JPS62108898A - Purification of nicotinamide adenine dinuclelotide (nad) - Google Patents

Abstract

PURPOSE:To quantitatively obtain nicotinamide adenine dinucleotide (NAD) in high purity, by adsorbing an NAD-containing solution containing inorganic salts, amino acids and unlceotides as impurities on a porous nonionic adsorbent resin and eluting the adsorbed NAD with water, etc. CONSTITUTION:A nicotinamide adenine dinucleotide (NAD)-containing solution containing the NAD and further inorganic salts, amino acids and uncleotides as impurities is adsorbed on a porous nonionic adsorbent resin consisting of a copolymer consisting essentially of styrene and divinylbenzene. The adsorbed NAD is then eluted with neutral - alkaline water or hydrous organic solvent to purify the NAD.

Description

[Detailed description of the invention] (Industrial application field) Nicotinamide adenine dinucleotide (NAD)
J! is an essential compound as a coenzyme for the oxidation-reduction reaction in the body, and plays an important role in energy metabolism and biosynthesis reactions in the body. It is widely used in the fields of biochemistry and clinical testing.

Furthermore, in recent years, NAD and ADP are being reused. It is a compound that is attracting attention because attempts have been made to produce useful substances such as amino acids using a bioreactor that includes a so-called coenzyme regeneration system.

(Prior art) NAD exists in small amounts in microorganisms, and is industrially produced by extraction from yeast or bacterial fermentation. Conventionally, NAD has been purified by adsorption using activated carbon. or precipitation method using metal ions, or using ion exchange resin alone or using ion exchange resin.

Combination method 1 For example, a method using a strongly basic anion exchange resin (H, Ijichi et, aL,
J, BioLChem, VoL 241.8701
(19(5i Ri), W -27898) has been widely used. There is also a method of combining ion exchange resins discovered by the present inventors and highly purifying the product and obtaining crystals of NADJ salt release (Unexamined Japanese Patent Publication No. Showa 57-675
9 (Problems to be solved by the invention) NAD-containing liquids produced by yeast extraction or bacterial fermentation contain large amounts of nucleotides, amino acids, and even inorganic salts. In the activated carbon adsorption method, other nucleotides and amino acids are also adsorbed at the same time, and highly pure NAD-
It is difficult to separate and purify NAD, and highly concentrated ammonia and a large amount of organic solvent are required to extract NAD. In the case of the ion exchange resin method that has been commonly used in the past, a large amount of resin and many types of resin are required due to the influence of coexisting inorganic salts and nucleotides. It requires acids and alkalis, and a further disadvantage is that the eluent is dilute and much energy is expended in concentrating it.

(Means for solving all problems) Selectively extract only NAD from an NAD-containing solution that is produced by yeast extraction method or bacterial fermentation method and contains large amounts of fifcNAD, inorganic salts, amino acids, and nucleotides. As a result of intensive research into methods that could separate the styrene and divinylbenzene with high yield, we found a porous and nonionic material made of a copolymer of styrene and divinylbenzene, or a partially halogenated copolymer of styrene and divinylbenzene. The adsorption resin adsorbs only NAD, and does not adsorb inorganic salts, amino acids, or nucleotides that coexist in large amounts in NAD-containing liquids, and can be removed by washing with water or diluted salts. The inventors discovered that NAD of extremely high purity could be obtained quantitatively by distilling NAD with dilute alcohols or dilute acetones containing these substances, and completed the invention.

Next, one aspect of the present invention will be explained in detail.

The NAD-containing liquid used in the present invention is a liquid containing NAD obtained by a biochemical reaction, such as an extract from microorganisms such as yeast, a reaction liquid obtained by salvage synthesis using bacteria, or a partially purified liquid thereof. If available, it can be used in either case.

71) What is the porous and nonionic adsorption resin that is a copolymer mainly composed of styrene and divinylbenzene?2For example, commercially available Amberlite XAD-2, XA
D-4 (manufactured by Rohm and Haas), Diaion HP-20, HP-30 (Mitsubishi Chemical Industries, Ltd., etc.),
Deolide S-861 (manufactured by Diamond Shamrotsuta Co., Ltd.) is a copolymer of Kaari, Sten and divinylbenzene with a gold main component, a part of which is halogenated, and is a porous and nonionic adsorption resin.For example, Seppapies 5P=206, 5P-207 (Mitsubishi Chemical Industries, Ltd.)
An example of this is Deolide ES-8610 (manufactured by Diamond Shamrotsuta Co., Ltd.). After adjusting the NAD-containing liquid to 7.0 or less.

5091 A porous and non-porous material made of a copolymer of styrene and divinylbenzene that has been regenerated with an organic solvent such as methanol and thoroughly washed with water, or a partially halogenated copolymer of styrene and divinylbenzene. When the liquid is passed through a column filled with ionic adsorption resin gold, inorganic salts and nucleotides are extracted.

Furthermore, amino acids pass through this resin without being adsorbed and become NAD.
is adsorbed. After passing through a small amount of water or dilute foundation column to remove attached nucleotides, etc., NA
Water or a dilute alkali, as well as dilute alcohols and dilute acetone containing them, are used as the zero bath release agent for eluting D. In order to prevent the contamination of coloring components that have a stronger affinity for resin than NAD and to maintain high purity of the NAD fraction.

For dilute alkali, the optimum pH is 10.5 or less, and when mixing dilute alcohols or dilute acetone, the optimum concentration is 20 or less.

Highly purified NAD can be obtained by directly using the NAD solution obtained by the above method or by further purifying the solution if necessary and then concentrating and crystallizing it.

(Example) Examples of the present invention will be shown below, but are not intended to limit the invention in any way.

In this example, the purity (HPLC purity) was measured by high performance dilute chromatography under the following conditions.

Column: TSK-GEL○DS-8 manufactured by Toyo Soda Co., Ltd.
0TM Mobile phase: 0.05M phosphate-potassium water bath solution containing 5% methanol Quantification: Measured by absorbance at 260 nm.

Purity by fermentation method (ADH purity) was measured by the alcohol dehydrogenation method shown below.

M, K Coingenberg, Methods
of Enzymatic Analysis
: 2nd edition, Vol, 4+20
49 (1974) Example 1 Brevibacterium ammoniagenes [AM 1
1,500 d of fermentation filtrate from 641 strains (NAD 8.112
1 containing, p) (6,5) f, Cehahis 5P-2
07 (manufactured by Mitsubishi Chemical Industries, Ltd.) 500- at 5V=2, and washed with 750-water.

Next, it was eluted with 2,000 ml of 10% methanol water.

NAD fraction 1.000 d (NAD 7.947 f
) obtained. After concentrating the obtained NAD fraction under reduced pressure,
As a result of cooling and crystallizing at ℃ for 24 hours, 6.785P of N
AD crystal (HPLCM degree 100%, ADH purity 99.1
%) Example 2 Candida utilis strain KJS-0582 (FB
RM-7396 strain) hot water extract from cultured bacterial cells 1.0
00ml (Contains NAD 662.4■.

(pH 1.0) was passed through a column filled with Amberlite XAD-4 (manufactured by Rohm and Haas) 500 ifc at 5 V = 2.0, and washed with water 500 ml. Subsequently, 750 ml of NAD fraction was eluted with 10% methanol water.
(containing NAD 648.9"j') 1-obtained.

The NAD fraction obtained by elution was concentrated under reduced pressure and incubated at 4°C for 24 hours.
It was cooled for an hour and crystallized. As a result, crystalline NAD5 5
0. OW CHPLCHFfl l 0 0%,
An ADH degree of 99.5% was obtained.

(Young Fruit of the Invention) In order to quantitatively obtain NAl') of extremely high purity in a simple process from an NAD solution coexisting with inorganic salts, amino acids, and nucleotides by the method of the present invention, Significant savings in drugs, energy and time are achieved.

Claims (2)

[Claims] (1) A porous solution made of a copolymer mainly composed of styrene and divinylbenzene, containing a nicotinamide/adenine/dinucleotide-containing liquid containing inorganic salts, amino acids, and nucleotides in addition to nicotinamide/adenine/dinucleotide. adsorbed on a neutral and nonionic adsorption resin,
A method for purifying NAD (nicotinamide adenine dinucleotide), which is characterized by elution with neutral to alkaline water or a water-containing organic solvent. (2) The method for purifying NAD according to claim 1, wherein the copolymer mainly composed of styrene and divinylbenzene is partially halogenated.