JPH0324198B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the basidiomycete, Trachyderma spp.
This invention relates to a novel bilirubin oxidase produced by and a method for producing the same. Bilirubin is a yellow substance produced in blood by the decomposition of hemoglobin, and rapid and accurate detection of bilirubin in serum is very important for medically diagnosing human disease conditions (e.g. jaundice). It is. That is, in patients with jaundice, bilirubin in serum increases abnormally, so the degree of jaundice can be diagnosed by measuring bilirunin in serum. Bilirubin oxidase is used to quantify bilirubin in serum, but this enzyme is also used to remove bilirubin, which can cause errors in measurement values when analyzing analytes other than bilirubin. Useful. When measuring glucose or cholesterol in serum, the most commonly used daily test method is the colorimetric method in which glucose oxidase, cholesterol oxidase, etc. are applied to the serum and the generated hydrogen peroxide is captured using peroxidase. . In particular, the coloring method using 4-aminoantipyrine-phenol has become the mainstream enzymatic method because it is simple and rapid, and the reagent is stable. Although this colorimetric method measures the red quinone pigment produced at 500 nm, the presence of bilirubin in serum gives a negative error. Therefore, if bilirubin oxidase is applied to serum in advance to remove bilirubin, and then glucose oxidase or cholesterol oxidase is applied to serum, accurate glucose or cholesterol values in serum can be obtained. Regarding bilirubin oxidase, R.
First reported by R. Brodersen and P. Bortels [Europ.J.Biochem, Vol. 10, No. 468]
Page (1969)]. They reported that bilirubin is oxidized to biliverdin by an insoluble bilirubin oxidase isolated from guinea pig brain. Furthermore, it has been reported that the fungal juice of Agaricus bisporus, a type of mushroom, has oxygen activity that oxidizes bilirubin and produces hydrogen peroxide (Japanese Patent Publication No. 11194/1982). moreover,
Recently, Myrothecium verrucaria strain MT-1 (Myrothecium verrucaria, which belongs to the genus Myrothecium)
MT-1) was found to produce bilirubin oxidase in the culture medium, and it has been reported that the purified enzyme oxidizes bilirubin to produce biliverdin [Agricultural &
Biological Chemistry (Agric.Biol.
Chem.) Vol. 45, p. 2385 (1981)]. The present inventors discovered that bilirubin produced by basidiomycetes
As a result of extensive research into oxidase, we discovered that a certain type of basidiomycete produces a significant amount of bilirubin oxidase in its culture solution, so we purified this enzyme and examined its properties. As a result, we came to the conclusion that this enzyme is a novel bilirubin oxidase that is different from previously reported bilirubin oxidases. The basidiomycetes used in the present invention are strains belonging to the genus Ebitake, such as Ebitake [trachyderma
Trachyderma tsunodae K-2593 (Trachyderma tsunodae K-
2593)]. This bacterial strain was isolated from a fruiting body growing on a dead beech trunk in Daisen, Tottori Prefecture, in August 1977. The morphological characteristics of the fruiting body and spores of this strain are as follows. A first-year-old, sessile, the cap is flat or low in the shape of a round mountain, semicircular, 5-15 x 3-15 cm, the surface is dark, dark red, and rough, with no luster and covered with fine wrinkles and granular hard protrusions. . Usually covered with cyokolate-colored spores. The flesh is flexible and tough while growing, and becomes extremely hard and woody when dried. The color of the flesh is almost white. The underside is almost white, later becoming dark brown. The tube hole is about 1.5 cm long and has a light yellow color.
The pores are minute. Spores are Ganoderma type, broadly ovoid and large, 20-24 x 14
~16.5μ, pale yellow, inner and outer membranes clear. Comparing the above characteristics with the descriptions in ``Illustrated Encyclopedia of Japanese Fungi in True and Continued Primary Colors,'' co-authored by Rokuya Imazeki and Tsuguo Hongo, published by Yokyusha Publishing, and ``Japanese Mycological Journal Vol. 2, No. 4'' by Seiya Ito, published in 1955 by Yokendo Publishing. , it is clear that this bacterium is Ebitake. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under microorganism accession number 387. To explain the present invention in more detail, the nutrient source added to the medium may be any one that can be used by the strain used, and the carbon source may include, for example, glycerol, glucose, starch, sucrose, maltose,
Lactose, dextrin, fats and oils, etc. can be used, and suitable nitrogen sources include yeast extract, peptone, cornstarch liquor, defatted soybeans, soy flour, and meat extract. In addition, inorganic and metal salts such as phosphates, potassium salts, and magnesium salts may be added. In addition, since the novel bilirubin oxidase of the present invention is a copper enzyme, adding copper sulfate to the medium significantly increases the amount of enzyme production. For example, by adding 5 ppm, 5 to 10 times more of the novel bilirubin oxidase according to the present invention is produced than when no addition is made. When culturing basidiomycetes, the production amount of the novel bilirubin oxidase according to the present invention varies greatly depending on the culture conditions, but in general, the culture temperature is 20 to 25℃.
℃, the pH of the medium is preferably 4 to 7, and the production of the novel bilirubin oxidase according to the present invention reaches its maximum after 3 to 15 days of aeration and agitation culture. It goes without saying that the culture conditions should be set so as to maximize the production of new bilirubin oxidase depending on the bacterial strain used, medium composition, etc. The novel bilirubin oxidase produced by the bacterium of the present invention is present in a culture solution, and the culture solution is mixed with an organic solvent such as alcohol, acetone, etc.
It is separated as a precipitate by adding 80% v/v or by adding a precipitant such as ammonium sulfate from 20 to 60% w/v. The obtained precipitate is desalted by dialysis or sepadex treatment to obtain a crude oxygen solution. To purify the obtained crude enzyme solution, it was buffered in advance with 0.03M phosphate buffer (PH7.0). The adsorbed material was adsorbed onto a column of DEAE-Sephadex (A-50), and the adsorbed material was dissolved in 0.1M phosphate buffer (PH
After washing with 7.0), elute with 0.3M phosphate buffer (PH7.0) and collect the active fraction. Next, this active fraction was concentrated by ultrafiltration, desalted, and then added to 0.03M phosphate buffer (PH
DEAE/Sepharose (CL−) buffered with 7.0)
6B), wash the adsorbed material with 0.1M phosphate buffer (PH7.0), elute with 0.2M phosphate buffer (PH7.0), and collect the active fraction. After concentrating this active fraction with a collodion membrane, 0.1M
Gel filtration was performed using a Sephacryl S-200 column buffered with phosphate buffer (PH7.0), and the obtained active fraction was dialyzed against 0.01M phosphate buffer (PH7.0), and then freeze-dried. Obtain purified enzyme powder. This enzyme powder is electrophoretically homogeneous on polyacrylamide gel discs. The enzymatic and physicochemical properties of the novel bilirubin oxidase obtained by the present invention are as follows. (1) Action: The enzyme of the present invention has the action of oxidizing bilirubin in the presence of oxygen to produce an almost colorless substance via biliverdin, a pale purple substance, but does not produce hydrogen peroxide. (2) Substrate specificity: Acts on bilirubin. It also has a slight effect on biliverdin, but it accounts for about 1% of the oxidation rate of bilirubin.
It is. It also acts on phenol, catechol, and hydroquinone, but not on hemoglobin or vitamin _B12 . (3) Enzyme activity measurement method: Enzyme activity was determined by decreasing the absorption of bilirubin at 460 nm. Namely, Omega High Bilirubin Control Serum (manufactured by Highland, Inc., USA)
0.1 ml, 300 μmol of phosphate buffer (PH7.0), 0.1 ml of appropriately diluted enzyme solution, and 3.0 ml of reaction solution.
Based on bilirubin, reacted for 10 minutes at 37°C
Absorption reduction at 460 nm was measured. One unit of new bilirubin oxidase per minute at 37℃
It is expressed as the amount of enzyme that reduces the absorption at 460 nm by 1.00. (4) Optimal PH and PH stability: The optimal PH of this enzyme was around PH5.5, as shown by the curve in Figure 1, and it had extremely high activity. This enzyme was incubated at 37°C at each pH of 60%.
Figure 3 shows the PH stability when treated for minutes. As is clear from Figure 3, this enzyme has a pH of 5~
Stable between pH9. (5) Optimal temperature and thermostability: The optimal temperature of this enzyme is around 50°C, as shown by the curve in Figure 2. Figure 4 shows the thermal stability of this enzyme when it was treated at pH 7.0 for 10 minutes at each temperature. This enzyme was stable up to 55°C. (6) Molecular weight: The molecular weight of this enzyme is approximately 83,000 when measured by gel filtration using Sephacryl S-200 (manufactured by Pharmacia).
It was hot. (7) Homogeneity: Disk electrophoresis was performed using 7.5% polyacrylamide gel (PH9.4). When two gels were run and one was stained for protein, a single stained band was observed. The other was subjected to activity staining. This enzyme has the effect of quantitatively developing red color from 4-aminoantipyrine and phenol. As a result, the positions of protein staining and activity staining completely matched. (8) Isoelectric point: The isoelectric point of the enzyme determined by focal electrophoresis using Pharmalite (PH3-10, manufactured by Pharmacia) was 3.92±0.05. (9) Effects of metal ions, inhibitors, etc.: As shown in Table ¹ , this enzyme
It was strongly inhibited by ascorbic acid. Also, dithiothreitol, potassium cyanide,
L-cysteine, sodium azide, EDTA,
It was also significantly inhibited by Fe ²⁺ etc. Table 1 Additive (1mM) Inhibition rate (%) Fe ²⁺ 57 Cu ²⁺ 100 Sodium azide 60 Thiourea 0 o-phenanthroline 34 α,α′-dipyridyl 30 L-ascorbic acid 100 Iodoacetic acid 10 Dithiothreitol 86 L-cysteine 74 2-mercaptoethanol 31 Potassium cyanide 84 Reduced glutathione 38 EDTA 59 (10) Visible absorption spectrum: When the visible absorption spectrum of this enzyme solution was measured, a maximum was observed at 610 nm, indicating that it was blue protein. (11) Sugar content: Perform disc electrophoresis using 7.5% polyacrylamide gel (PH9.4) and PAS staining (Analytical Biochem. Vol. 30, p. 148 (1969))
When this was performed, the same area as the protein staining position was stained. This indicates that this enzyme is a glycoprotein containing sugar. Therefore,
The sugar content was measured using the phenol/sulfuric acid method and was approximately 4.5%. (12) Copper content: When the amount of copper in this enzyme was determined using an atomic absorption spectrometer, it was found that 4 mol of copper was contained per 1 mol of oxygen. A comparison of the enzyme according to the present invention with conventional bilirubin oxidase is as shown in Table 2.

【table】

[Table] The method for producing the novel bilirubin oxidase according to the present invention is shown below with examples, but the present invention is not limited to the scope of the following examples. Example 1 Glucose 2%, Ebios 0.5% and Agar 1.5
Trachyderma tsunodae K-2593 was inoculated into a slant medium having a composition of % (Ebios medium), and cultured stationary at 25° C. for one week to prepare a seed culture. glycerol 2.0%,
Yeast extract 0.3%, peptone 1%, KH ₂ PO ₄ 0.3%,
_MgSO4・_7H2O0.1 % and _CuSO4・_5H2O5ppm
Dispense 100 ml of a medium with the composition into a 500 ml Erlenmeyer flask, sterilize it at 120°C for 20 minutes, cool it, inoculate it with the above inoculum, and shake at 100 revolutions per minute at 27°C for 10 days. Cultured. After the culture was completed, the bacterial cells were removed to obtain a filtrate. This new bilirubin
Oxidase activity was 15.0 units/ml. Example 2 Trachyderma tsunodae K-2593 cultured in the Ebios medium of Example 1 was cultured in a medium containing 2% glycerol, 0.3% yeast extract, 1% peptone, 0.3% KH ₂ PO ₄ and 0.1% MgSO ₄ 7H ₂ O. 100 ml was dispensed and inoculated into a 500 ml Erlenmeyer flask that had been sterilized (120°C, 20 minutes), and cultured at 27°C for 7 days to prepare a seed culture. Glycerol 2%, yeast extract 0.3%, peptone 1%, KH ₂ PO ₄ 0.3%, MgSO ₄ 7H ₂ O 0.1%,
Medium 20 containing 5 ppm of CuSO ₄ .5H ₂ O and 0.03% antifoaming agent (CB-442 manufactured by Nihon Yushi Co., Ltd.) was placed in a 30 volume jar fermenter and sterilized at 120° C. for 20 minutes. After cooling, inoculate 100ml of the above seed culture solution,
Culture was carried out at 27° C. for 7 days at an aeration rate of 20 rpm and a stirring rate of 250 revolutions per minute. After culturing,
The bacterial cells were removed by filtration to obtain a filtrate. The new bilirubin oxidase activity was 21.5 units/ml. Add 60% ammonium nitrate to this culture solution 17.
Add the ammonium sulfate precipitate to saturation and leave it overnight, then add the ammonium sulfate precipitate to a large amount of 0.03M phosphate buffer (PH7.0).
I underwent dialysis all day and night. The obtained crude enzyme solution was adsorbed onto a column (φ11.0cm x 10cm) of DEAE-Sephadex (A-50) buffered in advance with 0.03M phosphate buffer (PH7.0), and the adsorbed material was
The active fraction was washed with 0.1M phosphate buffer (PH7.0) and eluted with 0.3M phosphate buffer (PH7.0), concentrated by ultrafiltration, desalted, and washed with 0.03M phosphate buffer (PH7.0). .0)
DEAE-Sepharose (CL-6B) buffered with
Column (φ5.0cm x 5cm) to adsorb the adsorbed material.
After washing with 0.1M phosphate buffer (PH7.0), active fractions were collected by elution with 0.2M phosphate buffer (PH7.0).
After concentrating this active fraction using a collodion membrane, gel filtration was performed using a Sephacryl S-200 column (φ3.6 cm x 90 cm) buffered in advance with 0.1 M phosphate buffer (PH7.0), and the obtained active fraction was concentrated. After dialysis against 0.01M phosphate buffer (PH7.0), sucrose was added as a stabilizer to a final concentration of 0.1% and freeze-dried to obtain 760 mg of purified enzyme powder. The specific activity of this powder was 355 units/mg. This enzyme powder was uniform in polyacrylamide gel disc electrophoresis. The above purification steps are shown in Table 3.

【table】 [Brief explanation of the drawing]

FIG. 1 shows the relationship between PH and activity of the novel bilirubin oxidase obtained by the present invention, and FIG. 2 shows the relationship between temperature and activity. Figure 3 shows the relationship between PH and activity after the novel bilirubin oxidase was treated at 37°C for 60 minutes at each PH, and Figure 4 shows the relationship between PH and activity at PH7.0 and at each temperature.
It shows the relationship between temperature and activity after treatment for 10 minutes.

Claims (1)

[Claims] 1. A novel bilirubin with the following physical and chemical properties:
oxidase. (1) Action: Oxidizes bilirubin in the presence of oxygen,
Biliverdin has the effect of producing an almost colorless substance via a pale purple substance, but does not produce hydrogen peroxide. (2) Substrate specificity: acts on bilirubin, slightly acts on biliverdin, and also acts on phenol,
It also acts on catechol and hydroquinone. (3) Optimal PH and PH stability: The optimal PH is around 5.5, and when treated at 37°C for 60 minutes, it is stable between PH5 and PH9. (4) Optimal temperature and thermal stability: The optimal temperature is around 50°C, and it is stable up to 55°C when treated at PH7.0 for 10 minutes. (5) Molecular weight: Approximately 83,000 (gel filtration method) (6) Isoelectric point: 3.92±0.05 (7) Inhibitors: Inhibited by Cu ²⁺ , ascorbic acid, dithiothreitol, and potassium cyanide. (8) Visible absorption: Maximum absorption near 610 nm. (9) Sugar content: Contains approximately 4.5% sugar. (10) Copper content: Contains 4 moles of copper per mole. 2. A method for producing a novel bilirubin oxidase, which comprises culturing a novel bilirubin oxidase-producing bacterium belonging to the genus Ebitake, and collecting a novel bilirubin oxidase having the following physical and chemical properties from the culture. (1) Action: Oxidizes bilirubin in the presence of oxygen,
Biliverdin has the effect of producing an almost colorless substance via a pale purple substance, but does not produce hydrogen peroxide. (2) Substrate specificity: acts on bilirubin, slightly acts on biliverdin, and also acts on phenol,
It also acts on catechol and hydroquinone. (3) Optimal PH and PH stability: The optimal PH is around 5.5, and when treated at 37°C for 60 minutes, it is stable between PH5 and PH9. (4) Optimal temperature and thermal stability: The optimal temperature is around 50°C, and it is stable up to 55°C when treated at PH7.0 for 10 minutes. (5) Molecular weight: Approximately 83,000 (gel filtration method) (6) Isoelectric point: 3.92±0.05 (7) Inhibitors: Inhibited by Cu ²⁺ , ascorbic acid, dithiothreitol, and potassium cyanide. (8) Visible absorption: Maximum absorption near 610 nm. (9) Sugar content: Contains approximately 4.5% sugar. (10) Copper content: Contains 4 moles of copper per mole. 3. A novel bilirubin oxidase-producing bacterium belonging to the genus Ebitake is called Ebitake K-2593 (Trachyderma
tsunodae K-2593)
The method described in section.