JP2001226407A - Fucose sulfate-conteining polysaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fucose sulfate-containing polysaccharide that is useful as an apoptosis inducer that can be used as a medicine, a carcinostatic agent and a carcinogenic prophylaxis and provide a method of producing the same. SOLUTION: This fucose sulfate-containing polysaccharide has the following physical and chemical properties: (1) the constituent sugar: includes uronic acid; (2) when this polysaccharide is degraded with the fucoidan-degrading enzyme produced by Flavobacterium sp. SA-0082 (FERM BP-5402), a compound represented by the formula exemplified in the following formula I is formed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an apoptosis-inducing agent, a carcinostatic agent and a carcinogenesis preventive agent which can be used as a pharmaceutical. The present invention also provides an apoptosis induction method useful for elucidation of apoptosis mechanism, screening for an apoptosis induction inhibitor, and the like. The present invention further provides a sulfated-fucose-containing polysaccharide purified by the present invention and a decomposition product thereof, and a sulfated-fucose-containing polysaccharide-degrading enzyme useful for production of a decomposition product of a sulfated-fucose-containing polysaccharide and investigation of its structure.

[0002]

2. Description of the Related Art In recent years, with respect to the death of cell tissues, a form called apoptosis (also referred to as apoptosis; self-destruction or cell self-destruction) has attracted attention.

This apoptosis, unlike necrosis, which is a pathological cell death, is considered to be death that is originally incorporated into the cell's own gene. That is, some external or internal factors trigger the activation of a gene that programs apoptosis.Based on this gene, a programmed death gene protein is biosynthesized, and the generated programmed death protein degrades the cell itself, resulting in death. It is thought to lead.

[0004] If such apoptosis can be expressed in desired tissues and cells, unnecessary or pathogenic cells can be naturally eliminated from the living body, which is extremely significant.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to develop a highly safe compound having an action of inducing apoptosis, and to contain an apoptosis-inducing agent, an anticancer agent, a carcinogen-preventing agent, and a compound containing the compound. An object of the present invention is to provide a method for inducing apoptosis using the compound as an active ingredient. Another object of the present invention is to provide a compound-decomposing enzyme useful for producing a decomposition product of the compound of the present invention.

[0006]

SUMMARY OF THE INVENTION In general, the present invention relates to an apoptosis-inducing agent, which is characterized by containing a sulfated-fucose-containing polysaccharide and / or a decomposition product thereof.

The second invention of the present invention relates to a method for inducing apoptosis, which comprises using a sulfated-fucose-containing polysaccharide and / or a decomposition product thereof as an active ingredient.

A third invention of the present invention relates to an anticancer agent, characterized by containing the sulfated-fucose-containing polysaccharide of the fifth or sixth invention of the present invention and / or a decomposition product thereof.

[0009] A fourth invention of the present invention relates to a carcinogen-preventing agent, which is characterized by containing a sulfated-fucose-containing polysaccharide and / or a decomposition product thereof.

A fifth aspect of the present invention relates to a sulfated-fucose-containing polysaccharide having the following physicochemical properties. (1) Constituent sugar: contains uronic acid. (2) Flavobacterium sp.
Fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402) reduces the molecular weight and produces at least one compound selected from the compounds represented by the following formulas (I), (II) and (III). I do.

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A sixth aspect of the present invention relates to a sulfated-fucose-containing polysaccharide having the following physicochemical properties. (1) Constituent sugar: substantially free of uronic acid. (2) Flavobacterium sp.
Fucodyne-degrading enzyme produced by SA-0082 (FERM BP-5402) does not substantially reduce the molecular weight.

A seventh invention of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide according to the fifth invention of the present invention, comprising treating a sulfated-fucose-containing polysaccharide mixture with an agent capable of coagulating acidic polysaccharide in the presence of salts. The method includes a step of removing a precipitate.

An eighth invention of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide of the fifth invention of the present invention, wherein a sulfated-fucose-containing polysaccharide mixture is treated with an anion exchange resin in the presence of a divalent cation. And collecting a target polysaccharide.

A ninth invention of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide of the fifth invention of the present invention. A step of removing the substance using a polysaccharide substance or a substance having an anion exchange group.

A tenth aspect of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide according to the sixth aspect of the present invention, which comprises decomposing a sulfated-fucose-containing polysaccharide mixture having the ability to degrade a sulfated-fucose-sulfuric acid-containing polysaccharide containing uronic acid. A step of treating with an enzyme or a microorganism having the decomposing enzyme to collect a target polysaccharide.

An eleventh invention of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide according to the sixth invention of the present invention, which comprises converting a sulfated-fucose-containing polysaccharide mixture into an agent capable of coagulating an acidic polysaccharide in the presence of salts. A step of precipitating the polysaccharide.

A twelfth aspect of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide according to the sixth aspect of the present invention, wherein a sulfated-fucose-containing polysaccharide mixture is treated with an anion exchange resin in the presence of a divalent cation. And collecting a target polysaccharide.

A thirteenth invention of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide of the sixth invention of the present invention. A step of removing the substance using a polysaccharide substance or a substance having an anion exchange group.

A fourteenth aspect of the present invention relates to a method for producing a sulfated-fucose-containing polysaccharide mixture, and the seventh aspect of the present invention comprises the steps of:
When extracting the sulfated-fucose-containing polysaccharide mixture used in the invention of 8, 10, 11 or 12, an acetate ion and a calcium ion are made to coexist.

The fifteenth invention of the present invention relates to an endo-fucose sulfate-containing polysaccharide-degrading enzyme having the following physicochemical properties. (I) Action: acts on a sulfated-fucose-containing polysaccharide having the following physicochemical properties to lower the molecular weight of the sulfated-fucose-containing polysaccharide. (A) Constituent sugar: substantially free of uronic acid. (B) Flavobacterium sp.
The molecular weight is not substantially reduced by the fucoidanase produced by SA-0082 (FERM BP-5402).
It does not act on sulfated-fucose-containing polysaccharides having the following physicochemical properties. (C) Constituent sugar: contains uronic acid. (D) Flavobacterium sp.
The molecular weight is reduced by the fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402), and at least one compound selected from the compounds represented by the following formulas (I), (II) and (III) is produced. .

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Embedded image (Ii) Optimum pH: The optimum pH of the present enzyme is around 7-8. (Iii) Optimum temperature: The optimum temperature of the present enzyme is around 30 to 35 ° C.

The sixteenth invention of the present invention relates to an enzyme composition containing a calcium source and the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the fifteenth invention of the present invention.

The seventeenth invention of the present invention relates to a method for producing the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the fifteenth invention of the present invention, and the ability to produce the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the fifteenth invention of the present invention. Characterized by culturing an Alteromonas genus bacterium having, and collecting the enzyme from the culture.

The eighteenth invention of the present invention relates to a low-molecular-weight product of a sulfated-fucose-containing polysaccharide, wherein the endo-type sulfated-fucose-containing polysaccharide-degrading enzyme of the fifteenth invention of the present invention is replaced by the sulfated-fucose-containing polysaccharide of the sixth invention of the present invention. Characterized by being obtained by acting on

The present inventors have succeeded in obtaining various purified sulfated-fucose-containing polysaccharides and their degradation products, and then examined their biological activities. These substances induce apoptosis in cancer cells. And a strong anticancer effect. It has also been found that the sulfated-fucose-containing polysaccharide and / or its decomposed product exhibit a strong carcinogenesis-suppressing action. Furthermore, the fucose sulfate-containing polysaccharide-degrading enzyme useful for preparing the degradation product of the present invention was successfully isolated, and the present invention was completed.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described specifically. The sulfated-fucose-containing polysaccharide used in the present invention is not particularly limited, and for example, a product derived from Hibamata, a product derived from Gagome kelp, a product derived from Machu kelp, a product derived from Wakame, and a product derived from all brown algae plants are used. can do. Sea cucumber is also known to have a sulfated-fucose-containing polysaccharide on the body wall, but a sea cucumber-derived sulfated-fucose-containing polysaccharide can also be used in the present invention. In the present invention, a decomposition product of the sulfated-fucose-containing polysaccharide can also be used. Examples of the method for decomposing the sulfated-fucose-containing polysaccharide include a method for chemically decomposing the acid by treatment with acid, a method for physically decomposing the material by ultrasonic treatment, and a method for decomposing enzymatically.

The present inventors added various sulfated-fucose-containing polysaccharides and / or their degradation products to a culture solution of cancer cells as described above, and the cancer cells undergo apoptosis 1 to several days after the addition. I found that. It was also confirmed that the cells did not show toxicity to normal cells.

In the present invention, the sulfated-fucose-containing polysaccharide is a polysaccharide containing sulfated-fucose in the molecule, and is not particularly limited. For example, it is contained in brown algae plants, sea cucumber, etc. [Supervised by Tokio Soda and Fujio Egami Editing, Kyoritsu Shuppan Co., Ltd., published on December 15, 1955, Polysaccharide Chemistry, No. 3
19, page 321]. The sulfated-fucose-containing polysaccharides derived from brown alga plants are commonly called fucoidan, fucoidin, and fucan, and it is known that there are several molecular species, but these are often generically called fucoidan. For example, it has been reported that fucoidan manufactured by Sigma was divided into as many as 13 molecular species [Carbohydrate Research, Vol. 255, No. 213-
224 (1994)]. Among them, there are a group consisting mainly of fucose and a group consisting of several species of uronic acid and containing a large amount of fucose and mannose in the constituent sugars. Various biological activities such as enhancement of macrophage activity, suppression of cancer metastasis, and anticoagulation have been reported. However, since the sulfated-fucose-containing polysaccharide has molecular species, it is examined which molecular species has the main activity. For that purpose, it was necessary to separate and purify the sulfated-fucose-containing polysaccharide for examination. Examples of the sulfated-fucose-containing polysaccharide include those in which fucose is the main component of the constituent saccharide substantially containing no uronic acid, and those in which fucose or mannose is contained in the constituent saccharide containing several percent of uronic acid. Hereinafter, in the present specification, the one containing substantially no uronic acid is referred to as a sulfated-fucose-containing polysaccharide-F, the sulfated-fucose-containing polysaccharide containing uronic acid is referred to as a sulfated-fucose-containing polysaccharide-U, and a mixture of both is referred to as a sulfated-fucose-containing polysaccharide. Described as a mixture.

The known methods for separating sulfated-fucose-containing polysaccharide-F and sulfated-fucose-containing polysaccharide-U are molecular weight fractionation and separation using an anion exchange resin. It was difficult to prepare a large amount of functional food.

It is also known that it is difficult to completely remove the coloring substance from the sulfated-fucose-containing polysaccharide,
Commercially available fucoidan and the like also contain a coloring substance. Usually, this coloring substance is a polymer of polyphenol, which is extremely reactive and inhibits various enzyme reactions and cell growth, and irreversibly, for example, in contact with a resin or resin container. May be adsorbed. Therefore, in order to accurately examine the biological activity of the sulfated-fucose-containing polysaccharide and to prevent contamination of containers, resins, and the like, it is necessary to remove highly reactive coloring substances from the sulfated-fucose-containing polysaccharide.

Further, when soluble barium acetate, barium chloride or calcium chloride is used for extracting a sulfated-fucose-containing polysaccharide mixture from brown algae or a residue of alcohol washing of the brown algae, etc., mixing of alginic acid can be suppressed. Is known to be advantageous,
Soluble barium salt is not easy to treat waste liquid,
Also, calcium chloride changes its pH when mixed with seaweed, so pH adjustment is required to obtain a non-degradable sulfated-fucose-containing polysaccharide. During the pH adjustment, the seaweed powder may become viscous and aggregate, so that the subsequent extraction efficiency may decrease or solid-liquid separation, especially filtration may become difficult.

That is, despite the current industrial utility of the sulfated-fucose-containing polysaccharide, commercial products of sulfated-fucose-containing polysaccharide-F and sulfated-fucose-containing polysaccharide-U which are sufficiently separated by molecular species are commercially available. There is no report on its efficient manufacturing method. Further, the commercially available sulfated-fucose-containing polysaccharide contains a highly reactive coloring substance as described above.

Although the sulfated-fucose-containing polysaccharide has various activities, as described above, it is difficult to fractionate and prepare the sulfated-fucose-containing polysaccharide-F and the sulfated-fucose-containing polysaccharide-U. Did not.

However, the present invention provides a substantially purified fucose sulfate-containing polysaccharide-U, a simple extraction method thereof, and a method for producing the sulfated-fucose-containing polysaccharide-U.
In addition, according to the present invention, the sulfated-fucose-containing polysaccharide-U, which is usually free of a highly reactive coloring substance which is difficult to remove from the sulfated-fucose-containing polysaccharide and inhibits the enzymatic reaction and contaminates the resin.
Was provided.

Further, the present invention provides a substantially purified sulfated-fucose-containing polysaccharide-F, a simple extraction method thereof, and a process for producing the sulfated-fucose-containing polysaccharide-F.

Further, according to the present invention, there is provided a sulfated-fucose-containing polysaccharide-F from which a highly reactive coloring substance which is usually difficult to remove from a sulfated-fucose-containing polysaccharide and inhibits an enzymatic reaction and contaminates a resin is removed.

As the sulfated-fucose-containing polysaccharide used in the present invention, a sulfated-fucose-containing polysaccharide-containing material such as brown algae plants and sea cucumber may be used by drying and pulverizing as it is, for example.
Further, a sulfated-fucose-containing polysaccharide extract from a sulfated-fucose-containing polysaccharide-containing material, and a purified product from the extract may be used. The preparation method of the sulfated-fucose-containing polysaccharide extract and the purification method from the extract may be performed by a known method, and are not particularly limited.

The sulfated-fucose-containing polysaccharide decomposition product used in the present invention is obtained by decomposing a sulfated-fucose-containing polysaccharide by an enzymatic, chemical, or physical method. Enzymatic, chemical and physical methods can be used.

The sulfated-fucose-containing polysaccharide and the degraded sulfated-fucose-containing polysaccharide used in the present invention include pharmaceutically acceptable salts thereof.

Examples of brown alga plants containing sulfated-fucose-containing polysaccharides include, for example, Yukio Yamada, Muneyoshi Segawa, Hoikusha,
There are brown alga plants described on pages 22 to 52 of the Primary Color Japanese Seaweed Encyclopedia published in 1977, for example, Hibamata (Fucus evan).
escens), Gagome kelp (Kjellmaniella crassifolia),
Mackerel (Laminaria japonica), Seaweed (Undaria pinn)
atifida) can be used to prepare a sulfated-fucose-containing polysaccharide.

Sea cucumber containing a sulfated-fucose-containing polysaccharide includes, for example, sea cucumber described in JP-A-4-91027, for example, sea cucumber (Stichopus japonicus),
Nisekuro sea cucumber (Holothuria leucospilota) or the like can be used, and a sulfated-fucose-containing polysaccharide can be prepared by the method described in the publication.

The sulfated-fucose-containing polysaccharide has a sulfate group in the molecule, and this group reacts with various bases to form salts.
These sulfated-fucose-containing polysaccharides and their decomposition products are stable in a salted state, and are usually isolated in the form of salts such as sodium and / or potassium. By treating salts of these substances with a cation exchange resin such as Dowex 50W, it is possible to introduce free fucose sulfate-containing polysaccharides and free degradation products thereof. In addition, these can be further subjected to known and commonly used salt exchange as needed, and can be exchanged for various desired salts. As salts of sulfated-fucose-containing polysaccharides and their decomposed products, pharmaceutically acceptable salts are used, for example, alkali metal salts such as potassium and sodium, alkaline earth metal salts such as calcium, magnesium and barium, pyridinium and the like. And ammonium salts and the like with organic bases.

Brown alga plants, sea cucumber and the like containing the sulfated-fucose-containing polysaccharide can be dried and then subjected to a pulverization treatment to prepare a powdered sulfate-containing fucose-containing polysaccharide.

By subjecting the sulfated-fucose-containing polysaccharide-containing powder to hot water extraction and dilute acid extraction, a sulfated-fucose-sulfuric acid-containing polysaccharide extract can be prepared.

The extraction temperature and time from the fucosulfate-containing polysaccharide-containing material may be selected from the range of 0 to 200 ° C. and 1 to 360 minutes according to the purpose.
5 to 240 minutes, preferably 50 to 130 ° C, 10 to 180
It is better to select from the range of minutes.

As a means for purifying the extract to increase the content of the sulfated-fucose-containing polysaccharide, a method for fractionating the sulfated-fucose-containing polysaccharide using calcium chloride, barium acetate or the like, or an acidic polysaccharide coagulant such as cetylpyridinium chloride was used. There are a method for fractionating a sulfated-fucose-containing polysaccharide, a method for fractionating a sulfated-fucose-containing polysaccharide using an acidic polysaccharide coagulant in the presence of salts, gel filtration, ion exchange chromatography, and the like. It can be carried out.

As a method for decomposing the sulfated-fucose-containing polysaccharide, a method known as a method for decomposing a sulfated-fucose-containing polysaccharide, such as a method using a sulfated-fucose-containing polysaccharide-degrading enzyme, a method for performing acid decomposition, and a method for performing ultrasonic treatment, is used. The decomposition product can be purified by the above method.

Usually, brown algae contains a plurality of types of sulfated-fucose-containing polysaccharides, but the type of brown algae used in the present invention is not particularly limited. And those derived from sea kelp, seaweed and all other brown algae.

To produce the sulfated-fucose-containing polysaccharide, first, an extract of brown algae with an aqueous solvent is obtained.

The seaweed to be subjected to the extraction may be raw seaweed. Before obtaining the extract, the brown algae may be dried, dried, washed with 60 to 100% alcohol or acetone, formaldehyde, acetaldehyde, or the like. Immersion in an aqueous solution containing water, glutaraldehyde, ammonia, etc. is advantageous because the mixing of coloring substances into the sulfated-fucose-containing polysaccharide is greatly reduced.

In addition, when soluble barium acetate, barium chloride or calcium chloride is used to extract the sulfated-fucose-containing polysaccharide from the brown alga or the residue of alcohol washing of the brown algae, the mixing of alginic acid can be suppressed. Purification is advantageous, but for the above-mentioned reason, extraction is performed with a 50 mM calcium acetate solution of about 1 mM to 1 M.
It is preferred to extract at ~ 130 ° C.

When the seaweed is thick and the powder (particles) is large,
If 0.2 M or more of calcium acetate is used from the beginning, the extraction efficiency may be deteriorated. Therefore, it is only necessary to first add calcium acetate to water-extracted one to remove the resulting precipitate of alginic acid.

However, when it is desired to simultaneously extract the sulfated-fucose-containing polysaccharide with alginic acid or to obtain a product decomposed to some extent during the extraction, the solvent and the extraction conditions are not particularly limited, and water, salt, magnesium chloride, etc. Various concentrations of neutral salt aqueous solution, citric acid, phosphoric acid, hydrochloric acid, etc., various concentrations of acidic aqueous solution, sodium hydroxide, potassium hydroxide, etc. of various concentrations of alkaline aqueous solution can be used, and buffers and preservatives can be used. May be added. There are no particular restrictions on the pH, extraction temperature, extraction time, etc. of the extract,
In general, the sulfated-fucose-containing polysaccharide is weak against acids and alkalis, so that when an acidic solution or an alkaline solution is used, the molecular weight is easily reduced. By adjusting the heating temperature, time, pH, etc., any decomposed product can be prepared. For example, by adjusting the average molecular weight, molecular weight distribution, etc. of the decomposed product by gel filtration treatment, molecular weight fractionation membrane treatment, etc. Can be.

That is, the sulfated-fucose-containing polysaccharide of the present invention
The molecular weight and saccharide composition of U and the sulfated-fucose-containing polysaccharide-F vary depending on the harvesting period of the raw material of the sulfated-fucose-containing polysaccharide, the method of drying the raw material, and the method of storing the raw material. It depends on the pH conditions. For example, a sulfated-fucose-containing polysaccharide is hydrolyzed by an acid, and under alkaline conditions, β-elimination of uronic acid promotes molecular weight reduction. Therefore, the molecular weight and molecular weight distribution of the sulfated-fucose-containing polysaccharide-U and the sulfated-fucose-containing polysaccharide-F described in the present specification are only one example, and the molecular weight and the molecular weight distribution can be easily changed depending on the treatment conditions of the sulfated-fucose-containing polysaccharide. Can change. For example, at 100 ° C., 1
When heating and desalting using a molecular sieve membrane having a pore size of 300, the sulfated-fucose-containing polysaccharide-U and the sulfated-fucose-containing polysaccharide-F having a molecular weight distribution of about 1,000 to 10,000 are used.
Can be prepared, and the sulfated-fucose-containing polysaccharide-U and sulfated-fucose-containing polysaccharide-F of the present invention having arbitrary molecular weights and molecular weight distributions can be prepared depending on the conditions used.

In order to remove alginic acid and neutral sugars from the extract of the brown algae, for example, chloride is added in the presence of a salt such as sodium chloride at a concentration of 0.2 to 0.6 M until no more precipitate is formed. An acidic polysaccharide coagulant such as cetylpyridinium may be added and the precipitate may be collected.

If necessary, the precipitate is added to a concentration of 0.2 to 0.6M.
After washing with a salt solution such as sodium chloride, the cetylpyridinium chloride in the precipitate is washed away with a salt saturated alcohol,
A sulfated-fucose-containing polysaccharide mixture is obtained. In order to remove the dye from the sulfated-fucose-containing polysaccharide mixture thus obtained, the precipitate may be dissolved and then treated with an anion exchange resin or a polysaccharide resin, or subjected to ultrafiltration or the like. A freeze-dried product can be obtained by freeze-drying after desalting.

The present inventors have studied one type or two types of 0.6-3M.
It has been found that the sulfated-fucose-containing polysaccharide-F of the present invention and the sulfated-fucose-containing polysaccharide-U of the present invention exhibit completely different behaviors with respect to the acidic polysaccharide flocculant in the presence of more than one kind of salts.

For example, the sulfated-fucose-containing polysaccharide-U of the present invention can be separated from the aqueous solution of the sulfated-fucose-containing polysaccharide mixture by using the method of the present invention.

First, one or more salts are added to an aqueous solution of the sulfated-fucose-containing polysaccharide mixture, and the total concentration is adjusted to 0.6.
２2M. Salts to be added are, for example, sodium chloride,
It is not particularly limited by calcium chloride or the like.

In general, when the sulfated-fucose-containing polysaccharide-F of the present invention is separated from the sulfated-fucose-containing polysaccharide-U of the present invention:
The objective can be achieved with a salt concentration of about 5M (see the description of FIG. 1 described later). For example, after adjusting the salt concentration of the above salts to 1.5 M and adding an acidic polysaccharide flocculant such as cetylpyridinium chloride until no more precipitate is formed, the sulfated-fucose-containing polysaccharide-F forms a precipitate. A solution of the sulfated-fucose-containing polysaccharide-U of the present invention is obtained. If necessary, this solution is concentrated, and the sulfated-fucose-containing polysaccharide-U in the solution is precipitated with 4 times the amount of ethanol or the like, and the cetylpyridinium chloride in the precipitate is washed off with a salt-saturated alcohol. -U
Get. In order to remove the pigment from the sulfated-fucose-containing polysaccharide-U thus obtained, the precipitate may be dissolved and then subjected to ultrafiltration or the like. A freeze-dried product can be obtained by freeze-drying after desalting. Further, a preservative or the like may be added during the process.

Next, when it is desired to efficiently produce only the sulfated-fucose-containing polysaccharide-F of the present invention, when coagulating with cetylpyridinium chloride or the like, a salt concentration of, for example, 2 M instead of 0.2 to 0.6 M is used. At a salt concentration, the precipitate contains only the sulfated-fucose-containing polysaccharide-F of the present invention.

The present inventors have found that when divalent cations coexist when purifying a sulfated-fucose-containing polysaccharide with an anion exchange resin, the amount of the sulfated-fucose-containing polysaccharide adsorbed per unit amount of resin increases, and It has also been found that the separation of polysaccharide is improved. That is, when producing the sulfated-fucose-containing polysaccharide-U of the present invention using the method of the present invention, first, a drug serving as a divalent cation source, preferably 1 mM or more, is added to the sulfated-fucose-containing polysaccharide mixture. Next, the anion exchange resin is equilibrated with a solution containing preferably at least 1 mM of divalent cation, and the above-mentioned sulfated-fucose-containing polysaccharide mixture is adsorbed. After sufficiently washing the anion exchange resin with the equilibrated liquid, the sulfated-fucose-containing polysaccharide is eluted by, for example, a gradient of sodium chloride. When using this method, the concentration of the divalent cation to be added may be 1 mM or more.
For the purpose of adsorbing the sulfated-fucose-containing polysaccharide-U of the present invention onto a column, the concentration is preferably less than 0.5M. In addition, calcium salts and barium salts are particularly effective as chemicals serving as divalent cation sources used in the present method, but are not particularly limited, and magnesium sulfate, manganese chloride and the like can also be used. .

When a sulfated-fucose-containing polysaccharide mixture is produced from brown algae by an ordinary method, a highly reactive coloring substance is mixed as described above, thereby contaminating the resin or resinous container with which the mixture comes in contact, or enzymatic reaction. Or inhibit cell growth. It has been found that this coloring substance can be easily removed by bonding or adsorbing to a polysaccharide substance or a substance having an anion exchange group. That is, a solution containing a sulfated-fucose-containing polysaccharide, for example, Cellulofine, GCL-2
000 (manufactured by Seikagaku Corporation) and Sephacryl S-50
0, Sephadesc G-200, Sepharose CL-2
B (both manufactured by Pharmacia) and the like, or DEAE-Cellulofine A-800 (manufactured by Seikagaku Corporation), DEAE-Sepharose FF, DEAE-Sephadex A-50, QAE-Sephadex A-5
0, DEAE-Sephacel (both manufactured by Pharmacia),
Anions such as TSK-Gel DEAE-Toyopearl 650, TSK-Gel DEAE Toyopearl 550 (manufactured by Tosoh), amberlite-based anion exchange resin (sold by Organo), and chitopearl-based anion exchange resin (manufactured by Fuji Boseki) Remove after adding a substance having an exchange group, stirring,
Alternatively, if a solution containing a sulfated-fucose-containing polysaccharide is passed through a column packed with these, the highly reactive coloring substance can be easily removed. However, in the case of an anion exchange resin, a sulfated-fucose-containing polysaccharide can also bind, so that the salt concentration is preferably set to about 2 M when the coloring substance is adsorbed.

The sulfated-fucose-containing polysaccharide-U of the present invention can be prepared, for example, as described in Example 6. Hereinafter, the physicochemical properties of the sulfated-fucose-containing polysaccharide-U are shown, but the sulfated-fucose-containing polysaccharide-U of the present invention is not limited to this example.

The figure shows the precipitate forming properties of the sulfated-fucose-containing polysaccharide-U of the present invention and the sulfated-fucose-containing polysaccharide-F of the present invention obtained in Example 8 in the presence of an excessive amount of cetylpyridinium chloride at each sodium chloride concentration. It is shown in FIG.

In FIG. 1, the vertical axis represents the rate of precipitation (%), and the horizontal axis represents the concentration of sodium chloride (M). In the figure, solid lines and open circles indicate the rate of precipitation formation of the sulfated-fucose-containing polysaccharide-U of the present invention at each concentration of sodium chloride. In the figure, dotted lines and open triangles indicate each sodium chloride of the sulfated-fucose-containing polysaccharide-F of the present invention. The precipitate formation rate at the concentration (M) is shown.

The rate of precipitate formation was measured at a solution temperature of 37 ° C. as follows. The sulfated-fucose-containing polysaccharide-U and the sulfated-fucose-containing polysaccharide-F of the present invention are dissolved in water and 4M sodium chloride at a concentration of 2%, respectively, and mixed at various ratios to obtain various concentrations of sodium chloride. The dissolved sulfated-fucose-containing polysaccharide-U and sulfated-fucose-containing polysaccharide-F solutions were each prepared in an amount of 125 μl.
Next, cetylpyridinium chloride was dissolved in water and 4M sodium chloride at a concentration of 2.5% and dissolved in various concentrations of sodium chloride by mixing them.
A 5% cetylpyridinium chloride solution was prepared.

The sulfated-fucose-containing polysaccharide-U and the sulfated-fucose-containing polysaccharide-F of the present invention, which are dissolved in water at a concentration of 2%,
3.2 times by volume was required for complete precipitation with 25% cetylpyridinium chloride. Therefore, 2% fucose sulfate-containing polysaccharide-U dissolved in sodium chloride at each concentration.
400 μl of a cetylpyridinium chloride solution dissolved in sodium chloride at each concentration was added to 125 μl of each of the fucose sulfate-containing polysaccharide-F, and the mixture was stirred thoroughly.
After standing for 0 minutes, centrifugation was performed and the sugar content in the supernatant was determined by phenol-
Sulfuric acid method [Analytical C
hemistry), vol. 28, p. 350 (1956)], and the precipitate formation rate of each sulfated-fucose-containing polysaccharide under each concentration of sodium chloride was calculated.

The sulfated-fucose-containing polysaccharide of the present invention thus obtained is
When the molecular weight of U was determined by a gel filtration method using Sephacryl S-500, a molecular weight distribution centered at about 190,000 was shown (see FIG. 2). In FIG. 2, the vertical axis represents the sugar content in the sample measured by the phenol-sulfuric acid method.
The value is indicated by the absorbance at 80 nm, and the horizontal axis indicates the fraction number.

The conditions for gel filtration are shown below. Column size: 3.08 × 162.5 cm Solvent: 10 mM sodium phosphate buffer containing 0.2 M sodium chloride and 10% ethanol (pH 6.
0) Flow rate: 1.5 ml / min Sample concentration: 0.25% Sample liquid volume: 20 ml Molecular weight standard substance: Shodex STANDARD P-82 (manufactured by Showa Denko KK)

Next, the components of the obtained sulfated-fucose-containing polysaccharide-U of the present invention were analyzed. First, the Journal of
Biological chemistry (Journal of Biologica
Chemistry, Vol. 175, p. 595 (1948).

Next, the obtained sulfated-fucose-containing polysaccharide-U
Is dissolved in 1N hydrochloric acid at a concentration of 0.5%,
The mixture was treated at 110 ° C. for 2 hours, and hydrolyzed into constituent monosaccharides. Next, GlycoTAG (GlycoTAG) and Glycotag
The reducing end of the monosaccharide obtained by hydrolysis using a GlycoTAG Reagent Kit (both manufactured by Takara Shuzo Co., Ltd.) is pyridyl- (2) -aminated (PA-modified), and H
The ratio of constituent sugars was examined by PLC. The HPLC conditions were as follows. Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: Palpack type A (4.6 mm x 150 m)
m: manufactured by Takara Shuzo) Eluent: 700 mM borate buffer (pH 9.0): acetonitrile = 9: 1 Detection: fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) with excitation wavelength of 310 nm and fluorescence wavelength of 380 nm : 0.3ml / min Column temperature: 65 ° C

Next, the amount of uronic acid was determined according to the description in Analytical Biochemistry, Vol. 4, p. 330 (1962).

Next, a biochemical journal (Bioc
chemical Journal), 84, 106 (1962).
The sulfuric acid content was quantified according to the description.

As a result, the constituent sugars of the obtained sulfated-fucose-containing polysaccharide-U were fucose, mannose, galactose, glucose, rhamnose, xylose, and uronic acid. Other neutral sugars were not substantially contained. Further, the fucose: mannose: galactose: uronic acid: sulfate group of the main component is about 10: 7: 4:
5:20.

Next, when the IR spectrum of the calcium salt of the sulfated-fucose-containing polysaccharide-U was measured by a Fourier transform infrared spectrophotometer JIR-DIAMOND20 (manufactured by JEOL Ltd.), the spectrum shown in FIG. 3 was obtained. In FIG. 3, the vertical axis represents the transmittance (%), and the horizontal axis represents the wave number (c).
m ^-1 ).

Next, the sulfated-fucose-containing polysaccharide-U of the present invention is used.
The NMR spectrum of the calcium salt was measured with a 500 MHz nuclear magnetic resonance apparatus JNM-α500 type nuclear magnetic resonance apparatus (manufactured by JEOL Ltd.), and the spectrum shown in FIG. 4 was obtained.

In FIG. 4, the vertical axis represents the signal intensity, and the horizontal axis represents the chemical shift value (ppm). The chemical shift value in ¹ H-NMR was the chemical shift value in HOD at 4.65 pp.
m. ¹ H-NMR (D ₂ O) δ 5.27 (H at the first position of mannose), 5.07 (H at the first position of fucose), 4.49 (H at the third position of fucose),
4.37 (H at position 1 of glucuronic acid), 4.04 (H at position 4 of fucose), 3.82 (H at position 2 of fucose),
3.54 (H at position 3 of glucuronic acid), 3.28 (H at position 2 of glucuronic acid), 1.09 (C at position 5 of fucose)
H of H ₃₎

The specific rotation of the freeze-dried product of the sulfated-fucose-containing polysaccharide-U of the present invention was measured by a high-speed and high-sensitivity polarimeter SEPA-.
When measured with a 300 (manufactured by Horiba, Ltd.), -5
It was 3.6 degrees.

The present inventors have determined the structure of the sulfated-fucose-containing polysaccharide-U of the present invention obtained as described below.

Decomposition of the sulfated-fucose-containing polysaccharide-U by a degrading enzyme capable of degrading the sulfated-fucose-containing polysaccharide-U and purification of the degraded product The following endo-fucoidan-degrading enzyme is allowed to act on the purified sulfated-fucose-containing polysaccharide-U. The decomposition product was purified.

That is, 1% fucose sulfate-containing polysaccharide-
U solution (16 ml) and 50 mM phosphate buffer (pH 8.
0) 12 ml and 4 ml of 4 M sodium chloride and 32 mU
The mixture was mixed with 8 ml of an endo / fucoidan-degrading enzyme solution at a temperature of 25 ° C. for 48 hours. 2 with the progress of the reaction
It was confirmed that the absorbance at 30 nm increased, and it was found that the sulfated-fucose-containing polysaccharide-U was decomposed by the present enzyme. The reaction solution was desalted with a micro-acylizer G3 (manufactured by Asahi Kasei Corporation), and then separated and purified into three fractions (a), (b) and (c) by DEAE-Sepharose FF.

The above-mentioned endo-type fucoidanase is prepared by the following method.

The strain used for producing the endo-type fucoidan-degrading enzyme may be any strain as long as it has the ability to produce the endo-type fucoidan-degrading enzyme. Specific examples include, for example, Flavobacteri
um) sp. SA-0082 strain (FERM BP-54
02).

The present strain is a strain newly obtained by the present inventors from seawater in Aomori Prefecture, and its mycological properties are as follows. 1. Flavobacterium sp. SA-0082 strain a. Morphological properties (1) The bacterium is a short bacillus. Width 0.8-1.0 μm Length 1.0-1.2 μm (2) Presence or absence of spores None (3) Gram stain negative b. Physiological properties (1) Temperature range for growth It can be grown at 37 ° C or less. Suitable growth temperature is 15-28
° C. (2) Attitude to oxygen Aerobic (3) Catalase positive (4) Oxidase positive (5) Urease weak positive (6) Acid production D-glucose positive Lactose positive Maltose positive D-mannitol positive Sucrose negative Trehalose negative (7) Hydrolysis Degradation Starch Negative Gelatin Positive Casein Negative Esculin Positive (8) Nitrate Reduction Negative (9) Indole Production Negative (10) Hydrogen Sulfide Production Negative (11) Milk Coagulation Negative (12) Sodium Requirement Positive (13) Salts Requirement Growth on 0% saline medium Negative Growth on 1% saline medium Negative Growth on seawater medium Positive (14) Quinone menaquinone 6 (15) GC content of intracellular DNA 32% (16) OF-test O ( 17) Color tone of village Yellow (18) Mobility None (19) Glidability None

The strain was prepared using the method of Bergey's Manual of Sistmatic Bacteriology (Bergey's M.
anual of Systematic Bacteriology), Volume 1 (198
4) and Bergey's Manual of D. Bacteriology
eterminative Bacteriology), Vol. 9 (1994).
um aquatile) and Flavobacterium meningosepticum, which is considered to be a related bacterium.The former does not use sucrose to form acids, cannot degrade casein, and can degrade esculin. The difference is that gelatin can be liquefied and urease is positive, and the latter differs in that casein cannot be decomposed and that growth at 37 ° C. is slow. Therefore, this strain was identified as a bacterium belonging to Flavobacterium, and Flavobacterium sp. It was named SA-0082.

The above strain was obtained from Flavobacterium sp.
SA-0082 was displayed on the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry [1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan (zip code 305)] from March 29, 1995. 14872, and the FERMB was registered with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry.
P-5402 (Date of request for transfer to international deposit: February 1996
15).

The nutrient source added to the medium of the present strain may be any as long as it utilizes the strain used and produces an endo-type fucoidan-degrading enzyme. Examples of carbon sources include fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol , Glycerol, saccharose, maltose, lactose, starch and the like can be used. As the nitrogen source, yeast extract, peptone, casamino acid, corn steep liquor, meat extract, defatted soybean, ammonium sulfate, ammonium chloride and the like are suitable. In addition, inorganic salts such as sodium salts, phosphates, potassium salts, magnesium salts, and zinc salts, and metal salts may be added.

In culturing a bacterium producing the present endo-type fucoidan-degrading enzyme, the amount of production varies depending on the culture conditions.
Generally, the culture temperature is 15 ° C to 30 ° C, and the pH of the medium is 5 to
9 is best, and the production of the present endo-fucoidan-degrading enzyme reaches the maximum in aeration and agitation culture for 5 to 72 hours. It is natural that the culture conditions are set so as to maximize the production amount of the present endo-type fucoidan-degrading enzyme according to the strain used, the composition of the medium, and the like.

The present endo-fucoidan-degrading enzyme is present in both cells and culture supernatant.

The above Flavobacterium sp. SA
-0082 strain was cultured in a suitable medium, the cells were collected,
A cell-free extract can be obtained by disrupting cells by a commonly used cell disruption means, for example, ultrasonic treatment.

Next, a purified enzyme preparation can be obtained from the extract by a commonly used purification means. For example, purification is performed by salting out, ion exchange column chromatography, hydrophobic binding column chromatography, gel filtration, or the like, and the purified endo-fucoidan-degrading enzyme containing no other fucoidan-degrading enzymes can be obtained.

Also, since the present enzyme (extracellular enzyme) is present in a large amount in the supernatant of the culture solution from which the cells have been removed from the above-mentioned culture solution, it can be purified by the same purification means as the intracellular enzyme. it can.

An example of purification of an endo-type fucoidan-degrading enzyme will be described.

Flavobacterium sp. SA-00
82 (FERM BP-5402) with glucose 0.2
600 ml of a medium consisting of artificial seawater (manufactured by Jamarin Laboratories) pH 7.5 containing 5%, 1.0% peptone and 0.05% yeast extract was dispensed and sterilized (120 ° C, 2 ° C).
0 min) inoculate a 2 liter Erlenmeyer flask,
The cells were cultured for 4 hours to obtain a seed culture solution. Glucose 0.25
% Of peptone, 1.0% of peptone, 0.05% of yeast extract, and 0.01% of an antifoaming agent (manufactured by Shin-Etsu Chemical Co., Ltd., KM70) containing 0.01% of artificial seawater (manufactured by Jamarin Laboratory), pH 7.5. It was placed in a liter fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the above seed culture was inoculated and cultured at 24 ° C. for 24 hours under the conditions of aeration of 10 liters per minute and a stirring speed of 125 revolutions per minute. After completion of the culture, the culture was centrifuged to obtain cells.

[0095] The cells were transformed with 20 mM salt containing 200 mM.
The cells were suspended in a mM acetate-phosphate buffer (pH 7.5), sonicated, and centrifuged to obtain a cell extract. When the activity of the present endo-fucoidan-degrading enzyme in the cell extract was measured, an activity of 5 mU was detected in 1 ml of the medium.

Ammonium sulfate was added to the extract to a final concentration of 90% saturation, stirred and dissolved, followed by centrifugation. The precipitate was suspended in the same buffer as the above-mentioned bacterial cell extract.
20 mM acetate-phosphate buffer (p.
H7.5). After removing the precipitate generated by dialysis by centrifugation, the precipitate was adsorbed on a column of DEAE-Sepharose FF previously equilibrated with a 20 mM acetic acid-phosphate buffer (pH 7.5) containing 50 mM salt.
After sufficiently washing the adsorbed substance with the same buffer, the concentration is adjusted from 50 mM to 600
The active fraction was eluted with a gradient of mM saline and eluted. Next, sodium chloride was added to the active fraction so that the final concentration became 4 M, and the active fraction was adsorbed on a phenyl sepharose CL-4B column which had been equilibrated with a 20 mM phosphate buffer (pH 8.0) containing 4 M sodium chloride in advance. After sufficiently washing the adsorbate with the same buffer, the eluate was eluted with a gradient of 4 M to 1 M salt, and the active fraction was collected. Next, the active fraction was concentrated with an ultrafiltration device, and was previously added with 50 mM sodium chloride.
Sephacryl S-3 equilibrated with 0 mM phosphate buffer
Gel filtration was performed at 00 to collect the active fraction. The molecular weight of this enzyme determined from the retention time of Sephacryl S-300 was about 460,000. Next, this active fraction was
It was dialyzed against 10 mM phosphate buffer (pH 7) containing M salt. The enzyme solution was adsorbed on a Mono Q HR5 / 5 column which had been equilibrated with a 10 mM phosphate buffer (pH 7) containing 250 mM sodium chloride in advance, and the adsorbed material was sufficiently washed with the same buffer, followed by 250 mM And eluted with a gradient of 450 mM saline, collecting the active fractions,
A purified enzyme was obtained. Table 1 shows the above purification steps.

[0097]

[Table 1]

The activity of the present enzyme is measured as follows.

50 μl of a 2.5% fucoidan solution derived from Gagome kelp, 10 μl of the present enzyme, and 60 μl of 667
83 mM phosphate buffer pH containing mM sodium chloride
After mixing 7.5 and reacting at 37 ° C. for 3 hours, 105 μl of the reaction solution and 2 ml of water were mixed and stirred, and 230 n of the mixture was stirred.
The absorbance (AT) at m is measured. As a control, one prepared by reacting under the same conditions using only the above buffer in which the present enzyme was dissolved instead of the present enzyme and one prepared by performing reaction using only water instead of the fucoidan solution were prepared. The absorbance is measured in the same manner (AB1 and AB
2).

One unit of the enzyme is defined as an amount of an enzyme capable of eliminating a glycosidic bond between 1 μmol of mannose and uronic acid per minute in the above reaction system. The quantification of the cleaved bond is performed by calculating the molar extinction coefficient of the unsaturated uronic acid generated during the elimination reaction as 5.5. In addition,
The activity of the enzyme is determined by the following equation. (AT-AB1-AB2) × 2.105 × 120/5.
5 × 105 × 0.01 × 180 = U / ml 2.105: Volume of sample for measuring absorbance (ml) 120: Volume of enzyme reaction solution (μl) 5.5: Unsaturated uronic acid at 230 nm Millimol molecular extinction coefficient (/ mM) 105: Volume of reaction solution used for dilution (μl) 0.01: Enzyme solution volume (ml) 180: Reaction time (min)

The amount of the protein was determined at 280 nm of the enzyme solution.
This is performed by measuring the absorbance of the sample. 1 mg / m
Calculate the absorbance of 1 protein solution as 1.0.

The fucoidan derived from Gagome kelp as a substrate was prepared as follows.

The dried gagome kelp was pulverized with a free pulverizer M-2 (manufactured by Nara Machinery Co., Ltd.), treated at 70 ° C. for 2 hours in a 10-fold volume of 85% methanol, and then filtered.
Treat in twice the volume of methanol at 70 ° C. for 2 hours and filter. A 20-fold amount of water is added to the residue, the mixture is treated at 100 ° C. for 3 hours, and an extract is obtained by filtration. 400m salt concentration of the extract
After making the same as the sodium chloride solution of M, cetylpyridinium chloride is added until no more precipitate is formed and centrifuged. The precipitate was sufficiently washed with ethanol, and when cetylpyridinium chloride was completely removed, desalting and low-molecular removal were performed using an ultrafilter (excluded molecular weight of the filtration membrane: 100,000) (manufactured by Amicon). The resulting precipitate is removed by centrifugation. The supernatant is freeze-dried to obtain purified gagome kelp fucoidan.

Structural Analysis of Enzyme Reaction Product The above-mentioned endo-type fucoidanase decomposes the α1 → 4 bond between D-mannose and D-glucuronic acid present in the sulfated-fucose-containing polysaccharide-U by elimination. When acted on the obtained sulfated-fucose-containing polysaccharide-U, oligosaccharides having the structures of the following formulas (I), (II) and (III) were produced.

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The details will be described below.

The above three fractions (a), (b), and (c) separated and purified by DEAE-Sepharose FF were partially partially reduced with a glycotag and a glycotag reagent kit to remove the reducing end to pyridyl. −
(2)-amination (PA conversion), and each PA sugar (PA-
a), (PA-b) and (PA-c) were obtained. (PA
-A), (PA-b), and (PA-c) were analyzed by HPLC.

The HPLC conditions were as follows.

(A) HPLC using a molecular weight fractionation column
Analysis apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: SHOdex SB-803 (4.6 × 250)
mm) (manufactured by Showa Denko KK) Eluent: 0.2 M sodium chloride: dimethylsulfoxide = 9: 1 Detection: Fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) Detection at excitation wavelength of 320 nm, fluorescence wavelength of 400 nm Flow rate: 1 ml / Min Column temperature: 50 ° C

(A) HPLC analysis using a reversed-phase column Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: L-column (4.6 × 250 mm) [Chemical Inspection Association] Eluent: 50 mM Acetic acid-triethylamine (pH 5.
5) Detection: Fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) Detection at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 nm Flow rate: 1 ml / min Column temperature: 40 ° C.

FIGS. 5, 6 and 7 each show pyridyl-
(2) -aminated sugar compound (PA-a), (PA-
The elution patterns of HPLC of b) and (PA-c) are shown. In the figure, the vertical axis indicates the relative fluorescence intensity, and the horizontal axis indicates the retention time (minute).

The following formulas (I), (II) and (II)
It shows the physical properties of the compound represented by I), that is, (a), (b), and (c).

FIG. 8A, FIG. 9B, FIG.
FIG. 11 shows the spectrum of the mass (c), and FIG.
12, (b) and (c) of FIG. 13 show mass spectra. In each figure, the vertical axis represents relative intensity (%);
The horizontal axis shows the m / z value.

FIG. 14 is (a) and FIG. 15 is (b)
FIG. 16 shows the ¹ H-NHR spectrum of (c). In each figure, the vertical axis indicates the signal intensity, and the horizontal axis indicates the chemical shift value (ppm).

The chemical shift value of ¹ H-NHR was expressed assuming that the chemical shift value of HOD was 4.65 ppm.

Physical properties of (a) Molecular weight 564 MS m / z 563 [MH⁺]⁻ MS / MS m / z 97 [HSO₄]⁻, 157
[Unsaturated D-glucuronic acid-H₂O-H⁺]⁻,
175 [unsaturated D-glucuronic acid-H⁺]⁻225
[L-fucose sulfuric acid-H₂O-H⁺]⁻243
[L-fucose sulfuric acid-H⁺]⁻319 [unsaturated D
-Glucuronic acid combined with D-mannose-H
₂O-H⁺]⁻405 [M-unsaturated D-gluc
Ronic acid-H ⁺]⁻, 483 [M-SO₃−
H⁺]^{− 1} H-NMR (D₂O) δ 5.78 (1H, d, J = 3.7 Hz, 4 ″ -H),
5.26 (1H, d, J = 1.2 Hz, 1-H);
12 (1H, d, J = 4.0 Hz, 1'-H), 5.0
3 (1H, d, J = 6.1 Hz, 1 ″ -H), 4.47
(1H, dd, J = 3.4, 10.4 Hz, 3'-
H), 4.21 (1H, br-s, 2-H), 4.12.
(1H, m, 5'-H), 4.10 (1H, dd, J
= 3.7, 5.8 Hz, 3 "-H), 4.03 (1H,
d, J = 3.4 Hz, 4'-H), 3.86 (1H,
m, 3-H), 3.83 (1H, dd, J = 4.0,
10.4 Hz, 2'-H), 3.72 (1H, m, 4-
H), 3.72 (1H, m, 5-H), 3.70 (2
H, m, 5-CH₂H₂), 3.65 (1H, d
−d, J = 5.8, 6.1 Hz, 2 ″ −H), 1.08
(3H, d, J = 6.7 Hz, 5'-CH₃of
H₃) Sugar composition L-fucose: unsaturated D-glucuronic acid: D-
Mannose = 1: 1: 1 (1 molecule each) Sulfate 1 molecule (3rd position of L-fucose)¹The numbers of peak assignments in H-NMR are as follows:
It is as in the following formula (IV).

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Physical properties of (b) Molecular weight 724 MS m / z 723 [M−H ⁺ ] ⁻ , 361 [M
-2H ^{+] 2-} MS / MS m / z 97 [HSO _4] ^-, 175
[Unsaturated D-glucuronic acid-H ⁺ ] ⁻ , 243 [L
-Fucose sulfate-H ⁺ ] ^- , 321 [M-SO ₃
-2H ^{+] 2-,} 405 [M- unsaturated D- glucuronic acid -2SO ₃ -H ^{+] -,} 417 (M-L- fucose -2SO ₃ -H ^{+] _{- 1 H-NMR (D 2}} O) δ 5.66 (1H, d, J = 3.4 Hz, 4 ″ −H),
5.27 (1H, d, J = 7.3 Hz, 1 ″ -H),
5.22 (1H, d, J = 1.8 Hz, '-H);
21 (1H, d, J = 3.7 Hz, 1'-H), 4.5
0 (1H, d, J = 3.1 Hz, 4'-H), 4.32
(1H, q, J = 6.7 Hz, 5'-H), 4.27
(1H, dd, J = 3.7, 10.4 Hz, 2'-
H), 4.21 (1H, dd, J = 3.4, 6.7H)
z, 3 "-H), 4.18 (1H, dd, J = 1.
8, 11.0 Hz, H of 5-CH), 4.15 (1H,
br-s, 2-H), 4.10 (1H, dd, J =
5.8, 11.0 Hz, 5-CH H), 3.99 (1
H, dd, J = 3.1, 10.4 Hz, 3'-H),
3.90 (1H, m, 5-H), 3.82 (1H, m, 5-H)
3-H), 3.82 (1H, m, 4-H) 3.54 (1
H, br-t, J = 7.3 Hz, 2 "-H), 1.11
(3H, d, J = 6.7Hz , H 3 of the 5'-CH ₃₎ Sugar composition L- fucose: unsaturated D- glucuronic acid: D-
Mannose = 1: 1: 1 (1 molecule each) 3 molecules of sulfate (2nd and 4th position of L-fucose and 6th position of D-mannose) The number of peak assignment in ¹ H-NMR is represented by the following formula ( V).

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Physical properties of (c) Molecular weight 1128 MS m / z 1127 [MH ⁺ ] ^- MS / MS m / z 97 [HSO ₄ ] ^- , 175
[Unsaturated D-glucuronic acid-H ⁺ ] ⁻ , 225 [L
- fucose _-H 2 ^{O-H +] -,} 243 [L
-Fucose sulfuric acid-H ⁺ ] ^- , 371 [M-unsaturated D
- glucuronic acid -L- fucose _-SO 3 -2H ^+]
2, 405 [-H ⁺ that sulfated L- fucose and D- mannose bound] ^-, 721 [M-D- mannose -L--fucose _-SO 3 _-H 2 ^{O-H +] - 1} H- NMR (D ₂ O) δ 5.69 (1H, d, J = 3.7 Hz, (4) ″ −
H), 5.34 (1H, s, (1) -H), 5.16
(1H, s, 1-H), 5.10 (1H, d, J = 4.
0 Hz, (1) '-H), 5.50 (1H, d, J =
3.7 Hz, 1'-H), 4.93 (1H, d, J =
6.4 Hz, (1) ″ − H), 4.50 (1H, d−)
d, J = 3.4, 10.7 Hz, 3'-H), 4.47
(1H, dd, J = 3.4, 10.4 Hz, (3) '
-H), 4.39 (1H, d, J = 7.9 Hz, 1 "-
H), 4.33 (1H, br-s, (2) -H).
14 (1H, m, 2-H), 4.12 (1H, m, 2-H)
(3) "-H", 4.12 (1H, m, 5'-H),
4.12 (1H, m, (5) '-H), 4.04 (1
H, m, 4'-H), 4.03 (1H, m, (4) '-)
H), 3.85 (1H, m, 2'-H), 3.85 (1
H, m, (2) '-H), 3.82 (1H, m, 3-
H), 3.82 (1H, m, (3) -H), 3.73
(1H, m, 4-H), 3.73 (1H, m, 5-H)
H), 3.73 (1H, m, (4) -H), 3.70
(2H, m, of _{5-CH 2 H 2),} 3.70 (2
_{H, m, (5) H} 2 in -CH _2), 3.67 (1
H, m, 5 "-H), 3.62 (1H, m, 4"-)
H), 3.62 (1H, m, (2) "-H), 3.62
(1H, m, (5) -H), 3.51 (1H, t, J =
8.9 Hz, 3 ″ -H), 3.28 (1H, t, J =
7.9 Hz, 2 ″ −H), 1.09 (3H, d, J =
6.7Hz, (5) '- CH 3 of _H 3), 1.07
(1H, d, J = 6.7Hz , H 3 of the 5'-CH ₃₎ Sugar composition L- fucose: unsaturated D- glucuronic acid: D-
Glucuronic acid: D-mannose = 2: 1: 1: 2 (L-
Fucose and D- mannose 3-position of the two molecules with unsaturated D- glucuronic acid and D- glucuronic acid each molecule) sulfate 2 molecules (each L- fucose) In ^addition, the numbers of the peaks in 1 H-NMR is The following formula (VI) is used.

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When the above-mentioned endo-fucoidan-degrading enzyme is allowed to act on the obtained sulfated-fucose-containing polysaccharide-U, an elimination reaction occurs as the reaction proceeds, and the absorbance at 230 nm increases. The presence of a saturated hexuronic acid group in all of the main reaction products suggested the presence of a sugar chain in which hexuronic acid and mannose were alternately bonded in the molecule of the obtained sulfated-fucose-containing polysaccharide-U. Since most of the constituent sugars of the obtained sulfated-fucose-containing polysaccharide-U are fucose, the sulfated-fucose-containing polysaccharide-U is more easily decomposed into acids than general polysaccharides. On the other hand, it is known that the bond between hexuronic acid and mannose is relatively strong to acid. The present inventors have conducted carbohydrate research in order to clarify the type of hexuronic acid in the sugar chain in which hexuronic acid and mannose present in the molecule of the fucose sulfate-containing polysaccharide mixture of Gagome kelp are alternately bonded. 125 volumes, 283-2
First, referring to the method described on page 90 (1984), a sulfated-fucose-containing polysaccharide mixture is dissolved in 0.3 M oxalic acid, and
The mixture treated at 0 ° C. for 3 hours was subjected to molecular weight fractionation to give a molecular weight of 3
More than 000 fractions were collected, and the adsorbed fraction was further collected with an anion exchange resin. This material was freeze-dried, acid-hydrolyzed with 4N hydrochloric acid, adjusted to pH 8, converted to PA, and analyzed for uronic acid by HPLC. The HPLC conditions were as follows.

Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: Palpack type N (4.6 mm × 250 m)
m) (Takara Shuzo) Eluent: 200 mM acetic acid-triethylamine buffer (p
H7.3): Acetonitrile = 25: 75 Detection: Fluorescence detector Detected by F-1150 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 320 nm and a fluorescence wavelength of 400 nm Flow rate: 0.8 ml / min Column temperature: 40 ° C.

The standard substance of PA-hexyluronic acid is glucuronic acid manufactured by Sigma, galacturonic acid manufactured by Wako Pure Chemical Industries, and iduronic acid hydrolyzed 4-methylumbelliferyl α-L-iduronide manufactured by Sigma. Mannuronic acid and guluronic acid were obtained from Acta Chemica Scandinavica, Vol. 15, 139.
According to the method described on pages 7-1398 (1961), it was obtained by hydrolyzing alginic acid manufactured by Wako Pure Chemical Industries, Ltd., and separating it with an anion exchange resin to form PA.

As a result, it was found that the hexuronic acid contained in the sugar chain of the fucose sulfate-containing polysaccharide mixture was only glucuronic acid.

Further, glucuronic acid in the hydrolyzate of the sugar chain was separated from D-mannose by an anion exchange resin, and after freeze-drying, the specific rotation was measured. The glucuronic acid was dextrorotatory and the glucuronic acid was D-glucuronic acid. Turned out to be.

[0123] In addition, a mixture of fucose sulfate-containing polysaccharide derived from Gagome kelp, which was previously treated with the above endo-fucoidanase, was acid-hydrolyzed with oxalic acid in the same manner as above, but D-glucuronic acid and D-mannose were used. Was not detected. From this, the skeletal structure of the sulfated-fucose-containing polysaccharide cleaved by the above-mentioned endo-type fucoidanase by an elimination reaction is D-
It was found that glucuronic acid and D-mannose had a structure alternately bonded.

Further, in order to examine the bonding positions of D-glucuronic acid and D-mannose and the anomeric configuration of glycosidic bonds, the polymer obtained by oxalic acid decomposition was analyzed by NMR.

The results of NMR measurement of the polymer are shown below. However, the chemical shift value in ¹ H-NMR was 1.13 ppm of the chemical shift value of the methyl group of triethylamine.
In ¹³ C-NMR, the chemical shift value of the methyl group of triethylamine was expressed as 9.32 ppm.

¹ H-NMR (D ₂ O) δ 5.25 (1H, br-s, 1-H), 4.32 (1
H, d, J = 7.6 Hz, 1'-H), 4.00 (1
H, br-s, 2-H), 3.71 (1H, m, 5'-).
H), 3.69 (1H, m, H for 5-CH), 3.68
(1H, m, 3-H), 3.63 (1H, m, 5-CH
H), 3.63 (1H, m, 4'-H), 3.57
(1H, m, 4-H), 3.54 (1H, m, 3'-
H), 3.53 (1H, m, 5-H), 3.25 (1
H, t, J = 8.5 Hz, 2′-H) ¹³ C-NMR
(D ₂ O) δ 175.3 (C of 5′-COOH), 102.5
(1'-C), 99.6 (1-C), 78.5 (2-
C), 77.9 (4'-C), 77.0 (3'-C),
76.7 (5'-C), 73.9 (5-C), 73.7
(2'-C), 70.6 (3-C), 67.4 (4-
C), 61.0 (C of 5-CH ₂ OH) The numbers of peak assignments are as shown in the following formula (VII):

Embedded image

The configuration at the 1-position of D-glucuronic acid is β-D-glucuronic acid because its vicinal binding constant is 7.6 Hz.
It was determined to be glucuronic acid.

Since the configuration at the 1-position of mannose is 5.25 ppm from its chemical shift value, α-D
-Determined to be mannose;

The binding mode of the constituent sugars was determined using the HMBC method, which is a method for detecting heterogeneous nuclei with ¹ H detection.

DQF-COSY was assigned to ¹ H-NMR.
Law and HOHAHA ^method, H is the assignment of 13 C-NMR
The SQC method was used.

According to the HMBC spectrum, 1-H and 4'-
Cross peaks were observed between C, between 4'-H and 1-C, between 1'-H and 2-C, and between 2-H and 1'-C. From this fact, D-glucuronic acid is located at the second position of D-mannose by β bond, and D-mannose is D-mannose by α bond.
-It was revealed that they were respectively bonded to the 4-position of glucuronic acid.

Considering the above results, (a) shows that D-mannose, which is a reducing terminal residue, has a structure in which unsaturated D-glucuronic acid and L-fucose in which a sulfate group is bonded are bonded. (B) has a structure in which unsaturated D-glucuronic acid and L-fucose in which two sulfate groups are bonded to D-mannose which is a reducing terminal residue to which a sulfate group is bonded; D-mannose, which is the reducing terminal residue, has D
-L-fucose to which glucuronic acid and a sulfate group are bound, D-mannose is bound to the D-glucuronic acid, and L-fucose which is unsaturated D-glucuronic acid and a sulfate group is further bound to the D-mannose. -Fucose was found to have a bonded structure.

Thus, the obtained sulfated-fucose-containing polysaccharide-U
Has a structure in which D-glucuronic acid and D-mannose are alternately bonded, and has a structure in which L-fucose is bonded to at least one or more D-mannose.

Further, it has a partial structure represented by the following general formula (VIII) (provided that at least one alcoholic hydroxyl group in the formula is sulfated and n represents an integer of 1 or more).

Embedded image

According to the present invention, there is provided a sulfated-fucose-containing polysaccharide-U separated from the sulfated-fucose-containing polysaccharide-F of the present invention. The sulfated-fucose-containing polysaccharide-U of the present invention
Contains uronic acid as a constituent sugar, and contains Flavobacterium sp. SA-0082 (FERM BP-540
Fucoidan degrading enzyme produced by 2)
At least one compound selected from the compounds represented by formulas (I), (II) and (III) is produced. The molecular weight, molecular weight distribution, and sugar composition of the present invention do not limit the sulfated-fucose-containing polysaccharide-U of the present invention at all, and any molecular weight and sulfated-fucose-containing polysaccharide-U having a molecular weight distribution can be prepared. A sulfated-fucose-containing polysaccharide having clear physicochemical properties can be provided.

The sulfated-fucose-containing polysaccharide-U of the present invention does not have the strong anticoagulant activity of the sulfated-fucose-containing polysaccharide-F, and provides a sulfated-fucose-containing polysaccharide having substantially no anticoagulant activity. The sulfated-fucose-containing polysaccharide-U of the present invention can be used as a purified sulfated-fucose-containing polysaccharide as an anticancer agent, an anti-metastatic agent, a preventive agent for carcinogenesis, and the like, and is also useful as an antigen of a sulfated-fucose-containing polysaccharide antibody. Further, from the sulfated-fucose-containing polysaccharide-U, the above formulas (I), (II) and (II)
Oligosaccharides having structures such as I) can be produced, and are also useful for producing these novel compounds.

Next, the sulfated-fucose-containing polysaccharide-F of the present invention and a method for producing the same will be described. When producing the sulfated-fucose-containing polysaccharide-F of the present invention using the method of the present invention, a degrading enzyme capable of degrading sulfated-fucose-containing polysaccharide-U may be allowed to act on the sulfated-fucose-containing polysaccharide mixture. After the reaction is completed, the low molecular weight sulfated-fucose-containing polysaccharide-U may be removed by ultrafiltration or the like. The decomposing enzyme may be any enzyme as long as it can selectively decompose the sulfated-fucose-containing polysaccharide-U. Specific examples include, for example, Flavobacterium sp. S
The endo-type fucoidan-degrading enzyme described above produced by the A-0082 strain (FERM BP-5402) can be mentioned.

When the present enzyme is allowed to act, the substrate concentration, temperature, pH and the like may be set so that the enzymatic reaction proceeds advantageously. The substrate concentration is usually about 0.1 to 10%, and the temperature is 20%.
To about 40 ° C. and a pH of about 6 to 9.

Further, the sulfated-fucose-containing polysaccharide mixture is added to the medium, and a microorganism having a degrading enzyme-producing ability capable of degrading the sulfated-fucose-containing polysaccharide-U is cultured in the medium, and purified from the cultured medium. Is also good. The microorganism to be used may be any microorganism as long as it is a microorganism that produces a degrading enzyme having the ability to decompose sulfated-fucose-containing polysaccharide-U, and specifically, the above-described Flavobacterium s.
p. SA-0082 strain (FERM BP-5402)
Or Fucoidanobacter marinus (Fucoidanobacter
marinus) SI-0098 strain (FERM BP-540)
3).

The nutrient source to be added to the medium may be any as long as the strain used can produce the degrading enzyme. Examples of the carbon source include fucoidan, seaweed powder, alginic acid, fucose, glucose, mannitol, glycerol, saccharose, and the like. Maltose, lactose, starch and the like can be used, and as a nitrogen source, yeast extract, peptone, casamino acid, corn steep liquor, meat extract, defatted soybean,
Ammonium sulfate, ammonium chloride and the like are suitable. In addition, inorganic salts such as sodium salts, phosphates, potassium salts, magnesium salts, and zinc salts, and metal salts may be added.

The culturing conditions are, of course, set so as to maximize the decomposition activity of the sulfated-fucose-containing polysaccharide-U according to the strain to be used, the composition of the medium, and the like. 30 ° C., pH of the medium is 5-9,
The aeration and agitation culture may be performed for 5 to 72 hours. After completion of the culture, components other than the low molecular weight sulfated-fucose-containing polysaccharide-U and the components other than the sulfated-fucose-containing polysaccharide-F in the medium may be removed by ultrafiltration or the like.

[0142] The above-mentioned Fucoidanobacter marinus strain SI-0098 is a strain newly obtained by the present inventors from seawater in Aomori prefecture, and its bacteriological properties are as follows. . 1. Fucoidanobactor marinus SI-0098 a. Morphological properties (1) The bacterium is a diplococcus (short bacillus). Width 0.5-0.7 μm Length 0.5-0.7 μm (2) Presence or absence of spores None (3) Gram stainability negative b. Physiological properties (1) Temperature range for growth It can grow at 37 ° C. A preferred growth temperature is between 15 and 28 ° C. (2) Attitude to oxygen Aerobic (3) Catalase positive (4) Oxidase negative (5) Urease negative (6) Hydrolysis Starch positive Gelatin negative Casein negative Esculin positive (7) Nitrate reduction negative (8) Indole production negative (9) Generation of hydrogen sulfide positive (10) Milk coagulation negative (11) Sodium requirement positive (12) Salt requirement Growth on 0% saline medium Negative Growth on 1% saline medium Negative Growth on seawater medium (13) quinone menaquinone 7 (14) GC content of intracellular DNA 61% (15) Diaminopimelic acid in cell wall negative (16) Glycolyl test negative (17) Presence of hydroxy fatty acid positive (18) OF-test O (19 ) Color tone of settlement No characteristic pigment is formed (20) Motility available (21) Glidability not available (22) Flagella poles

The present strain is classified into Group 4 (Gram-negative aerobic bacilli and cocci) according to the basic classification described in the Virgie's Manual of Deterministic Bacteriology, Vol. 9, 1994. However, this strain has menaquinone 7 in the electron transport chain,
It is significantly different from the bacteria belonging to Group 4 in that the C content is 61%. Basically, Gram-negative bacteria have ubiquinone in the electron transport chain, and Gram-positive bacteria have menaquinone.

The Gram-negative bacteria, genus Flavobacterium and Cytophaga, exceptionally have menaquinone in the electron transport chain, but the GC content of the bacteria belonging to these genera is determined by the soil bacterium, cytofaga. Abensicola (Cytophaga arvensicola) is 43-46%, a marine bacterium, Cytophaga difluence (Cytophaga arvensicola).
diffluens), Cytophaga fermentans (Cytoph
aga fermentans), Cytophag Marina (Cytophag)
a marina) and Cytophaga uriginosa (Cytoph
ags uliginosa) is 42%, which is completely different from the properties of this strain. Furthermore, when the homology of the 16S rDNA sequence between the present strain and the identified strain was compared, the identified homologous strain Verrucomicrobium spinosum (Verrucomicrobium ssp.
pinosum), the homology was 76.6%. When the homology of the 16S rDNA sequence is 90% or less,
Since it is generally widely known that the genus of both bacteria is different, the present inventors concluded that this strain is a new genus of bacteria that does not belong to the existing genus, and therefore, named this strain Fucoidanobacter It was named Marinas SI-0098.

The above strain is Fucoidanobacter marinus
It is displayed as SI-0098, and it is FERM P- from March 29, 1995 to the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry [1-3 1-3, Higashi, Tsukuba, Ibaraki, Japan]. 14873, and was registered with FERMB by the Institute of Biotechnology and Industrial Technology,
P-5403 (Date of request for transfer to international deposit: February 1996
15).

As described above, the present inventors have found that the sulfated-fucose-containing polysaccharide-F of the present invention and the sulfated-fucose-containing polysaccharide-U of the present invention are coagulated with the acidic polysaccharide in the presence of one or more salts of 0.6 to 3M. It has also been found that the agent behaves quite differently.

For example, the sulfated-fucose-containing polysaccharide-F of the present invention can be separated from the aqueous solution of the sulfated-fucose-containing polysaccharide mixture by using the method of the present invention.

First, one or more salts are added to the aqueous solution of the sulfated-fucose-containing polysaccharide mixture, and the total concentration is adjusted to 0.6.
Ｍ3M. Salts to be added are, for example, sodium chloride,
It is not particularly limited by calcium chloride or the like. After adjusting the salt concentration in this manner, an acidic polysaccharide flocculant such as cetylpyridinium chloride is added until no more precipitate is formed, and the precipitate is collected to obtain the sulfated-fucose-containing polysaccharide-F of the present invention.
Is obtained.

However, care must be taken when the salt concentration is more than 2 M, since the sulfated-fucose-containing polysaccharide-F of the present invention hardly forms a precipitate due to cetylpyridinium chloride. For the purpose of separating the sulfated-fucose-containing polysaccharide-F and the sulfated-fucose-containing polysaccharide-U of the present invention, the purpose can usually be achieved at a salt concentration of about 1.5 M (see the description of FIG. 1).

Next, if necessary, the precipitate is washed, and the cetylpyridinium chloride in the precipitate is washed away with a saturated salt alcohol to obtain the sulfated-fucose-containing polysaccharide-F of the present invention.
In order to remove the pigment from the sulfated-fucose-containing polysaccharide-F of the present invention thus obtained, the precipitate may be dissolved and then subjected to ultrafiltration or the like. A freeze-dried product can be obtained by freeze-drying after desalting. Further, a preservative or the like may be added during the process.

As described above, the present inventors have found that the amount of sulfated-fucose-sulfuric acid-containing polysaccharide adsorbed per unit resin amount increases when divalent cations coexist when purifying the sulfated-fucose-containing polysaccharide with an anion exchange resin. Have also found that the separation of sulfated-fucose-containing polysaccharides is improved. That is, when producing the sulfated-fucose-containing polysaccharide-F of the present invention using the method of the present invention, first, a drug serving as a divalent cation source, preferably 1 mM or more, is added to the sulfated-fucose-containing polysaccharide mixture. Next, the anion exchange resin is replaced with a divalent cation, preferably 1
Equilibrate with a solution containing at least mM and adsorb the fucose sulfate-containing polysaccharide mixture. After sufficiently washing the anion exchange resin with the equilibrated solution, the sulfated-fucose-containing polysaccharide-F is eluted with, for example, a gradient of sodium chloride. When using this method, the concentration of the divalent cation to be added is 1 mM.
All that is required. In addition, calcium salts and barium salts are particularly effective as chemicals serving as divalent cation sources used in the present method, but are not particularly limited, and magnesium sulfate, manganese chloride and the like can also be used. .

The sulfated-fucose-containing polysaccharide-F of the present invention can be obtained, for example, as described in Example 8. Hereinafter, the physicochemical properties of the sulfated-fucose-containing polysaccharide are shown, but the sulfated-fucose-containing polysaccharide-F of the present invention is not limited to this example.

When the molecular weight of the obtained sulfated-fucose-containing polysaccharide-F was determined by gel filtration using Sephacryl S-500, a molecular weight distribution centered at about 190,000 was shown (see FIG. 17). In FIG. 17, the vertical axis represents the sugar content of the sample measured by the phenol-sulfuric acid method at 480.
The absorbance is shown in nm, and the horizontal axis represents the fraction number.

The conditions for gel filtration are shown below. Column size: 3.08 × 162.5 cm Solvent: 10 mM sodium phosphate buffer containing 0.2 M sodium chloride and 10% ethanol (pH 6.0).
0) Flow rate: 1.5 ml / min Sample concentration: 0.25% Sample liquid volume: 20 ml Molecular weight standard substance: Shodex STANDARD P-82 (manufactured by Showa Denko KK)

Then, the components of the obtained sulfated-fucose-containing polysaccharide-F of the present invention were analyzed. First, the Journal of
Biological chemistry (Journal of Biologica
Chemistry, Vol. 175, p. 595 (1948).

Next, the obtained sulfated-fucose-containing polysaccharide-F
Is dissolved in 1N hydrochloric acid at a concentration of 0.5%,
The mixture was treated at 110 ° C. for 2 hours, and hydrolyzed into constituent monosaccharides. Next, the reducing end of the monosaccharide obtained by hydrolysis using Glycotag and Glycotag Regent Kit is pyridyl- (2) -aminated (PA-modified), and HPL
C was used to determine the ratio of constituent sugars. The HPLC conditions were as follows.

Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: Palpack type A (4.6 mm × 150 m)
m) Eluent: 700 mM borate buffer (pH 9.0): acetonitrile = 9: 1 Detection: Detected with a fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 310 nm and a fluorescence wavelength of 380 nm. Flow rate: 0.3 ml / min Column temperature: 65 ° C

Next, the amount of uronic acid was determined according to the description in Analytical Biochemistry, Vol. 4, p. 330 (1962).

Next, a biochemical journal (Bioc
chemical Journal), 84, 106 (1962).
The sulfuric acid content was quantified according to the description.

As a result, the constituent sugars of the obtained sulfated-fucose-containing polysaccharide-F were fucose and galactose, and the molar ratio was about 10: 1. Uronic acid and other neutral sugars were substantially not contained. The molar ratio between fucose and sulfate groups was about 1: 2.

16 ml of a 1% fucoidan-F solution and 5
12 ml of 0 mM phosphate buffer (pH 8.0), 4 ml of 4 M sodium chloride and 32 mU / ml of the above-mentioned Flavobacterium sp. SA-0082 (FERM BP
Endo-fucoidan-degrading enzyme solution 8 derived from -5402)
The mixture was mixed and reacted at 25 ° C. for 48 hours. No generation of a decomposition product by the reaction was observed, and no reduction in the molecular weight was observed.

Next, the obtained sulfated-fucose-containing polysaccharide-F
The IR spectrum of the calcium salt was measured with a Fourier transform infrared spectrophotometer JIR-DIAMOND20 (manufactured by JEOL Ltd.), and the spectrum shown in FIG. 18 was obtained. In FIG. 18, the vertical axis indicates transmittance (%), and the horizontal axis indicates wave number (cm ^-1 ).

Next, the obtained sulfated-fucose-containing polysaccharide-F
The NMR spectrum of the sodium salt was measured with a 500 MHz nuclear magnetic resonance apparatus JNM-α500 type nuclear magnetic resonance apparatus (manufactured by JEOL Ltd.), and the spectrum shown in FIG. 19 was obtained.

In FIG. 19, the vertical axis indicates the signal intensity, and the horizontal axis indicates the chemical shift value (ppm). In addition, ¹ H-NMR
The chemical shift value of the HOD is 4.65 p.
pm. ¹ H-NMR (D ₂ O) 5.30 (H at position 1 of fucose), 1.19 (H of CH ₃ at position 5 of fucose)

Next, the obtained sulfated-fucose-containing polysaccharide-F
High-speed and high-sensitivity polarimeter SEPA
When measured with -300 (manufactured by Horiba, Ltd.), -1
It was 35 degrees.

According to the present invention, there is provided a sulfated-fucose-containing polysaccharide-F which has been separated and purified from the sulfated-fucose-containing polysaccharide-U of the present invention. The sulfated-fucose-containing polysaccharide-F of the present invention
Contains substantially no uronic acid as a constituent sugar, and contains Flavobacterium sp. SA-0082 (FERM BP
-5402) is not degraded by the fucoidan-degrading enzyme. The molecular weight, molecular weight distribution and sugar composition of the present invention do not limit the sulfated-fucose-containing polysaccharide-F of the present invention at all, and any molecular weight and sulfated-fucose-containing polysaccharide-F having a molecular weight distribution can be prepared. It is possible to provide a sulfated-fucose-containing polysaccharide-F which has clear physicochemical properties such as terminals and has a very high degree of sulfation.

The sulfated-fucose-containing polysaccharide-F of the present invention has a strong anticoagulant activity since it is separated from the sulfated-fucose-containing polysaccharide-U having substantially no anticoagulant activity. Sulfuric acid-containing polysaccharide-F and / or its decomposition product can be used as an anticoagulant as purified sulfated-fucose-containing polysaccharide,
It is also useful as an antigen for an anti-fucose sulfate-containing polysaccharide antibody.

When the sulfated-fucose-containing polysaccharide of the present invention or its decomposed product is added to a culture solution of cancer cells at a concentration of 1 μg / ml or more, the cancer cells undergo apoptosis one to several days after the addition. That is, the sulfated-fucose-containing polysaccharide of the present invention and its degradation product have a strong apoptosis-inducing effect. In addition,
These substances do not induce apoptosis in normal cells,
It does not show toxicity. In particular, the sulfated-fucose-containing polysaccharide derived from edible brown algae plants and sea cucumber, and its decomposition products are highly safe.

In order to formulate the apoptosis-inducing agent of the present invention, a sulfated-fucose-containing polysaccharide and / or a degradation product thereof may be used as an active ingredient, which may be combined with a known pharmaceutical carrier. Generally, the sulfated-fucose-containing polysaccharide of the present invention and / or
Or a decomposed product thereof is mixed with a pharmaceutically acceptable liquid or solid carrier, and if necessary, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant By adding an agent or the like, a solid preparation such as a tablet, a granule, a powder, a powder, a capsule, or the like, or a liquid preparation such as a normal liquid preparation, a suspension, or an emulsion can be obtained. Further, it can be made into a dried product which can be made into a liquid by adding an appropriate carrier before use.

The apoptosis-inducing agent of the present invention comprises an oral preparation,
Alternatively, it can be administered by any of parenteral preparations such as injections and infusions.

Pharmaceutical carriers can be selected according to the above administration forms and dosage forms. In the case of oral preparations, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like are used. You. In preparing an oral preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a flavor, and the like can be further added. Specific examples thereof include the following. (Binder) starch, dextrin, gum arabic powder,
Gelatin, hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, macrogol. (Disintegrant) Starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low-substituted hydroxypropylcellulose. (Surfactant) Sodium lauryl sulfate, soy lecithin, sucrose fatty acid ester, polysorbate 80. (Lubricant) Talc, wax, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol. (Fluidity promoter) Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

[0172] Oral liquid preparations can be suspensions, emulsions, syrups and elixirs. These various forms may contain flavoring agents, flavoring agents and coloring agents. .

On the other hand, in the case of parenteral preparations, the sulfated-fucose-containing polysaccharide and / or its decomposed product, which is the active ingredient of the present invention, is used as a diluent in distilled water for injection, physiological saline,
Glucose aqueous solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, dissolved or suspended in polyethylene glycol,
It is prepared by adding a bactericide, a stabilizer, an isotonic agent, a soothing agent and the like, if necessary.

The apoptosis-inducing agent of the present invention is administered by an appropriate administration route depending on the form of the preparation. The administration method is also not particularly limited, and may be internal use, external use and injection.
The injection can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, etc., and external preparations also include suppositories and the like.

The dosage of the apoptosis-inducing agent of the present invention is
The formulation, administration method, purpose of use and the age, weight, and symptoms of the patient to which the formulation is applied are appropriately set, and the amount of the active ingredient contained in the formulation is not constant but is generally 1 to 1000 mg per adult per day. , Preferably 10 to 20
0 mg. Of course, the dosage varies depending on various conditions, and thus, a dosage smaller than the above-mentioned dosage may be sufficient, or may be required to exceed the above range.

The sulfated-fucose-containing polysaccharide-U of the present invention, which has an anticancer effect, and the sulfated-fucose-containing polysaccharide-F of the present invention and / or a decomposed product thereof are used as active ingredients, and these are combined with a known pharmaceutical carrier to form a formulation. An anticancer drug can be manufactured.
The anticancer drug can be produced according to the above method. In general, the sulfated-fucose-containing polysaccharide of the present invention and / or its decomposition product is mixed with a pharmaceutically acceptable liquid or solid carrier, and if necessary, a solvent, a dispersant, an emulsifier, a buffer, a Solid, such as tablets, granules, powders, powders, capsules, etc., and usually liquids, suspensions, emulsions, etc., with the addition of agents, excipients, binders, disintegrants, lubricants, etc. be able to. In addition, it can be made into a dried product which can be made into a liquid form by adding an appropriate carrier before use.

The anticancer agent can be administered as an oral agent or a parenteral agent such as an injection or an infusion.

The pharmaceutical carrier can be selected according to the above-mentioned administration form and dosage form, and may be used according to the above-mentioned apoptosis-inducing agent.

The anticancer agent is administered by an appropriate route depending on the form of the preparation. There is no particular limitation on the administration method,
It can be by internal use, external use and injection. The injection can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, etc., and external preparations also include suppositories and the like.

The dose of the anticancer drug is appropriately determined depending on the form of the drug, the method of administration, the purpose of use, and the age, weight, and symptoms of the patient to which the drug is applied. The amount of the active ingredient to be used is 1 to 1000 mg, preferably 10 to 200 mg per adult day. Of course, the dosage varies depending on various conditions, and thus, a dosage smaller than the above-mentioned dosage may be sufficient, or may be required to exceed the above range. The agent of the present invention can be orally administered as it is, or can be added to any food or drink to be taken on a daily basis.

[0181] A carcinogen-preventing agent can be manufactured by combining a sulfated-fucose-containing polysaccharide having a carcinogenesis-inhibiting activity and / or a decomposed product thereof as an active ingredient and formulating it in combination with a known pharmaceutical carrier. Production of a carcinogen preventive agent can be carried out according to the above method. Generally, the sulfated-fucose-containing polysaccharide and / or its decomposition product is mixed with a pharmaceutically acceptable liquid or solid carrier, and if necessary, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, a stabilizer, Add tablets, binders, disintegrants, lubricants, tablets, granules, powders, powders,
Solid preparations such as capsules and liquid preparations such as ordinary liquid preparations, suspensions and emulsions can be prepared. In addition, it can be made into a dried product which can be made into a liquid form by adding an appropriate carrier before use.

The carcinogen-preventing agent can be administered as an oral agent or a parenteral agent such as an injection or an infusion.

The pharmaceutical carrier can be selected according to the above-mentioned administration form and dosage form, and may be used according to the above-mentioned apoptosis-inducing agent.

The carcinogen-preventing agent is administered by an appropriate route depending on the form of the preparation. The administration method is also not particularly limited, and may be internal use, external use and injection. Injectables can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, etc.
External preparations also include suppositories and the like.

The dose as a carcinogen-preventing agent is appropriately set depending on the form of the preparation, the administration method, the purpose of use, and the age, weight, and symptoms of the patient to which the preparation is applied, and is not fixed but is generally contained in the preparation. The amount of the active ingredient is 1 to 1000 mg, preferably 10 to 200 mg per adult day. Of course, the dose varies depending on various conditions, so a smaller amount than the above dose may be sufficient,
Or it may be necessary beyond the range. The agent of the present invention can be orally administered as it is, or can be added to any food or drink to be taken on a daily basis.

[0186] The sulfated-fucose-containing polysaccharide of the present invention and its decomposed product are substances of natural origin, and no toxicity is observed when administered orally to mice.

The drug of the present invention is expected to be used as a therapeutic agent for infectious diseases, reduction or enhancement of immune function, cancer diseases, viral diseases and the like. It can be used to maintain health as a carcinogen preventive agent. The method of inducing apoptosis according to the present invention is useful for studying biological defense mechanisms, immune functions or relationships with cancer, viral diseases, etc., and developing apoptosis-inducing inhibitors. In particular, if an edible brown algae plant or edible sea cucumber is used to prepare the sulfated-fucose-containing polysaccharide of the present invention, its decomposed product, these have a long history as foods, and the sulfated-fucose-containing polysaccharide prepared therefrom,
The degradation product is extremely safe when administered orally.

Next, the sulfated-fucose-containing polysaccharide is a sulfated polysaccharide having an extremely large molecular weight, and decomposes the sulfated-fucose-containing polysaccharide to some extent in order to improve antigenicity, homogeneity, anticoagulant activity, etc., when used as a pharmaceutical as it is According to the present invention, there is provided an enzyme capable of selectively decomposing only the sulfated-fucose-containing polysaccharide-F, and a low-molecular-weight product of the sulfated-fucose-containing polysaccharide-F obtained by acting the enzyme. .

The strain used in the present invention may be any strain as long as it belongs to the genus Alteromonas and has the ability to produce the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention. Specific examples of the strain having the ability to produce the endo-sulfated sulfate-containing polysaccharide-degrading enzyme include, for example, Alteromonas sp. SN-1
009 strains. If the endo-fucose sulfate-containing polysaccharide-degrading enzyme derived from the strain is allowed to act on the sulfated-fucose-containing polysaccharide, a low molecular weight product of the sulfated-fucose-containing polysaccharide-F of the present invention can be obtained.

The present strain is a strain newly obtained by the present inventors from seawater in Aomori Prefecture, and its bacteriological properties are as follows. a. Morphological properties (1) The bacterium is a bacillus. Width about 1 μm Length about 2 μm (2) Presence or absence of spores None (3) Gram stain negative b. Physiological properties (1) Temperature range for growth The preferred growth temperature is 15-30 ° C. 4 ℃ or 4
Cannot grow at 0 ° C. (2) Attitude to oxygen aerobic (3) catalase positive (4) oxidase positive (5) lipase positive (6) assimilation glucose positive mannose negative sucrose positive lactose negative cellobiose positive melibiose negative mannitol positive glycerin positive methanol negative DL-apple Acid negative succinic acid negative fumaric acid negative citric acid negative salicin negative (7) hydrolysis starch negative gelatin negative (8) nitrate reduction negative (9) denitrification reaction negative (10) alginic acid decomposition positive (11) β-hydroxybutyric acid (12) Accumulation of polyhydroxybutyric acid Negative (13) Sodium requirement positive (14) Salt requirement Growth on 0% saline medium Negative Growth on 1% saline medium Negative Growth on seawater medium Positive (15 ) Quinone ubiquinone 8 (1 ) GC content of 36% in the cells DNA (17) OF- test O (18) without generating a tone characteristic dye settlement (19) emitting negative (20) motility positive (21) flagella Gokutanke

This strain is described in the Vergie's Manual of Sistmatic Bacteriology, Volume 1, pages 343-352 (1984), and in the Verge's Manual of Deterministic Bacteriology, Volume 9, pages 75, 132-133. Page (199
It was identified as an Alteromonas genus bacterium described in 4). However, the physiological properties of this bacterium did not match any of the bacterial species described in the literature, and the GC content was low. Therefore, this strain was transformed into Alteromonas sp. SN
-1009.

The above strain was obtained from Alteromonas sp. SN
-1009, and the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry [1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan
(Postal code 305)] on February 13, 1996
Deposited as RM P-15436, and submitted to FERM BP-
No. 5747 (Request for transfer to international deposit: November 15, 1996).

The nutrient added to the culture medium of the strain used in the present invention may be any as long as it utilizes the strain used and produces the endo-fucose sulfate-containing polysaccharide degrading enzyme of the present invention. Polysaccharide, seaweed powder,
Alginic acid, fucose, galactose, glucose, mannitol, glycerol, saccharose, maltose, etc. can be used, and as a nitrogen source, yeast extract, peptone, casamino acid, corn steep liquor, meat extract,
Suitable are defatted soybeans, ammonium sulfate, ammonium chloride and the like. In addition, inorganic salts such as sodium salts, phosphates, potassium salts, magnesium salts, and zinc salts, and metal salts may be added.

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

In culturing the bacterium producing the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention, the production amount varies depending on the culturing conditions, but the culturing temperature is generally 15 ° C. to 30 ° C.
The pH of the medium is preferably 6 to 9, and the production of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention reaches the maximum in aeration and agitation culture for 5 to 72 hours.

It is a matter of course that the culturing conditions are set so as to maximize the production of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention in accordance with the strain to be used, the composition of the medium, and the like.

[0197] The endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention is present in both cells and culture supernatants.

The above Alteromonas sp. SN-1
009 is cultured in an appropriate medium, the cells are collected, and the cells are disrupted by a commonly used cell disruption means, for example, ultrasonic treatment, to obtain a cell-free extract.

Next, a purified enzyme preparation can be obtained from the extract by a commonly used purification means. For example, purification by salting out, ion exchange column chromatography, hydrophobic binding column chromatography, gel filtration, etc. can be performed to obtain the purified endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention which does not contain other fucoidan-degrading enzymes. .

[0200] Since the present enzyme is present in a large amount in the culture solution supernatant obtained by removing the cells from the above-mentioned culture solution, it can be purified by the same purification means as the intracellular enzyme.

The chemical and physicochemical properties of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention are as follows. (I) action: acting on a sulfated-fucose-containing polysaccharide having the following physicochemical properties, namely, a sulfated-fucose-containing polysaccharide-F, to reduce the molecular weight of the sulfated-fucose-containing polysaccharide-F; (A) Constituent sugar: substantially free of uronic acid. (B) Flavobacterium sp.
The molecular weight is not substantially reduced by the fucoidanase produced by SA-0082 (FERM BP-5402).
It does not act on the sulfated-fucose-containing polysaccharide having the following physicochemical properties, ie, sulfated-fucose-containing polysaccharide-U. (C) Constituent sugar: contains uronic acid. (D) Flavobacterium sp.
The molecular weight is reduced by fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402), and at least one or more compounds selected from the following formulas (I), (II) and (III) are produced.

Embedded image

Embedded image

Embedded image (Ii) Optimum pH: The optimum pH of the present enzyme is around 7 to 8 (FIG. 20). That is, FIG. 20 is a graph showing the relationship between the pH and the relative activity of the present enzyme. The vertical axis indicates the relative activity (%), and the horizontal axis indicates the pH. The solid line is a curve using the sulfated-fucose-containing polysaccharide-F (PA-FF) in which the reducing end is converted to PA as the substrate, and the dotted line is the curve obtained using the sulfated-fucose-containing polysaccharide-F described in (v)-(2) below. It is a curve in the case of using for. (Iii) Optimum temperature: The optimum temperature of the present enzyme is around 30 to 35 ° C. (FIG. 21). That is, FIG. 21 is a graph showing the relationship between the temperature and the relative activity of the present enzyme, where the vertical axis indicates the relative activity (%) and the horizontal axis indicates the temperature (° C.). The solid line is a sulfated-fucose-containing polysaccharide-F (PA-FF) having a reducing terminal PA.
Is a curve in the case where is used as a substrate, and the dotted line is the following (v)-
It is a curve at the time of using the fucose sulfate containing polysaccharide-F of (2) as a substrate. (Iv) Molecular weight: The molecular weight of the enzyme is determined by Sephacryl (Sephacryl).
It was about 100,000 as determined by gel filtration using acryl) S-200 (manufactured by Pharmacia).

(V) Method for Measuring Enzyme Activity: The activity of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention was measured as follows.

First, a sulfated-fucose-containing polysaccharide-F serving as a substrate for the endo-type sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention,
And PA-FF were prepared by the following steps (1) to (3).

(1) Preparation of Polysaccharide Mixture Containing Sulfuric Acid Containing Gagome Kombu Fucose 2 kg of dried gagome kelp was crushed by a free crusher Model M-2 (manufactured by Nara Kikai Seisakusho), and the mixture was dried at 80 ° C. in a 4.5-fold amount of 80% ethanol. After treatment for 2 hours, the mixture was filtered. For the residue
The above 80% ethanol extraction and filtration steps are further performed
This was repeated twice to obtain 1870 g of ethanol washing residue. 36 l of water was added to the residue, the mixture was treated at 100 ° C for 2 hours, and an extract was obtained by filtration. When the salt concentration of the extract is 40
After making the same as the 0 mM sodium chloride solution, 5% of cetylpyridinium chloride was added until no more precipitate was formed, and the mixture was centrifuged. The precipitate was repeatedly washed with 80% ethanol to completely remove cetylpyridinium chloride, dissolved in 3 liters of 2M sodium chloride, insoluble materials were removed by centrifugation, and equilibrated with 2M sodium chloride. 100 ml of DEAE-Cellulofine A-800 was suspended, stirred, and filtered to remove the resin. The filtrate was mixed with 100 ml of DEAE-Cellulofine A-800 equilibrated with 2M sodium chloride.
And the passing fraction was subjected to desalting and low molecular weight removal by an ultrafilter (excluded molecular weight of the filtration membrane: 100,000), and the precipitate formed at this time was removed by centrifugation. This supernatant was freeze-dried to obtain 82.2 g of a sulfated polysaccharide mixture containing purified sulfate of gagome kelp fucose.

(2) Preparation of sulfated-fucose-containing polysaccharide-F 6 g of the sulfated-fucose-containing polysaccharide mixture derived from Gagome kelp described above
20 m containing 600 ml of 0.2 M calcium chloride
M sodium acetate (pH 6.0), 3600 ml of DE previously equilibrated with 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride.
AE-Sepharose FF column, 20 mM sodium acetate containing 0.2 M calcium chloride (pH
After sufficiently washing the column with 6.0), elution was carried out with a gradient of 0 to 2 M sodium chloride. Fucose sulfate-containing polysaccharide eluted at a sodium chloride concentration of 0.75M or more
The F fraction was collected, concentrated and desalted with an ultrafilter equipped with an ultrafiltration membrane having an excluded molecular weight of 100,000, and then lyophilized to obtain 3.3 g of a lyophilized sulfated-fucose-containing polysaccharide-F sample.

(3) Preparation of PA-FF A freeze-dried preparation of the above-mentioned sulfated-fucose-containing polysaccharide-F 12 m
g was dissolved in 480 μl of water, dispensed into 36 tubes of 12 μl each, freeze-dried, and the reducing end was converted to PA using Glycotag and Glycotag Regent kit.
PA-FF was obtained. 15 ml of the obtained PA-FF
Was dissolved in a 10 mM ammonium acetate solution containing 10% methanol, and subjected to gel filtration with a Cellulofine GCL-300 column (40 × 900 mm) to collect a polymer fraction. The obtained high molecular fraction was sufficiently dialyzed using a dialysis membrane having a pore size of 3500, desalted, and then concentrated to 5 ml by an evaporator, and the PA for the substrate of the endo-fucose sulfate-containing polysaccharide-F-degrading enzyme of the present invention was used. -FF. In addition, PA-FF obtained in this manner was converted to commercially available pyridyl-
Quantification by comparison with the fluorescence intensity (excitation wavelength 320 nm, fluorescence wavelength 400 nm) of (2) -aminated fucose (manufactured by Takara Shuzo) was about 40 nmol.

When the activity of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention was measured using the sulfated-fucose-containing polysaccharide-F obtained by the above steps (1) and (2), the following procedure was used. .

That is, 12 μl of a 2.5% fucose sulfate-containing polysaccharide-F solution, 6 μl of a 1 M calcium chloride solution, 12 μl of a 1 M sodium chloride solution, and a buffer containing 72 μl of 50 mM acetic acid, imidazole and tris-hydrochloride. (PH 7.5) and 18 μl of the endo-fucose sulfate-containing polysaccharide-F-degrading enzyme of the present invention were mixed at 30 ° C.
After reacting for 3 hours, the reaction solution was treated at 100 ° C. for 10 minutes, and after centrifugation, 100 μl was analyzed by HPLC,
The degree of depolymerization was measured.

As a control, a reaction was carried out under the same conditions using a buffer in which the endo-fucose sulfate-containing polysaccharide degrading enzyme of the present invention was dissolved instead of the endo-fucose sulfate-containing polysaccharide degrading enzyme of the present invention. A solution prepared by using water instead of the sulfated-fucose-containing polysaccharide-F solution was prepared, and each was similarly analyzed by HPLC.

One unit of the enzyme is the amount of the enzyme that cuts the fucosyl bond of 1 μmol of the sulfated-fucose-containing polysaccharide-F per minute in the above reaction system. The quantification of the cleaved fucosyl bond was determined by the following equation. ｛(12 × 2.5) / (100 × MF)｝ × ｛(MF / M) -1｝ × ｛0.12 /
(180 × 0.01)｝ = U / ml (12 × 2.5) / 100: sulfated-fucose-containing polysaccharide-F (mg) added to the reaction system MF: average molecular weight of sulfated-fucose-containing polysaccharide-F substrate M: reaction Average molecular weight of product (MF / M)-1: 1 molecule of sulfated-fucose-containing polysaccharide-F
180: Reaction time (min) 0.01: Enzyme solution volume (ml) 0.12: Total reaction solution volume (ml)

The HPLC conditions were as follows. Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: OHpak KB-804 (8 mm × 300 m)
m) (manufactured by Showa Denko KK) Eluent: 25 m containing 5 mM sodium azide, 25 mM calcium chloride, and 50 mM sodium chloride
M imidazole buffer (pH 8) Detection: Parallax refractive index detector (Shodex RI-71,
(Showa Denko KK) Flow rate: 1 ml / min Column temperature: 25 ° C

For measuring the average molecular weight of the reaction product,
Commercially available pullulan of known molecular weight (STANDARD P-
82, manufactured by Showa Denko KK under the same conditions as in the above HPLC analysis, and the molecular weight of pullulan and OHpak KB-804 were analyzed.
Of the enzyme reaction product was represented by a curve, and the curve was used as a standard curve for measuring the molecular weight of the enzyme reaction product.

The activity of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention was measured using PA-FF obtained by the above steps (1) to (3) in the following manner.

That is, PA-FF of 8 pmol / μl
2 μl of solution, 5 μl of 1 M calcium chloride solution and 10 μl
μl of 1M sodium chloride solution, 23 μl of water, 5
0 μl of a buffer solution (pH 8.2) containing 50 mM acetic acid, imidazole and tris-hydrochloride, and 10 μl of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention were mixed with 30 μl of the mixture.
After reacting at 3 ° C. for 3 hours, the reaction solution was treated at 100 ° C. for 10 minutes, and after centrifugation, 80 μl was analyzed by HPLC to determine the degree of molecular weight reduction.

As a control, a reaction was carried out under the same conditions using a buffer in which the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention was dissolved instead of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention. And those prepared by performing a reaction using water instead of the PA-FF solution were prepared, and each was similarly analyzed by HPLC.

One unit of the enzyme is the amount of the enzyme that cuts the fucosyl bond of 1 μmol of the sulfated-fucose-containing polysaccharide per minute in the above reaction system. The quantification of the cleaved fucosyl bond was determined by the following equation. 16 × 10 ⁻⁶ × ｛(MF / M) -1｝ × ｛1 / (180 × 0.01)｝ = U
/ Ml 16 × 10 ⁻⁶ : PA-FF (μm) added to the reaction system
ol) MF: average molecular weight of the substrate PA-FF M: average molecular weight of the reaction product (MF / M)-1: 1 fucose sulfate-containing polysaccharide-F
180: Reaction time (min) 0.01: Enzyme solution volume (ml)

The HPLC conditions were as follows. Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: OHpak SB-803 (8 mm x 300 mm)
(Showa Denko) Eluent: 200 mM sodium chloride solution containing 5 mM sodium azide and 10% dimethyl sulfoxide Detection: 320 nm excitation wavelength and 400 nm fluorescence wavelength with fluorescence detector F-1150 (manufactured by Hitachi, Ltd.) detection. Flow rate: 1 ml / min Column temperature: 50 ° C

To determine the average molecular weight of the reaction product,
Commercially available pullulan of known molecular weight (STANDARD P-
82, manufactured by Showa Denko KK), and the reducing end was converted to PA using Glycotag and Glycotag Regent Kit.
To obtain PA-modified pullulan of various molecular weights. The obtained PA-modified pullulan of various molecular weights was analyzed under the same conditions as the above-mentioned HPLC analysis, and the molecular weight of pullulan and OHpak SB were analyzed.
The relationship between -803 and the retention time was represented by a curve, which was used as a standard curve for measuring the molecular weight of the enzyme reaction product.

The protein was quantified by measuring the enzyme solution at 280 nm.
The measurement was performed by measuring the absorbance. 1 mg /
The absorbance of ml protein solution was calculated as 1.0.

The present inventors have determined the mechanism of action of the endo-fucose sulfate-containing polysaccharide degrading enzyme of the present invention, as described below.

(1) Decomposition of Fucose Sulfate-Containing Polysaccharide-F with Endofucose Sulfate-Containing Polysaccharide-Degrading Enzyme and Preparation of Degraded Product The end-fucose sulfate-containing polysaccharide-F of the present invention is added to the purified fucose sulfate-containing polysaccharide-F derived from Gagome kelp. Degradation products were prepared by the action of a degrading enzyme.

First, a sulfated-fucose-containing polysaccharide-degrading enzyme was produced. That is, Alteromonas sp. SN-
1009 (FERM BP-5747) was prepared by adding 0.25% of glucose, 1.0% of peptone, and 0.05 of yeast extract.
% Artificial seawater (manufactured by Jamarin Laboratory) pH
Dispense and sterilize 600 ml of the medium consisting of 8.2 (1).
20 ° C, 20 minutes) inoculate a 2 liter Erlenmeyer flask,
The cells were cultured at 25 ° C. for 26 hours to obtain a seed culture solution. Artificial seawater pH containing 1.0% of peptone, 0.02% of yeast extract, 0.2% of sulfated-fucose-containing polysaccharide derived from Gagome kelp, and 0.01% of antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.)
Twenty liters of the medium consisting of 8.0 was placed in a 30 liter jar fermenter and sterilized at 120 ° C. for 20 minutes. After cooling, 600 ml of the above seed culture was inoculated and
24 hours at 10 ° C, aeration rate of 10 liters per minute and 250
The culture was carried out under the condition of rotation speed. After completion of the culture, the culture was centrifuged to obtain bacterial cells and culture supernatant. The obtained culture supernatant was concentrated by an ultrafilter having a molecular weight cutoff of 10,000 and then subjected to salting out with 85% saturated ammonium sulfate. The resulting precipitate was collected by centrifugation, and the concentration of 20 mM of artificial seawater containing 1/10 concentration of artificial seawater was collected. After sufficient dialysis against Tris-HCl buffer (pH 8.2),
0 ml of crude enzyme was obtained.

Of the crude enzyme thus obtained, 40 ml
And 44 ml of artificial seawater, 510 mg of the above-mentioned sulfated-fucose-containing polysaccharide-F and 36 ml of water were mixed, the pH was adjusted to 8, and the mixture was reacted at 25 ° C. for 48 hours.
Gel filtration was carried out using a 300, and the mixture was divided into four fractions, and F-Fd-1 (molecular weight: 25,000) in ascending order of molecular weight.
Super), F-Fd-2 (molecular weight 25000 to 12000)
Super), F-Fd-3 (molecular weight 12000-6500)
Super) and F-Fd-4 (molecular weight 6500 or less). These four fractions were desalted and freeze-dried, and the dried products were 170 mg, 270 mg, 300 mg and 340 mg, respectively.
mg.

Enzymatically decomposed product of sulfated-fucose-containing polysaccharide-F,
That is, FIG. 22 shows the result of gel filtration of a low molecular weight compound using Cellulofine GCL-300. In FIG. 22, the vertical axis represents the absorbance at 480 nm (the amount of color developed by the phenol-sulfuric acid method), and the horizontal axis represents the fraction number, one fraction being 10 ml. Column volume is 1075ml
And the eluate is a 0.2 M ammonium acetate solution containing 10% ethanol.

In FIG. 22, open circles indicate the results of gel filtration of sulfated-fucose-containing polysaccharide-F, and solid triangles indicate the results of gel filtration of sulfated-fucose-containing polysaccharide-F before enzymatic degradation.

From the results of the above Cellulofine GCL-300, it was found that the molecular weight distribution of the reaction product of the sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention was about 1,000 to 30,000.

(2) Analysis of the reducing terminal sugar and neutral sugar composition of the enzyme reaction product A part of the above-mentioned F-Fd-1, F-Fd-2, F-Fd-3 and F-Fd-4 The reducing end to PA using Glycotag and Glycotag Regent Kit.
And each of the obtained PA saccharides (PA-F-Fd-1),
(PA-F-Fd-2), (PA-F-Fd-3) and (PA-F-Fd-4) were converted to 4N hydrochloric acid at 100 ° C.
The mixture was hydrolyzed by treatment for 3 hours, and the reducing terminal sugar was examined by HPLC.

The HPLC conditions were as follows. Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: Palpack type A (4.6 mm × 150 mm) (manufactured by Takara Shuzo) Eluent: 700 mM borate buffer (pH 9): acetonitrile = 9: 1 Detection: fluorescence detection Detected by F-1150 (manufactured by Hitachi, Ltd.) at an excitation wavelength of 310 nm and a fluorescence wavelength of 380 nm. Flow rate: 0.3 ml / min Column temperature: 65 ° C

As a result, (PA-F-Fd-1), (P
The reducing terminal sugars of (AF-Fd-2), (PA-F-Fd-3) and (PA-F-Fd-4) were all fucose.

Also, F-Fd-1, F-Fd-2, F-
The neutral sugar composition of Fd-3 and F-Fd-4 was measured by the following method. In addition, after reducing the sulfated-fucose-containing polysaccharide-F used as the substrate by sulfuric acid hydrolysis, the reducing end of the constituent sugar was converted to PA using a glycotag and glycotag reagent kit, and the reducing end of the above enzyme reaction product was analyzed. When analyzed under the same HPLC conditions as before, only fucose and galactose were detected, and their steric configurations were L and D, respectively. Therefore, only L-fucose and D-galactose were measured in the product.

That is, in order to examine the content of D-galactose, which is one of the constituent sugars, a reaction system capable of measuring only D-galactose according to the instruction manual using F-kit lactose / galactose (manufactured by Boehringer Mannheim Yamanouchi) was used. F-Fd-1, F-Fd-2 and F-Fd- which were separately constructed and hydrolyzed with 4N hydrochloric acid at 100 ° C for 2 hours.
3, and F-Fd-4 were measured in this reaction system after neutralization.

In order to quantify L-fucose, which is the other constituent sugar, Clinical Chemistry (Clinic
al Chemistry), 36, 474-476 (19
90) According to the method described in the above, separately add 4N hydrochloric acid to 100
C, F-Fd-1, F-Fd-2 hydrolyzed for 2 hours,
F-Fd-3 and F-Fd-4 were measured in this reaction system after neutralization.

As a result, the ratio of L-fucose to D-galactose was F-Fd-1, F-Fd-2, F-Fd-
3, and about 100: 44, 1 for F-Fd-4, respectively.
00:27, 100: 5, and 100: 1.

To summarize the above results, the endo-type sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention acts on the sulfated-fucose-containing polysaccharide-F to hydrolyze fucosyl bonds, thereby producing a low-molecular-weight molecule having a molecular weight of about 1,000 to 30,000. It was found that the lower the molecular weight, the higher the molecular weight was, the higher the galactose content was. In addition, all the reducing terminals of the low molecular weight compound were L-fucose.

Next, to examine the substrate specificity of the present enzyme, the sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention was allowed to act on the sulfated-fucose-containing polysaccharide-U.

That is, 12 μl of a 2.5% fucose sulfate-containing polysaccharide-U solution, 6 μl of a 1 M calcium chloride solution and 12 μl of a 1 M sodium chloride solution, 72 μl of a 50 mM imidazole buffer (pH 7.5), 18
After mixing with μl of the endo-fucose sulfate-containing polysaccharide-degrading enzyme (1.6 mU / ml) of the present invention and reacting at 30 ° C. for 3 hours, the reaction solution was treated at 100 ° C. for 10 minutes, and centrifuged. 100 μl was analyzed by HPLC to determine the degree of molecular weight reduction.

As a control, a reaction was carried out under the same conditions using a buffer in which the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention was dissolved instead of the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention. And prepare H
Analyzed by PLC.

The HPLC conditions are as follows. Apparatus: Model L-6200 (manufactured by Hitachi, Ltd.) Column: OHpak KB-804 (8 mm x 300 mm)
(Showa Denko KK) Eluent: 25 m containing 5 mM sodium azide, 25 mM calcium chloride, and 50 mM sodium chloride
M imidazole buffer (pH 8) Detection: Parallax refractive index detector (Shodex RI-71,
(Showa Denko KK) Flow rate: 1 ml / min Column temperature: 25 ° C

As a result, the sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention did not reduce the molecular weight of the sulfated-fucose-containing polysaccharide-U at all.

As described above, the present invention relates to an enzyme composition containing the above-described sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention and a calcium source.

Examples of usable calcium sources include, in a solid composition, calcium salts such as calcium chloride, calcium carbonate and calcium acetate, calcium oxide, calcium hydroxide, and hydrates thereof. On the other hand, in a liquid composition dissolved, suspended or emulsified in a solvent such as water or alcohol, the calcium source may be a simple substance as described above, or may be in a state ionized by dissolution or the like. Is also good.

[0242] These calcium sources are effective because they activate or stabilize the enzyme.

Therefore, the enzyme composition may contain a usual additive depending on the use as long as the function and effect of the calcium source are not inhibited.

By reacting the sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention with a sulfated-fucose-containing polysaccharide-F-containing substance, a low-molecular-weight derivative of sulfated-fucose-containing polysaccharide-F can be prepared. As the sulfated-fucose-containing polysaccharide-F-containing material, for example, a sulfated-fucose-containing polysaccharide-F purified product may be used, the sulfated-fucose-containing polysaccharide mixture may be used, and an aqueous solvent extract of brown algae seaweed may be used. The dissolution of the sulfated-fucose-containing polysaccharide-F-containing substance may be performed by an ordinary method,
The concentration of the sulfated-fucose-containing polysaccharide in the dissolving solution may be the highest dissolving concentration, but is usually selected in consideration of its operability and enzyme titer.

The sulfated-fucose-containing polysaccharide-F solution may be selected from water, a buffer and the like depending on the purpose. The pH of the lysis solution is usually neutral, and the enzymatic reaction is usually performed at around 30 ° C. The molecular weight of the low molecular weight compound can be adjusted by adjusting the amount of the enzyme, the reaction time, and the like.

Next, the low molecular weight product is subjected to molecular weight fractionation, whereby a sulfated-fucose sulfate-containing polysaccharide having a more uniform molecular weight distribution is obtained.
F low molecular weight compound can be prepared. For the molecular weight fractionation, a commonly used method can be applied. For example, a gel filtration method or a molecular weight fractionation membrane may be used. The low molecular weight product may be further subjected to a purification operation such as an ion exchange resin treatment and an activated carbon treatment, if necessary. A dried product of the molecular compound can also be prepared.

[0247]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these descriptions. In the examples,% means% by weight.

Example 1 After thoroughly drying Gagome kelp, 2 kg of the dried product was pulverized with a free pulverizer (manufactured by Nara Kikai Seisakusho), and the obtained dried powder was suspended in 9 liters of 80% ethanol, and the suspension was heated at 80 ° C. for 2 hours. Processed. The residue was obtained by filtration through the paper after the treatment. This residue was subjected to the above-mentioned operations of washing with ethanol and filtration in 3 steps.
Repeatedly, ethanol washing residue was obtained. 40 of this residue
After suspending in 1 liter of water, the mixture was treated at 95 ° C. for 2 hours and filtered. The residue was washed with hot water to obtain 36 liters of an extract containing the sulfated-fucose-containing polysaccharide of Gagome kelp. 1.8 liters of the obtained extract was freeze-dried to obtain 15.4 g of a sulfated-fucose-containing polysaccharide preparation. Next, salt was added to the remaining extract to 0.4 M, and 5% cetylpyridinium chloride was further added until no more precipitate was formed, and the precipitate was collected by centrifugation. 3 liters of 0.4 M
After suspending in saline, the suspension was centrifuged and washed. After repeating this washing operation three times, 1 liter of 4M saline was added to the precipitate, and after stirring, ethanol was added to 80%, and after stirring, a precipitate was obtained by centrifugation. The operation of suspending the precipitate in 80% ethanol and centrifuging was repeated until the absorbance at 260 nm in the supernatant became 0. This precipitate was dissolved in 3 liters of 2M saline,
After removing insoluble matter by centrifugation, 100 ml of DEAE-Cellulofine A-800 equilibrated with 2M saline solution.
(Manufactured by Seikagaku Corporation) was added, and after stirring, the added resin was removed by filtration. The filtrate is applied to a DEAE-Cellulofine A-800 column equilibrated with 2M saline,
The non-adsorbed components were eliminated by ultrafiltration using an ultrafiltration apparatus equipped with a hollow fiber having a molecular weight of 100,000 or less, and after completely removing the coloring substance and salt, the insoluble substance was removed by centrifugation and filtration, followed by freeze-drying. . The freeze-dried sulfated-fucose-sulfuric acid-containing polysaccharide mixture weighed 76 g.

Example 2 After sufficiently drying the dried kelp, 2 kg of the dried product was pulverized with a free pulverizer (manufactured by Nara Kikai Seisakusho), and the obtained dry powder was suspended in 9 liters of 80% ethanol. Time processed. Filtration was performed on the treated paper to obtain a residue. The operation of washing with ethanol and filtering the residue was repeated three times to obtain an ethanol-washed residue. 40 of this residue
After suspending in 1 liter of water, the mixture was treated at 95 ° C. for 2 hours and filtered. The residue was washed with hot water to obtain 36 liters of an extract containing the sulfated-fucose-containing polysaccharide of sea kelp. Salt was added to the obtained extract to 0.4 M, and 5% cetylpyridinium chloride was further added until no more precipitate was formed, and the precipitate was collected by centrifugation. The precipitate was suspended in 3 liters of 0.3 M saline, centrifuged, and washed. After repeating this washing operation three times, 1 liter of 4M
A saline solution was added, and after stirring, ethanol was added to 80%, and after stirring, a precipitate was obtained by centrifugation. The operation of suspending the precipitate in 80% ethanol and centrifuging was repeated until the absorbance at 260 nm in the supernatant became 0. This precipitate was dissolved in 3 liters of 2M saline, and insolubles were removed by centrifugation. Then, 100 ml of DEAE-Cellulofine A-800 (manufactured by Seikagaku Corporation) equilibrated with 2M saline was added. After stirring, the added resin was removed by filtration. DEAE-Cellulofine A-800 in which the filtrate was equilibrated with 2M saline
It is applied to a column, and non-adsorbed components are eliminated.Ultrafiltration is performed using an ultrafiltration apparatus equipped with a hollow fiber having a molecular weight of 100,000 or less, and after completely removing coloring substances and salt, insoluble substances are removed by centrifugation and filtration. Lyophilized. The freeze-dried sulfated-fucose-sulfuric acid-containing polysaccharide mixture weighed 52 g.

Example 3 Extraction of Sulfuric Acid-Containing Fucose-Containing Polysaccharide Mixture of Japanese Kombu After sufficiently drying Japanese kombu, 2 kg of the mixture was pulverized with a free pulverizer (manufactured by Nara Machinery Co., Ltd.), and the obtained dry powder was mixed with 9 L of 80% ethanol. And treated at 80 ° C. for 2 hours.
The residue was obtained by filtration through the paper after the treatment. The above operation of washing with ethanol and filtration was repeated three times with respect to this residue to obtain an ethanol washed residue. This residue was suspended in 36 liters of a 0.2 M calcium acetate solution, treated at 95 ° C. for 2 hours, and filtered. The residue was washed with 4 liters of a 0.2 M calcium acetate solution to obtain 36 liters of an extract of a sulfated-fucose-containing polysaccharide mixture of sea kelp.

Example 4 Preparation of a Polysaccharide Mixture Containing Sulfuric Acid Fucose from Mackerel To the filtrate obtained in Example 3, 5% cetylpyridinium chloride was added until no more precipitate was formed, and the precipitate was collected by centrifugation. . 3 liters of this 0.3 liter
After suspending in M saline, the suspension was centrifuged and washed. After repeating this washing operation three times, 1 liter of 4M saline was added to the precipitate, and after stirring, ethanol was added to a concentration of 80%. After stirring, a precipitate was obtained by centrifugation. The operation of suspending the precipitate in 80% ethanol and centrifuging was repeated until the absorbance at 260 nm in the supernatant became 0. This precipitate was dissolved in 3 liters of 2M saline, and insolubles were removed by centrifugation. Then, 100 ml of DEAE-Cellulofine A-800 equilibrated with 2M saline was used.
(Manufactured by Seikagaku Corporation) was added, and after stirring, the added resin was removed by filtration. The filtrate is applied to a DEAE-Cellulofine A-800 column equilibrated with 2 M saline to remove non-adsorbed components and ultrafiltrate with an ultrafiltration apparatus equipped with a holofiber having a molecular weight of 100,000 or less, to obtain a colored substance and salt. After complete removal, insoluble substances were removed by centrifugation and filtration, followed by freeze-drying. The freeze-dried sulfated-fucose-sulfuric acid-containing polysaccharide mixture weighed 52 g. The sulfated-fucose-containing polysaccharide mixture did not contain a coloring substance adsorbed on the polysaccharide resin.

Example 5 Four 1 g of the freeze-dried product of the fucose-sulfuric acid-containing polysaccharide mixture described in Example 4 was weighed, and 4 g of water, 0.2 M sodium chloride, 0.2 M calcium chloride and 0.2 M chloride were weighed. Dissolved in magnesium. Next, 500 ml of DEA
Four columns of E-Sepharose FF were prepared, two of which were equilibrated with 0.2M sodium chloride, one with 0.2M calcium chloride, and one with 0.2M magnesium chloride. One of the columns equilibrated with 0.2 M sodium chloride was washed with 10 column volumes of water. The sulfated-fucose-containing polysaccharide mixture dissolved in water, sodium chloride, calcium chloride, and magnesium chloride, respectively, is applied to a DEAE-Sepharose FF column equilibrated with water, sodium chloride, calcium chloride, and magnesium chloride, respectively, and used for equilibration. Washed thoroughly with the solution and then eluted with a gradient of 0-4M sodium chloride. As a result, the total amount of the sulfated-fucose-containing polysaccharide mixture applied to the column only in the system using calcium chloride and magnesium chloride was adsorbed on the column. Only 0.4 g of the sulfated-fucose-containing polysaccharide was adsorbed on the column equilibrated with water and salt. In each column, the sulfated-fucose-containing polysaccharide-F of the present invention and the sulfated-fucose-containing polysaccharide-U of the present invention were substantially separated.

Example 6 After thoroughly drying Gagome kelp, 2 kg of the dried powder was pulverized with a free pulverizer (manufactured by Nara Kikai Seisakusho), and the obtained dried powder was suspended in 9 liters of 80% ethanol and treated at 80 ° C. for 2 hours. . The residue was obtained by filtration through the paper after the treatment. The above operation of washing with ethanol and filtration was repeated three times with respect to this residue to obtain an ethanol washed residue. This residue was suspended in 36 liters of a 0.2 M calcium acetate solution, treated at 95 ° C. for 2 hours, and filtered. The residue was washed with 4 liters of a 0.2 M calcium acetate solution to obtain 36 liters of a sulfated-fucose-containing polysaccharide mixture of Gagome kelp. The filtrate was concentrated to 2 liters by an ultrafilter equipped with an ultrafiltration membrane having an excluded molecular weight of 100,000. Then, sodium chloride was added to a final concentration of 1.5 M, and 5% cetylpyridinium chloride was added. Was added until no more precipitate formed. The resulting precipitate was removed by centrifugation. The obtained supernatant was concentrated to 1 liter by ultrafiltration, 4 liters of ethanol was added, and the resulting precipitate was collected by centrifugation. 1
After adding 00 ml of 4M saline and stirring well, ethanol was added to 80%, and after stirring, a precipitate was obtained by centrifugation. The operation of suspending the precipitate in 80% ethanol and centrifuging was repeated until the absorbance at 260 nm in the supernatant became 0. This precipitate was dissolved in 2 liters of 2M saline, and insolubles were removed by centrifugation. Then, 50 ml of DEAE-Cellulofine A-800 (manufactured by Seikagaku Corporation) equilibrated with 2M saline was added, and
After stirring, the added resin was removed by filtration. DEAE-Cellulofine A in which the filtrate was equilibrated with 2M saline
Exclude the non-adsorbed portion by applying -800 column and ultrafiltrate with an ultrafiltration device equipped with a hollow fiber having a molecular weight of 100,000 or less,
After completely removing the coloring substance and the salt, the insoluble substance was removed by centrifugation and filtration, followed by freeze-drying. The weight of the freeze-dried sulfated-fucose-containing polysaccharide-U was 15 g.
The sulfated-fucose-containing polysaccharide-U of the present invention did not contain a coloring substance adsorbed on the polysaccharide resin. The sulfated-fucose-containing polysaccharide-U produced oligosaccharides represented by the above formulas (I), (II) and (III) when the endo-fucoidan-degrading enzyme was allowed to act thereon.

Example 7 After thoroughly drying Gagome kelp, 2 kg of the dried powder was pulverized by a free pulverizer (manufactured by Nara Kikai Seisakusho), and the obtained dried powder was suspended in 9 liters of 80% ethanol and treated at 80 ° C. for 2 hours. . The residue was obtained by filtration through the paper after the treatment. The above operation of washing with ethanol and filtration was repeated three times with respect to this residue to obtain an ethanol washed residue. This residue was suspended in 36 liters of a 0.2 M calcium acetate solution, treated at 95 ° C. for 2 hours, and filtered. The residue was washed with 4 liters of a 0.2 M calcium acetate solution to obtain 36 liters of a sulfated-fucose-containing polysaccharide mixture of Gagome kelp. 5% cetylpyridinium chloride was added to the obtained filtrate until no more precipitate was formed, and the precipitate was collected by centrifugation. The precipitate was suspended in 3 liters of 0.4 M saline, centrifuged, and washed. After repeating this washing operation three times, 1 liter of 4M saline was added to the precipitate, and after stirring, ethanol was added to 80%, and after stirring, a precipitate was obtained by centrifugation. The operation of suspending the precipitate in 80% ethanol and centrifuging the suspension was repeated until the absorbance at 260 nm in the supernatant became zero. This precipitate was dissolved in 3 liters of 2M saline, and the insolubles were removed by centrifugation, and then 100 ml of DEAE-equilibrated with 2M saline.
Cellulofine A-800 (manufactured by Seikagaku Corporation) was added, and after stirring, the added resin was removed by filtration. The filtrate is applied to a DEAE-Cellulofine A-800 column equilibrated with 2 M saline to remove non-adsorbed components and ultrafiltrate with an ultrafiltration apparatus equipped with a holofiber having a molecular weight of 100,000 or less, to obtain a colored substance and salt. After complete removal, insoluble substances were removed by centrifugation and filtration, followed by freeze-drying. The weight of the freeze-dried sulfated-fucose-sulfuric acid-containing polysaccharide mixture was 90 g. The sulfated-fucose-containing polysaccharide mixture did not contain a coloring substance adsorbed on the polysaccharide resin. 7 g of the freeze-dried product of the sulfated-fucose-containing polysaccharide mixture is weighed,
Dissolved in 0.2 M calcium chloride. Next, 4000
ml of DEAE-Sepharose FF 0.2 ml
Of calcium chloride. The fucose sulfate-containing polysaccharide mixture dissolved in 0.2 M calcium chloride was subjected to DEAE.
-Applied to a column of Sepharose FF, washed thoroughly with 0.2M calcium chloride and then eluted with a gradient of 0-4M sodium chloride. A fraction having a sodium chloride concentration of 0.05 to 0.8 M in the eluted fraction was collected, desalted by dialysis, freeze-dried, and substantially sulfated-fucose-containing polysaccharide-
2.1 g of sulfated-fucose-containing polysaccharide-U separated from F was obtained. Further, the concentration of sodium chloride in the eluted fraction was 0.1.
Fractions of 9-1.5M were collected, desalted by dialysis and freeze-dried to obtain 4.7 g of sulfated-fucose-containing polysaccharide-F substantially separated from sulfated-fucose-containing polysaccharide-U.

Example 8 Production of sulfated-fucose-containing polysaccharide-F The sulfated-fucose-containing polysaccharide mixture obtained in Example 7 was prepared as follows.
2 g was weighed, dissolved in a 1.5 M sodium chloride solution to a final concentration of 0.2%, and a 1.25% cetylpyridinium chloride 1.5 M sodium chloride solution was added until no further precipitation occurred. . The resulting precipitate was collected by centrifugation, and the precipitate was suspended in 500 ml of 1.5 M saline, centrifuged, and washed. After repeating this washing operation three times, 1 liter of 4M saline was added to the precipitate, and after stirring, ethanol was added to a concentration of 80%. After stirring, a precipitate was obtained by centrifugation. 80% of this precipitate
The operation of suspending in ethanol and centrifuging was repeated until the absorbance at 260 nm in the supernatant became zero. This precipitate was dissolved in 500 ml of 2M saline, and the insoluble material was removed by centrifugation, and then 1 ml equilibrated with 2M saline.
Of DEAE-Cellulofine A-800 (manufactured by Seikagaku Corporation) was added, and after stirring, the added resin was removed by filtration. The filtrate is applied to a DEAE-Cellulofine A-800 column equilibrated with 2 M saline to remove non-adsorbed components and ultrafiltrate with an ultrafiltration apparatus equipped with a holofiber having a molecular weight of 100,000 or less, to obtain a colored substance and salt. After complete removal, insoluble substances were removed by centrifugation and filtration, followed by freeze-drying. The weight of the freeze-dried sulfated-fucose-containing polysaccharide-F is 710.
mg. The sulfated-fucose-containing polysaccharide-F did not contain a coloring substance adsorbed on the polysaccharide resin. This sulfated-fucose-containing polysaccharide-F was the sulfated-fucose-containing polysaccharide-F of the present invention containing no uronic acid and containing fucose as a main constituent.

Example 9 Enzymatic purification method of sulfated-fucose-containing polysaccharide-F The sulfated-fucose-containing polysaccharide mixture obtained in Example 7 was mixed with 10
g and weighed and dissolved in 500 ml of artificial seawater. SA-0082 (FERM B
P-5402) derived from endo-fucoidan-degrading enzyme
U was added and reacted at 25 ° C. for 50 hours. The reaction solution was subjected to ultrafiltration using an ultrafiltration apparatus equipped with a hollow fiber having an exclusion molecular weight of 100,000 or less to completely remove low-molecular substances, followed by centrifugation and filtration to remove insoluble substances and freeze-drying. The weight of the freeze-dried sulfated-fucose-containing polysaccharide-F was 6 g. The sulfated-fucose-containing polysaccharide-F did not contain a coloring substance adsorbed on the polysaccharide resin. Further, this sulfated-fucose-containing polysaccharide-F does not contain uronic acid, and contains the sulfated-fucose-containing polysaccharide of the present invention containing fucose as a main component.
It turned out to be F.

Example 10 Method for Purifying Sulfuric Acid-Containing Fucose-Containing Polysaccharide-F by Culture The polysaccharide mixture containing sulfuric acid for fucose obtained in Example 7 was mixed with 60
weighed and dissolved in 20 liters of artificial seawater, and then peptone 2
After adding 00 g and yeast extract 4 g, the mixture was sterilized in a 30-liter jar fermenter, and then the above-mentioned Flavobacterium sp. SA-0082 strain (FERM BP-540)
2) was inoculated and cultured at 25 ° C. for 24 hours. After removing the cells by centrifuging the culture solution, ultrafiltration is performed using an ultrafiltration device equipped with a hollow fiber having an exclusion molecular weight of 100,000 or less. After completely removing low-molecular substances, insoluble substances are removed by centrifugation and filtration. Was removed and lyophilized. The weight of the freeze-dried sulfated-fucose-containing polysaccharide-F was 36 g. The sulfated-fucose-containing polysaccharide-F did not contain a coloring substance adsorbed on the polysaccharide resin. The sulfated-fucose-containing polysaccharide-F was found to be the sulfated-fucose-containing polysaccharide-F of the present invention containing no uronic acid and containing fucose as a main constituent saccharide.

Example 11 Four 1 g of the freeze-dried product of the sulfated-fucose-containing polysaccharide mixture described in Example 7 was weighed, and 4 g of water, 0.2 M sodium chloride, 0.2 M calcium chloride, and 0.2 M chloride were obtained. Dissolved in magnesium. Next, 500 ml of DEA
Four columns of E-Sepharose FF were prepared, two of which were equilibrated with 0.2M sodium chloride, one with 0.2M calcium chloride, and one with 0.2M magnesium chloride. One of the columns equilibrated with 0.2 M sodium chloride was washed with 10 column volumes of water. The sulfated-fucose-containing polysaccharide mixture dissolved in water, sodium chloride, calcium chloride, and magnesium chloride, respectively, is applied to a DEAE-Sepharose FF column equilibrated with water, sodium chloride, calcium chloride, and magnesium chloride, respectively, and used for equilibration. The solution was washed thoroughly, then eluted with a gradient of 0-4M sodium chloride. As a result, the total amount of the sulfated-fucose-containing polysaccharide mixture applied to the column only in the system using calcium chloride and magnesium chloride was adsorbed on the column. Only 0.4 g of the sulfated-fucose-containing polysaccharide was adsorbed on the column equilibrated with water and salt. In each column, the sulfated-fucose-containing polysaccharide-F and the sulfated-fucose-containing polysaccharide-U were substantially separated.

Example 12 7 g of the sulfated-fucose-containing polysaccharide mixture described in Example 1 was weighed and dissolved in 800 ml of 0.2 M calcium chloride. Next, a column of 4 liters of DEAE-Sepharose FF was equilibrated with 0.2 M of calcium chloride, the above-mentioned sulfated-fucose-containing polysaccharide solution was applied to the entire column, washed with 8 liters of 0.2 M of calcium chloride solution, and then washed with 0 to 4 M
With a gradient of sodium chloride. Fraction in which uronic acid is detected in the eluted fraction (sodium chloride concentration: about 0.9 M or less: sulfated-fucose-containing polysaccharide-U), and fraction in which uronic acid is not detected (sodium chloride concentration: about 1.2 M)
Around: Each of sulfated-fucose-containing polysaccharide-F) was desalted and freeze-dried to obtain 1.4 g and 4.8 g of dried products, respectively.

Example 13 The mixture of sulfated-fucose-containing polysaccharide obtained in Example 1 was mixed with Flavobacterium sp. SA-0082 (FERM
When an endo-fucoidan-degrading enzyme produced by BP-5402) is allowed to act, an oligosaccharide having the following structure is produced.

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The present inventors performed the following enzymatic reaction to obtain the above oligosaccharide.

That is, 80 ml of the 2.5% fucose sulfate-containing polysaccharide mixture solution of Example 1, 60 ml of 50 mM phosphate buffer (pH 7.5), 20 ml of 4 M sodium chloride and 32 mU / ml of end-type fucoidan degradation 40 ml of the enzyme solution was mixed and reacted at 25 ° C. for 48 hours.

The reaction solution was treated with Cellulofine GCL-300.
(Seikagaku Corporation) was used to fractionate the molecular weight, and fractions having a molecular weight of 2000 or less were collected. These fractions were desalted with a micro-acylizer G3, then three fractions were separated with DEAE-Sepharose FF, desalted again, and lyophilized. Thus, 250 mg, 310 mg and 52 mg of the oligosaccharides of the above formulas (I), (II) and (III) were obtained, respectively.

Example 14 10 g of the sulfated-fucose-containing polysaccharide mixture obtained in Example 1
Was dissolved in 500 ml of 0.2 M citric acid and the pH was adjusted to 2.
After adjusting to 9, the mixture was treated at 100 ° C. for 3 hours. 150 ml of a 1 M calcium acetate solution was added to the hydrolyzate, and the resulting precipitate was removed by centrifugation.
Molecular weight fractionation by gel filtration through -25 (molecular weight 5000)
Above, 5000-3000, 3000-2000,
2,000-1000, 1000-500, 500 or less), GFd-Oli-
1, GFd-Oli-2, GFd-Oli-3, GFd
-Oli-4, GFd-Oli-5, and GFd-Ol
i-6. These 6 fractions were desalted and freeze-dried, and the dried products were 2.3 g, 1.7 g, 0.88 g, and 1.
8 g, 1.4 g, and 0.72 g were obtained.

Example 15 The sulfated-fucose-containing polysaccharide mixture obtained in Example 1 was mixed with 60
weighed and dissolved in 20 liters of artificial seawater, and then peptone 2
After adding 00 g and yeast extract 4 g, the mixture was sterilized in a 30-liter jar fermenter.
p. SA-0082 (FERM BP-5402) was inoculated and cultured at 25 ° C. for 24 hours. After the culture solution is centrifuged to remove the cells, it is subjected to ultrafiltration using an ultrafiltration device equipped with a hollow fiber having an exclusion molecular weight of 100,000 or less to completely remove low molecular substances, and then insoluble by centrifugation and filtration. Material was removed and lyophilized. The weight of the freeze-dried sulfated-fucose-containing polysaccharide-F was 36 g.

Example 16 5 kg of sea cucumber was disassembled, the internal organs were removed, and the body wall was collected. 500 ml of acetone was added per 200 g of body wall wet weight, treated with a homogenizer and filtered, and the residue was washed with acetone until there was no more colored substance. The residue was suction-dried to obtain 140 g of a dried product. Add 0.
After adding 2.8 liters of 4M saline and treating at 100 ° C. for 1 hour, the mixture was filtered and the residue was sufficiently washed with 0.4M saline to obtain 3.7 liters of an extract. To this extract, 5% cetylpyridinium chloride was added until no more precipitate was formed, and the formed precipitate was collected by centrifugation. This precipitate was suspended in 0.4 M saline and then centrifuged again. One liter of 4 M saline was added to the obtained precipitate, and after treatment with a homogenizer, 4 liters of ethanol was added with stirring. After stirring for a time, the mixture was filtered to obtain a precipitate.
The process of suspending the precipitate in 80% ethanol and then filtering was repeated until the absorbance of the supernatant at 260 nm became zero. The obtained precipitate was suspended in 2 liters of 2M saline, and insolubles were removed by centrifugation. The supernatant was subjected to ultrafiltration using an ultrafiltration apparatus equipped with a membrane having an excluded molecular weight of 30,000, completely desalted, and lyophilized to obtain 3.7 g of a sulfated-fucose-containing polysaccharide.

Example 17 After sufficiently drying Hibarata (Fucus vesiculosus), 2 kg of the dried product was pulverized with a free pulverizer (manufactured by Nara Machinery Co., Ltd.), and the obtained dry powder was suspended in 9 liters of 80% ethanol. C. for 2 hours. Filtration was performed on the treated paper to obtain a residue. The above operation of washing with ethanol and filtration was repeated three times with respect to this residue to obtain an ethanol washed residue. After suspending this residue in 40 liters of water,
Treated at 0 ° C. for 2 hours and filtered. Sodium chloride was added to the filtrate to 0.5 M, and 5% cetylpyridinium chloride was further added until no further precipitation occurred.
The precipitate was collected by centrifugation. The precipitate was suspended in 3 liters of 0.4 M sodium chloride, centrifuged, and washed. After repeating this washing operation three times, 250 g of sodium chloride was added to the precipitate, suspended in 3 liters of ethanol, and centrifuged to obtain a precipitate. The operation of suspending the precipitate in 80% ethanol and centrifuging was repeated until the absorbance at 260 nm in the supernatant became 0. This precipitate was dissolved in 3 liters of 2M sodium chloride, insoluble materials were removed by centrifugation, and then 100 ml of DEAE-Cellulofine A-800 equilibrated with 2M saline.
(Manufactured by Seikagaku Corporation) was added, and after stirring, the added resin was removed by filtration. The filtrate was applied to a DEAE-Cellulofine A-800 column equilibrated with 2 M sodium chloride to remove non-adsorbed components, and ultrafiltered with an ultrafiltration apparatus equipped with a holofiber having a molecular weight of 100,000 or less to remove the coloring substance and sodium chloride. After complete removal, insoluble substances were removed by centrifugation and filtration, and lyophilized. The weight of the freeze-dried sulfated-fucose-containing polysaccharide was 92 g.

Example 18 After thoroughly drying the seaweed (Undaria pinnatifida), 2 kg was crushed with a free crusher (manufactured by Nara Machinery Co., Ltd.), and the obtained dried powder was suspended in 9 liters of ethanol.
C. for 1 hour. Filtration was performed on the treated paper to obtain a residue. 9 L of 80% ethanol was added to the residue, stirred, treated at 80 ° C. for 1 hour, and filtered through a filter paper to obtain a residue. 80% of the above residue
The operation of washing with ethanol and filtration was repeated three times to obtain 1908 g of ethanol washing residue. 68 of this residue
After suspending 4 g in 9 liters of 0.2 M calcium acetate,
The mixture was treated at 95 ° C. for 1 hour and left standing for 24 hours to obtain a supernatant. 9 L of 0.2 M calcium acetate was added to the precipitate from which the supernatant had been removed, and the mixture was stirred. After standing for 1 hour, a supernatant was obtained and combined with the above supernatant. The supernatant thus obtained was filtered through a filter paper, and then ultrafiltered with an ultrafiltration device equipped with a hollow fiber having an excluded molecular weight of 10,000, and concentrated to 350 ml. The concentrate was centrifuged to remove the precipitate, and then subjected to ultrafiltration while adding 2 mM sodium chloride to completely remove calcium acetate and freeze-dried to obtain 3.2 g of a freeze-dried product. The weight of the sulfated-fucose-containing polysaccharide in the freeze-dried product is 3.
1 g.

Example 19 (1) Preparation of Polysaccharide Mixture Containing Sulfuric Acid Containing Gagome Kombu Fucose 2 Kg of dried Gagome kelp was pulverized with a free pulverizer M-2 type (manufactured by Nara Machinery Co., Ltd.), and 4.5 times as much as in 80% ethanol. At 80 ° C. for 2 hours, followed by filtration. For the residue
The above steps of 80% ethanol extraction and filtration were further repeated three times to obtain 1870 g of ethanol washing residue. 36 liters of water was added to the residue and treated at 100 ° C. for 2 hours.
An extract was obtained by filtration. 400 mM salt concentration of the extract
And then added 5% cetylpyridyl chloride until no more precipitate formed and centrifuged. The precipitate was repeatedly washed with 80% ethanol to completely remove cetylpyridinium chloride, dissolved in 3 liters of 2M sodium chloride, insoluble materials were removed by centrifugation, and equilibrated with 2M sodium chloride. 100 ml of DEAE-Cellulofine A
-800 was suspended, stirred and filtered to remove the resin.
The filtrate was equilibrated with 2M sodium chloride 100
ml of DEAE-Cellulofine A-800 column.
Desalting and low molecular weight removal were carried out according to (10), and the precipitate formed at this time was removed by centrifugation. The supernatant was freeze-dried to obtain a purified Gagome kelp fucose sulfate-containing polysaccharide mixture (82.2 g).
I got

(2) Preparation of sulfated-fucose-containing polysaccharide-F 6 g of the sulfated-fucose-containing polysaccharide mixture derived from Gagome kelp described above
20 m containing 600 ml of 0.2 M calcium chloride
M sodium acetate (pH 6.0), 3600 ml of DE previously equilibrated with 20 mM sodium acetate (pH 6.0) containing 0.2 M calcium chloride.
AE-Sepharose FF column, 20 mM sodium acetate containing 0.2 M calcium chloride (pH
After sufficiently washing the column with 6.0), elution was carried out with a gradient of 0 to 2 M sodium chloride. Fucose sulfate-containing polysaccharide eluted at a sodium chloride concentration of 0.75M or more
The F fraction was collected, concentrated and desalted with an ultrafilter equipped with an ultrafiltration membrane having an excluded molecular weight of 100,000, and then freeze-dried to obtain 3.3 g of a freeze-dried sulfated-fucose-containing polysaccharide-F sample.

(3) Preparation of Endofucose Sulfate-Containing Polysaccharide Degrading Enzyme Alteromonas sp. SN-1009 (FERM
BP-5747) containing 0.25% of glucose, 1.0% of peptone, and 0.05% of yeast extract in artificial seawater (manufactured by Jamarin Laboratories), pH 8.2;
0 ml was dispensed and inoculated into a sterilized (120 ° C., 20 minutes) 2-liter Erlenmeyer flask, and cultured at 25 ° C. for 25 hours to obtain a seed culture solution. 200 g of peptone, yeast extract 4
g, and 18 liters of a medium consisting of artificial seawater pH 8.0 containing 4 ml of an antifoaming agent (KM70 manufactured by Shin-Etsu Chemical Co., Ltd.)
120 ° C in a 0 liter jar fermenter
For 20 minutes. After cooling, another 20 g of Example 8 dissolved in 2 liters of artificial seawater sterilized at 120 ° C. for 15 minutes.
Inoculated with 600 ml of the sulfated-fucose-containing polysaccharide-F derived from Gagome kelp prepared using the method of
20 liters at 24 ° C., 10 liters per minute ventilation and 2 minutes per minute
The culture was performed under the condition of stirring speed of 50 rotations. After completion of the culture, the culture was centrifuged to obtain bacterial cells and culture supernatant. The activity of the sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention in the culture supernatant was measured using the sulfated-fucose-containing polysaccharide-F as a substrate, and was found to be 5 mU / ml · culture solution. The obtained culture supernatant was concentrated with an ultrafilter having a molecular weight cutoff of 10,000, the resulting precipitate was removed by centrifugation, and 85% saturated ammonium sulfate was precipitated, and the resulting precipitate was collected by centrifugation. 20 mM Tris containing artificial seawater (Jamarin S) at 1 / concentration
After sufficient dialysis against hydrochloric acid buffer (pH 8.2),
ml of crude enzyme were obtained.

The obtained crude enzyme solution was previously washed with a 20 mM Tris-HCl buffer solution (pH
DEAE-Cellulofine A-8 equilibrated in 8.2)
After that, the adsorbed substance was sufficiently washed with the same buffer, and then 100 mM, 2 mM,
Elution was performed with a solution containing 00 mM, 300 mM, 400 mM, and 600 mM sodium chloride, and the active fraction was collected. The activity of the obtained partially purified enzyme was measured using fucose sulfate-containing polysaccharide-F as a substrate.
(10.2 U). No other fucose-sulfuric acid-containing polysaccharide-degrading enzyme was mixed. A part of the obtained partially purified enzyme was previously equilibrated with artificial seawater (Jamarin S) at a concentration of 1/10 and 10 mM Tris-hydrochloric acid buffer (pH 8.0) containing 5 mM sodium azide. Gel filtration was performed at -200, and the molecular weight was determined to be about 100,000.

(4) Using the partially purified enzyme and PA-FF obtained in the above examples as enzyme sources and substrates, respectively, the concentration of calcium exerted on the activity of the enzyme was examined. As a buffer used for the enzyme reaction, a pH 7 buffer containing 50 mM acetic acid, imidazole, and Tris-HCl was used. Further, sodium chloride having a final concentration of 400 mM was dissolved in the reaction solution. When the concentration of calcium chloride in the reaction solution was changed from 0 to 100 mM and the enzyme activity was measured, the results shown in FIG. 23 were obtained. In FIG. 23, the vertical axis represents the relative activity (%), and the horizontal axis represents the calcium concentration (m
M). As a result, it was found that the activity of this enzyme was significantly enhanced in the presence of a calcium salt.

(5) The endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention was dialyzed against the following six kinds of buffers at 5 ° C.
, And the remaining activity was measured. 1.20 mM Tris-HCl buffer (pH 8.2) 20 mM Tris-HCl buffer (pH 8.2) containing 2.5 mM sodium azide 20 mM Tris-HCl buffer containing 3.5 mM sodium azide and 50 mM sodium chloride (PH 8.2) 20 mM Tris-HCl buffer containing 4.5 mM sodium azide and 500 mM sodium chloride (pH 8.2) 20 mM Tris-HCl buffer containing 5.5 mM sodium azide, 50 mM sodium chloride, and 10 mM calcium chloride Liquid (pH 8.2) 20 mM Tris-HCl buffer (pH 8.2) containing 6.5 mM sodium azide and 1/10 concentration of artificial seawater (Jamalin S) The sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention dialyzed against the liquid is inactivated, and 4, 5, and 6 Fucose-containing polysaccharide degrading enzyme of the present invention was dialyzed liquor activity was retained. From this, this enzyme is 5
It was found to be stabilized in the presence of 00 mM sodium chloride or 10 mM calcium ions.

(6) 5 g of the sulfated-fucose-containing polysaccharide-F prepared in the above example was weighed, and 471 ml of a 50 mM imidazole buffer (pH 8), 12.5 ml of 4 M sodium chloride, 6.25 ml of 4 M calcium chloride, and 10 ml (equivalent to 6 mU) of a partially purified product of the sulfated-fucose-containing polysaccharide-degrading enzyme of the present invention obtained in Example 19- (3) was mixed and reacted at 25 ° C. for 120 hours. A molecular product was obtained. The results of IR and NMR analyzes of the obtained low molecular weight compound were as shown in FIGS. 25 and 26, respectively. Further, when gel filtration was performed using Cellulofine GCL-300, the results shown in FIG. 24 were obtained. That is, the substance has a molecular weight of 1000-3.
0000. The sulfuric acid content of this substance was 46% as SO ₄ (molecular weight 96), and the neutral sugar composition was fucose: galactose = 100: 4.
Met. The vertical and horizontal axes in FIGS. 24 to 26 are the same as those in FIGS. 2 to 4.

Example 20 Human promyelocytic leukemia cell HL-60 (cultured at 37 ° C. in RPMI 1640 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes) ATCC CRL-1964) to ASF1
The cells were suspended in a medium 04 (manufactured by Ajinomoto Co., Inc.) at a concentration of 5 × 10 ⁵ cells / 9 ml. Prepare four 9 ml of this suspension,
To each suspension, a physiological saline solution of the sulfated-fucose-containing polysaccharide obtained in Examples 1, 12, and 15 (5 m
g / ml) of the filtered solution [pore size 0.20μ
m filtered through a cellulose acetate membrane (manufactured by Corning Incorporated) (the filter treatment was performed under these conditions)]
Is added at 37 ° C. in the presence of 5% carbon dioxide.
Cultured for hours. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were washed with 50 mM Tris-HCl buffer (pH: 10 mM) containing 10 mM ethylenediaminetetraacetate and 0.5% sodium lauroyl sarcosinate.
7.8) Suspend in 20 μl, add 1 μl of 10 mg / ml ribonuclease A (manufactured by Sigma), add 50 μl,
After treating for 30 minutes, 1 μl of 10 mg / ml proteinase K was added and treated at 50 ° C. for 1 hour. Using the cells after the treatment as a sample, electrophoresis was performed under a constant voltage of 100 V using a 2% agarose gel. After this gel was immersed in an ethidium bromide solution for 30 minutes, the state of DNA in the gel was confirmed using a transilluminator, and a DNA ladder unique to apoptosis was confirmed. For further confirmation, a 10 μg / ml solution of actinomycin D known as a reagent for inducing apoptosis was used in place of the above-mentioned sulfated-fucose-containing polysaccharide, and the same operation was carried out. The same DNA ladder as in the case was confirmed. These results revealed that HL-60 cells were induced to undergo apoptosis by the sulfated-fucose-containing polysaccharides obtained in Examples 1, 12, and 15. HL-60 (ATCC C
CL-240) to each of the sulfated-fucose-containing polysaccharide solutions obtained in Examples 1, 12, and 15 [30 mM Hepes buffer (pH 7.2) containing 120 mM sodium chloride to a concentration of 5 mg / ml]. Dissolve, 121 ° C, 20
), And the same results were obtained.

Example 21 Human promyelocytic leukemia cells HL-60 (cultured at 37 ° C. in RPMI 1640 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes) ATCC CCL-240) to ASF10
The cells were suspended in 4 culture media (manufactured by Ajinomoto Co.) at a concentration of 5 × 10 ⁵ cells / 9 ml. Example 1 was added to 9 ml of the suspension.
1 ml of a filtered solution of a physiological saline solution (5 mg / ml) of the sulfated-fucose-containing polysaccharide obtained in 5 was added,
The cells were cultured at 37 ° C. in the presence of 5% carbon dioxide for 20 hours. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were stained with Giemsa according to the “Apoptosis Experiment Protocol” (supervised by Shujunsha and Yasunori Tanuma, pp. 93-95, 1995). That is, the obtained cells were fixed on a slide glass using a Carnoy's fixative (acetic acid: methanol = 1: 3), stained with Giemsa dye (manufactured by Merck), and observed with an optical microscope. Nuclear fragmentation was observed. These results revealed that the HL-60 cells undergo apoptosis by the sulfated-fucose-containing polysaccharide obtained in Example 15. Example 1
5 sulfated-fucose-containing polysaccharide solution [5 mg / ml
30m containing 120mM sodium chloride so that
M Hepes buffer (pH 7.2),
Apoptosis-inducing effect of autoclave treatment for 20 minutes] was measured in the same manner as above, and similar results were obtained.

Example 22 Human promyelocytic leukemia cell HL-60 (cultured at 37 ° C. in RPMI 1640 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes) ATCC CCL-240) to ASF10
5 × 10 ⁵ cells / 4.5 ml in 4 media (manufactured by Ajinomoto Co.)
And suspended. Four 4.5 ml of this suspension were prepared, and each of the suspensions containing the sulfated-fucose-containing polysaccharide solution obtained in Examples 1, 15, and 18 [10 mg
/ Ml in 30 mM Hepes buffer (pH 7.2) containing 120 mM sodium chloride.
Autoclaved at 1 ° C for 20 minutes] and 12
0.5 ml of 30 mM Hepes buffer containing 0 mM sodium chloride was added, the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide, and the number of cells was counted by trypan blue staining 24 hours and 40 hours after the start of the culture. .

FIG. 27 shows the result. FIG. 27 shows HL-
FIG. 7 is a graph showing the relationship between the culture time and the number of viable cells in a culture solution when the sulfated-fucose-containing polysaccharide obtained in Examples 1, 15, and 18 was added to a culture solution of 60 cells so as to be 1 mg / ml. The horizontal axis indicates the culture time (hours), and the vertical axis indicates the number of living cells in the culture solution (× 10 ⁵ cells / 5 ml). FIG.
Medium, the type of sulfated-fucose-containing polysaccharide added to the medium is □
The mark is no additive (control), the + mark is Example 1, and the mark is Example 1.
5. The x marks indicate that the sulfated-fucose-containing polysaccharide obtained in Example 18 was used.

At this time, the dead cells exhibited a morphology unique to apoptosis such as cell shrinkage and fragmentation. That is, from these results, it was found that HL-60 cells were induced to undergo apoptosis by the sulfated-fucose-containing polysaccharide obtained in Examples 1, 15, and 18, and cell proliferation was significantly suppressed. Each of the sulfated-fucose-containing polysaccharide solutions obtained in Examples 1, 15 and 18 [120 m / ml
30 mM Hepes buffer (p.
H7.2), which had been filtered and treated], and the apoptosis-inducing effect was measured according to the above, and similar results were obtained.

Example 23 Human acute lymphoblastic leukemia cells were placed in 9 ml of RPMI1640 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes.
LT-3 (ATCC CRL-1552) was added to 5 × 10 ⁵
The cells were suspended so as to be 9 pieces / 9 ml. 9 ml of this suspension
Are prepared, and Examples 1, 2, 12, and 1 are
1 ml of a filtered solution of a physiological saline solution (5 mg / ml) of the sulfated-fucose-containing polysaccharide obtained in 6 was added,
The cells were cultured at 37 ° C in the presence of 5% carbon dioxide for 60 hours. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were mixed with 20 μl of 50 mM Tris-HCl buffer (pH 7.8) containing 10 mM of ethylenediaminetetraacetate and 0.5% of sodium lauroyl sarcosinate.
And 1 μl of 10 mg / ml ribonuclease A (manufactured by Sigma) was added and treated at 50 ° C. for 30 minutes. Then, 1 μl of 10 mg / ml proteinase K was added and treated at 50 ° C. for 1 hour. Using the cells after the treatment as a sample, electrophoresis was performed under a constant voltage of 100 V using a 2% agarose gel. After this gel was immersed in an ethidium bromide solution for 30 minutes, the state of DNA in the gel was confirmed using a transilluminator, and a DNA ladder unique to apoptosis was confirmed. For further confirmation, a 10 μg / ml solution of actinomycin D known as a reagent for inducing apoptosis was used in place of the above sulfated-fucose-containing polysaccharide, and the same operation was carried out. Same DN
A ladder was confirmed. From these results, Examples 1, 2, 1
The sulfated-fucose-containing polysaccharide obtained in Steps 2 and 16 is MOL
It was found to induce apoptosis on T-3 cells. Each of the sulfated-fucose-containing polysaccharide solutions obtained in Examples 1, 2, 12 and 16 [PBS was adjusted to 5 mg / ml.
(8 g sodium chloride, 0.2 g potassium chloride,
2.9 g of dihydrogen phosphate sodium dodecahydrate, and
2 g of potassium dihydrogen phosphate dissolved in 1 liter of water and autoclaved at 121 ° C. for 20 minutes] was measured in the same manner as described above, and similar results were obtained.

Example 24 Human acute lymphoblastic leukemia cells MOLT- cultured at 37 ° C. in an RPMI1640 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes. 3 (ATCC CRL-1552)
Was suspended in RPMI1640 medium to a concentration of 5 × 10 ³ cells / ml, and 1.8 ml was dispensed into each well of a FALCON 24-well plate. The fucose sulfate-containing polysaccharide mixture obtained in Example 1 and the sulfated-fucose-containing polysaccharide-F obtained in Example 12 were dissolved in PBS at a concentration of 0.5 mg / ml and sterilized by filtration. Then, 0.2 ml of each of the solutions of the sulfated-fucose-containing polysaccharide obtained in Examples 15 and 17 and dextran sulfate (molecular weight: 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) were added, and 37
The cells were cultured at 5 ° C. in the presence of 5% carbon dioxide. As a control, the same amount of PBS alone was added, and the cells were cultured in the same manner. The number of viable cells 2 days, 4 days, 6 days, and 8 days after the start of culture was counted by trypan blue staining. The result is shown in FIG. 28 shows that the sulfated-fucose-containing polysaccharide obtained in Example 1, the sulfated-fucose-containing polysaccharide-F obtained in Example 12, and the sulfated-fucose-containing sulfate obtained in Examples 15 and 17 were added to the culture solution of MOLT-3 cells. Polysaccharides and dextran sulfate are added to 0.
It is a figure showing the relationship between the culture time when added so that it may become 5 mg / ml, and the number of viable cells in a culture solution. The horizontal axis is culture time (day), and the vertical axis is the number of viable cells in a culture solution (× 10 ⁴ U /
2 ml). In FIG. 28, the types of sulfated polysaccharides added to the medium are as follows: o is no addition (control), ● is the sulfated-fucose-containing polysaccharide-F obtained in Example 12, and □ is the one obtained in Example 1.た represents the sulfated-fucose-containing polysaccharide obtained in Example 17, and ■ represents the sulfated-fucose-containing polysaccharide obtained in Example 15. Dextran sulfate showed substantially the same curve as that of the sulfated-fucose-containing polysaccharide obtained in Example 15. At this time, the dead cells exhibited a morphology unique to apoptosis such as cell shrinkage and fragmentation. That is, from these results, the MOLT-3 cells were obtained from the sulfated-fucose-containing polysaccharide obtained in Example 1, the sulfated-fucose-containing polysaccharide-F obtained in Example 12, and Example 15
In addition, it was found that the sulfated-fucose-containing polysaccharide obtained in Steps 1 and 2 and dextran sulfate induced apoptosis and significantly suppressed cell proliferation. The sulfated-fucose-containing polysaccharide obtained in Example 1, the sulfated-fucose-containing polysaccharide-F obtained in Example 12, the sulfated-fucose-containing polysaccharide obtained in Examples 15 and 17, and dextran sulfate (molecular weight: 50)
The apoptosis-inducing effect of each solution (manufactured by Wako Pure Chemical Industries, Ltd., dissolved in PBS to 0.5 mg / ml and autoclaved at 121 ° C. for 20 minutes) was measured according to the above, and the same results were obtained. I got

Example 25 Human promyelocytic leukemia cells HL-60 (cultured at 37 ° C. in RPMI1640 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes) ATCC CCL-240) to ASF10
5 × 10 ⁴ cells / 900 μl in 4 media (manufactured by Ajinomoto Co.)
And suspended. Nine 900 μl of this suspension were prepared, and for each suspension, physiological saline and the sulfated-fucose-containing polysaccharide preparation obtained in Example 1, F-Fd-1 to F-Fd-1 were used.
F-Fd-4 and the respective aqueous solutions of the three kinds of sulfated-fucose-containing oligosaccharides obtained in Example 13 in physiological saline (10 mg / m
l) Add 100 μl of the filter treatment solution,
The cells were cultured in the presence of% carbon dioxide for 40 hours. The cultured cells were observed under a microscope to examine the degree of proliferation and cell morphology. As a result, a sulfated-fucose-containing polysaccharide preparation, F-Fd-
All HL-60 cells supplemented with 1-F-Fd-4 and the three sulfated-fucose-containing oligosaccharides exhibited apoptotic characteristics such as cell shrinkage and cell fragmentation. The number of cells in HL-60 cells to which physiological saline was added increased about 4-fold, but the sulfated-fucose-containing polysaccharide preparations, F-Fd-1 to F-F-
HL-60 cells to which Fd-4 and three kinds of sulfated-fucose-sulfated oligosaccharides were added showed no increase in cell number.
The fucose sulfate-containing polysaccharide preparations, F-Fd-1 to F-Fd-4 and 3
It was found that the proliferation of HL-60 cells was suppressed by the apoptosis-inducing effect of the sulfated-fucose-containing oligosaccharides. For further confirmation, a 10 μg / ml solution of actinomycin D known as a reagent for inducing apoptosis was used in place of the sulfated-fucose-containing oligosaccharide, and the same operation was performed. As with the sugars, there was cell shrinkage and cell fragmentation. From these results, the HL-60 cells were obtained from the sulfated-fucose-containing polysaccharide preparation obtained in Example 1, F-Fd-1.
Apoptosis was found to be induced by -F-Fd- and the sulfated-fucose-containing oligosaccharide obtained in Example 13. Each solution of the sulfated-fucose-containing polysaccharide preparation obtained in Example 1, F-Fd-1 to F-Fd-4 and the three kinds of sulfated-fucose-containing oligosaccharide obtained in Example 13 [10 m
g / ml in 30 mM Hepes buffer (pH 7) containing 120 mM sodium chloride.
At 20.degree. C. for 20 minutes in an autoclave] was measured in the same manner as described above, and similar results were obtained.

Example 26 Lung cancer cells A-549 (ATCC CCL-185), S
V40 transformed lung cells WI-38VA13 (ATC
C CCL-75.1) and hepatoma cells HepG2 (A
TCC HB-8065) 10 ⁴ pieces / 1.
RPMI containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes to 8 ml
Suspended in 1640 medium. 1.8 ml of this suspension was dispensed, and the physiological saline, the sulfated-fucose-containing polysaccharide obtained in Examples 1 and 15 and the sulfated fucose obtained in Examples 1 and 15 were added to each suspension. 200 μl of a filter treatment solution of each of the 6 kinds of sulfated-fucose-containing oligosaccharides obtained in physiological saline (1 mg / ml) was added, and the mixture was cultured at 37 ° C. in the presence of 5% carbon dioxide for 6 days. The cultured cells were observed under a microscope to examine the degree of proliferation and cell morphology. As a result, the sulfated-fucose-containing polysaccharide obtained in Examples 1 and 15,
And lung cancer cells A-549 and WI-3 transformed with SV40 to which three fractions having a molecular weight of 2,000 or more among the six kinds of sulfated-fucose-containing oligosaccharides obtained in Example 14 were added.
8VA13 and hepatoma cells Hep G2 all exhibited apoptotic features such as cell shrinkage and cell fragmentation. In addition, although the number of cells in each of the cancer cells to which physiological saline was added significantly increased, the sulfated-fucose-containing polysaccharides obtained in Examples 1 and 15 and the six sulfated-fucose-containing oligosaccharides obtained in Example 14 were obtained. Among them, it was found that the number of various cancer cells to which three fractions having a molecular weight of 2,000 or more were added decreased, and the proliferation of various cancer cells was suppressed by the apoptosis-inducing action of the sulfated-fucose-containing polysaccharide and oligosaccharide. Apoptosis of autoclaved products of the fucose sulfate-containing polysaccharides obtained in Examples 1 and 15 and the six kinds of fucose sulfate-containing oligosaccharides obtained in Example 14 in PBS solutions (1 mg / ml) at 121 ° C. for 20 minutes. The provocation effect was measured according to the above, and similar results were obtained.

Example 27 Colon cancer cell HCT 116 (ATCC CCL-24)
7), and gastric cancer cells AGS (ATCC CRL-173)
9) such that the respectively become 10 ⁴ /1.8ml, 5
The suspension was suspended in McCoy's 5a medium (manufactured by Gibco) and Ham's F12 medium (manufactured by Gibco) containing 10% of fetal bovine serum (JRH Biosciences) treated at 6 ° C. for 30 minutes. This suspension was dispensed in 1.8 ml portions,
For each suspension, a physiological saline solution was used for each cancer cell, and the sulfated-fucose-containing polysaccharide obtained in Examples 1, 12, and 15 and four types of F-Fd-1 to F-Fd-4. Each physiological saline solution of sulfated fucose-containing oligosaccharide (10 mg / m
l) 200 μl of the filter treatment solution of
The cells were cultured in the presence of 5% carbon dioxide for 48 hours. The cultured cells were observed under a microscope to examine the degree of proliferation and cell morphology. As a result, the sulfated-fucose-containing polysaccharides obtained in Examples 1, 12, and 15 and 4 of F-Fd-1 to F-Fd-4 were obtained.
Colon cancer cells H supplemented with various fucose sulfate-containing oligosaccharides
CT 116 and gastric cancer cells AGS all exhibited apoptotic features such as cell shrinkage and cell fragmentation. In addition, although the number of cells of various cancer cells to which physiological saline was added significantly increased, the sulfated-fucose-containing polysaccharides obtained in Examples 1, 12, and 15 and 4 of F-Fd-1 to F-Fd-4 were obtained. It has been found that the number of various cancer cells to which various kinds of sulfated-fucose-containing oligosaccharides are added decreases, and the proliferation of various cancer cells is suppressed by the apoptosis-inducing action of these sulfated-fucose-containing polysaccharides and oligosaccharides. The sulfated-fucose-containing polysaccharides obtained in Examples 1, 12 and 15 and F-Fd-1 to FF
The apoptosis-inducing effect of the autoclaved solution of each of the four fucose sulfate-containing oligosaccharide solutions of d-4 in PBS (10 mg / ml) at 121 ° C. for 20 minutes was measured according to the above, and similar results were obtained.

Example 28 Human colon cancer cells HCT cultured at 37 ° C. in McCoy's 5a medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes
116 was suspended in McCoy's 5a medium at 5 × 10 ³ cells / ml, and 1.8 ml was dispensed into each well of a FALCON 24-well plate. For each suspension, 10 mg /
ml of the sulfated-fucose-containing polysaccharide sample obtained in Example 1,
Sulfated-fucose-containing polysaccharide mixture, sulfated-fucose-containing polysaccharide-F obtained in Example 12, sulfated-fucose-containing polysaccharide-U,
F-Fd-1, F-Fd-2, F-Fd-3, and F-
Fd-4, each solution of the sulfated-fucose-containing polysaccharide obtained in Example 15, and 5 mg / ml heparin (manufactured by Wako Pure Chemical Industries) and dextran sulfate (molecular weight: 5) dissolved in PBS.
Then, 0.2 ml of an autoclave treated at 121 ° C. for 20 minutes was added thereto and cultured at 37 ° C. in the presence of 5% carbon dioxide. As a control, the same amount of PBS alone was added, and the cells were cultured in the same manner. 1 day after culture, 2 days
The number of viable cells after 3 days, 3 days, and 4 days was determined by “Tissue culture technology” (second edition) (Asakura Publishing, edited by The Japanese Society for Tissue Culture, 1990)
Year)) according to the method described (pages 26 to 28). That is, measurement was performed by a method using trypan blue staining on a hemocytometer.

FIG. 29 shows the obtained result. 29 shows that the sulfated-fucose-containing polysaccharide sample obtained in Example 1, the sulfated-fucose-containing polysaccharide mixture obtained in Example 1, and the sulfated-fucose-containing polysaccharide obtained in Example 12 were added to the culture solution of HCT116 cells. U and sulfated-fucose-containing polysaccharide-F, F
-Fd-1, F-Fd-2, F-Fd-3, and FF
d-4. The culture time when the fucose sulfate-containing polysaccharide obtained in Example 15 was added to 1 mg / ml and heparin and dextran sulfate to 0.5 mg / ml, and the number of viable cells in the culture solution. It is a figure showing a relationship, a horizontal axis | shaft is culture time (day) and a vertical axis | shaft is the number of viable cells in a culture solution (* 10 < ⁴ >).
/ 2 ml). In FIG. 29, the types of sulfated-fucose-containing polysaccharides or oligosaccharides added to the medium are indicated by o (no control), solid circles indicate the sulfated-fucose-containing polysaccharide samples obtained in Example 1, and triangles indicate examples. 1 is a sulfated-fucose-containing polysaccharide mixture obtained in 1. The sulfated-fucose-containing polysaccharide-U and the sulfated-fucose-containing polysaccharide-F, FF obtained in Example 12.
d-1, F-Fd-2, F-Fd-3, and F-Fd-
4 and the sulfated-fucose-containing polysaccharide obtained in Example 15 showed substantially the same curve as that of the sulfated-fucose-containing polysaccharide mixture obtained in Example 1. Heparin and dextran sulfate showed substantially the same curves as those of the sulfated-fucose-containing polysaccharide preparation obtained in Example 1.

As a result, HCT 116 containing PBS was added.
Although the number of cells significantly increased, the sulfated-fucose-containing polysaccharide preparation obtained in Example 1, the sulfated-fucose-containing polysaccharide mixture, the sulfated-fucose-containing polysaccharide-F obtained in Example 12,
Sulfated-fucose-containing polysaccharide-U, F-Fd-1, F-Fd-
2, the number of cells of HCT116 cells supplemented with F-Fd-3 and F-Fd-4, the sulfated-fucose-containing polysaccharide obtained in Example 15, heparin, and dextran sulfate hardly increased or decreased. .

Further, the sulfated-fucose-containing polysaccharide preparation obtained in Example 1, the sulfated-fucose-containing polysaccharide mixture, Example 12
Sulfated-fucose-containing polysaccharide-F, sulfated-fucose-containing polysaccharide-U, F-Fd-1, F-Fd-2, F-Fd-
3, and HCT 116 cells supplemented with F-Fd-4, the sulfated-fucose-containing polysaccharide obtained in Example 15, heparin, and dextran sulfate all exhibited apoptotic characteristics such as cell shrinkage and cell fragmentation. That is, HCT is induced by the apoptosis-inducing action of these sulfated-fucose-containing polysaccharides and oligosaccharides, heparin, and dextran sulfate.
It was found that proliferation of 116 cells was suppressed. PB
10 mg / ml of the sulfated-fucose-containing polysaccharide sample obtained in Example 1, a mixture of the sulfated-fucose-containing polysaccharide obtained in Example 1, the sulfated-fucose-containing polysaccharide-F obtained in Example 12,
Sulfated-fucose-containing polysaccharide-U, F-Fd-1, F-Fd-
2, F-Fd-3 and F-Fd-4, a filter treatment solution of each solution of the sulfated-fucose-containing polysaccharide obtained in Example 15, and 5 mg / ml heparin dissolved in PBS,
The apoptosis-inducing effect of each filter treatment solution of dextran sulfate and dextran sulfate was measured according to the above, and similar results were obtained.

Example 29 Human colon cancer cells HCT cultured at 37 ° C. in McCoy's 5a medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes
116 was suspended in McCoy's 5a medium at 5 × 10 ³ cells / ml, and 1.8 ml was dispensed into each well of a FALCON 24-well plate. For each suspension, 20 mg /
Example 1 at ml, 30 mg / ml and 50 mg / ml
The solution of the sulfated-fucose-containing polysaccharide preparation obtained in
0.2 ml of autoclaved 20 ° C for 20 minutes
The cells were added and cultured at 37 ° C. in the presence of 5% carbon dioxide. As a control, the same amount of PBS alone was added, and the cells were cultured in the same manner. `` Tissue culture technology ''
(2nd edition) (Asakura Publishing, edited by The Japan Society for Tissue Culture, 1990)
Year) according to the method described on pages 26-28.
That is, measurement was performed by a method using trypan blue staining on a hemocytometer.

FIG. 30 shows the obtained result. That is, FIG. 30 is a diagram showing the relationship between the culture time and the number of viable cells in the culture solution when the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was added at various concentrations to the culture solution of HCT 116 cells. The horizontal axis shows the culture time (hours), and the vertical axis shows the number of living cells in the culture solution (× 10 ⁴ cells / 2 ml). In FIG. 30, the addition amount of the sulfated-fucose-containing polysaccharide preparation to the medium was as follows: ○ indicates no addition (control), ● indicates 2 mg / ml, ■ indicates 3 mg / ml,
The black triangle indicates 5 mg / ml.

As a result, HCT 116 containing PBS was added.
Although the number of cells significantly increased, the number of HCT116 cells to which the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was added decreased. All HCT 116 cells to which the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was added exhibited characteristics of apoptosis such as cell shrinkage and cell fragmentation. That is, the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was used at a concentration of at least 2 mg / ml in HCT.
It has been found that it has an apoptosis-inducing effect on 116 cells and can suppress cell proliferation. 20 mg / ml, 30 mg / ml, and 50 mg / ml dissolved in PBS
The apoptosis-inducing effect of the filtered solution of the solution of the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 in ml was measured in the same manner as described above, and similar results were obtained.

Example 30 Human gastric cancer cells AGS cultured at 37 ° C. in Ham's F12 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes at 37 ° C. were used. The suspension was suspended in sF12 medium to a concentration of 5 × 10 ³ cells / ml, and dispensed into each well of a FALCON 24-well plate in an amount of 1.8 ml. For each suspension, 20 mg / ml, 30
A solution of the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 in an amount of 0.2 mg / ml and 50 mg / ml was autoclaved at 121 ° C. for 20 minutes, and 0.2 ml of the solution was added.
The cells were cultured at 5 ° C. in the presence of 5% carbon dioxide. As a control, the same amount of PBS alone was added and cultured in the same manner. After the start of the culture, the number of viable cells was counted with time according to the method described in “Techniques of Tissue Culture” (second edition) (Asakura Publishing, edited by The Japan Society for Tissue Culture, 1990) (pages 26 to 28). That is, measurement was performed by a method using trypan blue staining on a hemocytometer.

FIG. 31 shows the obtained result. That is, FIG. 31 is a diagram showing the relationship between the culture time when the fucose sulfate-containing polysaccharide preparation obtained in Example 1 was added at various concentrations to the culture solution of AGS cells and the number of viable cells in the culture solution. The horizontal axis represents the culture time (hours), and the vertical axis represents the number of living cells in the culture solution (× 1).
0 shows the ⁴ co / 2 ml). In FIG. 31, the amount of addition of the sulfated-fucose-containing polysaccharide preparation to the medium is as follows: o, no addition (control), ●, 2 mg / ml, Δ, 3 mg / ml, black triangle, 5 mg / ml. .

As a result, the number of AGS cells to which PBS was added significantly increased, whereas the number of AGS cells to which the fucose sulfate-containing polysaccharide preparation obtained in Example 1 was added at a final concentration of 3 mg / ml or more was decreased. In addition, when 2 mg / ml was added, cell proliferation was remarkably suppressed. Example 1
To which the sulfated-fucose-containing polysaccharide preparation obtained in the above was added
All cells exhibited apoptotic characteristics such as cell shrinkage and cell fragmentation. That is, it was found that the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 had an apoptosis-inducing effect on AGS cells at a concentration of at least 2 mg / ml, and was able to suppress cell proliferation. 20 mg / ml, 30 mg / ml, and 50 mg / m in PBS
The apoptosis-inducing effect of the filtered solution of the solution of the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was measured in accordance with the above, and similar results were obtained.

Example 31 L-15 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes
Colon cancer cells SW480 (AT
CC CCL-228) in L-15 medium at 5 × 10
Suspend to ³ cells / ml and use FALCON 2
1.8 ml was dispensed into each well on a 4-well plate. For each suspension, 10
mg / ml, 30 mg / ml, and 50 mg / ml of the sulfated-fucose-containing polysaccharide preparations obtained in Example 1
After autoclaving at 21 ° C for 20 minutes, 0.2
Then, the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide. As a control, the same amount of PBS alone was added, and the cells were cultured in the same manner. After the start of culture, the number of viable cells was measured over time using “Tissue culture technology” (second edition) (Asakura Publishing, edited by the Japanese Society for Tissue Culture, 1
990) (pages 26 to 28). That is, measurement was performed by a method using trypan blue staining on a hemocytometer.

FIG. 32 shows the obtained result. That is, FIG. 32 is a diagram showing the relationship between the culture time and the number of living cells in the culture solution when the sulfated-fucose-containing polysaccharide preparations obtained in Example 1 were added at various concentrations to the culture solution of SW480 cells.
The horizontal axis shows the culture time (hours), and the vertical axis shows the number of living cells in the culture solution (× 10 ⁴ cells / 2 ml). In FIG. 32, the addition amount of the sulfated-fucose-containing polysaccharide preparation to the medium was as follows: ○ indicates no addition (control), ● indicates 1 mg / ml, ■ indicates 3 mg / ml,
The black triangle indicates 5 mg / ml.

As a result, the number of cells was significantly increased in the SW480 cells to which PBS was added. In addition, when 1 mg / ml was added, the proliferation of cells was remarkably suppressed. Also,
All SW480 cells to which the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was added exhibited characteristics of apoptosis such as cell shrinkage and cell fragmentation. That is, the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 had at least 1 mg /
It has been found that SW480 cells have an apoptosis-inducing effect at a concentration of ml and can suppress cell proliferation.
10 mg / ml, 30 mg / ml dissolved in PBS,
The apoptosis-inducing effect of the filtered solution of the solution of the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 and 50 mg / ml was measured in accordance with the above, and similar results were obtained.

Example 32 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes and NEAA (Dainippon Pharmaceutical)
In a DMEM medium (manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 1%
Colon cancer cells WiDr (ATCC CCL)
-218) was suspended in the above medium at a concentration of 5 × 10 ³ cells / ml, and 1.8 ml was dispensed into each well on a FALCON 24-well plate. For each suspension, 10 mg / ml of the sulfated-fucose-containing polysaccharide mixture obtained in Example 1 dissolved in PBS, Example 12
The solution of each of the sulfated-fucose-containing polysaccharide-F, F-Fd-3 and F-Fd-4 obtained in the above, and the sulfated-fucose-containing polysaccharide obtained in Example 15 were subjected to autoclaving at 121 ° C. for 20 minutes. .2 ml, and cultured at 37 ° C. in the presence of 5% carbon dioxide. As a control, the same amount of PBS alone was added, and the cells were cultured in the same manner. After the start of the culture, the number of viable cells was measured with time according to the method described in “Tissue Culture Techniques” (2nd edition) (Asakura Publishing, edited by the Japanese Society for Tissue Culture, 1990) (No. 26-
28). That is, measurement was performed by a method using trypan blue staining on a hemocytometer.

FIG. 33 shows the obtained result. That is, FIG. 33 shows a mixture of the sulfated-fucose-containing polysaccharide obtained in Example 1, the sulfated-fucose-containing polysaccharide-F obtained in Example 12, F-Fd-3 and F-Fd-4 in the culture solution of WiDr cells. 1 mg of the sulfated-fucose-containing polysaccharide obtained in Example 15
/ Ml is a graph showing the relationship between the culture time and the number of viable cells in the culture solution when added to the culture solution, and the horizontal axis represents the culture time (hours), and the vertical axis represents the number of viable cells in the culture solution (× 10 ⁴ U / 2m
1) is shown. In FIG. 33, the type of the sulfated-fucose-containing polysaccharide added to the medium is shown by the symbol ○ without addition (control), the symbol ■ with the sulfated-fucose-containing polysaccharide-F obtained in Example 12, and the symbol ● with the one obtained in Example 15. Is a sulfated-fucose-containing polysaccharide. FF
d-3 and F-Fd-4 showed substantially the same curves as those of the sulfated-fucose-containing polysaccharide obtained in Example 15.

As a result, the number of WiDr cells to which PBS was added significantly increased, but the sulfated-fucose-containing polysaccharide mixture obtained in Example 1 and the sulfated-fucose-containing polysaccharide-F and F-Fd obtained in Example 12 were obtained. The number of WiDr cells to which -3 and F-Fd-4 and the sulfated-fucose-containing polysaccharide obtained in Example 15 were added decreased. Further, the sulfated-fucose-containing polysaccharide mixture obtained in Example 1, the sulfated-fucose-containing polysaccharide-F obtained in Example 12, F-Fd-3, and F
All the WiDr cells to which Fd-4 and the sulfated-fucose-containing polysaccharide obtained in Example 15 were added exhibited characteristics of apoptosis such as cell shrinkage and cell fragmentation. That is,
The sulfated-fucose-containing polysaccharide mixture obtained in Example 1, the sulfated-fucose-containing polysaccharide-F and F-Fd obtained in Example 12
-3 and F-Fd-4, and the sulfated-fucose-containing polysaccharide obtained in Example 15 were found to have an apoptosis-inducing effect on WiDr cells and suppress cell proliferation. 10 mg / ml of the sulfated-fucose-containing polysaccharide mixture obtained in Example 1 dissolved in PBS, the sulfated-fucose-containing polysaccharide-F obtained in Example 12, F-Fd-3 and F-
The apoptosis-inducing effect of the filtered solution of the solution of Fd-4 and the sulfated-fucose-containing polysaccharide obtained in Example 15 was measured according to the above, and similar results were obtained.

Example 33 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes and NEAA (Dainippon Pharmaceutical)
In a DMEM medium (manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 1%
Colon cancer cells WiDr (ATCC CCL)
-218) was suspended in the above medium at a concentration of 5 × 10 ³ cells / ml, and 1.8 ml was dispensed into each well on a FALCON 24-well plate. For each suspension, 10 mg / ml, 30 m
g / ml and 50 mg / ml of the solution of the sulfated-fucose-containing polysaccharide sample obtained in Example 1 were autoclaved at 121 ° C. for 20 minutes, and 0.2 ml of the solution was added.
The cells were cultured at 5 ° C. in the presence of 5% carbon dioxide. As a control, the same amount of PBS alone was added, and the cells were cultured in the same manner. "Tissue culture technology" (second edition)
(Asakura Press, edited by the Japanese Society for Tissue Culture, 1990) (pages 26 to 28). That is,
It was measured by a method using trypan blue staining on a hemocytometer.

FIG. 34 shows the obtained result. That is, FIG. 34 is a diagram showing the relationship between the culture time and the number of viable cells in the culture solution when the fucose sulfate-containing polysaccharide preparation obtained in Example 1 was added at various concentrations to the culture solution of WiDr cells, The horizontal axis is the culture time (hours), and the vertical axis is the number of viable cells in the culture solution (×
10 shows a ⁴ co / 2 ml). In FIG. 34, the addition amount of the sulfated-fucose-containing polysaccharide preparation to the medium is as follows: 印 indicates no addition (control), ● indicates 1 mg / ml, ■ indicates 3 mg / ml, and black triangle indicates 5 mg / ml. .

As a result, the number of WiDr cells to which PBS was added increased remarkably, whereas the number of WiDr cells to which the fucose sulfate-containing polysaccharide preparation obtained in Example 1 was added at a final concentration of 3 mg / ml or more was decreased. However, the addition of 1 mg / ml also markedly suppressed cell growth. Further, W containing the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was added.
All iDr cells exhibited apoptotic characteristics such as cell shrinkage and cell fragmentation. That is, it was found that the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 had an apoptosis-inducing effect on WiDr cells at a concentration of at least 1 mg / ml, and was able to suppress cell proliferation. 10 mg / ml, 30 mg / ml, and 50 mg / ml in PBS
The apoptosis-inducing action of the filter-treated solution of the solution of the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 at mg / ml was measured according to the above, and similar results were obtained.

Example 34 Human promyelocytic leukemia cell HL-60 (cultured at 37 ° C. in RPMI 1640 medium (manufactured by Gibco) containing 10% fetal bovine serum (JRH Biosciences) treated at 56 ° C. for 30 minutes) ATCC CCL-240) to ASF10
5 × 10 ⁴ cells / 900 μl in 4 media (manufactured by Ajinomoto Co.)
And 4.5 ml was dispensed into each well on a FALCON 6-well plate. For each suspension, 10 m of a freeze-dried low-molecular-weight product of sulfated-fucose-containing polysaccharide-F described in Example 19- (6) was obtained.
g / ml in 30 mM Hepes buffer (pH 7) containing 120 mM sodium chloride, and 0.5 ml of the solution filtered and added at 37 ° C., 5%
Culture was performed in the presence of carbon dioxide. As a control, the same amount of the above buffer alone was added and cultured in the same manner. Start of culture 22
The number of viable cells after 48 hours and 46 hours was determined using a tissue culture technique (second
Edition) (Asakura Shuppan, edited by the Japanese Society for Tissue Culture) (pages 26 to 28). That is, measurement was performed by a method using trypan blue staining on a hemocytometer. As a result, the HL-60 cells were converted to the sulfated-fucose-containing polysaccharide-F described above.
It was found that apoptosis was induced by freeze-drying the low molecular weight compound of and the cell growth rate was suppressed.

Example 35 Fetal bovine serum (JRH Biosciences) obtained by treating human promyelocytic leukemia cells HL-60 at 56 ° C. for 30 minutes
5% in RPMI 1640 medium (manufactured by Gibco) containing 10%
× 10 ⁴ cells / 900 μl were suspended. Six suspensions were prepared, and 120
fucose sulfate-containing polysaccharide-F according to Example 19- (2), which was dissolved at 30 mg / ml in 30 mM Hepes buffer (pH 7) containing 10 mM sodium chloride,
F-Fd-1, F-Fd-2, F-Fd-3, and F-
100 μl of each Fd-4 filter treatment solution was added, and the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide for 46 hours. At 22 hours and 46 hours after the start of the culture, the number of viable cells in the culture solution was measured. In addition, human promyelocytic leukemia cells HL-
60, in ASF104 medium (Ajinomoto) 5 × 10 ⁴
Cells / 900 μl. Six suspensions were prepared, and each suspension was added to a 30 mM Hepes buffer (pH 7) containing 120 mM sodium chloride.
And sulfated-fucose-containing polysaccharide-F, F-Fd-1, F-Fd-2, and FF dissolved at 10 mg / ml in the same buffer.
100 μl of each of the filter treatment solutions d-3 and F-Fd-4 was added, and the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide for 40 hours. At 16 hours and 40 hours after the start of the culture, the number of viable cells in the culture solution was measured. The cells cultured in the above two types were observed under a microscope to examine the degree of proliferation and the morphology of the cells. As a result, in the cells cultured in the ASF104 medium, the sulfated-fucose-containing polysaccharide-F, FF
d-1, F-Fd-2, F-Fd-3, and F-Fd-
All the cells added with 4 exhibited characteristics of apoptosis such as cell shrinkage and cell fragmentation, and the number of viable cells hardly increased or almost completely died. The number of cells increased about three-fold in the medium to which only the buffer was added. On the other hand, in the cells cultured in the RPMI1640 medium, only the cells to which F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-4 were added all showed cell shrinkage and cell fragmentation. The cells were almost dead, exhibiting the characteristics of apoptosis. The number of cells increased about 3-fold in the medium to which only the buffer was added, and about 2.5 times in the medium to which the sulfated-fucose-containing polysaccharide-F was added. From the above results, although sulfated-fucose-containing polysaccharide-F has a strong apoptosis-inducing effect on cancer cells in a serum-free medium, F-Fd-1, F-Fd-2, F-Fd-3, and F-Fd-1 Fd-4 was found to have a very strong apoptosis-inducing effect in both serum-free medium and serum medium.

For further confirmation, RPMI containing 10% of fetal calf serum (manufactured by JRH Biosciences) obtained by treating human promyelocytic leukemia cells HL-60 at 56 ° C. for 30 minutes was used.
5 × 10 ⁴ cells / 900 in 1640 medium (manufactured by Gibco)
It was suspended so as to be μl. Two 9 ml suspensions were prepared, each containing 30 mM Hepes buffer (pH 7) containing 120 mM sodium chloride and 10 ml in the same buffer.
1 ml of the F-Fd-4 filter solution dissolved at mg / ml was added, and the mixture was cultured at 37 ° C. for 16 hours in the presence of 5% carbon dioxide. The cultured cells were separated from the supernatant by centrifugation. The obtained cells were suspended in 20 μl of 50 mM Tris-HCl buffer (pH 7.8) containing 10 mM ethylenediaminetetraacetate and 0.5% sodium lauroyl sarcosinate, and 10 mg
/ Ml ribonuclease A (manufactured by Sigma) was added at 1 μl, the mixture was treated at 50 ° C. for 30 minutes, and then 1 μl of 10 mg / ml proteinase K was added, and the mixture was treated at 50 ° C. for 30 minutes. Using the cells after the treatment as a sample, electrophoresis was performed under a constant voltage of 100 V using a 2% agarose gel. This gel was immersed in an ethidium bromide solution for 30 minutes, and then the DN in the gel was treated with a transilluminator.
When the state of A was confirmed, DN specific to apoptosis was observed.
A ladder was confirmed. For further confirmation, when actinomycin D known as a reagent for inducing apoptosis was used in place of F-Fd-4 in the same manner, the same DNA ladder as in the case of F-Fd-4 was confirmed. Was. That is, it was found that when fucose sulfate-containing polysaccharide-F is degraded by the endo-fucose sulfate-containing polysaccharide-F-degrading enzyme of the present invention, the effect of inducing apoptosis on cancer cells is enhanced.

Example 36 Vein blood of healthy humans of a 21-year-old woman and a 32-year-old man was collected.
100 mg of glucose per liter, CaCl ₂ ·
0.74 mg of 2H ₂ O, 19.92 m of MgCl ₂
g, 40.26 mg of KCl, 7371 m of NaCl
g, 2 times diluted with a solution containing 1756.5 mg of Tris-hydrochloric acid, and then gently overlaid the lymphocyte separation solution (Dainippon Pharmaceutical Co., Ltd.) in a centrifuge tube containing twice the volume of diluted blood in advance. The mixture was centrifuged at 18 to 20 ° C. and 400 g for 30 minutes. After centrifugation, the upper lymphocyte fraction of the lymphocyte separation solution was collected. The healthy lymphocytes thus obtained were 1.9 × 10
Add ⁵ plates to a 24-well plate, 1.8 ml
0.2 ml of 5 mg / m 2 in RPMI-1640 medium containing 10% fetal bovine serum (after treatment at 56 ° C. for 30 minutes).
l of a medium containing the sulfated-fucose-containing polysaccharide obtained in each of the above Examples and its decomposed product one by one, and then cultured at 37 ° C. As a control, physiological saline was added in place of the sulfated-fucose-containing polysaccharide solution. After the start of the culture, the morphological change of the cells in each well and the number of viable cells were measured with a microscope. As a result, there was no difference in the cell morphology between the wells to which various sulfated-fucose-containing polysaccharides and their degradation products were added and the control wells.
In addition, there was almost no difference in the number of living cells, and on the 13th day, both cells almost died. From these results, it was found that the sulfated-fucose-containing polysaccharide and its degradation product did not show toxicity to healthy cells even at a concentration that strongly induced apoptosis in cancer cells.

Example 37 Antitumor effect of sulfated-fucose-containing polysaccharide-U on solid tumors Mouse solid tumor MethA (4 × 10 ⁶ cells /
8 weeks old female BALB / c mouse (body weight about 2)
0 g) was injected subcutaneously in the abdomen. Thereafter, the sulfated-fucose-containing polysaccharide described in Example 6 was continuously placed in the same place for 10 days.
U (100 mg / kg / day) was injected subcutaneously. On the other hand, the control group was similarly injected subcutaneously with physiological saline. Two weeks later, the cancer tissue formed on the abdomen of the mouse was excised and its weight was measured. Table 2 shows the results. That is, the average cancer weight was 1.25 g in the control group, whereas the average cancer weight was 0.1 g in the fucose sulfate-containing polysaccharide-U administration group.
28 g, significant (p <0.0 with respect to the control group).
1) It exhibited an anticancer effect. The suppression rate was 77.6%.

[0310]

[Table 2]

Example 38 Carcinogenic Preventive Effect of Sulfate-Containing Fucose-Containing Polysaccharide (1) 7.4 mg / kg of azoxymethane (manufactured by Nacalai Tesque) was subcutaneously administered to 19 6-week-old Sprague-Dawley rats (male). Thereafter, once a week, subcutaneous administration to the back was performed until the 10th week. At the time of administration, azoxymethane was dissolved in a 0.1 M phosphate buffer (pH 6.5) containing 0.9% sodium chloride, and was dissolved in
The concentration of the solution was adjusted to be 0 μl. To 5 of the 19 mice, 70 ml of a hot water extract of Gagome kelp prepared according to the description in Example 1 was orally administered as drinking water every day simultaneously with the first administration of azoxymethane until the 30th week. This hot water extract contained 2 mg / ml of a sulfated-fucose-containing polysaccharide mixture, and 140 mg / kg of a sulfated-fucose-containing polysaccharide mixture was orally administered daily. The fucose sulfate-containing polysaccharide was not administered to 14 of the above 19 animals,
Tap water was provided as drinking water to serve as a control group. By 30 weeks, 14 out of 14 outbreaks of external ear nest cancer were observed in the control group, whereas 1 out of 5 rats in the fucose sulfate-containing polysaccharide mixture administration group had a marked carcinogenesis-suppressing effect. . By the 30th week, 3 animals died in the control group, but all the groups administered the sulfated-fucose-containing polysaccharide mixture survived. The average body weight of the control group at week 30 was 716 g, whereas the average body weight of the group administered the sulfated-fucose-containing polysaccharide mixture was 817 g. On the other hand, the average body weight of the azoxymethane non-administered rat group (5 rats) was 788 g, and the weight increase of the fucose sulfate-containing polysaccharide mixture administration group was equivalent to that of the azoxymethane non-administered rat group. Next, four control animals were selected, and from the 30th week onwards, 40 ml of the above-mentioned hot water extract of Gagome kelp (80 mg of a sulfated-fucose-containing polysaccharide mixture) was orally administered as drinking water. At the 36th week, the outer ear nest cancer of 2 out of 4 mice remarkably regressed, and the anticancer effect of the sulfated-fucose-containing polysaccharide was observed. By oral administration of the sulfated-fucose-containing polysaccharide,
The carcinogen-preventing action of the chemical carcinogen, the prevention of weight gain suppression by the chemical carcinogen, and the regression of cancer tissues were observed.

Example 39 Injection The sulfated-fucose-containing polysaccharide mixture produced in Example 1 was dissolved in distilled water for injection to prepare a 5% solution. This solution was filled into a vial for freeze-drying in one vial in an amount of 50 mg as a sulfated-fucose-containing polysaccharide, and freeze-dried. Separately, 2 ml of physiological saline was added as a solution.

Example 40 Injection An injection was prepared according to the following formulation. Fucose-sulfuric acid-containing polysaccharide-U [Example 12] 40 mg physiological saline 2 ml per 1 ampoule in the same manner An injection was prepared using the fucose-sulfuric acid-containing polysaccharide-F described in Example 12 in the same manner.

Example 41 Tablet A tablet was prepared according to the following formulation. Fucose sulfate-containing polysaccharide preparation (Example 1) 10 mg Corn starch 65 mg Carboxymethyl cellulose 20 mg Polyvinylpyrrolidone 3 mg Magnesium stearate 2 mg 100 mg per tablet

Example 42 Injection F-Fd-1 was dissolved in distilled water for injection to prepare a 5% solution.
This solution was filled into a vial for freeze-drying in one vial in an amount of 50 mg as a sulfated-fucose-containing polysaccharide, and freeze-dried. Separately, 2 ml of physiological saline was added as a solution.

Example 43 Injection An injection was prepared according to the following formulation. Lyophilized product of low molecular weight fucose sulfate-containing polysaccharide-F 2 obtained in Example 19- (6) 40 mg physiological saline 2 ml per 1 ampoule

Example 44 Tablet A tablet was prepared according to the following formulation. Lyophilized product of low molecular weight fucose sulfate-containing polysaccharide-F 2 obtained in Example 19- (6) 10 mg Corn starch 65 mg Carboxymethyl cellulose 20 mg Polyvinylpyrrolidone 3 mg Magnesium stearate 2 mg 100 mg per tablet

[0318]

Industrial Applicability The present invention has an apoptosis-inducing effect on unnecessary or pathogenic cells, and induces apoptosis in diseased cells in abnormally proliferating cell diseases such as cancer and viral diseases, thereby preventing or preventing such diseases. A therapeutically effective agent is provided. In particular, in the case of cancer of the digestive system such as colorectal cancer and stomach cancer, a cancer cell can be caused to undergo apoptosis by orally administering the agent of the present invention. The drug of the present invention containing the decomposed product as an active ingredient is an anticancer drug very suitable for gastrointestinal cancer. Moreover, the carcinogenesis by a chemical carcinogen etc. can also be prevented by its carcinogenic prevention effect. The drug of the present invention is excellent in that it can be supplied in large quantities at low cost using edible substances as raw materials, such as edible brown algae plants and edible sea cucumber, and is highly safe. In addition, health can be maintained and enhanced by daily ingesting a food or beverage containing the sulfated-fucose-containing polysaccharide and / or its decomposition product. In addition, the present invention provides a simple method for inducing apoptosis, and by using the method of the present invention, studies on elucidation of apoptosis mechanism, development of an apoptosis induction inhibitor, and the like can be performed.

Further, according to the present invention, the sulfated-fucose-containing polysaccharide of the present invention, which is substantially free of sulfated-fucose-containing polysaccharide-F and has a highly reactive coloring substance removed therefrom, is useful in the fields of sugar chain engineering, medicine and the like. U and its decomposition products were provided, as well as its efficient production method.

Further, according to the present invention, the sulfated-fucose-containing polysaccharide of the present invention which is substantially free of sulfated-fucose-containing polysaccharide-U and which is useful in the fields of sugar chain engineering, medicine, etc., from which highly reactive coloring substances have been removed. -F and its decomposition products were provided, as well as its efficient production method.

According to the present invention, sulfated-fucose-containing polysaccharide-F
Endofucose sulfate-containing polysaccharide-degrading enzyme which can be used for the production of a low molecular weight product of sulfated-fucose-containing polysaccharide-F useful for structural analysis and decomposition of sulfated-fucose-containing polysaccharide-F, and a method for producing the same, And sulfated-fucose-containing polysaccharide-F having a strong apoptosis-inducing effect on cancer cells
And a low molecular weight product of the enzyme.

The end-fucose sulfate-containing polysaccharide of the present invention, which has not been produced stably according to the present invention,
The F-decomposing enzyme can be produced extremely stably in the presence of calcium ions. Further, according to the present invention, the endo-fucose sulfate-containing polysaccharide-degrading enzyme of the present invention can work extremely efficiently in the presence of calcium ions.

[Brief description of the drawings]

FIG. 1 shows the precipitation rate of sulfated-fucose-containing polysaccharide.

FIG. 2 shows a molecular weight distribution of sulfated-fucose-containing polysaccharide-U measured by a gel filtration method using Sephacryl S-500.

FIG. 3 shows an IR spectrum of sulfated-fucose-containing polysaccharide-U.

Figure 4 is ¹ H-N of the sulfated-fucose-containing polysaccharide -U
It shows an MR spectrum.

FIG. 5 shows an elution pattern when the pyridyl- (2) -aminated saccharide compound (PA-a) of the saccharide compound (a) is separated by an L-column.

FIG. 6 shows an elution pattern when the pyridyl- (2) -aminated saccharide compound (PA-b) of the saccharide compound (b) is separated by an L-column.

FIG. 7 shows an elution pattern when the pyridyl- (2) -aminated saccharide compound (PA-c) of the saccharide compound (c) is separated by an L-column.

FIG. 8 shows the results obtained by mass analysis (negative measurement) of the sugar compound (a).

FIG. 9 shows the results obtained by mass analysis (negative measurement) of the sugar compound (b).

FIG. 10 shows the results obtained by mass analysis (negative measurement) of the sugar compound (c).

FIG. 11 shows the results obtained by mass-mass analysis (negative measurement) of the sugar compound (a).

FIG. 12 shows the results obtained by mass-mass analysis (negative measurement) of the sugar compound (b).

FIG. 13 shows the results obtained by mass-mass analysis (negative measurement) of the sugar compound (c).

FIG. 14 shows a ¹ H-NMR spectrum of the sugar compound (a).

FIG. 15 shows a ¹ H-NMR spectrum of the sugar compound (b).

FIG. 16 shows the ¹ H-NMR spectrum of sugar compound (c).

FIG. 17 shows a molecular weight distribution of sulfated-fucose-containing polysaccharide-F measured by a gel filtration method using Sephacryl S-500.

FIG. 18 shows an IR spectrum of sulfated-fucose-containing polysaccharide-F.

FIG. 19 shows ¹ H of sulfated-fucose-containing polysaccharide-F.
1 shows an NMR spectrum.

FIG. 20 is a graph showing the relationship between the pH and the relative activity of the endo-fucose sulfate-containing polysaccharide-degrading enzyme obtained by the present invention.

FIG. 21 is a graph showing the relationship between temperature and relative activity of the endo-fucose sulfate-containing polysaccharide-degrading enzyme obtained by the present invention.

FIG. 22 shows the molecular weight distributions before and after the decomposition of fucose sulfate-containing polysaccharide-F by the endo-fucose sulfate-containing polysaccharide-degrading enzyme obtained by the present invention, as measured by a gel filtration method using Cellulofine GCL-300. It is shown.

FIG. 23 is a graph showing the relationship between the relative activity and the calcium ion concentration in the reaction solution of the endo-fucose sulfate-containing polysaccharide-degrading enzyme obtained by the present invention.

FIG. 24 is a graph showing the sulfated-fucose-containing polysaccharide-F obtained by the present invention, which was measured by gel filtration using Cellulofine GCL-300 in Example 19- (6). 1 shows the molecular weight distribution of the product decomposed by the above method.

FIG. 25 shows an IR spectrum of a product obtained by decomposing sulfated-fucose-containing polysaccharide-F by the endo-form sulfated-fucose-containing polysaccharide-degrading enzyme obtained by the present invention.

FIG. 26 shows a ¹ H-NMR spectrum of a product obtained by decomposing sulfated-fucose-containing polysaccharide-F by the endo-form sulfated-fucose-containing polysaccharide-degrading enzyme obtained by the present invention.

FIG. 27 shows the culture time when the fucose sulfate-containing polysaccharide obtained in Examples 1, 15, and 18 was added to a culture solution of HL-60 cells at 1 mg / ml, It shows the relationship between the number of living cells.

FIG. 28 shows a sulfated-fucose-containing polysaccharide obtained in Example 1, a sulfated-fucose-containing polysaccharide-F obtained in Example 12, and a mixture obtained in Examples 15 and 17 in a culture solution of MOLT-3 cells. FIG. 2 shows the relationship between the culture time and the number of viable cells in a culture solution when a sulfated-fucose-containing polysaccharide and dextran sulfate are added.

FIG. 29 is a graph showing the sulfated-fucose-containing polysaccharide preparation obtained in Example 1, the sulfated-fucose-containing polysaccharide mixture obtained in Example 1, and the sulfated-fucose sulfate obtained in Example 12 in a culture solution of HCT 116 cells. -Containing polysaccharide-U and sulfated-fucose-containing polysaccharide-F, F-Fd-1, F-Fd-2, F-Fd-
3 shows the relationship between the culture time and the number of viable cells in the culture solution when F-Fd-4, the sulfated-fucose-containing polysaccharide obtained in Example 15, and heparin and dextran sulfate were added.

FIG. 30 shows the relationship between the culture time and the number of viable cells in the culture solution when the sulfated-fucose-containing polysaccharide preparations obtained in Example 1 were added at various concentrations to the culture solution of HCT 116 cells. It is something.

FIG. 31 shows the relationship between the culture time and the number of viable cells in the culture solution when the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was added at various concentrations to the culture solution of AGS cells. Things.

FIG. 32 shows the results obtained in Example 1 using the culture solution of SW480 cells.
1 shows the relationship between the culturing time and the number of viable cells in a culture solution when the sulfated-fucose-containing polysaccharide preparations obtained in Example 2 were added at various concentrations.

FIG. 33 shows a mixture of the sulfated-fucose-containing polysaccharide obtained in Example 1 and the sulfated-fucose-containing polysaccharide-F, F-Fd-3 and F obtained in Example 12 in a culture solution of WiDr cells.
10 shows the relationship between the culture time and the number of viable cells in the culture solution when adding the sulfated-fucose-containing polysaccharide obtained in Example 15 and Fd-4.

FIG. 34 shows the relationship between the culture time and the number of viable cells in the culture solution when the sulfated-fucose-containing polysaccharide preparation obtained in Example 1 was added at various concentrations to the culture solution of WiDr cells. Things.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 19/04 C12P 19/04 Z 19/14 19/14 Z (C12N 9/26 (C12N 9/26 C12R) 1:20) C12R 1:20) (C12N 9/26 (C12N 9/26 C12R 1:10) C12R 1:10) (31) Priority claim number Japanese Patent Application No. 8-171658 (32) Priority date 1996 June 12 (June 12, 1996) (33) Priority claiming country Japan (JP) (31) Priority claim number Japanese Patent Application No. 8-204187 (32) Priority date July 16, 1996 (1996. 7.16) (33) Countries claiming priority Japan (JP) (72) Inventor Hideo Kitano 82-4, Azaimachi, Oaza, Hirosaki City, Aomori Prefecture Inside Glycotechnology Research Institute, Inc. 82, Aibu-cho, Hirosaki-shi, Japan 4 Within Glycotechnology Research Laboratories Co., Ltd. 82-4, Azaicho, Hirosaki-shi, Aomori Prefecture Inside the Glycotechnology Research Institute, Inc. (72) Inventor Kaoru Kojima 82-4, Oazaimachi, Hirosaki-shi, Aomori Prefecture, Japan Glycotechnology Research Institute, Inc. Hitomi Kimura 82-4, Aibu-cho, Hirosaki-shi, Aomori Pref. Inside the Glycotechnology Institute, Inc. (72) Inventor Yoshikuni Nakanishi 82-4, Oaza-cho, Oaza, Hirosaki-shi, Aomori Pref. Inventor Kaoru Katayama 82-4, Aibu-cho, Hirosaki City, Aomori Prefecture Inside the Glycotechnology Research Institute, Inc. (72) Inventor Takao Tominaga 3-4-1 Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd. (72 ) Inventor Kazuo Shimanaka 3-4-1, Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd. (72) Inventor Katsushige Inoka 3-4-1 Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd. (72 Inventor Ikunoyuki Kato 82-4, Ozaimachi, Hirosaki City, Aomori Prefecture Inside the Glycotechnology Research Institute, Inc.

Claims (4)

[Claims] 1. A sulfated-fucose-containing polysaccharide having the following physicochemical properties. (1) Constituent sugar: contains uronic acid. (2) Flavobacterium sp.
The molecular weight is reduced by the fucoidan-degrading enzyme produced by SA-0082 (FERM BP-5402), and at least one compound selected from the compounds represented by the following formulas (I), (II) and (III) is produced. . Embedded image Embedded image Embedded image 2. The sulfated-fucose-containing polysaccharide according to claim 1, comprising a step of treating the sulfated-fucose-containing polysaccharide mixture with an agent capable of aggregating acidic polysaccharide in the presence of salts to remove a precipitate. Manufacturing method. 3. The fucose according to claim 1, further comprising a step of treating the sulfated-fucose-containing polysaccharide mixture with an anion exchange resin in the presence of a divalent cation to collect a target polysaccharide. A method for producing a sulfuric acid-containing polysaccharide. 4. The process for producing the sulfated-fucose-containing polysaccharide according to claim 1, comprising a step of removing a coexisting coloring substance using a polysaccharide substance or a substance having an anion exchange group. The method for producing a sulfated-fucose-containing polysaccharide according to claim 1.