Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1986-07-17
1987-06-30
Rosen, Sam
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435189, 435911, 435254, C12P 2100
Patent
active
046770623
DESCRIPTION:
BRIEF SUMMARY
Technical Field
The present invention relates to a novel bilirubin oxidase showing strong specific activities on bilirubin and biliverdin, a process for the production thereof using Basidiomvcetes and a method of quantitative determination for diagnostic purposes utilizing the enzyme.
BACKGROUND ART
Heretofore, a bilirubin oxidase produced by culturing a microorganism belonging to the genus Aqaricus is known, whose properties have not been clarified (Japanese Published Unexamined Patent Application No. 151193/79). Also, it is reported that a bilirubin oxidase is produced by culturing a microorganism belonging to the genus Myrothecium [N. Tanaka and S. Murao, Agric. Biol. Chem., 46, 2499-2503 (1982)]. This known enzyme shows specific activities on hydroquinone and catechol in addition to bilirubin and biliverdin. As a result of various studies of bilirubin oxidase, it has been found that a wide range of microorganisms of Basidiomycetes produce a novel bilirubin oxidase.
DISCLOSURE OF THE INVENTION
In further detail, the properties of the novel enzyme of the present invention are given.
The following method is adopted for the determination of the enzyme activity.
To 0.5 ml of a solution containing 0.01% bilirubin, 0.5 ml of 0.1M TES buffer solution (pH 7.0), 1.9 ml of H.sub.2 O and 0.1 ml of a solution of the bilirubin oxidase according to the present invention are added. The mixture is incubated at 37.degree. C. for 5 minutes with shaking and a decrease in absorption at 440 nm of bilirubin in the reaction solution is measured to determine the enzyme activity. One unit of enzyme activity is defined as the amount of the enzyme that oxidizes one .mu.mole of bilirubin per minute at a temperature of 37.degree. C. using 56.3 of the molecular extinction coefficient of bilirubin.
Furthermore, the amount of enzyme protein is determined according to Lowry's method [O. H. Lowry, N.J. Rosebrough, A. L. Fav and R. J. Randall, J. Biol. Chem, 193, 265 (1951)] using a Copper-Falin's reagent.
1. Action
The enzyme catalyzes the oxidation of bilirubin into water, not hydrogen peroxide in the presence of oxygen molecularity. FIG. 1 illustrates decrease with the elapse of time in the absorption of bilirubin in the visible region when bilirubin is decomposed with the enzyme. Also, the enzyme catalyzes the oxidation of biliverdin, and the rate at which the oxidation proceeds in slower than that of bilirubin. FIG. 2 illustrates the amounts of oxygen consumed in the initial stage of reactions in which bilirubin and biliverdin are used as the substrate.
2. Optimum pH
6-9
3. Stable pH range
Stable at a pH of 5-11 in an incubation at 37.degree. C. for 60 minutes.
4. Optimum temperature
50.degree.-60.degree. C.
5. Temperature stability
The enzyme keeps 90-95% of its activity in an incubation in 0.1M TES buffer solution (pH 8.0) at 60.degree. C. for 15 minutes.
6. Molecular weight
The molecular weight of the enzyme is calculated to be about 44,000 by the gel-filtration method in Sephadex G-100.
7. Isoelectric point
The isoelectric point of the enzyme is 3.98 as determined by the electrophoretic focussing method.
8. Absorption spectrum
The purified preparation of the enzyme exhibits its absorption maxima at 280 nm and 600 nm, and is a copper protein (FIG. 3).
According to the present invention, this novel bilirubin oxidase is obtained by culturing a microorganism belonging to the genus Coprinus, Trametes, Coriolus, Pholiota, Pleurotus, Lenzites or Fomitopsis and which is capable of producing the bilirubin oxidase, in a medium until the enzyme is formed and accumulated in the culture liquor and thereafter recovering the bilirubin oxidase therefrom.
Any microorganism may be used in the present invention so long as it belongs to the genus Coprinus, Trametes, Coriolus, Pholiota, Pleurotus, Lenzites or Fomitopsis and is capable of producing bilirubin oxidase.
Examples of the preferred strain include Coprinus micaceus HU8302 (NRRL 15400), Trametes hirsuta HU8311 (NRRL 15401), Trametes versicolor HU8312 (NRRL 15402), Coriolus consors HU8
REFERENCES:
patent: 4211844 (1980-07-01), Wu
patent: 4554249 (1985-11-01), Kosaka
Murao et al, (1981), Chemical Abstracts, 1982, vol. 96, No. 2, p. 229, Item #64651c.
Roesch et al, (1970), Chemical Abstracts, 1970, vol. 73, No. 25, p. 125, Item #128157f.
Gavrilova et al, (1983), Chemical Abstracts, vol. 99, No. 5, p. 319, Item #36017u.
Fedorov, (1970), Chemical Abstracts, 1971, vol. 74, No. 21, p. 109, Item #10846g.
Jackuliak, (1978), Chemical Abstracts, 1979, vol. 90, No. 9, p. 240, Item #68908r.
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Ando Mayumi
Uwajima Takayuki
Herald William J.
Kyowa Hakko Kogyo Co. Ltd.
Rosen Sam
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