CS266901B1 - Process for preparing nicotinamide adenine dinucleotide phosphate - Google Patents

Abstract

The method of preparing nicotinamide adenine dinucleotide phosphate by microbial phosphorylation consists in treating nicotinamide adenine dinucleotide with permeabilized cells of the species Brevibacterium stationis Breed or Arthrobacter globiformis Conn et Dimmick containing metaphosphate-dependent nicotinamide adenine dinucleotide kinase, which are grown on a medium containing yeast autolysate or other natural source of organic nitrogen, a source of carbon and minerals. The use of Brevibacterium stationis DBM 1073 and Arthrobacter globiformis CCM 193 cells is particularly preferred. The permeabilized cells can be immobilized. Phosphorylation can be carried out in the presence of inhibitors of the development of microbial contamination, for example sodium fluoride or sodium azide.

Description

The invention relates to a process for the preparation of nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), which is a coenzyme of a number of redox enzymes. In reduced form, many compounds are involved in the synthesis of living cells. In chemical, biological and clinical laboratories, it is used mainly in the enzymatic determination of glucose and in the metabolic activation of chemical compounds tested for their mutagenic and carcinogenic effects. It has other uses in biological and biochemical research.

Nicotinamide adenine dinucleotide phosphate was originally obtained by isolation from microbial cells similar to that of nicotinamide adenine dinucleotide (NAD ⁺ ). However, because nicotinamide adenine dinucleotide phosphate is present in cells at much lower concentrations than nicotinamide adenine dinucleotide, this procedure is very expensive to obtain. Improvement has been brought about by the phosphorylation of cheap nicotinamide adenine dinucleotide by the action of nicotinamide adenine dinucleotide kinase on permeabilized cells of various bacterial species, which is the subject of several patents.

However, said phosphorylation requires a relatively expensive adenosine triphosphate as a phosphate donor, which is converted into adenosine diphosphate. Some improvement has been brought about by the use of bacterial strains which, in addition to the enzyme nicotinamide adenine dinucleotide kinase, also contain the enzyme adenylate kinase (Watanabe T. et al., Jap. Patent 53-115891, 1978; Watanabe T. et al., Jap. Patent 53-115892, 1978). . A further improvement was the use of a combination of two species of microbial cells, one for phosphorylation of nicotinamide adenine dinucleotide in nicotinamide adenine dinucleotide phosphate and the other for regeneration of waste adenosine diphosphate in adenosine triphosphate using inorganic phosphate (Watanabe T. et al., Jap. Patent Y-14-142596, 1978, et al., U.S. Pat.

The use of permeabilized cells of a microbial type, which also contains metaphosphate-dependent nicotinamide adenine dinucleotide kinase, has significantly reduced the cost of the whole process, so that metaphosphate, which is much cheaper than adenosine triphosphate, is used as a phosphorus donor in phosphorylation. A suitable strain appeared to be Brevibacterium ammoniagenes JAM 1645, whose permeabilized cells were also used, similarly to processes using adenosine triphosphate, in immobilized form (Murata K., Biotechnol. Bioeng. 21, 887, 1979). However, the cells used for this process must be obtained by culturing in a medium containing a yeast extract free of phosphorylated organic compounds by a relatively complicated procedure according to Weimberger R. and Orton W. L. J. Bacteriol. 89, 740, 1963. The process described using metaphosphate dependen ^TNI • ^xn l. In addition, inamidadenine dinucleotide kinase requires aerobic conditions and is therefore very important

CS 266 901 Bl

Livý sensitive to the inhibitory effects of sodium azide, which is commonly used to suppress microbial contamination in technological enzyme reactions and in processes involving permeabilized or mobilized cells.

The process for the preparation of nicotinamide adenine dinucleotide phosphate by microbial phosphorylation according to the invention, which consists in treating nicotinamide adenine dinucleotide with permeabilized cells of Breevitbacterium stationis Breed or Arthrobacter globiformis Conn et Dimmick containing metaphosphate-dependent nicotinamidysinase kinase, which source of organic nitrogen, source of carbon and minerals. Particularly preferred is the use of Bravibacterium stationis DBM 1073 and Arthrobacter globiformis CCM 193 cells stored in the collection of microorganisms of the Department of Biochemistry and Microbiology of the University of Chemical Technology in Prague under numbers 1073 and 1075. Permeabilized cells can be mobilized. Phosphorylation can be performed in the presence of inhibitors of microbial contamination, such as sodium fluoride or sodium azide.

Extracellular phosphorylation of nicotinamide adenine dinucleotide by Brevibacterium stationis or by Arthrobacter globiformis according to the invention is carried out by propagating the cells of these microorganisms under aerobic conditions in a liquid medium containing a nitrogen source in the form of ammonium salt and yeast extract natural source of this element, as well as magnesium and potassium ions and phosphate. The cells are separated from the propagation medium, preferably by centrifugation, and washed with water as needed. Permeabilization of the cells is accomplished either by shaking with acetone or another solvent, or by treatment with a suitable surfactant (e.g., dodecyl sulfate, cetylpyridinium chloride, etc.) so that nicotinamide adenine dinucleotide kinase activity is maintained. A suitable gel such as polyacrylamide, agar, etc. can be used to mobilize the cells. Permeabilization of the cells can be performed before or after immobilization.

The reaction is carried out in a reaction solution of 2 to 20 mM nicotinamide adenine dinucleotide, 2 to 60 mM Mg ²⁺ and 0.05 to 5% by weight of metaphosphate, to which permeabilized cells, free or mobilized, are added in an amount of 5 to 60 mg dry matter / ml. optionally still glucose in a final concentration of 0.01 to 0.2 mol / l. The additions of sodium fluoride NaF (in a final concentration of 0.01 to 0.3 mol / l) and sodium azide NaN ₃ (in a final concentration of 0.1 to 3 mmol / l) are suitable for suppressing the development of microbial contamination. Both the starting materials and the activators and inhibitors are dissolved in a suitable buffer of pH 5.5 to 9.0, the reaction temperature being chosen in the range of 20 to 55 ° C.

After the reaction, nicotinamide adenine dinucleotide phosphate is separated from the reaction liquid by any of the known methods (e.g., by passing through a suitable ion exchanger) and further purified. Unreacted nicotinamide adenine dinucleotide can again be used for the reaction.

The invention is further elucidated on specific examples of a process for the preparation of nicotinamide adenine dinucleotide phosphate using Brevibacterium stationis DBM 1073 and Arthrobacter globiformis CCM 193.

Example 1

Brevibacterium stationis DBM 1073 is aerobically cultured at 30 ° C in a pH 7.5 medium containing in 1 liter: 20 g glucose, 1 g ammonium sulfate, 0.1 g MgSO ₄ . 7 H ₂ O, 8.7 g K ₂ HPO ₄ , 10 g yeast autolysate. After 18 h of culture, the cells were centrifuged at 800 g, washed with water and permeabilized with 3 volumes of chilled acetone for 15 minutes. The cells are then centrifuged, the acetone is decanted and its residues are removed with a stream of air. To a reaction mixture of 5 mM nicotinamide adenine dinucleotide, 20 mM MgCl ₂ , 1% w / w, metaphosphate and 1 mM NaN ₃ in Tris-HCl buffer pH 6.8, permeabilized cells were added at 20 mg cell dry matter / ml and the mixture was incubated with stirring. 2 h at 37 ° C. During this time, about 50% of the nicotinamide adenine dinucleotide is transformed into nicotinamide adenine dinucleotide phosphate. The incubation time can be extended up to 4 h with a corresponding increase in yield. The cells are separated from the reaction mixture by centrifugation at 800 g to 1000 g, and nicotinamide adenine dinucleotide phosphate is isolated from the supernatant by any known method.

Example 2

Arthrobacter globiformis CCM 193 microbial cells are cultured in the soil indicated in the example

A process for the preparation of nicotinamide adenine dinucleotide phosphate by microbial phosphorylation, characterized in that the nicotinamide adenine dinucleotide is treated with permeabilized cells of the species Brevibacterium stationis Breed or Arthrobacter globiformis Conn et Dimmick source of carbon and minerals.

1. 266% by weight of acrylamide monomer and 2% by weight of acrylamide monomer are added to a washed and centrifuged suspension of cultured cells in twice the volume of water. N, N‘-methylene-bis-acrylamide and, after cooling to 7-10 ° C, 3% by volume of a 5% (w / w) solution of dimethylaminopropionitrile and 5% (w / w) of potassium persulphate are added. After the polymerization, particles of 1 to 5 mm in size are prepared from the gel formed. An equal volume of acetone was added to these particles and shaken in the cold for 30 minutes to permeabilize the immobilized cells. The acetone is then decanted off, the particles are washed with water and charged to the reactor. After adding twice the volume of a reaction mixture of the same composition as in Example 1, the reaction mixture was stirred for 2 hours. After separation of the heterogeneous biocatalyst, nicotinamide adenine dinucleotide phosphate is isolated by any of the known methods. A new portion of the reaction mixture is added to a heterogeneous biocatalyst containing immobilized and permeabilized cells, and the biotransformation procedure is repeated. The yield of nicotinamide adenine dinucleotide phosphate is about 40%.

Example 3

Immobilized and permeabilized Brevibacterium stationis DBM 1073 cells prepared according to the procedure described in Example 1 and 2 were loaded onto a column, which was then passed through a reaction mixture of the same composition as in Example 1. The flow rate was chosen so that the total column packing was exchanged for 2 to 3 h. This continuous biotransformation process provides a yield of nicotinamide adenine dinucleotide phosphate of about 40%.

Claims (4)

OF THE INVENTION 2. The method of claim 1, wherein the nicotinamide adenine dinucleotide is treated with Brevibacterium stationis DBM 1073 and Arthrobacter globiformis CCM 193 cells. 3. The method of items 1 and 2, wherein the nicotinamide adenine dinucleotide is treated with immobilized permeabilized cells. 4. The process according to items 1 to 3, characterized in that the phosphorylation is carried out in the presence of inhibitors of the development of microbial contamination, for example sodium fluoride or sodium azide.