Flavin-binding glucose dehydrogenase

#The present invention relates to a flavin-binding glucose dehydrogenase useful for measurement of glucose concentration and a method for measuring glucose concentration by using the flavin-binding glucose dehydrogenase.

#Rapid and accurate measurement of the concentration of blood glucose is important for the diagnosis of diabetes. As examples of a method for measuring glucose concentration, a chemical method and an enzymatic method are known; among them the enzymatic method is preferable from the viewpoints of specificity and safety. Among the enzymatic methods, an electrochemical biosensor is advantageous from the viewpoints of reduction of the amount of a specimen, reduction of measuring time, and reduction of the size of a device.

#Glucose oxidase is known as an enzyme usable for such a biosensor. However, because glucose oxidase gives rise to the problem, for example, oxygen dissolved in blood causes measurement errors, therefore, some glucose dehydrogenases have been developed. Among glucose dehydrogenases, much attention is focused on flavin-binding glucose dehydrogenases as the enzyme for glucose biosensors because they need no addition of a coenzyme and are unaffected by dissolved oxygen (Patent Documents 1 to 7). These flavin-binding dehydrogenases include those which are superior in substrate specificity (Patent Document 5), those which exhibit an activity of 15% or more at 10° C., an activity of 30% or more at 20° C., and an activity of 70% or more at 60° C., when the activity at 50° C. is defined as 100% (Patent Document 6), and modified enzymes which are cell homogenates of recombinant Escherichia coli transformed by a gene encoding a FAD-dependent glucose dehydrogenase derived from Aspergillus oryzae and which exhibits improved relative activity at 25° C. when the activity at 37° C. was defined as 100% (Patent Document 7).

#FIG. 1 shows the absorption spectrums of glucose dehydrogenases (A) to (F) according to the present invention.

#FIG. 2 shows the heat stability of glucose dehydrogenases (A) to (D) according to the present invention.

#FIG. 3 shows stable pH ranges of glucose dehydrogenases (A) to (F) according to the present invention.

#The glucose dehydrogenase of the present invention is a flavin-binding glucose dehydrogenase and is an enzyme exhibiting activity when flavin is bound as a coenzyme. The glucose dehydrogenase of the present invention is an enzyme classified into EC1. 1. 99. 10 or EC1. 1. 99. 13, and preferably EC1. 1. 99. 10. Examples herein of the flavin include flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN).

#The glucose dehydrogenase of the present invention has the following properties (1) to (3) and especially the following (3). (1) Activity: which exhibits glucose dehydrogenase activity in the presence of an electron acceptor. (2) Substrate specificity: which shows an activity of 10% or less against maltose, D-galactose, D-fructose, sorbitol, lactose and sucrose, when the activity against D-glucose is defined as 100%. (3) Temperature characteristics: which has an activity range from 20 to 150% at 10 to 50° C., when the activity at 30° C. is defined as 100%.

#First, the glucose dehydrogenase of the present invention exhibits (1) glucose dehydrogenase activity in the presence of an electron acceptor. Specifically, the glucose dehydrogenase of the present invention catalyzes a reaction for oxidizing a hydroxyl group of glucose in the presence of an electron acceptor to yield glucono-δ-lactone. When a FAD-binding glucose dehydrogenase reacts with glucose, a coenzyme FAD is converted into FADH2. However, if a ferricyanide (for example, [Fe (CN)6]3−) is made to be present as the electron acceptor, FADH2 converts the ferricyanide into a ferrocyanide ([Fe(CN)6]4−) in this case and is itself returned to FAD. When potential is applied to a ferrocyanide, the ferrocyanide delivers an electron to the electrode and returns to a ferricyanide. Therefore, when such an electron transport material is used as an electron acceptor, this enables electrochemical signal detection.