38 results about "Glucose dehydrogenase activity" patented technology

The present invention relates to a glucose sensor (2) including an electrode (32) which includes a conductive component and glucose dehydrogenase immobilized to the conductive component. As the glucose dehydrogenase, use is made of a protein complex including a catalytic activity subunit which has glucose dehydrogenase activity, and an electron mediator subunit for supplying an electron donated from the catalytic activity subunit to the conductive component. Preferably, the glucose sensor (2) is designed to continuously measure the glucose level or successively measure the glucose level a plurality of times.

An object of the present invention is to provide a novel glucose dehydrogenase, a method for producing the glucose dehydrogenase, and applications of the glucose dehydrogenase. An isolated flavin-binding glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 80% or more identity to the amino acid sequence of SEQ ID NO: 1, and having glucose dehydrogenase activity is provided.

The present invention provides a novel glucose dehydrogenase that has excellent substrate specificity, specific activity, thermal stability, and the like, and that is suitable for use in SMBG sensors. The present invention provides a purified polypeptide comprising an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 1 and having glucose dehydrogenase activity.

The present invention relates to a protein-immobilized membrane (14) including a cell membrane homologous layer (14A) and a protein (14B) immobilized to the cell membrane homologous layer (14A), where the protein contains cytochrome or a cytochrome complex. The present invention also relates to a method for forming a protein-immobilized membrane (14), and an enzyme-immobilized electrode and a biosensor (X1) provided with a protein-immobilized membrane (14). Preferably, the cell membrane homologous layer (14A) may contain a phospholipid polymer, and the protein (14B) may be CyGDH including an α subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

An object of the present invention is to provide a novel glucose dehydrogenase, a method for producing the glucose dehydrogenase, and applications of the glucose dehydrogenase. An isolated flavin-binding glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 80% or more identity to the amino acid sequence of SEQ ID NO: 1, and having glucose dehydrogenase activity is provided.

A mutant glucose dehydrogenase having an amino acid sequence at least 80% identical to SEQ ID NO:3 and having glucose dehydrogenase activity, wherein amino acid residues corresponding to positions 326, 365 and 472 of said amino acid sequence are replaced with glutamine, tyrosine and tyrosine, respectively, and wherein said mutant glucose dehydrogenase shows an improved substrate specificity to glucose and a reduced reactivity to disaccharides.

Disclosed is a novel enzyme that exhibits glucose dehydrogenase activity. Furthermore, disclosed is a novel method pertaining to enzyme modification. The mutant enzyme comprises an amino acid sequence wherein one or at least two amino acids selected from the group consisting of (1)-(13) below are replaced with another amino acid in the amino acid sequence of a microorganism-derived glucose oxidase: (1) the amino acid corresponding to the amino acid at position 115 of the amino acid sequence indicated by SEQ ID NO: 1; (2) the amino acid corresponding to the amino acid at position 131 of the amino acid sequence indicated by SEQ ID NO: 1; (3) the amino acid corresponding to the amino acid at position 132 of the amino acid sequence indicated by SEQ ID NO: 1; (4) the amino acid corresponding to the amino acid at position 193 of the amino acid sequence indicated by SEQ ID NO: 1; (5) the amino acid corresponding to the amino acid at position 353 of the amino acid sequence indicated by SEQ ID NO: 1; (6) the amino acid corresponding to the amino acid at position 436 of the amino acid sequence indicated by SEQ ID NO: 1; (7) the amino acid corresponding to the amino acid at position 446 of the amino acid sequence indicated by SEQ ID NO: 1; (8) the amino acid corresponding to the amino acid at position 472 of the amino acid sequence indicated by SEQ ID NO: 1; (9) the amino acid corresponding to the amino acid at position 511 of the amino acid sequence indicated by SEQ ID NO: 1; (10) the amino acid corresponding to the amino acid at position 535 of the amino acid sequence indicated by SEQ ID NO: 1; (11) the amino acid corresponding to the amino acid at position 537 of the amino acid sequence indicated by SEQ ID NO: 1; (12) the amino acid corresponding to the amino acid at position 582 of the amino acid sequence indicated by SEQ ID NO: 1; (13) the amino acid corresponding to the amino acid at position 583 of the amino acid sequence indicated by SEQ ID NO: 1.

Provided is a flavin adenine dinucleotide-dependent glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 78% or more identity to the amino acid sequence of SEQ ID NO: 3, and having glucose dehydrogenase activity.

It is intended to highly efficiently produce a large amount of novel FAD-GDH capable of more accurately measuring a glucose level. Provided is a transformant in which a DNA encoding a flavin adenine dinucleotide-binding glucose dehydrogenase selected from the group consisting of (a) a DNA encoding the amino acid sequence of SEQ ID NO: 20; (b) a DNA consisting of the base sequence of SEQ ID NO: 19; (c) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 19 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity; (d) a DNA encoding the amino acid sequence of SEQ ID NO: 34; (e) a DNA consisting of the base sequence of SEQ ID NO: 33; and (f) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 33 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity has been introduced. Further, a method for producing a flavin adenine dinucleotide-binding glucose dehydrogenase using the transformant is provided.

A mutant glucose dehydrogenase having an amino acid sequence at least 80% identical to SEQ ID NO:3 and having glucose dehydrogenase activity, wherein amino acid residues corresponding to positions 326, 365 and 472 of said amino acid sequence are replaced with glutamine, tyrosine and tyrosine, respectively, and wherein said mutant glucose dehydrogenase shows an improved substrate specificity to glucose and a reduced reactivity to disaccharides.

The invention provides a flavin-binding glucose dehydrogenase exhibiting reduced fluctuation of activity depending on temperature environment, and a method for measuring glucose concentration using the flavin-binding glucose dehydrogenase. The flavin-binding glucose dehydrogenase has the following properties (1) to (3): (1) activity: which exhibits glucose dehydrogenase activity in the presence of an electron acceptor; (2) substrate specificity: which exhibits 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%; and (3) temperature characteristics: which exhibits lower fluctuation of activity in a wide temperature range of 10 to 50° C.

The present invention provides a protein derived from a thermophilic filamentous fungus having flavin adenine dinucleotide-dependent glucose dehydrogenase activity. The present invention provides an enzyme for glucose measurement that has better thermal stability than that of conventional enzymes while being unaffected by dissolved oxygen, not requiring the addition of a coenzyme, and maintaining the excellent properties of FADGDH in terms of having excellent substrate specificity (particularly low reactivity with maltose).

The invention provides a flavin-binding glucose dehydrogenase exhibiting reduced fluctuation of activity depending on temperature environment, and a method for measuring glucose concentration using the flavin-binding glucose dehydrogenase. The flavin-binding glucose dehydrogenase has the following properties (1) to (3): (1) activity: which exhibits glucose dehydrogenase activity in the presence of an electron acceptor; (2) substrate specificity: which exhibits 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%; and (3) temperature characteristics: which exhibits lower fluctuation of activity in a wide temperature range of 10 to 50° C.

The present invention relates to a glucose sensor (2) including an electrode (32) which includes a conductive component and glucose dehydrogenase immobilized to the conductive component. As the glucose dehydrogenase, use is made of a protein complex including a catalytic activity subunit which has glucose dehydrogenase activity, and an electron mediator subunit for supplying an electron donated from the catalytic activity subunit to the conductive component. Preferably, the glucose sensor (2) is designed to continuously measure the glucose level or successively measure the glucose level a plurality of times.

The purpose of the invention is to provide flavin-conjugated glucose dehydrogenase having little variation in activity in the typical biosensor measurement temperature range (10-40° C.) and a method for measuring glucose using same. The present invention relates to flavin-conjugated glucose dehydrogenase having the following properties (1)-(3) and the like: (1) action: shows glucose dehydrogenase activity in the presence of an electron acceptor; (2) substrate specificity: the activity value is 10% or less relative to maltose, D-galactose, D-fructose, sorbitol, lactose, and sucrose when the activity value relative to D-glucose is taken to be 100%; and (3) temperature characteristics: the range of the activity value at 10-40° C. is 20-150% when the activity value at 30° C. is taken to be 100%.

The present invention relates to isolated polypeptides having glucose dehydrogenase activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

The present invention provides a novel glucose dehydrogenase that has excellent substrate specificity, specific activity, thermal stability, and the like, and that is suitable for use in SMBG sensors. The present invention provides a purified polypeptide comprising an amino acid sequence having at least 80% identity to the sequence of SEQ ID NO: 1 and having glucose dehydrogenase activity.

The present invention provides a mutant-type glucose dehydrogenase having glucose dehydrogenase activity and having decreased reactivity with xylose, wherein said mutant-type glucose dehydrogenase comprises a mutant-type α-subunit comprising an amino acid sequence of 520 to 550 amino acids comprising an amino acid sequence of 520 to 550 amino acids comprising SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 in this order from N-terminus to C-terminus, except that one or more amino acid residue(s) selected from the group consisting of the glycine at position 10 in SEQ ID NO: 23, the histidine at position 4 in SEQ ID NO: 24, and the asparagine at position 4 in SEQ ID NO: 25 is / are substituted with another / other amino acid(s).

[Problems]To provide a glucose dehydrogenase, a polynucleotide encoding the enzyme, a method for manufacturing the enzyme, a method for measuring glucose using the enzyme, a measuring reagent composition, and a biosensor.[Solutions]A flavin-conjugated glucose dehydrogenase which is composed of proteins having the following amino acid sequence (a), (b) or (c), and having glucose dehydrogenase activity:(a) an amino acid sequence represented by SEQ ID NO: 2, 3, 5, 6, 8 or 9;(b) an amino acid sequence in which one or more amino acids are deleted from, replaced in or added to the amino acid sequence represented by SEQ ID NO: 2, 3, 5, 6, 8 or 9;(c) an amino acid sequence having at least 85% identity with the amino acid sequence represented by SEQ ID NO 2 or 3, at least 95% identity with the amino acid sequence represented by SEQ ID NO 5 or 6, or at least 80% identity with the amino acid sequence represented by SEQ ID NO 8 or 9.

Provided is a flavin adenine dinucleotide-dependent glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 78% or more identity to the amino acid sequence of SEQ ID NO: 3, and having glucose dehydrogenase activity.

The invention discloses a recombinant bacterium for producing purine nucleoside through fermentation as well as a construction method and application thereof. The invention relates to the field of microbial fermentation, in particular to recombinant bacteria for producing purine nucleoside as well as a construction method and application thereof. Compared with an original strain, the recombinant strain for producing purine nucleoside has a mutated purine operon regulation region, inactivated aldose transferase, enhanced 6-phosphate glucose dehydrogenase activity and weakened 6-phosphate glucose isomerase. The inosine yield of the recombinant bacteria provided by the invention is obviously improved. The invention develops a novel method for improving the fermentation yield of purine nucleoside, so that the method can be used for producing inosine, guanosine and adenosine by bacterial fermentation in practice.

A process for producing glucose dehydrogenases. This process comprises transferring a DNA containing a sequence represented by SEQ ID NO:1 which encodes an α subunit having a glucose dehydrogenase activity and a β subunit being an electron transfer protein into a microorganism belonging to the genus Pseudomonas to thereby construct a transformant, and culturing this transformant so as to allow the production of a first glucose dehydrogenase containing the above-described β subunit and a second glucose dehydrogenase free from the β subunit. The α subunit as described above has a molecular weight of about 60 kDa measured by, for example, SDS-polyacrylamide gel electrophoresis under reducing conditions, while the β subunit as described above has a molecular weight of about 43 kDa measured by, for example, SDS-polyacrylamide gel electrophoresis under reducing conditions.

An object is to provide a novel enzyme that exhibits glucose dehydrogenase activity. Furthermore, another object is to provide a novel method pertaining to enzyme modification. Provided is a mutated enzyme containing an amino acid sequence wherein one or at least two amino acids selected from the group consisting of (1) to (13) are substituted with another amino acid in the amino acid sequence of a microorganism-derived glucose oxidase: (1) the amino acid corresponding to the amino acid at position 115 of the amino acid sequence of SEQ ID NO: 1; (2) the amino acid corresponding to the amino acid at position 131 of the amino acid sequence of SEQ ID NO: 1; (3) the amino acid corresponding to the amino acid at position 132 of the amino acid sequence of SEQ ID NO: 1; (4) the amino acid corresponding to the amino acid at position 193 of the amino acid sequence of SEQ ID NO: 1; (5) the amino acid corresponding to the amino acid at position 353 of the amino acid sequence of SEQ ID NO: 1; (6) the amino acid corresponding to the amino acid at position 436 of the amino acid sequence of SEQ ID NO: 1; (7) the amino acid corresponding to the amino acid at position 446 of the amino acid sequence of SEQ ID NO: 1; (8) the amino acid corresponding to the amino acid at position 472 of the amino acid sequence of SEQ ID NO: 1; (9) the amino acid corresponding to the amino acid at position 511 of the amino acid sequence of SEQ ID NO: 1; (10) the amino acid corresponding to the amino acid at position 535 of the amino acid sequence of SEQ ID NO: 1; (11) the amino acid corresponding to the amino acid at position 537 of the amino acid sequence of SEQ ID NO: 1; (12) the amino acid corresponding to the amino acid at position 582 of the amino acid sequence of SEQ ID NO: 1; and (13) the amino acid corresponding to the amino acid at position 583 of the amino acid sequence of SEQ ID NO: 1.

It is intended to highly efficiently produce a large amount of novel FAD-GDH capable of more accurately measuring a glucose level. A transformant transfected with a DNA encoding a flavin adenine dinucleotide-binding glucose dehydrogenase selected from the group consisting of (a) a DNA encoding an amino acid sequence represented by SEQ ID NO: 20; (b) a DNA composed of a base sequence represented by SEQ ID NO: 19; (c) a DNA having a base sequence homologous to the base sequence represented by SEQ ID NO: 19 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenaseactivity; (d) a DNA encoding an amino acid sequence represented by SEQ ID NO: 34; (e) a DNA composed of a base sequence represented by SEQ ID NO: 33; and (f) a DNA having a base sequence homologous to the base sequence represented by SEQ ID NO: 33 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity is provided. Further, a method for producing a flavin adenine dinucleotide-binding glucose dehydrogenase using the transformant is provided.

The present invention relates to a protein-immobilized membrane (14) including a cell membrane homologous layer (14A) and a protein (14B) immobilized to the cell membrane homologous layer (14A), where the protein contains cytochrome or a cytochrome complex. The present invention also relates to a method for forming a protein-immobilized membrane (14), and an enzyme-immobilized electrode and a biosensor (X1) provided with a protein-immobilized membrane (14). Preferably, the cell membrane homologous layer (14A) may contain a phospholipid polymer, and the protein (14B) may be CyGDH including an α subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

The present invention relates to a protein having glucose dehydrogenase activity selected from: (a) an amino acid sequence represented by SEQ ID NO: 3; (b) an amino acid sequence in which 1 to 3 amino acids are deleted from, replaced in or added to the amino acid sequence represented by SEQ ID NO: 3; (c) an amino acid sequence which has at least 80% identity with the amino acid sequence represented by SEQ ID NO: 3 and whose N-terminus is SS; or (d) an amino acid sequence which has at least 80% identity with the amino acid sequence represented by SEQ ID NO: 3, and does not contain a sequence represented by SEQ ID NO: 8 at its N-terminus. The invention also includes a polynucleotide encoding the enzyme, a method for manufacturing the enzyme, a method for measuring glucose using the enzyme, a measuring reagent composition and a biosensor.