Kikkoman Corporation

Yosuke Masakari

Atsushi Ichiyanagi

Kozo Hirokawa

Seiichi Hara

Yasuko Tanabe

Airi Komatsuzaki

Haruka Hiraguchi

There is provided a novel quantification method for quantifying a concentration of EAP, which is a biomarker of depression, an enzyme for quantitation, a composition for quantitation, a kit for quantitation or a sensor for quantitation. There is provided a quantification method of ethanolamine phosphate by adding oxidoreductase to a sample containing ethanolamine phosphate. A mediator may be reduced by adding the oxidoreductase, and the reduced mediator may be reacted with a reagent to determine a concentration of ethanolamine phosphate. In addition, hydrogen peroxide produced by adding the oxidase as the oxidoreductase may be reacted with a reagent to determine a concentration of the ethanolamine phosphate.

Quantification method of ethanolamine phosphate, oxidoreductase for quantification, composition for quantification, kit for quantification and sensor for quantification

Provided is a method of continuous glucose monitoring (CGM) comprising using an FAD-GDH. The FAD-GDH is capable of retaining initial activity over a certain period of time. Also provided is a method for screening for an FAD-GDH suitable for use in CGM as well as a CGM device comprising an FAD-GDH.

Continuous glucose monitoring using an FAD-dependent glucose dehydrogenase

Provided is a composition by which glycated hemoglobin can be measured even in the presence of a stronger surfactant than a conventional case. Also provided is a buffer and/or stabilizer which maintains the residual activity of an amadoriase or lowers a reduction of residual activity. The present invention provides a composition for use in measuring glycated hemoglobin containing an amadoriase having substitution of one or more amino acid residues at a position(s) corresponding to an amino acid(s) selected from the group consisting of position 262, position 257, position 249, position 253, position 337, position 340, position 232, position 129, position 132, position 133, position 44, position 256, position 231 and position 81 of an amadoriase derived from the genusand represented by SEQ ID No: 1 or 3, and having residual activity even in the presence of a surfactant. The present invention also provides a composition and kit for use in measuring glycated hemoglobin, comprising a specific stabilizer and/or a buffer. The present invention can provide an enzyme and a composition for use in measuring glycated hemoglobin, excellent in storage stability even if they are exposed to a surfactant.

Modified amadoriase and method for producing the same, agent for improving surfactant resistance of amadoriase and composition for measuring HbA1c using the same

Provided are an amadoriase that is less likely to be influenced by oxygen concentration and a method and a reagent kit for measurement of HbA1c using such amadoriase. Provided are an amadoriase that is obtained via substitution of one or more amino acid residues at a position or positions corresponding to the position(s) selected from the group consisting of positions 280, 267, 269, 54, and 241 of the amadoriase derived from the genus, a method for measurement of HbA1c, a reagent kit for measurement, and a sensor using such amadoriase. The modified amadoriase according to the invention has a lowered oxidase activity and an enhanced dehydrogenase activity, and this enables the use of an electron mediator, and this reduces the influence of oxygen concentration. Thus, HbA1c can be measured with high sensitivity.

Amadoriase having enhanced dehydrogenase activity

A cytochrome b-glucose dehydrogenase fusion protein having modified electron transfer properties, and a glucose measurement method and measuring kit using the cytochrome b-glucose dehydrogenase fusion protein are provided. Provided are a cytochrome b-glucose dehydrogenase fusion protein in which glucose dehydrogenase having homology with SEQ ID NO: 1 or SEQ ID NO: 4 and cytochrome b are linked together, as well as a glucose measurement method, a measurement reagent kit and a sensor using the cytochrome b-glucose dehydrogenase fusion protein. The cytochrome b-glucose dehydrogenase fusion protein of the present invention has modified electron transfer properties, and can be used for measuring glucose in the presence of a free-form mediator in reduced concentration or in the absence of a free-form mediator, and can be used, for example, in continuous glucose monitoring.

Cytochrome-fused glucose dehydrogenase and glucose measurement method

The present invention provides a composition that enables measurement of glycated hemoglobin in the presence of a stronger surfactant than conventional surfactants. To this end, the present invention provides an amadoriase in which one or more amino acids have been substituted at positions corresponding to positions selected from the group consisting of positions 80, 71, 175, 172, 279, 12, 9, 77, 30, 28, 13, 3, 4, 286, 204, 338, 44, 340, and 194 of the amadoriase derived from the genushaving the amino acid sequence as shown in SEQ ID NO: 1 as well as a composition for measurement of glycated hemoglobin comprising an amadoriase that retains activity in the presence of an anionic surfactant. The present invention can provide an enzyme and a composition for measurement of glycated hemoglobin that sufficiently remain stable even when exposed to anionic surfactants.

Amadoriase having enhanced anionic surfactant tolerance

This invention provides an HbA1c dehydrogenase that is capable of directly acting on hemoglobin A1c and is less likely to be influenced by oxygen concentration and a method for measurement and a kit of assay reagents using such HbA1c dehydrogenase. The HbA1c dehydrogenase having dehydrogenase activity and capable of directly acting on HbA1c is obtained by substitution of one or more amino acid residues at positions corresponding to positions 280, 269, 54, 241, and 267 of the amadoriase that is capable of directly acting on hemoglobin A1c and is derived from, for example, the genus. This invention also provides a method for measurement of HbA1c, a kit of assay reagents, and a sensor using such HbA1c dehydrogenase. Such HbA1c dehydrogenase is capable of directly acting on hemoglobin A1c and has lowered oxidase activity and/or enhanced dehydrogenase activity. This not only eliminates the need for treatment of hemoglobin A1c with a protease but also enables the use of an electron mediator in the measurement of HbA1c, thereby reducing effects due to oxygen concentration, and enables HbA1c measured with high sensitivity.

HbA1c dehydrogenase

This invention provides an amadoriase that acts on the &#x3b2;-chain of hemoglobin A1c (HbA1c) and generates hydrogen peroxide, a method for measurement of HbA1c using such amadoriase, and a reagent kit for measurement of HbA1c using such amadoriase. The method for measurement of HbA1c involves the use of an amadoriase that has specific activity of 0.1 U/mg or greater to &#x3b1;F6P and oxidizes the HbA1c &#x3b2;-chain amino terminus so as to generate hydrogen peroxide, and the reagent kit for measurement of HbA1c comprises such amadoriase. The method and the kit for measurement of HbA1c enable quantification of HbA1c to be performed rapidly, simply, and accurately.

Hemoglobin A1c measurement method and measurement kit

A glucose dehydrogenase having modified electron transfer properties, and a glucose measurement method and measuring kit using the glucose dehydrogenase are provided. Provided are a glucose dehydrogenase having at least 1, 2 or 3 amino acid substitutions, for example, amino acid substitution with a polar amino acid or alanine, in the region corresponding to the 457th to 477th position in SEQ ID NO: 1 of a glucose dehydrogenase having homology with SEQ ID NO: 1, a glucose measurement method, a measurement reagent kit and a sensor using the glucose dehydrogenase. The electron transfer properties of the glucose dehydrogenase of the present invention is modified and can be used in measuring glucose in the presence of a mediator with reduced concentration or in the absence of a mediator, and can be used, for example, in continuous glucose measurement.

Glucose dehydrogenase having modified electron transfer properties, and glucose measurement method

This invention provides an amadoriase having high reactivity with &#x3b1;-fructosyl hexapeptide (&#x3b1;F6P) derived from the &#x3b2;-chain amino terminus of glycated hemoglobin (HbAlc), which enables satisfactory quantification of &#x3b1;F6P cleaved from HbAlc. A novel amadoriase is derived from the amadoriase of the genusby substitution of one or more amino acids at positions selected from the group consisting of positions 62, 63, 102, 106, 110, 113, 355, and 419. Such amadoriase enables rapid, simple, accurate, and satisfactory quantification of &#x3b1;F6P cleaved from HbAlc. With the use of such amadoriase, a method for measurement of HbAlc by an enzymatic method and a kit for measurement of HbAlc that are based on the same principle as used in the conventional standard method and excellent in terms of the relevance with the standard method.

Modified amadoriase reacting with fructosyl hexapeptide

This invention provides an amadoriase having high substrate specificity to fructosyl valyl histidine. Such amadoriase comprises substitution of one or more amino acid residues at positions corresponding to amino acids selected from the group consisting of position 98, position 259, position 154, position 125, position 261, position 263, position 106, position 103, position 355, position 96, position 66, position 67, position 70, position 100, position 110, position 113, position 114, and position 156 in the amadoriase derived from the genus. This invention enables accurate measurement of &#x3b1;-fructosyl valyl histidine derived from the &#x3b2;-chain amino terminus in glycated hemoglobin in the presence of &#x3b5;-fructosyl lysine.

Amadoriase having altered substrate specificity

An amadoriase having a substitution or a deletion of one or more amino acid residues at positions corresponding to amino acids selected from the group consisting of three amino acid residues from the carboxyl terminal and amino acids at positions 151, 43, 53, 267, 350, 185, 196, 299 and 323 in the amino acid sequence of amadoriase derived from thespecies indicated in SEQ ID NO: 1. The amadoriase having a heat resistance which is superior to that of a conventional amadoriase.

Amadoriase with improved thermostability, gene and recombinant DNA for the amadoriase, and method for production of amadoriase with improved thermostability

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Noda, Japan

Yosuke Masakari