JPH07100031B2 - NADH oxidase and method for producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to reduced nicotinamide adenine dinucleotide (NADH) oxidase and a method for producing the same. More specifically, the present invention relates to molecular oxygen.
NAD obtained from the bacterium Streptococcus mutans by oxidizing NADH in the presence of (O ₂ ) in a substrate-specific manner to generate hydrogen peroxide (H ₂ O ₂ ).
The present invention relates to NADH oxidase useful for measuring H and the like, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, H₂O₂・ Generating (H₂
O₂ generating) NADH oxidase
Tobacillus plantarum (Lactobacillus plantaru
m) Origin [Agricultural and Biolo
Zical Chemistry (Agricultural and Biologic
al Chemistry), 25, 876, 1961.
Year]; Accolepsezuma Raid Raui (Acholeplasma la
idlawii) From [European Journal
Bu Biochemistry (European Journal of Bio
Chemistry), 120, 329, 1981];
Iconostock Mescenteroides (Leouconostoc 
mesenterodes) Source [Journal of Bioke
Missry (Journal of Biochemistry), 97th
Vol. 127, 1985]; Bacillus megaterium
(Bacillus megaterium) From [Journal of
・ Biochemistry (Journal of Biochemistr
y), 98, 1433, 1985]; Bacillus
Rikeni Holmis (Bacillus licheniformis), Bacillus
・ Subtilis (Bacillus subtilis), Bacillus pumi
Lus (Bacillus pumilus), Bacillus anoirinorite
Ikas (Bacillus aneurinolyticus), Bacillus Sukh
Jericus (Bacillus sphaericus), Bacillus Amilo
Request Fence (Bacillus amyloliquefaciens),
Bacillus cereus (Bacillus cereus), Bacillus
Thuringiensis (Bacillus thuringiensis) Origin
[All of which are JP-A-63-251082]; and
And Brevibacterium ammoniagenes (Brevibacte
rium ammoniagenes), Corynebacterium flavum
Faciens (Corynebacterium flaccumfaciens), A
Lithrobacter Atrussianeus (Arthrobacter atro
cyaneus), Micrococcus flavus (Micrococcus f
ulvus), Pseudomonas aeruginosa (Pseudomonas 
 aeruginosa), Achromobacter Balplus (Achro m
obacter pulpulus), Agrobacterium radiobaku
Tar (Agrobacterium radiobacter), Flavobacterium
Umm Estella Romanicum (Flavobacterium este ra
romaticum), Streptomyces aureus (Stre pt
omyces aureus) Derived [JP-A-63-44882]
Gazette] etc. have been reported.

However, H ₂ derived from these microorganisms
O ₂ · Generating NADH oxidase has no substrate specificity and acts not only on NADH but also on reduced nicotinamide adenine dinucleotide phosphate (NADPH). Therefore, quantification of NADH in a mixture of NADH and NADPH Has the drawback that it cannot be used. In addition, these NADH oxidases are difficult to culture enzyme-producing bacteria and cannot be mass-produced (Acoleplasma raidlawi, etc.), and since they are membrane-bound enzymes, large-scale purification of the enzyme is difficult (Bacillus megaterium, etc.), It is unstable and does not endure long-term storage (Leuconostoc mesencentroides, etc.), and normally requires 5 to 40 hours of enzyme production time (Brevibacterium ammoniagenes, Corynebacterium flacamfaciens, Arthrobacter). -Atorocyaneus, Micrococcus flavus, Pseudomonas aeruginosa, Achromobacter parpras, Agrobacterium radiobactor, Flavobacterium esteroaromaticum, Streptomyces aureus, Bacillus licheniformis, Bacillus subtilis , Bacil Supurumisu, Bacillus anoillinolyticus, Bacillus spherikas, Bacillus
Amyloliquefaciens, Bacillus cereus, Bacillus thuringiensis, etc.).

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a NADH oxidase having a substrate specificity for NADH and capable of long-term storage, which is useful for quantifying NADH. Another object of the present invention is to provide a method for producing such NADH oxidase, which can be mass-produced and purified easily in a short time.

[0005]

In order to achieve the above object, the present inventors extensively searched for NADH oxidase suitable for industrial production. As a result, the facultative anaerobic bacterium Streptococcus mutans is specific to NADH only. It was found that H ₂ O ₂ -generating NADH oxidase that acts on the bacterium is produced, and the enzyme production can be easily carried out by transferring the anaerobic culture to aerobic conditions and agitating the culture for a short time (usually 3 to 4 hours). The present invention has been completed by clarifying that it is possible.

That is, the present invention is directed to NADH in the presence of oxygen molecules.
Is oxidized substrate-specifically to produce hydrogen peroxide, which has a molecular weight of 2
Provided is a NADH oxidase having 00000 to 220,000. Further, the present invention comprises culturing a bacterium Streptococcus mutans ,
There is also provided a method for producing the NADH oxidase, which comprises collecting the NADH oxidase from a culture.

The NADH oxidase of the present invention has the following enzymatic chemical properties.

Action and substrate specificity: NADH of the invention
Oxidase is molecular oxygen as shown in the following reaction formula.
(O_Two) To oxidize NADH to give NAD⁺And hydrogen peroxide
To catalyze the reaction producing. NADH + H⁺ + O₂ → NAD⁺ + H₂O₂  This enzyme acts only on NADH and acts on NADPH
Absent. Properties of this enzyme that cannot oxidize NADPH
Is NA in a biological sample in which NADH and NADPH are mixed.
There is an advantage that the DH amount can be selectively measured.

Molecular weight: The NADH oxidase of the present invention is a superprotein using ribonuclease A, chymotrypsinogen A, ovalbumin, bovine serum albumin, aldolase, catalase and ferritin (pharmacia high molecular weight and low molecular weight protein markers) as standard proteins. From the results of Rose 12 (FPLC system, Pharmacia) gel filtration chromatography, the molecular weight is estimated to be 200,000 to 220,000. In addition, from the results of polyacrylamide gel electrophoresis using sodium dodecyl sulfate using carbonic anhydrase, ovalbumin, bovine serum albumin, ovotransferrin, (BDH molecular weight marker), etc. as standard proteins, this enzyme has a molecular weight of 5.5. It is estimated to be composed of ˜60,000 identical proteins. That is, this enzyme is regarded as a tetramer composed of the same subunits having a molecular weight of 55 to 60,000.

The Km value ("Km" is defined as "the substrate concentration at which the ratio of enzyme reaction product formation rate is 1/2 of the maximum rate V"), and the smaller the value, the higher the affinity between the enzyme and NADH. High.): The Km value of this enzyme is in the range of 10 ⁻⁶ M to 10 ⁻⁵ M. This value indicates that the enzyme has a very strong affinity for NADH. The concentration of NADH in the body is μ
Conventional NADH oxidase, which has an M level and a weak affinity for 10 ⁻⁵ , cannot quantify NADH in a biological sample.

Requirement: The enzyme is activated by the addition of flavin adenine dinucleotide (FAD).

Optimal pH and pH stability: This enzyme has a pH of
Maximum activity at 5.5-7.0, especially around pH 6.0
(See Figure 1). The optimum pH of this enzyme being near neutral is suitable for the measurement of the biological sample itself. Furthermore, the activity of this enzyme is maintained at 90% at pH 5.0 to 8.0, especially pH 6.0 to 8.0, and the pH stability is large (see FIG. 4).

Suitable temperature range of action: phosphate buffer (50 m
M, pH 7.0)
Maximum activity at ~ 50 ° C, especially around 37 ° C-40 ° C
(See Figure 2). The optimum temperature for the action of the enzyme being around body temperature is suitable for measurement of the biological sample itself.

Thermostability: The thermostability of the enzyme was measured with a phosphate buffer (50 mM, pH 7.0). When heated at 50 ° C, about 60% of the activity was retained in 30 minutes (Fig. 3). Furthermore, add ethylenediaminetetraacetic acid to the phosphate buffer.
The heat resistance can be increased by adding (EDTA), NADH, FAD, and DTT.

As described above, the H ₂ O ₂ -generating NADH oxidase of the present invention is used in combination with the quantification method of H ₂ O ₂ to measure the activity of various dehydrogenases involved in NADH and various types of which use NAD as a coenzyme. It is a useful enzyme that can be used to measure the base mass of dehydrogenase.

The above NADH oxidase of the present invention is produced, for example, by culturing the enzyme-producing bacterium Streptococcus mutans and collecting it from the culture.

In one embodiment of the method for producing NADH oxidase of the present invention, Streptococcus mutans is used as the H ₂ O ₂ .generating NADH oxidase producing bacterium. Preferred strains include, for example, Streptococcus mutans NCTC10449, Streptococcus mutans ingbrid (Ingb
ritt) and Streptococcus mutans JC2.

In the medium used in the present invention, the strain used is NA
Any substance that produces DH oxidase may be used, but it is preferable to add carbonate to the medium because it requires carbonic acid for growth. Glucose, mannitol, sorbitol and the like are suitable as the carbon source for producing the enzyme, and casein, trypticase, peptone, yeast extract and the like can be used as the nitrogen source. In addition, inorganic salts such as phosphate, magnesium salt, manganese salt, iron salt and metal salts may be added, and vitamins and nucleic acid bases may be added.

In culturing the NADH oxidase-producing bacterium, the culturing temperature is usually within the range of 30 ° C to 40 ° C, preferably around 37 ° C. Since the strain used in the present invention is a lactic acid bacterium, the pH value is lowered due to the accumulation of acid in the medium as it grows. Therefore, the phosphate of the medium is added to potassium phosphate buffer (for example, 0.1M to 0.2M).
When used in the form of (degree), the productivity of the enzyme can be increased. The strain used in the present invention is a facultative anaerobic bacterium. That is, it can grow well in the anoxic state, but cannot produce the essential NADH oxidase. On the other hand, under aerobic conditions, NADH oxidase can be produced, but the growth is very slow. Therefore, NADH oxidase can be induced by culturing Streptococcus mutans under anaerobic conditions to some extent, then transferring to aerobic conditions, and performing aeration-agitation culture for 2 to 8 hours, preferably 3 to 4 hours. it can. At this time, the production amount of NADH oxidase can be set to the maximum by optimizing the conditions of aeration and stirring, the time of collecting the bacteria, and the like.

Since most of the NADH oxidase produced here is present in the microbial cells, cells are destroyed by a commonly used method after collecting and washing the culture bacterium to obtain a cell-free extract. Then, a purified enzyme can be obtained by a commonly used purification means. For example, purification by salting-out concentration, ion exchange chromatography, adsorption chromatography, gel filtration chromatography and the like can be performed to obtain a polyacrylamide gel electrophoretic homogeneous NADH oxidase sample.

NA of the present invention manufactured as described above
The enzyme activity titer of DH oxidase can be measured, for example, as follows. That is, a phosphate buffer containing NADH and EDTA are prepared in a quartz black cell for measuring ultraviolet light absorption. Add an appropriate amount of NADH to this reaction solution.
By adding oxidase, NADH is oxidized by oxygen in the solution to produce NAD ⁺ . The titer is measured by the time course of the decrease in absorbance at 340 nm specific for NADH. The enzyme activity titer is represented by 1 unit of the amount of enzyme required to oxidize 1 μmol NADH per minute. The titer at the time of adding FAD is determined by adding FAD to the reaction solution and reacting.

[0022]

EXAMPLES The present invention will be described in more detail with reference to examples.

(Example) Ammonium bicarbonate 0.2%, sodium glutamate 0.1%, yeast extract 0.1%,
Glucose 1%, 0.1M phosphate buffer (pH 7.0), inorganic salt solution (magnesium sulfate, manganese sulfate, ferrous sulfate, sodium chloride, etc.) and 0.01% as reducing agent
5L mini jar containing 3L of cysteine medium
The fermentor was inoculated anaerobically with 300 ml of Streptococcus mutans precultured for 12 to 15 hours, and 3
Static culture was performed at 7 ° C. When the growth rate of the fungus reached 0.2 to 0.3 at the absorbance of 660 nm, aeration-agitation culture was started. (Aeration rate: 3 l / min, stirring speed: 300 revolutions /
Minutes). The induced production of NADH oxidase reached a maximum with aeration and stirring for about 3 hours. The amount of cells obtained was 5 g / L. After suspending the cells in a 50 mM phosphate buffer (pH 7.0) containing 0.2 mM EDTA, the cells were disrupted and a crude enzyme solution was obtained by ultracentrifugation fractionation. The crude enzyme solution was subjected to DEAE Sepharose column chromatography preliminarily equilibrated with the same buffer, and eluted with a linear concentration gradient of KCl. H ₂ O ₂ · Generating NADH oxidase active fraction
It was eluted at a concentration of 0.2M to 0.3MKCl. The active fractions were collected and concentrated, and then 1.2 M ammonium sulfate was added.
It was subjected to butyl sepharose column chromatography equilibrated with 0 mM phosphate buffer (pH 7.0) and eluted with a linear concentration gradient of 1.2 M to 0 M ammonium sulfate, and the active fraction was eluted at a concentration of 0.5 M to 0.2 M. It was The active fraction was fractionated by mono Q column chromatography (FPLC system, Pharmacia), which was collected, and then re-chromatographed to separate and purify it as a uniform enzyme by SDS gel electrophoresis. This specific activity was 127 units / mg, and the yield from the crude enzyme was about 5%.

[0024]

INDUSTRIAL APPLICABILITY The present invention provides a production method advantageous for industrial production of NADH oxidase useful for measuring NADH.

[Brief description of drawings]

FIG. 1 is a NADH oxidase activity-pH correlation diagram.

FIG. 2 is a NADH oxidase activity-temperature correlation diagram.

FIG. 3 is a graph showing temperature stability of NADH oxidase activity.

FIG. 4 is a graph showing pH stability of NADH oxidase activity.

Claims (2)

[Claims] 1. An enzyme-producing bacterium, Streptococcus (Strepto)
coccus mutation) culture, optimum pH 5.5
~ 7, NADH oxidase that produces hydrogen peroxide by substrate-specific oxidation of NADH in the presence of oxygen molecules having a suitable temperature of action of 30 to 50 ° C and a molecular weight of 200,000 to 220,000. Wherein culturing the enzyme producing bacterium Streptococcus mutans (Streptococcusmutans), the N from the culture
The method for producing NADH oxidase according to claim 1, wherein ADH oxidase is collected.