DE2120411A1 - Process for obtaining high-purity glucose-6-phosphate from starch products - Google Patents

Description

Pafontanwiiie

ZELLENT-N υ. LUYKEN

30CG Munich 22
Zwsibrückensff. 6th

Ken Hayashibara April 26, 1971

Okayama / Japan SJ / Eu.

kho 7154-

Process for the production of highly pure glucose-6-phosphate from starch products »

The invention relates to a method for obtaining high purity Glucose-6-phosphate, which is an intermediate product of metabolism is in the human body and is used for medicinal purposes by phosphorylating starch or starch hydrolysates with phosphorylase and phosphoglucomutase.

Processes for the production of glucose-6-phosphate are known (hereinafter abbreviated to "G-6-P") by phosphorylation a mixture of starch or soluble starch and an inorganic phosphate by means of those present in potatoes Phosphorylase and conversion of the obtained glucose-6-phosphate ester by reacting with yeast or muscle phosphoglucomutase.

With the biosynthetic methods commonly used becomes a 10% aqueous slurry of potato or

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Sweet potato starch through. Gelatinized by heating, after adding clay 0.5 g (d © g starch) dibasic potassium phosphate completely dissolved and the cooled solution * kept ^{at 40 μg for 2 to 3 days.} However, this procedure gives G-6-P only in an extremely low yield, namely 50 to 60 Mo1% of the amounts of phosphate used. Since large amounts of polysaccharide remain in the reaction mixture, the G-6-P formed does not crystallize. Accordingly, it is necessary to separate off the remaining polysaccharides by precipitating with alcohol in the usual way; However, this precipitation method does not completely eliminate the polysaccharides, so that the G-6-P formed still does not crystallize. If a larger amount of ethanol is used for precipitation, the G-6-P also precipitates at the same time, so that its yield drops to a great extent.

In order to overcome these disadvantages and to facilitate the seaction process it has now been investigated whether a reduction in the viscosity of the reaction mixture does not affect the course of the process would make it easier if the branched amylopectin present in the starch by splitting off the

vlinearkettJKen j ^ i edriKmo leku degrades side chains with the help of alpha-1,6-gluco sidase to YMaltodexjbi = ¹¹ trines of the amylose type.

The examinations were carried out as follows:

5 to 10% strength starch suspensions were completely gelatinized by heating, then quickly ^{cooled to 50 to 60 °} C. and

* nac33. Addition of potato phosphorylase and yeast phosphoglucomutase _β ·.

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added as alpha-1,6-glucosidase pullulanase or isoamylase of the Pseudomonas genus, whereby the amylopectin side chains were broken down and low-viscosity solutions of short-chain amylose were obtained. The solutions obtained were mixed with phosphate buffer solution, raw potato phosphorylase and raw yeast phosphoglucomutase as well as toliiol and incubated for 2 days at 4-0 ⁰ O.

The obtained solution (a) was made with a gelatinized starch solution (b) as above, but without the use of alpha-1,6-glucosidase was treated, compared. It was found that the proportion of G-6-P in solution (a) exceeded 90 Mo1% and was about 65 #> higher than in solution (b). The results shows the table I.

Table I: Influence of isoamylase on glucose-6-phosphate formation.

attempt Treatment with isoamylase Yield mole% / p 1
7th there
there
there 90
92
93 4th
5 no
no 55
56

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Treatment with alpha-1,6-glucosidase became the following Phosphorylating process facilitated. The liberation of the Target product of glucose-1-phosphate (abbreviated with "G-1-P") was a consequence of treating the gelatinized starch with alpha-1,6-glucosidase and increasing the yield of G-6-P.

The object on which the present invention is based, to obtain highly pure G-6-P in high yield, thus became it according to the invention solved in that one

(A) starch slurries completely gelatinized by heating,

(B) treated the gelatinized starches with alpha-1,6-glucosidase,

(0) phosphorylates the solutions obtained in a manner known per se and

(D) from the reaction solutions after fractional precipitation of proteins and dextrins the target product falls as a barium salt).

The reaction solutions obtained are preferred according to the invention cleaned according to the enclosed scheme.

As noted above, treatment with alpha-1,6-glucosidase turns the starch into short chain, low molecular weight amy- loose and low molecular weight dextrins broken down. The latter are available in relatively small quantities and are difficult to fell, so that the precipitation caused with ethanol is small; because of absence

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Cleaning scheme reaction mixture

Protein precipitation by heating to 100 ° C

Level I.

sling

precipitated proteins, etc.

Precipitated dextrin liquid with dextrin precipitation alcohol

sling

step

Liquid /

Addition of 1.2 molar equivalent of BaOIp and alcohol level

sling

precipitation

Dissolve in hot water, heat with HGl (pH 1.5) at 10000 for 20 min

Cool, neutralize with NaOH

Cases with alcohol> ■
Spin liquid

Pälti

precipitation

Dissolve in hot water

Filter

Liquid

step

precipitation

sweetness

Crystallization by cooling

crystalline GOP-Ba-TH ₂ O mother liquor

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hext of G-6-P, the following precipitation process is easy. There in level 3 the simultaneous coprecipitation of sugars and dextrins in the G-6-P precipitating as the barium salt is small, the subsequent recrystallization of the barium salt is easy perform.

The pretreatment of the gelatinized starting starch with isoamylase and analogous enzymes thus facilitate the subsequent purification processes. Achieving a high purity G-6-P is only by the initial oxidation of the reaction solution brought about with alpha-1,6-glucosidase, the subsequent degradation and the Neutralization of the G-6-P as well as the repeated crystallization possible. Table III shows the essential differences in the yield and purity of G-6-P as a function from treatment with alpha-1,6-glucosidase or isoamylase.

Table II

Strength with
Isoamylase Yield of G-6-P
as Ba-SaIz
Mole% / P G-6-P-
purity
% salary
at G-1-P
below
% Inorganic
cal phos
phate un
below% treated
untreated 90 (medium)
48 (medium) 98
92 0.5
5 1
2

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According to the invention, the alpha-1,6-glucosidase is that from yeast recoverable isoamylase and that present in higher plants fi-enzyme can be used, - likewise also those from Aerobacter aerogenes pullulanase available, although it does not have the desired heat stability. The following enzymes are preferred:

Escherichia intermedia ATGG 21073

(described in Japanese PS 546 162)

Pseudomonas amyloderamosa ATGC 212162

(described in U.S. Patent 3,560 54-5),

also the enzymes according to the enclosed list.

Of those named are those of the Actinomycetes and Lactobacillus genera derived enzymes because of their good heat resistance.

It is preferable to purify the cell-bound cells obtained Enzymes and special cellular enzymes for the removal of cells and their precipitation by means of ammonium sulphate or organic Solvent.

The enzymes mentioned can preferably be used as follows:

A starch slurry of suitable concentration is ^{completely gelatinized by heating at 130 to 170 0} G, then rapidly cooled, adjusted to pH 4.5 to 7.0 and mixed with 10 to 50 units (per g of starch) alpha-6-glucosidase. Enzyme enhancement is evident when the mixtures are incubated for 5 to 60 hours.

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Streptomyces diastatochromogenes Actinomyces globisporus Nocardia asteroides Micromonospora melanospora Tnermonospora viridis

en Actinoplanes philippiifSis Streptosporangium roseum Agrobacterium tumefaciens Axotobacter indicus Bacillus cereus Erwinia aroideae Micrococcus lysodeikticus Mycobacterium phlei Sarcina albida Serratia indica Staphylococcus aureus Lactobacillus brevis Leuconostoc citrororum Pediococcus acMilactici Streptococcus faecalis Aerobacter aerogenes Corynecaterium sepedonicum Aeromonas hydrophila

Flavobacterium esteroaromaticum Acetobacter suboxydans Vibrio metsclmikovii Enterobacter aerogenes

Ii ¹ O 3337 IFO 12208 IFO 3384 IFO 12515 IFO 12207 ECO AOT- 0001 KGO AOT- 0005 IFO 3085 IFO 3744 IFO 5001 IFO 3057 IFO 3333 IFO 3158 I AM 1012 IFO 3759 IFO 3061 IFO 3345 ATGO 8081 IFO 3884 IFO 3128 ATGC 8724 IFO 3306 IFO 3820 IFO 3751 IFO 313O I AM 1039 ATGG 8724.

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After an incubation period of 1 to 2 hours at 4-0 to 60 ° C, the viscosity of the mixture has dropped and the iodine color has turned blue; this shows that the strength is increasing Amylose-type maltodextrins are broken down.

Potassium or is first added to the starch hydrolyzate solution obtained Sodium phosphate, then a raw potato extract as Enzyme source for phosphorylase and a raw yeast extract as a source for Phosphoj ^ glucomutase. Incubation of the mixture takes 2 days at pH 8.0 and 30 ° C. Analysis of the reaction mixture shows the formation of G-6-P from G-1-P.

The remaining polysaccharides, i.e. the remaining amylose, are then precipitated with an alkanol and removed. After adding a mole equivalent of barium salt, G-6-P is obtained as a barium salt and, after removal of the G-1-P, as a pure one G-6-P-Ba. In case of non-use of treatment with alpha-1,6-glucosidase the large proportion of residual dextrins or high molecular weight substances prevents crystallization and separation of the G-6-P.

Example";

1). Preparation of raw potato extract.

Peeled and homogenized potatoes are filtered through cheesecloth.

triert, the filtrate centrifuged in 10 min at 9000 and the liquid used for subsequent incubation. the

... 10 1 0 9 8 A 8/18 7 5

- ίο -

Enzyme activity of the extract is 6 units / ml.

2). Preparation of raw yeast extract.

15 g of baker's yeast are mixed with 15 ml of 0.1 M NaHCO ₅ and 15 ml

Toluene, the mixture shakes for 2 h at 40 ⁰ C and spun with G

in 10 min. The mixture of 10 ml of 0.1 M NaHCO ₅ , 10 ml of toluene and 10 g of baker's yeast is stirred for 6 h at room temperature

»Stirs or left to stand at 30 ^° C. for 18 h and then d

spun at 9OOO S ^ räai, the supernatant liquid was then adjusted to pH 5.0, treated for 15 min at 5O ⁰ C and 10 min with spun 3000th The supernatant liquid serves as an enzyme solution. The enzyme activity is 8 units / ml.

3). Quantitative G-6-P analysis.

10 ml of the reagent solution contained:

1 mg / ml NADT or water 5.0 ml

* 0.5 M "Tris" buffer solution, pH 7.8 3.0 ml

also containing 0.05 M EDTA ■ 1.5 ml

0.5 M MgCl ₂ 0.5 ml.

1.0 ml of a 5 mg / ml G-6-P dehydrogenase solution was added (collect 10 μΐ) .

The analysis was carried out by mixing 150 jU-1 of the above NADP solution with 200 μl of the solution to be tested. The density (optical density - 0.D ^) of 3 ^ 0 mji (at 0 hour) was measured

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The addition of 10 μl of G-6-P solution resulted in a volume of 360 μ, ϊ, the reading (read) of the blackening (Ö.D ^) of 34-0 ταμ, took place in 10 minutes determined according to the equation. (OD = optical density):

OD = OD ^ - 0.D. _O

Quantity G-6-P in FmMl = χ χ dil.

^{u J} 6.22 200

4). Quantitative G-1-P analysis:

The measurement was carried out as for G-6-P. The value of O.D.,. after adding 1 μ.1 phospho ^ glucomutase. Calculated became according to the equation:

_ _{x di: u}

6.22 200

Reading was carried out for Δ OD = OD-ODjj and Dxi. 360 pl was converted into 361 μΐ .

3). Treatment of the reaction drops.

(a). 175 ml of water were added to 14 g of sweet potato starch, the mixture was heated for 10 minutes in an autoclave at 120 ^° C. while stirring and then cooled quickly. Now at 55 ° G, 15 units (ge g starch) alpha-1,6-glucosidase, obtained from a Lactobacillus enzyme, were added and the mixture was stirred at pH 6.0. After drop of the viscosity of the mixture was allowed 30 h at 50 ⁰ G standing, seated thereon

... 109848/1875

0.34 g of KH ₂ PO ^ and 6.8 g of Na ₂ HPO ₄ were added. As soon as the solution occurred, the mixture was cooled with running water, then 75 ml of raw potato extract and 45 ml of raw yeast extract and, after stirring, 20 ml of toluene, and the mixture was incubated at 40 ° C. for 40 h.

(b). A cornstarch slurry was gelatinized at I50 to 160 ° C with stirring, then cooled to 60 ⁰ O and adjusted to pH 6.0. Then 15 units were added (per g of starch) of the asteroides Nocardia obtained from the enzyme, the mixture to 5O ⁰ C cooled and incubated 20th A low viscosity maltodextrin solution was obtained.

6). Cleaning the reaction solution.

After heating for 5 min at 100 ^° C., the deposited proteins were filtered off. then admitted alcohol. cooled and flung them

precipitated dextrins 10 min with 3000 # 5 ^ 8 »©. The supernatant liquid was mixed with 1.2 mol of BaCl ₂ , dissolved in a little water, and adjusted to pH 6.8. After adding alcohol, the barium salt of G-6-P precipitated. The cooled mixture was centrifuged, the material to be centrifuged was dissolved in hot water, the solution was adjusted to pH 1.5 and ^{heated at 100 °} C. for 20 minutes. After the G-1-P had decomposed, the mixture was neutralized, adjusted to pH 7-0, filtered and allowed to crystallize. The crystals obtained consisted mainly of pure barium salt of the formula G-6-P-Ba.7H ₂ O.

... 13 109848/1875

The yield was in all (a) 93 mol% / P, in case (b) 91 mol% / P, after recrystallization 45 mol% / P.

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Claims (2)

2120471 Patent attorneys M "!. · Ν ι ;. LUYKEN 80C0 Mi-, nc !, β η. 22nd April 26, 1971 SJ / Hu kho 7154 Claims Λ.I method for obtaining highly pure glucose-6-phosphate from starch products by phospharylation with phosphorylase and phosphoglucomutase, characterized in that one (A) starch slurries completely gelatinized by heating, (B) treated the gelatinized starches with alpha-1,6-glucosidase, (0) phasphorylates the solutions obtained in a manner known per se and (D) from the reaction solutions after fractional precipitation Of the proteins and dextrins the target product falls as the barium salt. 10984871875 2. Procedure according to. Claim. 1, characterized in that the through. Heat gelatinized starches at 130 to 17O ⁰ C after rapid cooling and adjust to pH 4, 5 to 7 O
at AO up to 60 ⁰ C 10 to 50 hours with 10 to 50 units /
g starch alpha-1,6-glucosidase treated and then phosphorylated for 2 days at pH 3 and 40 ° C. ;). Method according to claims 1 and 2, characterized in that
that the proteins are precipitated from the reaction solutions by heating, then the dextrins with alcohol and then the target product with BaCIo and alcohol, the barium salt dissolved in hot water is freed from glucose-1-phosphate by heating with hydrochloric acid, and the barium salt is again precipitated after neutralization with alcohol and its solution crystallizes by cooling. 109848/1875