JPS63208594A - Production of glucose-1-phosphoric acid - Google Patents

Abstract

PURPOSE:To obtain the titled phosphoric acid useful as medical antibacterial agent, antitumor agent and remedy for cardiopathy, with simple process at a low cost, by using a specific dextrin as alpha-glucan and reacting with an orthophosphoric acid salt and a phosphorylase. CONSTITUTION:The objective phosphoric acid can be produced by using (A) a dextrin having a DE value of 0.5-20 as alpha-glucan, adding (B) an orthophosphoric acid salt and (C) a phosphorylase to the component A and reacting e.g. at 25-40 deg.C. Preferably, the reaction is carried out e.g. by dissolving the component A in water at 20-40 deg.C and adding 0.01-4mol./l of the component B to the solution.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for producing glucose-1-phosphate, and more particularly to a method for producing glucose-1-phosphate, which is an initial compound of glycolysis, and is used, for example, as a medicinal antibacterial agent and an antibacterial agent. The present invention relates to an advantageous method for producing glucose-1-phosphate useful as a tumor agent (platinum complex) and a heart disease therapeutic agent (amine salt).

[Conventional technology and its problems]

Conventionally, glucose-1-phosphate (hereinafter abbreviated as "G-1-PJ") has been produced from α-glucan (various starches and glycogen) and orthophosphate using the enzymatic catalytic action of phosphorylase. For example, a method for producing glycodan from rabbit muscle extract as an enzyme solution (Cori et al. J, Biol, C.
ham, 121 465 (1937)), & A method for producing tet juice from starch as an enzyme solution (C, S, Ha
nes Proc, Roy, Soc, B 12917
4 (1940)) etc. Specifically, α-glucan and orthophosphate are used as substrates, G-1-P is synthesized by the action of phosporinase gold, and the unreacted orthophosphate is converted into MKNH, PO, B Hatake 5 (POa) *, Mlrs(P
After removing and disposing of Oa)ts Li5 (Pot) as an insoluble salt, unreacted glucan is removed by using an ion exchange resin or reprecipitating with alcohol to produce G-1-P.

The present inventors have already developed a method for separating and recovering orthophosphate by electrodialysis, which is cost-effective and allows the recovered orthophosphate to be reused in the production of G-1-P. Using this separation method, G-1 is more cost-effective.
-pl To produce G-1-P solution, G-
The higher the 1-P content, the cheaper the electrodialysis treatment cost, so it was necessary to produce G-1-Pl at a high concentration.However, in the conventional method, the substrate α-glucan Various types of starch, glycogen, etc. are used as such compounds, but these compounds themselves have problems such as low solubility in water and increased viscosity when made into a highly concentrated solution.As a result, G -1-P was difficult to produce.

Therefore, in the production of G-1-P from α-glucan and orthophosphate in the presence of phosphorylase, G-1-P
It has been desired to develop a reaction system that can produce at high concentrations.

[Means to solve the problem]

Under these circumstances, the present inventors have conducted intensive research on the reaction between α-glucan and orthophosphate in the presence of phosphorylase, and have found that certain dextrins have a high solubility in water, especially regarding the α-glucan used. Furthermore, the present invention was completed based on the discovery that the viscosity of the highly concentrated solution is not high, and therefore it can be suitably used as α-glucan in the above reaction.

That is, the present invention provides a method for producing G-1-P from α-glucan and orthophosphate using phosphorylase, in which α-glucan and dextrin having a DE value of 05 to 20 are used. The present invention provides a method for producing 1-P.

The dextrin used in the present invention has a 01 value of 05 to
20, particularly preferably 3 to 15. The DE(I)extroseEquival@nt) value used herein is an index indicating the degree of progress of saccharification, and is expressed by the following formula. The 01 value for dextrin is determined by the somegy method (J, Bl) on direct reducing sugars.
ol, Ch@m, 19519 (1952)).

The dextrin used in the present invention has a value of 01 as shown above. When the above dextrins are used, the enzymatic reactivity is poor, and in either case, a high yield of G-
This is because 1-P cannot be manufactured.

The dextrin having a DE value of 05 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.

Except that the method of the present invention uses 01 dextrin with a value of 05 to 20 as the α-glucan.

It can be carried out according to a known method for producing G-1-P using phosphorylase.

Specifically, for example, dextrin with a DE value of 05 to 20 is dissolved in water at 10 to 100°C, preferably 20 to 40°C, and an orthophosphate aqueous solution with a QOI of ~4 nol/l is added thereto. Dextrin t-10~
001-4 mol at 100°C, preferably 20-40°C
/l of orthophosphate aqueous solution or dextrin at 10-100°C, preferably 20-40°C.
After dissolving in a phosphoric acid solution of
Add phosphorylase obtained from animals, plants, microorganisms, etc. or its contents to the solution obtained by adjusting A, and add 10 to 5
j9G-1-P is produced by reacting at 0°C, preferably 25 to 40°C Other conditions in the above reaction: tb Reaction time %P"
% preservative, additives, presence or absence of stirring, etc. may be set depending on the purpose.

[Effects of the Invention] Since the method for producing G-1-P of the present invention uses dextrin with a valence of 01 and 05 to 20, the solubility of dextrin is higher than that of starch and glycogen, making it possible to react at high concentrations. -1-P production yield can be increased, which is advantageous in terms of cost. Furthermore, since the solubility of dextrin is high, there is no need to heat and dissolve dextrin, which is normally required, which simplifies the manufacturing process and is even more cost-effective.

〔Example〕

Next, an example will be given and explained. The dextrins of each DE value used in the following examples were prepared by the following method (preparation of dextrin). Suspend 100 ft of Tetodenden in 200 d of ion-exchanged water and turn into a paste in a boiling bath.

The pH is adjusted to 45K at 50°C, and α-amylase is added to carry out the reaction.The amount of direct reducing sugar is measured by sampling from time to time, and when the desired DE value is reached, the reaction is terminated by boiling.

Next, after filtering to remove the cinephile, the water is further evaporated in a boiling bath to form starch syrup.

Add 500 d of ethanol to precipitate dextrin,
Dextrin with a DE value of 82 for vacuum drying after filtration
I got ~95fi.

Example I 1KM of dextrin 15F with DE value a97, PO43
8 and 200 ml of HPO4a 6 f t-dissolved aqueous solution at 25°C. This is K.

? Obtained by crushing Tet 300f with a juicer and centrifuging it? 95w1 of Teto's sushi soup and 1d of toluene as a preservative were added, and after adjusting to 300W11 with ion-exchanged water, the mixture was reacted at 4.0°C for 48 hours. As a result, 11a
9 mmol/l of G-1-P was obtained.

Example 2 15t of dextrin with a DE value of 787 was mixed with 1K) I, Po,
38 f, K, I (Po, 56 f is dissolved in an aqueous solution of 200 tttl at 25 °C.
? Te) 95 d of potato sushi juice obtained by crushing 300F with a juicer and centrifuging it, and 1 d of toluene as a preservative.
i and adjusted to 300idK with ion-exchanged water at 40°C.
The reaction was allowed to proceed for 8 hours. As a result, 11 a 6 mmol
/t of G-l-Pt- was obtained.

Example 3 Dextrin 15f with a DE value of 11.26 was added to KM, PO
438 and HPo, 56 F'! ! -Dissolve in 200 d of dissolved aqueous solution at 25°C. This was obtained by crushing -te) 300F with a juicer and centrifuging it. Add 95 d of Teto sushi soup and toluene IIL as a preservative, adjust to 300 m with ion exchange water,
The reaction was carried out at 0°C for 48 hours. As a result, 1000 mm
1/l of G-1-P was obtained.

Example 4 Dextrin 3f with DE value a97 was added to KH, PO, 38F and HPO4a at t-
Dissolve in 201 L of aqueous solution at 27°C. to this? 1.5 d of concentrated solution (phosphorylase activity 2089 U/-) and preservatives extracted and purified from Nato 10kll by a known method (Nakanomado-1 Toshibe Fukui: Starch Science, 24.80 (1977)) 1 ml/l of toluene was added thereto, and after adjusting to 30 d with ion-exchanged water, the reaction was carried out at 40° C. for 24 hours. As a result, 210 mm
1/l of G-1-P was obtained.

Example 5 1) Dextrin 6t with an E value of a97, KM, PO4a
8 f, K, HPO4a 6 f (i-Dissolve in 20 iu of dissolved aqueous solution at 27°C. Add to this? Te) 1
The enzyme phosphorylase was extracted and purified from 0klF by a known method (Nakanomado-1 Toshibe Fukui: Starch Science, 24.80 (1977)), and a concentrated solution of 1.5 d (phosphorylase activity 2089 U/d) and a preservative were prepared. Add 1 ml of toluene and adjust to 30 d with ion-exchanged water.
The reaction was carried out at ℃ for 24 hours. As a result, 214mm+sl
/ L of G-1-Pi obtained Example 6 Dextrin 9f with a DE value of 11.26 was mixed with KM, PO4
Dissolve a 7 f and HPO4 & 4 f ft in an aqueous solution of 20 dK at 27°C. 1 for this? Tet 10
Known method from Yukara (Nakanomado-1 Toshibe Fukui: Starch Science, 2
4.80 (1977)) to extract the enzyme phosphorylase.
Add purified and concentrated solution L 25 d (phosphorylase activity 2418 U/Ml) and 1 d of toluene as a preservative, adjust to 30 d with ion-exchanged water and then heat to 40 °C.
The mixture was allowed to react for 24 hours. As a result, 230 mmol/
Example 7 Obtaining G-1-P of L Dextrin 3f with a DE value of a97 was ion-exchanged with 90
Dissolve dK at 28°C. 1KH, Po, 9.
Lo containing 15 F and HPo, 14 f
Mash the om aqueous solution and 300f potatoes with a juicer,
Add 100 g of ground juice of I-tet obtained by centrifugation and 1 g of toluene as a preservative, and boil for 300 m with ion-exchanged water.
After adjusting #4C, it was reacted at 40°C for 24 hours. Result 3
a 1 mmol / 1. G-1-P' (obtained.

Example 8 3 tons of dextrin with a DE value of 787 was ion-exchanged with 90
0K, Kl, PO49, dissolved in slJ at 28°C.
5f and 100m containing HPo, 14F
, potato sushi juice Loom obtained by crushing 300 tons of potatoes with a juicer and centrifuging them, and 1 d of toluene as a preservative were added, and 300 d of potato was added with ion-exchanged water.
After adjusting to
a 9 mmol / 1. G-1-PI was obtained.

Example 9 3 t of dextrin with a valence of 11.26 is dissolved in 90 d of ion exchange fishing rod at 28°C. To this, KM, Po,
9.5 and Loo containing HPO414f
Add 1001L of potato sushi juice obtained by crushing and centrifuging potato sushi juice with 100ml of potato aqueous solution with a 300tf Soyucer, and 1d of toluene as a preservative, and add 300ml of ion-exchanged water.
After adjusting the temperature, react at 40℃ for 24 hours.
A 3 mmol/L of G-1-P'ji was obtained.

Comparative example 1? The solubility, viscosity, and reactivity of Tetodengun and the dextrin used in the present invention were measured and compared. ? For tetodenzone, a first-class reagent manufactured by Wako Tsumugi Pharmaceutical Co., Ltd. was used.

Solubility Solubility was defined as the weight (f) that dissolves 100 dK of ion exchange water at 24°C.

Viscosity Viscosity was measured using a Type B viscometer at 40°C 〇 * Unmeasurable due to gluing Reactivity: Dextrin or ? Tetodengun 1-10%, orthophosphate 15 no 1/L
G -1-P 1 obtained when reacting under the conditions of
Comparison was made using Ik (mmol/t). in particular,
Dextrins 3 to 3 of the present invention were added to 150 MI of an aqueous solution containing KH, PO42a 6 and HPO44L8f.
Dissolve 309 at 28℃ and add it to this? Te) Obtained by crushing 300F with a juicer and centrifuging it? Tet's sushi soup 1
00d and toluene IMLl as preservative,
After adjusting 300iuK with ion-exchanged water, react at 40℃.
-1-P was produced. on the other hand,? Tetodenden is 3-30
t is suspended in about 100 d of ion-exchanged water, dissolved in a boiling bath, and cooled to room temperature. Add this to ion exchange water 1001
Ll.

Kn, PO42a 6 F and HPO44L 8
Add f'i to dissolve and serve) 300t was crushed with a juicer and centrifuged. Tet's sushi soup 100IL
1 and 1 d of toluene as a preservative were added, and the mixture was adjusted to 30011 iK with ion-exchanged water, followed by a reaction at 40°C. The obtained amount of G-1-P was measured over time and was taken as the value at which the amount of G-1-P did not change.

? Tetodenden 317 944 *
This dextry (DEa97) 4Q8 1
0a8 159.8 1000 bottles The reaction solution was glued and reaction was not possible.

From the above results, it is clear that using the dextrin of the present invention
9G-1-P yield increases and KG-1-P is cost-effective.
1-Pf: It is clear that it can be manufactured. In addition, by using dextrin, heat treatment is not required for dissolving α-glucan, making it possible to simplify the G-1-P production process.

Comparative Example 2 Dextrin with a DE value of 028 15 ft-1, Po, 3
Solution 20 in which HPo, 56 f was dissolved in 8f and
Suspended in 0mlF, dissolved in a boiling bath, cooled, and then added to this?
95al of Teto's sushi juice obtained by crushing TeI'300F with a juicer and centrifuging it, and toluene I as a preservative.
After adjusting the temperature to 300° C., the mixture was reacted at 40° C. for 48 hours. The amount of G-1-P obtained is as follows,
Even if a dextrin having a DE value outside the range of the present invention is used, good results cannot be obtained.The reaction solution becomes dull.

Comparative Example 3 Dextrin 15f with a DE value of 21 was added to 200d of a solution in which KH, PO438 and HPO456F were dissolved.
Dissolve at °C. to this? Obtained by crushing 300t of Tet with a juicer and centrifuging it? After adding 95 d of Tet's ground juice and 1,111 ml of toluene as a preservative to adjust the volume to 300 ML1, the mixture was reacted at 40° C. for 48 hours. Obtained G-1-P
The quantities are as follows.

From the above results, it was found that the use of dextrin with a DE value of 05 to 20 increased the 9 cm x-p yield.

that's all

Claims (1)

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