DE2151634C3 - Process for the preparation of nicotinic acid amide adenine dinucleotide phosphate - Google Patents

Description

It is already known. Nicotinic acid amide adenine dinucleotide phosphate. which is also called coenzyme II or triphosphopyridine nucleotide, by reaction of nieotinamide adenine dinucleotide with adenosine triphosphate in the presence of nicotinic acid adenine dinucleotide kinase to manufacture.

According to that from J. Biol. Chem. Vol. 182 (1950). S. 805 to 813 known method is used as nicotinamide adenine dinuclcotide kinase z. B. a Enzyme isolated from deaf cow or brewer's yeast is used. The phosphatase contained in the crude enzyme makes the action of nicotine acid camidadcnine dinucleotide kinase. namely the Phosphorvlierung. again reversed, so that only a slight bulge of nicotinic acid amide dinine dinucleotide phosphate is achieved. The purification of the mentioned kinase is * ° iedoch: chr difficult.

It is known that phosph; itase is present in enzyme systems extracted from microorganisms. whose phosphodiesterase activity is to be used. by using acid (pH 5) it can become inactive (cf. German Offenlegungsschrift 14 20 112) and that the phosphates contained in cell extracts can be inhibited by hydrogen or by cystein and glutathione (cf. Chargaf and Davidson, "The Nucleic Acids «. I) ·}". I. 1955, pp. 592 or 593). However, these measures have not yet been applied to enzyme systems which were used for the production of nieotinamide adenine dinueleotide phosphate, an acid amide adenine dinucleotide kinase, an acid amide adenine dinucleotide kinase produced by breeding Brevibacterium ammoniagenes AlCC 6872. Arthrobacter citreus ATCC 11624. Arthrobacter ureafaciens ATCC 7562 or Corynebactenum rathay.ATCC 13659 cell material obtained and that the reaction is carried out after previous treatment of this cell material with a mineral acid and / or in the presence of a fluoride and / or .n the presence of cysteine or glutathione. To obtain the cell material, the microorganisms mentioned are treated in the usual way under Vervvendu grown on a medium containing a carbon source such as carbohydrates, e.g. B. glucose or sucrose, organic acids or carbon nitrogen source, such as natural B. peptone. Meat extract. Embers _lu .cuuu a .. ^ rsanische nitrogen sources. z. B. NK (NH ₁ KSO,. Contains phosphates, such as potassium hydrogen phosphate and potassium dihydrogen phosphate. MgSO ₁ and _ao anoruanic cations such as iron, manganese, zinc, calcium. The respective microorganism is under aerobic conditions, e.g. under rubble by underwater culture with aeration and stirring at a temperature of 20 to 40 C. preferably grown to 9, preferably 7 to 8 of ^"1 S to 37 C. and at a pH of>. The culture is preferably performed for 6 to 48 h. If the cell material is to be treated with a mineral acid, the latter is added, for example in the form of HCl or 11, SO _{4, to} the breeding broth contained, so that a pH of 3 to 6. The breeding broth is then stirred for 1 to 24 hours DuTjewcil's breeding broth obtained can be used as such for the reaction, but it is also possible to use the microbial cells separated from the breeding broth, for example by centrifuging the separated cells bodies are preferably suspended in water. Special cleaning measures are not required.

As Fluoi Ul sodium fluoride or ammonium fluoride can be used, in a 5 ■ IO 'to -> * ()!' Is preferably 5 × 10 ² to 15 ■ 10 - molar concentration. The cysteine or glutathione is in an H) ^;) to IO '-. preferably 3 · 10 'to 5 · 10 ·' molar concentration added.

The reaction of nicotinic acid amide dinucleotide with adenosine triphosphate is at a pH of 4 to 8, preferably from 6 to 7.5. and at at a temperature of 35 to 43 ° C., preferably 36 to 38 ° C., in 1 to 24 hours. After completion the reaction is the nicotinic acid vimidadenine dinucleolide phosphate obtained from the reaction solution in the usual way, for. B. using of a strongly acidic ion exchange resin in combination

50

agile, one mumv miuh-u ιυ.ιι.ιι «ϋ« »^

It is also known. Nicotinic acid amide adenine with a strongly basic ion exchanger

nucleotide phosphate obtained by simply growing yeast with resin.

to produce conventional nutrients (cf. Japanese Patent Publication 24 S I I 64). Apart from the Difficult isolation, only low yields are achieved here too.

The invention is based on the object of a method / for the production of nicotinic acid amide adenine

find nucleotide phosphate with high yield / u, The method according to the invention relates to the

6o In the following the invention is explained by means of examples.

Example I.

Brevibacterium ammoniagenes ATCC 6872 was in a medium containing 14 "" glucose. 0.6 "" I meat extract. 20 mg ml E-Cvstein. 15 mg ml, .'-

■ laliren refers to aiii «.lic alanine. 0.01 "., Calcium chloride and 45 mg ml manganese

known reaction of Nicotinsäureamidadenindi- ⁶ 5 contained, at a pH of 7.5 and at a ' ^cm P ^e mieleotid with adenosine triphosphate in the presence of 30 C with stirring with 300 L; now in 36 Ii ν on nicotinic acid adenine dinucleotide kinase and
is characterized in that the nicotine

bred. Then wind up the broth with HCI adjusted to a pH value of 4 and left to stand for 3 h

sen. The microbial cells were then centrifuged separated from the breeding broth and at a concentration of about 20 mg / ml (calculated as Dry cell weight) suspended in water. This solution was adjusted to pH with sodium hydroxide solution set of 7.5 and there were 4 mg / ml nicotinic acid adenine dinucleotide and 4 mg / ml adenosine triphosphate added. The reaction was carried out at a temperature of 37 ° C. for 6 hours. In the Reaction solution was 0.8 mg / ml of nicotinic acid amide adenine dinucleotide phosphide educated.

Example 2

The reaction according to Example 1 was repeated with the difference that the reaction solution 4 mg / ml NaF were added, whereby 1.5 mg / ml nicotinic acid amide adenine in ucleotide phosphate produced became.

Example 3

The treatment described in Example 1 was repeated with the difference that the reaction solution was also mixed with 0.5 mg ml of cysteine. A yield of 0.85 mg, ml of nicotinic acid amiadenir.dinucleotide phosphate fell on.

Example 4

The reaction according to Example I was repeated with the difference that 4 mg ml of NIl ₁ F and 0.5 mg ml of cysteine were added, whereby 2.2 mg ml of nieotinic acid amide adenine dinucleotide phosphate were produced in the reaction solution.

Example 5

The reaction according to Example 4 was repeated, but with the difference that the additions of acid-treated, non-centrifuged ingredients were added. There were in the reaction solution 0.5 mg ml of nieotinamide adenine dinodine phosphate is generated.

Example 6

ßrevibactcrium ammoniagencs ATCC 6872 was grown in the manner described in Example 1 for 36 hours and centrifuged without acid treatment in order to separate the microbial cells. The separated cells were at a concentration on \ about 20 mg / ml (calculated as dry cell weight) were suspended in water. 4 mg ml of nicolinamide adenine dinucleotide, 4 mg ml of adenosine triphosphate, 4 mg ml of NaF and 0.5 mg ml of cysteine were added to this suspension. The pH was adjusted to 7.5 with sodium hydroxide solution. The reaction was carried out at 37 ° C. for 6 hours, whereby 1.1 mg / ml of nicotinic acid amide adenine dinucleotide phosphate were obtained in the reaction solution.

Example 7

A broth prepared in the same manner as in Example 1 became without acid treatment a cell suspension prepared and this in each case 4 mg / ml nicotinic acid adenine dinucleotide, adenosine triphosphate and NaF added. After adjusting the pH to 7.5 by means of sodium hydroxide solution, in 6 h at 37 C carried out the reaction, 1.0 mg / ml nicotinic acid adenine dinucleotide phosphate in the reaction solution revealed.

Example 8

Each of the following microorganisms was within 36 hours on the in Example I manner described: the one obtained in each case

»° Broth was used to separate the cell bodies centrifuged. The cell bodies were each suspended in water to give a cell suspension that was approximately 20 mg ml (calculated as dry cell weight) contained cell bodies. Then 4 mg ml of nicotine

* 5 acid amidad ^ nindinucleotide. 4 mg.ml adcnosine triphosphate and 4 mg ml NaF and 0.5 mg ml cysteine and adjusted the pH to 7.5 with sodium hydroxide solution one. Each reaction was carried out at 37 ° C. in 6 hours. The following amounts of nicotinic acid amide dinucleotide phosphate were used educated:

Microorganism

Cadinine nicolinic acid dinuclcotide phosphate (mg ml)

Arthrobacter citrcus ATCC 11 624 0.26

Arthrobacter ureafacicn • - ATCC 7562 0.76 Corynebactcrium ratha> i ATCC 13659 0.82

Example 9

Each of the following microorganisms was cultured and ^{treated in the same manner as in Example 7} except that the pH of the culture broth was adjusted to 4.0 with sulfuric acid. The following results were obtained:

Nicotinic acid cudcnindiiuiclcotidphiisphat (mg mil

Microorganism

Arhrobaeter citrcus ATCC 11 624 0.75

Arthrohacter ureafacicns ATCC 7562 1.20 Corvnebacterium rathayi ATCC 13659 0.95

Claims (1)

Claim: Process / for the preparation of nicotinic acid amide dinucleotide phosphate by reaction of nicotinic acid amide adenine dinucIeotKÜ with adenosine triphosphate in the presence of nicotinic acid adenine dinucleotide kinase, characterized in that that one as nicotinic acid amide dinine dinucleotide kinase by culturing from Brevibacterium ammoniagenes ATCC 6872, Arthrobacter citreus ATCC Π 624. Arthrobacter ureafaciens ATCC 7562 or Corynebacterium rathayi ATCC 13659 obtained cellular material used and that the reaction after previous treatment of this cell material with a Mineral acid and / or in the presence of an eluoride and / or in the presence of Cvstein or glutathione performs.