JPS5923797B2 - Separation and recovery method for nucleic acid-related substances and vitamins - Google Patents

Description

[Detailed Description of the Invention] Nucleic acid-related substances and vitamins are produced by various methods such as fermentation, enzymatic degradation, extraction, and synthesis. This means that the purity of the target substance in the liquid is low.

As a result, there were problems in that the separation and purification steps became complicated and the separation and purification yields were low.

The method of the present invention makes it possible to efficiently separate and recover nucleic acid-related substances and vitamins from such raw material liquids or from waste liquids from which target substances have been separated from raw material liquids.

The principles of the invention are based on the separation of amino acids, J. Am.
er, Chem, Soc, , Vol 95,
This is an application of what was disclosed in p.6108-6110 (1973).

1. As a result of repeated research on applying this principle to the separation and purification of nucleic acid-related substances and vitamins from an aqueous solution containing them, the present inventors found that
It was discovered that nucleic acid-related substances and vitamins can be easily separated and recovered, and that the purification effect is large.

The present invention will be explained in detail below.

Nucleic acid-related substances include purines such as hypoxanthine, guania, adenine, cytosine, and uracil, pyrimidines, nucleosides such as inosine, guanosine, cytidine, and uridine, 51-inosinic acid, 51-guanylic acid, 51-
Nucleotides such as cytidylic acid, precursor substances for purine biosynthesis such as 5-amino-4-imidazole carboxamide, and NAD.

It refers to coenzymes such as NADP, FMN, CoA, TPP, and CDP-choline, and vitamins include VA, VBl, and v
B2, VB6, nicotinic acid, folic acid, etc.

Aqueous solutions containing nucleic acid-related substances or vitamins include, for example, raw material liquids such as fermentation liquids, enzymatic decomposition liquids, extract liquids, and reaction liquids obtained for the purpose of producing these nucleic acid-related substances and vitamins, and target substances derived therefrom. These include various intermediate process liquids and waste liquids used to obtain

Depending on the type of carrier, an acid or alkali is added to the filtered aqueous solution containing the nucleic acid-related substance or vitamin to make it appropriately acidic or alkaline.

The acid may be hydrochloric acid, and the alkali may be a common one such as caustic soda or caustic potash.

In many cases, the concentration may be about 0.05 to 2N.

The carrier is a quaternary ammonium salt having a plurality of long-chain alkyl groups or an aromatic sulfonic acid and its salt, and has a low ability to form micelles.

R: Alkyl group of C, ~C18 R1: Methyl or benzyl These include aromatic hydrocarbons such as benzene, toluene, and xylene, chlorinated hydrocarbons such as ethylene dichloride and chloroform, aliphatic hydrocarbons such as hexane, and phosphoric acid esters such as tributyl phosphate.

The concentration of the carrier may be about 0.05 to 1.0M.

An acid or alkali is used for the receptor aqueous solution.

As the acid, hydrochloric acid, sulfuric acid, etc. of about 0.05 to 2N may be used, and as the alkali, caustic soda or caustic potash of about 0.05 to 2N may be used.

Separation of nucleic acid-related substances or vitamins is performed as follows.

First, the device has a tank 1 containing an aqueous solution containing a nucleic acid-related substance or vitamin and a carrier, and a tank 2 containing an aqueous receptor solution and a carrier, and is designed to be able to communicate with the carrier solution in both tanks. Use the one that is .

Schematic diagrams of several examples of devices are shown in FIGS. 1-4.

In the figure, A represents an aqueous solution containing a nucleic acid-related substance or vitamin, B represents a carrier solution, and C represents an aqueous receptor solution.

It is preferable to circulate each liquid in order to increase the separation rate.

The separation time varies depending on the contact area, temperature, liquid circulation rate, etc., but most nucleic acid-related substances and vitamins can usually be separated in several hours to several tens of hours.

In order to obtain the target substance from the nucleic acid-related substance/vitamin separation liquid (hereinafter referred to as separated liquid) obtained by transferring the nucleic acid-related substance/vitamin to the receptor aqueous solution, the separated liquid must be pH adjusted, concentrated, cooled, and decolorized. This may be done by appropriately combining known means such as the following.

When recovering from waste liquid, the waste liquid usually contains a wide variety of nucleic acid-related substances and vitamins, but it is sometimes possible to separate and recover individual substances by taking advantage of differences in movement speed.

Further, known separation means may be applied to the separated liquid.

It goes without saying that when a target substance is produced by an enzymatic reaction, a synthetic reaction, etc., unreacted substances and intermediate reactants can be recycled to each process.

In the method of the present invention, the migration speed varies considerably depending on the type of nucleic acid-related substance.

In general, the movement speed of purines and pyrimidines is extremely fast;
This is followed by nucleotides, whose migration speed is relatively slow.

Therefore, their mutual separation is possible.

1N Na so that each component is about 3 mmol/dl
Three mixed solutions obtained by dissolving in OH, namely a mixed solution of guanine, guanosine and 51-guanylic acid,
The results of measuring the migration speed of a mixed solution of hypoxanthine, inosine, and 51-inosinic acid, and a mixed solution of cytosine, cytidine, and 51-cytidylic acid are shown in FIGS. 5 to 7.

The volume of the mixed solution used was 750 ml, 750 ru4 of 1N hydrochloric acid for the receptor aqueous solution and 0.0 ml of 1N hydrochloric acid for the carrier solution.
．． 1 M trinormal octylmethylammonium chloride toluene solution 211 was used.

The apparatus is shown in Figure 2, and the column size is 5 x 10.
It is 0crn.

FIG. 5 shows a mixed solution of guanine, etc., FIG. 6 shows a mixed solution of hypoxanthine, etc., and FIG. 7 uses a mixed solution of cytosine, etc.

In each figure, the vertical axis shows the concentration of each component in the mixed solution and receptor aqueous solution, and the horizontal axis shows the circulation time.

Next, the results of using 1N hydrochloric acid as the solvent of the mixed solution and 1N NaOH as the receptor aqueous solution are shown in Figures 8 and 9.
As shown in the figure.

The mixed solution shown in FIG. 8 contains guanine, guanosine, and 51-guanylic acid, each containing about 2 mmol/dl, and the mixed solution shown in FIG.
Each component contains 3 mmol/cU of 1-inosinic acid.

The carrier solution was a 0.1 M sodium dinonylnaphthalene sulfonate toluene solution, and the other details were the same as those shown in FIGS. 5 to 7.

The mutual separation of nucleic acid-related substances due to the difference in migration speed is particularly useful as a means of separating and recovering unreacted guanosine during the process of producing 51-inosinic acid by phosphorylating guanosine. , therefore 51-
It is effective as a means for separating and recovering unreacted inosine during the process of producing inosinic acid.

Example 1 40 g of sodium hydroxide was added to liquid 11 (containing 30 g/l inosine) obtained by removing bacterial cells by centrifugation from the inosine fermentation liquid obtained by culturing Bacillus subtilis having the ability to produce inosine, and making it into an inosine stock solution. did.

A 0.1 M) toluene solution 21 of trin-octylmethylammonium chloride as a carrier solution and an IN hydrochloric acid aqueous solution 11 as an aqueous receptor solution were prepared and filled into the apparatus shown in FIG. 2.

That is, inosine stock solution was used as A, IN hydrochloric acid was used as C, and 0.1M tri-normal octylmethylammonium toluene solution was used as B.

Thereafter, the toluene layer was contacted with and circulated in the order of inosine stock solution and IN hydrochloric acid using a pump.

The flow rate of the pump is 100ml! / min. 1
After 5 hours, the inosine concentration at C was 27. Of! /l.

As a result, inosine was separated from the inosine stock solution at a yield of 90%.

The obtained separated liquid was concentrated, neutralized and cooled, and methanol was further added to obtain 22 g of inosine crystals (purity 99.5%).

Example 2 An inosine fermentation solution obtained by culturing Bacillus subtilis having the ability to produce inosine was centrifuged to remove bacterial cells, and 85 r/l of concentrated hydrochloric acid was added to solution 11 to obtain an inosine stock solution.

0.1 M of sodium dinonylnaphthalene sulfonate
Toluene solution 21 and IN sodium hydroxide solution 1
1 was prepared.

As in Example 1, the apparatus shown in FIG. 2 was used, and the inosine stock solution was used as A, the IN sodium hydroxide solution as C1, and the toluene solution as B.

The toluene solution was circulated by a pump at a rate of 100 TLl/min.

After 15 hours, the inosine concentration in C was 21 i/l.

Therefore, inosine could be separated from the inosine stock solution at a yield of 70%.

Example 3 The orotic acid fermentation liquid obtained by culturing Corynebacterium glutamicum having the ability to produce orotic acid was sterilized by centrifugation, and the sterilizing liquid (orotic acid 5 g/it') 111
40 g of sodium hydroxide was added to prepare an orotic acid stock solution.

In addition, 0.1 M l-luene solution 21 of tri-n-octylmethylammonium chloride and IN hydrochloric acid 11 were prepared.

These were filled into the apparatus shown in FIG. 2 in the same manner as in Example 1, and
A pump circulated the toluene solution.

After 12 hours, the total amount of orotic acid in C was 4.6 g.

This means that 92 orotic acids in the orotic acid stock solution were separated.

Example 4 Bacillus megaterium having the ability to produce 5-amino-4-imidazolecarboxamide riboside (AICA r ) was cultivated to produce AICA-R fermentation broth (AICA-r
2.2 g/rU) was obtained.

Next, Bacillus thiaminolyticus having the ability to produce AICA-r nucleosidase was cultured to obtain a fermentation liquid having AICA r nucleosidase activity.

For 10 parts (volume) of AICA-R fermentation liquid,
Mix 1 part of RIJ posidase fermentation liquid and incubate at 45℃ for 48 hours.
Allowed time to react.

This reaction solution was sterilized by centrifugation. Solution 11 (5-amino-4-imidazolecarboxamide (AICA) 10
g//l) and add 4.0 g of sodium hydroxide to the AI
It was used as a CA stock solution.

0,1 M-) linonemaroctylmethylammonium chloride toluene solution 21 and 0. IN hydrochloric acid 11 was charged into the apparatus shown in FIG. 2 in the same manner as in Example 1.

The toluene solution was circulated using a pump. After 16 hours, the AICA concentration of C was 9.0 g/11.

The separation yield was 90%.

The obtained AICA separated liquid was concentrated, neutralized, and cooled to a purity of 99.
7.9 g of AICA crystals were obtained.

AICA-r in the AICA stock solution was present at a ratio of 12% to AICA, or AICA-r in the obtained AICA separated solution was significantly reduced to 0.5% relative to AICA.

Example 5 Streptomyces hygroscopicus having the ability to produce angestomycin A and C was cultured to obtain a fermentation solution containing angestomycin A and C.

Liquid sterilized by filtration 11 (Angstomycin A;
112 m9/l, same C; 89 mp/A) was added with 20 g of sodium hydroxide to prepare an angestomycin stock solution.

Using Example 1 as the internal unit, and adding 2A of 0.1M toluene solution of normal octylmethylammonium chloride to the apparatus shown in Figure 2! , and IN hydrochloric acid 11, and the toluene solution was circulated using a pump.

After 16 hours, the concentration of angestomycin at C is angestomycin AA1101F? /l C77m9/11, and the separation yields were 98 and 85, respectively.

When 500 d of the obtained separated liquid was concentrated to dryness, 240 mfl of a solid product was obtained, which contained angestomycin A and C.
It contained 78% of

No inorganic substances were included.

Example 6 Inosine was phosphorylated using phosphorous oxychloride in trimethyl phosphate to give 51-inosinic acid.

Water was added to hydrolyze excess phosphorus oxychloride, and trimethyl phosphate was removed to prepare a 51-inosinic acid aqueous solution.

The 51-inosinic acid concentration of this solution is 10.1/V hydrochloric acid concentration IN.

Aqueous solution IA! Sodium 51-inosinate was separated in the same manner as in Example 1 using the apparatus shown in FIG. 2 using a 0.1 M sodium dinonylnafucrene sulfonate toluene solution 21 and an IN sodium hydroxide solution 11.

Eleven hours after starting the pump, the concentration of 51-inosinic acid separated in the sodium hydroxide solution was 9.6 g/l.

After concentrating this separated liquid, it was neutralized and 8.3 g of 51-inosinic acid 7 was added.
．． 5H20 crystals were obtained.

The purity was 99.

Example 7 Phosphorylation was performed using guanosine in exactly the same manner as in Example 6 to prepare a 51-guanylic acid aqueous solution.

The 51-guanylic acid concentration was 10.09/L hydrochloric acid concentration IN.

Guanylic acid was separated and recovered from this solution in exactly the same manner as in Example 6.

The separation yield was 92.7%.

Example 8 Adenosine 23g, sucrose 59g, dry brewer's yeast 150g
Dissolve g in 0.1M phosphate buffer (pH 7,0) and add 73
1 and phosphorylated adenosine at 37°C.

Insoluble matter in the reaction solution was removed by centrifugation, and a solution 17 (containing 11# of adenosine triphosphate (ATP)) was taken, and sodium hydroxide was added to adjust the pH to 13, a stock solution of 0.1M toI.
J normal octyl methyl ammonium chloride toluene solution 21H6 and 0. Separation operation was carried out in the same manner as in Example 1 using 11 N hydrochloric acid.

After 21 hours, the ATP concentration in the hydrochloric acid solution was 9.2 ji/l.

The separation yield was 92%.

This separated liquid was concentrated to dryness to obtain ATP powder with a purity of about 94.

Example 9 1 kg of pressed yeast (alcoholic yeast, moisture 66%) was suspended in water to give a suspension of 21, and after being kept at 85° C. for 10 minutes, insoluble materials were removed by centrifugation.

Take the supernatant 11 (containing NAD O, 5g/71) and add sodium hydroxide to give 0. 0.1M Torino V? in the apparatus shown in Fig. 2. ) Kanari methyl methyl ammonium chloride toluene solution 21 and 0. Charged as in Example 1 with 11 IN hydrochloric acid.

The toluene solution was circulated using a pump, and after 24 hours, the hydrochloric acid solution was analyzed by an enzymatic method.
It was 69/13.

The solid content was 72% pure (the solid content purity of the centrifuged supernatant was 0.2%).

NAD concentration *Solid content purity -, X100 Total solids concentration

[Brief explanation of drawings]

1 to 4 are diagrams showing an outline of an example of an apparatus used in the present invention. Figures 5 to 9 are diagrams showing the relationship between circulation time and concentration changes for various nucleic acid-related substances.

Claims (1)

[Claims] 1. When using a long-chain quaternary ammonium salt, contact a water-insoluble organic solvent solution of a long-chain quaternary ammonium salt or a long-chain alkyl aromatic sulfone hydrochloric acid with an aqueous solution containing a nucleic acid-related substance or vitamin and an aqueous receptor solution. , the aqueous solution containing the nucleic acid-related substance or vitamin is made alkaline and an acid is used for the receptor aqueous solution, and when a long-chain alkyl aromatic sulfonate is used, the aqueous solution containing the nucleic acid-related substance or vitamin is made acidic. Using an alkali in the receptor aqueous solution, the nucleic acid-related substance or vitamin is transferred from the aqueous solution containing the nucleic acid-related substance or vitamin to the receptor aqueous solution by contact, and the nucleic acid-related substance or vitamin is removed from the obtained nucleic acid-related substance or vitamin separated liquid. Or a method for separating and recovering nucleic acid-related substances and vitamins, which comprises separating and recovering vitamins.