuronidase of the present invention measured by the above-mentioned gel filtration method is about 120,000 to 180,000, and the molecular weight of the endo-α-L-fucosylase of the present invention is about 15 Million to 200,000. Example 7 The crude sulfated glucuronidated fucoidan portion described in Reference Example 1 (1) was purified by DEAE-Servin A · 800. That is, a column of 5 liters of DEAE-Serupine A-800 equilibrated with a 20 mM olfactory-hydrochloric acid buffer solution (ρΗ8.0) containing 50 mM sodium gas and 10% ethanol was deposited in advance, 5 g of the crude sulfated glucuronidated fucoidan described in Reference Example 1 (1) dissolved in the same buffer solution, washed with the same buffer solution, and then washed with 100 mM sodium chloride and Wash with 10% ethanol in 20 mM imidazole-hydrochloric acid buffer (pH 8.0) and elute with a gradient of 1Q0 mM to 2M sodium chloride. The dissolved fractions were divided into 500 ml portions, and the total sugar content was measured by a sulfuric acid test. The sulfated glucuronidated fucoid dissolved in the vicinity of 500 mM of dissolved salt concentration was collected as the main component, and was collected by an out-of-limit filtering device equipped with a hollow fiber with a molecular weight of 100,000. Desalted and freeze-dried to obtain 0.9 g of refined sulfated glucuronidated fucoidan. Review the adjustment of the glucuronic acid content of the above-mentioned refined sulfated glucuronidated fucoidan -99- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Invention Description (97) whole. First, construct the following reaction system. Reaction system 5 μl 50 mM imidazole-hydrochloric acid buffer (pH 6.6) 0.5 μl 4M sodium chloride 0.5 μl 1 M calcium vaporized 0.25 μl 5 mg / ml bovine serum albumin 3.35 μl Example 4 (2) 2.4 ml of the 1.25% refined sulfated glucuronidated fucoidan aqueous solution was mixed with all the above components and reacted at 25 ° C. The samples were taken at different times and the samples were fully dialyzed. Remove the cut-off glucuronic acid. Table 8 shows the amount of fucose, glucuronic acid, and their ratios contained in each sample. Table 8 Reaction time (hours) Fucose content (mM) Glucuronic acid content (mM) Molar ratio of fucose to glucuronic acid 0 6.1 1.4 4.4: 1 1 6.3 1.3 4.9: 1 2 6.9 1.2 5.8: 1 5 6.5 0.90 7.2: 1 10 6.9 0.70 9.9: 1 20 6.3-0.65 9.7: 1 -100- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the Invention (98) Based on the above results, it is determined that if the dissolution portion described in Example 4 (2) is used, the fucose can be adjusted by removing the glucuronic acid from the sulfated glucuronidated fucose. Molar ratio with glucuronic acid. Example 8 (1) Effect of calcium salt concentration on enzyme reaction When measuring the Γ sulfated glucuronidation fucoidan decomposition activity of the present invention ", if the concentration of gasified calcium contained in the reaction system is reduced, then A decrease in activity was seen. Therefore, for the α-D-glucosidase of the present invention and the -a-L-fucosidase of the present invention, the relationship between the concentration of calcium carbonate contained in the reaction system and the relative activity of the enzyme was also investigated. As a result, it was determined that both of these enzymes would be activated by calcium carbonate. Furthermore, it was found that this dienzyme was similarly activated by calcium acetate. (2) Effect of protein on enzyme reaction When the "sulfated glucuronidated fucoidan decomposition activity" of the present invention is measured, if bovine serum albumin is removed from the reaction system, the activity is reduced. Therefore, the relationship between the concentration of bovine serum albumin and the relative activity of the enzyme in the reaction system was also investigated for the "· -glucosidase" and "a_L-fucosidase" of the present invention. . As a result, it was determined that the second enzyme $ would be activated by bovine serum albumin. Furthermore, it was found that the protein produced by the fusarium SI-1234 strain degraded by the rock glycosaminoglycan of the present invention can be similarly activated. (3) Effect of sodium gasification on enzyme reaction When measuring the "sulfated glucuronidated fucoidan decomposition activity" of the present invention, if sodium gasification is removed from the reaction system, the activity is seen; low '. 101-This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) 1243853

Therefore, the relationship between the concentration of aerated sodium contained in the reaction system and the relative activity of the enzyme was also investigated for the a_D glucuronidase of the present invention and Nai-Jiang · L-shishensubo enzyme of the present invention. As a result, it was determined that the dienzyme was not activated by sodium vaporization when the low-molecular oligosaccharide was used as the base. From this, it can be confirmed that when sodium carbonate is a substrate in which the substrate of the enzyme is a polymerized sulfated glucuronide fucoidan ', both enzymes are activated. Example 9 Inoculation of the fucoidan strain SI-1234, which is a glycococcus lycoside, into the culture medium, the culture medium system will contain crude sulfated glucoaldehyde from Okinawa sea-grade algae prepared by the method of Reference Example 丨 (丨). 600 ml of culture medium consisting of acidified fucoidan fraction 0.2% and protein gland 1/0 artificial per water (pH 8.0) (manufactured by Gabriel Labradle) was prepared at 120. (: Obtained by 20-minute autoclave treatment. After inoculation, 'at 24 X: cultured for 72 hours as the culture broth. In a 30-liter shake flask fermenter, 200 g of protein gland and defoaming are included. Agent (Κλ17〇, manufactured by Shin-Etsu Chemical Industry Co., Ltd.) 18 liters of ρΗ8.0 culture medium formed by artificial seawater and 120 ° C 20-minute autoclave culture medium, and prepared from the method of Reference Example 1 (1) 40 grams of crude sulfated glucuronidated fucoidan from Okinawa seaweed was dissolved in 2 liters of artificial seawater and treated at 95 ° C for 1 hour, mixed, and then inoculated with the above culture medium And, at a rotation speed of 125 revolutions per minute, culture at 24 ° C for 72 hours. Furthermore, 'the pH value of the culture medium is automatically controlled to more than 7 in the culture. After the culture is complete,' the culture solution is centrifuged 'to obtain bacterial cells and Culture the supernatant. As described above, for the sulfated glucuronidated fucoidan added to the culture medium, the heating temperature is about 9 5 Ό and about -102- this paper size applies China National Standard (CNS) A4 Specification (210 X 297 PCT) 1243853 A7 B7 V. invention is described (1Q1)

Invest. Dermatol., 103: 306-309, 1994), the beard is separated from the hair follicle in a glass culture under a microscope. Take about 1 to 16 beards from one rat. Next, partially dissolve the crude sulfated glucuronidated fucoidan from the seaweed algae of Okinawa described in Reference Example 1 (1) in RPMI-1640 medium to prepare a concentration solution of 20 times the set concentration. Then add one-twentieth to the culture line. The same amount of medium was added to the control group. For beard culture, Falcon 3037 (manufactured by Becton Dickinson Labware) for tissue culture was used. 0.7 ml of 20% FCS RPMI-1 640 medium was added to the central acupoint, and a sterilized stainless steel mesh (Ikeda R & D) was placed on it. Co., Ltd.) and lens cleaning paper (made by Tishikesi Co., Ltd.). Hair was loaded thereon and cultivated. The sulfated glucose from the Okinawa Turtle was added to the culture medium in advance while the acidic fucoidan portion was added. The culture line was performed in the presence of 35 ° (: and 5% (: 02). The length of hair was measured under the microscope to a digit of 0.1 mm before and after the culture was started. For various sample concentrations The same group was measured using 3 to 5 hairs, and the length of elongation was expressed by the standard error of average soil. Furthermore, the significant difference test was performed by the student's t test, and the P 値 relative to the control group was determined. The results were compared. Shown in Table 9. -104- This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (1Q2 Table 9 Add sample concentration (mg / ml) Number of samples Hair elongation (standard mean soil error) (mm) P 値 Okinawa sea algae 0.01 5 0.9 soil 0.25 0.17 control 0 6 0.38 soil 0.23 As shown in Table 9, the sulfated glucuronidation of sea algae from Okinawa Compared to the control group, the fucoidan portion was confirmed to have the effect of elongating the hair of mice. Example 1 1 (1) Modulation of Enzymes The fucoidan prepared by the method of Example 9 will decompose the rock. Algae strain SI-1234 strain cell extract A 10 mM imidazole-hydrochloric acid buffer solution (pH 7.5) with 10 mM calcium carbonate was fully dialyzed, and the supernatant was obtained after centrifugation, that is, the pan-D-glucosaminidase and endo-π-L of the present invention. -A crude enzyme solution coexisting with fucosidase. The obtained crude enzyme solution was fed into a 500 ml DEAE-cellulose A-800 column equilibrated with the same buffer solution, and washed with the same buffer solution. With a gradient of 100 mM to 40 OmM sodium gaseous leaching, dissolve the dissolving fraction in 67 ml per bottle, and collect the sulfated glucuronidated fucoidan decomposing active fraction. The obtained sulfuric acid The glucuronidated fucoidan-decomposing active fraction was concentrated by an out-of-limit filter equipped with a hollow fiber with a molecular weight of 10,000, and then concentrated with a solution containing 50 mM sodium gas and 10 mM calcium gas. 10 mM imidazole-hydrochloric acid buffer solution (pH 7.5) was used to replace the buffer solution. The obtained enzyme solution was fed to an example of equilibration with the same buffer solution. -105- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (4 (1) 50 ml of sulfated glucuronidated fucoidan-celluloid After washing in the column with the same buffer solution, dissolve it in a gradient of 50 mM to 600 mM sodium gaseous solution. Dissolve the dissolution fraction in 10 ml per bottle, and measure the sulfated glucosyl in each fraction. Decomposition activity of acidified fucoidan. As a result, only the eluted portion could detect the activity, and no activity was found on the non-adsorbed portion. However, if the non-adsorbed portion was mixed with the eluted portion, the glucosyl sulfate was sulfated The acidified fucoidan decomposition activity will be approximately equal to the activity added to the column. The non-adsorbed portion was collected and concentrated to 100 ml by an external filter to obtain a non-adsorbed portion concentrate. On the other hand, the concentrated non-adsorbed portion was used to measure the sulfated glucuronidated fucoidan decomposition activity, and the active portion was concentrated to 200 ml by means of an out-of-limit filter to obtain a concentrated solution of the eluted portion. These enzymes were further refined in an attempt to prepare sulfated glucuronidated fuctooligosaccharides. First, a reaction system was constructed to measure the activity of each part. When the dissolving part and the non-adsorbing part mentioned above are used alone, they cannot act on the sulfated glucose fossil bath glycan 'and therefore cannot effectively generate oligosaccharides. However, if they coexist, the sulfated glucoaldehyde can be used. The acidified fucoid is decomposed, and it is efficiently made low molecular. In the state where the two enzymes coexist, the activity of decomposing sulfated glucoside acidified rock glycan is determined by the following activity measurement method. The method for measuring the activity of the non-adsorbed fraction was performed in the following manner. That is, 10 microliters of 1% crude sulfated glucose while acidifying a portion of the fucoidan solution, 5 3 microliters of 50 mM mimic-hydrochloric acid buffer (ρΗ6 · 6), 5 microliters of 4 M sodium chloride, 2 microliters of 1M calcium carbonate, 10 microliters of 5 mg / ml bovine serum albumin, 1 microliter of the dissolution portion concentrate and 19 microliters for measuring activity Paper size applies to China National Standard (CNS) A4 (210 X 297 mm) binding

Line 1243853 A7 B7 V. Description of the invention (1Q4) The enzyme solution from the non-adsorbed part is mixed, reacted at 30 ° C for 3 hours, and then treated at 100 ° C for 10 minutes. After centrifugation, use 90 microliters. HPLC analysis to determine the extent of low molecularization. As a control, the enzyme solution from the non-adsorbed portion was replaced by a buffer solution in which the enzyme solution was dissolved and reacted, and the crude sulfated glucuronidated fucoidan portion was reacted with water instead, and then The analysis was similarly performed by HPLC. 1 unit of sulfated glucuronidated fucoidan decomposition activity refers to the cleavage of the fucose bond of 1 micromole of sulfated glucuronidated fuctan in the above reaction system in 1 minute Amount of broken enzyme. The amount of the fucosyl bond that was cut off was obtained by the following formula. Mathematical formula 4 {(10xl000xl / 100) / MG} x {(MG / M) -l} x {l / (180x0.019)} = U / ml 10x1000x1 / 100: Crude sulfated glucose added to the reaction system Uronic acid fucoidan fraction (micrograms) MG ·· The average molecular weight of the sulfated glucuronidated fucoidan of the control reaction solution M: the average molecular weight of the reaction product (MG / M) -1: 1 molecule Number of sites where sulfated glucuronidated fucoidan is cut by enzyme 180: reaction time (minutes) 0.019: amount of enzyme solution (ml) Furthermore, the conditions of HPLC are as described below. Device · · L-6200 (manufactured by Hitachi) Column: OHpak SB-806HQ (8 x 3 00mm, manufactured by Showa Denko Corporation) -107- This paper size is in accordance with China National Standard (CNS) A4 (210 X 297) 1243853 A7 B7 V. Description of the invention (1Q5)

Eluate: 50 mM steel chloride containing 5 μM sodium azide. Detection: Parallax tortuosity detector (ShodexRI-71, manufactured by Showa Denko). Flow rate: 1 ml / min. Column temperature: 25 ° C. The average molecular weight of the reaction product was measured, and commercially available pullulan (Pullulan) (STANDARD P-82, manufactured by Showa Denko Co., Ltd.) having a known molecular weight was analyzed under the same conditions as the HPLC analysis described above, and the buds The relationship between the molecular weight and the retention time is represented by a curve, which is used as a standard curve for measuring the molecular weight of the reaction product. On the other hand, the method for measuring the activity of the eluted portion was performed as follows. That is, 10 microliters of a 1% crude sulfated glucuronidated fucoidan partial solution, 53 microliters of 50 mM imidazole-hydrochloric acid buffer (pH 6.6), and 5 microliters of 4M chlorination Sodium, 2 microliters of 1M calcium carbonate, 10 microliters of 5 mg / ml bovine serum albumin, 19 microliters of non-adsorbed fraction concentrate and 1 microliter of activity from the dissolution fraction The enzyme solution was mixed, reacted at 30 ° C for 3 hours, and then treated at 100 ° C for 10 minutes. After centrifugation, 90 microliters were analyzed by HPLC to determine the degree of low molecularization. As a control, the enzyme solution from the eluted portion was replaced with a buffer solution to dissolve the enzyme solution, and the crude sulfated glucuronidated fucoidan portion was reacted with water instead. It was then analyzed by HPLC in the same manner. 1 unit of sulfated glucuronidated fucoidan decomposition activity refers to the above reaction system which cuts off the fucose bond of 1 micromole of sulfated glucuronidated fuctan in 1 minute. The amount of enzyme. The amount of the fucosyl bond that was cut off was obtained by the following formula. -108- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (1Q6) Mathematical formula 5 {(ΙΟχΙΟΟΟχ 1/100) / MG} x {(MG / M) -l} x {1 / (18〇χ〇. 00 l)} = U / ml 10x1000x1 / 100: Crude sulfated glucuronidated fucose part (micrograms) added to the reaction system MG : Average molecular weight of sulfated glucuronidated fucoidan of the control reaction solution M: average molecular weight of reaction product (MG / M) -1: 1 molecule of sulfated glucuronidated fucoidan cut by enzyme Number of broken parts: 180. Response time (minutes) 0.001: Enzyme solution volume (ml) Purification of the concentrated solution without absorption is performed in the following manner. That is, 4 M sodium vaporization was added to the above non-adsorbed portion of the concentrated solution, adjusted to a concentration of 250 mM, and then fed to a 30 ml phenyl-cellulose column equilibrated with the same buffer solution. . After washing with the same buffer, it was eluted with a gradient of 250 mM to 0 mM sodium chloride, and then lysed with a 10 mM imidazole-hydrochloric acid buffer (pH 7.5) containing 10 mM calcium carbonate, followed by dissolution with 10% ethanol was dissolved with 10 mM imidazole-hydrochloric acid buffer solution (pH 7.5) of 10 mM calcium carbonate, and the dissolution portion was separated into 10 ml per bottle, and the sulfated content determined by the above method was collected. Glucuronidates the fucoidan-decomposing active moiety. The sulfated glucuronidated fucoidan-decomposed active fraction was decomposed with 10 mM Miye-hydrochloric acid buffer (pH7.) Containing 100 mM sodium gasification, 10 mM calcium chloride, and 5 mM sodium azide. 5) As the eluent, it was separated through a Sephacryl S-200 column (4.4 X 100 cm). Take 13.5ml per bottle -109- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Disclosure of the invention (1Q7). The desulfurized glucosino-acid-fucose-decomposing active part determined by the above method was used as the refined unadsorbed part. The purification of the eluted portion of the concentrated solution was performed in the following manner. That is, the above-mentioned eluted part of the concentrated solution was equipped with an out-of-limit filtration device with a partitioned molecular weight of 10,000 hollow fibers, and buffered with 10 mM imidazole-hydrochloric acid containing 100 mM sodium gas and 10 mM calcium gas. Solution (pH 7.5), then fed into a 4 5 ml DEAE-cellulose column equilibrated with the same buffer, washed with the same buffer, and then dissolved in a gradient of 100 mM to 400 mM sodium gaseous solution. Dissolve, dissolve the dissolving fraction in 10 ml per bottle, and collect the sulfated glucuronidated fucoidan decomposing active fraction determined by the above method. The obtained sulfated glucuronidated fucoidan-decomposing active fraction was decomposed with 10 mM micon-hydrochloric acid buffer (ρΗ7 ·, containing 100 mM sodium gas, 10 mM calcium gas, and 5 mM sodium azide). 5) As the eluent, it was separated through a Sephacryl S-200 column (4.4 × 100 cm). Dissolve the dissolution portion in 13.5 ml bottles. The desulfurized glucuronide-fucose-decomposing active part determined by the above method was used as the refined eluted part. (2) Preparation of Sulfated Glucuronidated Fucoidan Oligosaccharides The preparation of the sulfated glucuronidated Fucoidan oligosaccharides was performed using the above-mentioned purified unadsorbed portion and purified eluted portion. That is, 10 grams of crude sulfated glucuronidated fucoidan was partially dissolved in 1 liter containing 250 mM sodium gasification, 20 mM chloride # 5, 5 mM sodium azide, and 1 gram cattle After serum albumin 10 mM Mijun-hydrochloric acid buffer (pH 6.6), 100 mU of the refined dissolution portion and 600 mU of the purified unabsorbed portion were added and allowed to react at 30 ° C for 10 days. Centrifuge the reaction solution to obtain -110- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (1Q8) &quot; ^ ' There is a limit-exceeding / removing device that excludes hollow fiber with a molecular weight of 10,000, and recovers the polysaccharide fraction with a molecular weight of less than 10,000 as the sulfated glucuronidated fucoidan enzyme digestion part 3. (3) Purification of sulfated glucuronidated fucoidan oligosaccharides The above-mentioned sulfated glucuronidated fucoidan enzyme digestion part 3 was processed by a desalination device (micro-Asilaza-G3, Asahi Kasei Industrial company) desalination. To desalted sulfated gluconate to acidify the fucoidan digest part 3, add a bite to a concentration of 5 μM, and add sodium gasification to a concentration of 10 mM, and then feed it in advance An i-liter dEaE-cellulose A-800 column equilibrated with a 5 mM mimic-hydrochloric acid buffer solution (ρΗ7 · 0) containing 10 sodium vapors, washed with the same buffer solution, and then washed with 10 to 4 〇mM &lt; Gradient dissolution of sodium vaporization. The total sugar content of the eluted portion was measured by the phenol-sulfuric acid method, and the total uronic acid content was measured by the carbazole-sulfuric acid method. As a result, there were at least 5 distinct peaks in the dissolved solvent, and the peaks were collected as 'polysaccharides 3- (1) ~ (5)' and concentrated by an evaporator to 40 ml. After that, it was fed into a celluloid GCL-25 column preliminarily equilibrated with 10% ethanol, and dissolved and desalted with 10% ethanol. Thereby, the sulfated glucuronidated fucoidigosaccharides 3- (1) to (5) of the present invention can be obtained, that is, oligosaccharides 3_ (1) to (5). (4) Structural analysis of oligosaccharides For the desalted products of 3- (1) ~ (5) of the above-mentioned vermin, the terminal sugars and sugar composition were reduced by the fluorescent labeling method using 2-amine batches. During analysis, it was found that all the reducing terminal sugars of the oligosaccharide were fucose. Regarding sugar composition, oligosaccharides 3- (2) are formed only from fucose, and oligosaccharides 3- (1) and 3_ (3) ~ (5) are formed from fucose and glucuronic acid. By. Then, determine the sulfuric acid-111-this paper standard is applicable @ g 家 standard (CNS) A4 specification (⑽x297 public love) 1243853 A7 B7 V. Description of the invention (1Q9) content (by turbidimetric method using barium chloride) and The uronic acid content (by the carbazole-sulfuric acid method), and the mass was analyzed by a mass analyzer (API-III, manufactured by Parkinano Roussex Corporation). Furthermore, NMR analysis was performed using a JNM-α 500-type nuclear magnetic resonance apparatus (manufactured by Japan Electronics Corporation). The analysis sample was replaced with heavy water by a fixed method, and then the structure was analyzed. The method of combining sugars is performed using the HMBC method, which is a 1 Η-detection heterogeneous nuclear test method. The assignment of iH-NMR uses the DQF-COSY method and the ΗΟΗΑΗΑ method, and the assignment of 13C-NMR uses the HSQC method. The physical properties of the oligosaccharides 3- (2) to (5) are shown below. (A) The physical properties of the oligosaccharides 3- (2) are analyzed. As a result, it is determined that this substance is the same as the oligosaccharides 1 to (1). Of matter. (b) The physical property quality analysis of oligosaccharide 3- (3) and the assignment results of NMR analysis are shown below. The sulfated glucuronidated fucoidigosaccharide 3-(3) of the present invention Η-NMR The spectrum is shown in Figure 29, the 13 C-NMR spectrum is shown in Figure 30, and the mass spectrum is shown in Figure 31. In Figs. 29 and 30, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). Moreover, in FIG. 31, the vertical axis represents relative strength (%), and the horizontal axis represents m / z 値 0. Molecular weight: 1230 MS m / z 1273.4 [M + 2Na + -3H +]-, 625.2 [M + Na + -3H +] 2 ·, 409.2 [M-3H +] 3 · The results of analysis by 1H-NMR and 13C-NMR are shown in Table 10. -112- This paper size applies to China National Standard (CNS) A4 (210X297 mm) 1243853 A7 B7

V. Description of the invention (11Q Table 10 Chemical shift 値 (ppm) 13c-nmr [H-NMR Chemical shift 値, multiplicity, binding constant F1-1 96.9 4.47, d, 8.0 F1-2 70.7 3.44, dd, 8.0, 10.0 F1-3 78.4 3.57, dd, 3.0, 10.0 F1-4 68.5 3.85, d, 3.0 F1-5 71.3 3.66, q, 6.5 F1-6 16.5 1.13, d, 6.5 F2-1 96.2 4.96, d, 4.0 F2-2 67.1 3.81, dd? 4.0, 10.0 F2-3 75.6 3.90, dd, 3.0, 10.0 F2-4 69.2 3.93, d, 3.0 F2-5 66.8 4.06, q, 6.5 F2-6 16.5 1.09, d, 6.5 F3-1 96.5 4.98, d, 4.0 F3-2 67.4 3.84, dd, 4.0, 11.0 F3-3 77.5 3.97, dd, 2.5, 11.0 F3-4 80.4 4.65, d, 2.5 F3-5 67.2 4.29, q, 6.5 F3-6 16.5 1.13 , d, 6.5 F4-1 100.0 4.98, d, 4.0 F4-2 68.0 3.75, dd, 4.0, 11.0 F4-3 77.2 3.89, dd, 2.5, 11.0 F4-4 80.4 4.61, d, 2.5 F4-5 67.5 4.33, q, 6.5 F4-6 16.5 1.11, d, 6.5 F5-1 99.5 4.95, d, 4.0 -113- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 1243853 A7 B7 V. Description of the invention (111 Table 10 (continued) Chemical shift 値 (ppm) 13c-nmr b-NMR Chemical shift 値, multiplicity, binding constant F5-2 67.7 3.73, dd, 4.0, 11.0 F5-3 74.0 3.84, m F5-4 68.2 3.89, m F5-5 68.0 4.19, m F5-6 16.0 1.09, d, 7.0 F6-1 94.4 5.00, d, 4.0 F6-2 75.9 3.77, dd, 4.0, 10.5 F6-3 70.0 4.05, dd, 3.5, 10.5 F6-4 73.1 3.70, d, 3.5 F6-5 67.6 4.17, q, 6.5 F6-6 16.0 1.09, d, 6.5 GA1-1 99.5 5.115d, 4.0 GA1-2 71.3 3.45, dd, 4.0, 10.0 GA1-3 73.3 3.64, t, 10.0 GA1-4 72.0 3.35, t, 10.0 GA1-5 72.6 3.96, d, 10.0 GA1-6 176.7 Sugar composition L-rock bath sugar : D -glucose acid = 6: 1 sulfate group 2 molecules Further, the number of the peak assignment in 1 H-NMR and 13C-NMR is shown by the following formula (XIV). -114- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (112 GA 1

HCOCOOH

cn 1cn

〔X I VJ

CO

CO ΟΗ · Η -115- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X 297 mm) 1243853 A7 B7 5. Description of the invention (113) In addition, this substance is hereinafter referred to as 6Fuc-2S-lGlcUA. (c) The physical property quality analysis of oligosaccharide 3- (4) and the assignment results of NMR analysis are shown below. 1 H-NMR The spectrum is shown in Figure 32, the 13 C_NMR spectrum is shown in Figure 33, and the mass spectrum is shown in Figure 34. In Figs. 32 and 33, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In FIG. 34, the vertical axis represents relative strength (%), and the horizontal axis represents m / z 値. Molecular weight 1724 MS m / z 894. l [M + 3Na + -5H +] 2, 588.7 [M + 2Na + -5H +] 3-, 435.6 [M + Na + -5H +] 4 · By 1H-NMR and The results of 13C-NMR analysis are shown in Table 11. -116- This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (114 Table 1 1 Chemical shift 値 (ppm) 13c_nmr ^ -NMR chemical shift 値, multiplicity Binding constants FM 96.4 4.47, d, 7.9 F1-2 70.2 3.44, dd, 7.9, 9.9 F1-3 77.9 3.57, m F1-4 68.0 3.85, m F1-5 70.8 3.66, q, 6.7 F1-6 15.9 1.13, d, 6.7 F2-1 95.9 4.98, d, 4.0 F2-2 67.0 3.85, m F2-3 76.9 3.98, m F2-4 79.9 4.65, m F2-5 66.7 4.29, q, 6.7 F2-6 15.9 1.13, d, 6.7 F3-1 99.4 5.00, d, 4.0 F3-2 67.5 3.75, dd, 4.0, 10.4 F3-3 76.8 3.89, dd, 2.8, 10.4 F3-4 80.0 4.62, d, 2.8 F3-5 66.9 4.35, q, 6.7 F3-6 16.0 1.11, d, 6.7 F4-1 99.0 4.96, d, 4.0 F4-2 67.1 3.74, dd, 4.0, 10.4 F4-3 74.7 3.88, dd, 3.1, 10.4 F4-4 68.6 3.96, d, 3.1 F4 -5 67.4 4.21, q? 6.7 F4-6 15.5 U0, d, 6.7 F5-1 94.7 5.04, d, 4.0 -117- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Explanation of the invention (115 Table 1 1 (continued) Chemical shift 値 (ppm) 13c-nmr ^ -NMR Chemical shift 値, more Severe, binding constant F5-2 70.8 4.20, dd, 4.0, 10.4 F5-3 70.7 4.32, dd, 2.5, 10.4 F5-4 69.2 5.40, d, 2.5 F5-5 65.7 4.36, q, 6.7 F5-6 15.3 1.02, d, 6.7 Acetyl ch3 20.5 2.06, s Acetyl CO 173.9 F6-1 93.4 4.98, d, 4.0 F6-2 66.6 3.78, dd, 4.0, 10.4 F6-3 77.5 3.82, dd, 2.5, 10.4 F6 -4 79.8 4.55, d, 2.5 F6-5 66.5 4.35, q, 6.7 F6-6 16.0 1.13, d, 6.7 F7-1 99.7 4.96, d, 4.0 F7-2 67.6 3.71, dd, 4.0, 10.4 F7-3 75.6 3.89, dd, 2.8, 10.4 F7-4 79.6 4.64, d, 2.8 F7-5 66.7 4.25, q, 6.7 F7-6 16.2 1.11, d, 6.7 F8-1 97.9 4.96, d, 4.0 F8-2 68.7 3.57, dd , 4.0, 10.4 F8-3 70.2 3.76, dd, 3.1, 10.4 F8-4 72.3 3.66, d, 3.1 F8-5 67.4 4.20, q, 6.7 F8-6 15.4 1.08, d, 6.7

Binding

Line-118- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (116 Table 1 1 (continued) Chemical shift 値 (ppm) 13c-nmr ^- NMR chemical shift 値, multiplicity, binding constant GA1-1 99.5 5.19, d, 4.0 GA1-2 71.3 3.47, dd, 4.0, 9.8 GA1-3 73.3 3.58, t, 9.8 GA1-4 72.0 3.37, t, 9.8 GA1-5 72.6 3.81, d5 9.8 GA1-6 176.7 Sugar composition L · fucose: D-glucuronic acid = 8: 1 sulfate group 4 molecules acetamido 1 molecule Further, iH-NMR and 13C-NMR The number of the peak assignment is shown in the following formula (IX). -119- This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1243853 A7 B7 117 V. Description of the invention (i X3 OH Η GA 1

noox

V, * * j

The size of this paper applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (118) In addition, this substance is hereinafter referred to as 8Fuc-4S-lGlcUA_ 1 acetamido. (d) The physical property quality analysis of oligosaccharide 3- (5) and the results of NMR analysis are shown below. The 1H-NMR spectrum of the sulfated glucuronidated fucoidan 3- (5) of the present invention is shown below. As shown in Fig. 35, the 13C-NMR spectrum is shown in Fig. 36, and the mass spectrum is shown in Fig. 37. In Figs. 35 and 36, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). In FIG. 37, the vertical axis represents relative strength (%), and the horizontal axis represents m / z 値 0. Molecular weight: 2689 MS m / z 924.0 [M + 4Na + -7H +] 3 *, 687.3 [M + 3Na + -7H +] 4 ', 545.3 [M + 2Na + -7H +] 5, 450.5 [M + Na + -7H +] 6_ The results of analysis by 1H-NMR and 13C-NMR are shown in Table 12. -121-This paper size is in accordance with Chinese National Standard (CNS) A4 (210 x 297 mm) 1243853 A7 B7 V. Description of the invention (119 Table 12 Chemical shift 値 (ppm) 13c-nmr ^ -NMR Chemical shift 値, more Severity, binding constant F1-1 96.8 4.46, d, 7.9 F1-2 70.5 3.43, dd, 7.9, 10.0 F1-3 78.3 3.57, m F1-4 68.3 3.85, m F1-5 71.2 3.65, q, 6.7 F1-6 16.2 1.12, d, 6.7 F2-1 96.3 4.97, d, 4.0 F2-2 67.3 3.85, m F2-3 77.1 3.97, m F2-4 80.1 4.65, m F2-5 67.1 4.29, q, 6.7 F2-6 16.2 1.12 , d, 6.7 F3-1 99.7 4.98, d, 4.0 F3-2 67.9 3.74, m F3-3 77.1 3.88, m F3-4 80.3 4.62, m F3-5 67.3 4.35, q, 6.7 F3-6 16.3 1.11, d , 6.7 F4-1 99.3 4.94, d, 4.0 F4-2 67.4 3.73, m F4-3 75.0 3.87, m F4-4 69.0 3.95, m F4-5 67.7 4.20, q, 6.7 F4-6 15.9 1.10, d, 6.7 F5-1 95.0 5.03, d, 4.0

Binding

Line -122- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7

V. Description of the invention (12Q Table 1 2 (continued) Chemical shift 値 (ppm) 13c-nmr ^ -NMR Chemical shift 値, multiplicity, binding constant F5-2 71.1 4.20, m F5-3 71.1 4.31, m F5-4 69.6 5.39, m F5-5 66.1 4.36, q, 6.7 F5-6 15.6 1.01, d, 6.7 F5 ethynyl CH3 20.8 2.06, s F5 ethynyl CO 174.2 F6-1 93.7 4.97, d, 4.0 F6-2 66.9 3.79, m F6-3 77.8 3.82, m F6_4 80.1 4.55, m F6-5 66.9 4.35, q, 6.7 F6-6 16.3 1.12, d, 6.7 F7-1 99.9 4.94, d, 4.0 F7-2 67.9 3.70, m F7-3 75.9 3.88, m F7-4 79.9 4.64, m F7-5 67.1 4.24, q, 6.7 F7-6 16.5 1.11, d, 6.7 F8-1 99.3 4.94, d, 4.0 F8-2 67.4 3.73, m F8-3 75.0 3.87, m F8-4 69.0 3.95, m F8-5 67.7 4.20, q, 6.7 F8-6 15.9 1.10, d, 6.7 -123- This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (121 Table 1 2 (continued) Chemical shift 値 (ppm) 13c-nmr lH-NMR Chemical shift 値, multiplicity, binding constant F9-1 95.0 5.03, d, 4.0 F9-2 71.1 4.20, m F9-3 71.1 4.31, m F9-4 69.6 5.39, m F9-5 66.1 4.36, q, 6.7 F9-6 15. 6 1.01, d, 6.7 F9 ethynyl CH3 20.8 2.06, s F9 ethynyl CO 174.2 F10-1 93.7 4.97, d, 4.0 F10-2 66.9 3.79, m F10-3 77.8 3.82, m F10- 4 80.1 4.55, m F10-5 66.9 4.35, q? 6.7 F10-6 16.3 1.12, d, 6.7 F11-1 99.9 4.94, d, 4.0 F11-2 67.9 3.70, m F11-3 75.9 3.88, m F11-4 79.9 4.64, m F11-5 67.1 4.24, q, 6.7 F11-6 16.5 1.11, d, 6.7 F12-1 98.2 4.94, d, 4.0 F12-2 69.1 3.57, m F12-3 70.5 3.75, m F12-4 72.6 3.66, m F12-5 67.7 4.20, q, 6.7 -124- This paper size is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (122 Table 1 2 (continued) Chemical shift 値(ppm) 13c-nmr b-NMR Chemical shift 値, multiplicity, binding constant F12-6 15.8 1.07, d, 6.7 GA1-1 99.7 5.19, d, 4.0 GA1-2 71.6 3.47, dd, 4.0, 9.8 GA1-3 73.6 3.58, t, 9.8 GA1-4 72.3 3.37, t, 9.8 GA1-5 73.0 3.81, d, 9.8 GA1-6 177.0 GA2-1 99.7 5.19, d, 4.0 GA2-2 71.6 3.47, dd, 4.0, 9.8 GA2- 3 73.6 3.58, t, 9.8 GA2-4 72.3 3.37, t, 9.8 GA2-5 73.0 3.81, d, 9.8 GA2-6 177.0 Sugar composition L-rock Sugar: D - glucuronic acid = 12: 2-yl 6 molecules of acetylsalicylic acid molecules 2 Further, iH-NMR and a peak attributable to the number of 13C-NMR by the following formula (X) shown in FIG. -125- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7

This paper size applies to Chinese National Standard (CNS) A4 (210 x 297 mm) 1243853 A7 B7 V. Description of invention (124 §

* zy F 1 2 F 1 1 F 1 0 F 9 to -127-

This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) 1243853

Furthermore, this substance is hereinafter referred to as 12Fuc_6S_2G1cUA_2 ethenyl. (5) Modulation of Sulfated Glucuronidated Fucoidan Oligosaccharides Treated by Ultra-Bone Wave. Check the low molecular weight method of sulfated glucuronidated fucoidan treated by non-enzymatic method. Y is the 0.1-% aqueous solution of the crude sulfated gluconic acid-fucified fucoidan obtained from Okinawa ’s Izumi I, obtained by the method described in Reference Example 1 (1) of 1-week system, with a powerful ultrasonic generation device 201M (Manufactured by Kubota Corporation) at 2000w. This treatment solution was divided by an anion exchange resin, and each part of the knife was identified by mass analysis' to find a part having a substance with a molecular weight of 762. What ’s more? In the S part, by desalting and freeze-drying, the T sulfated glucose of the present invention can be obtained while acidifying fucoid oligosaccharides (_) (丨). Furthermore, by the same &lt; method &apos; as described above, the sulfated glucuronidated fuco-oligosaccharides of the present invention 1- (2) ~ (7), 2- (1) ~, and 3- (2) ~ (5). Example 1 2 (Ua-D -glucose: y: acidase and endo-L-fucosinase purification of fucoidan by the method of Example 9 Cultivate 3 times'. The bacterial cells obtained from each culture were prepared by the method of Example 9 to 10 mM imidazole-hydrochloric acid buffer (ρΗ7.5) containing 100 mM sodium vapor and 10 mM calcium vapor. ) After full dialysis and centrifugation, the supernatant is obtained, which is the crude enzyme solution of the present invention-glucose: y: acidase and endofucosidase coexisting. Hereinafter, the α-D-glucuronide of the present invention Enzyme activity was measured by the method described in Example 5 (3), and the inner-pan-L-rock deep glycosphinase activity of the present invention was measured by the method described in Example 5 (6), and each -128-) x 297 mm) 1243853 A7 B7 5. Description of the invention (126) Refined enzymes. The obtained crude enzyme solution was fed into a 3 liter DEAE-cellulose A-800 column equilibrated with the same buffer solution, washed with the same buffer solution, and subjected to a sodium chloride gradient of 100 mM to 400 mM. Dissolve, and dissolve the dissolution fraction by averaging 200 ml per bottle, and collect the a-D-glucosidase activity part of the present invention and the -a-L-fucosidase activity part of the present invention. . At this point, the two enzymes of the present invention exhibit almost the same behavioral characteristics and cannot be separated. The above two enzymes were concentrated with an out-of-limit filter equipped with a hollow fiber with a molecular weight of 10,000, and concentrated with a 10 mM imidazole · hydrochloric acid buffer solution containing 100 mM sodium chloride and 10 mM calcium carbonate ( pH 7.5) The buffer was replaced. The obtained enzyme solution was fed into a 240 ml DEAE-cellulose A-800 column equilibrated with the same buffer solution, washed with the same buffer solution, and then eluted with a gradient of 100 mM to 300 mM sodium chloride . The dissolution portion was divided into 25 ml bottles, and the a-D-glucosaminidase active portion of the present invention and the -a-L-fucosidase active portion of the present invention were collected. At this point, the two enzymes of the present invention exhibit almost the same behavioral characteristics and cannot be separated. 0 The active part of the two enzymes is concentrated by an external limit filter equipped with a hollow fiber with a molecular weight of 10,000. The buffer was replaced with a 10 mM imidazole-hydrochloric acid buffer (pH 7.5) containing 50 mM sodium chloride and 5 mM calcium chloride. The obtained enzyme solution was fed into a 50 ml sulfated celluloid (manufactured by Biochemical Industry Co., Ltd.) column equilibrated with the same buffer solution, and washed with the same buffer solution. CNS) A4 size (210 X 297 mm) 1243853 A7 ___ B7 Five I Invention Description (127) &quot; ~ ', then elute with 50 mM to 1 M sodium chloride gradient. The dissolution fraction was divided into 50 ml per bottle, and the α-glucosidase activity fraction of the present invention and the inside-from-L-fucosidase activity fraction of the present invention were collected. At this time, the two enzymes can be completely separated, and each enzyme is refined separately below. (2) Refinement of L-fucosylase; L-fucose enzyme is filtered from the active part of L-fucosyl-L-fucose fenase obtained in (1) above, and filtered with a molecular weight of 10,000 outside The membrane was concentrated and fed to a Sephacryl S-200 column (4) equilibrated with 10 mM imidazole-hydrochloric acid buffer (ρΗ7.5) containing 100 mM sodium vapor, 10 mM calcium vapor, and 5 mM sodium azide. 4 X 100 cm), dissolve in the same buffer. Dissolve the dissolving fraction 'in each bottle of 13.3 ml and collect the active fraction of endo-pan-L-fucose. The known content of the active part of a-L-lithokinosidase was fed into a mixture ratio of 10 mM imidazole · hydrochloric acid buffer (pH 7.5) containing ethanol with sodium mM sodium gas and 5 mM calcium gas and ethanol. A phenylcellulose column (2 · 4 X 44 cm) equilibrated with a buffer solution of 8 5 ·· 15 was eluted with the same buffer solution. Dissolve the dissolution portion in 30 ml 4 ways. The obtained active fraction showed homogeneous properties in an S d S -polyacrylamide electrophoresis test. In this way, within the present invention, -L-fucosidase purified product was obtained. (3) Purification of aD-glucosaccharinase The a-D-glucose of the present invention obtained in (1) above: y: acid enzyme active part, filtered with a molecular weight outside the limit of 10,000, membrane concentration , Fed into a Sephacryl S-200 column (4.4 X 100 cm) equilibrated with 10 mM imidazole-hydrochloric acid buffer (ρΗ7.5) containing 〇OOmM sodium vapor, 10mM calcium vapor, and 5mM sodium azide. Dissolve in the same buffer. Divide each bottle into 3 · 3 ml -130- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) 1243853

The eluted portion was taken, and then the active portion of the glucosidase of the present invention was collected. After knowing the a_D_glucoamidase part of the present invention, it was fully dialyzed with a 10 mM imidazole_hydrochloric acid buffer solution containing 3⑼mM sodium gas and 5mM calcium gas, and fed into 2 which was equilibrated with the same buffer. After being washed with the same buffer solution in a 0 liter sulfated-cellulose column, it was eluted with a gradient of 300 to 900 mM sodium vaporized sodium. Dissolve the dissolving portion in 10 ml per bottle, and then collect the active portion of the a-D-glucogluconic acid of the present invention. The obtained α-D-glucuronidase part of the present invention was fully dialyzed with 0-imidazole-hydrochloric acid buffer containing 200 g of sodium gas and 5 m of calcium gas, and fed into an equilibrium with the same buffer. After being washed with the same buffer solution in a 200 ml sulfated-cellulose column, the solution was eluted with a gasification nano gradient of 200 mM to 800 mM. The dissolution fraction was divided into 7 ml per bottle, and then the active fraction of a-D-glucosaminidase of the present invention was collected. The obtained active ingredients showed homogeneous properties in an S D S -polyacrylamide electrophoresis test. In this way, the α-D-glucose: y: acidase purified product of the present invention was obtained. Example 1 3 The endo-"-L-fucosidase purified product of the present invention obtained in Example 12 (2) and the -D-glucosaminidase purified product of the present invention obtained in Example 12 (3) were tested for sulfate Low molecularization of glucuronidated fucoidan. First, in order to investigate the effect of acetamyl on the decomposition of sulfated glucuronidated fucoidan, deacetylated sulfated glucuronidated fucoidan (1) Deacetylated Sulfated Glucuronidated Fucoidan Modified Sulfated Glucuronidated Fucoid -131, prepared by the method of Reference Example 1 (1)-This paper is applicable to this paper Chinese National Standard (CNS) A4 specification (210 × 297 mm) 1243853 A7 ____B7 V. Description of the invention (129) 200 mg of glycan was dissolved in 20 ml of 1N sodium hydroxide, and then treated at 25 ° C for 20 hours, and then A 20 mM imidazole-hydrochloric acid buffer (ρΗ6.6) containing 200 mM sodium vapor and 50 mM calcium vapor was thoroughly dialyzed to obtain deacetylated sulfated glucuronidated fucoidan. (2) Each Decomposition of the matrix: Sulfated glucuronidated fucoidan prepared by the method of Reference Example 1 (1) and by the method of (1) above The prepared deacetylated sulfated glucuronidated rock bath glycan is added with -D-glucose: y: acidase 150 &quot; u and within the present invention per 200 mg on average -α-L- Fucolytic enzyme 1 mU, decomposition reaction at 25 ° C. Furthermore, the final concentration of each component of these reaction solutions, sulfated glucoside acidified fucose is 6.7 mg / ml, bovine serum albumin is 0.1 mg / ml, sodium vaporized at 200 mM, calcium vaporized at 50 mM, imidazole at 20 mM, and the pH was adjusted to 6.6. As a result, sulfated gluconate acidified fucoidan and deacetylammonium at the beginning of the reaction The molecular weight of the sulfated glucuronidated fucoidan is approximately equal, but the average molecular weight after 2 days of reaction, the sulfated glucuronidated fucoidan is about 1.95 million, and the deacetylated sulfated The acidified fucose is about 27,000. That is, it can be assured that the α-D-glucosidase and the a-L-fucosinase of the present invention can be obtained with good efficiency. Sulfated glucuronidated fuco-oligosaccharides must be deacetylated. Furthermore, if the crude enzyme and partially purified enzyme described in Example 1 are used, Sulfated glucuronidation of fucoidan, the resulting glycans have almost no ethyl acetate, and if it is an example! (I) Refined unabsorbed and refined dissolution part of β It acts on sulfated glucuronidation of fucoidan to form an oligosaccharide with acetamyl group, which can be inferred -132-

1243853 A7

The fucoidan-decomposing strain Fugillella SI-1234 of the present invention can produce a fucoidan deacetylase which can free sulfated glucuronidated fucoidan from the acetyl group. Example 1 4 Fucoidan degrading fucoidan deacetylase produced by fucoidan-degrading Fucoidophila SI-1234 strain, which can free sulfated glucuronide to the acetyl group of fucoidan. (1) Purification of fucoidan deacetylase First, a crude enzyme for determining the fucoidan-degrading fucobacterium s I-1234 strain of the present invention prepared by the method of Example 11 (1) was established. Reaction system of fucoidan deacetylase activity present in solution. That is, 100 microliters of 50 mM imidazole-hydrochloric acid buffer (pH 75), 10 microliters of 4M sodium gasification, 1 microliter of 1M calcium gasification, and 40 microliters of 1% sulfated glucose while Acidified fucoidan and 29 μl of water were added with 20 μl of fucoidan deacetylase solution. After reacting for 3 hours at 30 t, a commercially available acetic acid quantitative kit (F- Acetate kit (manufactured by Rothschild Argus Stiks) was used to measure the free acetamidine. As a result, the fucoidan deacetylase activity of the present invention was about 2 mU per 1 ml of the culture medium. Another method is to use the 2-aminopyridine fluorescent marker as the reducing end of the 3- (4) oligosaccharide as the substrate, and measure the fucoidan deacetylase activity by the following reaction system. . Reaction system 75 microliters of 50mM sodium phosphate buffer solution (pH7.5) 28.5 microliters of water -133- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

Binding

Line 1243853 A7 _ __B7 _____ V. Description of the invention (131) 9 microliters of 4M sodium vaporization 15 microliters 3 mg / ml bovine serum albumin 15 microliters 4 picomoles / microliters of oligosaccharide 3- (4) 7.5 microliters of fluorescein fucoidan deacetylase solution mixed all the above components, reacted at 30 ° C for 1 hour, treated at 100 ° C for 10 minutes, and centrifuged. The supernatant was as follows: The conditions were analyzed by HPLC to determine the amount of deacetylation. Apparatus: L-6200 (manufactured by Hitachi, Ltd.). Column: L-column (4.6 x 250 mm, manufactured by Chemical Inspection Association). Eluent: 50 mM acetic acid-triethyl containing 0.5% butanol. Detection of amine buffer (ρΗ5.0): Fluorescence detector F-1150 (manufactured by Yori Manufacturing Co., Ltd.), excitation wavelength 320 nm, and light wavelength 400 nm.

Flow rate: 1 ml / min. Column temperature: 40 ° C. 1 unit of fucoidan deacetylase of the present invention refers to the above reaction system which cuts off 1 micromolar acetamyl bond in 1 minute. The amount of enzyme. Furthermore, if the acetamyl bond in the above reaction system is cut off, the same formation as the sulfated glucuronidated fuco-oligosaccharide obtained in Example 5 (1) 丨 (3) p A compound is formed. This structure is used to confirm the column retention time of the substrate in which the substrate is cut off. The amount of acetophenone that was cut off was obtained by the following formula. Mathematical formula 6 -134- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm ^ ^ ----- 1243853 A7 __—— ____B7 V. Description of the invention (132) ~ ~-DA / 6 〇x〇.〇15 = U / ml DA: the amount of acetamyl group cut off (micromolar) 6 〇 ·· reaction time (minutes) 0.015: the amount of enzyme solution (ml) ^ measured by the method described above In this case, the average activity of the fucoidan deacetylating enzyme of the present invention in 0.8 ml of culture medium is 0.8 mU / ml. 50 ml of the above-mentioned crude enzyme solution is fed into a gasification solution containing 100 mM. Steel 10 mM 1 # 5 and 5 mM sodium azide in 10 mM ton-hydrochloric acid buffer (ρΗ7.5) equilibrated Sephaeryl S200 column (4 4 x 〇〇〇〇, by the same Buffer solution dissolution. Dissolve the dissolution fraction in 13 ml per bottle, and determine the activity of fucoidan deacetylase of the present invention by the method described above. Calculate the rock of the present invention from the amount of the dissolution fluid. The molecular weight of fucoidan deacetylase is about 30,000 to 50,000. The above active fractions are collected, and the fucoidan deacetylated δ mother of the present invention is investigated. The results are shown in Figs. 1 to 2. That is, Fig. 1 shows the optimal ρ Η 値, and Fig. 2 shows the optimal temperature. As shown in Fig. 1, the fucoidan deacetylase of the present invention is the best. The pH is in the range of 6 to 9.1, and the optimal temperature is in the range of 23 to 45.0. (2) Using fucoidan deacetylase to deacetylate from various fucoidan The reaction builds the following reaction system to determine the amount of acetofluorene contained in various fucoidan. The fucoidan derived from seaweed shown in the following Table 13 is used in the reaction. The reaction system -135- ^ Paper scale applicable China National Standard (CNS) A4 (210 X 297 mm)

Binding

1243853 A7 B7 V. Description of the invention (133) 60 microliters 1% fucoidan 50 microliters 100mM sodium phosphate buffer (pH7.5) 10 microliters 4M sodium gasification 20 microliters 3 mg / ml bovine serum 30 microliters of albumin, 30 microliters of fucoidan deacetylase, mixed all the above components, and reacted at 30 ° C for 21 hours. The amount of acetic acid produced was determined by the above-mentioned acetic acid quantitative kit. Furthermore, each fucoidan was treated with 1N sodium hydroxide at 25 ° C for 18 hours, and the amount of acetic acid produced was measured by the above-mentioned acetic acid quantitative kit. The results are shown in Table 1 and 3. Table 1 3 Cases of using deacetylase in seaweeds Cases of using sodium hydroxide Okinawa sea drop 13.6 17.2 sea algae nd 1.74 cystic rock swing 0.91 15.4 nodular cyst leaf algae \ 0.69 7.74 Gagrim kumbu 1.27 4.40 11.3 Alamea 1.70 3.77 Wakame female 2.70 30.3 Black giant algae 3.80 22.9 Pyrococcus auricula 1.46 12.9 Duveriania antarctica 1.84 15.3 -136- This paper size applies to China National Standard (CNS) A4 (210 X 297 male) 1243853 A7 B7 V. Description of the invention (134) mg · Ethyl / g · fucoidan n.d .: Not determined As shown in Table 13, it can be confirmed that the Acetylase is an enzyme that acts on fucoidan derived from various seaweeds to hydrolyze acetamyl. Furthermore, by comparing with the case of non-specific deacetylation using sodium hydroxide, it can be confirmed that the fucoidan deacetylase of the present invention is effective against fucoidan from algae from Okinawa ( That is, the acetyl group of sulfated glucuronidated fucoidan is highly specific. Example 1 5 (1) The main structure analysis of the sulfated glucuronidated fucoidan of the present invention was determined to determine the complete structure and sulfated part of the refined sulfated glucuronidated fucoidan prepared in Example 7. The cut site of glucuronidated fucoidan was analyzed by NMR. The results of the NMR analysis are shown below. The 1 H-NMR spectrum of the sulfated glucuronidated fucoidan of the present invention is shown in FIG. 38 and the 13C-NMR spectrum is shown in FIG. 39. In Figs. 38 and 39, the vertical axis represents the signal strength, and the horizontal axis represents the chemical shift 値 (ppm). The infrared spectrum is shown in Fig. 40. In Fig. 40, the vertical axis represents the transmittance (%), and the horizontal axis represents the wave number (cm · 1). The analysis results by 1H-NMR and 13C-NMR are shown in Table 14. -137- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (135) Table 14 Chemical shift 値 (ppm) 13c-nmr ^ -NMR FM 94.8 5.03 F1 -2 71.0 4.10 F1-3 72.0 4.20 F1-4 69.4 5.31 F1-5 66.8 4.33 F1-6 15.4 1.01 Acetyl group ch3 20.5 2.07 Ethyl group group CO 175.0-ch3 20.5 2.07 F2-1 94.5 5.01 F2-2 67.0 3.85 F2-3 76.6 3.85 F2-4 80.0 4.63 F2-5 66.9 4.33 F2-6 16.0 1.10 F3-1 99.2 4.96 F3-2 67.0 3.80 F3-3 76.6 3.88 F3-4 80.0 4.63 F3-5 66.9 4.24 F3-6 16.0 1.10 F4-1 99.2 4.96 F4-2 67.2 3.73 F4-3 74.7 3.85 F4-4 68.7 3.95 F4-5 67.4 4.18 -138- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (136) Table 14 (continued) Chemical shift 値 (ppm) 13c_nmr ^ -NMR F4-6 16.0 1.10 GA1 -1 99.6 5.18 GA1-2 71.2 3.47 GA1-3 72.4 3.63 GA1-4 71.8 3.42 GA1- 5 72.6 3.87 GA1-6 177.0-With the attribution shown in Table 14, the sulfated glucuronidated fucoidan of the present invention is represented by the following formula (XV), the fucoid of F1 It is a π bond, while other sugars repeated 5 F4 lines of fucose bonded at position 3 persons. Furthermore, it was confirmed that the acetyl group has one residue per 5 sugars and is mainly bonded to the fourth position of F 1 fucose. That is, it can be judged that the sulfated glucuronidated fucoidan has a structure that overlaps the main skeleton shown below. -139- This paper size applies to China National Standard (CNS) Α4 size (210 X 297 mm) binding

1243853 A7 B7 V. Description of the invention (137)

-140-

This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1243853 A7 B7 V. Description of the invention (138) Possibility of industrial utilization The invention can provide novel fucoidan deacetylase, qD-glucosidase and endo-π-L-fucosinase, which can be used to analyze the structure and reproducibility of sulfated glucuronidate fucose and produce sulfated glucuronidation with good reproducibility Low molecular weight of fucoidan. The present invention also provides a method for producing the enzyme. Furthermore, by using the enzyme, various ratios of deacetylated deacetylated sulfated glucuronidated fucoidan, and various ratios of deacetylated degluconated degluconated deglycosylated can be provided. Sulfated glucuronidated fucoidan and sulfated glucuronidated fucoidan, which can be used as sugar chain engineering reagents. Furthermore, an additive is provided which can make the use of the enzyme efficient. Furthermore, there are provided microorganisms that produce enzymes capable of decomposing sulfated polysaccharides from various brown algae. SEQUENCE LISTING SEQ ID NO: 1: An oligonucleotide primer designed to amplify the 16S rDNA region. SEQ ID N0: 2: Oligonucleotide primers designed to amplify the 16S rDNA region. -141-This paper size applies to China National Standard (CNS) A4 (210X 297 mm)

Binding

1243853 A7 B7 V. Description of the Invention (139) Sequence Listing &lt; 110 &gt; Takala Xuzhou Company &lt; 120 &gt; Sulfated Glucuronidated Fucoidan &lt; 130 &gt; &lt; 150 &gt; JP 2000-186346 &lt; 151 〉 2000-06-21 &lt; 160 &gt; 3 &lt; 2101 &lt; 211 &gt; 20 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223> Designed to amplify the oligos of the 16S rDNA region甞 Acid primers. &lt; 400〉 1 agagtttgat cctggctcag &lt; 210〉 2 &lt; 211 &gt; 19

&lt; 212〉 DNA -142- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 1243853 A7 B7 V. Description of the invention (14) &lt; 213> Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Oligonucleotide primers designed to amplify 16S rDNA region &lt; 400> 2 ggctaccttg ttacgactt &lt; 210 &gt; 3 &lt; 211 &gt; 1535

&lt; 212 &gt; DNA &lt; 213 &gt; Fucoidan Decomposition Fucoidophile SI-1234 strain &lt; 400〉 3 gagattcttt gtattgaagc 60 taacacgtga gcaatctgcc 120 ggaLgtgata cgccaactca 180 gctcgcgcggatgaggat gatat gatat acctggagag 480 ggagactgca agcgttactc 540 gtcaggtgtg aaatctcggg 600 tattggaggg gtaagcggaa 660 agtgaacgct ctcggtggat ctaaagatcg tgttggtagt ttggtgaggt cacactggaa caatgggggc acctctgtca gaagcagtgg ggattcactg gctcaacctc ggcggcgtgg ttataaagat gaatagctcg attaaagcU aaaggctcac ctgagacacg aaccctgatg ccagggagca ctaactccgt ggcgtaaagg gaaactgcgc ttaagacatg gaaagtggca aggaaactcg gtaatggcgc caaggcaaag gtccagacac gtgcaacgcc acaagcaggt gccagcagcc gtgcgtaggc ctgaaactgt caagtcgaac aacgggtgcg aatLaatgcc tttaggagga acgggtagct ctacgggtgg gcgtgaggga tcatagcctg gcggtaatac ggatagatgt ctatctagag -143- This paper size applies to Chinese National Standard (CNS) A4 size (210x 297 mm) 1243853 A7 B7 V. Description of invention (141) tttctggtgt agcggtgaaa tgcgta gata tcagaaggaa caccaatggc gaaggcagct 720 tactggacaa atactgacgc Lgaggcacga aagcatgggt agcgaaaggg attagatacc 780 cctgtagtcc atgccgtaaa cgttgcacac taggtcttgg gggtttcgac cctttcagga 840 ccccagctaa cgcgataagt gtgccgcctg aggactacgg ccgcaaggct aaaactcaaa 900 ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa 960 gaaccttacc taggcttgac atgtaatgga cgattttcag agatgaattt ttcccttcgg 1020 ggctgttaca caggtgctgc atggccgtcg tcagctcgtg tcgtgagatg tttggttaag 1080 tccagcaacg agcgcaaccc tcgtccttag ttgccagcac gtaatggtgg ggactctaag 1140 gagacaaact ctcLttgaga gLgggaaggt ggggatgacg ctacaatgcc cggtacaata ggacgcaata ccgcgaggtg 1260 gagcaaatcc tcaaaaccgg gcccagttcg gattggagtc tgcaactcga ctccatgaag 1320 tcggaatcgc tagtaatgac gtatcagcta tgacgtcgtg aatacgttcc cgggccttgt 1380 acacaccgcc cgtcacatca tgaaagccgg ttttgcccga agtacgtgag ctatccctcg 1440 ggaggcagcg tcctaaggca gggctggLga ttgggatg 1478 -144- present scale paper tcaggtcagt atggccctta 1200 cgcctagggc tacacacgtg Applicable to China National Standard (CNS) A4 specification (210 x 297 mm)

Claims (1)

Patent application scope • -Specific fucoidan deacetylase 'is characterized by the following physical and chemical properties: ⑴ action on sulfur g double-glucose acidified rock steamed glycan and sulfur gt-glucose fermented rock oligosaccharide' The acetamidine is hydrolyzed to dissociate the acetic acid; (Π) The optimal p Η value is in the range of about 6 ~ 9 · 1; (Π) The optimal temperature is in the range of about 23 ~ 45t :. A-D-glucose acid enzyme, which is characterized by the following physical and chemical properties: (I) acts on deacetylated sulfated glucofurfurate acidified fucose and deacetylated sulfated glucuronide Fucoids collect glycans, and a-D-glucuronic acid bond is hydrolyzed to dissociate D-glucuronic acid; (II) The optimal p Η value is in the range of about 5 · 8 ~ 7 · 8 (in) The optimum temperature is in the range of about 14 ~ 29c. 3 · —Intraspecific _ a _ L-Fucose Chi: Enzymes, which are characterized by the following physical and chemical properties: (I) Acting on deacetylated sulfated glucuronidated fucoidan and deacetylated Sulfated glucuronidation of fucoidan, and the inward hydrolytic decomposition of ^ -L-fucosyl furfuryl 'to generate a polysaccharide that has L_fucose at the reducing end; (Π) Optimal pH It is in the range of about 4.5 to 7.5; (III) The optimal temperature is in the range of about 2 3 to 4 2 ° C. 4 · A method for manufacturing fucoidan deacetylase from the patent application No. 1 is characterized by culturing a microorganism having the enzyme production ability, and then obtaining the enzyme from its culture. CNS domain of this paper _χ 297 g g 1243853 丨 · • Patent application scope 5 .: Kind: The "_ method" of the second patent application scope is characterized by cultivation and right The owner obtains the enzyme from the culture of Gan Zhifang's formula. The glutinous compound is obtained from the 6th of a kind of manufacturing application patent within the scope of the "_" method, which is characterized by the cultivation of "" Get the enzyme. ', The microorganism of the moon dagger force, and then from 7--a method of deacetylating sulfated glucosamine acid fucoidan production method, characterized in that the application of the first scope of the patent scope of fucoidan deacetylation The amylase acts on sulfated glucuronidated fucoidan to obtain a deacetylated sulfated glucuronidated fucoidan that removes at least one molecule of ethylamyl. 8 · = The method for preparing the deacetylated sulfated glucuronidated fucoidan in the seventh target of the patent application in May, which is characterized in that sodium chloride, calcium salt and / or protein coexist. 9. A deacetylated sulfated glucuronidated fucoidan or a salt thereof, which is obtained by a method according to item 7 of the scope of patent application. A method for producing 10 # deacetylated deglucouronated sulfated glucuronidated fucoidan, which is characterized in that the α_D_glucose 4 acid in the second patent application scope acts on the Acetate the sulfated gluconate and acidify the fucoidan to obtain a deacetylated and deglucourated sulfated glucuronate fucoidan by removing at least one molecule of glucuronic acid residues. 11. The manufacturing method of deacetylation, deglucouration, sulfated glucuronidation, and fucoidan according to item 10 of the scope of the patent application, which is characterized by coexistence of sodium chloride, mother salt and / or protein. get on. 12. —A kind of deacetylated deglucouronated sulfated glucuronidated fucoidan 2 This paper size applies to China National Standard (CNS) A4 specification (210X297 public director) 1243853 Or its salt, which is obtained by the method of the scope of application of the patent application. 13. · A method for producing sulfated glucuronidated fucoidan, which is characterized in that fucoidan deacetylase, α_D-glucosaccharase and endo-α l_ fucoidan The furfur enzyme acts on sulfated glucuronidated fucoidan to obtain sulfated glucuronidated fucoidan. 14. A method for producing sulfated glucuronidated fucoidan, characterized in that a-D-glucose: y: acidase and endo_α. ^ Fucosinase act on alkali-treated Deacetylated sulfated glucuronidated fucose to obtain sulfated glucuronidated fucoidan. 15. The manufacturing method of sulfated gluconate acidified fucoid I sugar according to item 3 or 4 of Shenyan's patent scope is characterized in that it can be performed under the coexistence of gasification sodium, sodium salt and / or protein. 16. A sulfated glucuronidated fuco-oligosaccharide or a salt thereof, which is obtained by a method according to any one of claims 13 to 15 of the scope of patent application. 17. A sugar compound or a salt thereof having a chemical structure selected from the following general formulae (I) to (m): -3- This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) 1243853 VI. Scope of patent application * 〇 I_ This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm) 1243853 Patent application park I ~~ I ►—I X X This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm) 1243853 A8 B8 C8 D8 Patent application scope [I II; I This paper size applies the Chinese National Standard (CNS) A4 specification (210x 297 mm) 43 2 3 5 8 8 8 8 8 A BCD, patent application scope (where R is SO 'SO3H or CH3CO, and η is above 〇 (Integer) 18 · —A sulfated glucuronidated fucoidan or a salt thereof, which is characterized by the following physical and chemical properties: (1) Constituting sugar: contains fucose and glucuronic acid, and its molar ratio 3 5 ～ 4 4 ·· 1 0, (2) The sulfated bran represented by the following general formula (V111) is used as an essential component of sugar. · This paper size applies the Chinese National Standard (CNS) A4 specification (210 X (297 mm) 1243853 A8 B8 C8 D8 Patent Application Park This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) 8 8 8 8 A B c D 1243853 々, patent application scope (where R is Η, S03H or CH3CO). 19. The sulfated glucuronidated fucoidan according to item 18 of the patent application, characterized in that the η is in the range of 1 to 5000. This paper size applies to China National Standard (CNS) A4 (210X297 mm)