JPH10316698A - Purification method of fructose-2,6-bisphosphate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a synthetic by-product of fructose-2,6-bisphosphate, which is very difficult to separate from fructose-2,6-bisphosphate. And a method for purifying fructose-2,6-bisphosphoric acid from which fructose-2,6-bisphosphate can be obtained with high purity. SOLUTION: When purifying fructose-2,6-bisphosphate from a solution containing fructose-2,6-bisphosphate and fructose-1,6-bisphosphate,
Fructose-2,6-bisphosphate and fructose-
Aldolase and fructose-1,6- by the action of aldolase are added to a solution containing 1,6-bisphosphate.
A method for purifying fructose-2,6-bisphosphate, which comprises reacting an enzyme acting on a product generated from bisphosphate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a fructose-
2,6-bisphosphoric acid (hereinafter abbreviated as F2,6P2)
And a method for purifying.

[0002]

2. Description of the Related Art Regulation mechanisms of glucose metabolism in living organisms include, for example, regulation by insulin hormone, regulation by glucagon and adrenaline, regulation by cyclic adenosine 5'-monophosphate called second messenger, and adenosine 5'-monophosphate ( Below, AMP
Adenosine 5′-triphosphate (hereinafter A)
TP), regulation by citric acid, F2,6P2
Has been reported.

[0003] Glucose metabolism in living organisms is carried out by a series of pathways called glycolysis, in which phosphofructokinase 1 enzyme (EC 2.7.1.11:
Hereinafter, abbreviated as PFK1) is an important regulator.
It has recently been confirmed that F2 and 6P2 that activate PFK1 are important as factors relating to glucose metabolism.
Furthermore, it has been reported that F2,6P2 is useful as a therapeutic agent for diabetes and diabetic complications and as a hepatocyte proliferation promoter (see JP-A-2-202822).

Conventionally, as a method of manufacturing F2,6P2,
(1) Fructose-1,6-bisphosphate (hereinafter referred to as F
1,6P2) to obtain fructose-1,2-cyclic-6-bisphosphoric acid (hereinafter abbreviated as FCP) using dicyclohexylcarbodiimide as a catalyst, and further produce the FCP by partially decomposing it with an alkali. How to do [European Journal of
Biochemistry, 117, 319 (1981)
Journal of Biological Chemistry, 256, 8679 (1981);
No. 0-161994], and (2) polyethylene glycol precipitation from a yeast cell lysate, Sephacryl-blue resin, CM Sephadex (CM).
-Sephadex) resin purified using phosphofructokinase 2 enzyme (EC 2.7.1.105: hereinafter)
PFK2) to fructose-6-phosphate (hereinafter abbreviated as F6P) and ATP (Biochemistry, Vol. 32, 1
1147 (1993)].

However, among the above methods, the method (1) has a low conversion efficiency (decomposition by alkali) from FCP to F2,6P2 of 15%, and 85% of F is a by-product.
There was a problem that it was converted to 1,6P2 [European Journal of Biochemistry, 117, 320 pages (1981)]. Also,
Also in the method (2), if PFK1 is not sufficiently removed in the step of purifying PFK2 from the yeast cell lysate, P
There is a problem that FK1 synthesizes F1 and 6P2 from F6P and ATP. Thus, F2,6
Since F1 and 6P2 are produced as a by-product when synthesizing P2, F2 and 6P2 are required to obtain high-purity F2 and 6P2.
It is necessary to separate F1 and 6P2 from
1,6P2 is a positional isomer of F2,6P2, and both have very similar structures, so it is very difficult to separate them using a column or the like.

As a method of separating F1,6P2 from F2,6P2, F1,6P2 is separated from fructose-1,6-
A method of decomposing F2P and phosphoric acid by phosphatase and then separating and purifying F2,6P2 using an ion exchange resin is known [European Journal of Biochemistry, 117, 321 (198).
1 year)].

[0007]

However, in this method, since F2,6P2 in the solution inactivates fructose-1,6-phosphatase, the decomposition of F1,6P2 does not proceed sufficiently. There was a problem that F2,6P2 of purity could not be obtained. The present invention relates to F2,6
From a solution in which P2 and F1,6P2 coexist, high-purity F2
It is an object of the present invention to provide a method for purifying 6P2.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to solve such problems and found that F2,6P2
F1,6P2 can be decomposed efficiently by allowing aldolase and an enzyme acting on an aldolase decomposed product of F1,6P2 to act on F1,6P2 which is a by-product present in the synthesis solution of F1,6P2. And completed the present invention. That is, the present invention relates to F2
From a solution containing 6P2 and F1,6P2, F2,6P
In purifying 2, F2, a solution containing F2, 6P2 and F1, 6P2 is reacted with an aldolase and an enzyme acting on a product generated from F1, 6P2 by the action of the aldolase. The gist is a method for purifying 6P2.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

[0010] F2,6P2 is a sugar phosphate compound represented by the following general formula and recently discovered as a trace substance in animal tissues.

[0011]

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On the other hand, F1,6P2 is a positional isomer of F2,6P2 represented by the following general formula.

[0013]

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The aldolase used in the present invention is obtained by converting F1,6P2 into glyceraldehyde-3-phosphate (hereinafter abbreviated as GAP) and dihydroxyacetone phosphate (hereinafter abbreviated as DAP) as shown in the following reaction formula. ).

[0015]

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Aldolase is widely distributed in organisms having a glycolytic system. Examples of the aldolase used in the present invention include those extracted from baker's yeast, bovine liver, rabbit muscle and liver, and the like. Particularly, it is preferable to use aldolase extracted from rabbit muscle because of its high substrate specificity to F1,6P2.

In the present invention, F
In order to promote the 1,6P2 decomposition reaction, an enzyme that replaces GAP or DAP generated by the action of aldolase with another compound is simultaneously acted on, and the equilibrium of the reaction is adjusted to F1,
Tilt to the decomposition reaction of 6P2.

Among such enzymes, the enzyme acting on GAP is triosephosphate isomerase (hereinafter, referred to as "triose phosphate isomerase").
The enzyme acting on DAP is glycerol-3-phosphate dehydrogenase (hereinafter abbreviated as GDH).
Acts on GAP to produce DAP and GDH acts on DAP to produce glycerol-3-phosphate (hereinafter abbreviated as G3P).

[0019]

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In the present invention, the enzymes acting on GAP and DAP are not limited to these enzymes. In addition, the enzymes acting on GAP include glyceraldehyde-3-phosphate dehydrogenase and DAP. Enzymes that act include dihydroxyacetone phosphate phospholyase, dihydroxyacetone phosphate acyltransferase, and the like.

In the present invention, it is necessary that one or more of these enzymes coexist with aldolase. In particular, it is preferable to coexist both an enzyme acting on GAP and an enzyme acting on DAP.

The concentration of the aldolase used in the present invention is preferably 1 unit / liter or more, particularly preferably 10 units / liter to 50 units / liter, based on 1 mM F1,6P2. Further, the concentration of the enzyme acting on GAP or DAP used in the present invention is 2 mM with respect to 1 mM of F1,6P2.
Unit / liter or more, preferably 5
Unit / liter to 200 units / liter. When TIM and GDH are used, T
It is preferable that IM is 2 units / liter or more and GDH is 5 units / liter or more.
G from 0 units / liter to 100 units / liter
It is preferable that DH is 10 units / liter to 50 units / liter.

In addition, when GDH is used, G
To facilitate the reaction catalyzed by DH, NAD
It is preferable to add H at the same concentration or more as F1, 6P2, and it is particularly preferable to add H in an amount of 1 to 5 times.

The reaction temperature is preferably from 25 to 35 ° C., particularly preferably from 28 to 32 ° C. The reaction time is not particularly limited as long as F1, 6P2 is sufficiently decomposed, but is preferably 10 minutes or more, particularly preferably 20 to 60 minutes.
Minutes are preferred.

By such an enzymatic reaction, F in the solution
1,6P2 is almost completely decomposed, while F2,6P2
Is hardly decomposed. Thus, F1, 6P2
In order to further purify F2,6P2 from the solution from which F2, P2 is decomposed and removed, G2P, DAP and G3P, which are decomposition products of F1,6P2, are all compounds having one phosphate group, whereas F2,6P2 Is a compound having two phosphate groups, and can be easily carried out using an anion exchange resin by utilizing the fact that both have different adsorbing powers to an anion exchange resin.

The thus purified F2,6P2
May be further purified by electrodialysis or desalting using a reverse osmosis membrane or the like, or can be obtained in powder form by freeze-drying or the like.

[0027]

Next, the present invention will be described in detail with reference to examples.

Reference Example 1 (Purification of PFK2) PFK2 recombinant yeast Saccharomyces cerevisiae (Sa
ccharomyces cerevisiae) BJ2168 / pAB10
(FERM P-15210) was composed of 2% by weight of glucose (manufactured by Ishizu Pharmaceutical Co., Ltd., product number: 036-0064), 0.5% by mass of casamino acid (manufactured by Nissui Pharmaceutical Co., product number: 05834), and a nitrogen source containing no amino acid (manufactured by DIFCO) , Part number 0919-1
5-3) 0.67% by weight, L-leucine (manufactured by Ishizu Pharmaceutical Co., Ltd., product number 012-4021) 30 mg / liter, L-leucine
Tryptophan (Ishizu Pharmaceutical Co., Ltd., product number 015-331)
1) Medium containing 30 mg / liter (pH 5.2) 20
The mixture was inoculated to 1 liter and cultured at 30 ° C. using a 30 liter jar fermenter. Twenty liters of this culture solution was added to galactose (Ishizu Pharmaceutical Co., product number 012-330
3) 400% of a medium (pH 5.2) containing 2% by weight, 0.5% by weight of casamino acid, 0.67% by weight of an amino acid-free nitrogen source, 30 mg / L of L-leucine and 30 mg / L of L-tryptophan. 50 at 30 ° C
By culturing for 24 hours using a 0-liter jar fermenter, 4.2 Kg of cells were obtained.

Next, 200 g of the obtained cells were added to buffer A [50 mM Tris-HCl (Ishizu Pharmaceutical Co., Ltd., product number 325-2070), 20 mM KCl (Ishizu Pharmaceutical Co., product number 034-5283), 2 mM β-mercaptoethanol (mercaptoethanol, manufactured by Ishizu Pharmaceutical Co., product number 043-0461), 2 mM EDTA
PH (including Ishizu Pharmaceutical Co., Ltd., part number 042-0621)
7.5] in 500 ml, and suspend this suspension in D
Glass beads were crushed using a YNO-MILL crusher (type KDL, manufactured by Shinmaru Enterprises) and glass beads having a diameter of 0.25 mm (manufactured by Shinmaru Enterprises). PFK2 of the obtained cell lysate
The activity was 75 units. A polymer flocculant (UniFlocker UF-304, manufactured by Unitika Ltd.) is added to the cell lysate.
Was added in an amount corresponding to 12% of the volume of the crushed liquid. After stirring for 5 minutes, the solution was centrifuged (4 ° C., 5
8000 rpm), and the supernatant was recovered. The obtained supernatant was subjected to DEAE-Sepharose FF (manufactured by Pharmacia) and Blue Sepharose CL-6B (Blue
-Sepharose CL-6B, manufactured by Pharmacia) was packed in a column connected in series, and the KCl concentration of buffer A was increased to 2M.
The PFK2 was eluted by the above solution. PF at this time
The K2 activity was 8 units, and the activity of PFK1 was below the detection limit.

Reference Example 2 (enzymatic synthesis of F2,6P2) 50 mM Tris-HCl (manufactured by Ishizu Pharmaceutical Co., part number 32)
5-2070), a reaction solution (pH 9.0) containing 20 mM MgCl ₂ (manufactured by Ishizu Pharmaceutical Co., Ltd., product number 013-3043), 15 mM ATP (manufactured by Sigma Co., product number A-5394), and 10 mM F6P. Then, PFK2 purified in Reference Example 1 was added thereto at a concentration of 50 milliunits / milliliter and reacted at 30 ° C. After 4.5 hours from the start of the reaction, the reaction solution was boiled to stop the reaction. During this time, the amounts of F2,6P2 and F1,6P2 in the reaction solution were measured over time.

FIG. 1 shows the results. Figure 1 shows F2,6P
2 is a diagram showing the change over time in the synthesis amount of F2 and F1, 6P2, and the vertical axis shows the synthesis amount of F2, 6P2 and F1, 6P2;
The horizontal axis shows the reaction time. FIG. 1 shows that F2,6P2 was synthesized at a reaction yield of 41% 4.5 hours after the start of the reaction. In the absence of PFK1, F
It was intended to perform 2,6P2 synthesis, but F1,6P
It can be seen that 2 was synthesized with a reaction yield of 5%. This is probably because the synthesized F2,6P2 activated PFK1, which is present in a very small amount in the solution, and promoted the synthesis of F1,6P2.

Example 1 The pH of the F2,6P2 synthetic solution obtained by the method of Reference Example 2 was adjusted to 8.0 with HCl, and 1 mM NADH (Boehringer Co., product number 128023) was added. In addition, 1
0 units / liter aldolase (Boehringer, part number 102652), 20 units / liter T
IM (manufactured by Boehringer, part number 109754) and 10
Unit / liter GDH (manufactured by Boehringer, part number 127775) was added, and the reaction was carried out at 28 ° C. One hour after the start of the reaction, the reaction solution was boiled to stop the reaction.
During this time, the amount of F1,6P2 in the reaction solution was measured over time.

FIG. 2 shows the results. FIG. 2 is a graph showing the change over time in the amount of F1,6P2 remaining in the solution, with the ordinate indicating the remaining amount of F1,6P2 and the abscissa indicating the reaction time. From FIG. 2, aldolase and TIM were added to F1,6P2.
It turns out that F1,6P2 can be completely decomposed by the action of GDH.

A column was filled with 10 ml of activated carbon (Dia Hope S80, manufactured by Mitsubishi Chemical Corporation) and distilled water 100
After washing with milliliters, the reaction solution obtained by decomposing and removing the F1,6P2 diluted twice with distilled water was applied,
Elution of the F2,6P2-containing solution with 200 ml of distilled water allows adenosine, ATP, A
Compounds with adenosine such as MP were removed.

Further, anion exchange resin DIAION
WA10 (DIAION WA10, manufactured by Mitsubishi Chemical Corporation, O
This eluate was applied to a column packed with 5 ml of H-form), 20 ml of distilled water and 0.2 M of NaHCO ₃ (manufactured by Ishizu Pharmaceutical Co., Ltd., product number 034-5933)
After washing with 100 ml, 0.5 M NaHC
F2,6P2 was eluted with 100 ml of O ₃ . The result is shown in FIG. FIG. 3 is a diagram showing the results when the F2,6P2-containing liquid was purified using the anion exchange resin DIAION WA10, in which the vertical axis shows the concentration and the horizontal axis shows the fraction number. FIG. 3 shows that the use of the anion exchange resin DIAION WA10 results in F
It can be seen that the decomposition product of 2,6P2 and F1,6P2 and F6P can be efficiently separated, and high-purity F2,6P2 can be obtained.

The thus-purified F2,6P2-containing liquid was desalted and concentrated with distilled water using a reverse osmosis membrane (NTR-7250, manufactured by Nitto Denko Corporation) until the electric conductivity became 10 mmho. This concentrated solution was freeze-dried at 4 ° C. for 50 hours to obtain 43 mg of F2,6P2 powder.

[0037]

According to the present invention, F1,6P2, which is a by-product of synthesis of F2,6P2 and is very difficult to separate from F2,6P2, can be almost completely decomposed. As a result, F2,6P2 can be easily purified in high yield, and high-purity F2,6P2 can be obtained.

[Brief description of the drawings]

FIG. 1 is a diagram showing a change over time in the production amounts of F2, 6P2 and F1, 6P2 when F2, 6P2 is synthesized from F6P using PFK2.

FIG. 2: Aldolase, TIM and GD for F1,6P2
It is a figure which shows the time-dependent change of F1,6P2 residual amount when decomposed | disassembled with H.

FIG. 3 is a diagram showing the results of purification of F2,6P2 using anion exchange resin DIAION WA10.

Claims (1)

[Claims] 1. Purifying fructose-2,6-bisphosphate from a solution containing fructose-2,6-bisphosphate and fructose-1,6-bisphosphate,
Fructose-2,6-bisphosphate and fructose-
Aldolase and fructose-1,6- by the action of aldolase are added to a solution containing 1,6-bisphosphate.
A method for purifying fructose-2,6-bisphosphate, which comprises reacting an enzyme acting on a product generated from bisphosphate.