Coenzyme-binding glucose dehydrogenase
The present invention provides a microorganism-derived soluble coenzyme-binding glucose dehydrogenase which catalyzes a reaction for oxidizing glucose in the presence of an electron acceptor, has an activity to maltose as low as 5% or less, and is inhibited by 1,10-phenanthroline. The invention also provides a method for producing the coenzyme-binding glucose dehydrogenase, and a method and a reagent for measuring employing the coenzyme-binding glucose dehydrogenase. According to the invention, the coenzyme-binding glucose dehydrogenase can be applied to an industrial field, and a use becomes possible also in a material production or analysis including a method for measuring or eliminating glucose in a sample using the coenzyme-binding glucose dehydrogenase as well as a method for producing an organic compound. It became also possible to provide a glucose sensor capable of accurately measuring a blood sugar level. Therefore, it became possible to provide an enzyme having a high utility, such as an ability of being used for modifying a material in the fields of pharmaceuticals, clinical studies and food products.
1. An isolated soluble flavin dependent glucose dehydrogenase obtainable from Aspergillus terreus , or a mutant thereof which results from a deletion, substitution or addition of one amino acid residue, which:
(a) catalyzes a reaction for oxidizing glucose in the presence of an electron acceptor;
Co) has a 5% or less specific activity to maltose relative to the enzymatic activity to glucose; and
(c) is inhibited by 1,10-phenanthroline.
2. The flavin dependent glucose dehydrogenase of claim 1 , wherein its activity is inhibited by 50% or more in the presence of a 1 mM final concentration of 1,10-phenanthroline.
3. The flavin dependent glucose dehydrogenase of claim 1 , which oxidizes a hydroxyl group at the 1-position of glucose.
4. The flavin dependent glucose dehydrogenase or mutant thereof of claim 1 , wherein the flavin compound-binding glucose dehydrogenase is obtainable from the Aspergillus terreus deposited under the accession number FERM BP-08578.
5. An isolated soluble flavin dependent glucose dehydrogenase obtainable from Aspergillus terreus having:
(a) an optimum pH of pH 7.0 to pH 9.0;
(b) a pH for stability of pH 4.5 to pH 8.5;
(c) an optimum temperature of approximately 55° C.;
(d) a thermal stability at 50° C. or below;
(e) a molecular weight of about 130 kDa when measured by a gel filtration method, and about 85 kDa when measured by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
(f) a Km value of 49.7mM (D-glucose); and
(g) an isoelectric point (pl) of the coenzyme-binding glucose dehydrogenase measured by an isoelectric focusing of about 4.4.
6. An isolated Aspergillus terreus which is capable of producing the flavin dependent glucose dehydrogenase of claim 1 .
7. An isolated Aspergillus terreus as deposited under the Accession number FERM BP-08578.