Thermostable bilirubin oxidase and production process thereof
AssigneeToyo Jozo Kabushiki Kaisha
InventorsEiichi Yoshino, Shigeyuki Imamura, Kazuo Matsuura, Hideo Misaki
Life of this patent
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Abstract
Thermostable bilirubin oxidase having substrate specificity to at least bilirubin and capable of catalyzing a reaction in which biliverdin and water are formed from bilirubin and oxygen. It can be produced by culturing a bilirubin oxidase producing micro-organism belonging to the genus Bacillus, for example, Bacillus licheniformis and then preparing the resultant bilirubin oxidase from the cultured broth.
Claims9
A bilirubin oxidase having substrate specificity to at least bilirubin and being capable of catalyzing a reaction in which biliverdin and water are formed from bilirubin and oxygen, wherein said bilirubin oxidase has the following physical and chemical properties:
- (a) a molecular weight of 50,000.+-.10,000 as measured by gel filtration on "Sepharyl S-300";
- (b) an isoelectric point at about pH 3.35;
- (c) a Km value of about 2.86.times.10.sup.-5 M;
- (d) an optimum pH of about pH 8;
- (e) a stable pH of around pH 7 to 9;
- (f) an optimum temperature of about 80.degree. C.;
- and
- (g) stability at temperatures of up to about 70.degree. C.
A process for producing a bilirubin oxidase, which comprises:
- culturing a bilirubin oxidase- producing Bacillius licheniformis B-0891 strain (FERM BP-952);
- and
- isolating bilirubin oxidase produced in the cultured broth.
In an assay for determining the bilirubin content of a serum sample, the improvement comprising using a bilirubin oxidase having substrate specificity to at least bilirubin and being capable of catalyzing a reaction in which biliverdin and water are formed from bilirubin and oxygen, wherein said bilirubin oxidase is thermally stable for 10 minutes at a temperature of around 70.degree. C., wherein said bilirubin oxidase has the following physical and chemical properties:
- (a) a molecular weight of 50,000.+-.10,000 as measured by gel filtration on "Sephacryl S-300";
- (b) an isoelectric point at about pH=3.35;
- (c) a Km value of about 2.86.times.10.sup.-5 M;
- (d) an optmum pH of about pH 8;
- (e) a stable pH of around pH 7 to 9;
- and
- (f) an optimum temperature about 80.degree. C.
In a process for removing bilirubin from a serum sample to eliminate errors in the analysis of another biochemical substance in said serum sample, the improvement comprising using a bilirubin oxidase having substrate specificity to at least bilirubin and being capable of catalyzing a reaction in which biliverdin and water are formed from bilirubin and oxygen, wherein bilirubin oxidase is thermally stable for 10 minutes at a temperature of around 70.degree. C., wherein said bilirubin oxidase has the following physical and chemical properties:
- (a) a molecular weight of 50,000.+-.10,000 as measured by gel filtration on "Sephacryl S-300";
- (b) an isoelectric point of about pH=3.35;
- (c) a Km value of about 2.6.times.10.sup.-5 M;
- (d) an optimum pH of about pH 8;
- (e) a stable pH of around pH 7 to 9;
- and
- (f) an optimum temperature of about 80.degree. C.
Dependencies are inferred from the claim text.