JP2966513B2 - Method for producing peroxidase - Google Patents

Description

The present invention relates to a method for producing peroxidase by a peroxidase-producing bacterium belonging to the genus Flavobacterium.

[Conventional technology and problems]

Peroxidase is an enzyme that oxidizes the various compounds in the presence of H ₂ O _2, glycose, cholesterol, phospholipids, uric acid, etc. is used as a clinical diagnostic reagent various peroxidase is quantified these components to a substrate I have. Peroxidase has also been used as a labeling enzyme in an enzyme immunoassay.

Conventionally, horseradish, radish and the like have been known as sources of peroxidase [J. Biol. Chem., (196
6), 241 , 2166-2172 etc.]. However, peroxidase in these sources exists as isozymes with slightly different properties, so isozymes must be separated to obtain pure enzymes for use in clinical diagnostic reagents.
It takes a lot of trouble.

Many microorganisms are also known as sources of peroxidase. Among them, fungi include Arthromyces ramosus (Japanese
61-43987 and JP-A-63-219398), Coprinus cinereus [JP-A-61-104784, JP-A-61-254189 and J. Biochem. 103 , 693-699 (198)
8)], Alternaria (Alternalia) microorganism belonging to the genus, Kokurioborasu (Cochliobolus) microorganism belonging to the genus, Perikyuraria (Pellicularia) microorganism belonging to the genus, Curvularia (Curvulari
a) a microorganism belonging to the genus (more than 4 Shatoku Publication 58-5035), Oy audio dendron (Oidiodendron) microorganism belonging to the genus (JP-A-59-1790
75), Pellicularia filamentosa
filamentosa ) [Agricul. Biol. Chem. 45 , 1297-1299
(1981)] Saccharomyce
s cerevisiae) [J.Biol.Chem. 259 13027-13036 (19
84)] is known. However, these fungi take longer to culture than bacteria. In addition, peroxidases from Arthromyces ramos and Coprinus cinereus have weak effects on aniline dyes.

Bacillus stearothermophilus is a peroxidase-producing bacterium.
[J. Gen. Microbiol. (1988), 134 , 1971-1976], Shademonas fluorescens ( Pseudomonas fluores)
cens ) [J. Biol. Chem. 220 , 967-982 (1956)], Streptococcus faecali
s ) [J. Biol. Chem. 225 , 557-573 (1957)], Escherichia coli [J. Biol. Chem. 254 , 4]
245-4252 (1979)], Halobacterium halobium [J. Biochem. 98 , 1055-10].
61 (1985)], Rhode Pseudomonas capsulata ( Rh
odopseudomonas capsulata ) [J. Biol. Chem. 264 , 6871
-6876 (1987)].

An object of the present invention is to provide a method for producing a single peroxidase that does not exist as an isozyme and that has a strong effect on an aniline-based hydrogen donor in a shorter culture time than fungi.

[Means for solving the problem]

The present invention relates to a method for producing peroxidase, comprising culturing a peroxidase-producing bacterium belonging to the genus Flavobacterium in a medium and collecting peroxidase from the culture.

The strain used in the present invention belongs to the genus Flavobacterium, and may be any microorganism as long as it is a microorganism capable of producing peroxidase. A microorganism belonging to Flavobacterium meningosepticum , specifically, Flavobacterium meningosepticum T-2799 collected from wheat field soil in Odawara City, Kanagawa Prefecture is exemplified.

 Next, the bacteriological properties of the T-2799 strain will be described.

(1) Macroscopic characteristics a) Ordinary agar plate medium Hill-shaped, circular, smooth colonies are formed around, and the surface is smooth and shiny dark yellow.

b) Ordinary agar slant medium It grows well. It has a glossy dark yellow color.

(2) Microscopic characteristics Bacillus has no motility. No spores are formed.

(3) Growing well at 37 ° C, 40 ° C and 45 ° C.

(4) Physiological and biochemical characteristics Gram stain-OF test (+) catalase production + oxidase production + urease production + β-galactosidase production + cytochrome oxidase production + nitrate reduction-indole production-acid production from glucose- Use of glucose + Use of arabinose-Use of mannose (+) Use of mannitol + Use of N-acetylglucosamine-Use of maltose + Use of gluconic acid-The above is the culture result at 30 ° C for 2 days. The result of culturing for 7 days is added.

Use of Allantoin + Use of Glycerol-Indole Production-DNAase Production + Phosphatase Production-Based on the above properties, the International Journal of Systematic Bacteriology (Int.J.
Systematic Bacteriology) 213-216 (1989), Bergey's Manual of Systematic Bacterio
This strain was identified as belonging to the genus Flavobacterium in light of Vol.I, and was identified as belonging to Flavobacterium meningosepticum because it does not produce phosphatase. The strain was named Gosepticum T-2799 strain and deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microbial Laboratory Bacteria No. 11325.

As the medium used in the present invention, a medium containing a carbon source, a nitrogen source, an inorganic substance, and the like is used. As a carbon source, fructose, saccharose, xylose, maltose, glycerol and the like are used. As a nitrogen source, an inorganic nitrogen source such as ammonium sulfate or ammonium chloride can be used in addition to a natural nitrogen source such as malt extract, casamino acid and polypelton. In addition, salt, potassium chloride, magnesium sulfate, potassium phosphate monobasic, potassium phosphate dibasic, etc. are used as inorganic substances. In particular, 2'-deoxyguanosine is added to the medium at about 0.1 to 0.5%,
It is preferred to improve peroxidase productivity.

The culture temperature can be appropriately changed within the range in which the bacteria grow and produce peroxidase, but 10 to 45 ° C, preferably 25 to 45 ° C.
35 ° C. The pH of the medium is pH 4-10, preferably pH 6-9.

As a culture method, a usual culture method for producing an antibiotic, an enzyme, or the like may be used. The culture form may be liquid culture or solid culture. Industrially, it is preferable to inoculate a peroxidase-producing bacterial cell into a production medium and perform aeration-agitation culture.

The cultivation time varies depending on the conditions, but is usually about 15 to 50 hours, and the cultivation is terminated when the time when the peroxidase reaches the highest titer is determined.

Peroxidase is mainly contained and accumulated in the cells, and needs to be extracted. To illustrate the method of extracting peroxidase, for example, the culture is first solid-liquid separated, and the obtained wet cells are dispersed in a solution such as a phosphate buffer or a tris-hydrochloride buffer, and lysozyme treatment, ultrasonic treatment, and French press are performed. Various cell treatment means such as treatment are appropriately selected and combined to obtain a crude peroxidase-containing solution.

Purified peroxidase is obtained from the crude peroxidase-containing solution by using known protein and enzyme isolation and purification means. For example, peroxidase is precipitated and recovered by applying a fractional precipitation method using an organic solvent such as acetone, methanol, ethanol, or the like, or a salting-out method using ammonium sulfate, salt, aluminum sulfate, or the like to the crude peroxidase-containing liquid. The precipitate is further subjected to dialysis and isoelectric precipitation as necessary, and then purified by column chromatography using an ion exchanger, a gel filtration agent, an adsorbent, etc. until a single band is obtained by electrophoresis or the like. I do. For purification by column chromatography, for example, the above precipitate is dialyzed and then dissolved in a phosphate buffer or the like, which is then chromatographed using a gel filtration agent such as diethylaminoethyldextran gel, diethylaminoethylcellulose, triethylaminoethyldextran gel, or polyacrylamide gel. This is done by applying it to a graph. The above-mentioned purification means can be carried out in an appropriate combination. After purification, a freeze-stabilizing agent is appropriately added, followed by freeze-drying to obtain a purified powder of peroxidase.

Next, the physicochemical properties of the peroxidase obtained by the present invention will be described. In this regard, the activity of peroxidase was measured as follows.

Method for measuring peroxidase activity 0.2M acetate buffer (pH 5.5) 0.2m, 0.2% TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl)-
m-toluidine) 0.1m, 0.3% 4-aminoantipyrine 0.1m, 10mM hydrogen peroxide 0.1m, and 1m of reaction solution preliminarily heated at 37 ° C for 1 minute, then 20μm
And react for 10 minutes. After the reaction, 0.5
% SDS (sodium dodecyl sulfate) to stop the reaction, and within 5 minutes, using a cell having a layer length of 1.0 cm and a wavelength of 555 nm.
The absorbance at is measured (As). As a blind test, absorbance is measured by performing the same operation using 20 μl of distilled water instead of the enzyme solution (Ab). Absorbance using this enzyme (As)
The enzyme activity is determined from the absorbance (As-Ab) of the absorbance (Ab) and the blind absorbance (Ab). One unit of enzyme activity is 1 μmol of 4-aminoantipyrine per minute at 37 ° C. and the amount of enzyme that oxidizes TOOS.
The calculation formula is as follows.

Physicochemical properties (1) Action specificity The enzyme (the present enzyme) obtained in Example 1 catalyzes the oxidation of various compounds in the presence of hydrogen peroxide. The mechanism of action is as shown in the following equation.

(Where AH ₂ represents a hydrogen donor and A represents an oxidized hydrogen donor) (2) Specificity for hydrogen donor Table 1 shows the specificity of the present enzyme for various hydrogen donors. The relative activities were measured at the respective absorption maximum wavelengths.

ABTS: 2,2-azinosine [3-ethylbenzthiazoline sulfonic acid (Boehringer Mannheim / Yamanouchi Pharmaceutical) TOOS: (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine) (Dojindo Chemical Research) (3) Km value 9.5 × 10 ^-6 M (relative to H ₂ O ₂ ) (4) Isoelectric point pH 4.2 ± 0.2 (by electrophoresis using carrier-ampholite). There is no isozyme per single band.

(5) Molecular weight 220000 ± 20000 (Sephacryl S-300 and TSKG-3000 S
Gel filtration by W × L 4 subunits of 54000 ± 5000 (SDS polyacrylamide gel electrophoresis) (6) Optimum pH The optimum pH was determined according to the enzyme activity measurement method.
The result is shown in FIG. pH 3.5-5.0 is acetate buffer, pH 4.5-7.5 is phosphate buffer, pH 7.0-9.5 is Tris-HCl buffer, pH 9.5-10.5 is glycine-water It shows the activity value when a sodium oxide buffer was used, and the optimum pH was recognized to be 5.5 to 6.0 (phosphate buffer).

(7) pH stability 100 mM buffer solution (peroxidase 1 U / m) at 40 ° C
For 10 minutes, and the residual activity was measured according to the enzyme activity measurement method described above. The result is shown in FIG. pH 3.6-6.0
Is acetate buffer, pH 5.9-7.7 is phosphate buffer, pH 7.3-8.6
For Tris-HCl buffer, pH 8.3 to 10.0 for Glycine-Sodium hydroxide buffer. It showed the best stability in the pH range of 6.0 to 7.0.

(8) Optimum temperature The result of obtaining the optimum temperature by changing the temperature in the range of 25 ° C. to 65 ° C. according to the enzyme activity measurement method is as shown in FIG. 3, and the optimum temperature of the present enzyme is 50 ° C.

(9) Thermal stability 100 mM acetate buffer (pH 5.5) (peroxidase 1 U / m
) Was heat-treated at each temperature for 10 minutes, and the residual enzyme activity was measured according to the enzyme activity measurement method described above. As a result, it was stable up to 55 ° C. as shown in FIG.

(10) Influence of metal ions The results of examining the effects of various metal ions and the like (1 mM) on the activity of the present enzyme are shown in Table 2, and inhibition by Hg ions was observed.

Next, examples of the present invention will be described.

Example 1 1.0% sucrose, 1.0% polypeptone in test tubes,
0.3% of 2'-deoxyguanosine, 0.1% of K ₂ HPO ₄ and MgSO
₄ · 7H ₂ O consisting of 0.03% medium (pH 7.0) (120 ° C., 20 min sterilization) Flavobacterium main Nin meningosepticum Petit cam T-2799 strain in 10m of (FERM 11325) It was inoculated and cultured at 28 ° C for 24 hours to obtain a seed. The inoculum was then transplanted 10 m each to 100 Sakaguchi flasks containing 200 m of a medium having the same composition as described above, and cultured at 28 ° C. for 36 hours. After completion of the culture, the culture was centrifuged at 8,000 rpm for 30 minutes, and the obtained cells were washed with 5 pieces of water, and further 8,000 rp.
The cells were collected by centrifugation at m. for 30 minutes.

The cells were turbidized in 4.5 mM phosphate buffer (pH 7.0) of 4.5, and the cells were disrupted using a Dynomill. Thereafter, this was centrifuged at 8,000 rpm for 30 minutes to remove insolubles, thereby obtaining a supernatant (3,500 m (2.0 U / m) (7,000 U)). Then, ammonium sulfate was added to the supernatant so as to be 30% saturated (575
g), and the mixture was centrifuged at 8,000 rpm for 30 minutes to remove the precipitate. Further, ammonium sulfate was added (240 g) to obtain a 65% saturation, and the precipitate was recovered. This precipitate was dissolved in a 50 mM phosphate buffer (pH 7.0), and this was dissolved in a 10 mM phosphate buffer (pH 7.0).
Was dialyzed overnight in cellophane tubes. After dialysis,
A column (5.0 × 20 cm) of DEAE-Sephacel (manufactured by Pharmacia) equilibrated with 10 mM phosphate buffer (pH 7.0) was charged, and 10 mM phosphate buffer (pH 7.0) containing 0 to 1.5 M NaCl was added.
0) Elution was performed with a linear gradient of NaCl, and 0.85 to 0.95 M NaC
The eluted fraction (4,200 U) was collected at the 1-concentration elution. A column (2.5 × 2) of phenyl sepharose (manufactured by Pharmacia) equilibrated with 10 mM phosphate buffer (pH 7.0) containing 4 M NaCl by dissolving NaCl to a concentration of 4 M in this enzyme solution.
2 cm), and elution was performed with a linear NaCl concentration gradient of 10 mM phosphate buffer (pH 7.0) containing 4 M to 0 M NaCl, and an active fraction (1 to 0.5 M NaCl concentration) (3360 U) was collected. This was concentrated with Amicon PM-10 (manufactured by Amicon). Next, the concentrated solution was charged into a column (1.6 × 90 cm) of Sephacryl S-300 (manufactured by Pharmacia) previously equilibrated with 50 mM phosphate buffer (pH 7.0) containing 0.25 M NaCl,
Effluent was carried out with the same buffer (1 fraction: 1 m / tube), and fractions 73 to 87 were collected as active fractions. The active fractions were combined and lyophilized to obtain 56 mg (2350 U) of purified peroxidase powder.

〔The invention's effect〕

Flavobacterium-derived peroxidase (DEAE ion exchange chromatography, isoelectric focusing results)
Since there is no isozyme, the enzyme is superior to a conventional horseradish-derived peroxidase as a reagent for clinical diagnosis and a labeling enzyme in an enzyme immunoassay. In addition, since the peroxidase of the present invention is produced by bacteria, it is excellent in that it can be supplied more stably and in a larger amount than peroxidase derived from plants, fungi and the like. Furthermore, the peroxidase of the present invention has a strong action on aniline dyes.

[Brief description of the drawings]

 FIG. 1 shows the optimum pH of the peroxidase of the present invention. FIG. 2 shows the pH stability of the peroxidase of the present invention. FIG. 3 shows the optimum temperature of the peroxidase of the present invention. FIG. 4 shows the thermostability of the peroxidase of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 9/00-9/99 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A method for producing peroxidase, comprising culturing a peroxidase-producing bacterium belonging to the genus Flavobacterium in a medium and collecting peroxidase from the culture. 2. The method for producing peroxidase according to claim 1, wherein the peroxidase-producing bacterium belonging to the genus Flavobacterium is Flavobacterium meningosepticum. 3. The method according to claim 1, wherein the Flavobacterium meningosepticum is Flavobacterium meningosepticum T-.
3. The method for producing peroxidase according to claim 2, wherein the method is 2799 (No. 11325).