JPH06279483A - Production of d-xylulose 5-phosphate - Google Patents

Abstract

PURPOSE:To extremely simply and economically obtain the subject compound which is a raw material for hydroxy-ethyl-methyl-3(2H)furanone useful as a reagent, etc., for research for the metabolic pathway from a hydrolytic solution of a protein-containing raw material by hydrolyzing the raw material. CONSTITUTION:A protein-containing raw material such as soybean or wheat gluten is initially enzymically or chemically hydrolyzed. The objective compound is then separated from the resultant hydrolytic solution by a method for using the high-performance liquid chromatography, an ion exchange resin, etc. Furthermore, e.g. a proteolytic enzyme such as a protease or a plant cell wall-digesting enzyme such as a cellulase can be used as an enzyme used in enzymically hydrolyzing the raw material. In order to chemically hydrolyze the protein-containing raw material, an acid solution such as a 0.1-10% hydrochloric acid solution is added to the protein-containing raw material and the resultant mixture is then heated to >=70 deg.C and subsequently neutralized with an alkali such as sodium carbonate.

Description

Detailed Description of the Invention

[0001]

The present invention relates to 4-hydroxy-2 (or 5) ethyl-, which is expected to be used as a reagent for studying metabolic pathways in the field of biochemistry and as a flavor improving agent.
5 (or 2) methyl-3 (2H) furanone (hereinafter, HE
The present invention relates to a method for producing useful D-xylulose 5-phosphoric acid or a salt thereof (these are referred to as D-xylulose 5-phosphoric acid in the present invention) as a raw material of MF).

[0002]

2. Description of the Related Art Heretofore, a method for producing D-xylulose-5-phosphate has been hardly known, and the compound is very expensive.

[0003]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to obtain D-xylulose 5-phosphate by a new production method and economically.

[0004]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that an inexpensive or stable protein-containing raw material can be enzymatically or chemically hydrolyzed. It was found that the decomposed solution contained a large amount of D-xylulose-5-phosphate, and the present invention was completed based on this finding. That is, the present invention is characterized in that a protein-containing raw material is enzymatically or chemically hydrolyzed and D-xylulose 5-phosphoric acid is separated from the obtained decomposition solution. It is a method of producing an acid.

The present invention will be described in detail below. First, examples of the protein-containing raw material used in the present invention include soybean, defatted soybean, soybean isolated protein, wheat, wheat gluten, corn gluten, and other protein-rich raw materials.

Enzymes used when enzymatically hydrolyzing these protein-containing raw materials include proteolytic enzymes; starch degrading enzymes; plant cell wall-disintegrating enzymes such as cellulases, hemicellulases, and polygalacturonase; Inclusions: Koji such as soy sauce koji, rice koji and bran koji, and fermented foods such as natto and tempeh. You may use these together.

[0007] Enzymatic hydrolysis is carried out by subjecting the protein-containing raw material as it is, or by mixing the above-mentioned enzyme and the like with a heat-denatured product thereof, adding water, and then decomposing in the temperature range of action of the enzyme and the like.
The decomposition may be partial decomposition, but it is preferable to perform complete decomposition, for example, until the decomposed product becomes muddy.

Next, as a method for chemically hydrolyzing the protein-containing raw material, the protein-containing raw material is added to 0.1 by a conventional method.
This is carried out by a method of adding an acid solution such as a hydrochloric acid solution of about 10%, heating and decomposing at about 70 ° C. or higher, and then neutralizing by adding an alkali such as sodium carbonate.

Next, HP is used as a means for hydrolyzing the protein-containing raw material enzymatically or chemically and separating D-xylulose 5-phosphate from the resulting hydrolyzed solution.
Any method such as a method using LC or a method using an ion exchange resin can be mentioned. These may be used alone or in combination.

Thus, according to the present invention, D-xylulose 5-, which is useful as a reagent for studying metabolic pathways in the field of biochemistry and as a raw material for HEMF, which is expected to be used as a flavor improving agent, Phosphoric acid can be produced economically by a very simple method.

The present invention will be described more specifically by showing production examples of D-xylulose-5-phosphate and HEMF production examples (application examples) using the same.

[0012]

Example 1 (D-xylose from an enzymatic decomposition solution of soybean isolated protein)
Example of production of 5-phosphoric acid)

(1) Preparation of Enzymatic Degradation Treatment Solution 135 ml of distilled water was added to acid protease "Morijin Pharmaceutical Co., Ltd.
0.5% by weight of "Morcin", 0.5% by weight of neutral protease "Daiwa Kasei Co., Samoaze" and 0.5% by weight of cellulase "Meiji Seika Co., Ltd., Meiserase" were added and dissolved.
After sterilizing with 0.22 μm filter, 30 g of sterilized soybean isolated protein powder was added and dissolved, and the mixture was kept at 37 ° C. for 24 hours to prepare an enzyme-decomposed solution. This decomposed solution was centrifuged (3,000 rpm, 10 minutes) to obtain 94 ml of a supernatant.

(2) Separation of D-xylulose-5-phosphoric acid from the decomposed solution 10 μl of the supernatant of the decomposed solution was sampled and treated under the following high performance liquid chromatography (HPLC method) conditions: The eluent with a retention time of about 17 to 19 minutes is collected (about 2
ml), this was repeated 10 times in total, and about 20 ml of the obtained preparative solution was concentrated under reduced pressure by a rotary evaporator, and D
About 100 μl of a concentrate of xylulose 5-phosphate was obtained.

(3) High Performance Liquid Chromatography-Conditions Column: normal phase type column (manufactured by Tosoh Corporation, TSK gel)
Amide 80), inner diameter: 4.6 mm x 250 mm Monitor-: UV detector (210 nm) Flow rate: 1 ml / min Mobile phase A: acetonitrile (90%)-5 mM phosphoric acid (10%) Mobile phase B: acetonitrile (20%) ) -5 mM phosphoric acid (80%) Gradient conditions: According to Table 1 below.

[0016]

(3) Confirmation test for D-xylulose-5-phosphate A portion of the above-mentioned concentrated liquid was taken, and the concentrated liquid was used for SIMS of a mass spectrometer.
When analyzed in the mode, the obtained spectrum was compared with that of the standard D-xylulose 5-phosphate, and there was a perfect agreement. From this, it was confirmed that the compound obtained in this Example 1 was D-xylulose 5-phosphate.

From the above results, it is understood that according to the present invention, D-xylulose 5-phosphate can be easily obtained from inexpensive soybean isolated protein.

[0019]

Example 2 (Production Example of D-Xylulose 5-Phosphoric Acid from Soy Sauce Morobushi Juice, That is, Enzymatic Decomposition Solution of Protein-Containing Raw Material)

(1) Preparation of soy sauce mash juice before yeast fermentation at the initial stage of preparation In accordance with the usual soy sauce manufacturing method, defatted soybeans are sprinkled and then heat denatured, and fried and crushed wheat are mixed to obtain seeds. Koji is inoculated and cultivated to obtain soy sauce koji, which is then added to a saline solution to form a moromi mash, and this moromi mash before alcohol fermentation is filtered to obtain a moromi mash. It was

(2) D-xylose 5 from moromi juice
-Separation of Phosphoric Acid 10 μl of the above-mentioned Morobushi juice was collected and treated under the same high performance liquid chromatography (HPLC method) conditions as in Example 1 above, and the eluent having a retention time of about 17 to 19 minutes was collected ( About 2 ml), and this is repeated a total of 3 times, and about 6 ml of the obtained preparative solution is concentrated under reduced pressure with a rotary evaporator.
About 30 μl of a concentrated solution of D-xylulose 5-phosphate was obtained.

(3) Confirmation test of D-xylulose-5-phosphoric acid A part of the above concentrated liquid was taken, and this was put into SIMS of a mass spectrometer.
When analyzed in the mode, the obtained spectrum was compared with that of the standard D-xylulose 5-phosphate, and there was a perfect agreement.

From the above results, according to the present invention, D-xylulose 5-phosphate can be easily and economically obtained from a hydrolyzing solution of koji prepared from soybean and wheat as a raw material which can be stably supplied at a low cost. It turns out that it can be manufactured.

[0025]

[Application Example 1] (HEMF manufacturing example)

(1) Preparation of yeast nutrient medium (soy sauce koji digestive liquid medium) for HEMF production 100 g of soy sauce koji was put in a cloth bag, added to 1000 ml of distilled water and kept at 58 ° C. for 7.5 hours, then 5 The bag was suspended overnight at 0 ° C. to obtain 950 ml of digestive juice (pH 6.54). Next, this was boiled for 2 to 3 minutes and then filtered through a filter paper to obtain 885 ml of a filtrate. Glucose was added to this so as to be 5% by weight,
It was used as a nutrient medium (however, for soy sauce yeast, the salt content was adjusted to 17%).

(2) Preparation of D-xylulose-5-phosphate D-xylulose-5-phosphate obtained in Example 1 was used as it was.

(3) Yeast Culturing (HEMF Fermentation) The nutrient medium (soy sauce koji digestion solution) prepared in (1) above was added so that the D-xylulose 5-phosphate was added in the amount shown in Table 2. The medium was added to 1.2 ml to prepare a nutrient medium for yeast inoculation. (However, in the case of soy sauce yeast, the salt content was adjusted to 17%). Next, this prepared medium was placed in a vial bottle with a 5 ml screw cap, and 1 platinum loop of the slant was inoculated with the yeast described in Table 2 and statically cultured at 30 ° C. for 1 week while occasionally stirring. Then, as shown in Table 2, a culture solution containing a significant amount of HEMF was obtained.

Then, HEMF in the above culture solution was analyzed and quantified by gas chromatography (Journal of Agric).
ultural and Food Chemistry Vol.39, 934 (1991)).

For comparison, a culture solution was obtained and HEMF analysis was carried out in the same manner as in the above HEMF production method except that D-xylulose 5-phosphate was not added.
The results are shown in Table 2.

[0031]

[Table 2]

From the results shown in Table 2, it is understood that the D-xylulose 5-phosphate obtained in the present invention can be effectively used as a raw material for HEMF.

Claims (1)

[Claims] 1. A protein-containing raw material is enzymatically or chemically hydrolyzed, and D-xylrose is obtained from the resulting decomposition solution.
D-xylro-characterized by separating 5-phosphoric acid
Method for producing 5-phosphoric acid.