JP2001231592A - Method of producing mannose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of inexpensively producing mannose that is useful as a feed, a food and a raw material for medicines. SOLUTION: In this method of producing mannose, a cellulase originating from Aspergillus niger is characteristically allowed to act on mannan, glucomannan or galactomannan; or a natural product including mannan, glucomannan or galactomannan.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing mannose, which is useful as a feed for preventing infection of harmful bacteria or a raw material for synthesizing pharmaceuticals such as mannitol.

[0002]

2. Description of the Related Art Mannose is produced by acid or enzymatic decomposition of glucomannan contained in wood, konjac and the like. Further, a method of producing from glucose using molybdate as a catalyst is also known. further,
There has also been proposed a method of producing mannose by reacting mannose isomerase with fructose as a raw material to obtain a mannose-containing solution and then purifying the solution (JP-A-4-218370, JP-A-6-292578).
JP-A-8-9986). Of these,
The method using mannose isomerase is an excellent production method in that inexpensive fructose is used as a raw material, but has the disadvantage that the yield of mannose is low because the equilibrium of the reaction is biased toward the fructose side of the raw material. At present, it cannot be said that this method is a sufficient production method from the viewpoint of inexpensively producing.

[0003] Mannose is produced by the above-mentioned production method. However, in order to use it for applications such as feed for preventing the infection of harmful bacteria, the production cost is high, which is a factor hindering its practical use. Has become.

On the other hand, although no study has been made on an industrial production method, there is a report that copra meal can be used as a raw material for mannose (Zama et al., Tropical Agriculture 2).
9, No. 4, 221-225, 1985). According to this report, copra meal was defatted and treated with a weak acid, followed by extraction of mannan by caustic soda treatment, washing, and solvent precipitation to prepare copra mannan.
It is described that mannose can be prepared by allowing an extracellular mannanase derived from Aspergillus niger to act thereon.

In order to produce mannose by hydrolysis using mannan, glucomannan, galactomannan or a natural product containing mannan, glucomannan, galactomannan as a raw material, high-molecular polysaccharides such as mannan, glucomannan, and galactomannan are used. An enzyme that efficiently degrades is required.

[0006]

As described above, as an example known so far, it has been described that mannose can be prepared by allowing extracellular mannanase derived from Aspergillus niger to act on copramannan. (Zama et al., Tropical Agriculture Vol. 29, No. 4, 221-2)
25, 1985). However, when this enzyme is used, mannobiose, galactose, and glucose are by-produced together with mannose, and it is difficult to obtain high-purity mannose.

In order to efficiently produce mannose from various kinds of mannan, glucomannan and galactomannan,
Endo-type mannanase, exo-type mannosidase,
An enzyme containing glucosidase and galactosidase that release glucose and galactose present in the branched side chains is required.

The present inventors have proposed natural polysaccharides comprising mannose such as copra meal, copra mannan, locust bean gum, guar gum, konjac mannan,
Alternatively, an enzyme capable of efficiently and specifically releasing mannose is searched for a natural product containing a polysaccharide, and (1) action: acts on mannan to release mannose. (2) Optimum pH
And stable pH range: optimal pH is 4.0 to 5.0, pH 3 to
9 (20 ° C., 20 hours). (3) Range of suitable temperature for operation: Works between 45 and 80 ° C. (Japanese Unexamined Patent Publication No. 11-46787).
Reference).

The present inventors have also disclosed WO99 / 08544.
In the publication, it is also reported that mannose-containing copra meal obtained by reacting copra meal with a hemicellulase solution to release mannose can be used as a mannose-containing feed.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and have found that cellulase derived from Aspergillus niger has a very high desired activity. completed.

[0011] That is, the present invention provides a method for producing mannose, which comprises allowing a cellulase derived from Aspergillus niger to act on mannan, glucomannan or galactomannan or a natural product containing mannan, glucomannan or galactomannan. It is an abstract.

Hereinafter, the present invention will be described in detail. Examples of the cellulase derived from Aspergillus niger used in the present invention include Sumiteam AC-L (cellulase manufactured by Shin Nippon Chemical Co., Ltd., titer: 1,500 units / g). Although this enzyme is a cellulase preparation, it contains hemicellulase and acts on galactomannan. As for the enzymatic properties of this enzyme, it works practically at an optimum pH of 4 and between pH 3 and 8. The optimal temperature for the reaction is 60 ° C, 30 ° C
It works practically between ７５75 ° C. The enzyme activity of a commercially available product is 1,500 units / g as cellulase activity.

It goes without saying that the reaction conditions for releasing mannose may be selected under the optimum conditions according to the respective reaction substrates, but the reaction temperature is preferably carried out under conditions in which the enzyme is not inactivated. In order to prevent spoilage of the reaction solution, it is preferable that the reaction is performed at a temperature at which microorganisms do not easily proliferate. 20 ° C to 90 ° C, preferably 45 ° C to 80 ° C
C, more preferably 50C to 75C. Needless to say, it is desirable to carry out the reaction under the optimal action conditions of the enzyme as the pH of the reaction. pH 2 to 9, preferably pH 2.5 to 8, more preferably pH 3 to 6. The reaction time depends on the amount of the enzyme used, but is usually 3 hours.
It is preferable to set the time between 48 hours and 48 hours.

The amount of Sumizyme AC-L which is allowed to act on mannan, glucomannan, galactomannan or natural products containing mannan, glucomannan, galactomannan is 1 to 100 units, preferably 10 to 50 units per gram of substrate. Is appropriate.

The amount of water containing Sumizyme AC-L used in the reaction is preferably not more than 3 times by weight, more preferably 0.5 to 3 times, particularly preferably 0.5 to 3 times by weight of the substrate for feed. The amount is preferably 0.7 to 1.5 times. If the amount of water containing Sumiteam AC-L is more than 3 times,
The amount of water in the substrate increases, and various bacteria and the like easily grow, which makes it unsuitable for use as feed as it is.In addition, it takes time and cost to dry to make the amount of water suitable for use as feed. Not preferred. Also,
If the amount is less than 0.5 times, the enzyme solution containing Sumizyme AC-L does not contact uniformly, so that the amount of released mannose is not so large, which is not preferable.

On the other hand, in the case of a sugar solution, Sumiteam AC-L
Is preferably 0.5 to 10 times the weight of the substrate relative to the substrate. If the amount of water containing Sumizyme AC-L is large, the purification process takes a long time, which is not preferable.

As a method for bringing the enzyme solution containing Sumizyme AC-L into contact with the substrate, it is desirable to bring the enzyme solution into contact with the substrate so that the enzyme solution becomes uniform. There is a method in which an enzyme solution is added to a substrate and then pressure is applied thereto, and a method in which an enzyme solution is added to a granular substrate and water is naturally absorbed.

[0018]

The present invention will be described below in detail with reference to examples. Note that the present invention is not limited to these examples. Example 1 200 g of copra meal (manufactured by Fuji Oil Co., Ltd., fat content 1)
0%, water content 7.2%) were suspended in 2 L of water, and then sumitim AC-L (Cellular Co., Ltd., manufactured by Shin Nippon Chemical Co., Ltd.)
And a titer of 1,500 units / ml) were added and reacted at pH 3, 60 ° C., with stirring for 8 hours. After the reaction was completed, the mixture was allowed to stand, and the supernatant was filtered to obtain 1.9 L of a clear solution containing mannose. The sugar in this solution was analyzed by high performance liquid column chromatography. Analytical column is Aminex HP manufactured by Bio-Rad
X-87P was used. Column temperature 85 ° C, flow rate 0.6
It was eluted with water at a rate of ml / min. The sugar was detected using a differential refractometer, and the mannose content was determined from the quantitative value of the standard product. As a result of analyzing the solution after the above reaction,
42 g of mannose had accumulated in 1.9 L.

After the solution was degreased with ethyl ether, an anion exchange resin (Dowex SAR, OH ^- type, manufactured by Muromachi Chemical Co., bed volume 100 ml) and a cation exchange resin (Dowex HCRW, manufactured by Muromachi Chemical Co., Ltd.) were used.
2, H ⁺ type bed volume 100ml), activated carbon (Diahope S80 manufactured by Mitsubishi Chemical Corporation, bed volume 10)
0 ml) in this order, and a solution containing mannose was recovered. The collected solution was concentrated by an evaporator until it became Brix 70, and a sugar solution containing mannose was obtained.
The purity of mannose in this liquid is 82% (excluding water)
Met.

Example 2 200 g of locust bean gum (manufactured by Sanshin Co., Ltd.)
After suspending in water L, 2.1 ml of Sumitim AC-L (Cellulase, manufactured by Shin Nippon Chemical Co., Ltd., titer: 1,500 units / ml) was added, and the mixture was reacted at 60 ° C. with stirring for 25 hours. did. After completion of the reaction, the mixture was filtered to obtain 1.9 L of a clear solution containing mannose. The sugar in this solution was analyzed by high performance liquid column chromatography. Analytical column is Aminex HP manufactured by Bio-Rad
X-87P was used. Column temperature 85 ° C, flow rate 0.6
It was eluted with water at a rate of ml / min. The sugar was detected using a differential refractometer, and the mannose content was determined from the quantitative value of the standard product. As a result of analyzing the solution after the above reaction,
83 g of mannose had accumulated in 1.9 L.

Then, the solution was treated with an anion exchange resin (Dowex SAR, OH ^- type, bed volume 100 m).
l), cation exchange resin (Dowex HCRW2, H
⁺ Type bed volume 100ml), activated carbon (Diahope S80 manufactured by Mitsubishi Chemical Corporation, bed volume 100m)
The solution was passed through 1) in this order to recover a solution containing mannose. After concentrating the collected solution by an evaporator until it becomes Brix 70, hot ethanol was added to a final concentration of 85%.
, A small amount of crystalline D-mannose was added, and the mixture was allowed to stand at 4 ° C. By filtering this solution,
66 g of crystalline mannose were obtained. The melting point of the obtained mannose was 131.5 to 132.5 ° C.

Example 3 After suspending 200 g of guar gum (manufactured by Sanshin Co., Ltd.) in 2 L of water, Sumitim AC-L (cellulase manufactured by Shin Nippon Chemical Co., Ltd., titer: 1,500 units / ml) Was added and reacted at 60 ° C. for 9 hours with stirring.
After completion of the reaction, the mixture was filtered to obtain 1.9 L of a clear solution containing mannose. The sugar in this solution was analyzed by high performance liquid column chromatography. As an analytical column, Aminex HPX-87P manufactured by Bio-Rad was used. Column temperature is 85 ° C, flow rate is 0.6ml / min
And eluted with water. The sugar was detected using a differential refractometer, and the mannose content was determined from the quantitative value of the standard product. As a result of analyzing the solution after the above reaction, 97 g of mannose was accumulated in 1.9 L.

Next, this solution was used as an anion exchange resin (Muromachi
Dowex SAR, OH manufactured by Chemical Company ^-Mold, bed volume
100ml), cation exchange resin (Muromachi Chemical Co., Ltd.
Wex HCRW2, H⁺Bed volume 100
ml), activated carbon (Diahope S80 manufactured by Mitsubishi Chemical Corporation,
Liquid volume in this order.
The solution containing the north was recovered. Brick the recovered solution
Concentrate with an evaporator until it reaches 70
Was obtained. The purity of mannose in this sugar solution is 7
0% (excluding water).

Example 4 200 ml of Sumitim AC-L (Cellulase, manufactured by Shin Nippon Chemical Co., Ltd., titer 1,500 units / ml) was added to 6
It was added to 20 L of warm water at 0 ° C. Next, the tank (length 54c)
m, width 54 cm, height 50 cm) Palm kernel meal (Cargill, granular, mannan content 40%, fat content 1.0)
%, Water 7.5%), and 20 L of Sumizyme AC-L solution was added. The tank was left at 60 ° C. for 22 hours and then dried at 120 ° C. in a fluidized bed drier.
After drying for 5 hours, a mannose-containing feed was obtained. After suspending this feed in water and dissolving the sugar component in the feed in water, the sugar component was quantified by high performance liquid column chromatography. As a result, 165 g of mannose was accumulated in 1 kg of the sample. The water content of the feed was measured by the normal pressure drying method to be 8.3%.

Example 5 6 ml of Sumitim AC-L (Cellulase, manufactured by Shin Nippon Chemical Co., Ltd., titer: 1,500 units / ml) was added to 6
The suspension was suspended in 80 L of hot water at 0 ° C. Next, the copra flakes (manufactured by Fuji Oil Co., Ltd., having a particle size of 5 mm, centered on mannan content 30%, fat content 10%, water content 7.2) were placed in the tank.
%), And then 80 L of the enzyme solution was charged. After leaving at 60 ° C. for 24 hours, 1
After drying at 20 ° C. for 0.5 hour, a mannose-containing feed was obtained. After this feed was suspended in water to dissolve the sugar component in the feed water, the sugar component was analyzed. As a result, 124 g of mannose was accumulated in 1 kg of the sample. In addition, as a result of measuring the water content in the feed by an atmospheric pressure drying method, it was found that
5%.

Comparative Example 1 Cellulosin G was used instead of Sumizyme AC-L of Example 1.
0.6 g of M5 (mannanase manufactured by Hankyu Bio-Industry Co., Ltd., titer: 10,000 units / g) was added, the same enzyme reaction was performed, and the supernatant was recovered. As a result of analyzing the solution after the above reaction, 31 g of mannose was accumulated in 1.9 L. It can be seen that Smith-AC-L releases more mannose.

Comparative Example 2 Cellulosin G was used instead of Sumiteam AC-L of Example 2.
0.6 g of M5 (mannanase manufactured by Hankyu Bio-Industry Co., Ltd., titer 10,000 units / g) was added, the same enzyme reaction was performed, and the supernatant was recovered. As a result of analyzing the solution after the above reaction, 68 g of mannose was accumulated in 1.9 L.

Comparative Example 3 Cellulosin G was used instead of Sumizyme AC-L in Example 3.
1.5 g of M5 (mannanase manufactured by Hankyu Bio-Industry Co., Ltd., titer 10,000 units / g) was added, the same enzyme reaction was carried out, and the supernatant was recovered. As a result of analyzing the solution after the above reaction, 77 g of mannose was accumulated in 1.9 L.

Comparative Example 4 Cellulosin G was used instead of Sumiteam AC-L of Example 4.
60 g of M5 (mannanase manufactured by Hankyu Bio-Industry Co., Ltd., titer 10,000 units / g) was added,
A similar enzyme reaction was performed. As a result, 146 g of mannose was accumulated in 1 kg of the sample. The water content was 10.2%. It can be seen that Sumiteam AC-L has a larger amount of released mannose.

Comparative Example 5 Cellulosin G was used instead of Sumiteam AC-L of Example 5.
240 g of M5 (mannanase manufactured by Hankyu Bio-Industry Co., Ltd., titer 10,000 units / g) was added, and the same enzyme reaction was performed. As a result, sample 1k
In each g, 105 g of mannose had accumulated. The water content was 10.2%.

[0031]

According to the production method of the present invention, mannose useful as feed, food and pharmaceutical raw materials can be produced at low cost.

Continued on front page F-term (reference) 4B064 AF02 CA21 CB07 CC03 CD19 CD22 CD24 DA01 DA11 4C057 AA02 BB02

Claims (1)

[Claims] 1. A natural product containing mannan, glucomannan or galactomannan or mannan, glucomannan or galactomannan is added to Aspergillus.
A method for producing mannose, wherein a cellulase derived from a niger is allowed to act.