JPH0695942B2 - Method for producing glucose-1-phosphate - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing glucose-1-phosphate, more specifically a glycolytic initial compound such as a pharmaceutical antibacterial agent or an antibacterial agent. The present invention relates to an advantageous method for producing glucose-1-phosphate, which is useful as a tumor agent (platinum complex) and a therapeutic agent (amine salt) for heart disease.

[Conventional technology and its problems]

Conventionally, as a method for producing glucose-1-phosphate (hereinafter abbreviated as “G-1-P”), α-glucan (various starches, glycogen) is produced by the enzyme catalytic action of phosphorylase.
Various methods are known for the production from the orthophosphate. For example, a method for producing rabbit muscle extract from glycogen as an enzyme solution (Cori et al. J. Biol. Chem. 121 465 (193
7)), Method for producing potato juice from starch as enzyme solution (CSHanes Proc.Roy.Soc. B129 174 (1940))
Etc. Specifically, α-glucan and orthophosphate are used as substrates, phosphorylase is allowed to act on them to synthesize G-1-P, and unreacted orthophosphate is MgNH ₄ PO ₄ , Ba ₃ (PO ₄ ) ₂ , M
_{g 3 (PO 4) 2,} Li 3 (PO 4) removed after disposal in the insoluble salt ₂ such as the G-1-P, except the unreacted glucan and or alcohol reprecipitation or using ion-exchange resin It is manufactured.

The present inventors have already developed a method for separating or recovering orthophosphate by electrodialysis, in which the recovered orthophosphate can be reused in the production of G-1-P, which is also advantageous in terms of cost. In order to produce G-1-P more cost-effectively using this separation method, the higher the G-1-P content of the produced G-1-P solution, the lower the treatment cost of electrodialysis. , G-1-P had to be produced in high concentration.

However, in the conventional method, various starches, glycogen, etc. are used as the substrate α-glucan, but these compounds have low solubility in water per se or increase in viscosity when made into a high concentration solution. As a result, it was difficult to produce G-1-P at a high concentration.

Therefore, in the production of G-1-P from α-glucan and orthophosphate in the presence of phosphorylase, development of a reaction system capable of producing G-1-P at a high concentration has been desired.

[Means for solving problems]

In such a situation, the present inventors have conducted diligent research on the reaction between α-glucan and orthophosphate in the presence of phosphorylase, and as a result of diligent research on the α-glucan to be used, the specific dextrin has high solubility in water. Moreover, they have found that the high-concentration solution does not have a high viscosity, and thus can be suitably used as α-glucan in the above reaction, and thus completed the present invention.

That is, the present invention is a method for producing G-1-P from α-glucan and orthophosphate using phosphorylase, characterized in that a dextrin having a DE value of 0.5 to 20 is used as α-glucan. -P manufacturing method is provided.

The dextrin used in the present invention has a DE value of 0.5 to 20.
And those of 3 to 15 are particularly preferable. The DE (Dextrose Equivalent) value used here is an index showing the progress of saccharification, Indicated by. And the DE value for dextrin is
The direct reducing sugar was subjected to the Somogy method (J. Biol. Chem. 195 ).
19 (1952)).

The dextrin used in the present invention has the DE value shown above because the DE value has a viscosity of the reaction liquid of several% when the dextrin having a DE value of 0.5 or less is used.
Further, when a dextrin having a DE value of 20 or more is used, the enzyme reactivity is poor, and in either case, G-1-P cannot be produced in high yield.

The dextrin having a DE value of 0.5 to 20 used in the present invention may be one obtained by chemically decomposing starch or glycogen, or one obtained by decomposing it with an enzyme such as α-amylase or isoamylase.

The method of the present invention uses known phosphorylase except that dextrin having a DE value of 0.5 to 20 is used as α-glucan.
It can be carried out according to the method for producing G-1-P.

Specifically, for example, dextrin with a DE value of 0.5 to 20 is added to 10 to 1
Dissolve in water at 00 ℃, preferably 20 ~ 40 ℃, 0.01 ~ there
4 mol / l orthophosphate aqueous solution is added, or dextrin is added at 0.01 to 4 at 10 to 100 ° C, preferably 20 to 40 ° C.
After dissolving in a mol / l orthophosphate aqueous solution or dextrin in a phosphoric acid solution at 10 to 100 ° C., preferably 20 to 40 ° C., the solution obtained by adjusting the pH with an alkaline solution is used. , G-1-P is produced by adding phosphorylase obtained from plants, microorganisms or the like or a substance containing the phosphorylase and reacting at 10 to 50 ° C, preferably 25 to 40 ° C.

Other conditions in the above reaction, namely reaction time, pH,
The preservatives, additives, presence / absence of stirring, etc. may be set according to the purpose.

〔The invention's effect〕

Since the method for producing G-1-P of the present invention uses a dextrin having a DE value of 0.5 to 20, the solubility of dextrin is higher than that of starch and glycogen, and a reaction at a high concentration becomes possible.
This is a cost advantage because it can increase the production yield of 1-P. In addition, since the solubility of dextrin is high, it is unnecessary to heat and dissolve the dextrin which is usually required, the manufacturing process can be simplified, and the cost is further improved.

〔Example〕

Next, examples will be described. The dextrins of each DE value used in the following examples were prepared by the following method.

(Preparation of dextrin) 100 g of potato starch is suspended in 200 ml of ion-exchanged water and pasteurized in a boiling bath. Adjust this to 50 ° C, pH 4.5 and α-
Amylase is added and the reaction is carried out. The amount of reducing sugar is measured directly by sampling from time to time. When the target DE value is reached, the reaction is boiled to terminate the reaction. Then, insoluble matter is removed by filtration, and then water is evaporated in a boiling bath to form starch syrup. Add 500 ml of ethanol to precipitate dextrin, filter and dry in vacuum to obtain the target DE value of dextrin 82.
Got ~ 95g.

Example 1 15 g of dextrin having a DE number of 3.97 was mixed with 38 g of KH ₂ PO ₄ and K ₂ HPO _4.
Dissolve 56 g in 200 ml of an aqueous solution at 25 ° C. to this,
300 g of potato was crushed with a diuser, 95 ml of potato juice obtained by centrifugation and 1 ml of toluene as a preservative were added, adjusted to 300 ml with ion-exchanged water, and reacted at 40 ° C. for 48 hours. As a result, 11-8.9 mmol / l G-1-P was obtained.

Example 2 15 g of dextrin having a DE number of 7.87 was added to 38 g of KH ₂ PO _{4 and 4} 56 of K ₂ HPO.
Dissolve g in 200 ml of an aqueous solution at 25 ° C. To this, crush 300 g of potato with a juicer and centrifuge to add 95 ml of potato juice and 1 ml of toluene as a preservative,
After adjusting to 300 ml with ion-exchanged water, the mixture was reacted at 40 ° C for 48 hours. As a result, 116.6 mmol / l G-1-P was obtained.

Example 3 15 g of dextrin having a DE value of 11.26 was mixed with 38 g of KH ₂ PO ₄ and K ₂ HPO _4.
Dissolve 56 g in 200 ml of an aqueous solution at 25 ° C. To this, crush 300 g of potato with a juicer, add 95 ml of potato juice obtained by centrifugation and 1 ml of toluene as a preservative, adjust to 300 ml with deionized water, and then add 48 ml at 40 ° C.
Reacted for hours. As a result, 110.8 mmol / l G-1-P was obtained.

Example 4 3 g of dextrin having a DE number of 3.97 was mixed with 3.8 g of KH ₂ PO ₄ and K ₂ HPO _4.
Dissolve 5.6g in 20ml aqueous solution at 27 ℃. 1.5 ml of a solution obtained by extracting and purifying the enzyme phosphorylase from 10 kg of potatoes by a known method (Kenichi Nakano, Toshiro Fukui: Starch Science, 24 , 80 [1977]) (phosphorylase activity 2089 U / m
l) and 1 ml of toluene as a preservative were added, and the mixture was adjusted to 30 ml with ion-exchanged water and reacted at 40 ° C. for 24 hours. as a result
210 mmol / l G-1-P was obtained.

Example 5 6 g of dextrin having a DE number of 3.97, 3.8 g of KH ₂ PO _{4 and} 45 g of K ₂ HPO ₄ .
Dissolve 6 g in 20 ml of an aqueous solution at 27 ° C. The enzyme phosphorylase was extracted and purified from 10 kg of potatoes by a known method (Kenichi Nakano, Toshiro Fukui: Starch Science, 24 , 80 [1977]) and concentrated to 1.5 ml (phosphorylase activity 2089U / m).
l) and 1 ml of toluene as a preservative were added, and the mixture was adjusted to 30 ml with ion-exchanged water and reacted at 40 ° C. for 24 hours. As a result, 214 mmol / l G-1-P was obtained.

Example 6 9 g of dextrin having a DE value of 11.26, 5.7 g of KH ₂ PO ₄ and K ₂ HPO ₄
It is dissolved in 20 ml of an aqueous solution containing 8.4 g at 27 ° C. To this, a known method from 10 kg of potatoes (Kenichi Nakano, Toshiro Fukui:
Starch science, 24 , 80 [1977]) extracted and purified the enzyme phosphorylase and concentrated the solution 1.25 ml (phosphorylase activity
2418U / ml) and 1 ml of toluene as a preservative,
After adjusting to 30 ml with ion-exchanged water, the mixture was reacted at 40 ° C for 24 hours.
As a result, 230 mmol / l G-1-P was obtained.

Example 7 3 g of dextrin having a DE value of 3.97 was added to about 90 ml of deionized water at 28 ° C.
Dissolve in. To this, 100 ml of an aqueous solution containing 9.5 g of KH ₂ PO ₄ and 14 g of K ₂ HPO ₄ and 300 g of potatoes were crushed with a juicer, 100 ml of potato juice obtained by centrifugation, and 1 ml of toluene as a preservative. In addition, the mixture was adjusted to 300 ml with ion-exchanged water and reacted at 40 ° C for 24 hours. As a result 36.1 mmol / l
G-1-P was obtained.

Example 8 3 g of dextrin having a DE value of 7.87 was added to about 90 ml of deionized water at 28 ° C.
Dissolve in. To this, 100 ml of an aqueous solution containing KH ₂ PO ₄ 9.5 g and K ₂ HPO ₄ 14 g, potato 300 g was crushed with a juicer, 100 ml of potato juice obtained by centrifugation, and 1 ml of toluene as a preservative. Add 300 ml with deionized water
After adjusting to 40 ° C., the mixture was reacted at 40 ° C. for 24 hours. As a result 35.9 mmol
We obtained G-1-P of / l.

Example 9 3 g of dextrin having a DE value of 11.26 was added to about 90 ml of deionized water.
Melt at ℃. To this, 100 ml of an aqueous solution containing 9.5 g of KH ₂ PO ₄ and 14 g of K ₂ HPO ₄ and 300 g of potatoes were crushed with a juicer, 100 ml of potato juice obtained by centrifugation, and 1 ml of toluene as a preservative. After adjusting to 300 ml with ion-exchanged water, the mixture was reacted at 40 ° C for 24 hours. As a result 33.3 mmol / l
G-1-P was obtained.

Comparative Example 1 The solubility, viscosity and reactivity of potato starch and dextrin used in the present invention were measured and compared. As the potato starch, a first-class reagent manufactured by Wako Pure Chemical Industries, Ltd. was used.

Solubility The solubility was defined as the weight (g) that was dissolved in 100 ml of ion-exchanged water at 24 ° C.

Viscosity The viscosity was measured at 40 ° C. with a B-type viscometer.

Reactivity Regarding the reactivity, comparison was made with the amount of G-1-P (mmol / l) obtained when the reaction was carried out under the conditions of dextrin or potato starch 1 to 10% and orthophosphate 1.5 mol / l. Specifically, _{3 to} 30 g of the dextrin of the present invention was dissolved in 150 ml of an aqueous solution containing 28.6 g of KH ₂ PO _{4 and} 41.8 g of K ₂ HPO ₄ at 28 ° C., and 300 g of potato was crushed with a juicer and centrifuged. 100 ml of the obtained potato juice and 1 ml of toluene as a preservative were added, adjusted to 300 ml with ion-exchanged water, and reacted at 40 ° C. to produce G-1-P. On the other hand, potato starch is 3 to 3
0 g is suspended in about 100 ml of ion-exchanged water, dissolved in a boiling bath and cooled at room temperature. Add 100 ml of deionized water and KH ₂
PO ₄ 28.6 g and K ₂ HPO ₄ 41.8 g were added and dissolved, and potato 300 g was crushed with a juicer and centrifugally separated to obtain 100 ml of potato juice and 1 ml of toluene as a preservative, and
After adjusting to 300 ml with ion-exchanged water, the mixture was reacted at 40 ° C. The obtained G-1-P amount was a value measured when the G-1-P amount did not change with time.

From the above results, it is clear that the use of the dextrin of the present invention increases the G-1-P yield, and makes it possible to produce G-1-P in a cost-effective manner. In addition, the use of dextrin eliminates the need for heat treatment for the dissolution of α-glucan, which makes it possible to simplify the G-1-P production process.

Comparative Example 2 15 g of dextrin having a DE value of 0.28 was mixed with 38 g of KH ₂ PO ₄ and K ₂ HPO _4.
Suspended in 200 ml of a solution in which 56 g was dissolved, dissolved in a boiling bath and cooled, then crushed 300 g of potato with a juicer, added 95 ml of potato juice obtained by centrifugation and 1 ml of toluene as a preservative, After adjusting to 300 ml, the mixture was reacted at 40 ° C for 48 hours. The amount of G-1-P obtained is as follows, and good results cannot be obtained even if dextrin having a DE value outside the range of the present invention is used.

Comparative Example 3 15 g of dextrin having a DE value of 21 was mixed with 38 g of KH ₂ PO ₄ and 56 g of K ₂ HPO ₄
It is dissolved in 200 ml of the solution prepared by dissolving at 25 ° C. Potatoes on this 3
Add 95 g of potato juice obtained by crushing 00 g with a juicer and centrifuging, and 1 ml of toluene as a preservative, and add 300 ml.
After adjusting the temperature to 40 ° C., the reaction was carried out at 40 ° C. for 48 hours. The amount of G-1-P obtained is as follows.

From the above results, it was found that the G-1-P yield was increased by using the dextrin having the DE value of 0.5 to 20.

Claims (1)

[Claims] 1. A method for producing glucose-1-phosphate from α-glucan and orthophosphate using phosphorylase, wherein dextrin having a DE value of 0.5 to 20 is used as α-glucan. 1-Method for producing phosphoric acid.