JPH0733B2 - Purification method of dehydrogenase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for purifying dehydrogenase, and more particularly to a reduced nicotinamide adenine dinucleotide dehydrogenase or a reduced nicotinamide adenine dihydrogenase from a microbial cell. The present invention relates to a method for easily and highly purifying nucleotide phosphate dehydrogenase by affinity chromatography under a high salt concentration.

(Prior Art) Reduced nicotinamide adenine dinucleotide dehydrogenase or reduced nicotinamide adenine dinucleotide phosphate dehydrogenase [hereinafter abbreviated as NAD (P) H dehydrogenase] is
The following formula [NAD (P) represents NAD (nicotinamide adenine dinucleotide) or NADP (nicotinamide adenine dinucleotide phosphate), NAD (P) H represents NADH (reduced NAD) or NADPH (reduced NADP) Represent ], Which is an enzyme that catalyzes the oxidation of NAD (P) H in the presence of an acceptor, is also called NAD (P) H: acceptor oxidoreductase, and is widely used as one of the reagents for clinical tests. ing.

Specifically, the amount of change in the target quantitative product is
After formulating the amount of change in NAD (P) H (formula), NAD (P)
This is a method of reducing the acceptor using H dehydrogenase (formula) and colorimetrically quantifying the degree of coloration with visible light.

(Problems to be Solved by the Invention) NAD (P) H dehydrogenase is not directly utilized and NAD is directly used.
There is also a method (using the formula) of measuring the amount of change in (P) H at 340 nm in the ultraviolet region, but the amount of change in the target quantitative product is determined by NAD.
If the reaction equilibrium leading to the amount of change in (P) H is not in the direction of NAD (P) H production, accurate quantification of the target substance is difficult.

On the other hand, the reaction of NAD (P) H dehydrogenase is almost irreversible, so by combining the reaction (formula) of NAD (P) H dehydrogenase at the end of the reaction system, the reaction proceeds in the desired direction Can be accurately determined.

Furthermore, if a dye having a large molecular extinction coefficient is used as an acceptor, the sensitivity is increased accordingly. Thus, NAD
(P) H dehydrogenase is an important enzyme in clinical tests, and its demand has been increasing in recent years.

Methods for collecting NAD (P) H dehydrogenase from microbial cells have been known in the literature, for example, Atkinson et al., THE AMERICAN TYPE CULTURE COLLECTI.
Bacillus stearothermop in ON catalog
A method for purifying NAD (P) H dehydrogenase from hilus NCA 1503 (ATCC 7954) is reported. (Jounal
of Applied Biochemistry, Volume 1, 247, 1979).

Conventionally, in order to collect NAD (P) H dehydrogenase from microbial cells, after crushing the cells, extraction with a solution containing salt or surfactant, removal of nucleic acid, and fractionation by salting out with ammonium sulfate After that, column chromatography using various carriers has been carried out. Especially as column chromatography, ion exchange chromatography such as DEAE (diethylaminoethyl)-
Alternatively, a complicated process of various combinations of TEAE (triethylaminoethyl) -cellulose, adsorption chromatography such as hydroxyapatite or gel filtration chromatography such as appropriately cross-linked dextran gel or polyacrylamide gel has been involved. Therefore, it takes not only days until NAD (P) H dehydrogenase as a product is obtained, but also the recovery rate of NAD (P) H dehydrogenase decreases due to the increase in the number of times of chromatography.

Furthermore, since various substances derived from bacterial cells are mixed in the NAD (P) H dehydrogenase extract from the bacterial cells,
High-purity NAD (P) H is still obtained by the process described above.
Obtaining dehydrogenase was quite difficult. For example, NAD (P) H dehydrogenase from Clostridium kluyverii is an archive of Biochemistry and Biophysics, 13
As described in Volume 2, p. 91, 1969, its industrial products contain large amounts of heterologous proteins.
This industrial product is further purified by gel filtration chromatography and ion exchange chromatography. As a result, the degree of purification is improved 30 times, but still
It is only purified to a purity of about 90%.

Recently, Mains et al. NAD from Bacillus stearothermophilus.
We reported on the purification of (P) H dehydrogenase.
(The Biochemical Journal, 191, 457, 1980).

That is, after crushing the cells, sodium chloride was added and the mixture was treated at 70 ° C., followed by DEAE-cellulose chromatography, hydroxyapatite chromatography and Cibacron Blue 3GA (Reactive Blue 2 registered trademark of Ciba-Geigy)- Purification is performed by agarose chromatography. However, a small amount of heterologous protein still remains.

Thus, the purification of NAD (P) H dehydrogenase is complicated, and it is difficult to obtain highly pure NAD (P) H dehydrogenase.
Although (P) H dehydrogenase is expensive to produce, it is said to be important as a reagent for clinical examination, but it is a serious obstacle to its use. Therefore, high-purity NAD
The probability of a method for efficiently obtaining (P) H dehydrogenase on an industrial scale is strongly desired.

(Means for Solving the Problems) From these viewpoints, the present inventors have earnestly studied for the purpose of providing a method for efficiently obtaining highly purified NAD (P) H dehydrogenase by a simple purification operation. The present invention has been achieved by discovering that all of the above objects can be achieved only by performing affinity chromatography under high salt concentration as chromatography.

That is, according to the present invention, a crude enzyme solution containing NAD (P) H dehydrogenase obtained from a microbial cell is contacted with a specific adsorption carrier of NAD (P) H dehydrogenase in the presence of 0.5 M or saturated concentration of salts, NAD (P) H dehydrogenase is adsorbed on the adsorption carrier, and then adsorbed NAD (P) H
A method for purifying dehydrogenase, which comprises eluting dehydrogenase.

As the microbial cell used in the present invention, any microbial cell having an NAD (P) H dehydrogenase-producing ability can be used, and the origin is not limited,
Industrially, Bacillus stearothermophilus, which produces a large amount of thermostable NAD (P) H dehydrogenase, is preferable.
As a crude oxygen solution containing NAD (P) H dehydrogenase obtained from these microbial cells, for example, the microbial cells are suspended in water or a buffer solution and subjected to an autolysis method, a freeze-thawing method, a homogenizer method, a grinding method, a high pressure method. Alternatively, the supernatant liquid obtained by disrupting by osmotic shock method or the like and centrifuging may be used. Further, a solution obtained by treating this product with nucleic acid by protamine sulfate, streptomycin sulfate, polyethyleneimine, aqueous two-phase partitioning method or the like may be used, or further subjected to ammonium sulfate fractionation, pH treatment, heat treatment, etc. A solution may be used.

The NAD (P) H dehydrogenase-containing crude enzyme solution thus obtained and NAD (P) H dehydrogenase-specific adsorption carrier were mixed with 0.5
NAD (P) H dehydrogenase can be specifically adsorbed to the above carrier by simply mixing them or by passing them through the above carrier packed in a column in the presence of salts of M concentration or saturation concentration. At this time, it is necessary to carry out in the coexistence of salts at a concentration of 0.5 M to a saturation concentration in order to prevent adsorption of a heterologous protein other than NAD (P) H dehydrogenase to the carrier. For that purpose, for example, a salt having a concentration of preferably 1 M to a saturated concentration may be added to a crude oxygen solution containing NAD (P) H dehydrogenase in advance and used. Examples of the salt used include chloride ion, bromide ion,
Sulfate ion, phosphate ion, acetate ion, carbonate ion,
Examples thereof include those in which anions such as borate ions are appropriately combined with cations such as sodium, potassium, lithium, ammonium, and magnesium. Usually, sodium chloride, potassium chloride, ammonium sulfate, sodium phosphate, potassium phosphate. Are suitable.

Next, in the present invention, NAD (P) H adsorbed on the carrier is used.
It is necessary to elute the dehydrogenase from the carrier. For that purpose, first, a carrier having NAD (P) H dehydrogenase adsorbed thereon, preferably at a concentration of 0.5 M to a saturated concentration,
Particularly preferably, it is desirable to wash with a buffer solution containing the above-mentioned salts at a concentration of 1 M to a saturated concentration to remove the foreign protein not adsorbed on the carrier. The pH of this buffer is 5
10, particularly 6-9, and more preferably 7-9. After washing in this manner, NAD (P) H dehydrogenase can be eluted with an eluent described below, for example, to obtain NAD (P) H dehydrogenase of extremely high purity. As the eluent, for example, a buffer containing 0.2 mM to 10 mM, particularly preferably 0.5 mM to 1 mM NADH or NADPH is used. P of this buffer
H is preferably 5 to 10, particularly 6 to 9 and more preferably 7 to 9.

The elution method may be a so-called batchwise elution method in which a buffer solution is added all at once, or a so-called concentration gradient elution method in which the buffer solution is continuously raised to elute.

The specific adsorption carrier of NAD (P) H dehydrogenase used in the present invention may be, for example, a dye compound bound to a water-insoluble carrier and specifically adsorbing NAD (P) H dehydrogenase. Anything is acceptable. Among these, a water-insoluble carrier having Active Blue 2 (color index number 61211) as a ligand is preferable. Examples of the water-insoluble carrier include polysaccharides such as cellulose, dextran, agarose and starch, cellulose acetate, polyvinyl alcohol derivatives, polystyrene, polypropylene, polyethylene, polyvinyl chloride, poly (methylmethacrylic acid) ester, polybutene, Polypentene, polyvinylidene chloride, polyacrylonitrile, polymethacrylic acid, polyacrylic acid, polyaminostyrene, polybutadiene, polyisoprene, polymaleic acid monoester, crosslinked polyacrylamide, polymethacrylamide,
Polyvinylamine, poly (dialkylaminoethylmethacrylate), poly (dialkylaminomethylstyrene), poly (vinylpyridine), poly (vinylpyrrolidone), polyacrylic anhydride, polymethacrylic anhydride, polymaleic anhydride, Polymethacrylonitrile, poly (trifluoroethylene), poly (tetrafluoroethylene), poly (divinylbenzene), poly (α
-Methylstyrene), poly (N-vinylamine), poly (tetramethylene glycol divinyl ether), polyvinyl sulfone, polyvinyl sulfoxide, polyacrolein, polymethyl vinyl ketone, and other polymers containing unsaturated carbon-containing monomers, and polyphenylene. Oxoxide,
Polymethylene oxyad, polyethylene oxide, polytetramethylene oxide, and other polyethers, polyalanine, polyphenylalanine, and other polypeptides,
Nylon-3, Nylon-4, Nylon-5, Nylon-6, Nylon-7, Nylon-11, Nylon-12, Nylon-6.6,
Polyamide such as nylon-6.10, poly (m-phenylene-isophthalamide), poly (p-phenylene-terephthalamide), polycarboxylic acid such as terephthalic acid, isophthalic acid, adipic acid, maleic acid, fumaric acid, trimellitic acid, Polyesters derived from polyols such as ethylene glycol, propylene glycol, butylene glycol, pentaerythritol, and bisphenol A, polyesters derived from glycols, lactic acid, hydroxypyridalic acid, dimethylpolysiloxane, methylphenylpolysiloxane, Silicon rubber such as methyl vinyl polysiloxane, dianoalkyl methyl polysiloxane, fluoroalkyl methyl polysiloxane, triene diisocyanate, xylene diisocyanate, phenyle Polyurethanes and phenols derived from polyisocyanates such as diisocyanate, ethylene diisocyanate diphenylmethane diisocyanate and toluentisocyanate, and polyols such as polyethylene glycol, polypropylene glycol and polyesters having OH groups at both ends -Formaldehyde resin, xylene-formaldehyde resin,
Formaldehyde resins such as urea-formaldehyde resin and melamine-formaldehyde resin, polymers containing 4-membered rings such as polyimide, polybenzimidazole and polythiazole, polycarbonate, synthetic polymers such as polysulfone and glass, asbestos, clay, mica, hydroxyl Inorganic derivatives such as apatite, activated carbon, silica gel and alumina, and synthetic inorganic polymers such as polyphosphazene are listed. Of these, polysaccharide derivatives such as cellulose, dextran, agarose and starch are particularly preferable. This NAD
In order to obtain a specific adsorption carrier for (P) H dehydrogenase, the dye compound may be bound to the water-insoluble carrier by the method described in, for example, biochim.biophys.acta 358, 1-13 (1974).

Particularly preferred specific examples include commercially available Martrex Blue (manufactured by Amicon), Affi-Gel Blue (manufactured by Bio-Rad), and Blue Sepharose CL-6B (manufactured by Pharmacia).

(Effects of the Invention) According to the present invention, NAD (P) H dehydrogenase can be economically obtained with a simple operation and higher yield and purity than any known method.

In addition, to perform affinity chromatography,
Normally, it was necessary to consider the type, concentration, pH, etc. of the buffer solution, and therefore it was necessary to perform dialysis treatment or the like in advance with a fixed buffer solution, but in the present invention, such a complicated treatment is not necessary, and salt is added. All you have to do is add.

(Examples) Hereinafter, the present invention will be described more specifically with reference to Examples.

The enzyme activity of NAD (P) H dehydrogenase was 0.06m.
M-2.6-dichlorophenol indophenol and 1 mM
NAD (P) in 20 mM phosphate buffer (pH 7.5) containing NADH
It was determined by measuring the amount of decrease in the absorbance at 600 nm per unit time when H dehydrogenase was added, and the enzyme activity for decreasing 1 μmol of 2.6-dichlorophenolindophenol was defined as one unit.

Example 1, Comparative Example 1 1 kg of wet bacteria of Bacillus stearothermophilus NCA1503 (ATCC 7954) obtained by a known method was added to 25 mM phosphate buffer containing 2 M potassium chloride and 2 mM 2-mercaptoethanol. Suspend in 2 liters of liquid (pH 7.5) and use Dynomill (Willy
A.Bachofen Manufacuturing Engineers) was used to destroy the cells, and then the insoluble matter was removed by centrifugation to remove NAD.
A crude enzyme solution containing (P) H dehydrogenase was obtained. This crude enzyme solution was passed through a column with an inner diameter of 4.4 cm and a height of 25 cm filled with Martrex Blue A (Amicon), and then 2 M
Non-adsorbed heterologous protein was completely removed by passing 1 l of 25 mM phosphate buffer (pH 7.5) containing 2 mM of 2-mercaptoethanol and 2 mM of potassium chloride. When elution was carried out by a linear concentration gradient method using a buffer solution containing NADH, the target NAD (P) H dehydrogenase was eluted near the NADH concentration of 0.5 mM. The NAD (P) H dehydrogenase thus obtained had a yield of 78 mg and a yield of 96%, and the specific activity was 3,800 units per 1 mg of NAD (P) H dehydrogenase.

In addition, acrylamide disc electrophoresis showed a single band.

For comparison, the NAD (P) H dehydrogenase-containing crude enzyme solution obtained in the same manner as above was thoroughly dialyzed against 25 mM phosphate buffer (pH 7.5) containing 2 mM 2-mercaptoethanol, and then the same buffer was used in advance. The solution was passed through a Matrex Blue A packed column (internal diameter 4.4 cm, height 25 cm) that had been equilibrated with the liquid. NAD (P) H supplied to the column in the non-adsorbed eluate at this time and in the adsorbed eluate of the subsequent washing operation with a phosphate buffer (pH 7.5) containing 2-mercaptoethanol
NAD (P) H dehydrogenase activity of about 30% of the dehydrogenase activity was observed. Subsequently, elution was carried out by the linear concentration gradient method using the same buffer solution and a buffer solution containing 2 mM of NADH. NAD (P) was found to be near the concentration of 0.4 mM of NADH.
H dehydrogenase eluted. The yield was 60%, the specific activity was about 520 units / mg, and bands of NAD (P) H dehydrogenase and many other heterologous proteins were observed by acrylamide disk electrophoresis.

As described above, by performing chromatography in the presence of 2M potassium chloride, a surprisingly high purification efficiency was achieved compared to the conventional method.

Example 2, Comparative Example 2 500 g of wet bacteria of Bacillus stearothermophilus NCA 1503 (ATCC 7954) obtained by a known method was suspended in 2 l of 0.1 M phosphate buffer (pH 7.2), and French press After the cells were disrupted by using, the insoluble matter was removed by centrifugation to obtain a supernatant containing NAD (P) H dehydrogenase.
1 liter of a 1% aqueous solution of protamine sulfate was added to 2 liters of this supernatant, and after sufficiently stirring, the resulting precipitate was removed by centrifugation to obtain a protamine-treated supernatant. After adding 1 M ammonium sulfate to this supernatant, Blue Sepharose CL-
It was passed through a column having an inner diameter of 4.4 cm and a height of 10 cm filled with 6B (manufactured by Farmacia), and then a 0.1 M phosphate buffer solution (pH 7.2) containing 1 M ammonium sulfate was passed. Continued 1 mM NADH
NAD (P) H dehydrogenase was eluted with 0.1 M phosphate buffer containing The NAD (P) H dehydrogenase thus obtained had a yield of 36 mg and a yield of 90%, a specific activity of 3,600 units / mg, and showed a single band on acrylamide disk electrophoresis.

For comparison, the above-mentioned protamine-treated supernatant was dialyzed against 0.1 M phosphate buffer (pH 7.2) and then equilibrated with Blue Sepharose CL-6B column (inner diameter 4.4 cm, (10 cm in height), and then 0.1 M phosphate buffer (pH 7.2) containing 1 M ammonium sulfate was passed. NAD (P) H supplied to the column by these operations
Approximately 22% of dehydrogenase activity was shed. Subsequently, a 0.1 M phosphate buffer containing 1 mM NADH was passed through the column to elute the adsorbed NAD (P) H dehydrogenase. The yield of NAD (P) H dehydrogenase obtained was 5
5%, specific activity was 2,100 units / mg, and many bands were observed by acrylamide disk electrophoresis.

Example 3, Comparative Example 3 1 kg of wet cells of Bacillus stearothermophilus NCA 1503 (ATCC 7954) obtained by a known method was suspended in 2 l of 0.1 M potassium phosphate buffer (pH 7.5). , Dynomil was used to destroy the cells, and then treated with bovine pancreatic deoxyribonuclease (10 μg / ml) at 23 ℃ for 15 minutes, and the insoluble matter was removed by centrifugation to obtain crude oxygen containing NAD (P) H dehydrogenase. Got Sodium chloride was added to this crude enzyme solution to a concentration of 2M, and the mixture was stirred at 23 ° C for 1 hour, then at 70 ° C for 1 hour, cooled to 5 ° C, and centrifuged to remove insoluble proteins. Obtained NAD
The (P) H dehydrogenase solution is passed through a column having an inner diameter of 4.4 cm and a height of 25 cm packed with Martrex Blue A, and then 1 l of 50 mM potassium phosphate buffer (pH 7.5) containing 2 M potassium chloride is passed. After completely removing the non-adsorbed heterologous protein, the above buffer and 2 mM NAD were added.
Elution was performed by the linear concentration gradient method using a buffer solution containing H, and the target NAD (P) H was found near the NADH concentration of 0.5 mM.
Dehydrogenase was eluted.

The NAD (P) H dehydrogenase thus obtained is
The yield was 75 mg and the yield was 92%, and the specific activity was 3,750 units / mg. In addition, acrylamide disc electrophoresis showed a single band.

For comparison, the NAD (P) H dehydrogenase solution obtained in the same manner as above was treated with 50 mM potassium phosphate buffer (pH 7.
5) After sufficient dialysis, Martrex Blue A packed column (inner diameter 4.4c
m, height 25 cm). About 22% of the NAD (P) H dehydrogenase activity supplied to the column was detected in the non-adsorbed eluate and the eluate in the washing operation with phosphate buffer.
NAD (P) H dehydrogenase activity was confirmed. Subsequently, elution was carried out by the linear concentration gradient method using the same buffer and a buffer containing 2 mM of NADH, and NAD (P) H dehydrogenase was eluted near the concentration of 0.4 mM of NADH.

The NAD (P) H dehydrogenase thus obtained is
The yield was 63%, the inactivity was 500 units / mg, and bands of many heterologous proteins other than NAD (P) H dehydrogenase were recognized by acrylamide disk electrophoresis.

Claims (3)

[Claims] 1. A crude enzyme solution containing reduced nicotinamide adenine dinucleotide dehydrogenase or reduced nicotinamide adenine dinucleotide phosphate dehydrogenase obtained from microbial cells is treated with reduced nicotinamide adenine dinucleotide dehydrogenase or reducing agent nicotinamide adenine. The reduced nicotinamide adenine dinucleotide dehydrogenase or the reduced nicotinamide adenine dinucleotide phosphate dehydrogenase is contacted with a specific adsorption carrier of dinucleotide phosphate dehydrogenase in the presence of salts at a concentration of 0.5 M to a saturation concentration to obtain the adsorption carrier. Adsorbing to and then eluting the adsorbed reduced nicotinamide adenine dinucleotide dehydrogenase or reduced nicotinamide adenine dinucleotide phosphate dehydrogenase A method for purifying dehydrogenase, which comprises: 2. The purification method according to claim 1, wherein the microbial cell is Bacillus stearothermophilus. 3. A specific adsorption carrier for reduced nicotinamide adenine dinucleotide dehydrogenase or reduced nicotinamide adenine dinucleotide phosphate dehydrogenase is Reactive Blue 2 (color index number).
The purification method according to claim 1, which is a water-insoluble carrier having 61211) as a ligand.