JPH0147150B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing cholesterol oxidase. Various types of cholesterol oxidase-producing bacteria have been known. The present inventors collected Cellulomonas from the soil of a strawberry field in Ohito-cho, Tagata-gun, Shizuoka Prefecture.
A bacterium belonging to the genus B-0814 has substrate specificity for at least cholesterol and consumes 1 mol of oxygen per mol of cholesterol, 1 mol of cholestenone and 1 mol of cholesterol in its culture. We have discovered a new cholesterol oxidase-producing bacterium that produces cholesterol oxidase, which has the ability to catalyze the reaction that produces hydrogen peroxide. First, the characteristics of the culture of B-0814 on various media based on macroscopic and microscopic observations are as follows.
As described below. (A) Macroscopic observation (1) Broth agar plate medium (30°C, 40 hours) Forms round, flat or hill-like colonies, with smooth, moist, translucent surroundings, and a grayish-white to pale yellow color. No soluble pigments are produced. (2) Broth agar slant medium (30℃, 40 hours) Growth is rather weak, linear, moist and translucent, with a grayish-white to pale yellow color. No soluble pigments are produced. (3) Meat juice liquid medium (30°C, 40 hours) Growth is weak but uniformly cloudy. Produces precipitation. (B) Microscopic observation After culturing on ordinary agar medium at 30°C for 18 hours, the morphological characteristics were observed. (1) Cell shape and size Single, double, and occasionally short chains. It is rod-shaped with rounded edges and measures 0.4 x 0.8 to 1.5μ. (2) Presence or absence of polymorphism None. (3) Motility and flagella negative. (4) Spores (shape, position, size, swelling of bacterial bodies) None. (C) Physiological properties Nitrate reduction + denitrification reaction − MR test + VP test − Production of indole − Production of hydrogen sulfide (SIM medium) − Hydrolysis of gelatin + Hydrolysis of starch + Utilization of citric acid (Simmons medium) − (Christenzen medium) − Utilization of nitrates + Utilization of ammonium salts + Production of soluble pigments − Oxidase − Calatase + Urease (SSR medium) − Growth range Growth pH 5.5 to 8.0 Growth temperature 10 to 35 °C Attitude toward oxygen Facultability Anaerobic OF test (Hugh Leifson medium) F Aesculin degradation + Cellulose degradation + Casein degradation + Gram stain Positive or indeterminate Acid-fast stain Negative acid/gas production (Hugh-Leifson Base
Difco） Acid Gas Adonitol + − L(+) Arabinose + − Cellobiose + − Dulcitol − − Meso-erythol − − D(−) Fructose + − D(+) Galactose + − D(+) Glucose + − Glycerin − − Inositol − − Inulin − − Lactose + − Maltose + − D-Mannitol − − D-Mannose − − Raffinose + − L(+) Rhamnose + − Salicin + − L-Sorbose − − Sorbitol − − Starch + − Satucalose + − Trehalose + - D-xylose + - Based on the above mycological properties, this bacterium B-0814 is a Gram-positive or indeterminate bacillus and has the characteristic of decomposing cellulose, and is described in the Burgess Manual of Determinative Bacteriology ( Bergey, s Manual of Determinative
According to Bacteriologe, 8th edition (1974), this strain was recognized as belonging to the genus Cellulomonas. Furthermore, we compared the characteristics of this strain with the Burgess Manual of Determinative Bacteriology, 7th and 8th editions, but there was no mention of any bacterial species whose characteristics closely matched those of this strain. Cellulomonas sp.B.
It was identified and named 0814 (Cellulomonas sp.B-0814). Furthermore, this strain was designated by the Institute of Microbial Technology, Agency of Industrial Science and Technology as "FERM-P No. 6316 (FERM-P No.
6316)". The present invention was completed based on the above findings, and is a method for producing cholesterol oxidase, which comprises culturing cholesterol oxidase-producing bacteria belonging to the genus Cellulomonas in a medium, and collecting cholesterol oxidase from the culture. be. The above-mentioned Cellulomonas sp. B-0814 is one example of the bacteria used in the present invention, and not only this bacteria but also all bacteria belonging to the genus Cellulomonas that produce cholesterol oxidase can be used in the present invention. It can be used in In carrying out the present invention, cholesterol oxidase-producing bacteria belonging to the genus Cellulomonas are cultured by a conventional method for producing enzymes. The form of culture may be liquid culture or solid culture, and from an industrial perspective, it is advantageous to inoculate cells of cholesterol oxidase-producing bacteria into a production medium and perform deep aeration agitation culture. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, starch,
Examples include dextrin, molasses, waste molasses, and glycerin. The nitrogen source may be any available nitrogen compound, such as corn steep liquor, soybean flour, cottonseed flour, wheat gluten, peptone,
Meat extract, yeast extract, casein hydrolyzate, etc. are used. Others include phosphate, magnesium, calcium, potassium, sodium, zinc,
Salts such as iron, manganese, and halogens are used as necessary. Further, in order to induce the production of cholesterol oxidase, it is preferable to add about 0.1 to 1.5% cholesterol to the medium as an inducer. The culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce cholesterol oxidase.
The temperature is preferably 25 to 30°C, particularly about 26°C.
Cultivation time varies slightly depending on conditions, but is usually 12
~50 hours is enough, and 200~400rpm
The culture may be aerated with stirring under the following conditions, and the culture may be terminated at an appropriate time, taking into account the time when cholesterol oxidase reaches its maximum titer. In the culture of cholesterol oxidase-producing bacteria thus obtained, cholesterol oxidase is mainly contained within the cells. For example, when collecting this enzyme, the cells of the obtained culture are first collected by means such as filtration or centrifugation, and then the cells are subjected to ultrasonic treatment, French press treatment, or glass bead treatment. Destroy by mechanical destruction means such as lysozyme or enzymatic destruction means such as lysozyme, and if necessary, surfactant such as Triton Agents may also be added. The cholesterol oxidase-containing solution thus obtained is then concentrated or without concentration, salted out using a soluble salt such as ammonium sulfate, or a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. The enzyme may be precipitated using isopropanol or the like. Furthermore, this precipitate is dissolved in water or a buffer solution, and then dialyzed with a semipermeable membrane as necessary. Purification is performed by chromatography using an ion exchange resin such as carboxymethyl-Sephadex, or chromatography using a gelling agent such as molecular sieves such as Cephadex G200, Sepharose CL-6B, and Cephacryl S-200, followed by freeze-drying, etc. Purified cholesterol oxidase is obtained by this treatment. Next, a method for measuring the activity and properties of cholesterol oxidase of the present invention will be described. Activity measurement method 0.2M phosphate buffer (PH8.0) 0.25ml 0.3% 4-aminoantipyrine 0.1ml 0.2% Phenol 0.1ml 1% Triton X-100 0.3ml (50U/ml) Peroxidase 0.1ml 1mM Cholesterol 0.1ml Distillation 0.05ml water 1.0ml Preheat 1.0ml of the reaction solution having each of the above compositions to 37°C, then add 20μ of the enzyme solution to 37°C.
Incubate at ℃ for exactly 10 minutes. After the reaction, 2.0 ml of ethanol is added to stop the reaction, and the color development is then measured by absorbance at a wavelength of 490 nm. Enzyme activity is defined as 1 unit (1U), which is the activity that produces 1 μmole of hydrogen peroxide per minute. Calculation of titer follows the following formula. Enzyme activity (U/ml) = △A490×1.0/1
8 x 1/2 x 0.02 x 1/10 x dilution magnification (However, △A ₄₉₀ indicates the absorbance value at a wavelength of 490 nm) Action: For 1 mole of cholesterol, 1 mole of oxygen is consumed and 1 mole of oxygen is absorbed. It has the effect of catalyzing the reaction that produces cholestenone and hydrogen peroxide. Substrate Specificity In the activity measurement method described above, the following various substrates were used instead of cholesterol, and their activities were measured. The results were as shown in Table 1.

[Table] Optimal PH In the buffer solution used in the activity measurement method described above, the PH
The enzyme was prepared using acetate buffer (●-●) of 4-5.5, phosphate buffer (Γ-Γ) of PH5.5-8.5, and borate buffer (△-△) of PH8.5-10. Activity was measured.
As a result, as shown in FIG. 1, this enzyme was recognized to have an optimum pH around PH7. PH stability Enzyme solution containing cholesterol oxidase is mixed with various buffer solutions (acetate buffer with pH 4 to 5.5, pH 5.5 to 8.5).
Phosphate buffer, PH8.5~10 borate buffer) at 37
After heat treatment at ℃ for 60 minutes, the residual activity was measured based on the activity measuring method described above. As a result, as shown in FIG. 2, this enzyme was recognized to be stable in the pH range of 6 to 7. Thermostable cholesterol oxidase-containing enzyme solution,
10-70 min in 50mM phosphate buffer (PH7.0)
After heat treatment at ℃, the residual activity was measured based on the above-mentioned activity measurement method. As a result, as shown in Figure 3, this enzyme was found to be stable up to 50°C. Isoelectric point around PH4.8 (Measured by electrophoresis using carrier ampholite) Molecular weight Approximately 58,000 (Sephadex G-150) Approximately 58,000 (SDS polyacrylamide electrophoresis) Km value Approximately 2 x 10 ^-5 M ( Cholesterol) Based on the above properties of this enzyme, this enzyme is recognized as cholesterol oxidase. Furthermore, this cholesterol oxidase is used for quantifying samples that release cholesterol or samples that contain cholesterol. For example, by allowing this cholesterol oxidase to act on a cholesterol-containing sample, cholesterol can be quantified by quantifying the enzyme consumed or the hydrogen peroxide or cholestenone produced based on the enzymatic action of this cholesterol oxidase. It's something you get. In addition, the quantitative determination of oxygen and hydrogen peroxide may be carried out by electrical means using electrodes sensitive to the target components, such as oxygen electrodes and hydrogen peroxide electrodes, and enzymes may be measured by various known means. The immobilized enzyme may be used as an enzyme electrode fixed to an electrode surface used for electrical means. Furthermore, when measuring hydrogen peroxide, colorimetry is preferably performed using a system using one or more coloring agents that undergo a color change in the presence of hydrogen peroxide. For example, the amount of hydrogen peroxide produced is determined by the strength of the color using xylenol orange and a tetravalent titanium compound that reacts with the produced hydrogen peroxide to form a stable red color. Examples include a method of measuring a change in color tone by a reaction with phenol, 4-aminoantipyrine, and peroxidase. Also this 4
-4-aminofenazone may be used instead of aminoantipyrine. Furthermore, using a reagent containing diethyl-m-toluidine, 4-aminoantipyrine, and peroxidase, the amount of hydrogen peroxide produced may be measured by the intensity of its coloration. Other examples include quantitative measurement of hydrogen peroxide using a combination of 2.6-dichlorophenol indophenol and peroxidase, quantitative measurement of hydrogen peroxide using a combination of guaiac butter and peroxidase, quantitative measurement of hydrogen peroxide using a combination of homovanillic acid and peroxidase, etc. A quantitative method using peroxidase is preferred because it is simple and accurate. Furthermore, this reagent for quantifying hydrogen peroxide may be made into a simple laminated integral type by coating it on the surface of a carrier such as a film or paper. Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto. Example 1 Cholesterol 1.0%, meat extract 0.5%,
K ₂ HPO 0.1%, KCl 0.05%, MgSO _{4.7H 2} O 0.05
% containing medium into a 500ml Erlenmeyer flask,
After sterilization at 120℃ for 20 minutes, Cellulomonas sp.
B-0814 cells were inoculated with a platinum loop and cultured with shaking at about 26°C for 24 hours. Next, 200 ml (2 bottles) of this culture was inoculated into a 30-volume jar fermenter containing 20 medium of the same composition, and the mixture was heated at 26°C with a stirring speed of 200 rpm and an aeration rate of 20.
After 24 hours, the reaction was stopped and the bacterial cells were collected at 5000 rpm for 15 minutes. The collected bacteria contains 0.1% lysozyme and 5mM EDTA.
The enzyme was solubilized by dispersing it in 20mM phosphate buffer (PH7.5), 5.0, and lysing the bacterial cells at 37°C for 3 hours. After solubilization, insoluble materials were removed by centrifugation at 5,000 rpm for 15 minutes, and the resulting supernatant 4.8 (3.3/ml, 16,000
Ammonium sulfate fractionation was performed on u), and 0.54 to 0.7 saturated fractions were collected. This precipitated fraction was dissolved in 800 ml of 20 mM phosphate buffer (PH7.5), placed in a dialysis tube, and dialyzed against running water. Then remove the insoluble matter
Centrifugation was performed at 12,000 rpm for 10 minutes to obtain 760 ml of supernatant. (17,26u/ml, 13120u). Furthermore, the obtained supernatant was added to DEAE-Sepharose CL buffered with 20mM phosphate buffer (PH7.5).
-Charge to 6B (5 x 70cm), KCl concentration 0~
Elution was performed with a 0.5M concentration gradient, and the KCl concentration was 0.28~
Fractions eluted at 0.32M were collected (37.72u/ml,
12070u). This fraction was added to 10mM phosphate buffer (PH).
7.0) Dialyzed against 10, charged to a DEAE-Sepharose CL-6B (5 x 30 cm) column buffered with the same buffer, and dispensed with a KCl concentration gradient of 0.1-0.5M, 0.29-0.31M KCl. The fractions eluted with were collected (76.32u/ml, 10380). This fraction was concentrated using an instant filtration membrane (manufactured by Amicon) and charged to a Sephadex G-100 column (5 x 70 cm).
Elution was performed with 20mM phosphate buffer (PH7.5), active fractions were collected, and lyophilized (276mg, 8096.4u,
The yield was 50.6%, the specific activity of this preparation was 31.6 u/mg protein, and a single band was observed electrophoretically. Example 2 Optimal amount of cholesterol oxidase. 50mM phosphate buffer (PH7.5) 0.03% 4-aminoantipyrine 0.02% Phenol lipase (derived from producing bacteria belonging to the genus Chromobacterium) 200u/ml Peroxidase 2u/ml Cholesterol oxidase (2 types) ^* 0-1.67u/ ml ( ^* Two types of cholesterol oxidase were compared: one of the present invention and one derived from the genus Streptomyces.) Place 3 ml of the above composition solution in a small test tube, preheat it to 37°C, and then add 20μ of human serum. After heating at 37℃ for exactly 5 minutes
The reaction was carried out immediately after colorimetric determination at 500 nm.
The results are as shown in Figure 4, in which Γ-Γ indicates the case of cholesterol oxidase of the present invention, ●-● indicates the case of cholesterol oxidase derived from the genus Streptomyces, and Γ-Γ indicates the case of cholesterol oxidase derived from the genus Streptomyces. It was confirmed that while the cholesterol oxidase derived from the above requires about 5 per test, the cholesterol oxidase of the present invention requires only 0.5 per test, and is extremely fast in reaction.

[Brief explanation of drawings]

FIG. 1 shows the optimal pH curve for cholesterol oxidase of the present invention, FIG. 2 shows the PH stability curve, FIG. 3 shows the thermostability curve, and FIG. 4 shows the curve showing the optimal amount of cholesterol oxidase.

Claims (1)

[Scope of Claims] 1. A method for producing cholesterol oxidase, which comprises culturing cholesterol oxidase-producing bacteria belonging to the genus Cellulomonas in a medium, and collecting cholesterol oxidase from the culture. 2 Cholesterol oxidase-producing bacteria belonging to the genus Cellulomonas are Cellulomonas sp.
A method for producing a cholesterol oxidase-producing bacterium according to claim 1, which is B-0814 bacterium.