DE2048659B - Obtaining deoxyribonucleic acid and ribonucleic acid - Google Patents

Description

Ribonuclease treatment and similar phenol treatment. 0.7 g of purified deoxyribonucleic acid was obtained. It was found that the molecular weight of this preparation was greater than 5 · 10 ⁴ when it was obtained by means of the gradient sucrose centrifugation density method using deoxyribonucleic acid from "phages" as the standard (modified method by J. Marmur: J. Mol. Biol. 3, pp. 208, 1961).

After removing the fibrous precipitates, the fermentation liquor became 1 equivalent 95% ethanol was added and the resulting precipitates were collected and combined to give the crude ribonucleic acid. These became the deoxyribonucleic acid treatment and the phenol treatment subject. 2.0 g of purified ribonucleic acid were obtained. To obtain soluble ribonucleic acid, this sodium chloride (1 mol) was added and stirred. The supernatant was collected and it twice its volume of 95% ethanol was added. The resulting precipitation were dissolved in sodium chloride (0.1 mol) and further precipitated with isopropanol. 0.1 g was obtained soluble ri'oonucleic acid.

B e i s ρ i e 1 2

Micrococcus ureae ATCC 21288 was grown in a medium containing meat extract (1%), peptone (1%), NaCl (0.5%) and sorbitol (2%) and an adjusted pH of 7.2 (before sterilization The cultivation was carried out with shaking to obtain an inoculum culture within 24 hours. The inoculated culture was inoculated on a medium (3.01) containing meat extract (1%), peptone (1%), NaCl (0.5%). ) and sorbitol (10%) and had an adjusted pH of 7.2 (before sterilization). A 5-1 fermentation bottle with a volume ratio of 10% was used. Cultivation was carried out with stirring (400 rpm) and aeration ( 1 l / min.) was performed in 48 hours at 3O ⁰ C. The medium was adjusted with ammonia water to a pH of 6.5 to 7.5.

The fermentation liquor was treated similarly to the manner described in Example 1, and one obtained 0.5 g of purified deoxyribonucleic acid and 0.5 g of crude ribonucleic acid from 11 fermentation solution.

Example 3

Pseudomonas fluorescens ATCC 948 was grown in an inoculation medium similar to that described in Example 2 for 24 hours with shaking. A medium (3.01) containing KH ₃ PO ₄ (0.2%),
Na ₈ HPO ₄ (0.2%),
MgSO ₄ -7 H ₂ O (0.1%),
MnSO ₄ -4H ₂ O (0.002%),
FeSO ₄ -7 H.0 (0.2%),
ZnSO ₄ - 7 H, O (0.001%),
(NHJ ₂ SO ₄ (0.5%),
CuCl ₂ -4H ₂ O (0.0003%),

Corn steeple (0.1%), yeast extract (O ₁ I ¹ V ₁ "/ and η-paraffin (a mixture of C ₁₂ , Q ₃ , C ₁₄ and C ₁₅ ), -10% -v / v -containing, in a 5-1 fermentation bottle at a volume ratio of 10%.

The cultivation was similar to the manner described in Examples 1 and 2 taking 72 hours at 30 ° C Stirring (600 rpm) and aeration (3 l / min.) Carried out. The medium was made up with ammonia water adjusted to a pH of 7.0 to 7.5.

When the cultivation was completed, the η-paraffin was completely consumed. The microorganism cells were removed from the fermentation medium and the fermentation liquor became similar the manner described in Example 1 treated. 1.0 g was obtained from 11 fermentation liquors purified deoxyribonucleic acid and 1.8 g of crude ribonucleic acid.

Example 4

Aspergillus orycae ATCC 7252 was similar to that described in Example 3 at 25 ° C for 96 hours Wise grown using an inoculum similar to that described in Example 3 and one similar medium for fermentation.

The fermentation liquor was treated similarly to that described in Example 1 and yielded 0.4 g of purified deoxyribonucleic acid and 0.7 g of crude ribonucleic acid.

Example 5

Candida lipolitica ATCC 8861 was prepared in a manner similar to that described in Example 3 and using a seed culture similar to that described in Example 3 was grown.

The medium for the Fermantation was prepared similarly to the manner described in Example 3, a commercial nonionic surfactant was added.

The cultivation was carried out for 96 hours at 3O ⁰ C; the fermentation liquid obtained was treated similarly to the manner described in Example 1. From 1 liter of fermentation liquid, 0.2 g of a purified deoxyribonucleic acid and 0.7 g of crude ribonucleic acid were obtained.

Claims (1)

1 2 acid from the fermentation solutions mentioned is patent claim: commercially feasible. For the production of fermentation liquids By cultivating Arthro- a nutrient medium is used which contains a bacter paraffineus ATCC15590, Arthrobacter S hydrocarbon or a carbohydrate as the main simplex ATCC 15799, Arthrobacter hydrocarbo- carbon source and a nitrogen source, glutamicus ATCC15583, Brevibacterium keto- The hydrocarbon can be n-paraffin or hydrocarbon fractions containing n-Parafglutamicum ATCC15587, Micrococcus ureae fin are used-ATCC 21288, Corynebacterium hydrocarboclastus. Preferably «-. "en the n-Paraf fine IO to ATCC 15 592, Pseudomonas fluorescens ATCC94 $ IO 22, in particular 12 to 18 carbon atoms contain. Pseudomonas aeruginosa ATCC 15246, Ai-pcr- but also possible, other carbon sources eingillus orycae ATCC 7252 or Candida lipolitica, like any type of hydrocarbon-containing fermentation liquids that can be assimilated by the microorganism used in each case in the nutrient media used for this purpose, such as glucose, sorbitol, deoxyribonucleic acid and robin-15 and the like. The like, organic acids or alcohols, preferably small acids, C ₂ to C ₂₀ acids or alcohols and the like Nitrogen sources can use both inorganic and organic nitrogen compounds will. Other inorganic salts and growth ao substances are added to the nutrient medium. The nutrient medium is sterilized and then inoculated with one of the microorganisms mentioned. Culturing under aerobic conditions generally at 20 to 60 ⁰ C, preferably at 30 to 42 ° C. During the The invention deals with the production of 25 cultivation is to adjust to a pH of 4 to of deoxyribonucleic acid and ribonucleic acid. 10, preferably 6 to 9, urea solution, ammonia Deoxyribonucleic acid and ribonucleic acid are water, ammonia or ammonium carbonate solution so far supplied by extraction from cells of microorganisms. The fermentation is generally over after men - - both of higher animals and of about 7 days. It is preferable, however, to preserve the higher plants, which continue to ferment in various ways until the destiny has been destroyed in various ways. However, the above-mentioned nucleic acids indicate a maximum
the extraction and recovery processes of these. In the following, the invention is illustrated with reference to FIG known methods explained in more detail both as a result of the considerable examples:
Loss of these nucleic acids as well as due to the Presence of the residues of the destroyed cells 35 B e i s ρ i e 1 1 more or less difficult. The invention is based on the object to Arthrobacter simplex ATCC 15799 was in one give how deoxyribonucleic acid and ribonuclein medium cultured, which meat extract (1 ° / ₀ ), acidic more economical and easier to obtain, peptone (1 ° / ₀ ), NaCl (0.5%) and glucose (2%) contained. 4 ° and a set pH value of 7.2 (before the The invention consists in the use of sterilization). The cultivation was carried out with shaking in order to obtain Arthrobacter paraffineus ATCC 15590, an inoculum culture, in 24 hours. Arthrobacter simplex ATCC15799, Arthrobacter The inoculum was then used to inoculate a hydrocarboglutamicus ATCC 15583, Brevibacterium medium (3.0 1), the fish extract (l ° / ₀ ), ketoglutamicum ATCC 15587, Micrococcus ureae 45 peptone (1%), NaCl (0.5 ° / “) and glucose (10 ⁰ O) ent-ATCC 21288, Corynebacterium hydrocarboclastus hicit and an adjusted pH of 7.2 (before the Sterilization-ATCC 15592, Pseudomonas fluorescens ATCC 948, tion). A 5-1 fermentation bottle and a Pseudomonas aeruginosa ATCC 15246, Aspergillus volume ratio of 10 ° / _{0 were} used. The Züchorycae ATCC 7252 or Candida lipolitica ATCC 8861 .ung was dissolved in 30 hours at 30 ⁰ C with stirring in customary for this purpose nutrient media containing FER 50 (400 rpm) and aeration carried out (1 l / min.),
mentation fluids for the production of deoxy · The medium was diluted with ammonia water ribonucleic acid and ribonucleic acid. - The above- pH adjusted from 6.5 to 7.5. After the abovementioned microorganisms have ceased to exist in the »American breed, the glucose was almost used up.
Type Culture Collection «and for the public accessibility. SS removal of the microorganism cells from the fermentation The advantages achieved by the invention were obtained in the fact that the deoxyribonucleic acid and 500 ml of phenol were added, which was obtained from the fermentation chloride (0.15 mol) and sodium citrate (0.15 mol) by means of a sodium ribonucleic acid. 01 mol) was saturated after removal of the solution containing the microorganism cells. The treated solution was very easily isolated using known methods. It was shaken and centrifuged. After that it was able to. In contrast to the well-known cases, the accumulating upper layer is collected. After two drives, no cells are destroyed. In addition, repetitions were the top layers can be the FermentationsflUssigkeiten said united, to obtain an aqueous solution prepared also cheap, since strength for the growth of the overall which 1 equivalent 9S ° / _e ethanol (or Menannten microorganisms inexpensive hydrocarbon '65 ethanol) was added. substances or carbohydrates as raw materials for the fibrous precipitates obtained were Nutrient medium can be used. The profits won and yielded crude deoxyribonucleic acids. tion of deoxyribonucleic acid and ribonuclein- These have been purified by treating them twice