JPS6344882A - Production of nad(p)h oxidase - Google Patents

Abstract

PURPOSE:To obtain the titled substance from a culture mixture, by cultivating a bacterium belonging to the genus Brevibacterium, Corynebacterium, Arthrobacter, etc., capable of producing NAD(D)H oxidase and to use the substance to measure NAKH or NADPH. CONSTITUTION:A bacterium such as Brevibacterium ammoniagenes, Corynacterium flaccumfaciens, etc., belonging to the genus Brevibacterium, Corynebacterium, Arthrobacter, Micrococcus, Pseudomonas, Achromobacter, Agrobacterium, Flavobacterium or Streptomyces, capable of producing NAD(P) H, is cultivated. The culture is carried out at about 20-40 deg.C at pH about 6-8, a cell-free extracted solution is obtained from the mold, from which the aimed substance is obtained by chromatography, gel filtration, etc., industrially.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to reduced nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) and reduced nicotinamide adenine dinucleotide phosphate (hereinafter NA: [)PH and MAD(P)H produced by oxidation of peroxide
This invention relates to a method for producing oxidase using microorganisms.

[Conventional technology]

Nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) and nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as DP and T) are coenzymes of various dehydrogenases, and NADH and By fixing NADPH, it is possible to measure dehydrogenase activity or the amount of its substrate, and this measurement method is widely used in clinical analysis, food analysis, etc.

One way to measure NADH or NADPH is to use the unique absorption of NADH and NADPI (34).
There is a method of measuring the increase in 0 nm, or a method of colorimetrically fixing a formazan dye produced by reacting with a tetrazolium salt. But 340n! The method of measuring from the absorbance of No. 11 had a problem in sensitivity because the molecular extinction coefficients of NADH and NADPH were not very large.

Furthermore, in the method that leads to the production of formazan dye, because the formazan color patch is totally soluble, there are drawbacks such as the color chart precipitating or adhering to cells and tubes.

On the other hand, NAD(P)H oxidase □-ase, which oxidizes NADH and NADPH to generate hydrogen peroxide, is known. If this enzyme is used, hydrogen peroxide can be generated from NADH or NADPH, and a highly sensitive ratio can be obtained. The method can be applied to the color method and the fluorescence method, making it possible to perform measurements that eliminate the drawbacks of the above two methods. Conventionally, N generates hydrogen peroxide.
AD(P)H oxidase is derived from Lactobacillus plantarum (Agricultural and Biological Chemistry, Vol. 25, p. 876, 196
1 year), from Acholeplasma leiderai (European Journal of Fist Biochemistry, No. 12)
0, p. 329, 1981) and an enzyme derived from Bacillus megaterium (Journal Kenop Biochemistry, vol. 98, p. 1433, 1985).

[One problem to be solved by the invention] However, these microorganisms have a drawback of low enzyme productivity. There has been no report on a method for easily obtaining stable NAD (PIH oxidase) with high enzyme productivity.

Therefore, in view of the above-mentioned current situation, an object of the present invention is to provide a method for producing NAD(P)H oxidase that is stable and easily obtainable.

[Means for solving problems]

As a result of intensive study on NA(P)H oxidase which is advantageous for industrial production, the inventors of the tree discovered that the genus Brevibacterium and the genus Corynebaoterium -
, Arthrobacter spp.
, Miorooocous, Pseudomonas, Aohromobacter, Agrobaoterium,
Flavobacterium jig
ium), or Streptomyces (Strep
microorganisms belonging to P. tomyoes) have produced stable NAD (P
) H oxidase was discovered, and the present invention was completed.

□Therefore, the present invention relates to the genus Brevibacterium, the genus Corynebacterium, the genus Arthrobacter, the genus Microfuccus,
NAD consists of culturing NAD(P)H oxidase-producing bacteria belonging to the genus Pseudomonas, Achromobacter, Agrobacterium, Flavobacterium, or Streptomyces, and collecting NAD(P)H oxidase from the culture. This is a method for producing (P)H oxidase.

For the production of NAD(P)H oxidase according to the present invention, any strain belonging to the above genus and having the ability to produce NAD(P)H oxidase can be used. Examples of strains suitable for production include: , Brevibaoteriumammo
niageneo ) ■ AM 1645, Corynebacterium fracumfaciens (Q) rynebact
θriumflacaumfac1ens )AHU
1622, Arthrobacter atrocyaneus (
Arthrobacteratocyaneus)
Engineering AM 12339, Micrococcus flavus (M
icroooccua fravus ) engineering FO32
42, Pseudomonas @1 Aeruginosa (Pseud
omonas aeruginosa) AM115
6. Achromobacter bulbulas (Aohromob)
aoter (5I) parvulus) 1F013182, Agrobacterium radiobacter (Agrobaoteriu+
n radiobaat@r) 1F012664, Flavobacterium nisteroaromaticum (Flavo
bacterium eeteroaromatic
m) Engineering FO3751, Streptomyces aureus
9aureus) ■AM OO92.

The culture medium used in the present invention may be one that produces NAD(P)H oxidase, and the nitrogen source may include yeast extract, peptone, meat extract, cornstarch liquor, ammonium sulfate, chloride, etc. Examples of the carbon source include glucose, molasses, glycerol, sucrose, and sorbitol. In addition, inorganic salts and metal salts such as phosphates, calcium salts, and magnesium salts may be added, and further vitamins, growth promoting factors, etc. may be added.

When culturing NAD(P)H oxidase-producing bacteria, the culture temperature is usually in the range of 20°C to 40°C, preferably 3°C.
It is carried out at around 0℃. The initial pH is usually in the range of pu 6 to 8, preferably around pH 7, and the production of NAD(P)H oxidase reaches its maximum by aeration and agitation culture for 10 to 30 hours. It goes without saying that culture conditions should be set to maximize the production of NAD(P)H oxidase depending on the strain used, medium composition, etc.

Since most of the NAD(P)H oxidase produced in this way exists within the bacterial cells, the culture is solid-liquid separated and the resulting bacterial cells are destroyed by commonly used ultrasonication, enzyme treatment, homogenization, etc. , obtain a cell-free extract. Next, a purified enzyme preparation can be obtained from this extract by a commonly used 1'i# production method. For example, salting out, organic solvent precipitation, ion exchange column chromatography, column chromatography using an adsorbent, gel PJ
By performing purification by , freeze-drying, etc., it is possible to obtain a specimen of 1iAD, (P)H oxidase, which is stable in polyacrylamide gel disk electrophoresis.

The enzymatic chemical properties of NAD(P)H oxidase of the present invention are as follows.

(1) Action: This enzyme has N
ADH and NADPH oxidize to NAD or NAD
Generates P and hydrogen peroxide.

NAD(P)H 10H 1”Or-1AD(P) +
HxOt (2) Optimum pH% PH stability: When measured using Priton-Robinson buffer, this enzyme has a
．． The optimum pH was around 0 (first factor). In addition, using the same buffer, after treatment at 37°C for 60 minutes at each pH,
When its residual activity was measured, it was found to be 1) stable at H6 to 10.5 (Figure 3);

(3) Optimal temperature, thermostable potassium diphosphate buffer (1
)H7,0). However, this enzyme has approximately 4
It has an optimal temperature at 5°C (Figure 2), and when the residual activity was measured after treatment at each temperature for 10 minutes using the same loosening solution, it was stable up to 55°C (Figure 4). ).

The activity of this enzyme is measured as follows.

0.3M potassium phosphate buffer (TIH7,O)1. O
m! , 6mM NADHQ, 1 me and distilled water 1
．． After keeping the reaction solution consisting of 9d at 37°C in advance, the enzyme solution was
．． 1- is added, the reaction is carried out at 37°C, and the decrease in absorbance at 340 nm is measured. . Enzyme activity is 1 per minute
The enzyme sweetfish that oxidizes /' mol of NADH by 1 degree was defined as 1 unit.

〔Example〕

Next, the method for producing NAD(P)H oxidase according to the present invention will be explained with reference to Examples, but the present invention is not limited to the following Examples.

Example 1 Glucose 1.0%, meat extract 1.0%, peptone 1.
0%, KH Snow PO40, 1%, Mg5Oa” 7 H2
50% of medium 100-50% O, pH 7.0
Dispense into 〇-volume Erlenmeyer flasks, sterilize at 120°C for 15 minutes, and then remove Brevibacterium ammoniagenesae AM 1.
645f: Inoculated and cultured with shaking at 30°C for 24 hours, which was used as a seed culture solution.

Dispense 2 volumes of the same composition medium 500- into Erlenmeyer flasks, and
After sterilizing at 20°C for 15 minutes, the seed culture solution Tt20al'
The cells were inoculated and cultured with shaking at 30°C for 15 hours. After completion of the culture, solid-liquid separation was performed by centrifugation to obtain 68 f of wet bacterial cells from 5 t of culture solution.

This bacterial cell was added to 39mM potassium phosphate buffer (pH 7.0).
) After suspending in 150 centimeters of water, the bacterial cells were destroyed by ultrasonication,
The supernatant obtained by centrifugation was used as a crude fermentation solution. This crude enzyme was dissolved in 10mM glycine-NaOH buffer (pH 9,0).
), then DIOAIC- buffered in advance with the same buffer.
Sepharose (OL-6B) (manufactured by Pharmamia)
When passed through a column, activity was observed in the filtered solution.

Next, the pH'E of this flow-through solution was adjusted to 10.0 with NaOH, and then 1QmM glycine-NaOH buffer (pH
Q-Sepharose (1'?) buffered with 10,0) (
When passed through a column (manufactured by Pharmacia), NAD(P)
H oxidase was adsorbed. Therefore, 10mM to 1.0M
Elution was performed using a linear concentration gradient of Mail, the active fractions were collected, and after concentration in a collodion bag, gel was applied to a column of Sephacryl S-300 (manufactured by Pharmacia) equilibrated with roomM potassium phosphate buffer solution (pH 7, O). The active fraction was collected to obtain NAD(P)H oxidase 50M9.The specific activity was 2.1 units/'I9, and the yield from the crude enzyme solution was 42%. .

Example 2 The same medium 100 as in Example 1 was dispensed into a 500 m 1'# Erlenmeyer flask, and after killing at 120°C for 15 minutes, the strains shown in Table 1 were inoculated through a slant and incubated at 30°C for 24 hours. Cultured with shaking. The culture was separated into solid and liquid by centrifugation, and the resulting bacterial cells were added to 30 nM potassium phosphate buffer (pH 7.0).
It was suspended in 20 ml. Next, the suspension was subjected to ultrasonication and then centrifuged to obtain a clear solution. Each NA of the resulting supernatant
When the D(P)H oxidase activity was measured, the results shown in Table 1 were obtained.

〔Effect of the invention〕

The present invention provides an advantageous manufacturing method for industrial production of NAD(P)H oxidase useful for measuring NADH and NADPH.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between the activity and pH of NAD(P)H oxidase obtained by the present invention, Figure 2 is a glass showing the relationship between the activity of this enzyme and temperature, and Figure 3 is a glass showing the relationship between the activity of this enzyme and temperature. Figure 4 is a graph showing the thermostability of this enzyme.

Claims (1)

[Claims] 1. N belonging to the genus Brevibacterium, Corynebacterium, Arthrobacter, Micrococcus, Pseudomonas, Achromobacter, Agrobacterium, Flavobacterium or Streptomyces
A method for producing NAD(P)H oxidase, which comprises culturing AD(P)H oxidase-producing bacteria and collecting NAD(P)H oxidase from the culture.