DE2048659C - Obtaining deoxyribonucleic acid and ribonucleic acid - Google Patents

Description

known methods explained in more detail both as a result of the considerable examples:
Loss of these nucleic acids as well as a result of the

Ar essence 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 ribonucleic medium grown, which meat extract (1 ° / ₀ ), acidic more economical and easier to obtain, peptone (1 ° / ₀ ), NaCl (0.5%) and glucose (2%) contained. 40 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 (3.01), the meat extract (l ° / ₀ ), ketoglutamicum ATCC 15587, Micrococcus ureae 45 peptone (l ° / ₀ ) , NaCl (0.5 ° / ₀ ) and glucose (10%) ent-ATCC 21288, Corynebacterium hydrocarboclastus and had an adjusted pH of 7.2 (before the Sterilization-ATCC 15592, Pseudomonas fluorescens ATCC 948, tion) . A 5 liter bottle and a Pseudomonas aeruginosa ATCC 15246, Aspergillus volume ratio of 10 ° / _{0 were} used. The Züchorycae ATCC 7252 or Candida lipolitica ATCC 8861 tung 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 liquids 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 to 11 of the fermentation liquid, which is

accessibility. 55 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 by sodium ribonucleic acid from the fermentation chloride (0.15 mol) and sodium citrate (0.15 mol) mentioned. 01 mol) was saturated after removing the microorganism cell holding solution. The treated solution was very easily isolated using known methods and was shaken and centrifuged. After that it was able to. In contrast to the known methods, the accumulating upper layer is collected. After two drives, no cells are destroyed. In addition, repetitions were the top layers can be the fermentation liquids mentioned combined to give an aqueous solution, also manufactured at low cost, since low cost for culturing the overall wherein 1 equivalent of 95 ° / _o sodium ethanol (or Menannten microorganisms hydrocarbons 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 ribonucleic acid- These have been purified by treating them twice

Ribonuclease treatment and similar phenol treatment. 0.7 g of purified was obtained Deoxyribonucleic acid. It has been found that the molecular weight of this preparation is greater than 5 x 10 * was when it was using the gradient sucrose centrifugation density method using deoxyribonucleic acid from "phages" as a standard (modified method by J. M a r m u r: J. MoL Biol. 3, pp. 208, 1961).

After removing the fibrous precipitates, the fermentation liquor became 1 equivalent 95% ethanol added, and the resulting precipitates were collected and combined to give the crude ribonucleic acid. This became the deoxyribonucleic acid treatment and subjected to phenol treatment. It fell 2.0 g of purified ribonucleic acid on. Sodium chloride (1 mol) was added to this to obtain soluble ribonucleic acid and touched. The supernatant was collected and twice its volume of ao 95% ethanol added. The resulting precipitates were dissolved in sodium chloride (0.1 mol) and further precipitated with isopropanol. 0.1 g of soluble ribonucleic acid was obtained.

»5 Example 2

Micrococcus vreae ATCC 21288 was grown in a medium containing meat extract (1%), peptone (1%), NaCl (0.5%) and sorbitol (2%) and had an adjusted pH of 7.2 (before sterilization). Cultivation was carried out with shaking within 24 hours to obtain an inoculum. The inoculum was inoculated on a medium (3.01) which contained meat extract (1%), peptone (1%), NaCl (0.5%) and sorbitol (10%) and an adjusted pH of 7.2 (pre sterilization). A 5-1 fermentation bottle with a volume ratio of 10% was used. The cultivation was carried out with stirring (400 rpm) and aeration (1 l / min.) At 30 ^° C. for 48 hours. The medium was adjusted to pH 6.5 to 7.5 with ammonia water.

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 ₈ PO ₄ (0.2%),
Na, HPO ₄ (0.2%),
MgSO ₄ -7H ₄ O (0.1%),
MnSO ₄ -4H ₄ O (0.002%),
FeSO ₄ -7H ₂ O (0.2%),
ZnSO ₄ -7 H ₄ O (0.001%),
(NHJ ₄ SO ₄ (0.5%) _v
CuCl ₁ · 4H ₄ O (0.0003%),

Corn steep liquor (0.1 ¹ V ₀ ), yeast extract (0.1%) and α-paraffin (a mixture of C ₁₈ , C ₁₃ , C ₁₄ and C ₁₅ ), -10% -v / v -contained in one 5-1 fermentation bottle with a volume ratio of 10%.

Cultivation was carried out in a similar way to the manner described in Examples 1 and 2 for 72 hours at 30 ^° C. with stirring (600 rpm) and aeration (3 l / min.). The medium was adjusted to pH 7.0 to 7.5 with ammonia water.

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 liquid was similar to that in ^Be: treated in the manner described game 1 to give 0.4 g of purified Desoxyribonur'sinsäure 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 30 ^° C .; the fermentation liquid obtained was treated similarly to the manner described in Example 1. 0.2 g of a purified deoxyribonucleic acid and 0.7 g of crude ribonucleic acid were obtained from 11 fermentation liquors.

Claims (1)

acid from the fermentation solutions mentioned is patent claim: it is commercially feasible. For the production of the separation fluids Using by cultivating Arthro, a nutrient medium is used which contains a bacter paraffineus ATCC15590, Arthrobacter 5 hydrocarbon or a carbohydrate as the main simplex ATCC15799, Arthrobacter hydrocarbo carbon source and a nitrogen source, glutamicus ATCC15583, Brevibacterium keto-As a hydrocarbon or η can n-Parafglutamicum ATCC15587, Micrococcus ureae fin containing hydrocarbon fractions emge-ATCC 21288, Corynebacterium hydrocarboclastus. The n-paraffins should preferably contain 10 to ATCC15592, Pseudomonas fluorescens ATCC948, 10 22, in particular 12 to 18, carbon atoms. Pseudomonas aeruginosa ATCC15246, Asper- but also possible to add other carbon sources emgfllus orycae ATCC 7252 or Candida lipolitica, such as any type of microorganism, assimilable hydrocarbonaceous substances, and hydrocarbonaceous substances that are used in each case by the microorganism used for this purpose Carbohydrates, e.g. B. glucose, sorbitol, desoxyribonucleic acid and ribonucleic acid and the like, organic acids or alcohols, preferably small acid. wise C ₄ - to Cgo acids or alcohols and the like Both inorganic and organic nitrogen compounds can be used as sources of nitrogen 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. It is cultured under aerobic conditions in general at 20 to 6O ⁰ C, preferably at 30 to 42 ° C. During the The invention is concerned with the production 95 breeding is used to adjust to a pH of 4 to of deoxyribonucleic acids 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 nucleic acids mentioned indicate a maximum
the extraction and recovery processes of these. In the following, the invention is illustrated with reference to FIG