JP2001269193A - Method for producing mannose by enzymatic method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for increasing the isomerization rate from fructose to mannose to produce mannose in high yields. SOLUTION: This method is carried out by isomerizing fructose to mannose by the use of mannose isomerase in the presence of a reducing sulfur oxygen oxide or an ammonium compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to D-mannose (hereinafter referred to as "mannose") and D-fructose.
The present invention relates to a method for producing mannose from fructose in high yield using an enzyme for interconverting (hereinafter referred to as fructose) mannose isomerase. More specifically, the invention of this application relates to a method for producing mannose from fructose using mannose isomerase in the presence of a reducing sulfuric acid compound or an ammonium compound.

[0002]

2. Description of the Related Art Mannose is widely used as a raw material for mannitol and as a raw material for animal cell culture. It has also been reported that mannose has an effect of suppressing the growth of intestinal Salmonella, and it has been studied to add it to drinking water and feed of poultry such as chickens (RHBrown, F
eedstuff, June 12, 10 (1989)). In addition, mannose is also known for improving the flavor of various foods,
Use as food material is also considered.

Hitherto, as a method for producing mannose, there have been known a method of acid-decomposing glucomannan contained in plants such as wood and konjac, and a method of heat-treating glucose at a high temperature using molybdate as a catalyst. But,
Each of these methods has a problem that the cost is high due to a shortage of raw material resources, an expensive reagent and a high temperature. As a result, mannose has been very expensive and has not been provided in sufficient quantities.

[0004] The inventors of the present invention have enthusiastically aimed at establishing a technology for producing mannose using mannose isomerase using fructose as a raw material, and a technology for producing mannose directly from glucose using mannose isomerase and glucose isomerase. Research has been advanced.

[0005] Mannose isomerase was introduced in 1956 by Pall.
eroni and Doudoroff et al. for the first time in Pseudomonas saccharophila (Journal Biological Chemistry, Vol. 218, 535).
(1956)). Thereafter, the inventor of the present application proposed that Xanthomonas
Bacteria identified as Xanthomonas rubrilineans (Journal of the Japanese Society of Agricultural Chemistry, Vol. 37, 524 (1963), Ag)
ricaltural Biological Chemistry, Vol. 28, 601 (196
4)) and Streptomyces sp. (Reported by Institute of Fermentation, National Institute of Advanced Industrial Science and Technology, Vol. 28, 89 (1966)).

[0006] Furthermore, the inventor has searched for a mannose isomerase-producing bacterium having excellent thermostability for the purpose of industrially utilizing this enzyme (mannose isomerase). ) A thermostable mannose isomerase produced by a genus bacterium was confirmed and reported (JP-A-4-2183).
70, Japanese Patent Application No. 11-123870, etc.).

[0007]

However, in the conventional isomerization of fructose to mannose by mannose isomerase, the yield (isomerization ratio) is about 25%.
%, And it was difficult to separate and purify mannose from the reaction solution after the reaction. Therefore, there is a problem that purification requires cost.

Accordingly, the invention of this application has been made in view of the circumstances described above, and solves the problems of the prior art, increasing the isomerization rate of fructose to mannose, and achieving a high yield. It is an object to provide a method for producing mannose.

[0009]

Means for Solving the Problems The present invention solves the above-mentioned problems. First, in the presence of a reducing sulfuric acid compound or ammonium compound,
Provided is a method for producing mannose, which comprises isomerizing fructose to mannose using mannose isomerase.

Secondly, the invention of this application relates to a method for producing mannose in which fructose is isomerized to mannose using mannose isomerase in the presence of an ammonium compound of either ammonium carbonate or ammonium bicarbonate. provide.

Furthermore, the invention of this application is, thirdly, in the above-mentioned method for producing mannose, fructose isomerized to mannose using mannose isomerase in the presence of a reducing sulfuric acid compound or an ammonium compound. After that, by contacting the reaction solution with either a sulfite-type or bisulfite-type anion exchange resin,
Provided is a method for producing mannose to be purified by removing a reducing sulfuric acid compound or an ammonium compound.

Fourth, the invention of the present application provides a method for producing mannose as described above, wherein fructose is isomerized to mannose using mannose isomerase in the presence of ammonium carbonate or ammonium bicarbonate, and then carbonated. The present invention also provides a method for producing mannose to remove and purify ammonium or ammonium bicarbonate from a reaction solution by vaporizing the same.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below.
The invention of this application will be described in more detail.

According to the inventor's intensive studies, the invention of this application is based on the fact that the reaction for producing mannose from fructose using mannose isomerase is carried out in the presence of a reducing sulfuric acid compound or in the presence of an ammonium compound. And that the isomerization rate of fructose to mannose was remarkably increased.

Specifically, the reaction is carried out at a pH of 5.5 to 7.0 in the presence of sodium sulfite, sodium bisulfite, or sodium metabisulfite (also referred to as pyrosulfite) as a reducing sulfur oxygen compound. Thus, the isomerization rate of fructose to mannose can be reduced to 60 to 90%.
It has been confirmed that it can be increased.

By performing the reaction in the presence of, for example, ammonium carbonate or hydroxyammonium sulfate as an ammonium compound, the isomerization rate to mannose can be increased to about 30 to 85%, and mannose can be produced in a high yield. Was confirmed. Furthermore, it was confirmed that even when the reaction was carried out in the presence of an ion exchange resin treated with any of these compounds, the isomerization rate to mannose was significantly increased, and mannose could be produced with high yield. However, the presence of non-reducing sulfur oxyacids such as sodium sulfate did not increase the mannose yield.

The invention of this application has been made based on the above findings.

The mannose isomerase used in the method for producing mannose of the invention of the present application includes the aforementioned Xanthomonas and Pseudomonas.
udomonas), Agrobacterium
From bacteria of the genus Streptomyces and the like, for example, Journal of the Japanese Society of Agricultural Chemistry, Vol. 37, 524-528 (196
3), Agricultural Biological Chemistry, Vol. 28, No.
9, 601-604 (1964), Journal of Fermentation and Bio
engineering, Vol. 76, No. 3, 237-239 (1993), Institute of Industrial Science, Institute of Fermentation, vol. 28, p. 89 (1965), JP-A-4
In a liquid medium containing an organic nitrogen source such as polypeptone or an organic carbon source such as glucose, the cells can be obtained by culturing at 30 ° C. for 1 to 3 days according to the method described in US Pat. Mannose isomerase can be obtained as a free enzyme by sonication or autolysis of the cells after centrifugation of the cultured cells, or by extraction from the cells. Furthermore, mannose isomerase can also be used as an enzyme obtained by immobilizing cells or a free enzyme by a known method.

In the method for producing mannose of the invention of the present application, the yield of mannose is greatly affected by the type of reducing sulfuric acid compound or ammonia compound selected.

The reducing sulfur oxygen compounds include sodium sulfite (Na ₂ SO ₃ ) and potassium sulfite (K ₂ S
O ₃ ), sodium metabisulfite (Na ₂ S ₂ O ₅ ) (also called sodium pyrosulfite or sodium disulfite), potassium metabisulfite (K ₂ S ₂ O ₅ ) (also called potassium pyrosulfite or potassium disulfite) ), Sodium bisulfite (NaHSO ₃ ), potassium bisulfite (KHSO ₃ ), sodium bisulfite (Na ₂ S ₂ O ₄ ), and potassium bisulfite (K ₂ S ₂ O ₄ ). Of these, sodium sulfite, sodium bisulfite, and sodium metabisulfite are preferred because they give mannose at a high conversion. In terms of the isomerization rate, sodium metabisulfite is particularly preferred.

In the method for producing mannose according to the invention of the present application, an ion-exchange resin to which ions of the above-described reducing sulfuric acid compound are bonded may be used. The ion-exchange resin for binding such reducing sulfur oxygen anions is not particularly limited, but Amberlite IRA-400 (manufactured by Rohm & Haas), Dowex 1-X8 ( Examples thereof include a strongly basic anion exchange resin such as Dow Chemical Co., Ltd.), and a mixed resin thereof with a cation exchange resin (eg, Amberlite MB-1). The mixed resin is effective and is preferable for maintaining the pH of the reaction solution in a desired range. As described above, even when an anion exchange resin is used, treatment with sodium sulfite, sodium bisulfite, and sodium metabisulfite is preferable since mannose can be obtained in the highest yield.

The method of treating these anion exchange resins is not particularly limited. For example, after converting the anion exchange resin to OH type with sodium hydroxide, reduction of sodium sulfite, sodium bisulfite, sodium metabisulfite or the like is performed. A method of treating with an acidic sulfuric acid compound and washing with water. In addition, the size and the like of the anion exchange resin are not particularly limited. However, as the mesh is smaller, the base mass per resin can be increased, and mannose can be efficiently produced.

Various methods can be considered as a method for contacting the thus-obtained reducing oxygen-oxygen compound-bound anion exchange resin with a reaction solution containing fructose and mannose isomerase. For example, the column may be filled with a reducing sulfur-oxygen compound-bound anion exchange resin and the reaction solution may be passed through, or the reducing sulfur-oxygen compound-bound anion exchange resin may be immersed in the reaction solution. Is also good.

Further, in the method for producing mannose of the invention of the present application, instead of the reducing sulfuric acid compound,
An ammonium compound may be used. Examples of such ammonium compounds include ammonium chloride, ammonium sulfate, diammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), ammonium dihydrogen phosphate (NH ₄ H ₂ PO ₄ ), and ammonium hydrogen carbonate (N
H ₄ HCO ₃ ), ammonium carbonate ((NH ₄ ) ₂ CO ₃ ), hydroxylammonium (also called hydroxyamine, (NH ₂ O
H)), and compounds such as hydroxyammonium chloride and hydroxyammonium sulfate. Further, cation exchange resins treated with any of these compounds may be used.

The ion exchange resin to which ammonium compound ions are bound is not particularly limited.
Amberlite IR-120, Amberlite I
RC-50 (Rome & Haas Co., Ltd.), Dowex (D
owex) 50-X8 (manufactured by Dow Chemical Company) or a mixed resin of a cation exchange resin and an anion exchange resin.

The method of treating these cation exchange resins is not particularly limited, but the cation exchange resin is converted into Na form with sodium hydroxide or H form with hydrochloric acid, and then treated with the above ammonium salt or hydroxyammonium salt. A method of washing with water is exemplified. In addition, the size and the like of the cation exchange resin are not particularly limited, but a smaller mesh is preferable because a base mass per resin can be increased and mannose can be efficiently produced.

The method of contacting the ammonium compound-bound cation exchange resin thus obtained with a reaction solution containing fructose and mannose isomerase includes:
Various methods are conceivable. For example, the column may be filled with an ammonium compound-bound cation exchange resin and the reaction solution may be passed through, or the ammonium compound-bound cation exchange resin may be immersed in the reaction solution.

In the method for producing mannose of the invention of the present application, the yield of mannose can be increased by using hydrazine or a cation exchange resin treated therewith, in addition to reducing sulfur oxygen oxides and ammonium compounds. be able to. However, the above-described reducing sulfur oxyacid compounds and ammonium compounds are more preferable because they are used as food additives and are excellent in safety.

The amount of these compounds to be added varies depending on the type, the concentration of the starting substrate at the time of the reaction, or the desired isomerization rate to mannose. That is, as the amount of the compound added to the raw material substrate increases, the isomerization rate to mannose increases. For example, raw material substrate concentration 1
At 0% or more, the mannose content in the reaction solution can be increased from about 25% to 40 to 90% by adding the compound at a molar concentration of about 1 to 2 times the molar concentration of the substrate. .

In the method for producing mannose according to the invention of the present application, the timing of adding the compound as described above may be at the time when a certain amount of mannose (10 to 25%) is formed after the reaction is started, even when the reaction is started. It may be. In the method for producing mannose of the invention of this application, the inhibition of the mannose isomerase reaction by the compound to be added is not so large, so that mannose can be obtained in a high yield even if the compound is added from the beginning of the reaction.

Further, the pH of the reaction is not particularly limited, but when a reducing sulfur oxyacid compound is used, the pH significantly affects the mannose yield. The optimum pH of mannose isomerase is around pH 8,
When various reducing sulfur oxyacid compounds are used, the optimum pH is considered to be related to the stability of the resulting double salt, and differs depending on the compound to be added. It can be set appropriately according to the mannose yield. Preferably, the pH is around 5-8.

On the other hand, in the reaction using an ammonium compound, no remarkable influence by pH has been recognized, and its value is not particularly limited.
5.5 to 9.

In the method for producing mannose of the present invention, the reaction temperature also affects the mannose yield. Therefore, it is preferable that the reaction is carried out at 40 to 60 ° C. which gives a high yield. Of course, it may be performed in a temperature range other than this.

Further, in the method for producing mannose of the invention of the present application, the reaction may be carried out under an inert atmosphere as much as possible because the presence of oxygen may reduce the yield of mannose in a reducing sulfur oxyacid atmosphere. Is preferred. For example, the reaction can be performed in a nitrogen gas or helium gas atmosphere.

In the method for producing mannose of the invention of the present application, the added compound such as a reducing oxygen compound or ammonium compound is removed and purified.
Purity can be further increased.

There has been reported a method for separating and purifying mannose utilizing the property of forming a complex of mannose with sodium bisulfite (NaHSO ₃ ) (JP-B 63-2).
1678). This utilizes the property of sodium bisulfite to form a stable crystalline complex under the strong acidity (pH 3.5 or less) condition. Crystals are separated by centrifugation, and the weight is reduced by hydrogen peroxide. Oxidation of sulfurous acid to sulfuric acid gives free mannose.

According to the method for producing mannose of the invention of the present application, mannose can be obtained in a high yield without using a conventional strong acid condition. Specifically, p
By utilizing the weak affinity of reducing sulfur oxyacid compound and mannose observed near H6, mannose can be increased in the presence of mannose isomerase while converting fructose to mannose.

In the method for producing mannose of the invention of this application, as a method for separating and purifying the generated mannose and the added salts of reducing sulfur oxyacid compounds and ammonium compounds, for example, calcium chloride is added. A method in which salts are precipitated as calcium salts and removed as insoluble salts, a method in which the salts are removed by electrodialysis using a cation exchange resin or an anion exchange resin, or an ion exchange membrane, or a method in which metal ions such as calcium are bound. or ion exchange resins, sulfite (SO ₃ ^-) type or bisulfite (HSO ₃ ^-)
And column chromatography using a type of anion exchange resin. In addition, since a salt of a sulfur oxygen compound (sulfite) is generally hardly soluble in alcohol, the salt can be separated and removed by adding an alcohol to a reaction solution containing the salt of the sulfur oxygen compound. Among the various purification methods exemplified above, in particular, sulfite type,
Alternatively, a method using a bisulfite-type anion exchange resin is preferable because of high separation efficiency.

As a method for bringing the reaction solution into contact with a sulfite-type or bisulfite-type anion exchange resin, the reaction solution may be passed through a column filled with an anion exchange resin treated with the same compound as the compound added to the reaction solution. Alternatively, the ion exchange resin may be immersed in the reaction solution. Furthermore, the ion-exchange resin and immobilized mannose isomerase may be packed in a column, the pH may be adjusted, and fructose or a fructose-containing solution may be continuously flowed.By using such a method, mannose from fructose can be reduced. It is also possible to increase the transition to.

Further, in the method for producing mannose of the invention of this application, when highly volatile ammonium carbonate or ammonium bicarbonate is used as an ammonium compound in the reaction, the reaction solution is heated, , Only the ammonium compound can be removed and recovered. However, sugars such as fructose are easily decomposed and colored by heating, so that the treatment is preferably performed under reduced pressure. By reducing the pressure, it is possible to completely vaporize and remove the ammonium compound in a short time even at a relatively low temperature such as 40 ° C. At this time,
Although the removal efficiency of the ammonium compound varies depending on the degree of reduced pressure, a high degree of reduced pressure is not necessarily required.
The purpose can be sufficiently achieved even with a moderate degree of reduced pressure.

In the method for producing mannose of the invention of this application, the sulfites and ammonium salts separated by the above-mentioned method and the remaining raw material fructose can be reused. The reaction solution can be used as it is as a raw material for producing mannitol.

Further, in the method for producing mannose of the present invention, not only fructose but also a mixed sugar solution of fructose and mannose (fructose 75-100%, mannose 0-25%) or isomerized sugar A fructose-containing liquid such as invert sugar or the like can also be used.

In the method for producing mannose according to the invention of the present application, it is possible to carry out the reaction using not only mannose isomerase but also glucose isomerase as an enzyme. In other words, mannose can also be directly obtained by using a glucose-containing liquid such as glucose, isomerized sugar or invert sugar as a raw material substrate. Hereinafter, the present invention will be described in more detail with reference to Examples. It goes without saying that the invention of this application is not limited to the following embodiments.

[0044]

EXAMPLES In the following examples, the enzyme activity was measured by the following method. <Method of measuring enzyme activity> 0.1 M phosphate buffer (p
H7.0) 0.2M mannose solution 0.5m
To l, an appropriate amount of enzyme was added, and the total amount was adjusted to 1.0 ml with water, followed by reaction at 50 ° C. The reaction was stopped by adding a 0.5 M perchloric acid solution, and the generated fructose was quantified by the cysteine-carbazole method. Under these conditions, the amount of enzyme that produces 1 μM fructose per minute was defined as one unit. <Example 1> polypeptone 2%, glucose 1%, 0.2% yeast _{extract, K 2 HPO 4 0.3%,} MgSO 4 · 7H 2 O 0.
200 ml of a 3% medium (pH 6) was placed in a 1-L Erlenmeyer flask, sterilized by pressure at 121 ° C. for 15 minutes, and then subjected to Agrobacterium tumefaciens.
mefaciens) IFO12664 (ATCC-4718) and inoculated at 30 ° C.
Shaking culture was performed for a day.

After the culture, the cells were collected by centrifugation, washed with water, and suspended in distilled water. A part of the cells was crushed with a 20KC ultrasonic cell crusher, and the extracted mannose isomerase activity was measured. As a result, 0.7 unit of the enzyme was contained per 1 ml of the suspension.

Hereinafter, an experiment was performed using this cell suspension. Test Example 1 Mannose yield and reaction p in a reaction in the presence of sodium sulfite using the prepared mannose isomerase
The relationship of H was examined.

0.45 ml of a 40% fructose solution (1
60 mg, 0.9 M) and sodium sulfite (Na ₂ SO ₃ ) 1
00 mg (0.8 M) and 0.1 ml (0.07 units) of the above-mentioned mannose isomerase were added, and the total amount was
The reaction was carried out at 50 ° C. for 20 hours. The mannose content in the reaction solution was determined by high performance liquid chromatography. The results are shown in Table 1 and FIG.

[0048]

[Table 1]

As is clear from Table 1 and FIG. 1, the yield of mannose was significantly affected by the pH during the reaction, and it was confirmed that the optimum pH at which the yield of mannose was maximized was around 6.

The mannose yield of the reaction (pH 7.0, 50 ° C.) in the absence of sodium sulfite was 2%.
It was 5.4%. Test Example 2 As in Test Example 1, in the presence of sodium sulfite, isomerization of mannose from fructose by mannose isomerase was carried out, and the relationship between mannose yield and reaction temperature was examined.

The 40% substrate solution 0.4 used in Test Example 1
To 0 ml (160 mg, 0.9 M), 100 mg (0.8 M amount) of sodium sulfite (Na ₂ SO ₃ ) and 0.1 ml (0.07 unit) of mannose isomerase are added, and a total amount of about 1 ml (pH of about 6. 0), and the reaction was carried out at each temperature for 20 hours.

The produced mannose was quantified by high performance liquid chromatography. The results are shown in Table 2 and FIG.

[0053]

[Table 2]

Regarding the activity of mannose isomerase used in the examples, although the optimum temperature was about 60 ° C., from Table 2 and FIG. 2, the yield of mannose was also affected by the reaction temperature. , 45-55
A temperature of ° C has been shown to be optimal. Test Example 3 Mannose formation reaction in the presence of sodium sulfite was carried out under the same conditions as in Test Examples 1 and 2, and the effect of the concentration of the raw material substrate (fructose) was tested.

Fructose and sodium sulfite were added in Table 3
After adding 0.2 ml (0.14 units) of mannose isomerase to each of the concentrations shown in
H was adjusted to about 6.0, and the total amount was adjusted to about 1 ml with distilled water.
The isomerization reaction was performed at 0 ° C. for 20 hours.

The amount of mannose in the reaction solution was quantified by high performance liquid chromatography. Table 3 shows the results.

[0057]

[Table 3]

Each of the reactions is a reaction in which the raw material substrate (fructose) and sodium sulfite are present in an equimolar concentration, but the mannose content in the reaction solution tends to increase as the substrate concentration increases. That is, it was shown that performing the reaction under a high substrate concentration can increase the mannose yield per sodium sulfite. <Example 2> In the same manner as in Example 1, instead of sodium sulfite, sodium metabisulfite (also referred to as sodium pyrosulfite or sodium disulfite: Na ₂ S
_{Using 2} O ₅ ), the isomerization reaction was performed at pH 5.6 to 8.2.

0.45 ml of the substrate solution used in Example 1
(180mg), 270m of sodium metabisulfite
g and mannose isomerase 0.1 ml (0.07 units) were added, and the reaction was carried out at 50 ° C. for 45 hours.

The results obtained are shown in Table 4 and FIG.

[0061]

[Table 4]

As is clear from Table 4 and FIG. 1, in the reaction in the presence of metabisulfite, as in the case of sodium sulfite, the optimum pH was found around 6. <Example 3> The reaction was carried out in the same manner as in Examples 1 and 2, except that the amount of sodium sulfite relative to the raw material substrate (fructose) was changed.

To 0.45 ml (180 mg) of the 40% fructose solution used in Example 1 were added sodium sulfite (Na ₂ SO ₃ ) in the amounts shown in the table, and 0.1 ml (0.07 units) of mannose isomerase. Add about 1m
The reaction was carried out at pH 6.0 and 50 ° C. for 20 hours.

The results of quantifying the amount of mannose in the reaction solution by high performance liquid chromatography are shown in Table 5 and FIG.

[0065]

[Table 5]

From Table 5 and FIG. 3, it was shown that the amount of mannose increased as the amount of sodium sulfite relative to the raw material substrate increased. When sodium sulfite was added 1.5 times (molar concentration) to the substrate, about 55% of fructose was isomerized to mannose, and when added 2.0 times, about 59% of fructose was isomerized to mannose. Was done. Example 4 The same reaction as in Example 3 was carried out using sodium metabisulfite instead of sodium sulfite.

To 0.45 ml (180 mg) of the 40% fructose solution used in Example 1, sodium metabisulfite (Na ₂ S ₂ O ₅ ) was added in the amount shown in the table, and the mannose isomerase 0 prepared in Example 1 was added. 0.1 ml (0.07 units) was added, the pH was adjusted to about 6.0, and the reaction was carried out at 50 ° C. for 20 hours.

The results of quantifying the mannose content in the reaction solution by high performance liquid chromatography are shown in Table 6 and FIG.

[0069]

[Table 6]

As the amount of sodium metabisulfite added to the raw material substrate increases, the mannose content in the reaction solution gradually increases, and the amount of sodium metabisulfite is 1.5 times, 1: 1 for 1 mol of the substrate. When a 75-fold and 2.0-fold molar amount was added, the mannose content in the reaction solution was about 75%, about 80%, and about 83%, respectively.

The above results show that sodium metabisulfite can isomerize fructose to mannose at a higher isomerization rate than sodium sulfite. <Example 5> 0.2 ml (80 mg) of the 40% fructose solution used in Example 1 and 0.4 ml (160 m
g) or 0.8 ml (320 mg), mannose isomerase 0.1 ml (0.07 units) and distilled water were added to make a total volume of about 1 ml, and each was treated with sodium sulfite (Na ₂ SO ₃ ). Sulfurous acid type Dowex (Do
Wex) 1-X8 each 1 g (wet amount) was added,
The reaction was performed at 0 ° C. for 20 hours.

After the reaction, the amount of mannose in the reaction solution was quantified by high performance liquid chromatography, and the results are shown in Table 7.

[0073]

[Table 7]

In the reaction in the presence of a sulfite-type anion exchange resin, an increase in mannose was also observed. <Example 6> Amberlite MB-1 ion exchange resin (manufactured by Rohm & Haas Co., Ltd., sold by Organo Co., Ltd.), which is an equal mixture of a strongly acidic cation exchange resin and a strongly basic anion exchange resin, was treated with sodium metabisulfite. And packed in a column (diameter 2.0 cm x length 2.5 cm). To this, 2.5 ml of the 40% fructose solution used in Example 1 and 0.5 ml (0.35 units) of mannose isomerase were used. In addition, the reaction was carried out at 50 ° C. while adjusting the pH to 6.0 to 6.2.

After the reaction for 5 hours, the content of mannose in the reaction solution was quantified by high performance liquid chromatography. As a result, 38.5% of fructose was isomerized to mannose.

The ion exchange resin could be reused without regeneration. Example 7 Mannose was produced using glucose as a raw material substrate in the presence of mannose isomerase and glucose isomerase.

180 mg of glucose (1 M amount), 189 mg of sodium sulfite (1.5 M amount), 0.1 M MgSO ₄
0.05 ml (0.005M amount), commercially available immobilized glucose isomerase agent (manufactured by Nagase Seikagaku Corporation)
20 mg and 0.1 ml (0.07 units) of mannose isomerase prepared in Example 1 were added, and the total amount was adjusted to about 1 with distilled water.
ml, and reacted at pH 6.0-6.2 at 50 ° C. for 20 hours.

The contents of mannose, fructose and glucose in the reaction solution were determined by high performance liquid chromatography to find that they were 29.5% and 26.7%, respectively.
%, 43.8%. That is, compared to the mannose yield of 12.0% without sodium sulfite, 2%
A sugar solution containing mannose twice or more (29.5%) was obtained. <Example 8> 1 ml of a sugar solution of about 1M containing about 75% of mannose and about 25% of fructose prepared in Example 4
(SO ₃ ) type Dowex 1-X, which was kept at 40 ° C.
The solution was supplied to 8 columns (diameter 1.5 cm × length 25 cm) and eluted with water (flow rate: about 8 ml / hour, fractionation volume: about 2 m)
l). The results obtained are shown in FIG.

As is apparent from FIG. 4, salt and sugar could be almost completely separated.

FIG. 5 shows the sulfurous acid type Dowex 1-X8
The salt and the saccharide were separated using a bisulfite (HSO ₃ ) type Dowex 1-X8 column (diameter 1.5 cm × length 25 cm) in place of the above (flow rate: about 8 ml / hour, fractionation capacity: about 2 m).
l) It is a figure showing a result.

As is clear from FIG. 5, mannose could be almost completely separated from fructose and salts.

More complete separation can be achieved by using a bisulfite (HSO ₃ ) type anion exchange resin than by using a sulfite (SO ₃ ) type anion exchange resin column. confirmed.

Further, each of them could be similarly separated from sugar solutions containing oxyacid salts or ammonium salts of sulfur other than sulfites and bisulfites. Example 9 To 0.2 ml (160 mg) of an 80% sugar solution (mannose content 20%, fructose content 80%) consisting of 64% fructose and 16% mannose, 50 mg each of ammonium chloride, ammonium phosphate, or ammonium carbonate was added. , And 0.15 ml of the enzyme solution (0.
11 units), make up to about 1 ml with distilled water,
The reaction was carried out at 200C for 20 hours.

The mannose content in the reaction solution was quantified by high performance liquid chromatography, and the results are shown in Table 8.

[0085]

[Table 8]

As is evident from Table 8, the mannose yield was 25.5% when no ammonium compound was added, whereas the mannose yield was remarkable when any ammonium compound was added. Increased. <Example 10> 0.22 m of the substrate solution used in Example 9
1 (180 mg, 1.0 M amount), ammonium sulfate,
No addition of ammonium carbonate or ammonium bicarbonate, 0.25M, 0.5M or 1M amount and enzyme solution (0.15ml, 0.11 unit) are added, and the total amount is about 1ml with distilled water (pH about 7. 0), and the reaction was carried out at 50 ° C. for 20 hours. After the reaction, the produced mannose was quantified by high performance liquid chromatography, and the results are shown in Table 9.

[0087]

[Table 9]

As is evident from Table 9, the mannose yield increases as the amount of any of the ammonium compounds tested increases, and the mannose yield increases when 1.5 times the substrate concentration of ammonium carbonate is added. The yield is about 55
%Met. <Example 11> In this example, a case where a hydroxyammonium salt was used as an ammonia compound will be described.

0.2 ml of the substrate solution used in Example 9
(160 mg), 0.5M or 1.0M of hydroxyammonium chloride or hydroxyammonium sulfate
Amount and 0.15 ml of the mannose isomerase solution (0.
11 units), and adjusted to a total volume of about 1 ml with distilled water, and reacted at 45 ° C. for 20 hours.

The results obtained by quantifying the mannose content in the reaction solution by high performance liquid chromatography are shown in Table 10.

[0091]

[Table 10]

As is apparent from Table 10, the addition of the hydroxyammonium salt significantly increased the mannose yield, and a sugar solution having a mannose content of 60 to 75% was obtained. Example 12 A reaction was carried out using a cation exchange resin treated with ammonium chloride, hydroxyammonium or hydrazine as an ammonium compound.

Amberlite IR-120 (manufactured by Rohm and Haas Company, about 30 mesh) was made into H-form with 1N hydrochloric acid, treated with ammonium chloride, hydroxyammonium or hydrazine, and washed with distilled water.
g (wet amount) was placed in a test tube, 0.2 ml (160 mg) of the raw material substrate solution used in Example 9 and 0.15 ml (0.11 unit) of the mannose isomerase solution were added, and the total amount was about 1 m.
The reaction was carried out at 45 ° C. for 20 hours. Table 11 shows the results.
It was shown to.

[0094]

[Table 11]

As is clear from Table 11, any of the ammonium-type, hydroxyammonium-type, and hydrazine-type cation-exchange resins can be contacted with the reaction solution.
A marked increase in mannose yield was observed. <Example 13> 1 g (wet amount) of hydroxyammonium-treated Amberlite IR-120 used in Example 12, 0.1 ml (80 mg) of a raw material substrate solution, 0.2 ml (160
mg), 0.3 ml (240 mg), 0.4 ml (32
0 mg) or 0.5 ml (400 mg), and 0.15 ml (0.11 unit) of the enzyme solution.
The reaction was carried out at 45 ° C. for 20 hours.

Table 12 shows the obtained results.

[0097]

[Table 12]

From Table 12, it was confirmed that a sugar solution having a mannose content of about 59% can be obtained by adjusting the base weight per resin to an appropriate amount (about 80 mg of substrate per 1 g of wet resin weight). <Example 14> Amberlite IRC-50 (about 30 mesh, manufactured by Rohm & Haas) was made into H type with 1N hydrochloric acid, treated with ammonium chloride or hydroxyammonium hydrochloride, and washed with distilled water, each 1 g. (Wet amount) was placed in a test tube, and 0.2 ml (160 mg) of the raw material substrate solution used in Example 1 and 0.1 ml (0.
11 units) to make a total volume of about 1 ml.
A time reaction was performed.

The results are shown in Table 13.

[0100]

[Table 13]

From Table 13, it was confirmed that the yield of mannose can be remarkably increased also when an ammonium type or hydroxyammonium type weakly acidic cation exchange resin is used. <Example 15> Mannose 6.8%, fructose
2 ml of reaction solution containing 2% and 10.0% ammonium carbonate
Into a test tube (diameter 14 mm x length 100 mm).
Heated at 0, 55, 60, 65, 70, 80 or 100 ° C. Over time, a fixed amount was taken and the amount of remaining ammonium carbonate was determined by a densitometer. The results obtained are shown in Table 14 and FIG.

[0102]

[Table 14]

From Table 14 and FIG. 6, it was confirmed that by heating at a temperature of 60 ° C. or higher, ammonium carbonate could be vaporized and separated and removed from the product (mannose) solution. Example 16 Mannose 6.8 used in Example 15
%, Fructose 2.2% and ammonium carbonate 10.0
% Of the reaction solution was placed in a 100 ml concentrating flask, and concentrated at a temperature of 40, 50, 60, 70 and 80 ° C. under a reduced pressure of 30 mmHg.

When the water had evaporated, the same volume of distilled water was added, and the concentration was repeated. At each treatment (3-5 minutes)
With respect to the collected sample, the amount of remaining ammonium carbonate was determined by a densitometer.

Table 15 shows the results.

[0106]

[Table 15]

From Table 15, it was clarified that ammonium carbonate can be effectively vaporized at a relatively low temperature in a short time and effectively separated and removed from the reaction solution by this method.

[0108]

As described in detail above, according to the method for producing mannose of the invention of the present application, in the reaction using the conventional mannose isomerase, mannose which could be produced only at a low yield of about 25% was obtained. The yield can be increased to 90% or more.

Therefore, by using the method for producing mannose of the invention of this application, it is possible to produce mannose and mannitol from fructose in large quantities at low cost.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between reaction pH and mannose yield in Example 1 (Test Example 1) of the invention of this application.

FIG. 2 is a diagram showing the relationship between the reaction temperature and the mannose yield in Example 1 (Test Example 2) of the invention of this application.

FIG. 3 is a graph showing the relationship between the amount of sulfite added (molar concentration ratio) and the yield of mannose with respect to the substrate concentration in Examples 3 and 4 of the present invention.

FIG. 4 is a diagram showing separation of sulfites and sugars from sugar solutions containing mannose and fructose, respectively, using a sulfite-type anion exchange resin column in Examples of the present invention.

FIG. 5 is a diagram showing separation of a sulfite and each sugar from a sugar solution containing mannose and fructose by a bisulfite-type anion exchange resin column.

FIG. 6 is a diagram showing the influence of the treatment temperature on the vaporization and removal of ammonium carbonate from a reaction solution containing ammonium carbonate in Example 15 of the present invention.

Claims (4)

[Claims] 1. A method for producing mannose, wherein fructose is isomerized to mannose using mannose isomerase in the presence of a reducing sulfuric acid compound or an ammonium compound. 2. The method for producing mannose according to claim 1, wherein fructose is isomerized to mannose using mannose isomerase in the presence of an ammonium compound of either ammonium carbonate or ammonium bicarbonate. 3. The method for producing mannose according to claim 1, wherein fructose is isomerized into mannose using mannose isomerase in the presence of a reducing sulfuric acid compound or an ammonium compound, and then the reaction solution is sulfite-type or mannose-containing. A method for producing mannose, wherein a reducing sulfuric acid compound or an ammonium compound is removed by contact with any of bisulfite-type anion exchange resins to purify the mannose. 4. The method for producing mannose according to claim 2, wherein fructose is isomerized to mannose using mannose isomerase in the presence of ammonium carbonate or ammonium bicarbonate, and then ammonium carbonate or ammonium bicarbonate is vaporized. A method for producing mannose that is removed from the reaction solution and purified by the method described above.