FI85385B - FOERFARANDE FOER PRODUCERING AV RIBONUCLEINSYRA. - Google Patents

Description

1 85385

This invention relates to a method for producing ribonucleic acid (hereinafter referred to as RNA). More particularly, it relates to a method for isolating RNA from yeast grown in a medium such as molasses.

To produce RNA, a method is known in which RNA is extracted from yeast with hot saline, a solution containing the extracted RNA is treated with acid, and then RNA is isolated from the solution by sedimentation. Such a solution containing extracted RNA contains colloidal or suspended insoluble substances derived from yeast impurities. When RNA is sedimented by the addition of acid, insoluble matter sediments together with the RNA. Thus, insoluble substances contaminating the resulting RNA reduce its purity. To remove insoluble matter from a prepared solution to obtain RNA of high purity, it is proposed in JP 56-46837 to produce RNA by adding polyacrylate or similar protein coagulants to the RNA-containing solution, removing the insoluble matter from the solution by coagulating, then adding the acid to the clear solution thus obtained to acidify it, and then isolating the RNA from the solution by sedimentation.

On the other hand, in order to purify microbial products from solution, a method has been commonly used in which insoluble matter, which is also present in solution, is decomposed by adding protease to the solution, as a result of which 2,85,385 insoluble matter is removed therefrom. This method can also be applied to a solution of yeast-extracted RNA. Such a method is known, for example, from FI patent publication 53590, which describes a method in which biomass is treated with alkali, insoluble matter is separated after neutralization, proteins in solution are hydrolysed with a protease preparation to soluble hydrolysis products, then RNA is mixed with acid and finally the precipitated RNA-containing product is separated.

When RNA is extracted with hot saline from yeast grown by assimilating sugar in lignosulfonate-containing sulfite waste liquor, insolubles in the extracted RNA-containing solution can be removed relatively efficiently by adding 5 to 100 ppm of coagulant, such as sodium polyacrylate, to the solution. or by adding a protease to the solution to degrade insoluble substances.

However, with respect to a solution containing RNA extracted by heating saline from yeast grown in a broth that does not contain lignosulfonate, such as molasses, the insoluble substances therein cannot sufficiently remove the protease or coagulant, such as e.g. sodium polyacrylate, by adding. Thus, there is a problem that the RNA yield is significantly reduced compared to the case where RNA is isolated from a solution of RNA extracted from yeast separated from sulfite digestion broth.

3 85385 The main object of the present invention is therefore to provide an improved method for producing RNA in high yield.

According to the present invention, there is provided a method of producing ribonucleic acid, the method comprising the steps of a) adding 50-1000 ppm protease to a solution of ribonucleic acid extracted from yeast in heated saline and maintaining the temperature of the resulting solution at 30-80 ° C for 1-8 hours; b) adjusting the temperature of the solution containing ribonucleic acid extracted from yeast to between 50-90 ° C, adding a coagulant comprising 5-100 ppm of sodium polyacrylate and 40-500 ppm of a water-soluble metal salt selected from the group consisting of ferric salts and aluminum salts to remove insoluble matter of said solution by coagulating and sedimenting, and then adding acid to said solution clarified by removing insoluble matter to acidify the solution, and then isolating the resulting solution by sedimenting ribonucleic acid. The process according to the invention is characterized in that the steps described in a) above are carried out first and then the ribonucleic acid-containing solution obtained in a) is subjected to the steps described in b) above to obtain very pure ribonucleic acid in good yield.

The present invention preferably uses yeast cultured in a lignosulfate-free broth. The genus and species of yeast are not limited, although it is more preferable to use yeast with high RNA production capacity.

4 85385 RNA is extracted from yeast with heated saline. More specifically, the salt is added to the medium after the growth of the yeast, or the saline is added to the yeast separated from the medium. The solution is preferably prepared so that the dry matter of the yeast is between 7 and 14% by weight, the salt content is between 4 and 7% by weight, and the pH is between 7 and 8. The solution is then heated to 90-99 ° C, more preferably between 95-99 ° C for three to six hours. This results in the extraction of RNA from the yeast into the solution phase. After cooling, the resulting RNA solution is treated with protease.

According to the present invention, a protease is used which is active under less than neutral or slightly basic conditions. It is in accordance with this invention to use proteases such as those produced by trypsin, papain or the genus Bacillus, the genus Streptomyces, the genus Aspergillus and the genus Streptococcus. More preferably, the amount of protease added to the solution is between 50 and 1000 ppm.

The solution is treated with a protease at a starting pH of 4-8, at a temperature of 30-80 ° C, more preferably 40-75 ° C for one to eight hours. When the protease treatment is complete, the temperature of the solution is adjusted to between 50 and 90 ° C. Sodium polyacrylate and a water-soluble metal salt selected from the group consisting of a ferric salt such as ferric chloride and ferric sulfate and an aluminum salt such as aluminum sulfate are then added to the solution. The insoluble substances in the solution coagulate and thereby sediment.

According to the present invention, the sodium polyacrylate has a molecular weight of at least 5,000,000, more preferably at least 10,000,000. The effectiveness of the polyacrylate as a coagulant is worse if it has a molecular weight of less than 3,000,000. It is preferable to add polyacrylate and the above metal salt to the solution in amounts ranging from 5 -100 ppm and 40-500 ppm, respectively. When adding a water-soluble metal salt together with sodium poly- • V; with acrylate, the insoluble matter in solution coagulates more efficiently. However, if water soluble

II

5 85385 metal salts are added in excess, some of the RNA is lost as it co-sediments with insoluble substances. Although coagulation occurs significantly at room temperature, maintaining the temperature of the solution between 50-90 ° C favorably promotes the growth of aggregates.

As described above, coagulated and then sedimented insolubles are removed from the solution by centrifugation, filtration, or natural sedimentation, where the solution is allowed to stand at 50-90 ° C for one to four hours. In this way, the solution containing the extracted RNA is clarified.

The resulting clear solution is then cooled to 3-15 ° C, after which its pH is adjusted to about 1.5 by the addition of hydrochloric acid, causing RNA to precipitate.

This precipitate is collected and dissolved by neutralization with sodium hydroxide. The solution thus obtained is dried in a spray dryer or similar dryers to obtain RNA of high purity and high yield.

It can be considered that RNA of high purity can be obtained continuously in high yield according to the present invention on the following basis.

A portion of the RNA extracted with heated saline from yeast grown in a medium free of lignosulfonate forms a complex with the protein. This complex is characterized by the fact that it does not precipitate with the addition of acid. When hydrochloric acid is added to the RNA-containing solution, the RNA complexed with the protein does not precipitate. This may be the reason for the decrease in RNA yield when delivered according to conventional methods.

According to the present invention, in contrast, most of the RNA-protein complex described above is first separated into RNA and degraded protein by treating the solution with a protease. Thus, when a coagulant consisting of 6,85,385 sodium polyacrylates and a water-soluble metal salt is added to the solution in the next step, insoluble substances such as solution protein are effectively removed from the solution through coagulation and sedimentation without RNA losses. Therefore, by adding hydrochloric acid to the clear solution thus obtained to acidify it, both the RNA which was originally free and the RNA released from the complex can be recovered without loss. Therefore, according to the present invention, high purity RNA of high purity can be obtained continuously.

Also, solutions containing RNA isolated from yeast grown on the assimilation of sugar in sulfite waste liquor containing lignosulfonate can in some cases be obtained in purer and better yield than using RNA in combination with a coagulant. However, the effect in this case is not as significant as in a solution with RNA extracted from yeast grown in a broth without lignosulfonate.

The following examples illustrate the details of this invention.

Example 1

Saccharomyces cerevisiae, cultured in molasses, was separated by centrifugation and washed to give 1 kg of yeast cake. This cake was suspended in 3 L of water, and 150 g of sodium chloride was added for extraction. The solution thus obtained was heated at 95 ° C for four hours, during which time RNA was extracted. Next, the yeast was removed from the solution by centrifugation. The separated yeast was washed with 800 ml of water. Addition of the yeast wash water to the solution from which the yeast was separated gave 3.8 L of a solution of extracted RNA.

This solution was divided into two equal portions.

The second part of the divided solutions described above,

II

7 85385 was 1.9 L, the pH was adjusted to 6 with sodium hydroxide. Papain (protease) was then added to the solution at about 300 ppm, after which the solution was kept stirred at 40 ° C for six hours. Then 200 ppm ferric chloride was added to the solution heated to 70 ° C. The solution was stirred and an additional 40 ppm of sodium polyacrylate was added, as a result of which the insolubles in the solution coagulated and sedimented. After the coagulated slurry was removed from the solution by filtration, and the resulting clear RNA-containing solution was cooled to 5 ° C, the pH of the clear solution was adjusted to 1.5 with hydrochloric acid. This was followed by precipitation of RNA.

After the precipitated RNA was recovered by centrifugation, the recovered RNA was suspended in water and dissolved by adjusting the pH of the suspended solution to 5.6 with sodium hydroxide. The solution was then dried by spray drying to give 12 g of RNA. The yield of RNA obtained from the solution was 96% and its purity was 86.5%.

Comparative Example 1 Using a second portion of the 1.9 L portions prepared in Example 1, the procedure of Example 1 was repeated except that no papain was added to the solution to give 10.5 g of RNA. The yield of RNA obtained from the solution was 84% and its purity was 76%.

Example 2

Candida utilis, cultured with glucose, was separated by centrifugation and washed to obtain a 2 kg yeast cake.

This cake was suspended in 6 L of water, and 300 g of sodium chloride was added for extraction. The solution thus obtained was heated at 95 ° C for four hours, during which time the RNA was extracted. The yeast was then removed; from the solution by centrifugation. The separated yeast was washed with 1.6 L of water. Addition of the yeast wash water to the solution from which the yeast was separated gave 7.6 L of a solution of extracted RNA. This solution was divided into two equal portions.

s 85385

The pH of the second portion of the 3.8 L divided solutions described above was adjusted to 6 with sodium hydroxide. 300 ppm of Daiwa Kasei Co. was then added to the solution.

Ltd. supplied PROTIN PC-10 (protease) and then the solution was kept stirred at 75 ° C for six hours. Next, 150 ppm ferric chloride was added to the solution. The solution was stirred and an additional 30 ppm of sodium polyacrylate was added, as a result of which the insolubles in the solution coagulated and sedimented. After the coagulated slurry was removed from the solution by filtration, and the resulting clear RNA-containing solution was cooled to 5 ° C, the pH of the clear solution was adjusted to 1.5 with hydrochloric acid. This was followed by precipitation of RNA.

After the precipitated RNA was recovered by centrifugation, the recovered RNA was suspended in water and dissolved by adjusting the pH of the suspended solution to 5.6 with sodium hydroxide. The solution was then dried by spray drying to give 24.1 g of RNA. The yield of RNA obtained from the solution was 96.4% and its purity was 87%.

Comparative Example 2 Using a second portion of 3.8 L of the solutions prepared in Example 2, the procedure of Example 2 was repeated except that the solution was cooled to 5 ° C without the addition of ferric chloride and sodium polyacrylate to give 22.4 g of RNA. added. The yield of RNA obtained from the solution was 89.6% and its purity was 78%.

As described above, in accordance with the present invention, insoluble materials can be effectively removed by adding - to the solution alternately first a protease and then a coagulant with sodium polyacrylate, etc., under these special conditions, where high purity RNA can be continuously produced in high yield without losses.

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Claims (1)

A method for producing ribonucleic acid, the method comprising the steps of a) adding 50-1000 ppm protease to a solution of ribonucleic acid extracted from yeast in heated saline and maintaining the temperature of the resulting solution between 30-80 ° C for 1-8 hours, b) adjusting the temperature of the solution containing the ribonucleic acid extracted from the yeast to between 50-90 ° C, adding a coagulant comprising 5-100 ppm sodium polyacrylate and 40-500 ppm water-soluble metal salt selected from the group consisting of ferric salts and aluminum salts to remove the insoluble matter of said solution by acidification, and then adding acid to said solution, which had been clarified by removing insoluble matter, to acidify the solution, and then isolating the ribonucleic acid from the resulting solution by sedimentation, characterized in that the above The steps in (a) and then the ribonucleic acid solution obtained in (a) are followed by the steps in (b) above to give very pure ribonucleic acid in good yield. For the production of ribonucleic acid, the fineness of the ribonucleic acid is determined by: (a) a volume of 50-1000 ppm of the proteas and the addition of ribonucleic acid in the form of an additional salt from the salt and oceanic mixture . at a temperature between 30 and 80 ° C under a temperature range of 1 to 8 times, (b) the temperature between the temperature and the range of 50 to 90 ° C, with the addition of 5 to 100 ppm of sodium polyacrylates -500 ppm of wattage-free metal uptake by the Group of ferrous salt 10 8 5385 and aluminum salt for the recovery of the present material from the material genome coagulation and sediment ring, and with the addition of a mixture of water an analysis of the presence of material, in which case the analysis is carried out, and the isolation of the ribonucleic acid is isolated from the genome sedimentation ring, the results of which are shown in for Stegen under punkt b) ovan, varvid man erhäller ribonucleinsyra med hög renhet i hö gt utbyte. li