JP3655325B2 - Mannosyl-cyclodextrin - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a novel method for producing a mannosyl-cyclodextrin in which a mannosyl group is bonded to the glucosyl group of cyclodextrin by an α bond. Specifically, the present invention relates to an efficient method for producing mannosyl-cyclodextrin from an α-mannosyl sugar compound and maltooligosaccharide utilizing the reaction of α-mannosyltransferase and cyclodextrin synthase.
[0002]
[Background Art and Problems to be Solved by the Invention]
Cyclodextrins (hereinafter sometimes abbreviated as CD) are cyclic dextrins in which glucose is linked by α-1,4 bonds, and α-, β-, and γ-CD each consisting of 6, 7, and 8 glucoses. Is well known. Recently, in order to improve the solubility of CDs, branched CDs in which a glucosyl group or a maltosyl group is bonded to these CDs by α-1,6 bonds have been synthesized.
[0003]
These CDs and branched CDs have cavities inside the molecules, and the inside of the cavities are hydrophobic, so that they have an inclusion action and have the property of incorporating various oily substances. Since CD and branched CD have such properties, they are widely used in fields such as the food industry, cosmetic industry, and pharmaceutical industry.
[0004]
Recently, in the field of the pharmaceutical industry, in order to reduce the side effects of drugs, active attention has been paid to the cell recognition of carbohydrates and the use of this as a sensor for the labeled cells of drug carriers in drug delivery systems. Has been done. In particular, it is well known that galactose has a strong affinity for liver tissue, and mannose has a strong affinity for liver parenchymal cells, liver non-parenchymal cells and macrophages.
[0005]
Previously, in view of the above-mentioned situation, the mannosyl group was bonded to the 6-position hydroxyl group of the glucosyl group of CD by allowing α-mannosyltransferase to act on a solution containing CD and an α-mannosyl sugar compound. Mannosyl-CD has been successfully developed.
[0006]
However, the conventional methods for directly synthesizing mannosyl-CD using the transfer reaction of mannosyltransferase have a problem in yield, and development of a more efficient method for producing mannosyl-CD is desired. It was.
[0007]
[Means for Solving the Problems]
Therefore, as a result of intensive studies, the present inventor made a commercially available α-mannosyl transferase to act on an α-mannosyl sugar compound and malto-oligosaccharide such as maltopentaose to transfer a mannosyl group by α bond. -It was found that mannosyl-CD in which a mannosyl group was directly bonded to the glucose residue of CD could be synthesized efficiently by synthesizing maltooligosaccharide and allowing cyclodextrin synthase to act on this. We completed the present invention based on this finding.
[0008]
That is, the present invention provides a step of allowing α-mannosyltransferase to act on a solution containing malto-oligosaccharide and mannose and / or α-mannosyl sugar compound, and causing cyclodextrin synthase to act on the mannosyl-maltooligosaccharide produced in the step . The present invention provides a novel method for producing a mannosyl-cyclodextrin in which a mannosyl group is bonded to a hydroxyl group of a glucosyl group of cyclodextrin by an α bond.
[0009]
The branched CD obtained by the present invention can be represented by structural formulas I to III shown in FIG.
[0010]
The branched CD according to the present invention comprises a step of allowing α-mannosyltransferase to act on a solution containing malto-oligosaccharide and mannose and / or α-mannosyl sugar compound, and cyclodextrin synthesis on the mannosyl-malto-oligosaccharide obtained in this step. It can be produced by a process in which an enzyme is allowed to act.
[0011]
The present invention is described in detail below.
The malto-oligosaccharide used in the present invention is a series of oligosaccharides in which glucose is α-1,4-linked, and the chain length is not limited, but usually an oligosaccharide having up to about 10 carbon atoms is used. . Of these, maltotetraose, maltopentaose, maltohexaose and the like are preferable, and maltopentaose is particularly preferably used. These may be either a crude product or a purified product, or may be a mixture.
[0012]
Next, the α-mannosyl sugar compound which is a sugar donor used in the present invention is usually methyl-α-mannoside, ethyl-α-mannoside, phenyl-α-mannoside, paranitrophenyl-α-mannoside, α- Examples include glycosides and oligosaccharides containing an α-mannosyl group such as manno-oligosaccharides, polysaccharides, partially decomposed products thereof, and mixtures thereof (including mixtures with mannose).
[0013]
As the α-mannosyltransferase used in the present invention, when it is allowed to act on a solution containing a maltooligosaccharide and a sugar donor, that is, mannose or an α-mannosyl sugar compound, the α-mannosyl sugar compound is decomposed, and the α- Any compound that synthesizes mannosyl-malto-oligosaccharides by catalyzing the condensation reaction of mannose and malto-oligosaccharides by transferring the mannosyl group to malto-oligosaccharides with an α bond and synthesizing mannosyl-malto-oligosaccharides can be used. It can be used.
[0014]
This α-mannosyltransferase is widely distributed in nature. Usually, plant-derived enzymes such as red bean or almond, enzymes derived from animals such as turban shell and liver (bovine, rat, human), and enzymes derived from microorganisms such as Arthrobacter orescens and Aspergillus niger are often used. Although used, it is preferable to use an enzyme derived from a red bean.
[0015]
The mannosyl-malto-oligosaccharide produced by the action of the transferase is used for the synthesis of mannosyl-CD, and this can be used as a crude product, a purified product or a mixture.
Next, as the cyclodextrin synthase used in the present invention, those derived from microorganisms are used, and those derived from Bacillus stearothermophilus or Bacillus circulans are usually used.
[0017]
In the method of the present invention, the reaction for synthesizing mannosyl-malto-oligosaccharide is performed under the following conditions. The solution (aqueous solution or suspension) containing malto-oligosaccharide and sugar donor usually has a malto-oligosaccharide concentration of about 1 to 90% (W / W), preferably 2.5 to 50% (W / W). The donor concentration is about 1-90% (W / W), preferably 2.5-50% (W / W). The ratio (weight) of sugar donor to maltooligosaccharide varies depending on the type of sugar donor used, but is usually in the range of 0.1 to 50 times, preferably in the range of 0.3 to 2 times. Is appropriate.
Furthermore, the reaction for synthesizing mannosyl-CD is set so that the concentration of mannosyl-malto-oligosaccharide is usually about 1 to 90% (W / W), preferably 2.5 to 25% (W / W). Do it.
[0018]
The reaction for synthesizing mannosyl-maltooligosaccharide and the reaction for synthesizing mannosyl-CD are both carried out under conditions of pH 3 to 10, preferably 4 to 9, temperature 20 to 90 ° C, preferably 40 to 70 ° C. Is appropriate. Since the amount of enzyme used is closely related to the reaction time, the amount of enzyme used is usually such that the reaction is completed in 0.1 hour to 2 weeks, preferably 1 hour to 1 week, but is not limited thereto. It is not something.
[0019]
Next, the reaction product obtained by the above method is subjected to high performance liquid chromatography, and the reaction product is fractionated and fractionated, followed by enzymatic decomposition, molecular weight measurement by FAB-MS, and high performance liquid chromatography. As a result of elution time and structural analysis by ¹³ C-NMR, it was confirmed that the branched CD was represented by structural formulas I to III shown in FIG.
[0020]
【Example】
EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
Example 20 g of methyl-α-mannoside and 10 g of maltopentaose were dissolved in 42.5 ml of 10 mM acetic acid buffer (pH 4.5), 11 units of α-mannosidase (manufactured by Sigma) from Tachinama bean was added, and 55 ° C. For 1 week. After completion of the reaction, a part of the reaction liquid is a high-speed liquid using an amino column (Tosoh, TSK-GEL Amide-80, 4.6 × 250 mm, solvent 65% acetonitrile, flow rate 1 ml / min, column temperature 30 ° C.) The results of analysis by chromatography are shown in FIG. The yield of the transfer product was 15% of the maltopentaose used.
[0021]
Next, the enzyme-heat-inactivated solution was subjected to high performance liquid chromatography (Showa Denko, guard column; Asahipak NH2P-130G: main column; Asahipak NH2P-50, solvent 68% acetonitrile, flow rate 2.5 ml / min, column temperature. 30 g), 1.5 g of the transfer product (yield 5%: raw material basis) was collected. This transfer product was digested with glucoamylase and then decomposed into maltopentaose, maltotetraose, maltotriose and mannose by α-mannosidase derived from the red bean. Further, as shown in FIG. 3, the result of ¹³ C-NMR analysis shows that a low magnetic field shift is observed at the 6-position carbon of glucose. Therefore, 6-mannosyl-maltopentaose in which mannose is bonded to the 6-position of glucose. (Mixture of 6 ⁵ -O-, 6 ⁴ -O-, 6 ³ -O-mannosyl-maltopentaose).
[0022]
After dissolving 60 mg of this mannosyl-maltopentaose in 2.1 ml of 10 mM acetate buffer (pH 5.2), 33 units of cyclodextrin synthase derived from Bacillus stearothermophilus (produced by Hayashibara Biochemical Laboratories) In addition, the mixture was reacted at 40 ° C. for 10 minutes. A portion of the reaction solution before and after the reaction was subjected to high performance liquid chromatography using an ODS column (YMC, YMC-Pack ODS-AP302, 4.6 × 250 mm, solvent 3% methanol, flow rate 1 ml / min, room temperature). The analysis results are shown in FIGS. When all the products A, B and C were combined, the production rate was 16%.
After completion of the reaction, the enzyme-heat-inactivated solution was used at high speed using an ODS column (YMC, YMC-Pack ODS-AP302, 4.6 × 250 mm, solvent 3% methanol, flow rate 1 ml / min, room temperature). The products A, B and C purified by liquid chromatography were fractionated 4 mg, 4 mg and 1 mg, respectively (yields were 7%, 7% and 2%, respectively).
[0023]
Product A isolated by the above method was found to have a molecular weight of 1134 by FAB-MS analysis. Further, it was completely decomposed into equimolar mannose and α-CD by α-mannosidase derived from red bean. Furthermore, as shown in FIG. 6, the result of ¹³ C-NMR analysis is confirmed to be a compound (structural formula I in FIG. 1) in which a mannosyl group is bonded to the 6-position hydroxyl group of α-CD by an α bond. It was done.
[0024]
Product B isolated by the above method was found to have a molecular weight of 1296 by FAB-MS analysis. In addition, it was completely decomposed into equimolar mannose and β-CD by α-mannosidase derived from red bean. From this, based on the above results, it was confirmed that the product B was a compound in which a mannosyl group was bonded to the hydroxyl group at the 6-position of the glucosyl group of β-CD by an α bond (Structural Formula II in FIG. 1). It was.
[0025]
Further, the product C isolated by the above method was found to have a molecular weight of 1458 by FAB-MS analysis. Further, it was completely decomposed into equimolar mannose and γ-CD by α-mannosidase derived from red bean. From this, based on the above results, it was confirmed that the product C was a compound in which a mannosyl group was bonded to the 6-position hydroxyl group of the glucosyl group of γ-CD by an α bond (Structural Formula III in FIG. 1). It was.
[0026]
【The invention's effect】
According to the present invention, mannosyl-CD in which a mannosyl group is bonded to a hydroxyl group of a glucosyl group in a CD molecule with an α-1,6 bond is efficiently obtained using mannosyltransferase and cyclodextrin synthase. Can do. This mannosyl-CD is expected to be widely used in the fields of food, cosmetics, etc. in addition to the pharmaceutical field.
[Brief description of the drawings]
FIG. 1 shows the structure of mannosyl-CD obtained by the present invention.
FIG. 2 is a high performance liquid chromatograph of a transfer reaction solution (after addition of α-mannosidase) of an example.
FIG. 3 is a ¹³ C-NMR spectrum of a transfer product of an example.
FIG. 4 is a high-performance liquid chromatograph of the synthesis reaction liquid of Example (before addition of cyclodextrin synthase).
FIG. 5 is a high-performance liquid chromatograph of the synthesis reaction solution of Example (after addition of cyclodextrin synthase).
FIG. 6 is a ¹³ C-NMR spectrum of product A in the example.

Claims (3)

  A step of allowing α-mannosyltransferase to act on a solution containing malto-oligosaccharide and mannose and / or α-mannosyl sugar compound, and a step of causing cyclodextrin synthase to act on the mannosyl-maltooligosaccharide produced in the step. A novel method for producing a mannosyl-cyclodextrin in which a mannosyl group is bonded to the hydroxyl group of a glucosyl group of cyclodextrin by an α bond. The production method according to claim 1, wherein the α-mannosyltransferase is allowed to act under conditions of pH 3 to 10 and temperature 20 to 90 ° C. The production method according to claim 1, wherein the cyclodextrin synthase is allowed to act under conditions of pH 3 to 10 and temperature 20 to 90 ° C.

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