JPS61130297A - Separation of oligosaccharide - Google Patents

Abstract

PURPOSE:To separate a large amount of an oligosaccharide in high purity, by treating the oligosaccharide by column chromatography using a synthetic adsorbent free from ion exchange group as the filler. CONSTITUTION:A sugar syrup composed of e.g. an oligosaccharide having a glucose polymerization degree of 1-10 (G1-10) is adjusted to a concentration of preferably 5-15%, and passed through a column packed densely with a synthetic adsorbent having preferably fine and uniform particle size, free from ion exchange group, and substituted with pure water. The amount of the sugar syrup to be passed through the column is preferably 5-10vol% of the amount of the adsorbent. Thereafter, water or an alcohol (e.g. ethanol) having a proper concentration is passed through the column to elute the adsorbed oligosaccharide successively from the one having lower polymerization degree.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for separating oligosaccharides, and particularly to a method for separating oligosaccharides in which oligosaccharides are fractionated with high purity based on molecular weight differences using column chromatography. .

[Prior Art] By hydrolyzing starch with acid or decomposing it with enzymes such as amylase, oligosaccharides consisting of α-1,4 bonds of D,-glucose units, so-called malto-oligosaccharides, are obtained. Due to their unique physical properties, such oligosaccharides are substances whose demand is expected to increase dramatically in the fields of medicine, diagnostic substrates, and the like. Currently, for example, high-purity maltopentaose with a degree of polymerization of zero 5 (GS) is available.

It is used as a substrate for measuring amylase activity for diagnosing acute pancreatitis. In addition, even oligosaccharides separated with a slightly lower grade of purity, especially maltose with a degree of polymerization of 2 (G2) and maltopenose with a degree of polymerization of 3 (G3), are used in the food industry as sweets with an elegant flavor. It is being used as a filler and excipient.

Precise separation (hereinafter referred to as "isolation") of oligosaccharides with different degrees of polymerization has conventionally been performed using gel filtration, adsorption chromatography using activated carbon (activated carbon method), solvent precipitation, and other methods.
Chromatography method using a cation exchange resin (GPC mode) disclosed in No. 9-148794 (cation exchange resin method)
is considered.゛ [Problems to be solved by the invention] However, all of the conventional isolation methods have problems, as described in Moreover, no industrial method has been established that allows for easy fractionation.For this reason, the use of oligosaccharides has not been fully developed, and a preparative method has not been established.

There was a strong desire to expand its uses and increase demand.

■ Gel filtration method It is considered to be the most suitable method for isolating oligosaccharides among the conventional techniques, but its biggest disadvantage is that the packing material is very expensive, making it difficult to scale up the equipment. It is. In addition, since the particle size of the filler is small, the pressure loss through liquid flow is large.

■ Activated carbon method The adsorption power is too strong and a large amount of alcohol (ethanol, etc.) is required to elute the adsorbed material. Furthermore, it is difficult to elute oligosaccharides with a high degree of polymerization.

Moreover, if the alcohol concentration of the eluent is increased too much, the oligosaccharide to be treated will precipitate.

■ Solvent precipitation method Requires a large amount of organic solvent beyond activated carbon.

Furthermore, the separation property is inferior to other methods, and it is not suitable for isolation.

■ The cation exchange resin method itself has advantages such as less increase in differential pressure because the filler has a larger particle size than gel, is easy to operate, is inexpensive, and is easier to scale up than gel. , the separation properties are insufficient to isolate oligosaccharides. Therefore, it is necessary to use a method of circulating the liquid through dozens of cycles using a column several tens of meters long, and the recovered liquid becomes very dilute.
Subsequent concentration of oligosaccharides is not easy.

[Means for Solving the Problems] The present invention has been made to solve the above-mentioned conventional problems and to provide a method for separating oligosaccharides that has good separation properties and is suitable for isolation. That is, the present invention uses a synthetic adsorbent that does not have an ion exchange group as a packing material in a chromatographic separation method using a column similar to the conventional gel filtration method, activated carbon method, or cation exchange resin method, thereby reducing the molecular weight of oligosaccharides. A method for separating oligosaccharides by column chromatography, which enables effective separation based on differences, characterized in that the column is filled with a synthetic adsorbent packing material that does not have an ion exchange group. The gist is a method for separating oligosaccharides.

The present invention will be explained in detail below.

In the present invention, the stock solution to be separated includes a sugar solution made of oligosaccharides with a degree of glucose polymerization of about 1"lO (G+"Glo). (Note that since sugar (glucose) with a degree of glucose polymerization of 1 (Gl) is a monosaccharide, it is not included in oligosaccharides in the narrow sense. However, in the present invention, this glucose is also targeted for separation. Therefore, the oligosaccharide referred to in the present invention has a broad meaning that includes glucose.) Such a sugar solution can be easily obtained by hydrolyzing starch with an acid or decomposing it with an enzyme. If you specifically want to isolate one specific type of these oligosaccharides (for example, maltopentaose (Gs)), you can use a special enzyme (in this case, maltopentaose-generating amylase) during the decomposition of starch. It is desirable to prepare a sugar solution containing a large amount of the target substance and use it as a stock solution. In the method of the present invention, it is possible to treat the oligosaccharide concentration of the stock solution up to about 30%, but preferably 5 to 15%.

In the present invention, such a stock solution is passed through a column filled with a synthetic adsorbent packing material having no ion exchange group.

Examples of fillers include porous polymers (high porous polymers) such as Diaion HP20, Diaion 5P207 (all manufactured by Mitsubishi Chemical Industries, Ltd.), and Amberlite XAD (manufactured by Rohm and Haas) series. , in terms of adsorption power, especially Diamond Ion 5P2.
07 is preferred. It is preferable that the particle size of the filler is fine and uniform, but separation can be effected satisfactorily even if a commercially available filler is used as it is.

The column may be of any material as long as it can withstand a pressure of several tens of kg/c rn', and there are no restrictions on its material.The packing material may be packed into the column by a conventional method such as a tap method, a vibration method, or a slurry filling method. Fill densely.

The direction of flow of the stock solution and eluent may be either upward flow or downward flow, and the flow rate is not particularly limited, but SVo, 2
~3, Oh r-' (1) range, particularly preferably S
Vo, about 4-1, 0 hr- is suitable.

The processing temperature is 50°C to improve separation and prevent spoilage of the sugar solution.
The temperature is preferably 0.degree. C. or higher, but room temperature may be used as long as proper antiseptic measures are taken.

The liquid passing method may be the same as that used in ordinary column chromatography, in which the stock solution is first introduced by a pump into a packed column that has been replaced with pure water.

The amount of this stock solution supplied is 20% by volume based on the amount of column packing material.
Although it is possible to increase the amount up to a certain level, it is preferably 5 to 10% by volume.1 Next, the column supply liquid is switched from the stock solution to the eluent by switching the valve, etc., and the eluent is flowed at the same flow rate as the stock solution to complete the adsorption. The oligosaccharide adsorbed on the agent is extruded and expanded. As a result, the particles are eluted in order from those with a low degree of polymerization (that is, those with a low molecular weight) to those with a high degree of polymerization (that is, those with a large molecular weight).

Water, preferably pure water, is usually used as the eluent; however, if the degree of polymerization is 4 (maltotetraose) or higher, it will not be easily eluted with water due to strong adsorption to the filler. Therefore, in order to elute oligosaccharides with a higher degree of polymerization, a dilute aqueous solution of alcohol such as ethanol is used. Since the higher the alcohol concentration of the eluent, the stronger the elution power, the alcohol concentration is changed as appropriate depending on the degree of polymerization of the substance to be eluted. As a measure of alcohol concentration, for example, an oligosaccharide (maltopentaose) with a polymerization degree of 5 can be sufficiently eluted with an ethanol aqueous solution with a polymerization degree of about 3% by weight. When changing the alcohol concentration of the eluent within one cycle, it is effective to employ a stepwise method or a gradient method, which are common liquid chromatography techniques. Furthermore, as the eluent, in addition to the above, water-soluble organic solvents 1 having hydrophobic water properties such as acetonitrile, ethers, dioxy acids, etc. can be used.

In this way, oligosaccharides are chromatographically separated from the column using the eluent, and the eluate that flows out is used to detect the sugar content using an RI (differential refractometer), etc., and then collect it using a fraction collector, etc. The fraction containing only the target substance (or the target substance and other components within an acceptable range) is concentrated by removing water or alcohol aqueous solution by the usual evaporation method or reverse osmosis membrane method, and then concentrated as necessary. After drying, it is used for uses such as pharmaceutical substrates and food materials.

Although the substance other than the target substance (or the mixed liquid of the target substance and adjacent components) may be discarded as is, it is preferable to return it to its original solution and reprocess it.

The synthetic adsorbent used in the present invention does not require treatment such as regeneration and can be used any number of times.

[Function] The separation mechanism of the present invention is based on the size exclusion effect of molecules such as gel filtration method and cation exchange resin method (larger molecular size that cannot penetrate into the inside of the filler, i.e., the larger one molecular weight). It is completely different from the elution characteristics or effects, and is thought to be based on the affinity and adsorption power between the oligosaccharide and the filler, similar to the activated carbon method. Therefore, the larger the molecular weight, that is, the larger the degree of polymerization, the greater the affinity and adsorption force with the packing material. Therefore, those with a smaller molecular weight, that is, those with a smaller polymerization degree, are eluted and separated in order, and fractionated from the column. Ru.

However, since the adsorption power of oligosaccharides adsorbed on synthetic adsorbents that do not have ion exchange groups is relatively weak, it is not possible to almost completely isolate and recover them by using water or an aqueous alcohol solution as an eluent. It is possible.

Note that the method of the present invention can be applied not only to isolation but also to crude separation.

[Examples] The present invention will be explained in more detail by examples below.
The present invention is not limited to the following examples unless it exceeds the gist thereof.

Example 1 Isolation of malto-oligosaccharides by the method of the present invention using a mixture of equal amounts of malto-oligosaccharides G + "G 4 (each 2.5% by weight, total concentration 10% by weight) as a stock solution as an oligosaccharide. Diaion 5P207 was used as a commercially available packing material, and this was packed into a column (20 mmφ, 400
0 mmH, volume 1256 ml). The stock solution was flowed into this column at a ratio of 7% by volume based on the amount of packing material, and then pure water was added as an eluent at an Sv concentration of 0.6
The liquid was passed through the tube at a flow rate of 2 hr'''' (LV-2, 48 m/hr). The treatment temperature was room temperature (20°C).

When the chromatogram of the eluate was measured, the elution curve shown by the broken line in FIG. 1 was obtained. Further, the elution curves of each malto-oligosaccharide of G I'' G 4, which were determined by analyzing fractions of the eluate every 5 minutes using a high performance liquid chromatograph, were as shown by the solid line in FIG.

In such an isolation process, fractions of the eluate from 156 minutes to 220 minutes were collected and water was removed.
was recovered with a recovery rate of approximately 70%.

Example 2 A mixture of equal amounts of malto-oligosaccharides G3 to G5 as a stock solution (
! Isolation was carried out in the same manner as in Example 1, except that a 2% by weight aqueous ethanol solution was used as the eluent.

The measurement results of the chromatogram of the eluate at this time are shown as broken lines in Figure 2, and the elution curves of each malto-oligosaccharide obtained by analyzing the fraction of the eluate every 5 minutes with a high performance liquid chromatograph are shown as the broken line in Figure 2. This is shown by the solid line in Figure 2.

In the isolation process of this example, fractions of the eluate from 194 minutes to 310 minutes were collected.

When ethanol and water were removed, 100% pure G5 (maltopentaose) was recovered at a recovery rate of about 63%.

[Effects] As detailed above, the method for separating oligosaccharides of the present invention uses a column packed with a synthetic adsorbent packing material that does not have ion exchange groups, and elutes with water or an aqueous alcohol solution.

■ Uses a much cheaper filler than the gel filtration method.

■ Elution can be performed with water or an aqueous alcohol solution.

- Unlike maltose purification methods, several components can be isolated in one cycle.

[Phase] The device used has an extremely simple configuration.

Because of these various advantages, malto-oligosaccharides can be isolated with high purity and high recovery rate, which is extremely advantageous economically and industrially.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are diagrams showing chromatograms obtained in Example 1 and Example 2, respectively.

Claims (1)

[Claims] (1) A method for separating oligosaccharides by column chromatography, characterized in that the column is filled with a synthetic adsorbent packing material having no ion exchange group.