Method for quantitative determination of 1,5-anhydroglucitol

A method for the quantitative determination of 1,5-anhydroglucitol in a sample, comprising the steps of:

• selecting an enzyme having activity that is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner, wherein said enzyme does not use 1,5-anhydroglucitol as a substrate,
• mixing the sample with said enzyme,
• thereafter measuring activity of the enzyme, and
• comparing the activity with a calibration curve previously obtained from known amounts of 1,5-anhydroglucitol, wherein the enzyme having activity that is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner is trehalase.

A method for the quantitative determination of 1,5-anhydroglucitol in a sample, comprising the steps of:

• selecting an enzyme having activity that is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner, wherein said enzyme does not use 1,5-anhydroglucitol as a substrate,
• mixing the sample with said enzyme,
• thereafter measuring activity of the enzyme, and
• comparing the activity with a calibration curve previously obtained from known amounts of 1,5-anhydroglucitol, wherein the enzyme having activity that is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner is trehalose phosphorylase.

The purified enzyme trehalase which is obtained from Nocardia sp. NK-2067 (FERM BP-5359), said enzyme having:

• a Ki value of 0.33 mM or less for 1,5-anhydroglucitol;
• a Km value for trehalose of 6.7 mM;
• an optimum pH of 5-6 and stability at a pH range of 5-10, when treated at 50.degree. C. for 30 minutes;
• an optimum temperature of about 45.degree. C. and stability up to 50.degree. C. when treated at pH 5.0 for 30 minutes;
• a molecular weight, as measured by gel filtration, of about 400,000;
• and
• a substrate specificity for trehalose;
• wherein said enzyme is inhibited by metal chelating agents, SH blocking reagent, hydroxylamine and nickel sulfate.

A kit for quantitative determination of 1,5-anhydroglucitol in a sample comprising:

• an enzyme having activity that is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner and does not use 1,5-anhydroglucitol as a substrate, wherein said enzyme is isolated from said sample prior to use;
• a substrate for the enzyme, wherein said substrate and said enzyme are mutually isolated in said kit prior to use;
• and
• a reagent for quantitative determination of a product formed by the enzyme activity,
• wherein the components of the kit are within a container and said kit is admixed with the sample to conduct said determination.

A kit for quantitative determination of 1,5-anhydroglucitol in a sample comprising:

• an enzyme having activity that 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 activity,
• wherein the enzyme is isolated in said kit from the substrate for the enzyme before use, and wherein the enzyme having activity that is inhibited by 1,5-anhydroglucitol in concentration-dependent manner is the trehalase derived from Nocardia sp. NK-2067 (FERM BP-5359).

A kit for quantitative determination of 1,5-anhydroglucitol in a sample comprising:

• an enzyme having activity that is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner and does not use 1,5-anhydroglucitol as a substrate;
• a substrate for the enzyme, wherein said substrate and said enzyme are mutually isolated in said kit prior to use;
• a reagent for quantitative determination of a product formed by the enzyme activity;
• and
• a known amount of 1,5-anhydroglucitol,
• wherein said enzyme and said substrate are admixed with the reagent in the presence of the sample or in the presence of said 1,5-anhydroglucitol.