JPS6018395B2 - Method for producing all-trans polyprenyl alcohol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing all-trans polybrenyl alcohol, and more specifically, the present invention relates to a method for producing all-trans polybrenyl alcohol, and more specifically, it has the general formula (D) (where n is 2 to
General formula (1), characterized in that all-trans polyprenyl pyrophosphate represented by the general formula (representing an integer of 10) is hydrolyzed by the action of phosphatase isolated from potato sprouts (where n is ) has the same meaning as in )) and general formula (m) (where 1 is 2
(representing an integer of ~4) and isobentenyl pyrophosphate are reacted by the action of polyprenyl pyrophosphate synthetase of microbial origin (separated from microorganisms) to form the general formula (〇′). (In the formula, m represents an integer of 8 or 9) All-trans pyrophosphate is synthesized, and then the pyrophosphate is hydrolyzed by the action of phosphatase isolated from potato sprouts. The present invention relates to a method for producing all-trans polyprenyl alcohol represented by formula (1') (where m has the same meaning as in general formula (')).

General formula (1) or (1') obtained by the method of the present invention
The all-trans polyprenyl alcohol represented by ) is useful as a synthetic intermediate for upiquinone-n (Tachibana Enzyme Q1n).

For example, a polyprenyl alcohol such as octaprenyl alcohol or decaprenyl alcohol represented by the above general formula (1) and 2,3-dimethoxy-5-methylhydroquinone are reacted in the presence of an alcoholic acidic condensing agent such as trifluoride trifluoride. Then, this condensation product is oxidized with an oxidizing agent such as silver oxide to obtain coenzyme Q-n (the following general formula (W)
) can be obtained (Hakama Kosho 39-1751
(See Publication No. 3). This reaction is represented diagrammatically as follows. Regarding coenzyme Q-n, the all-trans form of coenzyme Q-n in terms of the configuration of its side chain portion is particularly considered to be an in vivo active type of coenzyme Q-n.

In order to synthetically obtain this in vivo-active coenzyme Q-n, synthesis of all-trans polyprenyl alcohol is naturally required. Various methods for synthesizing polyprenyl alcohol are known; for example, the decaprenol synthesis method by Isler et al. is shown below [Helv. Chim.
Ac. 42.2616 (1959)]. In addition, Sato et al. proposed the following method for synthesizing polyprenyl alcohol (see Tokusekki Sho 51-141804).
. However, when polybrenyl alcohol with a long carbon chain is synthesized stepwise by conventional methods, the synthesis process is extremely long and complicated, and the production rate of the desired trans isomer is extremely low, or it is expensive. This method requires reagents and is not suitable for industrially obtaining all-trans polyprenyl alcohol.

On the other hand, attempts have also been made to synthesize polyprenyl pyrophosphate by enzymatic reactions using various prenyl pyrophosphate synthetases and to convert this into polyprenyl alcohol.

However, in previous studies, the three-dimensional structure of the obtained polyprenyl pyrophosphate with a long carbon chain has not been clarified, and no method has been found to easily convert the polyprenyl pyrophosphate into polybrenyl alcohol. Not yet.

The present inventors previously attempted to hydrolyze polyprenyl pyrophosphate using commercially available yeast-derived phosphatase or phosphatase extracted from pig kidney, but the corresponding polyprenyl alcohol could not be obtained [Bioc
hem. andBiophys. Res. Commu.
, 4, 251 (1971)].

In addition, Block et al. described the hydrolysis of polyprenyl birophosphate using trichloroacetic acid, but the yield of polyprenyl alcohol produced was extremely low [J. Bi
ol. Chem. , 24 Ko 8), 1895 (1967)
]. As a result of continued intensive research into an advantageous method for producing all-trans polyprenyl alcohol, the inventors of the present invention have now discovered that phosphatase isolated from potato sprouts has been found to be an all-trans polyprenyl alcohol of the general formula (0). It has been found that the present invention has excellent enzymatic activity for hydrolyzing brenyl pyrophosphate into the corresponding all-trans polyprenyl alcohol of general formula (1). The present inventors also discovered that polyprenyl pyrophosphate obtained by reacting isobentenyl pyrophosphate with all-trans pyrophosphate of the general formula (m) by the action of polyprenyl pyrophosphate synthetase of microbial origin is an all-trans pyrophosphate. By discovering that it is octaprenyl pyrophosphate or/and nonaprenyl pyrophosphate, and combining this knowledge with the above-mentioned knowledge regarding the action of phosphatase isolated from potato sprouts, all-trans pyrophosphate of general formula (m) (i.e., geranyl pyrophosphate, phalnesyl pyrophosphate, or geranylgeranyl pyrophosphate) and isobentenyl pyrophosphate are reacted by the action of polyprenyl pyrophosphate synthetase of microbial origin, and the general formula (ro'
) and hydrolyzing the all-trans polyprenyl pyrophosphate of general formula (D') by the action of phosphatase isolated from potato sprouts. Moreover, we succeeded in obtaining all-trans polyprenyl alcohol of general formula (1') in good yield.

The phosphatase used in the present invention can be obtained by treating potato sprouts by conventional techniques to separate and obtain the enzyme.

For example, potato sprouts have a pH of 5.0 to 8. If the mixture is grated in a buffer solution of preferably 6.5 to 8.0, the juice is squeezed out and centrifuged, a phosphatase-containing solution is obtained as a supernatant solution. In the present invention, the above-mentioned supernatant solution can be directly subjected to the hydrolysis reaction of all-trans polyprenyl pyrophosphate. The physicochemical properties of the isolated phosphatase are shown below. Optimum pH: 7.4 Optimum temperature: 370 Substrate specificity: Hydrolyzes all-trans polyprenyl phosphate of general formula (D).

In the present invention, the hydrolysis reaction of all-trans polybrenyl pyrophosphate represented by general formula (b) with phosphatase is carried out to convert all-trans polybrenyl pyrophosphate in a buffer such as Tris-HCl buffer. This is done by the action of phosphatase.

This reaction is generally carried out at a pH of 5.0 to 8.5, preferably 6.5.
~8.0 Reaction temperature 25-60qo, preferably 30-40qo
00 for about an hour. The optimal reaction conditions vary slightly depending on the type of substrate used, so it is best to select them appropriately. The condensation reaction of all-trans pyrophosphate represented by general formula (m) and isobentenyl pyrophosphate by polyprenyl pyrophosphate synthetase of microbial origin is carried out by the hydration of all-trans polyprenyl pyrophosphate by the above-mentioned phosphatase. The reaction can be carried out under almost the same reaction conditions as for the decomposition reaction.

The polyprenyl pyrophosphate synthetase used in the present invention may be polyprenyl pyrophosphate synthetase isolated from any microorganism, and a representative example is Micrococcus lithodecticus (MicrMocc).
uslysodeiktjcus) bacterial body (ATCC46
98).

Isolation of polyprenyl pyrophosphate synthetase from Micrococcus lysodiecteicus cells can be carried out, for example, by the following method. The method of Block et al. [J. Biol. Chem. 242,
1895 (1967)] (supernatant liquid obtained by centrifugation for 1 hour at 60,000 yen) is fractionated with ammonium sulfate at a saturation of 30 to 50%, and the precipitate is collected.

The precipitate was dissolved in 0.09M phosphate buffer pH 6.8 (referred to as buffer 1) containing 0.08M potassium chloride, and the column of Sephadex G-25 equilibrated with buffer 1 (Sweden) was prepared. (manufactured by Pharmacia) and equilibrated, or equilibrated to Buffer 1 by dialysis. This solution was equilibrated with buffer 1 to DEAE-Sephadex A-
50 column (1.5 x 20, manufactured by Feilmacia, Sweden), and after the unadsorbed portion was removed, the concentration of potassium chloride contained in the buffer solution was adjusted from 0.08M using buffer solution 1. The protein is eluted in a gradual linear manner up to 108M. During the fractionation, the fraction
Using a collector, aliquot the amount of buffer solution into 5' portions. The total volume of buffer used is 500 skins. Polyprenylpi. Phosphate synthetase is eluted between fraction numbers 40 and 5 (potassium chloride concentration 0.45 to 0.59M). The physicochemical properties of polyprenyl pyrophosphate synthetase obtained by this method are shown below. Molecular weight: 78,000 Optimum base: 7.0-7.6 Optimum temperature: 370 Substrate specificity and products; Trans-octaprenyl pyrophosphate and nonaprenyl pyrophosphate are produced in a ratio of 5:3.

Enzyme reaction rate: geranyl pyrophosphate > phalnesyl pyrophosphate > geranylgeranyl pyrophosphate, the enzyme reaction rate ratio is 100:25:20.

The polyprenyl pyrophosphate production process and the polyprenyl pyrophosphate hydrolysis process of the present invention can be carried out either continuously or batchwise, but it is preferable to use a continuous process in which the enzyme used does not inhibit the product. desirable.

As a continuous production method, an immobilized enzyme method is particularly preferred.
The immobilized coenzyme may be prepared by any of the conventionally known entrapment methods, ionization methods, and covalent bonding methods. Below, one embodiment of the method for preparing phosphatase used in the present invention is shown in Reference Example 1, and the present invention will be specifically explained using Examples and Comparative Examples.

Reference example 1 Preparation of phosphatase Young sprouts (5 to 10 skins) of potatoes sprouted in the basement 9
．． 10 containing lmM 3-mercabutethanol.
mM Tris-HCl buffer 7. 7.6 Grate the juice and squeeze out the juice with gauze.

This squeezed juice is centrifuged for 2 minutes at 500 °C. The obtained supernatant solution 10' is a phosphatase enzyme solution. Example
1 geranyl pyrophosphate 20 M, isobentenyl pyrophosphate 50 M, magnesium chloride 10 M
Micrococticus lithodecticus cells (A
When a reaction mixture containing 20 units of enzyme solution of polybrenyl pyrophosphate synthetase isolated from TCC 46 Nada) was reacted at 370°C for 3 hours, all-trans octaprenyl pyrophosphate and nonaprenyl pyro were detected. A polyprenyl pyrophosphate mixture of phosphates (composition ratio 5:3) was obtained in a yield of approximately 50% (based on isobentenyl pyrophosphate).

Next, 10 parts of the phosphatase enzyme solution obtained in Reference Example 1 was added to the obtained polyprenyl birophosphate mixture, and a nonionic surfactant (Triton x-100
) was added to give a concentration of 0.2% M/V, and the mixture was reacted at 370° C. for two hours. Thereafter, the reaction solution was made slightly alkaline with a 6N aqueous sodium hydroxide solution, and extracted with n-hexane and petroleum ether to obtain all-trans octaprenol and nonaprenol in a ratio of about 5:3. This was chromatographed (high performance liquid chromatography, packing material: Hitachi gel #3011, solvent: methanol/n-hexane)
It was separated and purified by The yield from the hydrolysis reaction was almost quantitative. Comparative Example 1 In place of the phosphatase used in Example 1, commercially available phosphatase (yeast origin, manufactured by Boehringer Mannheim [E
．． C. 3, 6, 1, 1]), the same hydrolysis reaction as in Example 1 was attempted, but the corresponding polyprenyl alcohol was not produced.

Example 2 Geranylgeranyl pyrophosphate 50 M, magnesium chloride low M and Tris-HCl buffer pH 7.4
, 10w of a phosphatase enzyme solution obtained in the same manner as in Reference Example 1 was added to a mixture of 100mM and 100mM, and a nonionic surfactant (Triton x 1
00) to 0.2% M/V and 3 at 370.
When the reaction was allowed to proceed for several hours, geranylgeraniol was obtained almost quantitatively.

Example 3 Preparation of Oka Sadai Q Enzyme Polyprenyl pyrophosphate synthetase: 60 ml of enzyme solution of polyprenyl pyrophosphate synthetase isolated from Micrococcus lingicticus cells was diluted to 0.
．． After dialyzing potassium chloride using 0.09M potassium chloride as an external solution, it was equilibrated with a phosphate buffer containing 0.09M potassium chloride, and a DEAE-Sephadex A-50 column (1.5 x 20 The immobilized enzyme was prepared by adsorbing the enzyme to the molecule (Arc).

Phosphatase: To 10 parts of the phosphatase enzyme solution obtained in the same manner as in Reference Example 1, 15 parts of acrylamide monomer, 8 parts of N·N'-methylenebisacrylamide, 10 parts of 5% 8-dimethylaminopropionitrile,
Add 10 ml of 2.5% potassium persulfate and mix to make 37 q.
0 and allowed to gel.

The resulting gel was crushed using a Waring blender,
A granular immobilized enzyme was prepared. The immobilized polyprenyl pyrophosphate synthetase and immobilized phosphatase obtained above were packed into separate columns, and the columns were connected.

Claims (1)

[Scope of Claims] 1. All-trans polyprenyl pyrophosphate represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (wherein n represents an integer from 2 to 10) isolated from ginger sprouts A method for producing all-trans polyprenyl alcohol, which is characterized by its hydrolysis due to the action of phosphatase, which is represented by the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (wherein n has the meaning above). 2 All-trans pyrophosphate and isopentenyl pyrophosphate represented by the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (in the formula, 1 represents an integer from 2 to 4) All-trans pyrophosphate represented by the general formula ▲Mathematical formula, chemical formula, table, etc.▼ (in the formula, m represents an integer of 8 or 9) is synthesized by reacting with the action of tase, and then the pyrophosphate is injected into a potato. General formula ▲ characterized by hydrolysis by the action of phosphatase isolated from buds
There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, m has the meaning above) A method for producing all-trans polyprenyl alcohol.