JPS61233697A - Production of nicotinamide adenine dinucleotide phosphate - Google Patents

Abstract

PURPOSE:To obtain inexpensively and efficiently the titled compound useful as a biochemical analysis reagent, etc., from nicotinamide adenine dinucleotide, acetyl phosphate and adenosine 5'-monophosphate, by using a bacterum having specific enzymatic activity. CONSTITUTION:A solution such as phosphoric acid buffer solution, etc. is blended with 0.5-20 mg/ml nicotinamide adenine dinucleotide(NAD), 0.003-1.0 mg/ml adenosine 5'-monophosphate(AMP) and 0.14-5.8 mg/ml acetyl phosphate(ACP) in preferable concentrations (usually AMP/NAD is <=1/10 equivalent) and reacted with a mold of bacterium [e.g., Brevibacterium ammoniagenes(ATCC15312), etc.] having enzymatic activities of NAD kinase, acetate kinase and adnylate kinase under preferable conditions at 5.0-9.0 pH at 15-50 deg.C to give the aimed compound.

Description

Detailed Description of the Invention (Field of Industrial Application) Nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NAI) is used in vivo together with nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD). Enzyme 1 that governs redox reactions It is an important substance that has long been known as a coenzyme, such as glucose 6-phosphorus dehydrogenase, glutamate dehydrogenase, and 6-phosphogluconate dehydrogenase. It is widely used not only as chemical analytical reagents and diagnostic reagents for clinical test strips, but also for measuring glucose and gluconic acid in food. Furthermore, today, bioreactors are used to produce useful substances such as amino acids. ) and NADP regeneration systems, and it is a useful substance that is attracting attention.

(Prior Art) (1) N A D P has conventionally been produced by an extraction method from yeast.

(2) In recent years, microorganisms with NAI) kinase activity have been used to synthesize NAD and adenosine triphosphate (AT).
Method for producing NADP from (abbreviated as P)
46858. % Kaisho 50-18.5290. Tokukai Showa 5O
-142792) was developed. (Reaction formula) (3) There is also a method using an energy coupled system using adenylate kinase (Japanese Unexamined Patent Publication No. 58-11-5891). (Reaction formula 2) (4) On the other hand, as a system for regenerating ATP, (Reaction formula 3)
As shown in Figure 2, a method using ACP and acetate kinase has also been reported to be effective. (Maruo, FF
``Enzyme Handbook'' supervised by Inomiya.

Asakura Shoten, P367.1983) Furthermore, this reaction'
(!7NA]') 'Myawaki et al. reported that they used NA and D kinases and acetate kinases isolated and purified from chicken liver and Escherichia coli, respectively, as examples of their use in P production reactions. (0sato M 1yaia
kiet, at, , Agric, f3ioL
Chem,, Vol, 46+ 2725-2788
゜1.982) (Problems to be solved by the invention) (]) With the extraction method from yeast, it is difficult to obtain a large amount because there is little accumulation in the bacterial body □ (2) N A D and A' In the method of making from rP, NAI
) and require equimolar amounts of expensive ATP.

(3) Even when using an energy conjugated system, NA and D1
0.5 mole of ATP per mole is required, which is not particularly advantageous.

(4) The method of isolating and using each enzyme is complicated and lacks stability, so it cannot be said to be industrially advantageous. (Means and effects for solving the problem) The present inventors have identified the above drawbacks. As a result of intensive studies to solve this problem, we found that when biochemically synthesizing NADP from NAD and ATP through the reaction of NAD kinase, it is possible to do so by using bacteria that simultaneously have the enzymatic activities of NAD kinase, adenylate kinase, and acetate kinase. We have discovered that it is possible to perform the NADP synthesis reaction and the ATP regeneration reaction simultaneously by separating and purifying each enzyme. In other words, AMP is converted to A by the action of the adenylate kinase of the bacterial cell itself.
ACP is first generated by acetate kinase.
Generates ATP from and ADP, and also generates ATP from this and
was used in the synthesis reaction to produce NADP, and the resulting A D
P can be converted into A TP again by the action of A, CP and acetate kinase, and the advantage is that the expensive ATP is not necessarily required to initiate the reaction, and the cheaper AyaIP can be used.

Furthermore, they discovered that the reaction proceeded even when the amount was added at a level lower than 1/10 of a molar equivalent, leading to the completion of the present invention.

Next, two aspects of the present invention will be described in detail. NA, D according to the present invention
Examples of Bacterium I77 having enzyme activities of kinase, acetate kinase, and adenylate kinase include Bacillus and Previbacte quintatum.

Examples include Corynebacterium, alkaline earth metals, Arthrobacillus, Escherichia, Proteus, Shutomonas, Sarcinus, and Serratia. Furthermore, two or more types of these bacteria may be combined.

For example, Brevibacterium ammoniagenes (ATCC 15812) is cultured according to a conventional method to obtain bacterial cells. The cells are washed if necessary, and used as they are, or as immobilized cells that have been immobilized on a gel such as acrylamide or alginic acid.

At this time, the bacterial cells or immobilized bacterial cells may be brought into contact with an organic bath agent or a surfactant to increase the enzymatic activity of the bacterial cells before use. The substrate concentration in the reaction solution is NAD 0.5
~20 mVml, AMP 0.008-1.
OTng, ACPO, a range of 14 to 5.84 odor is desirable, and an enzyme activator such as magnesium or manganese salt may be added as necessary. As the ACP, ACP produced by reacting acetic anhydride with a phosphate may be added to one portion of the reaction solution. The bacterial cell concentration is preferably in the range of 5 to 50 to 1
The reaction is carried out using a suitable buffer 9, such as a phosphate buffer. Reaction pH 5.0-90, reaction temperature 15-50°C
is promising. For anti-cheating, bacterial cells are separated and purified using an ion exchange resin or the like according to a conventional method.

(Example) Nine examples of the present invention are shown below, but the present invention is not limited to the present invention in any way.

Example l Previbacterium ammoniagenes (ATCo
15812) with 1% glucose, 1% yeast extract,
Culture #L at 80°C for 124 hours in a medium containing 1% peptone. Collect bacteria by centrifugation, and add 0.2 M 17 phosphoric acid buffer containing 20 mM manganese chloride so that the dry cell mass is 20 to 4. # Suspend in solution (pH 7.5) (Make 7 mol/l
concentration of 1 sip] M indicates a concentration of 1000. ). To this solution, add 41% toluene to the solution, stir for 1 hour at room temperature, collect bacteria by centrifugation, and add 02M phosphoric acid buffer H7 containing 20+nM manganese chloride so that the dry cell mass is 20 x 41.゜5).

N A, I) 4.0 mvlnl, A, MP 0
．． 03”!/me, A CP 1.21n7! was added and reacted at 87r for 6 hours. NAD generated in the reaction solution
P was 8.91^e.

Example 2 17v of Brevibacterium ammoniagenes (ATCC15B 1.2) (as a wet bacterial cell) obtained in the same manner as in Example 1 was suspended in a 1°C saline solution in a concave portion, and the suspension was heated to 4°C (after heating to 1°C). , 3% 1 (-1% saline solution containing carrageenan d'
The mixture was mixed at a liquid ratio of 1:1 and added dropwise to a 0°3M potassium chloride aqueous solution to form a gel. This immobilized bacterial cell was dissolved in a 1.2M phosphate buffer (pH 7.5) containing 20mM manganese chloride.
and treated with toluene in the same manner as in Example ① to obtain 68 fixed cells.

Home country 9 (1 [body eNA D 4.Oq/mz, A M
P O,08nVrn1. A CP 1.2 mt4
0.2 M containing e and 20 mM manganese
Suspend in II phosphate buffer and react at 37°C for 6 hours. N A i) P produced after the reaction is 2.5 m
gAne '11' Atsuta.

Example 8 2.5M 21potassium phosphate solution #'40- to water 20-
4. Adjust the pH to 6 with potassium hydroxide at the specified concentration.
1 ml of ice-free acetic acid was gradually added dropwise while adjusting the concentration to 0 to 7.0. After stirring for 1 hour under water cooling, water was added ■00
- adjusted. This reaction solution contains 80 w of A and CP.
vmt, contained.

Separately, Brevibacterium ammoniagenes (ATCC
5812) and Escherichia coli Bi (as moist bacterial cells cultured in a medium containing 1% glucose, 1% peptone, and 1% yeast extract) were mixed together, and 5°4 ml 1.83ml of 30% potassium chloride was mixed under water cooling. 5% acrylamide 5.4ml 1. 5% β-dimethylaminopropionate 1.8d, 6.5% ammonium persulfate 2.1 +
++/ was added and heated to solidify, leaving 802 fixed bacterial cells.

The bacterial cells were suspended in 5 mM')') HCl buffer (pH'7.0) containing 8% toluene, and incubated at 25°C.
Activated by shaking for an hour.

This immobilized bacterial cell is NAI) 4.0 mg/me, A-
M P 0.08^e, ACP synthesis reaction solution obtained above (
80 m@/me) 0.0 ], 5q as ACP
ptJ/atl and 2 OmMfg formed after the MAG reaction was completed.

(Effects) As described above, in the present invention, instead of using a combination of separated enzymes, by using bacteria that simultaneously have the necessary enzyme activity, the expensive A
By using AMP, A, and CP, which are cheaper even without using TP, silicon AMP can be added at less than 1/10 mole per mole of N A D and only one mariyasu is added.

And @Rateyo (NAI) Pi! It is possible to build J.

Claims (4)

[Claims] (1) Nicotinamide adenine dinucleotide, acetyl phosphate and adenosine 5'- A method for producing nicotinamide adenine dinucleotide phosphate from monophosphoric acid. (2) A method for producing nicotinamide adenine dinucleotide phosphate according to claim 1, in which bacterial cells are used as they are in suspension or are immobilized on a carrier. (3) Nicotinamide adenine dinucleotide phos according to claim 1, characterized in that as the acetyl phosphoric acid, a product produced by reacting acetic anhydride and a phosphate is used as a reaction solution. How to make fetes. (4) Nicotinamide adenine dinucleotide according to claim 1, characterized in that adenosy 5'-monophosphoric acid is added in an amount of 1/10 molar equivalent or less to nicotinamide adenine dinucleotide.・Method of producing phosphate.