JP2639221B2 - Sugar production method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing saccharides having a fixed sugar chain length such as glucose, maltose, maltooligosaccharide and the like in a single product with high purity.

BACKGROUND ART As a method for obtaining a saccharide having a specific sugar chain length in a single product with high purity, a glucan such as starch is decomposed with a single or two or more kinds of appropriate amylases, and this is decomposed into various known column chromatography. For example, by separating them from oligosaccharides and monosaccharides not intended for other purposes (eg, Starch Science Handbook, p452, 1987, JP-A-57-209000, JP-A-62-9
No. 19210, Japanese Patent Publication No. 2-17158) or a method of further leading it to crystallization and separating it from other oligosaccharides or monosaccharides which are not contained in a trace amount and which are not intended (for example, Handbook of Starch Science, p456, 1987).

However, any of the above methods essentially involves the production of glucose, uncleaved dextrin, and other undesired oligosaccharides, and it has been virtually impossible to remove these undesired saccharides from the reaction system.

It has also been found that the coexistence of a trace amount of a non-target saccharide significantly prevents crystallization of the target saccharide having a specific sugar chain length.

Furthermore, if an attempt is made to obtain an oligosaccharide having as high a purity as possible by the above-mentioned known method, the production process becomes complicated, and there are problems in terms of yield and cost.

DISCLOSURE OF THE INVENTION Accordingly, the present inventors believe that there is likely to be room for improvement in known methods for producing saccharides, particularly glucose, maltose, substantially free of unintended saccharides that are not suitable for pharmaceutical use.
An attempt was made to construct a method for producing only a saccharide having a specific sugar chain length, such as maltooligosaccharide, as a single product.

The gist of the present invention is to continuously perform the following series of operations.

That is, a substance capable of being substantially separated from a target saccharide having a specific sugar chain length (hereinafter, referred to as a “separable substance”) is directly or via an intermediate from another sugar chain source by a glycosyltransferase. The sugar chain is transferred, and the resulting oligosaccharide is subjected to an enzyme (hereinafter referred to as "exo-type cleavage enzyme") that cuts a sugar chain of a specific length into an exo-type with respect to the oligosaccharide, thereby causing a specific specific target to be obtained. This is a method of isolating a sugar having a sugar chain length.

 The present invention is described in detail below.

The separable substance used in the present invention is chitosan beads, an ion exchange resin, a synthetic polymer resin, or a carrier used as an immobilized carrier or the following general formula [I] (Wherein R represents hydrogen, lower alkyl, hydroxyalkyl, phenylalkyl, phenylalkenyl, phenylalkynyl, phenoxyalkyl, phenoxyalkenyl, or phenoxyalkynyl, where phenyl includes substituted ones). The compounds represented by the formula (hereinafter referred to as "moranolines"), nojirimycin, aminocyclitol, aminocyclitol derivative or glucuronic acid, or saccharides having polarities which can be adsorbed to ion exchange resins such as glucosyl or oligoglycosyl (hereinafter referred to as "moranolines") "Polar sugar"). The carrier may be in the form of beads, a film, a fiber, or the like. Moranolins can be obtained from mulberry bark, actinomycetes, and the like (for example, Japanese Patent Application Nos. 54-159417, 55-76838, and 5-5).
No. 6-9919, Japanese Patent Application No. 57-93997, Japanese Patent Publication No. 59-27337
No.). Glucosyl and oligoglucosyl forms of moranolines can be produced by the methods already disclosed (eg, Agricultural and Biological Chemistry, 49 , 2159 (1985)). A method for obtaining aminocyclitol from validamycins is disclosed (for example, Japanese Patent Application No. 55-128157), and aminocyclitol can be prepared from a commercially available validamycin preparation.

In order to transfer a sugar chain to a carrier having no residue capable of transferring a sugar chain by a sugar chain transferase such as glucosyl or a polar sugar at the terminal, the sugar chain can be transferred to the carrier in advance by a sugar chain transferase. It is necessary that the intermediate having the residue is bound directly or via a spacer. Here, examples of the intermediate include polar sugars, monosaccharides such as glucose, and oligosaccharides such as maltose and maltotriose. In order to bind the intermediate to the carrier, for example, maltose as an intermediate is reduced to a carrier such as sodium cyanoborohydride (NaBH ₃ CN) on a chitosan bead carrier to which a spacer having an amino group at the terminal is bound. And the like. Here, as the spacer, oxirane, glutaraldehyde (GLA)
And monofunctional and bifunctional substances used in the immobilization technique.

Glycosyltransferases used to transfer sugar chains from other sugar chain sources include, for example, cyclodextrin glycosyltransferase (CGTase), α-amylase and the like.

CGTase has been reported to be produced from several strains, for example, Bacillus macerans, Bacillus megaterium, Bacillus circulans, alkalophilic Bacillus, production from heat-resistant Bacillus, etc. I have. Each cyclodextrin produced has its own characteristics, but the CGTase used in the present invention
Can adequately serve its purpose with any of these types of CGTases. It is also known that some strains other than enzymes directly form glycosyltransfer products (for example, Japanese Society of Agricultural Chemistry, 4N-2, Showa 5).
(Issued March 10, 2006), which can also serve the purpose of the glycosyltransferase of the present invention.

The pH and reaction temperature in the transglycosylation reaction are slightly different depending on the glycosyltransferase used, for example, CGTase
Is used, the pH is in the range of 4.0 to 11.5, preferably pH 5.5.
The reaction temperature is suitably in the range of 20 to 85 ° C, preferably in the range of 45 to 65 ° C. When CGTase is used as a transferase, the time required for the sugar chain transfer reaction varies considerably depending on the pH, the reaction temperature, the enzyme concentration, and the concentration of the sugar chain source, but is usually several hours to 4 days. CG
It is preferable that the amount of Tase used is to transfer as many sugar chains as possible to the residues to which sugar chains can be transferred by the sugar chain transferase, so that the ratio of the enzyme to the starch-based polysaccharide of the sugar chain source is large. Better results are obtained. Specifically, it depends on the starch polysaccharide of the sugar chain source used, pH, and reaction time,
A suitable amount is about 500 to 5,000 units (BV method), preferably 1,000 to 2,000 units, per gram of the starch-based polysaccharide.

Here, examples of the sugar chain supply source include starch, starch-based saccharide, cyclodextrin, and the like. As the starch, any commercial starch that is commercially available can be used. As the starch-based saccharide, any of various intermediate hydrolysates such as dextrin, amylose and amylopectin, or those having a high, medium or low degree of polymerization can be used. The concentration of the starch-based polysaccharide varies depending on the action time and the amount of transferase according to the requirements in production, but is suitably 5 to 60%, preferably 10 to 20%. For dextrins, up to 30-50% is practical. However, when starch is used at a high concentration as a sugar chain source, its viscosity becomes extremely high.
Generally, starch is liquefied with CGTase, α-amylase or the like. Further, in order to increase the utilization rate of the starch-based polysaccharide to be added, it is preferable that the relative amount of the starch-based polysaccharide is slightly lower than the amount of the transferase, for example, the sugar chain transfer per 1 g of the starch-based polysaccharide. An enzyme amount of 2000-3000 units is preferred.

When a polar sugar is used as the separable substance, its concentration can be increased to, for example, about 15% in the case of moranoline. However, it is effective that the weight ratio of the sugar chain source to moranolin is about 2 to 20 times.

When a carrier is used as a separable substance, the sugar chain-extended carrier is usually sufficiently washed after the completion of the sugar chain transfer reaction. Further, after washing, for example, heating at 100 ° C. for 5 minutes to inactivate the remaining glycosyltransferases, and further sufficient washing can be performed. The glycosyltransferase used was ultrafiltration (hereinafter "U
F ”)) method.

When a polar sugar is used as the separable substance, after the sugar chain transfer reaction is completed, the polar sugar and the glucose oligomer transferred to the sugar are adsorbed to the ion exchange resin. In this case as well, the glycosyltransferase used can be easily recovered by the UF method or the like.If the glycosyltransferase is not recovered, the reaction solution is directly used. Apply to a suitable ion exchange resin. Here, as the ion exchange resin, Dowex 50W-X2 (registered trademark),
Diaion SA-11A (registered trademark), Amberlite IR
-120 (registered trademark) and the like, which are appropriately selected depending on the polarity of the polar sugar. It goes without saying that the amount of the ion exchange resin must be increased or decreased depending on the amount of the polar sugar used in the reaction. In addition, after the polar saccharide and the glucose oligomer transferred to the saccharide are adsorbed on the ion exchange resin, they are usually sufficiently washed. After washing, these are generally eluted from the ion-exchange resin, for example, eluted with 1N ammonia water or the like.

Subsequently, at a pH suitable for the exo-type cleavage enzyme, the exo-type cleavage enzyme is usually treated at a reaction temperature of 30 to 55 ° C. for several hours to 2 hours.
The exo-type cleavage enzyme includes glucoamylase, β-amylase, maltooligosaccharide-forming enzyme and the like. For example, when producing maltose, β-amylase can be used, but pH 4.5 to 6.0 suitable for the β-amylase to be used, for example, pH 4.8
It is appropriate to adjust the reaction solution, add β-amylase to the reaction solution, and react at a reaction temperature of 30 to 60 ° C. for 1 to 2 days. The amount of the exo-type cleavage enzyme to be added depends on the type of the exo-type cleavage enzyme and the separable substance to be used, but when the separable substance is a carrier, if 20 to 100 units are added per 1 ml of the carrier,
Cutting can be completed within one working day of industry.

When polar sugar is used as the separable substance and eluted from the ion-exchange resin in the previous operation, the exo-type cleavage enzyme is separated as it is after the action of the exo-type cleavage enzyme or by a separation method such as an appropriate UF membrane method. By applying the polar sugar again to an ion-exchange resin that adsorbs the used polar sugar, it is possible to substantially isolate only the target saccharide having a specific sugar chain length.
The amount of resin needs to be increased or decreased mainly depending on the amount of polar sugar charged.

When a polar saccharide is used as the separable substance and the exo-type cleavage enzyme is allowed to act without being eluted from the ion exchange resin in the previous operation, or when a carrier is used as the separable substance, after the exo-type cleavage enzyme is acted on, By separating the exo-type cleavage enzyme by a suitable separation method such as filtration or a UF membrane method, it is possible to substantially isolate only the target saccharide having a specific sugar chain length.

The exo-type cleavage enzyme isolated as described above can be reused.

Concentration of the final solution can be achieved by vacuum evaporation using a rotary evaporator, reverse osmosis (RO) membrane method, etc.
It can be carried out by a commonly used concentration means.

When a polar sugar is used as the separable substance, the glycosyltransferase and the exo-type cleavage enzyme can be used in the form of a solution, but the immobilized enzyme prepared by a method for producing a widely used known immobilized enzyme is used. The reaction can also be carried out in the form of In particular, when a crude enzyme is used, using an immobilized enzyme can reduce impurities and is advantageous in the purification step.

The reaction of maltose produced by the production method according to the present invention is schematically shown as follows.

(In the formula, A represents a separable substance, G represents glucose, and B represents β-amylase.
Are not necessarily the same. ▼ indicates a cleavage site of β-amylase. ) As in the above formula, β-amylase leaves a disaccharide or trisaccharide without cleavage due to its properties (in the case of the above formula, GG, A-
G and AGG). Therefore, only maltose (GG), which is a solution and a neutral sugar, is separated from solid or polar sugars (A, AG and AGG) by filtration or ion exchange resin. Can be easily isolated.

Then, after separating maltose, glycosyltransferase can extend sugar chains on other residues again, and ultimately, continuous maltose production can be achieved by repeating transglycosylation, cleavage, and separation. Becomes

From the above, it can be said that the production method according to the present invention is a novel and revolutionary production method having continuity and capable of substantially obtaining only a saccharide having a specific target sugar chain length as a single product. it can. Further, according to the present invention, not only the advantage that only a saccharide having a specific sugar chain length can be obtained, but also the production process is extremely simple as compared with the conventional method, and the cost, labor, and the like are significantly reduced. And the yield can be sufficiently increased.

The saccharides obtained by the production method of the present invention, for example, maltose are particularly useful for pharmaceuticals requiring high purity, and examples of the form of use include instillation. It can also be used for food. Maltotriose, maltotetraose, maltopentaose, maltohexaose and the like can be used as diagnostic agents and the like. Further, the crystallization of maltitol, which is a sweetener produced by reducing maltose, is better as the raw material maltose is pure, so that maltose produced by the production method of the present invention has the following disadvantages. Is also very effective.

Examples of applying the saccharide according to the present invention for medical use include, for example, infusion. When applied to infusion, for example, maltose is known to be more beneficial than glucose or the like because its 10% solution is isotonic and has twice the energy value of an equivalent amount of glucose ( "Anesthesia and Resuscitation," Vol. 20, No. 3, page 163. 1984
Similarly, a 15% solution of isotonic maltotriose is also more effective, having an energy value three times that of glucose.
When the saccharide according to the present invention is applied for medical use, for example, those having the following composition can be applied, but generally, saccharides (maltotriose, maltotetraose,
Maltopentaose, maltohexaose, etc.) alone or simultaneously with glucose or maltose, and may appropriately contain an inorganic salt such as sodium chloride, potassium chloride or sodium acetate.

(Example of infusion composition) 15 g of maltotriose, 0.03 g of potassium chloride, 0.02 g of calcium chloride, 0.6 g of sodium chloride and 0.31 g of sodium lactate are mixed to make a composition of 100 ml of infusion.

BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described in more detail and specifically with reference examples and examples, but the present invention is not limited to these examples.

Reference Example 1 Preparation of immobilized CGTase beads First, Bacillus macerans CGTase (Contizyme,
200 ml (manufactured by Amano Pharmaceutical Co., Ltd.) was dialyzed against a 0.01 M acetate buffer (pH 6.0). Separately, 50 ml of Chitopearl BCW-3010 (registered trademark, manufactured by Fuji Spinning Co., Ltd., the same applies hereinafter) was gently shaken at room temperature for 24 hours in 0.1 M acetate buffer (pH 5.0) containing 2.5% glutaraldehyde, and then sufficiently shaken. Washed with water. Then, the supernatant obtained by centrifuging the above inner solution of dialysis was added to the activated chitopearl, and after binding the enzyme while gently shaking at 4 ° C. for 16 hours, the plate was thoroughly washed with water. by this,
The immobilized enzyme of 23.8 mg protein / ml beads was obtained.

Reference Example 2 Preparation of Immobilized β-Amylase First, 4.8 ml of sweet potato-derived β-amylase (manufactured by Sigma, ammonium sulfate suspension) was dialyzed against 0.01 M acetate buffer (pH 5.0). Separately, 10 ml of Chitopearl BCW-3010 2.
0.1M acetate buffer (pH 5.0) containing 5% glutaraldehyde
Gently at room temperature for 24 hours, followed by thorough washing with water.
Then, the supernatant obtained by centrifuging the above inner solution of dialysis was added to the activated chitopearl, and after binding the enzyme while gently shaking at 4 ° C. for 16 hours, the plate was thoroughly washed with water. This yielded 6.44 mg protein / ml beads of the immobilized enzyme.

Reference Example 3 Preparation of carrier having maltose bound to chitosan beads 5 ml of chitopearl BCW-3010 and 1.8 g of maltose as chitosan beads were added to 20 ml of a mixed solvent of methanol / water = 1/1, and maltose was dissolved. Sodium boron was added, the pH was adjusted to 3 to 4 with acetic acid, and the mixture was reacted for 3 days. After filtering the beads, the beads were sufficiently washed with water to obtain the present carrier.

Reference Example 4 Preparation of immobilized glucoamylase 35 g of glucoamylase "Gluczyme NL-3" (manufactured by Amano Pharmaceutical Co., Ltd.) was diluted with 35 l of ion-exchanged water, and the column was filled with an enzyme concentrate obtained by purification by the UF method. Quito Pearl
It was introduced into 33 l of BCW-3010 and adsorbed. That is, this enzyme solution was adsorbed by circulating a solution finally contained in 56 liters of 0.05 M acetate buffer (pH 5.0) at 8 ° C. for 24 hours, and then a 0.05 M acetate buffer (pH 5) was added. .0) was washed with 15 l, and immobilization was completed by circulating 120 l of 0.05 M acetate buffer (pH 5.0) containing 2.5% glutaraldehyde through the column. Excess glutaraldehyde was removed by passing 550 l of water. This yielded 24.4 mg protein / ml beads of the present immobilized enzyme.

Example 1 A 50 ml Erlenmeyer flask was charged with 300 mg each of moranolin, N-methyl moranoline, N-benzyl moranoline, N-hydroxyethyl moranoline, N-butyl moranoline, and glucosyl moranoline, each of which contained soluble starch 120 mg.
0 mg was added, and the mixture was heated and dissolved in 10 ml of water, and 5 ml of the immobilized CGTase beads prepared by the method of Reference Example 1 was added. The mixture was shaken at 40 ° C. for 3 days to perform a transfer reaction. Then, 3 ml of each of these reaction solutions was taken, applied to 10 ml of strongly acidic ion exchange resin Dowex 50W-X2, washed sufficiently with water and 60 ml of 1N aqueous ammonia.
And concentrated to dryness with a rotary evaporator.
The weight of this transglycosylation is shown in Table 1, and the TLC
(Kieselgel 60F _254, Merck; eluent: n-propanol / aqueous ammonia / water = 6/2/1, coloring agent: After spraying 10% ethanol solution of sulfuric acid, heated on a flame, the same hereinafter.) The As shown in FIG.

Next, the transglycosylated product was dissolved in water so as to have a concentration of 50 mg / ml, the pH was adjusted to 5 to 6 with 2N hydrochloric acid, and 200 particles of the immobilized β-amylase prepared by the method of Reference Example 2 were added thereto. , 37 ℃
For 5 hours. TLC of this β-amylase reaction solution
Is shown in FIG.

It is clear from FIG. 2 that maltose was generated.

Subsequently, each of the β-amylase reaction solutions was applied to 10 ml of strongly acidic ion exchange resin Dowex 50W-X2, and the passed solution was neutralized with 2N sodium hydroxide solution and concentrated to dryness with a rotary evaporator. The obtained maltose weights are shown in Table 1.

The TLC of these maltose fractions is shown in FIG.

It is apparent from FIG. 3 that only maltose is produced.

Example 2 In a 50 ml Erlenmeyer flask, 60 mg validinamine and 1200 soluble starch were used.
of the CGTase beads prepared by the method of Reference Example 1 and added thereto.
The mixture was shaken at 3 ° C. for 3 days to carry out a transfer reaction. Then, take 3 ml from this reaction solution, apply to 10 ml of strongly acidic ion exchange resin Dowex 50W-X2, wash thoroughly with 1N ammonia water 60m
The mixture was eluted with l and concentrated to dryness on a rotary evaporator. The weight of the transglycosylated product was 274 mg, and the TLC of the transglycosylated product is shown in FIG.

Next, 5.5 ml of water was added to the transglycosylated product, and the pH was adjusted with 2N hydrochloric acid.
Was adjusted to 5 to 6, and 200 particles of the immobilized β-amylase prepared by the method of Reference Example 2 were added thereto and reacted at 37 ° C. for 5 hours. The TLC of this β-amylase reaction solution is shown in FIG.

It is clear from FIG. 5 that maltose was generated.

Subsequently, the β-amylase reaction solution was applied to 10 ml of strongly acidic ion exchange resin Dowex 50W-X2, and the passing solution was 2N.
After neutralization with caustic soda, the mixture was concentrated to dryness using a rotary evaporator. The resulting maltose weight was 113 mg. The TLC of this maltose fraction is shown in FIG.

It is apparent from FIG. 6 that only maltose is produced.

Example 3 N- (1,3-dihydroxy-2-propyl) valiolamine 60 mg, enzyme dextrin "Amicol" (registered trademark, manufactured by Nippon Starch) 240 mg, and CGT
Add 1mg of ase “Contizyme” (manufactured by Amano Pharmaceutical Co., Ltd.), and add 2m
Then, water was added to the mixture, and the mixture was subjected to a transfer reaction while shaking at 50 ° C. for 48 hours. The whole reaction solution was thoroughly washed with water through about 10 ml of strongly acidic ion exchange resin Dowex 50W-X2, eluted with 60 ml of 1N aqueous ammonia, and the fraction was concentrated to dryness with a rotary evaporator. The weight of the obtained transglycosylation product was 118.
It was 6 mg.

To this transglycosylation product, 10 ml of 0.1 M acetate buffer (pH 4.8) was added to dissolve, followed by addition of 100 μl of β-amylase (derived from sweet potato, ammonium sulfate suspension, manufactured by Seikagaku Corporation), followed by addition of 37 μl.
The reaction was carried out at 16 ° C for 16 hours. Then, the β-amylase reaction solution was added to 10 ml of strongly acidic ion exchange resin Dowex 50W-X2.
Then, the flow-through section was neutralized with 2N sodium hydroxide together with the washing solution, and concentrated to dryness with a rotary evaporator to obtain 129.
8 mg of maltose were obtained.

Example 4 4.7 g of oligoglucosyl moranoline was dissolved in 170 ml of water, adjusted to pH 4.9 with 1N hydrochloric acid, and then β-amylase (manufactured by SERVA, derived from sweet potato, 848 U / mg protein, recrystallized three times) Product, 5 mg / 1 ml), and reacted at 37 ° C. for 20 hours. The reaction solution was treated with a strong basic ion exchange resin Diaion SA-11A 30.
Then, the mixture was passed through the plate and washed sufficiently with ion-exchanged water. And
The passing solution and the washing solution were combined, passed through 50 ml of strongly acidic ion exchange resin Dowex 50W-X2, and sufficiently washed with ion exchange water. Subsequently, the flow-through liquid and the washing liquid are combined and concentrated to dryness.
4 g were obtained.

The maltose content was determined by high performance liquid chromatography (column: Nucleosil 5NH ₂ , 5 μm, 4 mm id × 25; mobile phase: acetonitrile / water = 70/30; detection: differential refractometer)
As a result of the analysis, it was 98.3%.

Example 5 15 g of soluble starch was dissolved in 100 ml of water by heating, and 5 g of moranoline was dissolved. Then, after adding water to make the total volume 170 ml, the immobilized CGTase beads prepared by the method of Reference Example 1 were used.
20 ml was added and reacted at 55 ° C. for 42 hours. Then, the immobilized CGTase beads were removed by filtration, and after washing with water, the filtrate and the washing liquid were combined, and 50 m of strong acid ion exchange resin Dowex 50W-X2 was used.
l passed. After washing with water, this was eluted with 1N aqueous ammonia, and the eluate was freeze-dried to obtain 11.3 g of a powder.

This powder was dissolved in 250 ml of water, the pH was adjusted to 5.0 with 2N hydrochloric acid, and 10 ml of immobilized β-amylase prepared by the method of Reference Example 2 was added, followed by reaction at 37 ° C. for 3 hours. The reaction solution was filtered to remove the immobilized β-amylase. After washing with water, the filtrate and the washing solution were combined, passed through 30 ml of a strongly basic ion exchange resin DIAION SA-11A, and sufficiently washed with water. Subsequently, the combined passing solution and washing solution were concentrated under reduced pressure to about 20 ml, and 100 ml of the strongly acidic ion exchange resin Dowex 50W-X2 was added.
Passed through. After gently washing with water, the passing solution and the washing solution were combined and freeze-dried to obtain 800 mg of maltose powder.

As a result of analyzing 10 μl of this product having a maltose concentration of 85 mg / ml under the same high performance liquid chromatography conditions as in Example 4, as shown in FIG. 7, this product was 100% maltose.

Example 6 To 5 ml of maltose-bound chitopearl prepared in Reference Example 3 was added 3 g of the enzyme dextrin "Amicol" (registered trademark, manufactured by Nisse Chemical Co., Ltd.) and 10 ml of water. The mixture was adjusted to pH 6.0 with 2N hydrochloric acid, and then CGTase (contizyme) was added. (Manufactured by Amano Pharmaceutical Co.) was added, and the mixture was gently shaken at 50 ° C. for 24 hours. Then, the carrier beads were filtered, washed sufficiently with water, heated in boiling water at 100 ° C. for 5 minutes, and further thoroughly washed with water.

Separately, as a control, untreated chitopearl was reacted similarly in place of maltose-bound chitopearl.

10 ml of 0.2 M acetate buffer was added to 5 ml of the carrier beads thus obtained, and β-amylase (Selva (SB)
RVA) (50 μl) and gently shaken at 37 ° C.

Sample 500 μl of the reaction solution over time, add 1 ml of 3,5-dinitrosalicylic acid reagent, heat in boiling water at 100 ° C. for 10 minutes, cool with water, add 5 ml of water and remove maltose released at 535 nm absorbance. It was measured. The result is shown in FIG.
It was shown to.

Compared with untreated chitopearl, maltose-bound chitopearl clearly showed maltose release. The supernatant after the β-amylase treatment was concentrated and confirmed by TLC. As a result, only maltose was detected, and no other small sugars were mixed.

Example 7 In a 50 ml Erlenmeyer flask, the maltose-bound chitopearl used in Example 6 after cleavage with β-amylase and the control chitopearl were heat-treated at 100 ° C for 5 minutes, washed thoroughly with 5 ml, and the enzyme dextrin “Amicol” ( 3 g of registered trademark (manufactured by Nippon Starch Co., Ltd.), 10 ml of water, and 5 ml of CGTase “Contizyme” (manufactured by Amano Pharmaceutical Co., Ltd.) were added, and the reaction was performed again while shaking at 50 ° C. for 24 hours. Then, after the beads were filtered and washed, they were heated at 100 ° C. for 5 minutes and further sufficiently washed with water.

5 ml of these beads was added to 10 ml of 0.2 M acetate buffer (pH 4.8), and 50 μl of β-amylase (derived from sweet potato, ammonium sulfate suspension, manufactured by Seikagaku Corporation) was added and incubated at 37 ° C. 0.5 ml was sampled and added with 1,5-dinitrosalicylic acid reagent 1 ml, heated in boiling water at 100 ° C. for 5 minutes, cooled with water, added 5 ml of water and measured the released maltose at an absorbance of 535 ml, Fig. 8-B shows the change in absorbance.
It was shown to.

As is clear from FIG. 8-B, similarly to the first reaction of Example 6, the formation of maltose was sufficiently recognized as compared with the control.

Thus, the production of maltose can be repeated even when maltose-bound beads are used. That is, the possibility of continuous production was suggested.

Example 8 149 mg of a glycosyl transfer product of moranoline obtained in the same manner as in Example 1 was dissolved in 3 ml of water, and adjusted to pH 5.0 with 2N hydrochloric acid.
200 immobilized glucoamylase beads prepared in Reference Example 4 were added, and the mixture was shaken at 40 ° C. for 4 hours to release glucose. This reaction solution was applied to 10 ml of Dowex 50W-X2, and the fraction that appeared without being adsorbed was neutralized with 2.5N sodium hydroxide solution, and then concentrated and dried with a rotary evaporator to obtain 58.3 mg of pure glucose. .

 FIG. 9 shows the TLC.

Example 9 Each of N-propylmoranoline and a transglycosylation product of N-benzylmoranoline obtained in the same manner as in Example 1 was prepared.
2.5 mg was dissolved in 250 μl of water, adjusted to pH 6.0 with 2N hydrochloric acid, and added to Streptomyces griseus NA-468 (FER
250 μl of a maltotriose-forming amylase solution prepared from MP-2227) (95 units / 0.25 mg protein; 1 unit is an amount of an enzyme that produces a reducing sugar corresponding to 1 mg of maltotriose per hour), and then added at 40 ° C. For 2 hours to produce maltotriose. FIG. 10 shows the reaction mixture.
TLC was indicated.

Further, the reaction solution was applied to Dowex 50W-X2, and the fraction that appeared without being adsorbed was neutralized with 2.5N sodium hydroxide. FIG. 11 shows the TLC of this maltotriose fraction.

From FIG. 11, it is clear that only maltotriose is produced.

In addition, when quantified based on the maltotriose standard by the 3,5-dinitrosalicylic acid method, 5.2 mg and 5.7 mg, respectively, were obtained.
mg of maltotriose was confirmed.

Example 10 N-propylmoranoline and N-benzylmolanoline glycosyl transfer product obtained in the same manner as in Example 1
2.5mg Pseudomonas Stats
stutzeri) 250 μl of a maltotetraose-forming amylase solution prepared from IFO-3773 strain (26.25 units / 0.24 mg)
Protein; 1 unit is an amount of an enzyme that produces a reducing sugar equivalent to 1 mg of maltotetraose per hour).
Shake gently at 2 ° C. for 2 hours to produce maltotetraose.

Further, this reaction solution was applied to Dowex 50W-X2 to obtain a fraction that was not adsorbed, and the TLC of the maltotetraose fraction at the 20th minute of the reaction is shown in FIG.

From FIG. 12, it is clear that only maltotetraose is produced.

In addition, when quantified based on a maltotetraose standard by the 3,5-dinitrosalicylic acid method, 2.0 mg each,
1.6 mg of maltotetraose was confirmed.

Example 11 N-propylmoranoline and N-benzylmolanoline glycosyl transfer product obtained in the same manner as in Example 1
2.5mg Pseudomonas sp.
250 μl of maltopentaose-forming amylase solution prepared from KO-8940 (FERM P-7456) (10.13 units / 1.85 m)
g protein; 1 unit is an amount of an enzyme that produces a reducing sugar equivalent to 1 mg of maltopentaose per hour).
Shake gently at 2 ° C. for 2 hours to produce maltopentaose.

Further, this reaction solution was applied to Dowex 50W-X2 to obtain a fraction that was not adsorbed, and a TLC at the 90th minute of the reaction of the maltopentaose fraction from N-benzylmolanoline was shown in FIG.
3 is shown.

From FIG. 13, it is clear that only maltopentaose is produced.

When quantified by the 3,5-dinitrosalicylic acid method based on a maltopentaose standard, 0.6 mg,
0.8 mg of maltopentaose was confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a TLC of a transglycosylated product in Example 1.
In the figure, 1 is for molanolin, 2 is for N-methylmoranoline,
3 is N-benzylmoranoline, 4 is N-hydroxyethylmoranoline, 5 is N-butylmoranoline, 6 is glucosylmoranoline, each of which is a sugar chain transfer product, and a, b,
c, d, e, and f represent the above moranolines as standards.

FIG. 2 shows the TL of the β-amylase reaction solution in Example 1.
C. 1, 2, 3, 4, 5, and 6 are the same as those described in FIG. M represents maltose.

FIG. 3 is a TLC of the maltose fraction in Example 1. 1, 2, 3, 4, 5, and 6 are the same as those described in FIG. M represents maltose.

FIG. 4 is a TLC of the transglycosylated product in Example 2.
7 indicates a transglycosylation product of validamin, and Ba indicates validamin.

FIG. 5 shows the TL of the β-amylase reaction solution in Example 2.
C.

7 indicates a glycosyl transfer product of validinamine, and M indicates maltose.

FIG. 6 is a TLC of the maltose fraction in Example 2. 7 indicates a glycosyl transfer product of validinamine, and M indicates maltose.

FIG. 7 is a high performance liquid chromatogram of the maltose powder obtained in Example 5. In the figure, A indicates the peak of the solvent, and B indicates the peak of maltose.

FIG. 8-A shows the result in Example 6. ● indicates the case where maltose-bound chitopearl was used, and ○ indicates the case where untreated chitopearl was used. The vertical axis represents the absorbance of the solution, and the horizontal axis represents time (hour).

FIG. 8-B shows the result in Example 7. ● indicates the case where maltose-bound chitopearl was used, and ○ indicates the case where untreated chitopearl was used. The vertical axis represents the absorbance of the solution, and the horizontal axis represents time (hour).

FIG. 9 is a TLC of the glucose fraction in Example 8. In the figure, 8 is a glucose fraction, G is glucose,
Shown respectively.

FIG. 10 is a TLC of a maltotriose-forming amylase reaction solution in Example 9. In the figure, 9 indicates N-propylmoranoline, 10 indicates N-benzylmolanoline transfer product, and T indicates maltotriose.

FIG. 11 shows the TL of the maltotriose fraction in Example 9.
C. In the figure, 9 indicates a maltotriose fraction from N-propylmoranoline, 10 in the figure indicates a maltotriose fraction from N-benzylmoranoline, and T indicates maltotriose.

FIG. 12 shows the maltotetraose fraction in Example 10.
TLC. In the figure, 11 indicates the maltotetraose fraction from N-propylmoranoline, 12 in the figure indicates the maltotetraose fraction from N-benzylmoranoline, and Te indicates maltotetraose.

FIG. 13 shows the maltopentaose fraction in Example 11.
TLC. In the figure, 13 is a maltopentaose fraction from N-benzylmoranoline, Pe is maltopentaose,
Shown respectively.

Claims (2)

(57) [Claims] Claims: 1. A sugar chain is transferred from a different sugar chain source to a substance which can be substantially separated from a target sugar having a specific sugar chain length, directly or via an intermediate, by a sugar chain transferase; The obtained oligosaccharide is subjected to an enzyme that cuts a sugar chain of a specific length into an exo-form on the oligosaccharide to isolate a saccharide of a specific sugar chain length of interest. Manufacturing method. 2. A substance which can be substantially separated from a target saccharide having a specific sugar chain length is a carrier or the following general formula [I]: (Wherein R represents hydrogen, lower alkyl, hydroxyalkyl, phenylalkyl, phenylalkenyl, phenylalkynyl, phenoxyalkyl, phenoxyalkenyl, or phenoxyalkynyl, where phenyl includes substituted ones). The method for producing a saccharide according to claim 1, which is a compound represented by the formula, nojirimycin, aminocyclitol, an aminocyclitol derivative, glucuronic acid, or a glucosyl or oligoglucosyl derivative thereof.