DE1442260B - Process for the preparation of 5 Phos pho D nbosyl 1 pyrophosphate - Google Patents

Description

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

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 filing date 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. 20% barium acetate solution was added to the concentrate treated in this way in an amount corresponding to 1.2 times the calculated amount and alcohol (4 times Amount) for the deposition of the barium salt of the PRPP. The deposits were reduced Pressure dried, with 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. This was followed by cultivation as a shaking culture ^{at 28 ° C. for 48 hours.} PRPP was enriched in the fermentation medium in the amounts given in the table.

Composition of the fermentation medium:

Glucose 10%

Urea 0.4%

NH ₄ Cl 0.2%

K _{2 HPO 4} l.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 ATCC14621

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 4} casein amino acids, fish extracts and the like and inorganic 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 40 ° C under aerobic conditions, e.g. 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-1-pyrophosphate liquid and the microorganism in the cells, in which certain D-ribose phosphate-forming men are formed. Microorganisms are grown. After the fermentation has ended, the process described in the German patent 1,295,505 5-phospho-D-ribosyl-1-pyrophosphate in the manner described in the ren for the biotechnological production of D-ribose-5 examples by 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 example 1
Ribose-5-phosphate and small amounts of 5-phospho- D-ribosyl-1-pyrophosphate can be enriched. 35,300 cc of a sterilized aqueous nutrient medium A for industrial implementation suitable procedure the 2 ° / "glucose, 1 ° / ₀ peptone, 1 ° / ₀ meat extract, drive for the biotechnological preparation of 5-phospho 0.25 ° / ₀ NaCl and 0.2% urea contained, D-ribosyl-1-pyrophosphate is not previously 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 Fermentastoff- and nitrogen sources and inorganic salts tion medium was mixed with 10 percent by volume inoculum containing nutrient medium containing phosphate ions in 45 based on the amount of the fermentation medium, an amount of 0.2 to 2.0 ° / ₀ and magnesium ions in inoculated. The fermentation medium used had / contains an amount of 0.01 to 0.2 ° _0, Ge has the following composition by
indicates that as D-ribose phosphate forming sucrose 15% / Microorganisms Micrococcus glutamicus ATCC / χτττ ^ _n η λο / 14995, Micrococcus glutamicus ATCC 14305, ash- 50 ΚΗΡΟ ". '.'.". '.'. '. Υ.'. '.'. '. 0 8 ° / richia coli ATCC 14621, Aerobacter aerogenes ATCC _K HPO * 08 ° / ° 14304, Bacillus subtilis ATCC 14618 or Brevibacte- MgSO- 7HO 6 6 ⁰ I rium ammoniagenes ATCC 15137 can be used. Biotin ^{4 2} 30v / l ° The 5-phospho-D-ribo-PeDton obtainable according to the invention 0 2 ° / syl-1-pyrophosphate is used as a biochemical reagent and 55 rr „cn 7ti ή n'nm ° o / as a starting material for the production of nucleotides 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 holds 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 are created after a cultivation time of 48 hours at 34 ° C. and preparations containing such nitrogen sources, calcium hydroxide was given, services such as B. peptone, yeast extract, meat extract, and the separated inorganic phosphorus