JP2002112766A - Bilirubin dehydrogenase and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a bilirubin dehydrogenase and a method for producing the bilirubin dehydrogenase derived from a fungus belonging to the genus Aspergillus, which are characterized by using an artificial electron acceptor. SOLUTION: The appearance of an enzyme acting on bilirubin, which has an assay precision not reduced by an oxygen concentration in a reaction solution and various kinds of decomposition products and is not affected by various interference substances in a serum to be a specimen in an assay system using an optical system and is usable in an assay system having a selectivity to conjugation type bilirubin or liberation type bilirubin, is expected. The method for producing the enzyme is also expected. Consequently, a bilirubin dehydrogenase which is not affected in an assay precision by an oxygen concentration in a reaction solution in an assay of bilirubin in a specimen in a clinical diagnosis field, provides a single reaction product (e.g. bilirubin) having an absorption in a long wavelength region and exhibits selectivity to conjugation type or liberation type bilirubin can be provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for culturing a bilirubin dehydrogenase and a bilirubin dehydrogenase-producing bacterium belonging to the genus Aspergillus, wherein the enzyme uses an artificial electron acceptor, and collecting bilirubin dehydrogenase from the culture. And a method for producing bilirubin dehydrogenase.

[0002]

2. Description of the Related Art As an enzyme acting on bilirubin,
Bilirubin oxidase is well known. Bilirubin oxidase is an enzyme that catalyzes a multi-step oxidation reaction of bilirubin using oxygen as a hydrogen acceptor. Bilbergine has been identified as one of the reaction intermediates. Conventionally, the genus Myrosesium (JP-A-60-12032), the genus Bacillus (JP-A-61-209587), the genus Suehirotake (JP-A-59-135886), the genus Coprinus, the genus Trametes, and the genus Coriorama Genus, Foliota, Pseirotus, Rangedes, Fusidopsis (JP-A-59-198971), Solanaceae,
Enzymes derived from Musaceae, Liliaceae (JP-A-60-78580), Penicillium (JP-A-63-309187) and the like have been reported.

[0003] Bilirubin is a yellow substance formed in blood by the decomposition of hemoglobin, and is a main pigment of bile produced in the liver. There are conjugated and free bilirubin in serum. An increase in serum conjugated bilirubin reflects the conjugation and transport system of bilirubin from liver microsomes to the duodenum, whereas an increase in serum free bilirubin may cause the onset of hemolytic anemia caused by various causes. As reflected, quantifying conjugated and / or free bilirubin is of great clinical importance.

[0004]

The conventionally known bilirubin oxidase catalyzes a multistage oxidation reaction of bilirubin via bilubeldin using oxygen as a hydrogen acceptor. When bilirubin is measured using this bilirubin oxidase, it is affected by the measurement accuracy due to the oxygen concentration in the reaction solution, the measurement accuracy decreases due to the large number of degradation products, and the measurement system using an optical system. Has drawbacks such as the need to rely on changes in absorbance around 450 nm, which are susceptible to various interfering substances in the serum to be tested, and the ambiguity of selectivity for conjugation and free bilirubin. .

[0005] An object of the present invention is to provide a single reaction product (eg, virbergine) having an absorption in the long wavelength region without being affected by the measurement accuracy due to the oxygen concentration in the reaction solution in the determination of bilirubin. The purpose of the present invention is to provide a bilirubin dehydrogenase having selectivity for free and free bilirubin.

[0006]

Means for Solving the Problems In order to achieve the above object, the present inventors have screened a wide range of bilirubin-operating enzymes that utilize compounds other than oxygen as hydrogen acceptors from nature and found that Yoshida of Sakyo-ku, Kyoto-shi A strain belonging to the genus Aspergillus aspergillus ocraceus IB-3 isolated from soil collected from a mountain produces bilirubin dehydrogenase using an artificial electron acceptor. Furthermore, the present bilirubin dehydrogenase produces bilirubin in a single reaction. The present inventors have found that the compound has specificity for ditaurobilirubin, which is a model compound of conjugated bilirubin, and completed the present invention.

[0007] The present invention has been completed based on the above findings, and characterized in that a bilirubin dehydrogenase and a bilirubin dehydrogenase-producing bacterium belonging to the genus Aspergillus, which are characterized in that the enzyme uses an artificial electron acceptor, are cultured in a medium. And a method for producing bilirubin dehydrogenase, comprising collecting bilirubin dehydrogenase from a culture. Hereinafter, the present invention will be described in detail. First, the bilirubin dehydrogenase producing bacterium of the present invention is not particularly limited as long as it is a microorganism capable of producing bilirubin dehydrogenase, and may be a variant or mutant having the ability to produce bilirubin dehydrogenase, Preferably, Aspergillus ocraceus IB-3 (FERM P-18035) strain belonging to the genus Aspergillus is exemplified.

[0008] Aspergillus ocraceus IB
-3 (FERM P-18035) strain was submitted to the Research Institute of Biotechnology, Industrial Science and Technology, Ministry of International Trade and Industry, located at 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture on September 13, 2000, under the accession number FERM P-18035. And is available to anyone.

The taxonomic properties of the novel bilirubin dehydrogenase producing bacteria of the present invention are as follows. 1. Growth state in each medium (1) Tzapek agar medium (Cz) Growth is fast. The basal mycelium is flat, hard and thick. Conidium formation is good and light ocher. Odor is mushroom smell. Produces sclerotium well and is scattered in single brown. (2) Wort agar medium The growth is fast and the basal hypha layer is flat and thin. 37 conidia
Does not grow at ℃. 2. Physiological properties Growing pH: 1.5 to 10.5 Growing optimal pH: 3.5 to 8.5 Growing temperature: 17 to 37 ° C Growing optimal temperature: 28 to 34 ° C Morphological characteristics under a microscope The conidium is spherical, 200-300 μm in diameter, and ocher. The conidiophore is 1-1.5 mm in length, 10-15 μm in diameter, and yellow. The walls are thick and the surface is ancestral. The tip is enlarged and forms a sac. Apical sac, spherical, 30-80μ in diameter, colorless, thin wall. Metre is formed from the whole sac, 10-25x
3-5 μm. The phialide is 7 to 10 × 2 to 3 μm. Conidia are subspherical, 3-4 μm in diameter, and have a fine rough surface. The sclerotium is ovoid, 400-700 μm in diameter, white to pale pink. Based on the growth and morphological characteristics indicated by the above results, the IB-3 strain was determined to belong to Aspergillus oculaseus, and this strain was named Aspergillus oculaseus IB-3 strain.

[0010] In producing the bilirubin dehydrogenase of the present invention, the bilirubin dehydrogenase-producing bacterium is cultured by an ordinary method for producing an enzyme or the like. The form of culture may be either liquid culture or solid culture, but industrially, it is advantageous to inoculate the cells for the production of bilirubin dehydrogenase-producing bacteria into a culture medium for the production and to perform deep aeration and stirring culture. As the medium composition for culturing bilirubin dehydrogenase, those commonly used for culturing microorganisms, particularly Aspergillus, are widely used. Any available nitrogen compound may be used as the nitrogen source, and examples thereof include corn steep liquor, peptone, casein, soybean flour, yeast extract, and various meat extracts. The carbon source may be any assimilable carbon compound, and for example, molasses, glucose, sucrose, dextrin and the like are used.

Other potato extract is also a suitable medium component, and various inorganic salts such as salt, potassium chloride, magnesium sulfate, monopotassium phosphate, dipotassium phosphate and magnesium sulfate are used as necessary.
The cultivation temperature can be appropriately changed within a range in which the bacterium grows and produces bilirubin dehydrogenase, but is 20 to 37 ° C, preferably 25 to 30 ° C, particularly 28 ° C. The cultivation time varies slightly depending on the conditions, but is usually 2 to 8 days, especially 4 to 8 days.
About a day. However, it is a matter of course that the culture is terminated at an appropriate time in view of the time when the bilirubin dehydrogenase reaches the highest titer.

After completion of the cultivation, the enzyme can be collected from the culture using conventional enzyme collecting means. Bilirubin dehydrogenase is mainly contained and accumulated in the cell membrane fraction, and may be extracted from the cell membrane.
As an example of the extraction method, cells are separated from the culture by centrifugation or the like, and the cells are treated with a surfactant (TX) for membrane solubilization.
After suspension in a buffer such as a phosphate buffer or Tris-HCl buffer containing -100), the suspension is crushed with ultrasonic waves, glass beads or the like and centrifuged, and the soluble fraction is recovered as a crude enzyme solution. The crude bilirubin dehydrogenase-containing solution thus obtained is treated with a known protein and enzyme isolation and purification means, whereby a purified bilirubin dehydrogenase can be obtained.

General enzyme purification methods such as fractional precipitation with an organic solvent such as acetone or ethanol, salting out with ammonium sulfate, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, etc. Select as appropriate,
Purified bilirubin dehydrogenase can be obtained in combination with a stabilizer such as sucrose or glycerol in an amount of about 5 to 50% and an amino acid or coenzyme in an amount of 0.01 to 50%.
It may be freeze-dried by adding about 0.1%.

As artificial electron acceptors, phenazine methosulfate (PMS), phenazine ethosulfate (PES), methoxatin, 1,4-benzoquinone (B
Q), 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Qo), 2,6-dimethyl-1,4-benzoquinone (2,6-DMBQ), 2,6-dichloro-1,
4-benzoquinone (2,6-DCBQ), 1,2-naphthoquinone (NQ), 1,2-naphthoquinone-4-sulfonic acid (NQ-4-sulfonic acid), K ₃ Fe (CN) ₆ ,
N, N-dimethyl-p-phenylenediamine and N,
N, N, N-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and the like.

Next, the physicochemical properties of the bilirubin dehydrogenase obtained by the present invention and the method for measuring the enzyme activity will be described below. Method for measuring enzyme activity of bilirubin dehydrogenase Measuring reagent 100 mM acetate buffer (pH 4.0) 0.05 mM Ditaurobilirubin 0.1 mM PMS

After heating, 5 ml after adding 0.1 ml of enzyme solution
After a minute, the absorbance at 450 nm (A) is measured. As a blank, the absorbance (B) at 450 nm is measured 5 minutes after the addition of 0.1 ml of purified water instead of the enzyme solution. The enzyme activity is determined from the difference (A) between the absorbance (A) and the absorbance (B). The enzyme activity of 1 U is defined as the amount of the enzyme that reduces the absorbance at 450 nm in an optical path length of 1 cm by 1 per minute under the above reaction conditions, and the calculation formula is as follows. Enzyme activity (U / ml) = [(B)-(A)] × 11 × dilution factor of enzyme Further, the same measurement can be performed based on an increase in absorbance at 660 nm caused by the generation of biliverdin.

Physicochemical properties (1) Enzymatic action Enzymatic action using ditaurobilirubin or bilirubin as a substrate is shown below. (1) Zitaurobilirubin + PMS → Zitaurobiliverdin + PMSH ₂ (2) Bilirubin + PMS → Biliverdin + PMS
H _{2 Under the} above enzyme activity measurement conditions, the activity of (2) is 5% of the activity of (1). (2) Substrate specificity The following compounds serving as laccase substrates did not act at all. The activity was determined by examining the coupling reaction with 4-aminoantipyrine.

Laccase substrate: TOOS (N-ethyl-
N- (2-hydroxy-3-sulfopropyl) -3-methylaniline), dimethylaniline, diethylaniline, N, N-dimethyl-p-phenylenediamine, catechol, resorcinol, hydroquinone, phenol,
Guaiacol, pyrogallol, p-hydroxybenzoic acid, caffeic acid, hydrocaffeic acid, o-cresol, p-toluidine, o-chlorophenol, m-chlorophenol, p-chlorophenol, 2,4-dichlorophenol, 2, , 6-dichlorophenol, 2,4
6-trichlorophenol, 2,6-dimethoxyphenol, ABTS (2,2-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, propyl gallate. Under the above-mentioned activity measurement conditions, PMS was converted to various electron acceptors (0.1
mM) and the activity was measured. Table 1 shows the relative activity values when the enzyme activity without the addition of the electron acceptor was defined as 100%.

[0019]

[Table 1]

(5) Optimum pH FIG. 1 shows the results of the examination of the optimum pH. Buffer pH 1.8
4 to 3.87 are sodium acetate-hydrochloric acid buffer, pH 3.
45 to 5.93: sodium acetate-acetic acid buffer, pH
6.58 to 8.18 are phosphate buffer, pH 8.22 to
9.62 used Tris-HCl buffer. The substrate was measured using ditaurobilirubin. As a result, the optimal pH of the bilirubin dehydrogenase of the present invention was 3.87 (sodium acetate-acetic acid buffer). (6) Optimum temperature Fig. 2 shows the results of the examination of the optimum temperature. The optimum temperature of the bilirubin dehydrogenase of the present invention was 60 ° C. (7) pH stability FIG. 3 shows the results of the examination of pH stability. The buffer is pH 3.
03-4.21 is sodium acetate-hydrochloric acid buffer, pH
4.67 to 5.63 are sodium acetate-acetate buffer, p
H6.12 to 7.46 is a phosphate buffer, pH 7.7 to
9.35 is Tris-HCl buffer, pH 10.06-1.
1.26 used boric acid-NaOH buffer. The relative activity after treatment in various buffers at 37 ° C. for 30 minutes is shown. As a result, p of the bilirubin dehydrogenase of the present invention
H was stable in the range of 3.03 to 11.26. (8) Thermal Stability FIG. 4 shows the results of the study on thermal stability. The residual activity after treatment at various temperatures for 10 minutes was shown. As a result, the bilirubin dehydrogenase of the present invention was stable up to 50 ° C.

It is also possible to measure bilirubins (such as conjugated bilirubin or free bilirubin) in a test solution using the bilirubin dehydrogenase of the present invention and an artificial electron acceptor. When measuring bilirubins in a test solution, decrease the absorbance specific to bilirubins at around 450 nm in the presence of bilirubin dehydrogenase and an artificial electron acceptor, or measure the absorbance specific to bilurubins of the reaction product at around 660 nm What is necessary is just to measure the increase of. In addition, it is also possible to prepare an electrode by immobilizing bilirubin dehydrogenase and an artificial electron acceptor on a carbon paste and measure a current generated when the electrode is brought into contact with a test solution.

In this case, a buffer which does not adversely affect the enzyme reaction system, for example, an acetate buffer, a citrate buffer, a phosphate buffer, a Tris-HCl buffer, a mono- or diethanolamine buffer, a glycine buffer or a good buffer This is performed using a buffer. The pH of the reaction solution when measuring bilirubin may be a pH at which bilirubin dehydrogenase and various bilirubins react, and the pH of the reaction solution when measuring bilirubin may be a pH from a neutral region to an alkaline region, preferably The pH is 6.5 to 8.5. The pH when measuring ditaurobilirubin may be a pH in a weakly acidic region, and is preferably from 3.5 to 6.0.

The amount of bilirubin dehydrogenase to be added to the reaction solution may be any amount at which the measurement of bilirubin can be performed smoothly, for example, 0.01 to 1000 U / ml, preferably 0.1 to 100 U / ml. . The test liquid to be measured includes a biological sample containing bilirubin, and includes, for example, serum, plasma, urine, and peritoneal fluid. The reaction is carried out by using the above-mentioned reaction system using 2 to 1000 μl of such a test solution, and the reaction temperature is, for example, 15 to 45 ° C., preferably 20 to 40 ° C.
The reaction time may be 1 to 60 minutes, preferably 1 to 10 minutes in the case of the endpoint method, and within the time when the reaction time is linearly performed in the rate assay, preferably 1 to 60 minutes. Measure for ~ 3 minutes.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to examples, but the method of the present invention is not limited thereto.

[0025]

Example 1 5% glucose, 0.5% polypeptone, 0.1% yeast extract, 0.1% KH ₂ PO ₄ ,
0.1% K ₂ HPO ₄ , 0.1% MgSO ₄ .7H ₂ O
100 ml of a liquid medium (pH 6) containing the mixture is dispensed into 20 500 ml Erlenmeyer flasks, sterilized by heating at 120 ° C. for 20 minutes, and then added to Aspergillus oculaseus IB.
-3 strains were transplanted by removing one platinum loop from the slant and cultured at 28 ° C. for 72 hours with shaking.

The cells obtained by filtering 2 L of the culture solution with a paper filter are mixed with 1 L of 0.1% Triton X-10.
10 mM Tris-HCl buffer containing 0 (pH 7.5)
, And crushed using a cell crusher Dynomill (manufactured by WILLY A. BACHOFEN) using glass beads.
Centrifugation was performed at 00 rpm for 60 minutes to obtain a solubilized fraction. The solubilized fraction was concentrated to 10 ml with an ultrafiltration device (fraction molecular weight: 100,000) manufactured by Amicon, and then 0.25 M
20 mM Tris-HCl buffer containing NaCl (pH
Sephacryl S-200 equilibrated in 7.5)
HR column (1.5 × 90cm) (Pharmacia)
And eluted with the same buffer to collect an active fraction. The obtained enzyme solution was dialyzed against 10 L of 20 mM Tris-hydrochloric acid (pH 7.5) at 4 ° C. overnight, and then lyophilized to obtain an enzyme preparation (Table 2).

[0027]

[Table 2]

[0028]

Example 2 Measurement of Ditaurobilirubin Using Bilirubin Dehydrogenase of the Present Invention Using PMS as an Artificial Electron Acceptor Into a spectrophotometer cell having a 1 ml capacity and a 1 cm optical path length, various concentrations (0, 2, 5; (10, 20, 40 μM) in a 950 μl reaction solution containing 100 mM acetate buffer (pH 4.0), incubated at 37 ° C. for 2 minutes, and then subjected to initial absorbance (450 nm and 660 n).
m) is measured. Thereafter, 50 μl of a bilirubin dehydrogenase solution was added to start the reaction, and after incubation at 37 ° C. for 10 minutes, the absorbance (at 450 nm and 660 nm) was measured.
nm). FIG. 5 shows the measurement results. The white circles in the figure indicate the absorbance at 450 nm, and the black circles indicate the absorbance at 660 nm. As shown in the figure, the change in absorbance before and after the reaction was proportional to the concentration of ditaurobilirubin.

[0029]

Example 3 Measurement of bilirubin using the bilirubin dehydrogenase of the present invention using PMS as an artificial electron acceptor Various concentrations (0, 2, 5, 10 and 10) were placed in a spectrophotometer cell having a capacity of 1 ml and a path length of 1 cm. (20, 40 µM) bilirubin and 950 µl of a reaction solution containing 100 mM Tris-HCl buffer (pH 8.4), incubated at 37 ° C for 2 minutes, added 50 µl of bilirubin dehydrogenase solution, and reacted at 37 ° C. Start. 30 minutes after the start of the reaction, the absorbance change for 1 minute (450 nm and 660 n
m) is measured. FIG. 6 shows the results. The white circle in the figure is 45
The absorbance at 0 nm and the filled circles indicate the absorbance at 660 nm. As shown in the figure, the change in absorbance before and after the reaction was proportional to the bilirubin concentration.

[0030]

Example 4 Measurement of Ditaurobilirubin Using a Combination of the Bilirubin Dehydrogenase of the Present Invention and a Hydrogen Peroxide Electrode A peroxidase was immobilized on a dialysis membrane having a molecular weight cutoff of 100, and a bilirubin electrode kneaded with ferrocene as a mediator was used. An attempt was made to quantitatively determine hydrogen peroxide generated from reduced PMS and dissolved oxygen generated by the dehydrogenase reaction, and to quantify ditaurobilirubin.

2 ml of 100 containing 230 μM PMS
100 μl of a bilirubin dehydrogenase solution was added to a mM acetate buffer (pH 4.0), and monitoring of a change in current was started with a hydrogen peroxide electrode. After the current stabilizes,
When ditaurobilirubin was added so that the final concentrations became 4.5 μM, 9.0 μM, and 13.5 μM, as shown in FIG. 7, an increase in the current value depending on the concentration of ditaurobilirubin was confirmed. 3. Each time at the time indicated by the arrow in the figure.
5 μl of ditaurobilirubin was added.

[0032]

According to the present invention, in the field of clinical diagnosis, a single reaction product having absorption in a long wavelength region (for example, Bilbergine) is not affected by the measurement accuracy due to the oxygen concentration in the reaction solution in the measurement of a bilirubin in a test solution. ) To provide a bilirubin dehydrogenase that is selective for conjugated and free bilirubin.

[Brief description of the drawings]

FIG. 1 shows an optimal pH curve of the bilirubin dehydrogenase of the present invention.

FIG. 2 shows an optimal temperature curve of the bilirubin dehydrogenase of the present invention.

FIG. 3 shows the p-value of the bilirubin dehydrogenase of the present invention.
3 shows an H stability curve.

FIG. 4 shows a thermostability curve of the bilirubin dehydrogenase of the present invention.

FIG. 5 shows the measurement results of ditaurobilirubin using the bilirubin dehydrogenase of the present invention and PMS as an artificial electron acceptor.

FIG. 6 shows the measurement results of bilirubin using the bilirubin dehydrogenase of the present invention and PMS as an artificial electron acceptor.

FIG. 7 shows the measurement results of ditaurobilirubin obtained by combining the bilirubin dehydrogenase of the present invention with a hydrogen peroxide electrode.

Claims (7)

[Claims] 1. A bilirubin dehydrogenase, wherein the enzyme uses an artificial electron acceptor. 2. An artificial electron acceptor comprising phenazine methosulfate, phenazine ethosulfate, methoxatin,
1,4-benzoquinone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone, 2,6-dimethyl-1,4
-Benzoquinone, 2,6-dichloro-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-
4-sulfonic acid, potassium ferricyanide, N, N,
The enzyme according to claim 1, which is N ', N'-tetramethyl-p-phenylenediamine dihydrochloride or N, N-dimethyl-p-phenylenediamine. 3. The enzyme according to claim 1, wherein the enzyme has the following physicochemical properties. (1) The residual activity after heat treatment at 50 ° C. for 30 minutes is at least 80% or more. (2) The optimum temperature is 60 ° C. (3) It is stable in the range of pH 3 to pH 11. 4. The enzyme according to claim 1, wherein the enzyme has the following substrate specificity.
An enzyme as described. (1) No laccase activity. (2) Bilbergine is produced from bilirubin as a single reaction product and does not act on biliverdin. (3) It shows higher specificity for ditaurobilirubin, which is a model compound of conjugated bilirubin, than free bilirubin. 5. The method according to claim 1, which is obtained from the genus Aspergillus.
The enzyme according to any one of claims 4 to 4. 6. A method for producing bilirubin dehydrogenase, comprising culturing a bilirubin dehydrogenase-producing bacterium belonging to the genus Aspergillus in a medium, and collecting the bilirubin dehydrogenase from the culture. 7. The method for producing bilirubin dehydrogenase according to claim 5, wherein the bacteria producing bilirubin dehydrogenase belonging to the genus Aspergillus is Aspergillus oculaseus IB-3 (FERM P-18035).