JPH06237782A - Method for separating and obtaining mannose - Google Patents

Abstract

PURPOSE:To industrially obtain mannose by fractionating a mannose-containing saccharide liquid according to chromatography using an ion exchange resin and then obtaining the mannose fraction. CONSTITUTION:A mannose-containing saccharide liquid is used as a raw material liquid and the mannose and other saccharides are fractionated according to the chromatography using an ion exchange resin to obtain a mannose fraction. A fructose fraction formed as a by-product is reutilized for producing the mannose.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating and obtaining mannose. More specifically, a mannose-containing sugar liquid is used as a raw material liquid, and mannose and other sugars in the raw material liquid are fractionated by chromatography using an ion exchange resin to obtain a mannose fraction. Regarding separation acquisition method.

Mannose is expected to be applied as a feed additive for poultry such as chickens and as a functional food material.

[0003]

2. Description of the Related Art Mannose has hitherto been very expensive because it has been produced by decomposing mannan contained in plants such as wood and konjak with hydrochloric acid.
Therefore, although mannose was used as a reagent, it was hardly used for food or feed.

Recently, attempts have been made to produce mannose-containing sugar solutions from fructose using mannose isomerase and from glucose using glucose isomerase and mannose isomerase. , It wasn't established yet.

[0005]

Although mannose-containing sugar solutions could be produced from fructose by using mannose isomerase, or from glucose by using glucose isomerase and mannose isomerase, respectively, however,
This mannose-containing sugar solution is a mixture of mannose and other sugars (fructose, glucose and oligosaccharides), and the composition ratio of mannose in the mannose-containing sugar solution is at most 25% of the solid content of sugar. It was nothing more than a thing.

Therefore, when mannose is used in foods, this mannose-containing sugar solution is still insufficient,
Furthermore, it was necessary to separate and acquire mannose from the sugar solution containing mannose.

[0007]

[Means for Solving the Problems] Therefore, the present inventors have made extensive studies on a method for separating mannose from a mannose-containing sugar solution. Then, after fractionating mannose and other sugars in the mannose-containing sugar solution by chromatography using an ion exchange resin, a mannose fraction is obtained and the by-produced fructose fraction is reused for mannose production. As a result, we succeeded in efficiently obtaining mannose and completed the present invention.

The mannose-containing sugar liquid as the raw material liquid of the present invention means a mixed liquid of mannose and other sugars, and the production method thereof is not limited at all.
For example, a mannose-containing sugar solution is produced from fructose using mannose isomerase, or from glucose using glucose isomerase and mannose isomerase. As mannose isomerase used for isomerization of fructose,
Any mannose isomerase can be used regardless of its origin, but those of microbial origin and thermostable are preferable.

For example, the novel thermostable mannose isomerase described in JP-A-4-218370 can be preferably used.

The enzyme-producing strain was newly found in soil,
It has been isolated and its mycological properties are as follows.

(1) Morphology Cell shape and size: 0.6-0.8 × 1.0-2.0μ rod bacillus Presence / absence of cell polymorphism: Not observed Motility: Motile with two or more polar flagella Presence or absence of spores: Not formed Gram stainability: Negative Acidity: Negative

(2) Growth state in each medium Meat broth agar plate culture: Colonies are cultured at 37 ° C. for 48 hours and then diameter
0.5 ~ 0.8mm round, smooth surface, semi-lens-like ridge, yellow-yellowish brown on all sides, translucent, resin-like gloss Meat agar slope culture: good growth, straight upheap growth,
Yellow-brown with metallic luster and green-brown water-soluble pigment diffused into the agar at the bottom of the slope Broth liquid culture: No pellicle formation, medium turbidity, yellowish litmus milk culture: liquefaction Does not solidify

(3) Physiological properties Nitrate reduction: Negative Gas from nitrate: Negative MR test: Negative VP test: Negative Indole formation: Negative Hydrogen sulfide formation: Negative Starch hydrolysis: Negative Utilization of citric acid: Simmons and Krisden medium used together Pigment formation: No pigment formation in King A and King B medium Urease: Negative oxidase: Negative (weak if any) Catalase: Positive OF test: Oxidized arginine hydrolase: Positive acyl Amidase: Positive amino acid decarboxylase (lysine, ornithine):
Negative DNase: Negative Growth temperature range: Does not grow above 41 ° C Attitude toward oxygen: Aerobic growth against salt: 0-4 (%)

Presence or absence of acid production from sugars: galactose + glucose + saccharose + trehalose + L-arabinose + cellobiose + xylose + lactose + maltose + mannose + mannitol + fructose + salicin + starch-

Degradation of gelatin (Fraizer method): Positive TSI agar medium (slope acid) :-( High-layer acid):-SS agar medium: No growth NAC agar medium: No growth MacConky agar medium: Growing Triphenyl Tetro Zolium (0.1%) Agar medium: Acid production from 10% lactose that grows: positive Esculin degradation: positive (weak) phenylalanine deaminase: negative Tween80 degradation: positive 0.1% methylene blue milk reaction: reduced liquefaction ONPG reaction: Positive Levan production: Negative Mucoid colonization occurrence: Negative

Regarding the above-mentioned mycological properties, Bergey's Man
Comparing the properties with reference to the nual of Systematic Bacteriology, Volume 2 (1986), this bacterium is a bacillus that produces yellow pigment and has polar flagella. Nitrate reduction negative, oxidase negative, Tween80 decomposition positive, Since the salt tolerance is low and the produced pigment is water-soluble, this bacterium was recognized as a bacterium belonging to the genus Pseudomonas. In addition, this bacterium has physiological characteristics such as negative levan-producing ability, positive arginine hydrolase, negative phenylalanine deaminase, negative starch degradation, positive acylamidase, and a bacterial width of 0.3.
~ 0.6μ.

Based on the above, this strain was determined to be a new strain and was named Pseudomonas sp. AM-9582. This strain was submitted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology
Deposited as No. 3207 (FERM BP-3207).

First, a method for culturing this strain to obtain mannose isomerase will be described below.

The fish meat extract 2.5%, glucose 2.5%, fructose _{1.25%, K 2 HPO 4 0.2} %, MgSO 4 · 7H 2 O 0.05
%, CuSO ₄ 6 × 10 ⁻⁴ M in medium (pH 7.0) 20 L 30
The mixture was placed in an L-volume jar fermenter, sterilized by a conventional method, inoculated with Pseudomonas sp. AM-9582 (FERM BP-3207), and cultured with aeration and stirring at 30 ° C for 2 days. After culturing, the cultured cells were treated in the presence of 0.05% cetyltrimethylammonium bromide at pH 8.0 at 30 ° C. for 6 hours and then centrifuged to extract the enzyme. The enzyme solution thus obtained is
60% ammonium sulfate was salted out to obtain a precipitate, which was further dialyzed to obtain DEAE.
Sepharose column chromatography and gel filtration with Sephadex G150 were performed 3 times. The purified mannose isomerase obtained was electrophoretically single and had a specific activity of about 290 times.

The enzymatic chemistry of the novel thermostable mannose isomerase (EC 5.3.1.7) thus obtained is as follows.

(A) Action Mannose is isomerized to fructose and fructose is isomerized to mannose. Also, acting on D-lyxose, D
Isomerized to xylulose.

(B) Substrate specificity Acting on D-mannose and D-lyxose, D-rhamnose, D-fucose, D-glucose, D-ribose, D-xylose, D-arabinose, L-xylose, L- It does not substantially act on arabinose, L-rhamnose and L-fucose.

(C) Working pH and optimum pH It works in a pH range of about 6 to 11, but the optimum pH was found around 8 (reaction under 0.1 M phosphate buffer at 50 ° C. for 30 minutes. ).

(D) Working temperature and optimum temperature It works up to about 70 ° C., but the optimum temperature was found to be about 55 ° C. [0.1 M mannose, 0.05 M Tris buffer (pH 7.0) 30 Minute reaction].

(E) Thermostability The residual activity when heated at 60 ° C. for 10 minutes in 0.05 M Tris buffer (pH 7.0) was about 68%.

(F) pH stability After standing for 3 hours at room temperature (25 ° C.) in a 0.1 M buffer solution (acetic acid or phosphate buffer solution), the residual activity was measured. As a result, it was stable in the range of pH 6-9.

(G) Inhibitor It was strongly inhibited by 5 × 10 ⁻³ M HgCl ₂ , FeSO ₄ , AgNO ₃ , AlCl ₃ , p-chloromercuribenzoate (pCMB) and the like.

The activity was measured by the following method. 0.2
0.1 M phosphate buffer (pH 7.0) in which M mannose was dissolved.
Add an appropriate amount of enzyme to 5 ml, make the total volume 1.0 ml with water, and heat at 50 ° C.
React with. Then, the produced fructose is quantified by the cysteine-carbazole method. Under this condition, the amount of enzyme that produces 1 micromol of fructose per minute is defined as 1 unit.

The glucose isomerase used in the present invention is not particularly limited, and any glucose isomerase can be used. For example, glucose isomerase of the genus Streptomyces (manufactured by Carter Co., Finland, Japan Commercially available glucose isomerases (sold by Carter Co., Ltd.) and the like.

In the measurement of glucose isomerase activity, the amount of the enzyme that reacts with 0.1 M glucose and 0.01 M MgSO ₄ at 60 ° C. to produce 1 micromol of fructose per minute was defined as 1 unit.

Next, a method for producing a mannose-containing sugar solution and a method for separating and obtaining mannose from a mannose-containing sugar solution from fructose using mannose isomerase and from glucose using glucose isomerase and mannose isomerase will be described.

When producing a mannose-containing sugar solution from fructose using mannose isomerase, the fructose-containing solution used as a substrate has a fructose concentration of 5 to 70 W / V%, preferably 10 to 50 W / V%. .
The high fructose fraction by-produced by the method of the present invention contains fructose in this concentration range, and thus can be reused as a substrate as it is.

When producing a mannose-containing sugar liquid from glucose using glucose isomerase and mannose isomerase, the concentration of glucose in the glucose-containing liquid used as a substrate is also 5 to 70 W / V%, preferably 1
It is 0 to 50 W / V%.

The mannose isomerase and glucose isomerase used in the reaction may be either crude enzyme products or purified products, and the microbial cells themselves having the enzyme activity or the cells may be immobilized. It may be one that has been converted.

The mannose isomerase activity in the case of obtaining a mannose-containing sugar solution from fructose using mannose isomerase is 0.01 to 1000 units, preferably 0.1 to 100 units per 1 g of the substrate fructose.
The glucose isomerase activity when a mannose-containing liquid is obtained from glucose using glucose isomerase and mannose isomerase is
It is 0.01 to 1000 units, preferably 0.1 to 100 units, and the mannose isomerase activity is 0.01 to 1000 units, preferably 0.1 to 100 units, per 1 g of substrate glucose.

In any case, the enzyme reaction conditions are pH
The reaction may be performed at 4 to 10 at a temperature of 20 to 80 ° C for 5 to 200 hours, preferably at a pH of 6 to 8 and a temperature of 40 to 60 ° C for 4 to 15
It is advisable to carry out the reaction for 0 hours.

The mannose-containing sugar solution thus obtained is
Although it can be used as it is as the raw material liquid of the present invention, it is more efficient to use it as the raw material liquid of the present invention after concentration.

Next, the inventors of the present application examined the mannose from the mannose and other sugars mixed in the raw material liquid by carrying out chromatography using the raw material liquid and using various ion exchange resins. As a result, they have found that it is possible to fractionate mannose and other sugars in various ion exchange resins, that is, in both cation exchange resin and anion exchange resin.

As the cation exchange resin used for the separation, either a strong acid cation exchange resin or a weak acid cation exchange resin can be used, but a strong acid cation exchange resin is preferable, and a particularly preferable one is It is a calcium type strong acid cation exchange resin. Specific examples thereof include DIAION FRK series and DIAION "Unibeads" series calcium-type strongly acidic cation exchange resins. Note that Diaion is a product name of Mitsubishi Kasei Co., Ltd.

As the anion exchange resin used for the separation, both a strong basic anion exchange resin and a weak basic anion exchange resin can be used, but a strong basic anion exchange resin is preferable, Preferred are sulfite type strong basic anion exchange resin, hydrochloride type strong basic anion exchange resin and carbonate type strong basic anion exchange resin.

As a specific example of the sulfite type strong basic anion exchange resin, Dowex 1-X8 (trade name of Dow Chemical Co.) is a specific example of the hydrochloride type strong basic anion exchange resin. DIAION SA-10A, and Amberlite IRA-400 (trade name of Organo Co.) are specific examples of the carbonate type strongly basic anion exchange resin.

The raw material liquid is passed through the column at 20 to 90 ° C., preferably 40 to 70 ° C. for 0.1 to 50 hours, preferably 0.5 to
Do it for 10 hours.

The separation operation of the present invention comprises four separation towers (shown in FIG. 1) in which a mannose-containing sugar liquid consisting of a mixed liquid of mannose and fructose is used as a raw material liquid and calcium type strong acid cation exchange resin is filled. The case of fractionation by a chromatography device will be described in more detail below.

First, the mannose-containing sugar solution is circulated in the packed bed apparatus from the first tower to the second tower, the third tower and the fourth tower to the fourth tower, and then the mannose-containing sugar solution is separated into four towers. To the third column to allow the compartment to flow down, and during this time, the second stage of extracting the mannose-rich solution flowing out from the compartment to the outside of the system, and further eluting water to the first tower to separate the compartment. A third step in which the fructose-rich solution is allowed to flow out and during this period the fructose-rich solution flowing out of the compartment is withdrawn out of the system;
Also, the eluting water is supplied to the first tower to flow down the section,
The effluent of the first tower is allowed to flow into the second tower, the effluent of the second tower is allowed to flow into the third tower, and the mannose-rich solution flowing out of the third tower is withdrawn to the outside of the system. Mannose and fructose in the raw material liquid are fractionated into a mannose section and a fructose section, respectively, by a separation operation that is repeated in sequence (a fractionation diagram is shown in FIG. 2).

A high-purity mannose fraction of about 80 to 90% can be obtained by this separation operation, and the by-produced residual fructose fraction is directly reacted with mannose isomerase to produce a mannose-containing sugar solution. Therefore, it can be reused as a raw material liquid.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0047]

【Example】

Example 1 (1) Production of mannose-containing sugar solution Pseudomonas was added to 1.0 L of a 30 W / V% aqueous solution of fructose.
Add mannose isomerase (1200 units) derived from SP AM-9582 (FERM-BP 3207), pH 7.0, 50 ℃
And reacted for 20 hours to obtain a mannose-containing sugar solution (1.0 L).

(2) Separation and acquisition of mannose 1.0 L of the mannose-containing sugar solution obtained in (1) was filtered with activated carbon, and then ion-exchange resin [K tower: Diaion SK
1B (Mitsubishi Kasei product), Tower A: Diaion WA30
(Product of Mitsubishi Kasei Co., Ltd.), MB tower: Amberlite IRA-
411 (manufactured by Organo) + Diaion SK1B (manufactured by Mitsubishi Kasei)] and concentrated to obtain a raw material liquid (0.5 L) for fractionation of mannose and fructose. The properties of this raw material liquid were as follows. In the properties, ds is an abbreviation for dry sugar, that is, dry matter sugar.

Concentration 60% pH 5.5 Coloring 0.007 Turbidity 0.000 Sugar composition Fructose 75.8 (% / ds) Mannose 23.0 Glucose 0.7 Oligosaccharide 0.5

Using the raw material liquid having the above composition, a chromatographic fractionation apparatus consisting of four separation towers packed with a calcium-type strongly acidic cation exchange resin (Diaion UBK555, Mitsubishi Kasei Co., Ltd., trade name) was used at 60 ° C. The liquid is passed through to separate the mannose fraction and the fructose fraction, and the high-purity mannose liquid (0.78 L) and the high-purity fructose liquid (1.
13 L) was obtained. The properties of this product liquid were as follows.

[0051]

[Table 1]

Example 2 (1) Production of mannose-containing sugar solution 1.5 L of fructose category (solid content 21.0%, fructose content 94.8%) produced as a by-product in Example 1
In addition, Pseudomonas SP AM-9582 (FERM-BP 320
250 g of cultured mannose isomerase derived from 7)
(1750 units) at pH 7.0 and 50 ℃ for 16 hours, then 85
The mixture was heat treated at 25 ° C. for 25 minutes and centrifuged to obtain a mannose-containing sugar solution (1.4 L).

(2) Separation and acquisition of mannose The mannose-containing sugar solution (1.4 L) obtained in (1) was filtered with activated carbon, followed by purification and concentration with an ion exchange resin.
The raw material liquid (0.53 L) was used for fractionation. The properties of this raw material liquid were as follows.

Concentration 60.2% pH 5.4 Coloring 0.006 Turbidity 0.000 Sugar composition Fructose 75.5 (% / ds) Mannose 24.0 Glucose 0.7 Oligosaccharide 0.8

Using the raw material liquid having the above composition, a chromatographic fractionation apparatus consisting of four separation towers packed with a calcium type strongly acidic cation exchange resin (Diaion UBK555, Mitsubishi Kasei Co., Ltd., trade name) was used at 60 ° C. The mannose fraction and the fructose fraction were fractionated into a high-purity mannose solution (0.91 L) and a high-purity fructose solution (1.
22L) was obtained. The properties of this product liquid were as follows.

[0056]

[Table 2]

Example 3 (1) Production of sugar solution containing mannose 15 g glucose, Pseudomonas sp. AM-9582
(FERM-BP 3207) derived mannose isomerase 1
5 units, glucose isomerase 7.5 units, MgSO ₄ 0.01
The mixture (50 ml) containing M was maintained at pH 7.0 while maintaining the temperature at 50 ° C.
The reaction was carried out for 48 hours. After completion of the reaction, it was treated with H-type Amberlite IR-120 and OH-type Amberlite IRA-400 and then concentrated under reduced pressure at 60 ° C to contain mannose consisting of about 5.5% mannose, about 19.5% fructose, and about 24.5% glucose. A sugar solution was obtained.

(2) Separation and acquisition of mannose Dowex 1-X8 column (diameter 2 cm x length) of bisulfite type strongly basic anion exchange resin in which 2 ml of the mannose-containing sugar solution obtained in (1) was kept at 40 ° C. 15 cm) and then flushed with water at a flow rate of 9.9 ml / h. The effluent was fractionated in 3 ml fractions with a fraction collector. The results obtained are
As shown in FIG. 3, first, fructose and then glucose, and finally mannose flowed out, and mannose could be recovered in a yield of about 105 mg.

[0059]

INDUSTRIAL APPLICABILITY The present invention is a method for efficiently separating and obtaining mannose by fractionating mannose and other sugars in the raw material solution with a mannose-containing sugar solution as a raw material solution, which is inexpensive. It provides a simple manufacturing method. By the method of the present invention, the use of mannose in the food field and the feed field is greatly expanded.

[Brief description of drawings]

FIG. 1 shows a schematic view of four separation columns of an ion exchange chromatography fractionation device of the present invention.

FIG. 2 shows a fractional diagram of a mannose-containing sugar solution prepared by the calcium-type strongly acidic cation exchange resin of the present invention. -□ in the figure
− Represents the elution curve of fructose, − + − represents mannose, and − ◯ − represents the respective elution curves of glucose and oligosaccharide.

FIG. 3 shows a fractional diagram of a mannose-containing sugar solution prepared by the bisulfite type strongly basic anion exchange resin of the present invention. In the figure, the solid line shows fructose, the broken line shows glucose, and the dotted line shows mannose.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Masao Asano Masao Asano 24-13 Kitahama-cho, Chita-shi, Aichi Japan Nippon Kogyo Co., Ltd. 51 Amano Pharmaceutical Co., Ltd. Central Research Institute (72) Inventor Ryuichi Oya Nishinoharu-cho, Aichi Pref. Nishinoharu, Kunotsubo Nishijoya 51 30

Claims (7)

[Claims] 1. A mannose fraction is obtained by using a mannose-containing sugar liquid as a raw material liquid and fractionating mannose and other sugars in the raw material liquid by chromatography using an ion exchange resin. How to get separated. 2. The method for separating and obtaining mannose according to claim 1, wherein the mannose-containing sugar liquid is a fructose isomerization liquid using mannose isomerase. 3. The method for separating and obtaining mannose according to claim 1, wherein the mannose-containing sugar liquid is a glucose isomerase and a glucose isomerization liquid using mannose isomerase. 4. The method for separating and obtaining mannose according to claim 1, wherein the ion exchange resin is a strongly acidic cation exchange resin. 5. The method for separating and obtaining mannose according to claim 1, wherein the ion exchange resin is a strongly basic anion exchange resin. 6. The method for separating and obtaining mannose according to claim 4, wherein the strongly acidic cation exchange resin is a calcium type strongly acidic cation exchange resin. 7. The strongly basic anion exchange resin is any one of hydrogen sulfite type strongly basic anion exchange resin, carbonate type strongly basic anion exchange resin and styrene type strongly basic anion exchange resin. Item 5. The method for obtaining and separating mannose according to Item 5.