WO1984004328A1 - Process for preparing bilirubin oxidase - Google Patents

Process for preparing bilirubin oxidase Download PDF

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Publication number
WO1984004328A1
WO1984004328A1 PCT/JP1984/000223 JP8400223W WO8404328A1 WO 1984004328 A1 WO1984004328 A1 WO 1984004328A1 JP 8400223 W JP8400223 W JP 8400223W WO 8404328 A1 WO8404328 A1 WO 8404328A1
Authority
WO
WIPO (PCT)
Prior art keywords
bilirubin
enzyme
oxidase
sample
bilirubin oxidase
Prior art date
Application number
PCT/JP1984/000223
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
Takayuki Uwajima
Mayumi Hamamatsu
Original Assignee
Kyowa Hakko Kogyo Kk
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Hakko Kogyo Kk filed Critical Kyowa Hakko Kogyo Kk
Priority to DE8484901804T priority Critical patent/DE3479519D1/de
Publication of WO1984004328A1 publication Critical patent/WO1984004328A1/ja

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
    • C12Y103/03005—Bilirubin oxidase (1.3.3.5)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi

Definitions

  • the present invention relates to a novel bilirubin oxidase exhibiting strong specificity for bilirubin, biliperdin, a method for producing the same using basidiomycetes, and a method for use in the analysis of clinical diagnostic agents.
  • the measurement of the enzyme activity is performed as follows.
  • 0.1 ml of 0.1 MTES buffer (pH 7.0), 0.1 ml of H20.1 and 0.1 ml of the enzyme solution were added to 0.5 ml of the 0.1% bilirubin solution.
  • the reaction was carried out by shaking at 37 "C for 5 minutes.
  • the enzymatic activity was determined by measuring the decrease in the absorbance of bilirubin in the reaction solution at 44 nro. Using the molecular absorbance number 56.3, the amount of 1 umol of bilirubin per minute at 37 t was defined as one unit.
  • the amount of enzyme protein is determined by using a low-cost reagent using ⁇ -Folin reagent.
  • Fig. 1 shows the temporal decrease in the absorption of pyrilvin in the visible region when this enzyme was allowed to act. At that time, it does not generate hydrogen peroxide but produces water and also oxidizes pyriperdin, but the oxidation rate is slower than that of bilirubin.
  • Figure 2 shows the enzyme consumption in the early stage of the reaction when using bilirubin and biliverdin as substrates.
  • the molecular weight of the enzyme measured by gel filtration using Sephadex G-100 is 44,000.
  • the novel bilirubin oxidase belongs to the genus Coprinas, the genus Trametes, the genus Choriolas, the genus Foliota, the genus Pleirotus, the genus Lenpetes or the genus Phymitopsis, It is obtained by culturing a microorganism having the ability to produce oxidase in a medium, producing and accumulating bilirubin oxidase in the culture, and collecting the enzyme from the culture.
  • Microorganisms used in the present invention belong to the genus Coprinus, the genus Trametes, the genus Choriolas, the genus Pholiota, the genus Pirorotus, the genus Rangetes or the genus Fumitopsis, and have an ability to produce pyrilvinoxidase. Any one that you have is used.
  • strains include Coprinus micaseus HU8302 (NRRL1540000), Trametes hirsata HU8311 (NRRL15401), Trametes persicola HU8 3 1 2 (NRRL 1 5 4 0 2), Coliath Consols HU 8 3 1 3 (NRRL 1 5 4 0 3), Foliota Nameko HU 8 3 2 1 (NRRL 1 5 4) 0 4), Proitas * o Steatus HU 8 3 3 1 (NRRL 1 54 0 5), Rangetes Stella Cina HU 834 1 (NRRL 1 5 4 0 6), Fumitopsis Kasuna Nea HU 8 3 5 1 (NRL 1 540 7).
  • any of a synthetic medium and a natural medium can be used as long as the medium contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients.
  • a carbon source sugars such as glucose, sucrose and molasses, and sugar alcohols such as glycerol, sorbitol and mannitol can be used.
  • Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium phosphate, and the like, nitrogen compounds such as urea, leptone, yeast extract, casey.
  • Nitrogen-containing organic substances such as hydrolysates, cloves soybeans, or digests thereof can be used.
  • the inorganic substance salts of metals such as sodium, potassium, manganese, magnesium, potassium, and the like, and salts such as phosphoric acid, sulfuric acid, nitric acid, and hydrochloric acid can be used.
  • the culturing temperature is usually in the range of 20 to 40 * C, preferably in the range of 25 to 30 * C.
  • the pH at the start of the culture is usually in the range of 5.5 to 7.5, preferably around 6. Under such conditions, if 4 to 6 shake cultures or deep agitation cultures are performed, bilirubin oxidase is produced in a large amount in the culture.
  • the enzyme can be obtained from the culture using a conventional enzyme collecting means. Although this enzyme accumulates in the cells, it accumulates in large quantities in the culture solution. Usually, cells are separated and removed from the culture, and the resulting crude enzyme solution is purified, for example, by the following method. Add ammonium sulfate to the crude solution until 80% saturation and leave at 4 overnight. Add 3 ⁇ 4 the auxiliary agent and collect the sedimentation section. After sufficient dialysis with a 0.1 M phosphate buffer (pH 7.0), centrifuge the inner dialysis solution to obtain a supernatant. This is done by ion exchange chromatography using DAE-cellulose, QAE-Sephadex, etc., gel filtration method using Sephadex G-200, Sepharose 6B, etc. and adsorption using hydroxyapatite.
  • a phosphate buffer pH 7.0
  • Bilirubin can be quantified using the enzyme of the present invention.
  • the amount of bilirubin in the reaction solution can be determined by measuring the absorbance at 450 nm of the reaction solution by decomposing the bilirubin by applying this rich component to a sample of bilirubin.
  • Bilirubin-old oxidase is dissolved in a suitable buffer, for example, phosphate buffer, to prepare 0.01 to 2 units / ml, which is added to the sample and added to the sample for 15 to 45 t, 5 to 6 Let react for 0 minutes.
  • a suitable buffer for example, phosphate buffer
  • the enzyme also eliminates the interference of bilirubin present in the sample in the determination of substrate and enzyme activity.
  • Hydrogen peroxide is generally quantified by reacting it with a chromogen to form a dye and measuring the absorption of the colored reaction solution.
  • Phenols such as 4-amino pyridine and phenol are often used as the chromogen used here.
  • conventionally known bilirubin oxidase acts on phenol, which is not convenient.
  • the bilirubin oxidase of the present invention is used to decompose bilirubin, it does not act on phenols, so that the substrate in the sample can be more accurately quantified.
  • a more accurate enzyme activity can be obtained by performing the same procedure as above except that a substrate is added to the sample.
  • a more accurate enzyme activity can be obtained by performing in the same manner as described above except that the substrate is added to the sample.
  • Cholesterol, triglyceride, uric acid, etc. are exemplified as substrates to be quantified.
  • the purpose can be achieved by decomposing bilirubin in a sample by the above-mentioned method and then applying a known method. Immediately by adding a reagent solution containing reagents, substrates, chromogens, surfactants, etc. necessary for the measurement of substrate or enzyme activity, reacting the solution, and measuring the absorption of the reaction solution by measuring the absorption of the chromogen at a large wavelength. You can achieve your goals.
  • FIG. 1 shows the temporal decrease in the absorption of bilirubin when this enzyme acts on bilirubin.
  • Fig. 2 shows the amount of oxygen absorbed by this enzyme when bilirubin and bilibenodine were used as substrates.
  • FIG. 3 shows the absorption spectrum of this enzyme.
  • Coprinus micaseus HU83002 is glucose 3%, shake mouth 2%, soy protein powder 1.5%, CSL (corn steep liquor) 0.5% K2HPO 4 0.1 %, MgS0 « ⁇ 7H 2 0 0 0 5%, FeC-6H 2 0 lOmg / i Vitamin B 2 2 ng / medium (PH 6.5) Inoculate 3 Erlenmeyer flasks and shake culture at 28 for 4 days.
  • the seed culture thus obtained is inoculated into a sterilized medium 15 described above in a jar fermenter of 30 volumes in advance. 2 8 t: 4 say rush, airflow 15 Incubate at 250 rpm / min.
  • the cells are removed using a Buchner funnel to obtain a culture solution 10.5 ⁇ (4.0 8 units / ml).
  • the obtained sulfate (a crude enzyme solution) of ammonium sulfate in a concentration of 0 to 80% saturated precipitate is dissolved in 0.01 M phosphate buffer (pH 7.0), and a cellulose tube is used as a dialysis membrane. Dialyze overnight. Next, the precipitate formed in the dialysate is removed by centrifugation. Ammonium sulfate was added to the obtained supernatant, and the fraction which precipitated at 50 to 70% saturation was dialyzed sufficiently with the above buffer, and then equilibrated with a 0.01 M phosphate buffer (pH 7.0).
  • Example 3 Culture is performed in the same manner as in Example 1 except that Trametes' Hirsata HU8311 is used as the microorganism in Example 1, and a culture solution 10.2 containing 0.24 units / ml of bilirubin oxidase activity is obtained from the 15i medium. This was further purified in the same manner as in Example 1 to obtain a powdery purified 13 ⁇ .
  • Example 3
  • the enzyme activity of the cultured SS [ ⁇ shown in Table 1 is obtained. 1 strain ⁇ Enzyme activity (U / ml) Trametes persicola HU8312 0.3 1
  • (B) Reagents for determination of circulating fatty acids Commercially available Detamina NEFA kit (manufactured by Kyowa Medettas). The reagent composition was acyl CoA synthetase, acyl CoA oxidase, oxidase, ATP, CoA, methylcarbamoyl-3,7-dimethyl-amino-10H-phenothiazine and a good buffer (pH 6.7 5).
  • a serum sample was prepared by adding 20 mg / dg of bilirubin (manufactured by Sigma), and this was used as a test sample for measurement by the method of the present invention and the conventional method.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
PCT/JP1984/000223 1983-04-28 1984-04-27 Process for preparing bilirubin oxidase WO1984004328A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DE8484901804T DE3479519D1 (en) 1983-04-28 1984-04-27 Process for preparing bilirubin oxidase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP58075587A JPS59198971A (ja) 1983-04-28 1983-04-28 ビリルビンオキシダ−ゼの製造法

Publications (1)

Publication Number Publication Date
WO1984004328A1 true WO1984004328A1 (en) 1984-11-08

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ID=13580477

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1984/000223 WO1984004328A1 (en) 1983-04-28 1984-04-27 Process for preparing bilirubin oxidase

Country Status (5)

Country Link
US (1) US4677062A (en, 2012)
EP (1) EP0148950B1 (en, 2012)
JP (1) JPS59198971A (en, 2012)
DE (1) DE3479519D1 (en, 2012)
WO (1) WO1984004328A1 (en, 2012)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4746606A (en) * 1986-05-27 1988-05-24 Eastman Kodak Company Bilirubin-specific enzyme and its analytical use

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4921804A (en) * 1983-09-01 1990-05-01 Eastman Kodak Company Method for extracting bilirubin-specific enzyme preparation
JPH0653069B2 (ja) * 1985-02-05 1994-07-20 旭化成工業株式会社 耐熱性ビリルビン・オキシダーゼの製造法
DE3620817A1 (de) * 1986-06-21 1987-12-23 Boehringer Mannheim Gmbh Verfahren zur spezifischen bestimmung des serumfructosamingehalts sowie hierfuer geeignetes reagenzgemisch
JP2578430B2 (ja) * 1987-06-10 1997-02-05 旭化成工業株式会社 新規なビリルビン．オキシダーゼおよびその製造法
DE3732688A1 (de) * 1987-09-29 1989-04-13 Boehringer Mannheim Gmbh Verfahren zur spezifischen bestimmung des serumfructosamingehalts sowie hierfuer geeignetes reagenzgemisch
DE4406379A1 (de) * 1993-03-24 1994-09-29 Boehringer Mannheim Gmbh Bilirubinoxidase aus Alfalfa und Verwendung des Enzyms
WO1996019570A1 (en) * 1994-12-22 1996-06-27 Novo Nordisk A/S An enzyme preparation with cellulytic activity
FR2957934B1 (fr) 2010-03-24 2014-10-17 Centre Nat Rech Scient Bilirubine oxydase de bacillus pumilus et ses applications
FR2975704B1 (fr) 2011-05-24 2015-02-20 Centre Nat Rech Scient Bilirubine oxydase de magnaporthe oryzae et ses applications

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57159487A (en) * 1981-03-30 1982-10-01 Amano Pharmaceut Co Ltd Preparation or bilirubin oxidase

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4211844A (en) * 1978-05-19 1980-07-08 Eastman Kodak Company Bilirubin-specific fungal enzyme preparation
IT1130252B (it) * 1980-02-04 1986-06-11 Elvi Spa Metodo per l'eliminazione dell'interferenza da biliribuna nel dosaggio di perossido di idrigeno mediante una reazione di trinder modificata
DE3239236A1 (de) * 1982-02-18 1983-09-01 Amano Pharma Co Ltd Verfahren zur quantitativen bestimmung von physiologischen komponenten in biologischen fluessigkeiten oder gasen
DK68983A (da) * 1982-02-18 1983-08-19 Milchem Inc Fremgangsmaade ved boring under havoverfladen og borevaeske til brug ved udoevelse af fremgangsmaaden
JPS59135886A (ja) * 1983-01-25 1984-08-04 Takara Shuzo Co Ltd ビリルビン・オキシダ−ゼの製造法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57159487A (en) * 1981-03-30 1982-10-01 Amano Pharmaceut Co Ltd Preparation or bilirubin oxidase

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4746606A (en) * 1986-05-27 1988-05-24 Eastman Kodak Company Bilirubin-specific enzyme and its analytical use

Also Published As

Publication number Publication date
JPS59198971A (ja) 1984-11-10
JPH0440987B2 (en, 2012) 1992-07-06
US4677062A (en) 1987-06-30
EP0148950B1 (en) 1989-08-23
EP0148950A4 (en) 1986-05-16
DE3479519D1 (en) 1989-09-28
EP0148950A1 (en) 1985-07-24

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