JP2014030399A - Purification method of n-acetylsucrosamine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide simple, efficient, low cost and safe purification of N-acetylsucrosamine from a glucide-mixed solution exemplified by a synthetic reaction solution of N-acetylsucrosamine by transglycosylation reaction of β-fructofuranosidase with sucrose and N-acetylglucosamine as a substrate.SOLUTION: A purification method of N-acetylsucrosamine from a glucide-mixed solution, comprises at least the following steps A to C. Step A: cultivating yeast in a glucide-mixed solution comprising N-acetylsucrosamine; step B: separating the yeast from the solution after cultivation; and step C: obtaining an N-acetylglucosamine purification fraction by column chromatography.

Description

  The present invention relates to a method for purifying N-acetylscrosamine.

Conventionally, monosaccharides and oligosaccharides that exhibit various types of physiologically useful functions have been developed. Most of these sugars are used as functional sweeteners, many of which are prepared by enzymatic degradation of biomass polysaccharides or enzymatic conversion of oligosaccharides that are abundant in nature. Recently, regarding N-acetyl-D-glucosamine (GlcNAc), which is a monosaccharide obtained by hydrolysis of chitin, which is a biomass polysaccharide, intake of this saccharide may increase production of mucopolysaccharides such as hyaluronic acid and chondroitin. Confirmed by animal experiments. For this reason, the intake of this monosaccharide is expected to prevent skin aging and improve osteoarthritis.
As an oligosaccharide containing GlcNAc as a constituent sugar, N-acetylscrosamine [β-D-fructofuranosyl- (2 → 1) -α-N-acetyl-D, which is a disaccharide composed of D-fructose and GlcNAc, is used. -Glucosaminide] is known. The present inventors have reported a method of synthesizing N-acetylscrosamine using a Koji fungus-immobilized carrier holding β-fructofuranosidase and using GlcNAc and sucrose as substrates (Non-patent Documents 1 and 2). .
However, in order to purify N-acetylscrosamine from this synthesis reaction solution, it is necessary to combine a plurality of column chromatography such as activated carbon column chromatography and medium pressure column chromatography, which is costly and troublesome. There was a problem. In particular, medium-pressure column chromatography is not suitable for mass purification of oligosaccharides at low cost because a large amount of high-concentration organic solvent (ethanol, isopropanol, etc.) must be used and the resin is very expensive. was there.

Hirano et al., “Synthesis of N-acetylscrosamine using Aspergillus oryzae dry mycelia as a whole cell catalyst”, Journal of Applied Glycoscience, Applied Glucose Science, Vol. 1, No. 3, pp. 39-40 (2011 ) Hirano et al. catalyst ', Carbohydrate Research, 353, 27-32 (2012)

  The present invention provides a simple, efficient, low-cost and safe solution from a carbohydrate mixture solution exemplified by N-acetylscrosamine synthesis reaction solution by transglycosylation reaction with β-fructofuranosidase using sucrose and GlcNAc as substrates. Another object is to purify N-acetylscrosamine.

In order to solve the above-mentioned problems, the present inventors examined a method for treating a carbohydrate mixed solution using yeast. In the examination, first, the assimilation ability of yeast to the above monosaccharides and oligosaccharides was examined. As a result, N-acetylscrosamine is not assimilated by yeast (Saccharomyces cerevisiae), but it becomes clear that most of the contaminating carbohydrates contained in the carbohydrate mixed solution are assimilated, thereby completing the present invention. It came.
The present invention provides the following method for purifying N-acetylscrosamine.
(1) A method for purifying N-acetylscrosamine from a carbohydrate mixed solution, comprising at least steps A to C below;
Step A: culturing yeast in a mixed carbohydrate solution containing N-acetylscrosamine,
Step B: separating yeast from the cultured solution
Step C: A step of obtaining a purified N-acetylscrosamine fraction by column chromatography.
(2) The N-acetyl sucrose according to (1) above, wherein the saccharide mixed solution is a N-acetyl scrosamine synthesis reaction solution by transglycosylation with β-fructofuranosidase using sucrose and GlcNAc as substrates. Purification method of samine.
(3) N-acetyl sucrose according to (2) above, wherein the synthesis of N-acetyl scrosamine by transglycosylation with β-fructofuranosidase using sucrose and GlcNAc as substrates includes at least the following steps a and b. Purification method of samine;
Step a: contacting sucrose and GlcNAc with a Koji fungus hyphae immobilization carrier holding β-fructofuranosidase,
Step b: A step of performing N-acetylscrosamine from sucrose and GlcNAc by performing a transglycosylation reaction with β-fructofuranosidase held in the mycelium in the Koji mycelium-immobilized carrier after the contact.
(4) The method for purifying N-acetylscrosamine according to any one of (1) to (3) above, wherein the column chromatography in Step C is activated carbon column chromatography.

According to the present invention, N-acetylscrosamine can be purified from a saccharide mixed solution simply, efficiently, at low cost and safely. In particular, in the synthesis reaction solution obtained as a result of synthesizing N-acetylscrosamine using a Koji fungus-immobilized carrier that retains β-fructofuranosidase and GlcNAc and sucrose as substrates, there are many contaminating carbohydrates. Although N-acetylscrosamine can be singly purified only by activated carbon column chromatography, most of the contaminating carbohydrates are assimilated by yeast. This operation is very simple and is an excellent purification method capable of mass processing and at low cost. Yeast can be used repeatedly, and since pure GlcNAc can be recovered in the course of manipulation, it can be reused.
By using this method, the production cost of N-acetylscrosamine is drastically reduced.

A. Schematic of the transglycosylation reaction between sucrose and GlcNAc by granules consisting of dried oryzae NBRC100959 strain mycelium and Celite 535. In the figure, the amount, concentration and yield of the compound are examples. Continuous production of N-acetylscrosamine using a column reactor. A: A diagram of the column reactor apparatus is shown. B: Quantitative result by HPLC of N-acetylscrosamine concentration in the reaction solution. HPLC analysis data of carbohydrates contained in reactor column eluate for N-acetylscrosamine production Crystal of N-acetylscrosamine Results of assimilation tests of various carbohydrates using yeast HPLC analysis data of carbohydrates contained in the solution after the yeast treatment of the reactor column eluate for N-acetylscrosamine production HPLC analysis data of N-acetylscrosamine purified by activated carbon column chromatography Scheme for production and purification of N-acetylscrosamine

1. N-acetylscrosamine purification method The N-acetylscrosamine purification method of the present invention is a method for purifying N-acetylscrosamine from a carbohydrate mixed solution, which comprises at least the following steps A to C.
Step A: culturing yeast in a mixed carbohydrate solution containing N-acetylscrosamine,
Step B: separating yeast from the cultured solution
Step C: A step of obtaining a purified N-acetylscrosamine fraction by column chromatography.
The method for purifying N-acetylscrosamine of the present invention is for purifying N-acetylscrosamine from a saccharide mixed solution containing at least one saccharide and N-acetylscrosamine assimilated by yeast. Available. The present inventors have found for the first time that sucrose, glucose, fructose, and fructooligosaccharide are assimilated by yeast, but N-acetylscrosamine is not assimilated at all, as described in Examples below. The synthesis reaction of N-acetylscrosamine by transglycosylation with β-fructofuranosidase using sucrose and GlcNAc as substrates is carried out by, for example, a method for synthesizing N-acetylscrosamine using a Koji fungus mycelium immobilization carrier described later. be able to. The synthesis reaction solution obtained by the method for synthesizing N-acetylscrosamine using Koji mycelium immobilized carrier includes N-acetylscrosamine and GlcNAc added in excess, as well as sucrose and sucrose degradation products as substrates. Some glucose and fructose and some fructooligosaccharides, which are byproducts of the transglycosylation reaction, are included. Therefore, this synthesis reaction solution is suitable as a carbohydrate mixed solution in the present invention.
Although the kind of yeast to be used is not particularly limited, edible yeast is particularly preferable from the viewpoint of safety. The yeast may be a budding yeast or a fission yeast. Examples of the budding yeast include baker's yeast (Saccharomyces cerevisiae), beer yeast, wine yeast, sake yeast, and the like. Examples of fission yeast include Shizosaccharomyces pombe.
The yeast culture conditions are not particularly limited as long as the yeast grows to such a degree that the contaminating sugar can be removed to a desired level. For example, the culture is performed in a buffer solution at about 30 ° C. for 12 to 24 hours. The
In the case of an N-acetylscrosamine synthesis reaction solution by transglycosylation with β-fructofuranosidase using sucrose and GlcNAc as substrates by the above-described yeast culture treatment, the sugar present in the yeast treatment solution is converted to GlcNAc. Only N-acetylscrosamine can be used.
Next, the yeast is separated from the yeast treatment solution. The separation method is not particularly limited, and can be performed, for example, by filtration or centrifugation. The separated yeast can be reused in Step A.
In Step C, a purified fraction of N-acetylscrosamine is obtained by column chromatography. In the case where the sugar mixed solution is a reaction solution for synthesizing N-acetylsucrosamine by transglycosylation with β-fructofuranosidase using sucrose and GlcNAc as substrates, a mixed solution of GlcNAc and N-acetylscrosamine is obtained by yeast treatment. can get. From this mixed solution, GlcNAc can be removed by column chromatography to obtain a purified N-acetylscrosamine fraction. The column chromatography is preferably activated carbon column chromatography which is effective for purifying oligosaccharides in large quantities and at low cost. The activated carbon does not adsorb monosaccharides such as GlcNAc, glucose, and fructose, but adsorbs oligosaccharides such as sucrose, N-acetylscrosamine, and fructooligosaccharide. These adsorbed oligosaccharides can be desorbed with, for example, a 5% (v / v) aqueous isopropanol solution. That is, monosaccharides and oligosaccharides can be easily separated using activated carbon column chromatography. Therefore, if oligosaccharides other than N-acetylscrosamine contained in the column reactor eluate can be removed in the previous stage of the activated carbon column chromatography purification operation, the isolation of N-acetylscrosamine will be dramatically improved. Simplified.
2. Method for synthesizing N-acetylscrosamine using Koji mycelium-immobilized carrier The synthesis of N-acetylscrosamine using Koji mycelium-immobilized carrier includes at least the following steps a and b.
Step a: contacting sucrose and GlcNAc with a Koji fungus hyphae immobilization carrier holding β-fructofuranosidase,
Step b: A step of performing N-acetylscrosamine from sucrose and GlcNAc by performing a transglycosylation reaction with β-fructofuranosidase held in the mycelium in the Koji mycelium-immobilized carrier after the contact.
In step a, Koji mycelium-immobilized carrier means a carrier in which Koji mycelium is easily reacted with a porous material having irregularities on its surface, such as diatomaceous earth and pearlite particles, and includes, for example, a carrier. It can be produced by culturing Koji fungi in a medium, separating a Koji mycelium-immobilized carrier from the cultured medium, and drying a Koji mycelium-immobilized carrier.
For example, when diatomaceous earth is used as a carrier, Koji mycelium-immobilized carrier is prepared by adding a suspension of Koji fungal spores to a diatomaceous earth-containing culture solution, allowing the mycelia to grow at a suitable temperature, Immobilizing diatomaceous earth), filtering the culture solution, separating Koji mycelium-immobilized diatomaceous earth, performing dehydration treatment and drying treatment exemplified by acetone treatment, etc. to produce a Koji mycelium immobilization carrier it can.
The method for bringing the sugar substrate into contact with the Koji mycelium-immobilized carrier is not particularly limited, and examples thereof include a batch method and a column reactor method. From the viewpoint of mass production of N-acetylscrosamine by continuous production, a column reactor method is particularly desirable. In this case, it is desirable to use a physically strong carrier such as pearlite particles.
In step b, for the enzyme reaction, for example, a buffer solution exemplified by sodium citrate buffer (pH 5.5) is appropriately selected as the enzyme reaction solution, and a sugar substrate and a Koji mycelium immobilization carrier are added to the enzyme reaction solution. Can be carried out by coexisting under appropriate conditions. The amount of the Koji mycelium immobilization carrier added to the substrate solution can be appropriately adjusted by those skilled in the art depending on the reaction time. The conditions for the enzyme reaction are not particularly limited, but can be appropriately selected by those skilled in the art, for example, by gently shaking at 25 to 30 ° C.
FIG. 1 shows an example of a method for synthesizing N-acetylscrosamine using a Koji mycelium-immobilized carrier produced using Celite.
The present inventors have described GlcNAc according to the above method in Japanese Journal of Applied Glycoscience, Applied Glycoscience, Vol. 1, No. 3, 39-40 (2011), Carbohydrate Research, 353, 27-32 (2012). The synthesis method of was reported. Any of the saccharide mixed solutions containing N-acetylscrosamine obtained by the synthesis method published by these reports is preferable as an application target of the N-acetylscrosamine purification method of the present invention. The present inventors also flow a solution containing high-concentration GlcNAc and sucrose through a column reactor filled with immobilized mycelia of Aspergillus oryzae containing transglycosyl β-fructofuranosidase. The present inventors succeeded in efficiently producing N-acetylscrosamine over a long period of time by the transglycosylation reaction of the enzyme. For the continuous production of N-acetylscrosamine using the column reactor method, see Example 1 described later.
Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited thereto.

Continuous production of N-acetylscrosamine using a column reactor (1) Preparation of Koji fungus hyphae immobilization support spore suspension of oryzae NBRC4290 0.1 mL of 9.9 mL medium containing 1 g of pearlite granules (weight / volume 2% sucrose, 0.5% peptone, 0.2% NaNO ₃ , 0.1% K ₂ HPO ₄ Liquid medium containing 0.05% MgSO ₄ .7H ₂ O, 0.05% KCl and 0.001% FeSO ₄ .7H ₂ O), and the mixture is shaken at 80 rpm for 3 days at 28 ° C. Incubated. After the incubation, pearlite grains on which A. oryzae mycelia had propagated were collected by suction filtration using No. 5A filter paper (Kiriyama Seisakusho, Tokyo, Japan). Next, this was immersed in 50 mL of cold acetone for 30 minutes and collected by suction filtration using filter paper. This operation was repeated twice. The obtained beads with attached dehydrated A. oryzae mycelia were dried under reduced pressure at room temperature using an aspirator. oryzae NBRC4290 mycelium-immobilized perlite granules were obtained. The content of dry mycelium in the dried mycelium-immobilized perlite was 20%.
(2) Continuous production of N-acetylscrosamine 20 mM sodium citrate containing 10% (weight / volume) sucrose and 19.4% (weight / volume) GlcNAc in a column (diameter 2 cm × height 20 cm) packed with oryzae NBRC4290 mycelium-immobilized perlite grains A buffer solution (pH 5.5) was flowed at a flow rate of about 100 mL / day, and N-acetylscrosamine was continuously produced by a column reactor. FIG. 2A shows a diagram of the column reactor apparatus. N-acetylscrosamine in the reaction solution was quantified by high performance liquid chromatography (HPLC). As a result, the production amount of N-acetylscrosamine was about 2.5 to 4.0 g / day even in the small column, and stable continuous production for at least 30 days was possible (FIG. 2B). This shows that mass production of N-acetylscrosamine is possible by using this system.
In the reactor column eluate containing N-acetylscrosamine, excessively added GlcNAc as a raw material, unreacted sucrose, glucose and fructose as sucrose degradation products, and fructooligosaccharides (1-kestose and Nystose). FIG. 3 shows HPLC analysis data of carbohydrates contained in the reactor column eluate.
It was confirmed by HPLC quantitative analysis that 42.1 mg of N-acetylscrosamine was dissolved in the reactor column eluate. The HPLC analysis conditions are shown below.
(HPLC analysis conditions)
Pump; LC-10AS (Shimadzu Corporation)
Detector: Shodex RI-71 (Showa Denko)
Column: COSMOSIL Sugar-D (column size: φ4.6 × 250 mm, manufactured by Nacalai Tesque)
Mobile phase solvent; 77% (v / v) acetonitrile aqueous solution flow rate; 0.8 mL / min

When N-acetylscrosamine was crystallized from hot methanol, very elongated needle-like crystals were obtained as shown in FIG. 4A. When crystallized from hot ethanol, slightly thick needle-like crystals were obtained as shown in FIG. 4B. When melting | fusing point was measured about these crystals, all were 156-158 degreeC. The solubility of this disaccharide in water was extremely high, as was sucrose. The specific rotation [α] _D ²³ (c 0.97) of the disaccharide when water was used as a solvent was 80.7 ^° . The stability of N-acetylscrosamine at pH 3 to pH 9 and stability to heat were compared with sucrose. As a result, both disaccharides were not decomposed at all even when left at 100 ° C. for 1 hour in a buffer solution of pH 5, 7, 9 but when left at 75, 100 ° C. for 1 hour in a buffer solution of pH 3 Was decomposed into monosaccharides. However, the degree of degradation of N-acetylscrosamine was much lower than that of sucrose. This indicates that N-acetylscrosamine is more stable in acidic solution than sucrose. Next, the degradability of N-acetylscrosamine to various sucrose hydrolases was examined. Examples of the enzyme include yeast β-fructofuranosidase (manufactured by Mitsubishi Chemical Foods), rat small intestine sucrase (manufactured by Sigma) and Carbohydrate Research, 353, 27-32 (2012). The glycosyltransferase β-fructofuranosidase and the hydrolyzed β-fructofuranosidase were used. As a result, sucrose was rapidly hydrolyzed by all the enzymes used in dilute solutions of these disaccharides, but N-acetylscrosamine was hydrolyzed only by the sugar-transferred β-fructofuranosidase of Aspergillus oryzae. Decomposition was confirmed. This indicates that N-acetylscrosamine is not a substrate for sucrase or hydrolyzed β-fructofuranosidase.

10 mg of dry yeast powder derived from Saccharomyces cerevisiae (manufactured by Nisshin Foods, trade name: Super Camellia Dry Yeast) was added to a 20 mM sodium citrate buffer solution in which each sugar was dissolved at a concentration of 146 mM, and 100 rotations / Culturing was performed at 30 ° C. with shaking for minutes. The state of assimilation of each sugar in the culture solution by yeast was confirmed over time by thin layer chromatography (TLC) and HPLC.
The conditions of TLC and HPLC are shown below.
(TLC analysis conditions)
TLC plate; silica gel 60, developing solvent manufactured by Merck & Co .; n-butanol / pyridine / water 8: 3: 1 (v / v / v)
Number of deployments; 2 deployments; 30 ° C
Sample color development: After spraying an aqueous solution containing 2.4% phosphomolybdic acid, 5% sulfuric acid, and 1.5% phosphoric acid, the sample spot was colored by heating with a heat gun.
(HPLC analysis conditions)
Pump; LC-10AS (Shimadzu Corporation)
Detector: Shodex RI-71 (Showa Denko)
Column: COSMOSIL Sugar-D (column size: φ4.6 × 250 mm, manufactured by Nacalai Tesque)
Mobile phase solvent: 77% (v / v) acetonitrile aqueous solution flow rate: 0.8 mL / min As a result of the above experiment, as shown in FIG. 5-A, glucose, fructose, sucrose, 1-kestose, and nystose Although it was assimilated by yeast at 24 hours in culture, it was found that GlcNAc and N-acetylscrosamine remained even at 24 hours in culture. Further, when HPLC quantitative analysis was performed on GlcNAc and N-acetylscrosamine, these carbohydrates were not reduced at all even after 24 hours of culture (FIG. 5-B).
Based on the above experimental results, it was examined that dry yeast was added to the reactor column eluate and all saccharides other than GlcNAc and N-acetylscrosamine were assimilated by the yeast.

2 g of dried yeast derived from Saccharomyces cerevisiae (manufactured by Nisshin Foods, trade name: Super Camellia Dry Yeast) was added to 100 mL of the reactor column eluate obtained by the same method as in Example 1, and rotated at 100 rpm. Culturing was performed at 30 ° C. with shaking. After 24 hours of culture, the yeast cells were removed by filtration using celite as a filter aid, and the carbohydrates in the obtained filtrate were confirmed by HPLC.
(HPLC analysis conditions)
Pump; LC-10AS (Shimadzu Corporation)
Detector: Shodex RI-71 (Showa Denko)
Column: COSMOSIL Sugar-D (column size: φ4.6 × 250 mm, manufactured by Nacalai Tesque)
Mobile phase solvent: 77% (v / v) acetonitrile aqueous solution flow rate: 0.8 mL / min As a result of the above experiment, carbohydrates (glucose, fructose, sucrose) in the reactor column eluate other than GlcNAc and N-acetylscrosamine 1-kestose, nystose, and unidentified oligosaccharides) were found to be assimilated by yeast at 24 hours in culture (FIG. 6).
Next, N-acetylscrosamine was purified from the obtained filtrate by activated carbon column chromatography.

The filtrate obtained in Example 4 was loaded on an activated carbon column (φ5.5 × 18 cm, mobile phase solvent; water), and all GlcNAc was eluted by flowing water, and then the N− adsorbed on the activated carbon. Acetyl scrosamine was desorbed and eluted by flowing a 5% (v / v) aqueous isopropanol solution. The obtained N-acetylscrosamine solution was concentrated and dried by an evaporator. Thereafter, a small amount of water was added to dissolve N-acetylscrosamine again, and lyophilization was performed to obtain white powdery N-acetylscrosamine.
When the purity of the obtained N-acetylscrosamine eluate was confirmed by performing HPLC analysis, it was confirmed to be single as shown in FIG.
(HPLC analysis conditions)
Pump; LC-10AS (Shimadzu Corporation)
Detector: Shodex RI-71 (Showa Denko)
Column: COSMOSIL Sugar-D (column size: φ4.6 × 250 mm, manufactured by Nacalai Tesque)
Mobile phase solvent: 77% (v / v) acetonitrile aqueous solution flow rate: 0.8 mL / min In addition, 3.08 g of N-acetylscrosamine powder could be obtained by the above experiment. Since it was confirmed by the previous HPLC quantitative analysis that 3.4 g of N-acetylscrosamine was dissolved in 100 mL of the reactor column eluate used, it was confirmed that Example 4 and By the operation of Example 5, N-acetylscrosamine could be isolated with a molar yield of 90.6%. It was also possible to recover pure GlcNAc. The above operations are summarized in FIG.
A. 4.08 g of N-acetylscrosamine was isolated in the same manner as in Examples 4 and 5 using the eluate of the column reactor of oryzae NBRC100537 mycelium immobilized carrier.
By assimilating most of the contaminating carbohydrates contained in the eluate of the reactor column for producing N-acetylscrosamine with yeast, N-acetylscrosamine could be purified to a single substance only by activated carbon column chromatography. . This operation is very simple and is an excellent purification method capable of mass processing and at low cost. Yeast can be used repeatedly, and since pure GlcNAc can be recovered in the course of manipulation, it can be reused.
By using this method, the production cost of N-acetylscrosamine was drastically reduced.

  The present invention can be used for simple, efficient, low-cost and safe purification of N-acetylscrosamine.

Claims (4)

A method for purifying N-acetylscrosamine from a mixed carbohydrate solution, comprising at least the following steps A to C;
Step A: culturing yeast in a mixed carbohydrate solution containing N-acetylscrosamine,
Step B: separating yeast from the cultured solution
Step C: A step of obtaining a purified N-acetylscrosamine fraction by column chromatography. The N-acetylscrosamine according to claim 1, wherein the carbohydrate mixed solution is a N-acetylscrosamine synthesis reaction solution by a transglycosylation reaction with β-fructofuranosidase using sucrose and N-acetylglucosamine as substrates. Purification method. The N-acetylscrosamine according to claim 2, wherein the synthesis of N-acetylscrosamine by transglycosylation with β-fructofuranosidase using sucrose and N-acetylglucosamine as substrates comprises at least the following steps a and b. Purification method of
Step a: contacting sucrose and N-acetylglucosamine with a Koji fungal mycelium-immobilized carrier holding β-fructofuranosidase,
Step b: A step of generating N-acetylscrosamine from sucrose and N-acetylglucosamine by performing a transglycosylation reaction with β-fructofuranosidase held in the mycelium in the Koji mycelium-immobilized carrier after the contact. The method for purifying N-acetylscrosamine according to any one of claims 1 to 3, wherein the column chromatography in Step C is activated carbon column chromatography.