Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1989-10-04
1992-10-20
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 26, 435 28, 435 41, 435136, 4352534, 435874, 435 15, 435 18, 435 4, C12A 126, C12A 132, C12P 100, C12N 120
Patent
active
051569558
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a novel enzyme for catalyzing the oxidation reaction of nucleosides, and more particularly to a novel nucleoside oxidase YT-1 for causing a neucleoside to react with molecular oxygen to form a nucleoside-5'-carboxylic acid via a nucleoside 5'-aldehyde without forming hydrogen peroxide as a by-product, a process for producing the enzyme, a novel microorganism capable of producing the enzyme, and a novel assay method utilizing the enzyme.
BACKGROUND ART
Known enzymes active on nucleosides include those effecting hydrolysis or deamination thereof, and enzymes (nucleoside oxidases) for catalyzing the oxidation reaction thereof represented by the following equations. ##STR1##
Nucleoside oxidase is usually isolated from microorganisms typical of which is, for example, Pseudomonas putida, and the oxidase as purified is used for preparing nucleoside-5'-carboxylic acids, quantitative determination of nucleosides and systems for determining the activity of enzymes or the like wherein a nucleoside is produced or the amount thereof descreases due to a reaction.
However, in the oxidation reaction system wherein the oxidase is used, H.sub.2 O.sub.2 is inevitably produced, so that in producing the nucleoside-5'-carboxylic acid, the resulting H.sub.2 O.sub.2 needs to be decomposed by adding catalase to the reaction system. Further when the amount of H.sub.2 O.sub.2 produced in the reaction system is measured to quantitatively determine the nucleoside, the method has the disadvantage that peroxidase must be used. The method further has the disadvantage, for example, of necessitating a complex determination system (see, for example, Unexamined Japanese Patent Publications SHO 57-58883, SHO 57-68794 and SHO 57-94300).
We have conducted extensive research on nucleoside oxidases derived, for example, from microorganisms and found that a strain belonging to the genus Pseudomonas and newly isolated from soil has ability to produce an enzyme which catalyzes an oxidation reaction different from those involving the activity of the conventional nucleoside oxidases. We have also succeeded in isolating and purifying the enzyme, clarifying the characteristics thereof and developing novel assay or determination techniques utilizing the enzyme. Thus, the present invention has been accomplished.
DISCLOSURE OF THE INVENTION
The present invention provides an enzyme for catalyzing oxidation reaction of a nucleoside, i.e., a novel nucleoside oxidase YT-1 characterized in that the oxidase causes the nuceloside to react with molecular oxygen to produce a nucleoside-5'-carboxylic acid via a nucleoside-5'-aldehyde without producing hydrogen peroxide, a process for producing the enzyme, a novel microorganism having ability to produce the enzyme, and a method of assaying a system containing or for producing a nucleoside by causing the nucleoside oxidase YT-1 to act on a mixture of the system and a chromogenic reagent forming a color on oxidation and measuring the degree of color formation by the chromogenic reagent which is proportional to the quantity of nucleoside in the system.
The nucleoside oxidation reaction catalyzed by the enzyme of the invention is represented by the equations (1) and (2) given below and distinctly differs from the known oxidation reactions effected by nucleoside oxidases. Of course, no nucleoside oxidase is known which catalyzes the reaction represented by the equations (1) and (2). ##STR2##
Furthermore, the nucleoside oxidase YT-1 of the present invention effects the nucleoside oxidation reaction of the above equations and, at the same time, exhibits laccase-like activity in accordance with the amount of nucleoside as the unique characteristics of the enzyme. The present enzyme is entirely different from the conventional nucleoside oxidases also in this feature. With the assay method of the invention, the degree of color formation is measured, for example, in terms of absorbance utilizing the laccase-like activity which is unique to the enzyme. Such assay can not be
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Hoshino Masami
Isono Yoshikazu
Escallon Miguel
Otsuka Foods Co., Ltd.
Wax Robert A.
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