JPH0919298A - Assay using new bilirubin oxidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a usage of bilirubin oxidase catalyzing the reaction to produce biliverdin and water from bilirubin and oxygen. SOLUTION: Bilirubin oxidase is made to act on a specimen to convert the bilirubin in the specimen into biliverdin, which is then quantified, or the oxygen consumed is determined, thus assaying the bilirubin in the specimen. The bilirubin oxidase has the following characteristics: having substrate specificity to bilirubin; having no substrate specificity to at least biliverdin, catechol and hemin; and catalyzing an enzymatic reaction to produce two molecules of biliverdin and two molecules of water from two molecules of bilirubin and one molecule of oxygen. Therefore, the bilirubin oxidase is useful for diagnosing hepatopathy and scavenging bilirubin when the bilirubin in a specimen during clinical diagnosis hampers the assay of other substance(s), and can also be used for decoloring human waste treatment water because of its extremely high activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel assay method using bilirubin oxidase and a method for eliminating bilirubin in a sample.

[0002]

Bilirubin oxidase is an enzyme that catalyzes the reaction of producing biliverdin and water from bilirubin and oxygen, and has been conventionally found in the genus Milocesium (JP-A-60-12032) and the genus Bacillus (JP-A-61-12032). -20
9587) and the genus Suehirotake (Japanese Patent Laid-Open No. 59-1358).
86), Coprinus genus, Trametes genus, Coriorama genus, Foliota genus, Proirotas genus, Rangedes genus, Fusidopsis genus (Japanese Patent Laid-Open No. 59-198971), Solanaceae, Basaceae, Lily family (Japanese Patent Laid-Open No. 60-78580). )
The enzyme derived from is known.

Bilirubin is a yellow substance formed in the blood by the decomposition of hemoglobin and is the main pigment of bile produced in the liver. Conjugated bilirubin and free bilirubin were present in the serum, and increased serum conjugated bilirubin, impaired bilirubin delivery system from conjugation to duodenum in liver microsomes, and increased serum free bilirubin Indicates that hemolytic anemia or the like caused by various causes has occurred, and it is extremely important clinically to determine conjugated and / or free bilirubin. Furthermore, bilirubin oxidase is very useful for actively reducing bilirubin levels in analyte samples.

[0004]

The above-mentioned conventionally known bilirubin oxidase has a low substrate specificity,
Therefore, there was a problem in using it in the field of clinical chemistry, for example. That is, the above-mentioned known microbial-derived bilirubin
All oxidases also had substrate specificity for biliverdin and were not suitable for bilirubin analysis. In addition, plant-derived enzymes are not industrially advantageous because there are various restrictions such as seasonal factors in the raw materials, and the solanaceae of the Solanaceae family are limited to biliverdin and hemoglobin. Does not work, but does not disclose anything about the other substrates. Thus, known bilirubin
Since oxidase acts on a substance having a tetrapyrrole structure or a phenol-based substance, it has a drawback that it is greatly affected by other contaminating compositions when used in blood biochemical tests in the clinical laboratory field.

[0005]

The inventors of the present invention have conducted various studies on bilirubin oxidase, which is free from the drawbacks of these conventionally known enzymes, and as a result, an incomplete bacterium isolated from the soil of Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture. Strain M5613 belonging to the genus Penicillium produces bilirubin oxidase which catalyzes the reaction of bilirubin and oxygen to produce bilirubin and water, and the bilirubin oxidase exhibits substrate specificity for bilirubin, and biliverdin, catechol, hemin, etc. Have found that the enzyme has an excellent property of not showing substrate specificity.

The present invention has been completed based on the above findings, and has a substrate specificity for bilirubin and at least no bispecific for biliverdin, catechol and hemin, and two molecules of bilirubin and The present invention relates to the use of bilirubin oxidase which catalyzes an enzymatic reaction that produces two molecules of biliverdin and two molecules of water from one molecule of oxygen. That is, according to the present invention, the analyte has a substrate specificity for bilirubin, and at least has no substrate specificity for biliverdin, catechol and hemin.
A bilirubin oxidase that catalyzes an enzymatic reaction that produces two molecules of biliverdin and two molecules of water from one molecule of bilirubin and one molecule of oxygen is caused to act, and bilirubin in a test substance is converted into viruverdin, and the amount of biliverdin produced or The present invention relates to a method for measuring the amount of bilirubin in a subject by measuring the amount of oxygen consumed. Furthermore, the present invention provides that a sample has a substrate specificity for bilirubin, and at least has no substrate specificity for biliverdin, catechol and hemin, and has two molecules of bilirubin and one molecule of oxygen and two molecules of biliverdin and two molecules. The present invention relates to a method for erasing bilirubin in a sample by causing bilirubin oxidase, which catalyzes the enzymatic reaction that produces water, to act.

The taxonomical properties of the novel bilirubin oxidase-producing bacterium of the present invention are as follows. I. Growth condition in each medium 1. Czerbek agar (Cz) When grown at 25 ° C, the growth is normal and the diameter is 36-40 in 7 days.
mm. The flora is flat and fluffy. Pale yellow
low (Method 3A3)]. No leachate is produced, or a little colorless when it is released. Does not emit diffusible dyes. The back side is grayish yellow [4C
5)].

2. Yeast extract-containing Cavék agar (CYA) At 25 ° C, the growth is fast, and the diameter is 45 to 49 m in 7 days.
m. The flora is slightly thick and fluffy. It has 4 to 5 wrinkles in a radial pattern and is a mixture of pale red [pale red (9A3)]-green gray [greenish gray (27A3)]. The surrounding area is all green. The leachate is large and light red [pal
e red (9A3)]. Diffusible dye is pale red [pal
e red (9A3)]. The back side is purple brown [violet
brown (11E8)].

3. Wort agar medium (HA) At 25 ° C, the growth is fast and the diameter is 48 to 51 m after 7 days.
m. The flora is flat and fluffy. Gray green [greyish g
reen (29C3)]. The surrounding area is all green. No leachate or diffusible dye is produced. The back is olive brown [oliv
e brown (4D7)].

II. Physiological properties Viable pH 1.5 to 10.5 Optimum pH 3.5 to 8.5 Viable temperature 17 to 37 ° C Optimal growth temperature 28 to 34 ° C

III. Morphological characteristics under a microscope No sexual generation was observed, and the cells were proliferated by conidia. Conidia peduncles are derived from aerial hyphae or aerial hyphae and are relatively long.
00 to 250 × 2.0 to 2.8 μm, with smooth or slightly rough wall. Conidia formation is a filophora type. Penicillin is monocyclic or divergent. The metre spreads over a wide angle, 1
1 to 15 × 2.5 to 2.8 μm, 2 to 3 rings, smooth or slightly rough surface. Fear rid is a tokuri type, 7 to 10 ×
2 to 2.5 μm, 4 to 7 walnuts, conidia are subspherical or elliptical, and slightly pear-shaped mixed 2.3 to 3.0 × 2.0
~ 2.5 μm, smooth or slightly rough surface.

From the formation of exogenous conidia, M5
Strain 613 belongs to an incomplete bacterium. The conidia is of the phialophora type and belongs to the genus Penicillium because it forms conidia from the tip of the fialid. So, "Thegenus penicil
ium "Academic press.Londo
n p434 (1979) and "A manual.
of the penicillia "The Will
liams and Wilkins, Baltimo
Re, p875 (1949). Furthermore, because Penicillium is a unicycle or divergent state, grows rapidly in each medium, and the rear surface of CYA is markedly purple-brown, Penicillium jancinerum (Penicillum) is used.
Lium janthinellum) and Penicillium simplicitium (Penicillium si)
mplicissium), but penicillium symplecticum has a conidia peduncle of 400-800.
It can be distinguished from the fact that the wall of conidia peduncle is rough and the back surface is not purple-brown, etc., and the M5613 strain was identified as Penicillium janthinellum. The color of the growth state in each medium is indicated by Kornerup. A. and J. H. Wansch
er. 1978. Methen handbook
ofcolor. 3rd ed. Eyer Meth
According to the notation of Uen and London.

The strain of the present invention was found in Penicillium Jancineram M5613 at the Institute of Microbial Biotechnology, Institute of Industrial Science and Technology.
(Penicillium janthinellum
M5613) Micro Engineering Research Institute No. 9167 (FERM P-
9167). This strain was transferred to a deposit under the Budapest Treaty,
Deposited as 674 (FERM BP-1674).

In producing the bilirubin oxidase of the present invention, the bilirubin oxidase-producing bacterium is cultured by a usual method for producing an enzyme and the like. The form of culture may be liquid culture or solid culture, but it is industrially advantageous to inoculate the production medium with cells of the bilirubin oxidase-producing bacterium and perform deep aeration agitation culture.
As a medium composition for culturing bilirubin oxidase, those commonly used for culturing microorganisms, particularly penicillium, are widely used.

Any nitrogen compound can be used as a nitrogen source, for example, corn steep liquor, peptone,
Casein, soybean flour, yeast extract, various meat extracts and the like are used. Any carbon compound that can be assimilated may be used as the carbon source, and for example, sugar condensate, glucose, sucrose, dextrin and the like are used. In addition, potato extract is also a suitable medium component, and various inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, potassium monophosphate, and dipotassium phosphate are used as necessary. The culturing temperature may be appropriately changed within a range in which the bacterium grows and produces bilirubin oxidase, but it is preferably 20 to 37 ° C., preferably 2
It is 5 to 30 ° C, especially 28 ° C. The culturing time varies depending on the conditions, but is usually 2 to 8 days, especially about 4 days. However, it is natural to terminate the culture at an appropriate time, considering the time when bilirubin oxidase reaches its maximum titer.

After culturing, the usual enzyme collecting means can be used to collect the present enzyme from the culture. Although the present enzyme is present in both the cells and the culture solution phase, it is usually obtained from the culture solution phase in which the cells are separated and removed from the culture due to problems such as collection efficiency. The enzyme may be collected using a usual means for collecting the enzyme, and used in combination with the enzyme obtained from the culture solution phase. The crude enzyme solution thus obtained is further purified by a known isolation and purification means for proteins, enzymes, etc.
An oxidase is obtained. For example, crude bilirubin
The crude enzyme may be recovered by precipitating the enzyme in the oxidase-containing solution using a fractional precipitation method using an organic solvent such as acetone or methanol, a salting-out method using ammonium sulfate, sodium chloride, aluminum sulfate or the like, and recovering the crude enzyme.

Then, for further purification, if necessary, the precipitate is dissolved in a solvent such as Tris-hydrochloric acid buffer solution, and this is dissolved in carboxymethyl-cellulose, carboxymethyl-dextran gel, sulfopropyl-dextran gel, etc. Ion-exchange resin and dextran gel or polyacryl amide gel, etc. are combined and purified by an appropriate combination of adsorption chromatography using gel filtration agents, and then dried by a technique such as freeze-drying to obtain purified bilirubin oxidase. . The physicochemical properties of the bilirubin oxidase thus obtained are as described below.

(1) Method for measuring activity Preparation of substrate solution: 6 mg of unconjugated unbound bilirubin (manufactured by Wako Pure Chemical Industries, Ltd.) was placed in a 100 ml beaker and 5N Na was added.
Wet with 0.5 ml of OH solution, add 10 ml of 1M Tris-HCl buffer and mix well, add 70 ml of water and p.
After preparing H to 8.4, make up to 100 ml.

Preparation of enzyme solution: 50 mg of lyophilized product of bilirubin oxidase obtained according to the examples described below was accurately weighed, dissolved in 65 ml of distilled water and adjusted to 37 ° C. Activity measurement: 3 ml of the substrate solution was placed in a small test tube and allowed to stand in a water bath at 37 ° C for 2 minutes, and then 0.1 ml of the enzyme solution was added,
The reaction was started. Next, after reacting exactly at 37 ° C. for 10 minutes, the absorbance at 680 nm was measured using a spectrophotometer (A ₁ ), and 0.1 ml of water was added instead of 0.1 ml of the enzyme solution as a control. It was measured (A ₂ ).

Bilirubin oxidase activity (U / m
l) 11.9 * ¹ : biliverdin dihydrochloride (manufactured by Sigma, l
ot. 43F-8020) (8.3 mg) was dissolved in 1 M Tris-HCl buffer (10 ml), and water (70 ml) was added to the solution to give 5N.
Adjust the pH to 8.4 with NaOH solution and 100
Made up to ml. This was diluted 2, 5, 10 times with 0.1 M Tris-hydrochloric acid buffer (pH 8.4),
Total absorption at 750 to 350 nm was measured using a spectrophotometer (UV-210A, manufactured by Shimadzu Corporation). As shown in FIG. 5, the doubling diluted biliverdin solution at 680 nm
Absorbance is 0.73, 0.3-1 when diluted 5 times
The one diluted 0 times showed a value of 0.155. From these results, the absorbance of a 1 mM solution of biliverdin at 680 nm was set to 11.9.

(2) Substrate specificity: Specificity for each substrate was examined from the oxygen consumption rate using an oxygen electrode meter. At this time, HgCl ₂ was added to the substrate solution to a concentration of 0.1 mM. The substrate was 0.1 mM, and the enzyme was 0.1 ml of the enzyme solution used for the above-described activity measurement. As a result, as shown in Table 1, substrates other than bilirubin did not react. Therefore, the bilirubin oxidase of the present invention has very high bilirubin specificity.

[0022]

[Table 1]

(3) Enzymatic action: It catalyzes the following reaction. Bilirubin + 1 / 2O ₂ → biliverdin +
H ₂ O

(4) Thermal stability: 0.5 M of 0.2 M potassium phosphate buffer (pH 6.5) to which 0.5 ml of the above enzyme solution was added was added at 40 to 80 ° C. at 30 ° C.
The mixture was heated for each minute, and the residual activity was measured based on the activity measuring method. The result was stable up to 50 ° C. as shown in FIG.

(5) Optimum temperature: The activity of the bilirubin oxidase of the present invention measured at each temperature of 40 to 70 ° C. using the substrate solution in the activity measurement method is shown in FIG.
As shown in, the optimum temperature was 55 to 60 ° C.

(6) pH stability: 0.5 of the above enzyme solution
0.5 ml of 0.2 M pH buffer was added to each ml, and the mixture was heated at 70 ° C. for 30 minutes, and the reaction mixture was added to 0.1 ml.
The residual activity was measured in ml based on the activity measuring method, and the influence of pH was examined. The result is as shown in FIG.
[In the figure: Acetate buffer (pH 4-6) as buffer ●-
●, potassium phosphate buffer (pH 6 to 7) △-△, Tris-hydrochloric acid buffer (pH 7 to 8) ○-○], pH stability of the bilirubin oxidase of the present invention is pH 4.
It was recognized as around 0 to 4.5.

(7) Optimum pH: The substrate solution prepared by the activity measuring method was adjusted to pH 7.5, 8.0, 8.5, 9.0, 9.5, 10 with 5N HCl or 5N NaOH. Then, the optimum pH was measured using this. However, it was assumed that the absorbance of biliverdin was not changed by the change of pH. The results are shown in Fig. 4, and the pH
It had an optimum pH of 8.5 to 9.0.

(8) Inhibition and activation: The activity of bilirubin oxidase was measured by adding 1 mM, 1 mM and 0.05% of metal ion, EDTA and surfactant to the substrate solution based on the activity measurement method. The relative activity when the activity without addition was defined as 100 was determined. The results are shown in Table 2, and include mercury ions, lead ions,
It was markedly activated by cetyltrimethylammonium chloride and markedly inhibited by copper and cobalt ions.

(9) Molecular weight: 500,000 ± 50,0
00 (by gel filtration method using Sephadex G-200. Molecular weight marker; using catalase, ferritin and thyroglobulin). (10) Isoelectric point: The result measured by the focus electrophoresis apparatus has an isoelectric point at pH 4.7 ± 0.5.

[0030]

[Table 2] * ¹ ; Cetyltrimethylammonium chloride * ² ; Cetyltrimethylammonium bromide

(11) Km value: 2,4,6,8,10,15,20 mg of a bilirubin standard product (manufactured by Wako Pure Chemical Industries, Ltd.) was taken and dissolved according to the adjustment of the substrate solution in the activity measuring method, Km using 0.1 ml of the enzyme solution used to measure the activity of
As a result of measuring the value, the Km value for bilirubin under this measurement condition was 1.1 × 10 ⁻⁴ M. Comparing these properties with known bilirubin oxidase, it is clear that they are different from any of the enzymes.

In order to measure the amount of bilirubin in a sample using the bilirubin oxidase of the present invention, known bilirubin measuring methods can be applied. That is, the amount of bilirubin in the test sample is measured by converting bilirubin in the test sample into bilirubin by the action of the bilirubin oxidase of the present invention, and measuring the amount of biliverdin produced or the amount of oxygen consumed in the reaction. I can do it.

When the concentration of bilirubin in a sample is measured, the amount of the bilirubin oxidase of the present invention used is 0.02 as the bilirubin oxidase activity.
Although it is 1 to 10 units, it may be appropriately adjusted according to the measurement time, and reaction accelerating substances such as sodium dodecyl sulfate, sodium cholate, potassium ferricyanide, etc. and other buffers, enzyme stabilizers, etc. may be used as necessary. All you have to do is add.

Further, in the case of separately measuring conjugated bilirubin and unconjugated bilirubin in a sample, the bilirubin oxidase of the present invention may be applied to a known method. For example, conjugated bilirubin is analyzed at a pH of less than 6. The total amount of bilribin can be detected by
The unconjugated bilirubin can be analyzed in 10
It is determined by subtracting the amount of conjugated bilirubin from the total amount of bilirubin.

Furthermore, by appropriately adding the bilirubin oxidase of the present invention, a system for measuring a substance other than bilirubin in a sample in clinical diagnosis, for example, a system using glucose oxidase-peroxidase in glucose measurement, cholesterol, In the measurement, bilirubin, which is a measurement substance, can be eliminated in a system using costerol oxidase-peroxidase and the like.

Examples and reference examples of the present invention will be given below. However, it goes without saying that the present invention is not limited to these examples and reference examples. Reference Example 1 To 200 g of sliced potatoes, 1 liter of distilled water was added, and after boiling for 1 hour, 20 g of glucose was added to the supernatant filtered with a cloth.
g, yeast extract 1.5 g, KH ₂ PO ₄ 2.0 g, Mg
It was added SO ₄ · 7H ₂ O 0.5g, and 1 liter added after dissolution of distilled water. 500 ml of 100 ml of main culture
The mixture was dispensed into an Erlenmeyer flask and sterilized by heating at 121 ° C. for 20 minutes. To this Penicillium Jancineram M561
3 platinum (FERM BP-1674) was inoculated with 1 platinum loop from the preserved agar slant medium, and shake-cultured at 28 ° C. for 4 days.

10 ml of culture broth for 20 minutes at 3 ° C. and 15K
The cells were sonicated to destroy the cells. This was repeated 5 times to obtain 50 ml of crude enzyme solution (20u). This was kept for 20 minutes at 5 ° C. and 15,000 rpm. p. m. The pH of the supernatant after centrifugation was adjusted to 5.0 with 1N hydrochloric acid,
Minutes, 5 ° C., 15,000 r. p. m. And centrifuged. This precipitate was added to 20 mM potassium phosphate buffer (pH
6.5) Dissolve in 50 ml, 20 minutes, 15,000
r. p. m. Centrifugation was carried out with to obtain a supernatant (11.3
u). Sephadex G-100 obtained by buffering 5 ml of this with 20 mM potassium phosphate buffer (pH 6.5)
Gel filtration was performed using a (2.8 x 60 cm) column. This was repeated 10 times to obtain 50 ml of enzyme solution (7.2 u). To this, 100 mg of sucrose was added, and freeze-dried according to a conventional method to obtain 320 mg of a freeze-dried product of 0.018 u / mg.

Example 1 3 ml of the substrate solution prepared according to the above-mentioned activity measuring method / preparation of substrate solution was placed in a 3 ml volume quartz cell and 10 mM H 2
30 μl of gCl ₂ was added. To this, 50 mg of the lyophilized product of bilirubin oxidase obtained in Example 1 was accurately weighed and dissolved in 6.5 ml of distilled water.
Add 50 μl and 100 μl of oxidase solution,
The sample was set on a spectrophotometer (UV-210A, manufactured by Shimadzu Corporation), and the change with time of the absorbance at 680 nm at 37 ° C was drawn on a chart (Fig. 6). As is clear from FIG.
When 50 μl of the enzyme solution was used, the reaction of bilirubin oxidase was completed in 6 minutes, and when 100 μl of the enzyme solution was used, the reaction was completed in 3.5 minutes. From this, it is possible under this condition to measure bilirubin in a short time by measuring the production of biliverdin, and elimination of bilirubin is also possible under conditions that do not affect other crude products due to its high substrate specificity. It was

Example 2 Human sera of 0.1, 0.2, 0.3, 0.4, 0.5,
0.6 and 0.7 ml of each was taken, 0.1 M Tris-hydrochloric acid buffer was added to make 3 ml, and 0.1 U of the bilirubin oxidase of the present invention was added, followed by incubation at 37 ° C. for 30 minutes, and the reaction The liquid was set in a spectrophotometer (UV-210A, manufactured by Shimadzu Corporation), and the absorbance at 680 nm was measured at 37 ° C. The result is shown in FIG.
As shown in Table 1, extremely good linearity was obtained in serum solutions diluted with various ratios.

Example 3 Preparation of calibration curve (1) Preparation of test solution: Buffer solution: 0.1 M Tris-hydrochloric acid buffer solution (pH 8.
4) Enzyme reaction solution: 0.1 M Tris-hydrochloric acid buffer solution (pH 8.
The bilirubin oxidase of the present invention was added to 4) at a rate of 0.1 U / test for adjustment. (2) Preparation of standard bilirubin solution: Accurately weigh 5.9 mg of unconjugated unbound bilirubin (manufactured by Wako Pure Chemical Industries, Ltd.), moisten it with 0.5 ml of 5N NaOH solution, and 1M Tris-hydrochloric acid buffer solution ( After dissolving with 10 ml of pH 8.4),
The solution diluted to 100 ml was used as a 100 μM bilirubin solution, which was used as a 0.1 M Tris-hydrochloric acid buffer solution (p.
H8.4) and diluted to 2,4,6,8,10,14μ
M standard bilirubin solution was prepared.

(3) The above enzyme reaction solution was added to 3 ml each of the above standard bilirubin solution, and the mixture was kept at 37 ° C. for 3 minutes.
After incubating for 0 minutes, the reaction solution was set in a spectrophotometer (UV-210A manufactured by Shimadzu Corporation),
The absorbance at 680 nm was measured at 37 ° C. The results are shown in FIG. 8, which showed extremely good linearity.

[0042]

INDUSTRIAL APPLICABILITY The bilirubin oxidase of the present invention has a substrate specificity for bilirubin, is advantageous for diagnosing liver diseases, and when bilirubin in a sample in clinical diagnosis becomes an inhibition in the measurement of other substances. , It is useful for the elimination of bilirubin and has a very high activity, and therefore it is expected to be used for decolorization of treated human waste water.

[Brief description of the drawings]

FIG. 1 shows the thermostability of bilirubin oxidase of the present invention.

FIG. 2 shows the optimum temperature of bilirubin oxidase of the present invention.

FIG. 3 shows pH stability of the bilirubin oxidase of the present invention.

FIG. 4 Optimum pH of bilirubin oxidase of the present invention
It shows.

FIG. 5 shows the absorption curve of biliverdin.

FIG. 6 shows the reaction course of bilirubin oxidase of the present invention.

FIG. 7 shows a correlation diagram between the amount of blood sample and the activity using the enzyme of the present invention.

FIG. 8 is a calibration curve for bilirubin.

Claims (2)

[Claims] 1. A test sample having a substrate specificity for bilirubin and at least no substrate specificity for biliverdin, catechol and hemin, and two molecules of bilirubin and 1
The bilirubin oxidase, which catalyzes an enzymatic reaction that produces two molecules of biliverdin and two molecules of water from molecular oxygen, is made to act, and the bilirubin in the test substance is converted to bilberdin, and the amount of biliverdin produced or the amount of consumed oxygen is changed. A method for measuring the amount of bilirubin in a subject by measuring the amount. 2. A specimen has a substrate specificity for bilirubin, and at least has no substrate specificity for biliverdin, catechol and hemin, and has two molecules of bilirubin and 1 molecule.
A method of erasing bilirubin in a sample by causing bilirubin oxidase, which catalyzes an enzymatic reaction that produces two molecules of biliverdin and two molecules of water, from oxygen of a molecule to act.