DE1442260C - Process for the preparation of 5-phospho-D-ribosyl-1-pyrophosphate - Google Patents

Description

bindings were removed by centrifugation. The obtained supernatant liquid was through a with ion exchangers known under the trade name "Dowex-1" on the filing date of the present invention, in the formic acid form, loaded column, whereby the ribose phosphates were adsorbed. Then, in the order given, 0.2 molar, 0.4 molar and finally 0.5 molar Sodium formate buffer solutions (pH = 5.0) eluted.

The PRPP was contained in the eluate obtained with 0.5 molar sodium formate buffer solution. the individual fractions were then passed through a column that was used on the day of the present invention ion exchanger (Η-form) known under the trade name "Diaion SK No. 1" was charged, neutralized to pH = 7.0 and then concentrated under reduced pressure.

The concentrate was treated with a small amount of activated charcoal and the pH value with the help of Sodium hydroxide adjusted to 8.3. To that on this »o Treated concentrate was 20% barium acetate solution in an amount corresponding to that 1.2 times the calculated amount and alcohol (4 times the amount) to separate the barium salt of the PRPP given. The deposits were under reduced as dried under pressure, whereby PRPP in a yield of 41.2 g - calculated as anhydrous barium salt - was obtained.

Example 2

30th

From Micrococcus glutamicus ATCC 14305, Escherichia coli ATCC 14621, Aerobacter aerogenes ATCC 14304, Bacillus subtilis ATCC 14618 and Brevibacterium ammoniagenes ATCC 15137 were as in Example 1 described inoculum produced.

In each case 40 ml of a fermentation medium of the composition given below were sterilized and inoculated with 10 percent by volume of one of the inoculation cultures. Then it was shaken for 48 hours at 28 ° C bred. PRPP was enriched in the fermentation medium in the amounts given in the table.

Composition of the fermentation medium:

Glucose 10%.

Urea 0.4% / ₀

NH ₄ Cl 0.2%

K ₂ HPO ₄ 1.0%

KH ₂ PO ₄ 1.0%

MgSO ₄ -7H ₂ O 0.6%

Yeast extract 0.4%

Peptone 0.2%

FeSO ₄ -7H ₂ O 0.002%

pH value before sterilization 7.8

Vaccination culture

Micrococcus glutamicus ATCC14 305

Escherichia coli ATCC 14621

Aerobacter aerogenes ATCC 14304 ..

Bacillus subtilis ATCC 14618 ...

Brevibacterium ammoniagenes
ATCC 15137

PRPP

(mg / ml)

5.0 4.9 7.0 3.2

8.0

Claims (1)

1 2 Claim- casein amino acids, fish extracts and the like. And inorganic ones Salts, e.g. B. phosphates such as potassium phosphate Process for the production of 5-phospho-D- and ammonium phosphate, magnesium salts such as maribosyl-1-pyrophosphate by growing D-ribogagnesium chloride and other salts that make up the wax phosphate forming microorganisms in a 5 turn of the microorganisms indispensable elements contain assimilable carbon and nitrogen sources, e.g. B. salts of potassium, calcium, and nutrient medium containing inorganic salts, iron, manganese, zinc, copper, des the phosphate ion in an amount of 0.2 to 2% boron and the molybdenum. and magnesium ions in an amount of 0.01 to The phosphate and magnesium ions are in the Contains 0.2%, characterized in-io nutrient medium at concentrations of 0.2 to 2.0 ° / ₀ net that as D-ribose phosphates forming micro- or 0.01 to 0.2 ° / ₀ contain. If the growth Organisms Micrococcus glutamicus ATCC 14995, a microorganism due to the high concentration Micrococcus glutamicus ATCC 14305, Escherichia these salts is inhibited in the nutrient medium coli ATCC 14621, Aerobacter aerogenes ATCC these in small amounts during fermentation 14304, Bacillus subtilis ATCC 14618 or Brevi- 15 can be added. bacterium ammoniagenes ATCC 15137 used The fermentation is carried out at temperatures of will. 20 to 4O ⁰ C under aerobic conditions, for example. B. as Shake or submerged culture with aeration and Stirring, carried out. After 2 to 7 days of growing 20 surprisingly high amounts of 5-phospho- The invention relates to a biotechnological process D-ribosyl-1-pyrophosphate (PRPP) in fermentation Production of 5-phospho-D-ribosyl-l-pyrophosphate and in the cells of the microorganism, in which certain D-ribose phosphate-forming men are formed. Microorganisms are grown. After the fermentation has ended, the process described in German Patent 1,295,505 is 5-phospho-D-ribosyl-1-pyrophosphate in the manner described in the examples for the biotechnological production of D-ribose-5 Absorption on phosphate, with the Micrococcus glutamicus ATCC a suitable strongly basic ion exchanger and 15455 or Brevibacterium Ammoniagenes ATCC fractionated elution.
6872 is cultivated in a suitable nutrient medium. In the following examples, the implementation of the proposed and from J. Gen. Appl. Microbiol. 30 described method according to the invention.
(Tokyo), 9, pp. 457-8 (1963), CA. 61 (1964), 12349c, The percentages given are weight is known that by growing an unidentified volume percent,
th microorganism in the fermentation liquid B eisdie 1 1
Ribose-5-phosphate and small amounts of 5-phospho- D-ribosyl-1-pyrophosphate can be enriched. 35,300 cc of a sterilized aqueous Nährraediums, A for industrial implementation suitable procedure the 2 ° / ₀ glucose, 1% peptone, l ° / ₀ meat extract, drive for the biotechnological preparation of 5-phospho 0.25% NaCl and 0.2 % Urea contained, D-ribosyl-1-pyrophosphate is not yet known- in a 2-1 Erlenmeyer flask with a culture of become. Micrococcus glutamicus ATCC 14995 inoculated. Then the subject of the invention is now a process for 40 was grown for 24 hours at a pH of 7.5 and the production of 5-phospho-D-ribosyl-l-pyrophosphate at a temperature of 28 ° C,
by cultivating D-ribose phosphates forming The inoculum thus obtained was used for inoculating microorganisms in an assimilable charcoal fermentation medium. The fermentation material and nitrogen sources and inorganic salts were inoculated with 10 volume percent inoculum, nutrient medium containing phosphate ions in 45 based on the amount of fermentation medium, an amount of 0.2 to 2.0% and magnesium ions in. The fermentation medium used contained an amount of 0.01 to 0.2%, thereby giving the following composition. indicates that as D-ribose phosphate forming ς., _Λ τ ,,. _η ., _1S o / ._ .... ... . . rr ^^ - i ^^ oaccnarose υ in Microorganisms Micrococcus glutamicus ATCC / _ΝΗ \ vr \ 0 2 ° / 14995, MicTococcus glutamicus ATCC 14305, ash-50 KH PO "". 0 8 ° / ° richia coli ATCC 14621, Aerobacter aerogenes ATCC _κ ηρο * 08 ° / ° 14 304, Bacillus subtilis ATCC 14 618 or Brevibacte-MgSO · 7HO 66 ⁰ I. rium ammoniagenes ATCC 15137 can be used. Biotin * ² 30v / l The 5-phospho-D-ribo-Perjton obtainable according to the invention 0 2 ° / syl-1-pyrophosphate is as a biochemical reagent and 55 FeSO · 7H 0 0002 ° / as starting material for the production of nucleo- ^{4 2} ' tiden valuable. pH value: Adjusted to 7.3 before sterilization The fermentation used in the process of the invention was carried out using 3 l Culture medium corresponds to that for the production of the fermentation medium, which is in a 5-1 fermentation D-ribose phosphates known nutrient medium. Entation containers had been sterilized when submerged maintains suitable quantities of carbon sources for this purpose, cultivation is carried out with aeration and stirring, such as B. glucose, sucrose, saccharides, starch, During the cultivation the pH was at 7.0 Starch hydrolysates, molasses, gluconates, acetates etc. are kept. Nitrogen sources, e.g. B. inorganic nitrogen sources to 2 1 of the filtrate, which after separating the such as ammonium salts and nitrates or organic cells from the fermented fermentation medium Nitrogen sources such as urea, amino acids, peptides after a cultivation time of 48 hours at 34 ⁰ C and preparations containing such nitrogen sources, calcium hydroxide was given, services such as B. peptone, fleece extract, meat extract, and the separated inorganic phosphorus