JPS63251082A - Production of nadh oxidase - Google Patents

Abstract

PURPOSE:To collect NADH oxidase, by cultivating Bacillus sphaericus, Bacillus amyloliquefaciens, Bacillus cereus or Bacillus thuringiensis in a proper medium. CONSTITUTION:Bacillus licheniformis, Bacillus subtilis, Bacillus aneurinolyticus, Bacillus sphaericus, Bacillus amyloliquefaciens, Bacillus cereus or Bacillus thuringiensis is cultivated in a proper medium. The culture temperature is usually about 25-40 deg.C and the culture time is usually about 5-40hr. A cell is separated from the prepared culture mixture by a means such as filtration or centrifugation, the cell is ground by ultrasonic treatment or various mechanical means, solubilized into an aqueous solution, separated and collected.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel method for producing NADH oxidase, particularly H2O2-generating NADH oxidase.

As a NADH oxidase, H2O2
There are known cases of outbreaks and cases of H20 outbreaks.

The former enzyme, H2O2-generating NADH oxidase, is an enzyme that catalyzes the reaction of the following formula, and quantitatively generates H2O from NADH.

generate.

NADH+H"+02→NAD+1H2O2+02), H2O2-generating NADH okindase can be used in conjunction with H2O2 quantitative methods to determine the activities of various dehydrogenases involving NADH and the amount of substrate for various dehydrogenases that use NAD as a coenzyme. It is a useful enzyme that can be used for measurements.

Conventionally, H2O2-generating NADH oxidase has been developed using Bacillus megaterium.
gaterium) [Journal of Biochemistry (J, Bi
oehem, ), 98. 1433-1440 (1
985)). However, the amount of the enzyme produced by Bacillus megathyrium is extremely small (see Comparative Examples below), and therefore the enzyme is difficult to obtain and practical for enzyme analysis and the like.

Therefore, there is a demand for inexpensive and stable supply of the enzyme.

Means for Solving the Problems In order to meet the above-mentioned demands, the inventors of the present invention have developed a H2O2-generating N
We have conducted extensive research into methods for easily producing ADH oxidase in large quantities. As a result, the inventors discovered that a specific species of the genus Bacillus produces a significant amount of the enzyme, leading to the completion of the present invention.

That is, the present invention provides Bacillus licheniformis, Bacillus
subtilis, Bacillus pumilus, Bacillus aneurinolyticus, Bacillus spheericus, Bacillus
The present invention relates to a method for producing H2O2-generating N'ADH oxidase, which comprises culturing B. amyloliquefacens, Bacillus cereus, or Bacillus theuringiensis in a medium, and collecting H2O2-generating NADH oxidase from the culture.

In the present invention, Bacillus licheniformis (Bacillus
illus licheniformis), Bacillus subtilis (Bacillus 5ubtilis)
), Bacillus pum
illus), Bacillus aneurinolyticus (Bac
illus aneurinolyticus), Bacillus sphaericus (Bacillus 5phaer)
icus), Bacillus amyloliquefaciens (
Baci ] l usamyloliliquefaci
ens), Bacillus cereus
It is necessary to use a strain belonging to any of the species Bacillus thuringiensis and Bacillus thuringiensis.

For example, preferred strains include Bacillus licheniformis IAM11054, Bacillus subtilis IFO3
007, Bacillus φpumilus IF012086, Bacillus aneurinolyticus AHU1354, Bacillus
Sphericus IF03341, Bacillus amyloliquefaciens IFO14141, Bacillus cereus I
F03132 and Bacillus theuringiensis I
F03951 can be mentioned. These strains are
All of these bacteria are preserved in public preservation institutions and can be easily obtained through distribution. The preservation institutions are IAM, Institute of Applied Microbiology, University of Tokyo, IFO, Fermentation Research Institute, and AHU, Faculty of Agriculture, Hokkaido University. It goes without saying that various mutant strains of these strains can also be suitably used.

These strains can be cultured in the same manner as conventional Bacillus strains, using appropriate carbon sources, nitrogen sources,
Cultivate in a nutrient medium containing minerals and other nutrients as necessary. The carbon source may be any assimilable carbon source, such as glucose, sucrose, mannitol, and the like. The nitrogen source may be any available nitrogen source (for example, inorganic nitrogen compounds such as ammonium sulfate, ammonium phosphate, ammonium nitrate, peptone, meat extract, yeast extract, casein hydrolyzate, corn steep liquor, etc.). Further, other phosphates, various salts of metals such as magnesium, calcium, potassium, iron, manganese, zinc, vitamins, etc. can be added as necessary.

Culture may be either solid culture or liquid culture, but liquid culture is usually convenient.

The culture temperature may be any temperature suitable for the growth of Bacillus strains, and is usually about 25 to 40°C.

The culture time is not particularly limited, and the desired H2
The culture may be terminated at an appropriate time when O2-generating NADH oxidase reaches its maximum yield, which is usually about 5 to 40 hours.

In order to collect H2O2-generating NADH oxidase from a culture, since this enzyme mainly exists within the bacterial cells, first, the bacterial cells are separated from the obtained culture by means such as filtration or centrifugation. Next, this bacterial cell is subjected to suitable bacterial cell crushing means,
For example, it is crushed and solubilized by ultrasonication, various mechanical methods, etc., and separated and collected as an aqueous solution.

Purification of the target oxidase thus obtained from the crude enzyme solution is carried out by appropriately combining methods used for the purification of ordinary enzymes.

For example, methods such as salting out, organic solvent precipitation, dialysis, ion exchange chromatography, gel filtration, and affinite chromatography can be used in combination.

To give a more specific example, for example, after disrupting the bacterial cells, they are centrifuged and mixed with a crude enzyme solution to obtain a supernatant.

The crude enzyme solution is salted out with ammonium sulfate to obtain an N A D H oxidase active fraction. After removing ammonium sulfate contained in the precipitate by dialysis or gel filtration, DEAE Sepharose CL-
Adsorb and elute on 6B ion exchanger (manufactured by Pharmacia). After concentrating the active fraction, Ultrogel ACA34 (L
KB) gel filtration was performed, and the active fraction was further filtered from 5' to
Isolation and purification can be carried out by performing affinity chromatography using AMP-Sepharose (manufactured by Pharmacia).

H2O2-generating NADH obtained by the production method of the present invention
The enzymatic chemical and physicochemical properties of oxidase are shown below.

(1) Effect: Oxidize 1 mol of NADH with 1 mol of 02 to produce 1 mol of NAD+ and 1 mol of H20□.

(2) Coenzyme: FAD is used as a coenzyme and is contained in the enzyme protein, but the activity is increased several tens of times by adding FAD separately to the reaction solution.

(3) Substrate specificity: It acts on both NADH and NADPH, but as shown in Table 18 below, F
By adding AD, the activity against NADH increases about 35 times, but the activity against NADPH increases only about 5 times -2, and in the reaction in the presence of FAD, the activity against NADH increases by about 5 times. Shows approximately 7 times more activity than NADH.

Table 1 (4) Km value for two NADIl: is approximately 3.2
X10'M, and the K m value for FAD is approximately 6.7
It is X10-6M.

(5) Molecular weight = approximately 116,000 by gel filtration using Sephadex G-150J (manufactured by Pharmacia) and approximately 56,000 by 5DS-polyacrylamide gel electrophoresis. It is a two-tone form.

(6) Optimum pH: As shown by the curve in Figure 1, approximately p
It has high activity in H6.5-8.0.

(7) Optimal temperature: As shown by the curve in Figure 2, pH
7.0, which is around 45 degrees Celsius.

(8) pH stability: 24 hours at 30°C, each pH
Figure 3 shows the pH stability when left in H. As is clear from FIG. 3, it is stable between pH 7.0 and .9.0.

(9) Thermal stability: Figure 4 shows the thermal stability when treated at pH 7 and 2 for 0 minutes at each temperature. As is clear from Figure 4, it is stable up to 30°C. Furthermore, it is stable up to 40°C in the presence of 0.1% bovine serum albumin.

(10) Inhibitor: Inhibited by heavy metal ions such as Ag", Hg", and Cu4+. KCN and PCMB (p-chloromercury-henshade) are partially inhibited.

The properties of the H2O2-generating NADH oxidase obtained by the present invention are described in the Journal of Biochemistry, 98.1433-1440 (1985).
Compared to the properties of similar NADH oxidase described in
5) They are also similar in terms of molecular weight.

Enzyme activity measurement method: Enzyme activity in the present invention is determined by measuring NAD in 1 minute under the following conditions.
The amount of enzyme required to oxidize 1 μmol of H is defined as 1 unit, and the amount is determined by measuring the decrease in the absorbance of NADH at 340 nm.

Reagents: (A) 0.25M potassium phosphate buffer pH
7,0 (B) 2mM NADH aqueous solution (C) 1mM FAD aqueous solution (D) IM NaN3 aqueous solution (E) Enzyme solution: Prepare IM NaN3 aqueous solution (D) in advance so that the concentration of NaN3 is 0.1M. 1710
Add amount.

duj determination procedure: Into a cuvette with an optical path length of 10 mm, add the above potassium phosphate buffer (A) 0. 6ml, substrate solution (B
) 0.3ml, coenzyme solution (C) 0. 1011, I
M NaN3 aqueous solution (D) 0. Add 3 ml of enzyme solution and 1.6 ml of distilled water and mix. After prewarming at 30°C for 10 minutes, add 0.1 ml of enzyme solution (E) and mix immediately.
Start the reaction. 340nm of substrate NADH at 30°C
The initial rate of decrease in absorbance (ΔA34o) at is measured, and the amount of oxidized NADH is calculated according to the following formula. The enzyme concentration is 3 after the start of the reaction.
ΔA34o for 1 minute from 0 seconds to 90 seconds is 0.035 to 0
．． Measurement is performed by adjusting the amount of change to be approximately 0.050.

6.22 H2O2 measurement method: Under the enzyme activity measurement conditions described above, add enough enzyme to completely oxidize NADH and carry out the reaction. When the decrease in absorbance at 340 nm reaches 0, the reaction completed solution is The amount of H2O2 produced in the reaction solution was determined using the oxidative color method (4-aminoantipyrine-N,
N'-dimethylaniline-peroxidase system).

Reagents: (A) 0.25M potassium phosphate buffer pH
7,0 (B) 0.3% 4-aminoantipyrine aqueous solution (C) 0.2% N,N'-dimethylaniline-0,
0IN HCI solution (D) Peroxidase solution: 50u/Peng's 30mM potassium phosphate buffer pH 7.2 Measurement procedure: Add the above potassium phosphate buffer (A) to a test tube
0. 2ml, 4-aminoantipyrine aqueous solution (B)
O, 1 ml, N, N'-dimethylaniline solution (C)
0. 2rrvQ and H2O2 quantitative sample solution 0.
Add 8mG, mix, and add peroxidase solution (
D) 0. Add 1auQ, mix and leave at room temperature for 10 minutes, then add 1.6mm of distilled water and leave the final solution ff13. 0I
The absorbance at 565 nm is measured as TIQ. H
The 2O2 content is determined by measuring a known amount of H2O in advance by the above measurement method.
H corresponding to the measured absorbance from the standard line prepared for 2.
Read and determine the amount of 2O2.

Effects of the Invention According to the present invention, especially by using a specific species of the genus Bacillus, H2O2-generating NADH oxidase can be easily produced in large quantities, and the enzyme can be stably supplied at low cost. .

EXAMPLES The method for producing NADH oxidase according to the present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples.

Examples 1 to 9 and comparative examples Medium composition Glucose 1.0%, peptone 1.0%, yeast extract 0.2%, sodium citrate 0.1%, (N
Ha)2SOa 0.2%, KH2PO40.6
%, K2HPO410.4%, M g S O4 7H
200.02%, pH 7.2.
Dispense into a 17.120'C flask with a cotton plug.
It was sterilized in an autoclave for 115 minutes. First, inoculate various bacterial strains from slant culture into 5 ml of test tube culture, and
A seed culture was prepared by shaking culture at 0'C1 for about 20 hours.

Next, 50 m of seed cultures of these various strains! The cells were inoculated into a Q-volume flask medium and cultured with shaking at 30°C for about 20 hours. After culturing, collect the bacterial cells by centrifugation, suspend the viable bacterial cells to a concentration of approximately 10% (weight/volume in percent) in 30 m~1 potassium phosphate buffer pH 7.2, and treat with ultrasonication under water cooling. (20KC15 minutes). This ultrasonic treatment 7
1k was centrifuged and the supernatant was used as a crude enzyme solution to measure the activity.

In addition, as a comparative example, Bacillus megaterium AHU1
240 was used for culturing, enzyme purification, and measurement in the same manner.

The measurement results are shown in Table 2.

In Table 2, the NADH oxidase activity is the enzyme activity of the crude enzyme solution converted per 1 mQ of the original culture solution. The H20□ generation rate represents the ratio of the measured value of H20□ in the reaction solution to the theoretical value generated when NADH or NADH oxidase is oxidized according to the reaction formula.

Example 10 A 50 Q jar fermenter containing 30 Q of medium prepared in the same manner as in Example 1 was steam sterilized at 1-20°C for 20 minutes. Seed culture 3 of Bacillus licheniformis IAMIIO54 cultured in advance in the same manner as in Example 1-7
00ml inoculated, 30°CC 1300rp, aeration rate 3
The cells were cultured for 6 hours at 0min. NA of the obtained culture solution
DH oxidase activity was 0.43 u/mQ. '
? 3 Centrifuge the ii solution 3 (l) and collect the bacterial cells from 30 m~
Suspended in potassium phosphate buffer pH 7, 23, 2Q,
The cells were crushed using a bacterial cell crusher Dynomil KDL (manufactured by Lyle Roux Hakkofuen Co., Ltd.).

The cell suspension was subjected to continuous centrifugation (1200 rpm) to obtain a supernatant of 3200 ITII2. Add ammonium sulfate to the supernatant, salt out ammonium sulfate, dissolve in 35% ammonium sulfate, and precipitate in 60% ammonium sulfate. Collect the two parts, dissolve in 30mM potassium phosphate buffer, dialyze against the same buffer, and add to the same buffer. It was adsorbed onto an equilibrated DEAE Sepharose CL-6B column 1゛2, and 0.2M
It was eluted with NaCQ.

The active fraction of the eluate was salted out with ammonium sulfate, and both fractions precipitated at 70% saturation with ammonium sulfate were added to 100 mM potassium phosphate buffer 1 (kpH 7).
, 2 and equilibrated in the same buffer.
Gel filtration was performed using A34.

The active elution fraction was concentrated by ultrafiltration, dialyzed against 30 mM potassium phosphate buffer pH 7.2, adsorbed on a 5'-AMP-Sepharose 4B column equilibrated with the same buffer, and the non-adsorbed fraction was washed. After elution, the active fraction was eluted with the same buffer containing 0.5M NaCQ, and this active fraction was further added to Sephacryl S-200 (manufactured by Pharmacia) equilibrated to 100mM potassium phosphate buffer pI ('7.2). )
The active eluate fraction was concentrated by ultrafiltration to isolate and purify NADH oxidase. Water-purified NADH oxidase showed a single band on 5DS-polyacrylamide electrophoresis, with a specific activity of 56.
4. u/mg-protein, the specific activity was increased 137 times, and the recovery rate of activity was 44%. The properties of this enzyme are as described above.

[Brief explanation of the drawing]

FIG. 1 shows H2O2-generating NAD obtained by the present invention.
It is a graph showing the optimum pH of H oxidase. FIG. 2 is a graph showing the optimum temperature of the oxidase. FIG. 3 is a graph showing the pH stability of the oxidase. FIG. 4 is a graph showing the thermostability of the oxidase. (Above) Figure 1 pH Figure 2 Temperature (°C) Figure 3 Figure 4

Claims (1)

[Claims] (1) Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus anoirinolyticus, Bacillus sphaericus, Bacillus amyloliquefaciens, Bacillus cereus or Bacillus cereus
thuringiensis in a medium, and H_ from the culture.
A method for producing H_2O_2-generating NADH oxidase, which comprises collecting 2O_2-generating NADH oxidase.