JP2003339393A - Method for producing oligosaccharide (salt) - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an oligosaccharide (salt), capable of producing the oligosaccharide or its salt in a high yield, without causing discoloration of the oligosaccharide (salt), nor change in chemical structure thereof due to oxidation, by controlling reverse reaction of enzymolysis thereof. <P>SOLUTION: In this method for producing the oligosaccharide (salt), a first space and a second space are arranged in such a manner that the spaces are separated from each other by a partition having a permeable membrane through which oxygen is not permeated but the produced oligosaccharide (salt) is permeated, then a mucopolysaccharide or its salt is decomposed by an enzyme in the first space, and further the oligosaccharide (salt) which is formed by the decomposition and travels to the second space through the permeable membrane is recovered. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an oligosaccharide (salt) for producing an oligosaccharide or an oligosaccharide salt by decomposing at least one of mucopolysaccharide and a salt of the mucopolysaccharide. .

[0002]

2. Description of the Related Art An oligosaccharide is a substance produced by combining several (10 or more) monosaccharides according to the literature (Dictionary of Molecular and Cellular Biology 1997 Tokyo Kagaku Dojin). Further, oligosaccharides are highly useful substances that are widely used as raw materials for medicines, cosmetics, foods and the like.

Among the oligosaccharides, those having high utility include, for example, hyaluronic acid oligosaccharides (“hyaluronic acid oligosaccharides” are appropriately referred to as “oligohyaluronic acid”). Note that hyaluronic acid, which is one of the mucopolysaccharides, is shown in formula (1).

[0004]

[Chemical 1]

Further, it has been known that oligohyaluronic acid obtained by degrading the above hyaluronic acid, such as hyaluronic acid tetrasaccharide, is effective as an apoptosis inhibitor, and that its derivative exhibits anticoagulant activity. .
Therefore, oligohyaluronic acid such as hyaluronic acid tetrasaccharide has been attracting attention as a substance showing physiological activity.

A method applicable to the production of the above oligohyaluronic acid is, for example, JP-A-63-5760.
No. 2 (publication date: March 12, 1988), Japanese Patent Laid-Open No. 2
-245193 (publication date: September 28, 1990)
Sun) and Japanese Patent Laid-Open No. 11-124401 (publication date: May 11, 1999).

The method disclosed in Japanese Patent Laid-Open No. 63-57602 is a method for producing a low molecular weight hyaluronic acid by treating a hyaluronic acid-containing raw material with an alkali and further treating with an acid. In addition, JP-A-2-245193
The method disclosed in the publication is a method of obtaining a low-polymerization hyaluronate by treating hyaluronic acid obtained by a fermentation method with a chlorine-based oxidizing agent. In addition, JP-A-11
The method disclosed in Japanese Patent No. 124401 is a method of obtaining oligohyaluronic acid using an ultrafiltration membrane after degrading hyaluronic acid with a degrading enzyme.

[0008]

However, in order to produce oligohyaluronic acid having an average molecular weight of less than 10,000 by the method disclosed in Japanese Patent Laid-Open No. 63-57602, the pH in the acid treatment is as described in that publication. Must be set to an extremely acidic condition such as a value of less than 2.8. Further, the above-mentioned method for producing oligohyaluronic acid having an average molecular weight of less than 10,000 under such extremely acidic conditions has a problem that the yield of the oligohyaluronic acid is lowered. Further, the above method under the extremely acidic condition as described above has a problem that discoloration of oligohyaluronic acid occurs.

Further, in the method of treating hyaluronic acid with a chlorine-based oxidizing agent such as hypochlorous acid, as in the method disclosed in JP-A-2-245193, hyaluronic acid is decomposed by oxidation. Therefore, there is a problem that oligohyaluronic acid, which is not produced by hydrolysis with an enzyme or the like, is produced by the above method of treating with an oxidizing agent. That is, there is a problem that the chemical structure of oligohyaluronic acid is changed by the oxidizing agent. Specifically, the above method of treating with an oxidant produces oligohyaluronic acid having a new chemical structure at the reducing end and the non-reducing end. Oligohyaluronic acid having a terminal of such a new chemical structure, pharmaceuticals, cosmetics,
It is also a problem when oligohyaluronic acid is used in food products.

Further, in the method disclosed in JP-A-11-124401, an endo-type glucosidase is used as a degrading enzyme. It is known that such a decomposition reaction using an endo-type glucosidase is accompanied by a glycosyl rearrangement reaction in parallel with the decomposition reaction.

The above-mentioned transglycosylation reaction will be specifically described. For example, oligohyaluronic acid is produced by degrading hyaluronic acid with an endo-type glucosidase, hyaluronidase. Further, as the decomposition progresses, hyaluronic acid decreases and oligohyaluronic acid increases. But,
When decomposition progresses and the amount of oligohyaluronic acid increases, there is a problem in that the enzyme that used to decompose until now causes a reaction reverse to the decomposition. The reverse reaction is a reaction for synthesizing hyaluronic acid from oligohyaluronic acid. Further, there is a problem that oligohyaluronic acid is reduced due to the opposite reaction.

In the method using an acid / alkali and the method using an oxidizing agent, the decomposition reaction proceeds only in the direction of decomposition. However, in the method using an enzyme as described above, in addition to the decomposition direction, the reaction proceeds in the opposite direction to the decomposition, that is, in the synthesis direction. That is, the method using an enzyme has a problem that it is difficult to control the decomposition reaction, and oligohyaluronic acid cannot be efficiently obtained. In particular, the literature (Kinoshita M et al., Electrophoresi
s (2001), 22, p3458-3465, and Takagaki K et al., Bi
According to Ochem. (1994), 33, p6503-6507), the reverse reaction as described above, when using high concentration of hyaluronic acid and a large amount of enzyme to industrially prepare oligohyaluronic acid, More noticeable.

The reverse reaction as described above can be prevented to some extent by making the concentration of the reaction substrate hyaluronic acid as low as possible, for example 1% or less. However, such a method for producing an oligohyaluronic acid by preventing the reverse reaction requires a step of concentrating the reaction solution in a large amount when obtaining the oligohyaluronic acid. Therefore, it cannot be said to be practical as a method for industrially producing oligohyaluronic acid (Kakehi, J. Chromatogr.
(1994), 630, p141).

The present invention has been made in view of the above-mentioned conventional problems, and an object thereof is to use an enzyme without causing discoloration of an oligosaccharide or an oligosaccharide salt and a change in chemical structure due to oxidation. An object of the present invention is to provide a method for producing an oligosaccharide (salt) with high yield by suppressing a reaction opposite to decomposition.

[0015]

Means for Solving the Problems The present inventors have made extensive studies in order to solve the above problems. As a result, oligosaccharides (salts) with a molecular weight below a certain level were obtained by the enzymatic decomposition reaction of mucopolysaccharides (salts) in a space partitioned by a partition that has a membrane that separates molecules with a certain molecular weight.
Thought that it would go out of the partition from the above membrane. And
Oligosaccharides (salts) with a certain molecular weight or less that came out of the partition
The present invention has been completed by finding that it can be selectively fractionated while being prevented from being used in the reaction opposite to decomposition.

The method for producing an oligosaccharide (salt) of the present invention is a method for producing an oligosaccharide (salt) in which a mucopolysaccharide or a salt thereof is decomposed with an enzyme to produce an oligosaccharide (salt) without passing through the enzyme. A first space and a second space which are partitioned by a partition having a passage membrane that allows the oligosaccharide (salt) to be produced to pass therethrough
A space is prepared, and the mucopolysaccharide or its salt is decomposed with an enzyme in the first space, and the oligosaccharide (salt) produced by the decomposition and moved to the second space through the passage membrane is recovered. I am trying.

According to the above method, the mucopolysaccharide or its salt is enzymatically decomposed in the first space partitioned by the partition. Examples of the passage membrane include a membrane that separates molecules by molecular weight. Furthermore, examples of the membrane that separates molecules by their molecular weight include dialysis membranes and ultrafiltration membranes. For example, assume that the oligosaccharide (salt) to be produced has a molecular weight of 750. At that time, a membrane which can pass a molecule having a molecular weight of 750 or less and cannot pass a molecule having a large molecular weight such as mucopolysaccharide (salt) and enzyme can be used as the above-mentioned passage membrane.

The partition having a passage membrane that allows the oligosaccharide (salt) to be produced, but not the enzyme, to be passed may be such that at least a part of the partition is provided with the passage membrane. At that time, the partition portion other than the passage membrane is composed of, for example, an enzyme, mucopolysaccharide (salt), oligosaccharide (salt), or the like that cannot pass through. In addition, of course, as for the said partition, all of the partition may be comprised by the said passage membrane.

When the mucopolysaccharide (salt) is enzymatically decomposed in the first space partitioned by the partition, the decomposition produces an oligosaccharide (salt) that can pass through the passage membrane. The oligosaccharide (salt) that can pass through the passage membrane passes through the passage membrane in the partition and exits into the second space.

For example, mucopolysaccharide (salt), degrading enzyme,
A buffer suitable for the reaction of the enzyme is put into the dialysis membrane closed on one side. The other side of the dialysis membrane is then closed and the dialysis membrane placed in the liquid. Examples of the liquid into which the dialysis membrane is placed include buffer solutions suitable for decomposition by enzymes. When the dialysis membrane is put into such a buffer solution, the pH inside the dialysis membrane (in the first space) is kept constant, and the decomposition by the enzyme inside the dialysis membrane is stabilized. When the mucopolysaccharide (salt) inside the dialysis membrane (in the first space) is decomposed in such an environment, the oligosaccharide (salt) whose molecular weight has passed the dialysis membrane is outside the dialysis membrane, that is, outside the partition. Go to (2nd space).

As described above, the oligosaccharide (salt) that has passed through the passage membrane (dialysis membrane) and has come out of the partition leaves the enzyme inside the partition (first space). Further, since the enzyme cannot be released to the outside of the partition (second space), the oligosaccharide (salt) that has been released to the outside of the partition does not bind to the enzyme. Therefore, the oligosaccharide (salt) that has exited into the second space is not affected by the enzyme. That is, the oligosaccharide (salt) that has gone out of the partition is not further decomposed by the enzyme, nor is it subjected to a reaction opposite to the decomposition. In this way, since the reaction opposite to the decomposition by the enzyme can be avoided, the yield of the oligosaccharide (salt) is higher than that of the conventional method for producing the oligosaccharide (salt) using the enzyme. To rise. Further, according to the above method, it is not necessary to separate the enzyme and the oligosaccharide (salt) after the completion of the decomposition reaction.

Further, according to the above method, since the decomposition reaction by the enzyme is used, the mucopolysaccharide (salt) can be decomposed within the range where the enzyme does not lose its activity. In other words, avoid environments with extremely low pH and environments with extremely high pH,
It is possible to decompose mucopolysaccharides (salts). Moreover, the mucopolysaccharide (salt) can be decomposed without using an oxidizing agent. As a result, the production of high-yield oligosaccharides (salts) is suppressed by suppressing the discoloration of oligosaccharides or oligosaccharide salts and the change in chemical structure due to oxidation, and suppressing the reaction opposite to the enzymatic decomposition. A method can be provided.

The method for producing an oligosaccharide (salt) of the present invention is characterized in that, in addition to the above method, the passage membrane allows only a substance having a molecular weight equal to or lower than the molecular weight of the oligosaccharide (salt) to be produced to pass through. There is.

According to the above method, the passage membrane allows only a substance having a molecular weight equal to or lower than that of the oligosaccharide (salt) to be produced to pass through. That is, a substance having a molecular weight higher than that of the oligosaccharide (salt) to be produced, such as mucopolysaccharide (salt), cannot pass through the passage membrane.

As a result, the raw material mucopolysaccharide (salt),
The step of separating the oligosaccharide (salt) produced by decomposing the mucopolysaccharide (salt) can be omitted. Furthermore, when the oligosaccharide (salt) obtained as a result of the decomposition passes through the passage membrane and exits into the second space, the concentration of the oligosaccharide (salt) in the first space decreases, so that the enzymatic reaction in the first space occurs. Can be promoted in the direction of decomposition.

Further, in addition to the above-mentioned method, the mucopolysaccharides of the present invention include the hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparin, heparan sulfate, chitin, and It is characterized by being a polysaccharide selected from the group consisting of chitosan.

According to the above method, the mucopolysaccharide is a polysaccharide selected from the above group, and each of these mucopolysaccharides has an enzyme that decomposes.

As a result, in addition to the above effects, many kinds of oligos obtained by enzymatically degrading hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparin, heparan sulfate, chitin and chitosan. Sugar (salt) can be produced.

Further, the method for producing an oligosaccharide (salt) of the present invention uses an oligohyaluronic acid or oligohyaluronic acid consisting of 4 or more sugars by using hyaluronic acid as a mucopolysaccharide and endo-type hyaluronidase as an enzyme. It is characterized by producing salt.

According to the above method, by using endo-type hyaluronidase, hyaluronic acid (salt) having a minimum unit of 4 sugars having hexosamine as a reducing end can be obtained as an oligosaccharide (salt) in a high yield. it can. Examples of the endo-type hyaluronidase include bovine or ovine testis (testis) -derived hyaluronidase, actinomycete-derived hyaluronidase, and the like. Further, for example, when a membrane having a molecular weight cutoff of 1000 is used as a passage membrane, hyaluronic acid tetrasaccharide (salt) can be selectively obtained.

As a result, in addition to the above effects, oligohyaluronic acid (salt) (oligosaccharide (salt) of hyaluronic acid), for example, tetrasaccharide hyaluronic acid (salt) can be selectively and efficiently produced. .

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION An embodiment of the present invention will be described. In the method for producing an oligosaccharide (salt) of the present embodiment, the mucopolysaccharide or a salt thereof is decomposed with an enzyme to produce an oligosaccharide or a salt of the oligosaccharide. In addition, the first space and the second space partitioned by a partition having a passage membrane that allows the oligosaccharide (salt) to be produced to pass therethrough without passing through the enzyme are prepared, and the mucopolysaccharide or the salt thereof is provided in the first space. Is decomposed with an enzyme. Further, the oligosaccharide (salt) generated by the decomposition and moved to the second space through the passage membrane is collected. In the method for producing an oligosaccharide (salt) of the present embodiment, this recovery produces an oligosaccharide (salt).

That is, the oligosaccharide (salt) of the present embodiment
In the method for producing, first, as a passage membrane, for example, a membrane that separates molecules at a constant molecular weight is used, and in a space divided by a partition containing the passage membrane, the mucopolysaccharide (salt) is enzymatically decomposed. . The oligosaccharide (salt) having a molecular weight that can pass through the passage membrane is released to the outside of the space, so the oligosaccharide (salt) released to the outside of the space is collected to obtain a high-purity oligosaccharide. It is a method for producing (salt). In particular, the method for producing an oligosaccharide (salt) of the present embodiment, as shown below, is an oligosaccharide of mucopolysaccharide (oligomucopolysaccharide).
Is useful as a method for producing.

Conventionally, in the method of obtaining oligomucopolysaccharide, a method of simultaneously performing enzymatic decomposition, prevention of a reaction opposite to the decomposition, and fractionation of oligosaccharide (salt) having a certain molecular weight has not been known. . However, in the method of the present embodiment, the decomposition using the enzyme is performed in the first space, and the oligosaccharide (salt) produced in the first space is discharged to the second space. Is a method of simultaneously preventing the reverse reaction and fractionating oligosaccharides (salts) having a constant molecular weight.

(1) Mucopolysaccharide (salt) First, the mucopolysaccharide (salt) will be described. The mucopolysaccharide (salt) is a naturally occurring high molecular polysaccharide having hexosamine, which is a sugar having an amino group, as a constituent sugar in its structure. Examples of the mucopolysaccharide (salt) include hyaluronic acid, chondroitin sulfates, heparin,
Heparan sulfate, chitin, chitosan and the like can be mentioned.

Examples of hyaluronic acid include those derived from microorganisms and those derived from tissues (cows).

[0037]

[Chemical 2]

Further, hyaluronic acid is generally represented by the formula (1)
Is a compound represented by. In addition, n of Formula (1) shows a positive integer. Further, hyaluronic acid has a carboxyl group (—COOH) as shown in formula (1). Therefore, hyaluronic acid often exists as a salt with a metal such as sodium (hyaluronate).

Examples of chondroitin sulfates include chondroitin 4-sulfate, chondroitin 6-sulfate,
And dermatan sulfate and the like. Also usually
In nature, the mucopolysaccharide is in the form of a metal salt,
It exists especially in the form of salts of alkali metals such as sodium and potassium.

The mucopolysaccharide that can be used in the production method of the present embodiment is a mucopolysaccharide that can be decomposed with an enzyme.

(2) Oligosaccharide (salt) An oligosaccharide is usually a substance in which several monosaccharides are bound, and includes a substance in which ten or more monosaccharides are bound. The oligosaccharide (salt) in the present embodiment is an oligosaccharide (salt) obtained by decomposing a mucopolysaccharide (salt) with an enzyme.

As an example, an oligosaccharide of hyaluronic acid (oligohyaluronic acid) will be described. In the formula (1) showing hyaluronic acid, when n is 1, the number of sugars constituting hyaluronic acid is four, which is an oligosaccharide called hyaluronic acid tetrasaccharide. Similarly, when n is 2,
Hyaluronic acid is composed of eight sugars, which is an oligosaccharide called hyaluronic acid octasaccharide. Other than that, oligohyaluronic acid also includes 6-, 10-, or 12-sugars constituting the hyaluronic acid hexasaccharide, hyaluronic acid decasaccharide, hyaluronic acid dodecasaccharide, and the like.

(3) Enzyme that Decomposes Mucopolysaccharide or a Salt Thereof and Conditions for Degrading It Next, an enzyme that decomposes a mucopolysaccharide (salt) will be described. From the viewpoint of substrate specificity, examples of the enzyme that decomposes the mucopolysaccharide include hyaluronidases, chondroitinases, heparinases, heparitinase, chitinase, chitosanase and the like.

Among the hyaluronidases, there are those derived from the testicles (testes) of cattle or sheep. Hyaluronidase derived from it is hyaluronic acid, chondroitin 4
Sulfuric acid and chondroitin 6-sulfate can be decomposed. In addition, hyaluronidases include actinomycetes, bacteria,
And leech derived from them, and hyaluronidase derived from them decomposes hyaluronic acid.

Further, as the chondroitinase, there is one that decomposes hyaluronic acid and chondroitin sulfates (chondroitinase ABC, etc.). Heparinase is heparin, heparinase is heparan sulfate,
Chitinase decomposes chitin and chitosanase decomposes chitosan.

Since the mode of decomposition differs depending on the enzyme selected, it is preferable to select the above enzyme in accordance with the production of the desired oligosaccharide (salt). For example, if you want to obtain oligohyaluronic acid with the minimum unit of hexosamine as the reducing end and a tetrasaccharide, testicles (testis) of cattle and sheep.
It is preferable to select an endo-type hyaluronidase such as a hyaluronidase derived from or a hyaluronidase derived from actinomycete.

Next, the conditions for enzymatic decomposition will be described. The above-mentioned enzymatic decomposition is carried out, for example, under conditions in which the mucopolysaccharide or its salt can be decomposed by the enzyme, for example, under conditions in which the mucopolysaccharide or its salt and the enzyme are fluid. The conditions under which the mucopolysaccharide or salt thereof and the enzyme have fluidity include, for example, conditions under which a mucopolysaccharide or salt thereof dissolved, suspended or emulsified in a liquid and an enzyme are present. Is. However, the conditions for enzymatic degradation need not be limited to such conditions. For example, the decomposition with the above-mentioned enzyme may be carried out with the enzyme immobilized on an insoluble carrier.

The conditions under which the mucopolysaccharide or salt thereof dissolved and suspended or emulsified in the liquid and the enzyme are dissolved, suspended, or in a liquid such as water, or The presence of an enzyme and an emulsion of mucopolysaccharide or a salt thereof may be mentioned. Of course, a buffer solution having a pH suitable for enzymatic decomposition may be used as the liquid. The liquid is not limited to water or the like. For example, the above liquid may be other solvent instead of water as long as the enzyme can decompose mucopolysaccharide (salt) and the oligosaccharide (salt) to be produced can pass through the passage membrane.

When the mucopolysaccharide (salt) is decomposed by an enzyme, it is generally decomposed under the optimum conditions of the enzyme, for example, optimum pH and optimum temperature. For example,
The mucopolysaccharide (salt) is decomposed in a buffer solution which has the optimum temperature and optimum pH for the decomposition reaction. However, in addition to the above optimum conditions, the mucopolysaccharide (salt) may be decomposed under conditions under which the enzymatic decomposition reaction proceeds gently in order to increase the polymerization degree of the obtained oligomucopolysaccharide (salt). Usually, the reaction conditions are selected from the range of pH 4 to 10 and reaction temperature of 4 ° C to 60 ° C.

When it is expected that the decomposition by the enzyme takes a long time and the growth of microorganisms and the like is expected, the liquid may contain an antiseptic agent in an amount that does not affect the decomposition reaction by the enzyme. Examples of such preservatives include methylparaben and the like.

(4) Passing Membrane and Partition Next, a partition having a passing membrane that does not allow the enzyme to pass but allows the oligosaccharide (salt) to be produced to pass will be described.

Examples of the passage membrane include membranes that separate molecules according to a certain molecular weight. Also,
Membranes that separate molecules according to a certain molecular weight include, for example, dialysis membranes and ultrafiltration membranes. The passage membrane is not limited to the dialysis membrane and the ultrafiltration membrane.

As the passage membrane, it is preferable to use a membrane that allows passage of only a substance having a molecular weight equal to or lower than that of the oligosaccharide (salt) to be produced. When such a membrane is used, substances having a molecular weight higher than that of the oligosaccharide (salt) to be produced, such as mucopolysaccharide (salt), do not pass through the passage membrane. Therefore, when obtaining an oligosaccharide (salt), the mucopolysaccharide (salt) that is the raw material and the mucopolysaccharide (salt)
The step of separating the oligosaccharide (salt) produced by decomposing the can be omitted. Furthermore, when the oligosaccharide (salt) obtained as a result of the decomposition passes through the passage membrane and exits into the second space, the concentration of the oligosaccharide (salt) in the first space decreases, so that the enzymatic reaction in the first space occurs. Can be promoted in the direction of decomposition.

As described above, in order to select a passage membrane that cannot pass the mucopolysaccharide (salt) and the enzyme but the oligosaccharide (salt) to be produced, the molecular weight of the oligosaccharide (salt) to be produced and It is necessary to select the passage membrane while paying attention to the molecular weight of the mucopolysaccharide (salt) and the molecular weight of the enzyme.

The reason for decomposing inside the partition having at least a part of the passage membrane is to separate the oligosaccharide (salt) which can pass through the passage membrane from the enzyme. The reason for separating the oligosaccharide (salt) from the enzyme in this way is that when the enzyme and the oligosaccharide (salt) coexist, a reaction reverse to the decomposition occurs and the oligosaccharide (salt) yield is low. Further, if there is a step of removing the enzyme as in the conventional manufacturing method, the manufacturing process becomes complicated.

Further, as described above, since oligosaccharides are usually substances in which several monosaccharides are bound, the molecular weight of oligosaccharides (salts) is the maximum although it varies somewhat depending on the type and number of functional groups. But it will be about 2000. Therefore, when a membrane that separates molecules by a certain molecular weight is used as the passage membrane in the present embodiment and the oligosaccharide (salt) to be produced has a maximum molecular weight of 2000, the molecular weight cutoff is 2000 or less. Select the membrane of.

The cut-off molecular weight of the membrane may be determined according to the molecular weight of the oligosaccharide (salt) to be produced. For example, in the case of an oligosaccharide (salt) composed of 10 or more sugars, the molecular weight of the oligosaccharide (salt) is about 3000 to 3500. When producing such oligosaccharides (salts) consisting of 10 or more sugars, the molecular weight cutoff of the membrane is adjusted to 3000 to 3500 accordingly.

When a membrane that separates molecules according to a certain molecular weight is used as the passage membrane as described above, the enzyme that decomposes the mucopolysaccharide (salt) is an enzyme that has a molecular weight that does not pass through the passage membrane.

The molecular weight of the oligosaccharide to be obtained can be changed by changing the type of the passage membrane. For example, when a membrane having a molecular weight cutoff of 2000 is selected as a passage membrane and a membrane having a molecular weight cutoff of 1000 is selected as a passage membrane, the molecular weight cutoff is 200.
When the membrane of 0 is selected, oligosaccharides having a large molecular weight can be obtained.

The partition in the present embodiment means
At least a part of the partition has the passage membrane. In the case where the partition has the above-mentioned permeable membrane as a part thereof, the partition other than the permeable membrane is configured so as not to allow the liquid and molecules in the liquid to pass through. In addition, as for the said partition, all of the partition may be comprised by the said passage membrane.

Next, the manner of the passing membrane and the partition will be described. As the partition, for example, the entire partition may be composed of a dialysis membrane that is a passage membrane.
Specifically, a solution in which a mucopolysaccharide (salt) is dissolved or suspended in an aqueous buffer solution is put into a dialysis membrane (dialysis tube) whose one side is closed. Next, an enzyme that decomposes mucopolysaccharide (salt) is put into the dialysis membrane, and the remaining one side of the dialysis membrane is closed. Next, the dialysis membrane is put into the same aqueous buffer solution as described above to decompose the mucopolysaccharide while keeping the temperature constant (eg, the optimum temperature for decomposition). That is, the mucopolysaccharide is decomposed in the space (first space) closed by the dialysis membrane. Then, the oligosaccharide (salt) that has emerged outside the dialysis membrane (second space) is collected to produce the oligosaccharide (salt).

Further, the partition may be constructed by sealing both sides of a tube made of resin or the like with a dialysis membrane (passage membrane). In this case, first place the dialysis membrane (passage membrane) on one side of the tube.
Seal with. Next, a solution in which mucopolysaccharide (salt) is dissolved or suspended in an aqueous buffer solution is put into a tube. Next, an enzyme that decomposes mucopolysaccharide (salt) is put into a tube, and the remaining side of the tube is sealed with a dialysis membrane (passage membrane). In this case, the space closed by the tube and the dialysis membrane is the first space. Then, an aqueous buffer solution is flown through the tube to collect the oligosaccharide (salt) flowing out from the dialysis membrane into the second space, thereby producing the oligosaccharide (salt).

(5) Method for Acquiring (Recovering) Oligosaccharide (Salt) Next, a method for acquiring (recovering) the oligosaccharide (salt) that has passed through the passage membrane and is outside the partition will be described. As described above, the oligosaccharide (salt) that has passed through the passage membrane and has come out of the partition exists in the liquid outside the partition. Therefore, the oligosaccharide (salt) is recovered from the liquid outside thereof.
For example, the oligosaccharide (salt) can be recovered by precipitation using an organic solvent such as alcohol or acetone, lyophilization, and adsorption by an ion exchange resin or the like. In addition, as said alcohol used when precipitating, ethanol, isopropanol (2-propanol) etc. are mentioned, for example.

When an oligosaccharide (salt) is recovered by forming a precipitate using an organic solvent such as alcohol or acetone, the organic solvent may be selected according to the oligosaccharide (salt) to be produced. For example, it is preferable to use ethanol when recovering oligohyaluronic acid.

In the conventional method for producing an oligosaccharide (salt), after decomposing mucopolysaccharide, the target oligosaccharide is
A method of separating by a chromatographic method such as gel filtration has been used. However, in the chromatographic method, the operation is generally complicated and the yield is poor. Therefore, the chromatographic method cannot be said to be a method for efficiently obtaining the target oligosaccharide. The method of the present embodiment does not require a complicated operation such as a chromatographic method, and the yield of oligosaccharide (salt) is good.

In the conventional method for producing oligosaccharides (salts), as a method not using a chromatographic method, hyaluronic acid is decomposed with an enzyme into oligohyaluronic acid,
There is also a method of using an ultrafiltration membrane having a molecular weight cut-off of 10,000 or less to collect oligohyaluronic acid passing through the membrane. However, in this method, an enzyme causes a reaction reverse to the decomposition (that is, a synthetic reaction), so that an oligosaccharide produced by the decomposition and a sugar having a larger molecular weight than the oligosaccharide are also produced. As a result, the method of collecting oligosaccharides with an ultrafiltration membrane after degrading hyaluronic acid with an enzyme cannot efficiently obtain oligosaccharides having a molecular weight according to the manufacturer's requirements.

Further, as described above, when ultrafiltration is carried out after the decomposition, naturally one step of the ultrafiltration step is added. However, before using the liquid after the decomposition reaction for ultrafiltration, further treatment for increasing the filtration rate in ultrafiltration is required. The treatment for increasing the filtration rate is a treatment for preventing clogging of the filtration membrane by using the supernatant obtained by centrifugally separating the solution of the decomposition reaction for filtration. As described above, since the step of centrifuging to obtain the supernatant is required, the above conventional method using an ultrafiltration membrane after decomposing hyaluronic acid with an enzyme complicates the manufacturing process.

In the production method of the present embodiment, a passage membrane that allows an oligosaccharide (salt) to be produced but not an enzyme to pass therethrough, for example, a passage that allows only a substance having a molecular weight equal to or lower than the molecular weight of the target oligosaccharide to pass therethrough. Use a membrane. for that reason,
There is no need for an ultrafiltration step for separating a desired oligosaccharide (salt) and an oligosaccharide (salt) having a larger molecular weight after the decomposition reaction, which is required in the conventional method. Centrifugation to raise is also unnecessary.

[0069]

Example 1 As an example of a method for producing an oligosaccharide (salt), hyaluronic acid is used as a mucopolysaccharide and hyaluronidase is used as an enzyme, and oligohyaluronic acid or oligohyaluronic acid salt, particularly hyaluronic acid 4 is used. An example of producing sugar (salt) will be shown.

10 m containing 0.05% methylparaben
In 50 ml of M acetate buffer (pH 5.3), sodium hyaluronate which is a salt of mucopolysaccharide (manufactured by Nakarai Techs,
5 g of special grade, microbial origin and endo-type hyaluronidase (hyaluronidase derived from ovine testis (Bi
ozyume)) 250,000 U (National Institute of Health Standard hyaluronidase conversion value) was added to prepare a hyaluronic acid solution. Moreover, the hyaluronic acid solution is 30 ° C to 40 ° C.
While maintaining the temperature at 1, the mixture was stirred for 7 days.

Next, 100,000 U of the above hyaluronidase was added to the hyaluronic acid solution, and the hyaluronic acid solution was further treated with a dialysis membrane having a molecular weight cut off of 1000 (Spectra /
PorMWCO1000) tube and the end of the tube was closed and sealed. Next, the sealed dialysis membrane was put in 900 ml of the acetate buffer solution and dialyzed for 7 days while keeping the entire buffer solution at 30 to 40 ° C.

Next, after concentrating the dialysate obtained by the above dialysis, sodium acetate was dissolved in the dialysate to a concentration of 10%. Next, 9 times amount (V / V) of ethanol was added to the dialysate to deposit a precipitate. Next, the precipitate was obtained by filtration, and the obtained precipitate was washed with 2-propanol and acetone. After washing, the precipitate was put in a desiccator together with phosphorous pentoxide, and the precipitate was dried under reduced pressure. The weight of the precipitate (oligohyaluronic acid) after drying was 3.9 g. The sodium hyaluronate initially used was 5 g, and the obtained precipitate (oligohyaluronate) was 3.9 g, so the yield was 78%.
Met.

The molecular weight of oligohyaluronic acid is about 1000 when it is a hexasaccharide. Therefore, in the precipitate obtained above, as oligohyaluronic acid, 6
Sugars are expected to contain oligosaccharides below.

Next, the precipitate (oligohyaluronic acid) obtained above was analyzed by capillary electrophoresis. The conditions of the capillary electrophoresis at this time are shown below. The apparatus used was a Beckman P / ACE5010 type. The capillary is
A fused silica capillary (inner diameter 50 μm, effective length 40 cm) was used. As the buffer, 50 mM borate buffer (pH 9.3) containing 100 mM SDS was used. In addition, SDS is sodium dodecyl sulfate (sodium dod
ecyl sulfate). The applied voltage is 20k
V, analysis temperature was 30 ° C., and ultraviolet absorption of 200 nm was used for detection. The result of this capillary electrophoresis is
As shown in FIG.

As a result of the capillary electrophoresis shown in FIG.
As expected above, it was found that the resulting precipitate contained hyaluronic acid tetrasaccharide as the main component as oligohyaluronic acid. Further, it was found that the obtained precipitate also contained hyaluronic acid hexasaccharide, and also contained very small amount of hyaluronic acid octasaccharide. That is, it was found that oligohyaluronic acid was obtained in a high yield of 78% by the above method.

Further, by further precipitating the above oligohyaluronic acid with ethanol and purifying it, the hyaluronic acid tetrasaccharide can be easily obtained in a high yield.

First, 1 g of 3 g of the above oligohyaluronic acid was added.
It was dissolved in 30 ml of 0% aqueous sodium acetate solution. Next, 90 ml of ethanol was added to the solution in which the oligohyaluronic acid was dissolved, and the mixture was centrifuged (3,000 rpm, 1
0 minutes). Next, the supernatant after centrifugation was recovered, and 270 ml of ethanol was added to the recovered supernatant to obtain a precipitate. Next, the precipitate was obtained by filtration, and the obtained precipitate was washed with 2-propanol and acetone. After washing, the precipitate was put in a desiccator together with phosphorous pentoxide, and the precipitate was dried under reduced pressure. The dried precipitate (hyaluronic acid tetrasaccharide) was 1.25 g.

From 3 g of oligohyaluronic acid to 1.2
Since 5 g of a precipitate (hyaluronic acid tetrasaccharide) was obtained, the yield calculated from oligohyaluronic acid was about 40% (41.67).
%).

Furthermore, the above-mentioned precipitation (hyaluronic acid tetrasaccharide)
Was analyzed by capillary electrophoresis. The results are shown in Figure 2. The analysis conditions for capillary electrophoresis were the same as above. From the data shown in FIG. 2, it was found that the precipitate purified by the above operation was hyaluronic acid tetrasaccharide having a purity of 97% or more.

(Example 2) An example of a method for producing an oligosaccharide (salt) using a dialysis membrane having a molecular weight cut off larger than that in Example 1 will be described. Acetic acid buffer solution 5 ml (pH 5.3), sodium hyaluronate 5 g, hyaluronidase 250,000
U was added to prepare a hyaluronic acid solution. Next, the hyaluronic acid solution was applied to a dialysis membrane (SpecSpec
(tra / Por MWCO2000) tube and sealed at both ends. Next, the tube of the sealed dialysis membrane is put in 300 ml of acetate buffer, and the whole buffer is heated at 30 ° C to 30 ° C.
It was dialyzed for 2 days while keeping it at 40 ° C. The same acetate buffer, sodium hyaluronate, and hyaluronidase as in Example 1 were used.

Next, after concentrating the dialysate obtained by the above dialysis, sodium acetate was dissolved in the dialysate to a concentration of 10%. Next, 9 times amount (V / V) of ethanol was added to the dialysate to deposit a precipitate. Next, the precipitate was obtained by filtration, and the obtained precipitate was washed with 2-propanol and acetone. After washing, the precipitate was put in a desiccator together with phosphorous pentoxide, and the precipitate was dried under reduced pressure.

The molecular weight of oligohyaluronic acid is about 2000 in the case of 12 sugars. Therefore, a molecular weight cutoff of 2000
The above-mentioned precipitate obtained using the dialysis membrane of No. 1 is expected to be oligosaccharides having 12 or less sugars.

Next, the precipitate was analyzed by capillary electrophoresis. The result is shown in FIG. As shown in FIG. 3, it was found that the amount of sugars of 14 sugars or more contained in the precipitate was very small. Moreover, it was found that the above-mentioned precipitate was an oligosaccharide having a sugar of 12 sugars or less as a main component as expected.

Further, as can be seen by comparing Example 1 and Example 2, it is possible to selectively obtain oligosaccharides having different molecular weights by changing the type of the passage membrane, that is, the dialysis membrane in Example. Is possible.

Example 3 An example using chondroitin tetrasulfate as the mucopolysaccharide will be shown. 1.5 g of chondroitin tetrasulfate (manufactured by Wako Pure Chemical Industries, Ltd. (chondroitin sulfate A)) and 5000 U of hyaluronidase (the same as in Example 1) were dissolved in 15 ml of an acetate buffer (the same as in Example 1) to prepare a solution. Was prepared. Moreover, the solution is 30 ° C. to 40 ° C.
The temperature was kept at ° C and the reaction was carried out for 3 days.

Next, 5000 U of the above hyaluronidase was added to the solution, and the solution was further subjected to a dialysis membrane with a molecular weight cut off of 1000 (Spectra / Por MWCO1000).
, And the end of the tube was closed and sealed. Then, the sealed dialysis membrane was washed with the above acetate buffer (15
0 ml), the whole buffer solution was kept at room temperature, and dialyzed for 7 days.

Next, after concentrating the dialysate obtained by the above dialysis, sodium acetate was dissolved in the dialysate to a concentration of 10%. Next, 9 times amount (V / V) of ethanol was added to the dialysate to deposit a precipitate. Next, the precipitate was obtained by filtration, and the obtained precipitate was washed with 2-propanol and acetone. After washing, a precipitate was put in a desiccator together with diphosphorus pentoxide, and the precipitate (oligochondroitin sulfate) was dried under reduced pressure.

Chondroitin sulfate is N-acetyl D-
It consists of repeating sugar units of glucosamine and D-glucuronic acid. In chondroitin sulfate, the average hydroxyl group of N-acetyl D-glucosamine is about 1
The individual is a sulfate ester. Therefore, since the molecular weight of chondroitin sulfate is approximately 4 saccharides, the precipitation obtained above is expected to be oligosaccharides having 4 saccharides as a main component.

Next, the precipitate was analyzed by capillary electrophoresis. The result is shown in FIG. As shown in FIG. 4, when analyzed by capillary electrophoresis, multiple peaks were obtained and the composition was not clearly shown.

The reason why the composition is not clear as described above is considered as follows. Among chondroitin sulfates, those in which the 4-position hydroxyl group of N-acetyl D-glucosamine, which is a repeating sugar unit, is a sulfate ester is called chondroitin 4-sulfate. But,
The so-called chondroitin tetrasulfate includes a sugar unit having no sulfate group in the same molecule and a sugar unit having a sulfate group at the 6-position. That is,
What is called chondroitin tetrasulfate has a heterogeneous structure. Therefore, when analyzed by capillary electrophoresis, multiple peaks were obtained, and it is considered that the composition was not clearly shown.

Therefore, the above-mentioned precipitate was subjected to mass spectrometry (MALD
I-TOFMS: Matrix assisted laser-desorption io
nization time-of-flight mass spectrometry). The result is shown in FIG. As shown in FIG. 5, five peaks are remarkable. Among them, m / z = 958, m
The peaks near / z = 980 and m / z = 1002 are peaks derived from chondroitin sulfate tetrasaccharide. The peaks near m / z = 856 and m / z = 879 are those of chondroitin tetrasulfate having no sulfate group. Therefore, it was found that the main component of the precipitate (oligochondroitin sulfate) was the tetrasaccharide as expected above.

(Comparative Example 1) The conditions of each enzyme reaction such as the concentration of hyaluronic acid used as a substrate at the time of reaction, the concentration of enzyme, the reaction temperature, and the reaction time were the same as in Example 1, but no dialysis membrane was used. Then, an operation of obtaining a precipitate (oligohyaluronic acid) was carried out. Further, the precipitate was examined by capillary electrophoresis. The results of Example 1 and the results of Comparative Example 1 are shown in FIG. 6 and Table 1.

In Comparative Example 1, decomposition was performed under the same conditions as in Example 1 except that a dialysis membrane was not used, and after the decomposition, precipitation was performed using a large amount of ethanol without using a dialysis membrane ( Oligo hyaluronic acid) was obtained. This operation and the result of this operation are referred to as Comparative Example 1-1. In addition, in FIG. 6, the result of Comparative Example 1-1 is shown in FIG.
Shown in.

Further, in Comparative Example 1, an operation of decomposing under the same conditions as in Example 1 except that a dialysis membrane was not used, and after the decomposition was dialyzed with a dialysis membrane to obtain a precipitate was also performed. This operation and the result of this operation are referred to as Comparative Example 1-2. In FIG. 6, Comparative Example 1-
The result of No. 2 is shown in FIG.

[0095]

[Table 1]

In Example 1, as shown in Table 1, the yield of the precipitate (oligohyaluronic acid) was 3.9 g. Further, as shown in Table 1 and FIG. 6 (a), oligohyaluronic acid was mainly composed of tetrasaccharides, also contained hexasaccharides, and contained only a few octasaccharides.

On the other hand, the results of Comparative Example 1-1 are shown in Table 1.
As shown in Fig. 6 (b), the proportion of octasaccharide is high. It also contains decasaccharide, decasaccharide, etc., which are larger than octasaccharide. Furthermore, according to the result of FIG.
Larger molecules than sugar are also found. In addition, Comparative Example 1-1
The result is that the yield is 2.5 g, which is smaller than the yield of Example 1. Therefore, as in Comparative Example 1-1, when oligohyaluronic acid is produced without using a dialysis membrane, it is found that oligohyaluronic acid having a small yield and a large variation in molecular weight is produced.

Further, in Comparative Example 1-2, as shown in Table 1, since the dialysis membrane was used after the decomposition, oligohyaluronic acid containing most of tetrasaccharide and hexasaccharide can be produced.
However, as shown in Table 1, in Comparative Example 1-2, the yield was 1.3 g, which is considerably lower than that in Example 1.

Therefore, as in this example, inside the partition having the passage membrane such as the dialysis membrane (first space), the mucopolysaccharide (salt) such as hyaluronic acid is decomposed and the outside of the partition (the second space). The method of collecting oligosaccharides (salts) in the space is
Oligosaccharides (salts) with high yield and little variation in molecular weight
Can be said to be a method of manufacturing.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the present invention.

[0101]

The method for producing an oligosaccharide (salt) of the present invention comprises:
As described above, the first space and the second space that are partitioned by the partition having the passage membrane that allows the oligosaccharide (salt) to be produced to pass therethrough without passing the enzyme are prepared, and the mucopolysaccharide or the mucopolysaccharide in the first space is prepared. This is a method in which the salt is decomposed with an enzyme, and the oligosaccharide (salt) produced by the decomposition and moved to the second space through the passage membrane is recovered.

Therefore, an oligosaccharide (salt) having a high yield can be obtained by suppressing the discoloration of the oligosaccharide or the oligosaccharide salt and the change in the chemical structure due to the oxidation and suppressing the reaction opposite to the decomposition.
There is an effect that the manufacturing method of can be provided.

In addition to the above method, the method for producing an oligosaccharide (salt) of the present invention is a method in which the passage membrane allows only a substance having a molecular weight equal to or lower than the molecular weight of the oligosaccharide (salt) to be produced to pass through. Is.

Therefore, the mucopolysaccharide (salt) as the raw material,
It is possible to omit the step of separating the oligosaccharide (salt) produced by decomposing the mucopolysaccharide (salt). Furthermore, the effect of promoting the equilibrium of the enzymatic reaction in the first space in the decomposition direction is exerted.

Further, in addition to the above-mentioned method, the mucopolysaccharides of the oligosaccharides (salts) of the present invention include hyaluronic acid, chondroitin tetrasulfate, chondroitin 6-sulfate, dermatan sulfate, heparin, heparan sulfate, chitin, and The method is a polysaccharide selected from the group consisting of chitosan.

Therefore, in addition to the above-mentioned effects, many kinds of oligosaccharides obtained by enzymatically degrading hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparin, heparan sulfate, chitin and chitosan. The effect is that sugar (salt) can be produced.

[0107] In addition to the above-mentioned method, the method for producing an oligosaccharide (salt) of the present invention further comprises adding hyaluronic acid to the mucopolysaccharide.
In this method, endo-type hyaluronidase is used as an enzyme to produce oligohyaluronic acid or oligohyaluronate composed of four or more sugars.

Therefore, in addition to the above effects, oligohyaluronic acid (salt) (oligosaccharide of hyaluronic acid (salt)), for example, tetrasaccharide hyaluronic acid (salt) can be selectively and efficiently produced. .

[Brief description of drawings]

1 is an example of the present invention, wherein hyaluronic acid is used as the mucopolysaccharide and a dialysis membrane with a cut-off molecular weight of 1000 is used as the passage membrane to obtain an oligosaccharide, and the obtained oligosaccharide (oligohyaluronic acid) is used. 2 is a chart showing the results of analysis by capillary electrophoresis.

FIG. 2 shows the result of purifying the oligosaccharide (oligohyaluronic acid) showing the result of FIG. 1 and analyzing the purified oligosaccharide (oligohyaluronic acid) by capillary electrophoresis in the example of the present invention. It is a chart.

[Fig. 3] In the example of the present invention, hyaluronic acid was used as a mucopolysaccharide, and a dialysis membrane with a cut-off molecular weight of 2000 was used as a passage membrane to obtain an oligosaccharide, and the obtained oligosaccharide (oligohyaluronic acid) was used. 2 is a chart showing the results of analysis by capillary electrophoresis.

[Fig. 4] In the example of the present invention, chondroitin tetrasulfate was used as the mucopolysaccharide, and the cut-off molecular weight was 1000 as the passage membrane.
2 is a chart showing the results of analyzing the obtained oligosaccharide (oligochondroitin sulfate) by capillary electrophoresis using the dialysis membrane of 1. above.

[Fig. 5] In the example of the present invention, chondroitin tetrasulfate was used as the mucopolysaccharide, and the cut-off molecular weight was 1000 as the passage membrane.
Oligosaccharide was obtained using the dialysis membrane of No. 3, and the obtained oligosaccharide (oligochondroitin sulfate) was treated with MALDI-T.
It is a spectrum figure which shows the result analyzed by OFMS.

FIG. 6 (a) shows the same result as FIG. 1, but FIG.
In Comparative Example 1, after decomposition of hyaluronic acid, oligohyaluronic acid was obtained using a large amount of ethanol without using a dialysis membrane, and the result of analyzing the oligohyaluronic acid by capillary electrophoresis was shown in FIG. (C) is a chart showing the results of analyzing oligohyaluronic acid by dialysis with a dialysis membrane after decomposition of hyaluronic acid in Comparative Example 1 and analyzing the oligohyaluronic acid by capillary electrophoresis.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B064 AF21 BA09 BH09 CA21 CB07                       CD19 CE06 DA01 DA10                 4C057 AA04 AA30 BB04 CC03 EE03

Claims (4)

[Claims] 1. A method of producing an oligosaccharide (salt), which comprises decomposing mucopolysaccharide or a salt thereof with an enzyme to produce an oligosaccharide or a salt thereof, wherein the enzyme is not passed but the oligosaccharide (salt) to be produced is passed. A first space and a second space partitioned by a partition having a permeable membrane are prepared, and mucopolysaccharide or a salt thereof is decomposed by an enzyme in the first space, and the second space is produced by the decomposition and passes through the permeable membrane. A method for producing an oligosaccharide (salt), which comprises recovering an oligosaccharide (salt) that has moved to a space. 2. The method for producing an oligosaccharide (salt) according to claim 1, wherein the passage membrane passes only a substance having a molecular weight equal to or lower than the molecular weight of the oligosaccharide (salt) to be produced. 3. The mucopolysaccharide is a polysaccharide selected from the group consisting of hyaluronic acid, chondroitin 4 sulfate, chondroitin 6 sulfate, dermatan sulfate, heparin, heparan sulfate, chitin, and chitosan.
Or the method for producing an oligosaccharide (salt) according to 2. 4. An oligohyaluronic acid or oligohyaluronic acid salt comprising 4 or more sugars is produced by using hyaluronic acid as a mucopolysaccharide and endo-hyaluronidase as an enzyme. 2. The method for producing an oligosaccharide (salt) according to 2.