Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1997-10-16
2000-11-28
Prats, Francisco
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435 18, 435100, 435872, C12N 924, C12Q 134
Patent
active
061534196
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for enzymatic determination of 1,5-anhydroglucitol, a reagent for use in the method, novel trehalase which is suitable for use in the method for enzymatic determination of 1,5-anhydroglucitol, and a process for producing the novel trehalase. The determination of the 1,5-anhydroglucitol concentration is useful in diagnosis of diabetes.
2. Description of Related Art
It is known that 1,5-anhydroglucitol is present in human cerebrospinal fluid and blood plasma and that the level of 1,5-anhydroglucitol changes in patients of some diseases, especially diabetes, and thus it is important as a diagnostic marker for diabetes. Previous methods for quantitatively determining 1,5-anhydroglucitol include a method using a special analytical instrument such as gas chromatography, a method using an enzyme which specifically oxidizes 1,5-anhydroglucitol (Japanese Published Unexamined Patent Application No. 79780/87), and a method which comprises converting substances such as glucose in a sample to other substances by using various enzymes, and then determining the amount of 1,5-anhydroglucitol remaining in the sample by using pyranose oxidase or L-sorbose oxidase (Japanese Published Unexamined Patent Application No. 185397/88).
Generally, instrumental analyses such as liquid chromatography and gas chromatography involve complicated pretreatment of samples, requires expensive special instruments, and is time-consuming; therefore, this method is not suitable for the analysis of a large number of samples. In the method using an enzyme which specifically acts on 1,5-anhydroglucitol, it is not easy to prepare the enzyme and to lead to an appropriate detection system. The method using pyranose oxidase has the defect that the substrate specificity of pyranose oxidase is insufficient.
SUMMARY OF THE INVENTION
The method of the present invention for the quantitative determination of 1,5-anhydroglucitol in a sample comprises (1) mixing a sample and an enzyme, wherein the activity of the enzyme is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner, and (2) measuring the activity of the enzyme. The present invention also provides a reagent for quantitative determination of 1,5-anhydroglucitol composed of an enzyme, wherein the activity of the enzyme is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner, a substrate for the enzyme, and a reagent for quantitative determination of a product formed by the enzyme. Preferred enzymes useful in the present invention include trehalase and trehalose phosphorylase.
The enzyme trehalase may be prepared by a process of the present invention by culturing a microorganism belonging to the genus Nocardia, having the ability to produce trehalase, and recovering the trehalase from the culture. The trehalase useful in the present invention preferably: has a Ki value of 0.33 mM or less for 1,5-anhydroglucitol; has a Km value for trehalose of 6.7 mM; has an optimum pH of 5-6 and is stable at a pH range of 5-10 when treated at 50.degree. C. for 30 minutes; has an optimum temperature of about 45.degree. C. and is stable up to 50.degree. C. when treated at pH 5.0 for 30 minutes; has a molecular weight as measured by gel filtration of about 400,000; has a substrate specificity for trehalose; and is inhibited by metal chelating agents.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the pH-activity curve of trehalase derived from Nocardia sp. NK-2067.
FIG. 2 shows the pH-stability curve of trehalase derived from Nocardia sp. NK-2067.
FIG. 3 shows the thermostability curve of trehalase derived from Nocardia sp. NK-2067.
FIG. 4 shows the temperature-activity curve of trehalase derived from Nocardia sp. NK-2067.
FIG. 5 shows the calibration curve obtained by using as an index the decomposition activity of trehalose phosphorylase derived from Catellatospora ferruginea FERM BP-4329. The numbers on the ordinate indicate the enzyme activity inhibition rate (%) and those on the abscis
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Aisaka Kazuo
Ando Katsuhiko
Ochiai Keiko
Tazoe Sakae
Kyowa Hakko Kogyo Co. Ltd.
Prats Francisco
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