JPS5834118B2 - Nicotinamide Adenine Dinucleotide Phosphate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides nicotinamide adenine dinucleotide
The present invention relates to a method for producing phosphate (hereinafter abbreviated as NADP), and more specifically relates to a method for producing NADP from nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) and metaphosphoric acid or polyphosphoric acid using microorganisms.

NADP is a physiologically important substance and is useful as a pharmaceutical (eg, an antihypertensive agent) or a biochemical reagent.

Conventionally, a method for producing NADP from NAD and adenosine triphosphate using a microorganism containing nicotinamide adenine dinucleotide kinase has been known. No.-46353, Japanese Unexamined Patent Publication No. 135290).

However, in these methods, the substrate adenosine triphosphate is expensive, and means for inhibiting adenosine triphosphate degrading enzyme must be used, and NAD
P cannot be manufactured at low cost.

The present inventors have conducted various studies to produce NADP in large quantities at low cost. As a result, certain microorganisms belonging to the genus Micrococcus, Corynebacterium, Brevibacterium, Achromobacter, and Acetobacter have been found to produce NADP in large quantities at low cost.
It was discovered that NADP has the ability to be produced from metaphosphoric acid or polyphosphoric acid that is supplied at low cost, and if divalent metal ions are allowed to coexist when reacting the enzyme source with the substrate. It was discovered that the amount of NADP produced was significantly increased, and the present invention was completed.

That is, the present invention relates to a culture solution of a microorganism belonging to the genus Micrococcus, Achromobacter, Corynebacterium, Brevibacterium, or Acetobacter and having the ability to produce NADP from NAD and metaphosphoric acid or polyphosphoric acid; or the bacterial cells or treated bacterial cells obtained from the culture solution in the presence or absence of divalent metal ions,
This is a method for producing NADP, which consists of reacting NAD with metaphosphoric acid or polyphosphoric acid.

The microorganisms used in the present invention belong to the above genera,
Any microorganism that has the ability to produce NADP from NAD and metaphosphoric acid or polyphosphoric acid can be used, such as Micrococcus flavano 0UT8276,
Micrococcus glutamicus ATCC13761, Micrococcus rhizodiicticus IF03333, Achromobacter butiri 0UT8004 (Feikoken Bacterium No. 6979), Corynebacterium cepedonicum F
O3306 (February Institute of Fine Arts and Technology Research No. 6980), Brevibacterium flavum ATCC14067, Brevibacterium ammoniagenes IAM1645, Acetobacter xylinum 0UT8302 (February 2011, Microtechnology Research Institute No. 6981)
No.) etc. are preferably used.

In culturing the above-mentioned microorganisms, a commonly used medium is used, for example, glucose, sucrose, glycerol, etc. are used as a carbon source, and peptone, meat extract, yeast extract, corn steep liquor, urea, etc. are used as a nitrogen source. Using an organic nitrogen source such as ammonia or an inorganic nitrogen source such as ammonia or ammonium sulfate, inorganic salts such as potassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, compounds necessary for bacterial growth, etc. A medium to which an appropriate amount is added is preferably used.

The culture is preferably carried out under aerobic conditions such as aerated culture.

Further, the culture temperature is preferably about 20 to 40°C, and the culture time is usually sufficient for 10 to 70 hours.

As the enzyme source used in the present invention, the culture fluid obtained as described above, bacterial cells isolated from this culture fluid, and further processed products of the bacterial cells are used.

Examples of treated bacterial cells include bacterial cells washed with physiological saline, treated with surfactant, sonicated, or immobilized bacterial cells wrapped in acrylamide-based polymer Q gel. Can be mentioned.

According to the present invention, NADP is reduced by allowing the enzyme source as described above to act on NAD and metaphosphoric acid or polyphosphoric acid.
can be manufactured.

This enzyme reaction is carried out at pH 5.5 to 9.0, especially pH 6.0.
~7.5, preferably at a temperature of 20 to 60°C, especially 35 to 50°C.

Furthermore, during this enzymatic reaction, if divalent metal ions are allowed to coexist in the reaction system, the amount of NADP produced can be significantly increased.

Examples of divalent metal ions used here include magnesium ions, manganese ions, cobalt ions,
Examples include nickel ions, calcium ions, copper ions, etc., and the concentration of these divalent metal ions added is 5 x 10-
Approximately 3M to 5X10''M is preferable.

After the reaction is completed, the produced NADP can be easily isolated and purified using common purification means such as chromatography using an ion exchange resin.

The method of the present invention will be specifically explained below with reference to Examples.

Example 1 Glucose 0.5%, yeast extract 1.0%, meat extract 0.
Brevibacterium 5%, peptone 1.0%, salt 0.5%, magnesium sulfate 0.1%, potassium phosphate 0.5% (1) H7.0) Lo Inoculate with ammoniagenes IAM1645,
Culture with shaking at 30°C for 20 hours.

Bacterial cells are collected from the resulting culture solution, washed twice with physiological saline, and suspended in physiological saline again.

Surfactant cation-8 was added to 10 mg/1 nl (dry cell weight) of the bacterial cell suspension prepared in this manner at a concentration of 2 mg/ml, and the mixture was shaken at 37°C for 30 minutes.

The product thus prepared was used as an enzyme source, and NAD4.
Each component was added to give a concentration of 2 ml/ml, 2% metaphosphoric acid (manufactured by Katayama Chemical Industry Co., Ltd., the same hereinafter), 10-2 M of magnesium ion, and 0.1 M of Tris-HCl buffer (pH 7.0). React at ℃ for 3 hours.

The amount of NADP thus generated in the reaction solution was 0.72 m9.
It was 7m1.

Example 2 10 my of bacterial cell suspension prepared in the same manner as in Example 1
/rnl (dry bacterial weight) N A D 4.2 m
y/rnl, metaphosphoric acid 2%, magnesium ion I
C)”M.

Tris-HCl buffer (1) H7,0) O, 1M and surfactant cation -82yn9/rnl were added to each component, and the mixture was shaken and reacted at 37°C for 3 hours.

The amount of NADP thus generated in the reaction solution was 0.68 m9/
It was TLl.

Example 3 Bacterial cells were collected from the culture solution obtained in the same manner as in Example 1,
After washing twice with physiological saline, it was suspended in 0.002M)'JS hydrochloric acid buffer (pH 7.0) and washed for 90.00 cycles.
Sonicate for 0 minutes.

This sonicated product was used as an enzyme source (protein 10 my/m0
To this were added 97 ml of NAD 4.2, 2% metaphosphoric acid, 101 M of divalent metal ions shown in Table 1 below, and 0.1 M of Tris-HCl buffer (pH 7.0). , react at 37°C for 3 hours.

The amount of NADPO thus produced in the reaction solution was as shown in Table 1 below.

Example 4 Enzyme source obtained in the same manner as Example 3 (protein 10 m
9/ml), NAD4.2m9/Ttl, polyphosphoric acid (
(manufactured by Katayama Chemical Industry Co., Ltd.) 0.05%, magnesium ion 10'M and Tris-HCl buffer (pH 7.0)
Each component was added to a concentration of 0.1 M, and the mixture was reacted at 37°C for 3 hours.

The amount of NADP thus generated in the reaction solution was 0.2 m9/
rrLl Te Atsuta.

Example 5 Achromobacter butyri 0UT8004 (Feikoken Bacterial Serial No. 6979) was added to a medium having the same composition as in Example 1.
Cultivate in the same way.

Bacterial cells are collected from the obtained culture solution, washed twice with physiological saline, and suspended in physiological saline again.

4ml of this bacterial suspension! (contains 11 wet bacterial cells) and 50 ml of acrylamide. N-N'-methylene-bis-
Add 40 m of acrylic acid amide, 0.5 ml of 9.5% β-(dimethylamino)-propionitrile and 0.5 TrLl of 2.5% potassium persulfate, and react for 20 minutes at room temperature.

After the reaction is completed, the resulting gel is crushed.

The thus prepared immobilized bacterial cells were added to 0.2 M acetate buffer (
pH 5,5) to 2.5 TIll, and to this add NAD 4.
2m97m1! , 2% metaphosphoric acid and 10 −2 M of magnesium ions were added, and the mixture was reacted at 37° C. for 3 hours.

The amount of NADP thus generated in the reaction solution was 0.1 m97.
m1 tea tsuta.

Example 6 Immobilized bacterial cells prepared in the same manner as in Example 5 are immersed in 100% acetone for about 3 hours, and then reacted in the same manner as in Example 5.

・NADP thus generated in the reaction solution is 0.9 yn
It was 9/ml.

Example 7 In the same manner as in Example 1, the strains shown in Table 2 below are cultured in a medium having the same composition as in Example 1.

Bacterial cells are collected from the culture solution, and a sonicated product is prepared in the same manner as in Example 3.

To this sonicated product, each component was added so that NAD 4.2 my/rul, metaphosphoric acid 2%, magnesium ion 10-2M, and Tris-HCl buffer (pH 7,0) 0.1M were added, and the mixture was incubated at 37°C for 3 hours. Allow time to react.

The amount of NADPO thus produced in the reaction solution was as shown in Table 2 below.

Claims (1)

[Scope of Claims] 1. Nicotinamide adenine dinucleotide and metaphosphoric acid or polyphosphoric acid, which belong to the genus Micrococcus, Achromobacter, Corynebacterium, Brevibacterium or Acetobacter. A culture solution of a microorganism having the ability to produce dinucleotide phosphate, or bacterial cells or a bacterial cell treatment product obtained from the culture solution, is allowed to act on nicotinamide adenine dinucleotide and metaphosphoric acid or polyphosphoric acid. A method for producing nicotinamide adenine dinucleotide phosphate, which is characterized by: 2. Belongs to the genus Micrococcus, Achromobacter, Corynebacterium, Brevibacterium or Acetobacter, and produces nicotinamide adenine dinucleotide phosphate from nicotinamide adenine dinucleotide and metaphosphoric acid or polyphosphoric acid. A culture solution of a microorganism that has the ability to produce, or bacterial cells or a bacterial cell treatment product obtained from the culture solution, is treated with nicotinamide adenine dinucleotide and metaphosphoric acid or polyphosphoric acid in the presence of divalent metal ions. A method for producing nicotinamide adenine dinucleotide phosphate, which is characterized by causing nicotinamide adenine dinucleotide phosphate to act.