JP2001292792A - Method for recovering n-acetylglucosamine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for recovering N-acetylglucosamine from an N-acetylglucosamine solution containing glucose in which the N- acetylglucosamine with a low glucose content usable for synthesizing substances on an industrial scale using the N-acetylglucosamine as a raw material can simply be recovered. SOLUTION: This method for removing the glucose from the N- acetylglucosamine solution containing the glucose and recovering the N- acetylglucosamine is characterized by treating the solution with a glucose oxidase and separating and removing a glucose oxidase reactional product from the treated solution. The cost can be reduced by reutilizing the recovered N-acetylglucosamine and the method is useful for environments.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for recovering N-acetylglucosamine from an N-acetylglucosamine solution containing glucose.

[0002]

2. Description of the Related Art N-acetylglucosamine is a complex carbohydrate of animals and plants and microorganisms, particularly chitin, glycosaminoglycan,
It is widely distributed as a constituent component of glycoproteins and glycolipids.

[0003] N-acetylglucosamine is used as a raw material for synthesizing N-acetyllactosamine. N-acetyllactosamine is a biochemically important disaccharide present in the sugar chains of human milk oligosaccharides, lipopolysaccharides, various glycoproteins and glycolipids, and is a kind of intestinal bacterium, Bifidobacterium Because of its proliferative activity and excellent intestinal regulation, its use in highly nutritional foods such as infant formula has attracted attention. Furthermore, in recent years, the physiological activity of complex carbohydrate sugar chains containing N-acetyllactosamine has also attracted attention. For example, it has been drawing attention as a raw material for synthesizing sialyl Le ^X sugar chains having cell adhesion inhibitory activity.

As a method for synthesizing this N-acetyllactosamine, a chemical synthesis method and an enzyme synthesis method are known. However, since the target substance can be obtained by a one-step reaction, the synthesis method on an industrial scale is required. There has been extensive research on enzyme synthesis methods using exclusively β-galactosidase or galactosyltransferase. Among them, most of the enzyme synthesis methods using β-galactosidase are methods using a substrate containing N-acetylglucosamine and lactose as reaction raw materials. In the reaction solution after completion of the enzymatic reaction using this substrate, unreacted N-acetylglucosamine, unreacted lactose, and reaction by-products such as glucose are present together with the generated N-acetyllactosamine. . It is desired to efficiently recover N-acetylglucosamine from this reaction solution and reuse it as a reaction raw material for environmental measures and cost reduction of N-acetyllactosamine production on an industrial scale.

[0005] Further, glucose is known to have an action of inhibiting the galactose transfer activity of β-galactosidase, so that the recovered N-acetylglucosamine can be reused for N-acetyllactosamine production as much as possible. It is necessary to obtain high-purity N-acetylglucosamine having a low glucose content.

[0006] However, it is difficult to separate N-acetylglucosamine and glucose, which are monosaccharides, from each other by chromatography that can be used on an industrial scale such as an activated carbon / celite column which is conventionally used. N-fractionated from the reaction solution obtained by the production of N-acetyllactosamine using an activated carbon / celite column
The acetylglucosamine fraction contains about 20% glucose. In order to further remove this glucose and obtain N-acetylglucosamine having a low glucose content such that it can be reused in the above-mentioned enzyme synthesis of N-acetyllactosamine, more complicated purification operations and expensive purification materials are required. I need.

[0007]

SUMMARY OF THE INVENTION Accordingly, the present invention provides an N-acetylglucosamine solution containing glucose.
A method for recovering acetylglucosamine, wherein N-acetylglucosamine having a low glucose content, which can be used for substance synthesis on an industrial scale using N-acetylglucosamine as a raw material, can be recovered simply and at low cost, and The aim is to provide a beneficial way to the environment.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, glucose oxidase is allowed to act on glucose-containing N-acetylglucosamine solution to selectively produce glucose. It has been found that high-purity N-acetylglucosamine can be easily obtained by conversion to gluconic acid using a commonly used separation method, and the present invention has been completed.

[0009] Accordingly, the present invention relates to N-containing glucose-containing compounds.
A method for recovering N-acetylglucosamine by removing glucose from a -acetylglucosamine solution, comprising treating the solution with glucose oxidase and separating and removing a glucose oxidase reaction product from the treated product. A method of characterizing is provided.

As the N-acetylglucosamine solution containing glucose used in the method of the present invention, a reaction mixture obtained in the enzyme synthesis of N-acetyllactosamine using lactose and N-acetylglucosamine as substrates is preferably used. be able to.

When N-acetylglucosamine is recovered from the reaction mixture obtained in the enzyme synthesis of N-acetyllactosamine, at least N-acetyllactosamine is separated and removed from the reaction mixture after the enzyme synthesis reaction. It is preferable that the fraction be a fraction composed of monosaccharides collected after the enzyme synthesis reaction.

[0012] Glucose oxidase used in the method of the present invention includes Aspergillus or Penicillium.
Glucose oxidase derived from a microorganism belonging to the genus can be preferably used, and glucose oxidase derived from Aspergillus niger can be particularly preferably used.

In the method of the present invention, glucose is converted into gluconic acid by treating the solution with glucose oxidase, and gluconic acid is separated and removed from the treated solution by anion exchange resin treatment to recover N-acetylglucosamine. Is preferred.

In the method of the present invention for converting glucose into gluconic acid and separating and removing gluconic acid from the solution by anion exchange resin treatment to recover N-acetylglucosamine, the method according to the present invention has a purity of 90% or more. Can be obtained.

According to another aspect of the present invention, there is provided a method for producing N-acetyllactosamine, comprising reacting N-acetylglucosamine obtained by the method of the present invention with lactose in the presence of β-galactosidase. Provided.

Further, N-acetylglucosamine and lactose are reacted in the presence of an enzyme capable of transferring a galactose residue from lactose to N-acetylglucosamine to produce N-acetyllactosamine,
A method of obtaining a monosaccharide fraction containing N-acetylglucosamine and glucose by treating the reaction mixture with activated carbon, treating the fraction with glucose oxidase, and separating and removing the produced gluconic acid to obtain N-acetylglucosamine. And a method for producing N-acetyllactosamine characterized by adding lactose to a solution containing N-acetylglucosamine obtained thereby and subjecting the solution to the above-mentioned N-acetyllactosamine synthesis reaction.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The glucose-containing N-acetylglucosamine solution used in the method of the present invention may be a solution containing at least both, and the method for producing the solution and the origin are not particularly limited. Such a solution is preferably a solution containing more N-acetylglucosamine than glucose.

Examples of such a solution include a reaction mixture obtained in the production of N-acetyllactosamine by enzyme synthesis using lactose and N-acetylglucosamine as substrates. The reaction mixture is not limited as long as it is obtained in the production of N-acetyllactosamine by the enzyme synthesis. It is usually a solution, but may be a solid in which the solvent is removed from the solution. In the production of N-acetyllactosamine by this enzyme synthesis, specifically, an enzyme having an action of transferring a galactose residue from lactose to N-acetylglucosamine is added to a substrate solution containing lactose and N-acetylglucosamine. To synthesize N-acetyllactosamine. After completion of the reaction, N-acetylglucosamine and glucose are usually collected by obtaining a solution from which N-acetyllactosamine has been separated and removed by ion chromatography or gel filtration, or by collecting only the monosaccharide fraction. Can be obtained. Such a monosaccharide fraction usually contains about 70 to 90 mol% of N-acetylglucosamine and about 10 to 30 mol% of glucose.

A galactose residue is converted from lactose to N-acetyl used in the enzymatic synthesis of N-acetyllactosamine.
The enzyme having an action of transferring to acetylglucosamine is not limited in its form and origin as long as it has such a catalytic action.For example, only a so-called enzyme itself such as a purified enzyme or a partially purified enzyme is used. Rather, quiescent cells, dried cells, crushed cells and the like containing the enzyme are also included in the category of the enzyme in the present invention. Specific examples of such enzymes include Bacillus circulans and the like.
Us microorganisms, Rhodotorula minuta and Rhodotorula lactos
Rhodotorula microorganisms such as a, a purified enzyme or partially purified enzyme or enzyme preparation of β-galactosidase derived from a microorganism belonging to the genus Sterigmatomyces such as Sterigmatomyces elviae, and in particular, β-1,4 derived from Bacillus circulans.
Β-galactosidase with high specificity for glycosidic bonds is preferred.

The enzyme may be extracted and purified from a natural product such as a microorganism capable of producing the enzyme, or a recombinant cell or the like into which a gene DNA encoding the enzyme has been introduced; It may be produced by genetic engineering techniques such as extraction and purification. Further, the enzyme may be a polypeptide containing an active site of an enzyme having a galactosyltransfer reaction, and such a polypeptide can be produced by a genetic engineering technique.

The conditions for the above enzyme synthesis reaction are not particularly limited, and the reaction can be carried out according to known conditions for the reaction of the synthetic enzyme to be used, and can be appropriately selected by those skilled in the art. It is preferable to carry out the reaction under conditions of pH and optimum temperature. The substrate mixture such as a solution containing the substrate is not limited as long as it does not affect the reaction by the enzyme to be used, but is preferably a mixture in a buffer solution.

As the reaction mode, conventional means of enzymatic reaction can be adopted, and the substrate and the enzyme may be allowed to act while the enzymatic reaction proceeds. For example, the enzyme may be brought into contact with the substrate solution, or vice versa. A method in which such an enzyme to be used is immobilized on an appropriate carrier and the substrate solution is brought into contact with the substrate solution may be employed. More specifically, for example, it is also possible to immobilize the enzyme to be used on a bead-like carrier, fill the column, and continuously perform synthesis while passing a mixture of lactose and N-acetylglucosamine. Alternatively, a batch method in which the immobilized enzyme is added to the substrate solution, and the two are sufficiently contacted and reacted by stirring or the like, and the immobilized enzyme is separated after the reaction, may be employed.

The reaction mixture thus obtained contains:
In addition to N-acetyllactosamine which is the target substance of the reaction by the enzyme, unreacted substrates such as N-acetylglucosamine and lactose, glucose which is a by-product and galactosyl-N-acetyllactosamine are contained. .

The method for fractionating the monosaccharide fraction from the reaction mixture is not particularly limited as long as the method is capable of fractionating the monosaccharide fraction. For example, if necessary, the reaction mixture is subjected to preliminary purification such as decolorization / desalting treatment with pulverized coal or an ion exchange resin,
If the concentration of the component is low, it is further concentrated under reduced pressure, etc., and then separated by chromatography using a strongly acidic cation exchange resin, gel filtration separation, molecular sieving, activated carbon / celite ( The monosaccharide fraction can be easily collected by a known method such as adsorption chromatography using an adsorbent such as a trade name column.

As an example, the method of fractionation using an activated carbon / celite column will be described in more detail. Column capacity of 2
Feeding 6% (W / V) of the synthesis reaction mixture to a column,
The eluate is flowed at a flow rate of 60 to 200 ml / hour to elute saccharides. The monosaccharide fraction containing N-acetylglucosamine and glucose is eluted with water. The sugar concentration of the synthesis reaction end mixture to be supplied to the column is suitably about 20 to 65% (w / w). If the sugar concentration is higher than this, the separation pattern is disturbed. In the above method, N-acetyllactosamine is eluted with 14 to 20% methanol, so that N-acetyllactosamine can be separated.

Glucose oxidase used for removing glucose from a glucose-containing N-acetylglucosamine solution and recovering N-acetylglucosamine has an action of converting glucose to gluconic acid and is substantially converted to N-acetylglucosamine. Enzymes that do not have any effect (here, the term "enzyme" is a concept that includes not only enzymes such as purified enzymes and partially purified enzymes, but also static cells, dried cells, and crushed cells containing the enzymes) If so, its origin is not particularly limited. For example, Aspergil
lus niger and other Aspergillus genus, Penicilliu
and those derived from the genus Penicillium such as m notatum.
In particular, those derived from Aspergillus niger are preferred.

The enzyme may be extracted and purified from a natural product such as a microorganism having an ability to produce an enzyme capable of converting glucose to gluconic acid. Gene DNA encoding such an enzyme may be used. It may be produced by genetic engineering techniques such as growing, extracting and purifying a recombinant cell or the like into which is introduced. Furthermore, a polypeptide containing an active site of glucose oxidase may be used as long as it has an action of converting glucose to gluconic acid and does not substantially affect N-acetylglucosamine. It can be manufactured by engineering techniques.

The conditions for performing the glucose oxidase treatment are not limited as long as they do not hinder the reaction of converting glucose to gluconic acid and do not substantially affect N-acetylglucosamine, and can be appropriately selected by those skilled in the art. However, it is preferable that the reaction is carried out under the optimum pH and optimum temperature conditions of the glucose oxidase to be used. Aspergil
When using lus niger-derived glucose oxidase, the reaction is preferably carried out at a pH of 5.7 to 6.2 and about 30 ° C.

After the treatment with glucose oxidase, the treated product can be used as a source of N-acetylglucosamine as a substrate in a reaction for producing N-acetyllactosamine by an enzymatic method. Usually, gluconic acid, which is a reaction product of glucose oxidase, is separated and removed from the treated product containing N-acetylglucosamine by a known and commonly used method.

In general, gluconic acid is known to exist as an equilibrium mixture of glucono-1,4-lactone, glucono-1,5-lactone and free gluconic acid in an aqueous solution. Gluconic acid produced by glucose oxidase is present in aqueous solution as an equilibrium mixture of glucono-1,4-lactone, glucono-1,5-lactone and free gluconic acid. Therefore, in the present invention, "gluconic acid" is used as a term including such gluconolactone in addition to gluconic acid itself, and the above separation / removal method can also remove gluconolactone. Preferably, it is a method.

Gluconic acid (gluconolactone) is separated and removed from the solution after the glucose oxidase treatment,
The method for fractionating -acetylglucosamine is not particularly limited as long as N-acetylglucosamine can be obtained. For example, Muromakku Cl ^- type (Muromachi Kagaku Kogyo, styrenic strongly basic resins having a quaternary ammonium group), or Diaion SA10A or 11A or the like of the SA series or PA series (Mitsubishi Chemical, styrene having a quaternary ammonium group There are chromatography using an anion exchange resin such as a strong basic resin) and electrodialysis. In particular, gluconic acid is adsorbed and removed by chromatography using an anion exchange resin, and it is a non-adsorbed fraction. N-
A method for obtaining acetylglucosamine is simple and preferred.

According to the method of the present invention, N-acetylglucosamine having a purity of 90% or more, preferably 95% or more is obtained. When the method of the present invention is carried out in combination with the production of N-acetyllactosamine, N-acetyllactosamine is eluted with 14 to 20% methanol in the fractionation using the activated carbon / celite column. Samin can be recovered. The N-acetylglucosamine recovered by the method of the present invention is very useful because it can be reused as a raw material for the enzyme synthesis of N-acetyllactosamine. Make up one form.

When the method of the present invention is carried out in combination with the production of N-acetyllactosamine, for example,
Perform an enzyme synthesis reaction of N-acetyllactosamine and subject the reaction mixture to chromatography using a strongly acidic cation exchange resin, gel filtration, activated carbon / celite column, etc. Monosaccharide fractions containing glucosamine and glucose are each fractionated, and glucose oxidase is allowed to act on the monosaccharide fraction,
The produced gluconic acid can be removed and N-acetylglucosamine can be recovered. However, the present invention is not limited to this. For example, N-acetyllactosamine, which is the target substance, may be separated and removed from the enzyme synthesis reaction end solution, and the method of the present invention may be applied to the separated mixture. Then, the method of the present invention may be applied to an unfractionated solution after the enzymatic reaction.

[0034]

EXAMPLES The present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

[0035]Reference Example 1  Method for measuring N-acetylglucosamine In the examples described later, the purity of N-acetylglucosamine was measured.
Stands for High Performance Liquid Chromatography (hereinafter referred to as HPLC)
And under the following conditions. Column: Asahipak NH2P-50 column (Asahi Kasei, 4.6 x 250 nm) Mobile phase: 70% acetonitrile aqueous solution Flow rate: 0.7 ml / min Temperature: 37 ° C Detection: UV210 nm (absorption of N-acetyl group) Differential refraction (saccharides) Sample: 5 ml Recover N-acetylglucosamine by this HPLC analysis method
The tension time was 7.8 minutes.

[0036]Reference example 2  Screening of enzyme sources Lipomyces lipofe, a yeast capable of lactose utilization
r (IFO 0673), Rhodotorula minuta (IFO 0928), Rho
dotorula lactosa (IFO 1423), Sterigmatomyces elviae
(IFO 1843) (The above is from RIKEN Microbial Strain Preservation Facility.
To 28 ° C on a Malt's extract agar slant medium.
And cultured for 6 days to prepare a preservation culture.

Medium containing lactose as the sole carbon source (lactose; 2%, peptone; 1%, yeast extract; 1%, (NH ₄ ) _{2
SO 4; 0.5%, K 2} HPO 4; 0.3%, KH 2 PO 4; 0.1%, MgSO 4 · 7H
₂ O; 0.05%, pH 7.0) into a 500 ml Sakaguchi flask.
ml, and autoclaved at 121 ° C. for 15 minutes. One platinum loop was inoculated with each of the above preservation cultures and cultured at 28 ° C. for 4 days. After completion of the culture, the cells were collected by centrifugation, washed twice with the same amount of physiological saline as the culture solution, and then collected and stored frozen at -20 ° C.

Lactose (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a 100 mM sodium acetate buffer solution (pH 6.0) so that the washed cells prepared by the above method had a final concentration of 500 mg / ml.
And N-acetylglucosamine (manufactured by Nacalai Tesque) were suspended in a substrate solution in which the concentrations were 0.5 M and 1 M, respectively, and reacted at 60 ° C. for 17 hours. Thereafter, the reaction supernatant obtained by centrifugation was heated in a boiling bath (5 minutes) to inactivate the enzyme, and then diluted 1/10 with distilled water.
The reaction products were analyzed by HPLC. Table 1 shows the results.
The results synthesized by the method of Usui et al. (JP-A-3-49692) are also described.

[0039]

[Table 1]

Washed cells using yeasts of the genus Rhodotorula or Sterigmatomyces also produce N-acetyllactosamine, and particularly when the yeasts of the genus Rhodotorula are used, Bacillus
N-acetyllactosamine productivity equivalent to that of enzyme synthesis using β-galactosidase from circulans was obtained. Therefore, washed cells of Rhodotorula minuta were used in the following experiments.

[0041]Example 1  Of N-acetyllactosamine using the immobilized yeast cell method
Recovery of N-acetylglucosamine in continuous production method
Law

(1) Enzyme Synthesis (Immobilized Yeast Cell Method) Rhodotorura minuta cells prepared according to Reference Example 2 were prepared by a conventional method (Japanese Society for Biotechnology, edited by Biochemical Engineering, First Edition, Second Edition).
Imprint, 316 (1993), Baifukan) and immobilized with calcium alginate. Specifically, it was cooled in advance 2.
Cell concentration of 500 mg / ml in 8% sodium alginate aqueous solution
The suspension was dropped into a cooled 0.5% aqueous calcium chloride solution using a Pasteur pipette, and stored in a cool and dark place for 24 hours.

As a preliminary experiment, a batch method using immobilized cells (immobilized cell concentration 0.44 ml / ml) (substrate: lactose
0.5 M, N-acetylglucosamine 1.0 M, reaction conditions: pH 6.
(0, 60 ° C.), it was confirmed that 60 mM N-acetyllactosamine was obtained in the reaction solution.

The immobilized cells were placed in a glass column (1.6 × 10 c
m) to give an immobilized cell column. A substrate solution obtained by dissolving lactose and N-acetylglucosamine in 100 mM sodium acetate buffer (pH 6.0) to 0.5 M and 1 M, respectively, was heated to 60 ° C., and supplied to the column at a flow rate of 2 ml / hour. N-acetyllactosamine synthesis reaction was continuously performed.

The reaction solution (40 ml) passed through the immobilized bacterial cell column was applied to an activated carbon-celite (1: 1, Wako Pure Chemical Industries) column (8 × 13 cm), and the column was washed with water three times the amount of the column. The extract was washed, and eluted at a flow rate of 0.82 ml / min and a fraction volume of 20 ml by a gradient method of linearly increasing the methanol concentration using water (3000 ml) and 40% methanol (3000 ml).

The eluted fraction was 210 nm (acetyl group absorption)
Determined by measuring UV absorption at The results are shown in Figure 1. Peak 1 is a monosaccharide fraction (glucose, N-acetylglucosamine), peak 2 is a disaccharide fraction (N-acetyllactosamine, N-acetylallolactosamine), and peak 3 (not shown) is a trisaccharide fraction ( Galactosyl-N-acetyllactosamine). Fractions having an absorbance of at least 0.8 at 210 nm with respect to peak 1 were pooled to obtain an N-acetylglucosamine fraction containing glucose.

The obtained N-acetylglucosamine fraction was freeze-dried to obtain 8.3 g of pure white powder. Purity assay by HPLC showed that the powder was 78% pure N-acetylglucosamine. The glucose content in the N-acetylglucosamine fraction containing glucose obtained here was about 22%.

(2) Purification by treatment with glucose oxidase derived from Aspergillus niger The crude N-acetylglucosamine obtained in (1) was treated with 100 mM
Dissolve in sodium acetate buffer (pH 6.0) and add 100 mg / ml
10 ml of a crude N-acetylglucosamine aqueous solution was prepared. Glucose oxidase derived from Aspergillus niger (Kurabo Industries, specific activity 156 units / mg) was added thereto to a final concentration of 5 U / m.
After the addition, the enzyme reaction was carried out at 30 ° C. for 24 hours while controlling the pH of the reaction solution to pH 5.7 to 6.2 with 15% aqueous ammonia to convert glucose to gluconic acid.
The obtained reaction solution is applied to an anion exchange resin (Muromac 1 × 4
Chromatographed using ^- (Cl type, 20 ml, Muromachi Kagaku Kogyo) was), gluconic acid is adsorbed to the resin, the non-adsorbed fraction was lyophilized to give a pure white powder 700 mg. HP
As a result of analysis by LC, this powder was N-acetylglucosamine having a purity of 95%.

[0049]Example 2  N-acetate using recovered N-acetylglucosamine
Synthesis of tyllactosamine Rhodotorur prepared in Reference Example 2
a minuta washing cells (final concentration 500 mg / ml) and 100 mM sodium acetate
0.5M lactose in thorium buffer solution (pH 6.0)
And N-acetone containing glucose obtained in Example 1 (1)
Til glucosamine fraction, purified from Example 1 (2)
N-acetylglucosamine fraction, or as a control
N-acetylglucosamine (made by Nacalai Tesque) sold
It will be 1M as N-acetylglucosamine
Reaction temperature 60 ° C, reaction time 1
N-acetyllactosami by batch method under the condition of 7 hours
Enzyme synthesis. Table 2 shows the results.

[0050]

[Table 2]

The amount of N-acetyllactosamine produced when the N-acetylglucosamine fraction recovered in Example 1 (2) was used was almost the same as that when commercially available N-acetylglucosamine was used. However, the amount of N-acetyllactosamine produced when the N-acetylglucosamine fraction of Example 1 (1) collected without performing the glucose oxidase treatment was compared with the case where commercially available N-acetylglucosamine was used. N-
The rate of acetyl allolactosamine production increased.

[0052]

According to the present invention, N containing glucose is mixed.
-High-purity N-acetylglucosamine having a low glucose content can be easily and at low cost recovered from -acetylglucosamine. In particular, in the production of N-acetyllactosamine by enzymatic synthesis, application of the method of the present invention makes it possible to reuse N-acetylglucosamine remaining in the reaction-finished solution as a substrate, which is an expensive raw material. N-acetylglucosamine can be used without waste, and production costs can be reduced. Gluconic acid produced by the glucose oxidase treatment can also be used as a raw material for producing pharmaceuticals.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing a sugar elution pattern in activated carbon chromatography of a mixture obtained in a continuous production of N-acetyllactosamine by an immobilized cell method.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1: 685) C12R 1: 685) (C12P 19/26 (C12P 19/26 C12R 1:80) C12R 1 : 80) F term (reference) 4B064 AE01 AF03 CA05 CA06 CA21 CA36 CB11 CB30 CE09 CE10 CE20 DA16 4D017 AA07 BA04 CA13 CA17 DA03 EB03 4G066 AC14B AD06B AE10B CA56 DA07 EA01

Claims (10)

[Claims] 1. A method for recovering N-acetylglucosamine by removing glucose from an N-acetylglucosamine solution containing glucose, wherein the solution is treated with glucose oxidase, and glucose oxidase is recovered from the thus treated solution. A method comprising separating and removing a reaction product. 2. The method according to claim 1, wherein the solution is a reaction mixture obtained in the enzymatic synthesis of N-acetyllactosamine using lactose and N-acetylglucosamine as substrates. 3. The method according to claim 2, wherein the reaction mixture obtained in the enzyme synthesis of N-acetyllactosamine is a mixture obtained by separating and removing at least N-acetyllactosamine after the enzyme synthesis reaction. 4. The method according to claim 2, wherein the solution is a monosaccharide fraction obtained from a reaction mixture obtained in the enzymatic synthesis of N-acetyllactosamine. 5. The method according to claim 5, wherein the glucose oxidase is Aspergillus.
The method according to any one of claims 1 to 4, which is a glucose oxidase derived from a microorganism belonging to the genus s or Penicillium. 6. Glucose oxidase is Aspergillus
The method according to any of claims 1 to 4, which is derived from niger. 7. The method according to claim 1, wherein glucose is converted to gluconic acid by treating the solution with glucose oxidase, and gluconic acid is separated and removed from the treated solution by anion exchange resin treatment to recover N-acetylglucosamine. The method according to any one of claims 1 to 6. 8. After the enzymatic synthesis reaction of N-acetyllactosamine using lactose and N-acetylglucosamine as substrates, the reaction mixture is treated with activated charcoal, and the resulting monosaccharide fraction containing N-acetylglucosamine and glucose is treated. A method of converting glucose into gluconic acid by treating with glucose oxidase, and separating and removing gluconic acid from the solution by treating with an anion exchange resin to recover N-acetylglucosamine. 9. The method according to claim 1, wherein the purity of the obtained N-acetylglucosamine is 90% or more. 10. An N-acetyllactosamine obtained by transferring N-acetylglucosamine and lactose obtained by the method according to claim 1 from lactose to a galactose residue to N-acetylglucosamine. A method for producing N-acetyllactosamine, characterized in that the reaction is carried out in the presence of an enzyme having the action of producing acetyl.