JPH0467954B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing NAD synthase. [Conventional technology] Conventionally, NAD synthetase (NAD ⁺ synthetase)
Rat liver [J.Biol.Chem., 233 , 493-500
(1958)], Pig Liver [J.Biol.Chem., 236 , 525~
530 (1961)] Yeast [J.Biol.Chem., 247 , 4794~
4802 (1972)], E.Coli [J.Biol.Chem., 236 , 1494
~1497 (1961)], J.Biol.Chem., 242 , 385-392
(1967), their existence is known. and,
In terms of enzyme classification, Mg ⁺⁺ ATP + Deamide - NAD + NH ₃ AMP + ppi +
NAD synthase (EC6.3.1.5) that catalyzes the NAD reaction
and Mg ⁺⁺ ATP + deamide − NAD + Gln + H ₂ OAPM +
NAD synthase (EC6.8.5.1) that catalyzes the reaction of ppi + NAD + L-glutamic acid
All of them can utilize amide of NH ₃ or Gln, and the difference between them is determined by whether or not they are inhibited by azaserine. To measure the activity of this enzyme, the reduced NAD produced by the reaction is measured using alcohol dehydrogenase (E.
C.1.1.1) and measuring the absorbance of the generated NADH at 340 nm or measuring the generated NAD using a fluorescence method have been reported. but,
Based on this activity measurement method, ATP, deamido-NAD
Even if attempts were made to quantify NH ₃ or Gln, the sensitivity was low, and conventional enzymes were extremely difficult to use as measurement reagents. The present inventors performed an amplification reaction using a coenzyme cycling reaction by combining a redox reaction system using NAD produced by the reaction of the NAD synthase as a coenzyme and a redox reaction system using reduced NAD as a coenzyme. We focused on this by quantifying the components consumed or produced. In the NAD synthase of the present invention, Bacillus
The enzyme produced by Licheniformis B-0844 was newly discovered by the present inventors, and is different from the previously known enzyme.
It is more stable than NAD synthase and is useful as a diagnostic enzyme, and the present invention relates to the enzyme belonging to the genus Bacillus.
This is a method for producing NAD synthase, which is characterized by culturing NAD synthase-producing bacteria in a medium and collecting NAD synthase from the culture. First, the Bacillus licheniformis B-0844 strain was isolated from compost in Ono, Shuzenji-cho, Tagata-gun, Shizuoka, and its mycological properties are as follows. a. Morphological characteristics Using an ordinary agar slant medium, the cells were cultured at 30° C. for 18 to 24 hours and then microscopically observed. (1) Shape and arrangement; The ends are rounded, straight or slightly curved rods, singly or sometimes in short chains. (2) Size: 0.6-0.8 x 1.5-3.0 μm (3) Motility: Moves with pericytium (4) Spore: Formed near the center or edge. The size is 0.8 x 1.5 μm, and the cells sometimes swell. b Growth status on each medium (50°C) (1) Ordinary agar plate medium Gray-white, circular periphery forms flat colonies with a wavy edge. Wrinkles may appear on the surface. No soluble color is produced. (2) Ordinary agar slant medium Grows well in an echnulate shape. It appears grayish white. No soluble pigments are produced. (3) Bouillon medium Grows uniformly and with good turbidity. Later, a bacterial membrane is formed and a fluffy precipitate is produced. (4) BCP milk After 1 to 2 weeks, it solidifies and becomes partially peptonized. c Physiological properties (+: positive, -: negative) Gram staining + catalase production + oxidase production + urease production (SSR medium) - 〃 (Chris, medium) - gelatin hydrolysis + starch hydrolysis + casein hydrolysis - (3 day) Aesculin hydrolysis + Cellulose hydrolysis - Indole production - Hydrogen sulfide production + Acetoin production + MR test + (weak) Nitrate reduction + Denitrification reaction - Citrate utilization + Growth in 7.0% NaCl supplemented medium + Growth at 50℃ + Growth at 20℃ + Production of acids from sugars* (no gas production) Adonitol - L(+)arabinose + cellobiose + dulcitol - meso-erythritol - fructose + fucose - galactose + glucose + glycerin + inositol + inulin - lactose + maltose + mannitol + mannose + melezitose - melibiose + raffinose + L (+) rhamnose + salicin + L sorbose - sorbitol + starch + satucalose + trehalose + xylose + OF test (Huker's method) NT (no change) OF test (modified method) *F (fermentation) *Basal medium (NH ₄ ) ₂ HPO ₄ 1.0g KCl 0.2g MgSO ₄ 7H ₂ O 0.2g Yeast ex 1.0g Agar 3.0g BTB 0.02g Distilled water 1000ml PH7.0 Use Test (as carbon source) D-alanine - L-alanine + fructose - propanol - ethanol - ethylamine - lactate + α-aminobutyrate - glucose + inositol - sucrose + cellobiose - L(+)arabinose - mannose + maltose + rhamnose - trehalose + Acetate - Propionate + DNA cytosine guanine content (%) 45.6%
(Tm modified method) Based on the above mycological properties, this B-0844 strain is
It is a rod-like bacterium with rounded ends, straight or slightly curved, and is characterized by being Gram-positive, forming spores, and decomposing sugars by fermentation. The taxonomic position of this fungus with these characteristics
Bergey's Manual 8th edition, 1974, Medical Bacteria Identification Guide 2nd edition, 1974 and Agriculture Handbook,
427, The genus Bacillus, this bacterium forms spores and can grow under aerobic conditions, so it is determined that it belongs to the genus Bacillus.
Therefore, as bacterial species that can grow at 50℃, (a) Bacillus coagulans, (b) Bacillus licheniformis, (c) Bacillus subtilis, (d) Bacillus brevis, and (v) Bacillus stearothermophilus are listed. It will be done. Comparing and contrasting these properties is as follows [+: positive, -negative, d: reactions differ depending on the strain]

[Table] From the comparison and contrast described above, this strain closely matches Bacillus licheniformis, and the results of comparative examination of other properties also show that it matches well with Bacillus licheniformis. Bacillus licheniformis (Bacillus licheniformis) strain 0844
lichniformis) B-0844. In addition,
This strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as "FERM BP-601".
The above-mentioned Bacillus licheniformis B-0844 is an example of a synthetic enzyme-producing bacterium, and not only this strain, but any bacterium belonging to the genus Bacillus that produces NAD synthase can be used in the present invention. In the present invention, first, NAD-producing bacteria belonging to the genus Bacillus are cultured using a conventional method for enzymatic production.
The form of culture may be liquid culture or solid culture, but
It is desirable to carry out deep aeration agitation culture industrially. As the nutrient source for the medium, those commonly used for culturing microorganisms are widely used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, starch, dextrin, molasses, blackstrap molasses, and glycerin. The nitrogen source may be any available nitrogen compound, such as corn, stew, liquor,
Soybean flour, cottonseed flour, wheat gluten, peptone, meat extract, yeast extract, casein hydrolyzate, etc. are used. Other salts such as phosphate, magnesium, calcium, potassium, sodium, zinc, iron, manganese, and halogen are used as necessary. The culture temperature is such that NAD synthetase producing bacteria grow.
It may be changed as appropriate within the scope of producing this enzyme, but 26
~50°C is preferred. Although the culture time varies depending on the culture conditions, it is usually about 15 to 40 hours. The culture should be terminated at an appropriate time by determining the time when the enzyme reaches its maximum titer.If aeration and agitation are used,
A condition of 200 to 400 rpm is sufficient. NAD synthase is collected from the culture of the NAD synthase-producing bacteria obtained in this way, but since this enzyme is mainly contained within the bacteria,
The resulting culture is collected by means such as filtration or centrifugation, and the bacteria are destroyed using mechanical destruction methods such as ultrasonication, French press treatment, or glass bead treatment, or enzymatic destruction methods such as lysozyme. Also, if necessary, use Triton X-100 (Triton
A surfactant such as Adecatol SO-120 (trade name) may be added. The NAD synthase-containing solution obtained in this way is either concentrated or
Alternatively, without concentrating, the enzyme may be salted out using a soluble salt such as ammonium sulfate, or the enzyme may be precipitated using a hydrophilic organic solvent such as methanol, ethanol, acetone, isopropanol, etc. After dissolving this precipitate in water or a buffer solution, it is dialyzed with a semi-permeable membrane as necessary, and then DEAE-cephadedex, DEAE-cepharose, or DEAE
- Chromatography and Sephadex using ion exchange resins such as cellulose, carboxymethyl-cellulose, carboxymethyl-Sepharose, carboxymethyl-Sephadex, etc.
G200, Cepharose CL-6B, Cephacryl S-
The NAD synthetase produced can be obtained by purification by chromatography using a gel filtration agent such as molecular sieve 200, followed by treatment such as freeze-drying. Next, the properties of the NAD synthase obtained by the present invention will be described. (1) Molecular weight: Approximately 62,000 (by Cephadex G-150 gel filtration) (2) Isoelectric point: around PH4.6 (by electrophoresis using ampholite) (3) Action: Mg ⁺⁺ ATP + Deamide-NAD + NH ₃ →AMP＋ppi＋
NAD (4) Substrate specificity: In addition to _NH3 , it also acts on L-Gln and L-Asn to catalyze the following reaction. ATP + deamino-NAD + Gln (or A Mg ⁺⁺ sn) → AMP + ppi + NAD + Glu (or Asp) (5) Extract PH The buffer solution of reaction solution 1 for enzyme activity assay was acetate buffer (PH 3.8 to 6.6), dimethyl glutaric acid. - The results of measuring enzyme activity in addition to sodium hydroxide buffer (PH5.1-6.8) and Tris-HCl buffer (PH6.5-8.8) are shown in FIG. PH
It has an optimal pH around 8.0 to 8.7. (6) PH stability This enzyme was dissolved in 50mM acetate buffer (PH3.9-6.8),
Dimethylglutaric acid-dissolved in sodium hydroxide buffer (PH4.1-7.1), phosphate buffer (PH6.3-7.9) and Tris-HCl buffer (PH6.4-8.9) and treated at 37°C for 60 minutes. After that, the residual activity was measured according to the enzyme activity measurement method.
As shown in the figure. Stable within the pH range of 5.5 to 9.0. (7) Thermostability After dissolving this enzyme in 50mM Tris-HCl buffer (PH6.8) and heating it at each temperature for 10 minutes, the residual activity was measured according to the enzyme activity assay method. As shown in Figure 3. Stable up to 40℃. (8) Effect of surfactant In the enzyme activity measurement method, the surfactant listed in Table 1 was added to the reaction solution at a concentration of 0.1%, and after heating to 37%, 5μ of the enzyme solution was added. 37
After reacting at °C for 10 minutes, add 0.8 ml of reaction solution,
After reacting at 37℃ for exactly 5 minutes, add 0.1N hydrochloric acid.
Stop the cycling reaction by adding 2.0 ml of
Colorimetric determination was performed at 550 nm. The results are shown in Table 1. It was not affected by nonionic surfactants, but was inhibited by cationic and anionic surfactants.

[Table] (9) Effect of metal ions In the enzyme activity measurement method, reaction solution (20mM
After adding various metal salts to a final concentration of 1 ml (containing _MgCl2 ), the enzyme activity is expressed as relative activity according to the enzyme activity measurement method.
As shown in the table.

[Table] (10) Enzyme activity measurement method Activity measurement reaction solution 50mM Tris-HCl buffer PH8.0 20mM KCl 20mM MgCl ₂ 0.05% Bovine serum albumin 2mM ATP 0.5mM Deamide-NAD 25mM (NH ₄ ) ₂ SO ₄ reaction Solution 50mM Tris-HCl buffer PH8.0 10U Diaphorase/ml (Toyo Jozo, derived from Bacillus genus producing bacteria) 8% Ethanol 10U Alcohol dehydrogenase/ml (Toyobo, derived from yeast) 0.025% NTB (Nitrotetrazolium blue) 0.1% Take 10.2 ml of Triton was stopped and the cycling reaction was started. The cycling reaction was carried out at 37°C for exactly 5 minutes.
After the cycling reaction was stopped by adding 2.0 ml of 0.1NHCl, the absorbance at 550 nm was measured, and the enzyme activity was determined from the value at that time. The activity was calculated according to the following formula. NAD synthase activity (mU/ml) = Δ550/Δ550×1.0/0.005×f/10 Δ550: Absorbance of sample Δ550: Absorbance of standard solution (0.1mMNAD) 0.005: Sample volume (ml) 10: Reaction time f: Dilution Magnification Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto. Example 1 1% peptone, 1% meat extract in an 80-volume jar,
Liquid medium containing 0.2% yeast extract and 0.3% NaCl (PH
7.3) After sterilizing at 120℃ for 20 minutes, inoculate 200ml of seed culture pre-cultured in a medium with the same composition as above, and stirring at 50℃ for 16 hours with aeration rate of 20/min.
Aerated culture was performed at 300 rpm/min. After culturing, cells are collected by centrifugation, and the collected cells are added to 10mM Tris-HCl buffer (PH8.0) containing 0.1% lysozyme.
2 and reacted at 37°C for 30 minutes to lyse the bacteria. This solution was centrifuged at 500 r.p.m for 10 minutes, and the supernatant was 1.9
I got it. Ammonium sulfate was added to this supernatant to perform ammonium sulfate fractionation (0.44 to 0.54 saturation), and this precipitate was dissolved in 200 ml of 10 mM Tris-HCl buffer (473 U) and dialyzed against this buffer 12. Insoluble matter generated in the dialysate was removed by centrifugation (12000 rpm, 10 minutes). Supernatant (462U) was diluted with 10mM Tris-
Charge a DEAE-Sepharose CL-6B column (5 x 30 cm) buffered with HCl buffer pH 8.0,
Elution was performed using a concentration gradient method from 0 to 0.5M NaCl. 0.15
Collect the fractions eluted at ~0.2 M NaCl (120 ml,
388U) After concentration using Amicon ultrafiltration membrane PM-10, Sephadex G-150 (3.6 x 80cm)
The active fractions were collected and used as a purified sample (86 ml, 324 U). In addition, bovine serum albumin, glucose, maltose, mannitol, sucrose, and fructose were added to each of these preparations at a concentration of 1%, and the mixture was freeze-dried. The product without additives had an activity yield of 86%, but the product with additives showed no decrease in activity and was stable. Reference example 1 Reaction solution 50mM Tris-HCl buffer (PH8.0) 20mM KCl 20mM MgCl ₂ 0.05% Bovine serum albumin 1mM Deamino-NAD 50mM (NH ₄ ) ₂ SO ₄ 400mU NAD-synthase/ml Reaction solution 50mM Tris- HCl buffer (PH8.0) 20U Diaphorase/ml 3% Ethanol 20U Alcohol dehydrogenase/ml 0.05% NTB 0.1%, Triton
Add 5μ of each of 0, 5, 10, 20, 30, and 40μM ATP solutions, react at 37°C for 10 minutes, add 0.3ml of the reaction solution, react at 37°C for exactly 5 minutes, and then add 0.1 Add 2.0ml of NHCl to stop the reaction,
Absorbance was measured at 550 nm. The results are shown in FIG. 4, and good linearity was obtained. The cycling reaction rate in this reaction was 4800 revolutions/hour. Reference Example 2 Equal amounts of the reaction solution shown in Reference Example 1 and the reaction solution were mixed and heated to 37°C. 1.0 ml of this mixture was added to a quartz cell (1.0
ml) and the same concentration as used in Example 1.
5μ of ATD solution was added, and the change in absorbance was measured at 550 nm for 2 minutes from 5 minutes to 7 minutes after addition.
The results are shown in Figure 5, and high sensitivity and good linearity were obtained even in the kinetics method. The rotation rate at this time was approximately 2800 revolutions/hour. Reference Example 3 Various concentrations of ammonium sulfate, L-glutamine, and L-asparagine were added to the reaction solution shown in Reference Example 1, in which 5mM ATP was added instead of 50mM ammonium sulfate, and the same operation as in Example 2 was performed. . The results are shown in FIG. 6, and good linearity was obtained with ammonium sulfate, L-glutamine, and L-asparagine. 〇-〇: Ammonium sulfate, ●-●: L-glutamine, Δ-
Δ: L-asparagine.

[Brief explanation of the drawing]

Figure 1 shows the optimal PH curve of NAD synthase produced by Bacillus lichenformis B-0844, Figure 2 shows the PH stability curve of the enzyme, Figure 3 shows the thermostability curve of the enzyme, and Figure 4 5 shows the ATP quantification curve according to the end point method according to the present invention, FIG. 5 shows the ATP quantification curve according to the kinetics method, and FIG. 6 shows the L-glutamine and L-asparagine quantification curve according to the end point method.

Claims (1)

[Scope of Claims] 1. A method for producing NAD synthase, which comprises culturing an NAD synthase-producing bacterium belonging to the genus Bacillus in a medium, and collecting NAD synthase from the culture. 2. The production method according to claim 1, wherein the NAD synthase producing bacterium is Bacillus licheniformis B-0844 (FERM BP-601).