JP2007300818A - Pyranose oxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pyranose oxidase, a method for producing the same, a gene encoding the enzyme, a recombinant vector containing the gene, a transformant inserted with the gene, a method for measuring sugar using the enzyme, a reagent composition for measuring sugar, a biosensor for measuring sugar and a method for producing ketosugar. <P>SOLUTION: The method for producing the pyranose oxidase is to insert a gene expressed by a specific base sequence into a microorganism belonging to the genus Escherichia to form a transformant, culture the same and harvest the pyranose oxidase. The pyranose oxidase produced by the method, the gene encoding the enzyme, the recombinant vector containing the gene, the transformant inserted with the gene and expressing the gene, the method for measuring sugar using the enzyme, the reagent composition for measuring the sugar containing the enzyme, the biosensor for measuring the sugar using the enzyme and the method for producing the ketosugar using the enzyme are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention includes a pyranose oxidase, a gene encoding the same, a recombinant vector containing the gene, a transformant into which the gene is inserted, a method for producing the pyranose oxidase, a method for measuring a saccharide using the pyranose oxidase, The present invention relates to a saccharide-measuring reagent composition containing the pyranose oxidase, a biosensor for saccharide measurement using the pyranose oxidase, and a method for producing a ketosaccharide using the pyranose oxidase.

Pyranose oxidase is an enzyme that catalyzes a reaction of oxidizing 2-carbon of D-glucose and other aldopyranose to produce a 2-keto product and hydrogen peroxide.
Pyranose oxidase is described as being applicable to the production of antibiotics (Non-Patent Document 1) and the industrial production of fructose (Non-Patent Document 2). Pyranose oxidase is expected to be used as a glucose biosensor. By quantifying the hydrogen peroxide generated by pyranose oxidase by conjugating peroxidase, catalase, etc., and then subjecting it to an appropriate color development system, enzymatic measurement of glucose in the sample can be performed. Specifically, it can be used for measurement of glucose in foods and body fluids. In particular, it is expected to be useful as a blood glucose test method in clinical situations such as diabetes diagnostic tests.

The advantages of pyranose oxidase compared to conventionally used glucose oxidase are high substrate affinity (Non-patent Document 3) and not only β-D-glucose but also α-D-glucose at the same time. Is also necessary, so that it is not necessary to add mutarotase (Non-patent Document 4). Furthermore, since pyranose oxidase uses 1,5-anhydroglucitol, which is attracting attention as a blood glucose control marker for diabetes, as a substrate (Non-patent Document 5), it is used as a diagnostic agent for diabetes.
Koths et al. (1992) Carbohydr. Res. 232: 59-75 Shaked and Wolfe (1988) Methods Enzymol. 137: 599-615 Nishimura et al. (1996) J. Biotechnol. 52: 11-20 Taguchi et al. (1985) J. Appl. Biochem. 7: 289 Yabuuchi et al. (1989) Clin. Chem. 35: 2039-2043

An object of the present invention is to provide a novel pyranose oxidase.
The present invention also relates to a gene encoding such a pyranose oxidase, a recombinant vector containing the gene, a transformant in which the gene is inserted, a method for producing the pyranose oxidase, and a method for measuring a saccharide using the pyranose oxidase An object of the present invention is to provide a saccharide-measuring reagent composition containing the pyranose oxidase, a biosensor for measuring saccharides using the pyranose oxidase, and a method for producing ketosaccharides using the pyranose oxidase.

  The present inventors have found that a pyranose oxidase produced by a specific basidiomycete is a novel enzyme having an excellent pyranose oxidase activity, and it is possible to measure saccharides and ketosaccharides using such an enzyme. The present inventors have found that a biosensor for saccharide measurement can be produced using oxidase.

That is, in the invention described in claim 1, the gene represented by SEQ ID NO: 1 is inserted into a microorganism belonging to the genus Escherichia to form a transformant, which is cultured and pyranose oxidase is collected from the culture. The present invention provides a method for producing pyranose oxidase characterized by the following.
The invention described in claim 2 provides a pyranose oxidase produced by such a production method.
The invention according to claim 3 provides the pyranose oxidase according to claim 2, comprising the amino acid sequence represented by SEQ ID NO: 2.
The invention according to claim 4 is the amino acid sequence represented by SEQ ID NO: 2, comprising one or several amino acid sequences deleted, substituted or added, and having pyranose oxidase activity. The described pyranose oxidase is provided.
In addition, the invention according to claim 5 is based on Basidiomycetous fungi No.1. The pyranose oxidase according to any one of claims 2 to 4, which is derived from 52 strains (FERM P-10106).
The invention according to claim 6 provides a gene encoding the pyranose oxidase according to any one of claims 2 to 5.
The invention according to claim 7 provides the gene according to claim 6 represented by SEQ ID NO: 1.
The invention according to claim 8 provides a gene encoding a protein having 60% or more homology with the gene represented by SEQ ID NO: 1 and having pyranose oxidase activity.
The invention according to claim 9 provides a recombinant vector containing the gene according to any one of claims 6 to 8.
The invention according to claim 10 provides a transformant in which the gene according to any one of claims 6 to 8 is inserted and the gene is expressed.
The invention according to claim 11 is the transformant according to claim 10, wherein the host is a microorganism belonging to the genus Escherichia, the genus Bacillus, the genus Saccharomyces or the genus Pichia. It is to provide.
The invention described in claim 12 provides a method for producing pyranose oxidase, comprising culturing the transformant according to claim 10 or 11 and collecting pyranose oxidase from the culture.
The invention described in claim 13 provides a method for measuring a saccharide characterized by using the pyranose oxidase according to any one of claims 2-5.
The invention according to claim 14 provides the method for measuring a saccharide according to claim 13, wherein the saccharide is glucose or 1,5-anhydroglucitol.
The invention described in claim 15 provides a saccharide-measuring reagent composition containing the pyranose oxidase according to any one of claims 2-5.
The invention described in claim 16 provides the reagent composition for measuring a saccharide according to claim 15, wherein the saccharide is glucose or 1,5-anhydroglucitol.
The invention described in claim 17 provides a biosensor for measuring saccharides, characterized in that the pyranose oxidase according to any one of claims 2 to 5 is used.
The invention according to claim 18 provides the biosensor for measuring sugar according to claim 17, wherein the sugar is glucose or 1,5-anhydroglucitol.
The invention described in claim 19 provides a method for producing a ketosaccharide, characterized by using the pyranose oxidase according to any one of claims 2-5.

  By using the pyranose oxidase of the present invention, saccharides can be measured with high accuracy.

  The pyranose oxidase of the present invention consists of the amino acid sequence represented by SEQ ID NO: 2. In addition, it is well known that natural proteins include mutant proteins having one or several amino acid mutations due to gene mutations due to differences in the species of organisms that produce them or differences in ecotypes. It is. Accordingly, the pyranose oxidase of the present invention includes a pyranose oxidase having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having pyranose oxidase activity. . In general, the structure and amount of the sugar chain that binds to the enzyme differ depending on the production method of the enzyme. In a protein having many sugar chain bonds, it is preferable to remove the sugar chain in order to use it as a biosensor because the sugar chain part prevents movement of electrons. The pyranose oxidase obtained by the production method of the present invention has substantially no sugar chain to be bound, and is extremely useful as a biosensor.

The pyranose oxidase of the present invention preferably has the following physicochemical properties.
(1) Substrate specificity: Shows the highest specificity for D-glucose, and also acts on 6-deoxy-D-glucose, δ-D-gluconolactone, L-sorbose and D-xylose.
(2) Optimal pH and pH stability: It has the highest activity in the range of pH 5.5 to 8.0, and is stable between pH 5.5 and 9.0 after treatment at 50 ° C. for 30 minutes.
(3) Optimal temperature and thermal stability: Optimal temperature is 60 ° C and no deactivation is observed up to 50 ° C, more than 90% after treatment at 60 ° C for 30 minutes, 50% after treatment at 70 ° C for 30 minutes It has the above remaining activity.
(4) Molecular weight: The molecular weight measured by the gel filtration method is about 300,000.
(5) Isoelectric point: 6.2
(6) Inhibitor specificity: Inhibited by Ag ⁺ , Cu ⁺⁺ , Hg ⁺⁺ , Ni ⁺⁺ , PCMB (p-chloromercury benzoate). Not inhibited by Fe ⁺⁺ . Not inhibited by high concentration NaCl.
(7) Km value: 3.1 mM (D-glucose)

  The gene of the present invention is a gene encoding such a pyranose oxidase, and is preferably a gene consisting of the base sequence represented by SEQ ID NO: 1. Furthermore, the homology with the base sequence represented by SEQ ID NO: 1 is preferably 60% or more, more preferably 70% or more, particularly preferably 80% or more, most preferably 90% or more, and the present invention. And a gene encoding a protein having the characteristics of pyranose oxidase. In addition, in the base sequence represented by SEQ ID NO: 1, the sequence of 1 to 1929 is a sequence encoding the amino acid sequence represented by SEQ ID NO: 2, and the sequence of 1930 to 1932 is a sequence corresponding to the stop codon.

  As long as the pyranose oxidase of the present invention has the characteristics described in the present specification, its origin, production method and the like are not particularly limited. Natural proteins, proteins expressed from recombinant DNA by genetic engineering techniques, or chemical synthesis Any of the proteins may be used. Among these, the pyranose oxidase of the present invention is collected from the Basidiomycetaus fungi strain 52, is easy to express by genetic engineering techniques, has a high yield, and the amount of sugar chains to be bound is low. It is preferable in that it is substantially zero.

An example of a method for producing the pyranose oxidase of the present invention using a genetic engineering technique will be described below.
(Acquisition of mRNA)
A basidiomycete, preferably Basidiomycetaus fungi 52 strain, is pulverized and extracted with a buffer solution, followed by filtration. Lithium chloride is added to the supernatant to precipitate, and the precipitate is recovered and dissolved in DEPC (diethyl pyrocarbonate) treated water, then treated with DNase, and then the supernatant is recovered and dissolved in DEPC treated water. mRNA can be obtained.

(RT-PCR)
Using the obtained mRNA as a template, RT-PCR is performed using primers designed with restriction enzyme sites upstream and downstream of the pyranose oxidase gene to amplify the pyranose oxidase gene.

(Creation of recombinant vectors)
An expression vector for preparing a recombinant vector is one in which the protein to be expressed is produced as it is, or one in which the histidine tag is added to the N-terminus or C-terminus, or N-terminus or C It is appropriately selected from those produced by fusing maltose-binding protein and GST peptide at the end. A recombinant vector can be prepared by cleaving an expression vector using the same restriction enzyme as described above to form a linear molecule, and then introducing a pyranose oxidase gene obtained by RT-PCR.

(Creation of transformants)
Introduction of a recombinant vector incorporating the gene of the present invention into a host cell (transformation (transfection)) can be performed using a conventionally known method. For example, in the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA., 69, 2110, 1972), protoplast method (Mol. Gen. Genet., 168, 111, 1979) and competent methods (J. Mol. Biol., 56, 209, 1971) In the case of yeast (Saccharomyces cerevisiae, Pichia pastoris, etc.), for example, the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA., 75, 1927, 1978) or the lithium method (J Bacteriol., 153, 163, 1983), in the case of animal cells, for example, by Graham's method (Virology, 52, 456, 1973), In the case of insect cells, for example, Summers (Summe rs) et al. (Mol. Cell. Biol., 3, 2156-2165, 1983). The transformation need not be expressed by the above recombinant vector. For example, a means for directly inserting the pyranose oxidase gene of the present invention into the chromosomal DNA of the host cell and expressing it may be used. In the present invention, the host is preferably a microorganism belonging to the genus Escherichia.

(Expression and collection of pyranose oxidase)
The protein of the present invention can be obtained by culturing transformed cells containing the expression vector prepared as described above in a nutrient medium and collecting the expressed pyranose oxidase. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, sucrose, methanol, and the like. However, when a nutrient that is a substrate of the pyranose oxidase of the present invention is used, hydrogen peroxide is generated, which is the amount of the host cell. It is also expected to affect the properties. Examples of the inorganic nitrogen source or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like. In addition, other nutrients (for example, inorganic salts (for example, sodium chloride, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)) are included as desired. You may go out. The culture is performed by a method known in the art. The culture conditions such as temperature, medium pH and culture time are appropriately selected so that the protein of the present invention is produced in large quantities.

  The saccharide measurement reagent composition of the present invention contains the pyranose oxidase of the present invention as an essential component and is suitable for measurement of pyranose, particularly glucose and 1,5-anhydroglucitol. 1,5-Anhydroglucitol is present in human cerebrospinal fluid and plasma, and it is known that the concentration in plasma decreases particularly in the case of diabetes. Therefore, if this 1,5-anhydroglucitol is quantified as a marker, diabetes can be diagnosed.

  The saccharide measurement reagent composition of the present invention is preferably combined with a reagent for removing saccharides other than the saccharide to be measured (saccharide removal agent) and a hydrogen peroxide detection reagent. Hereinafter, 1,5-anhydroglucitol will be described as an example of the saccharide to be measured. As the saccharide removal agent, a combination of a boric acid binding resin, an anion exchange resin and a cation exchange resin, or a combination of a strongly basic anion exchange resin and a cation exchange resin is preferable. When the specimen is treated with a strongly basic anion exchange resin or boric acid, saccharides are selectively removed without any removal of 1,5-anhydroglucitol, which contains 1,5-anhydroglucitol. A sample is obtained.

  Examples of the hydrogen peroxide detection reagent include peroxidase or peroxidase-like active substance and chromogenic substrate or chromogenic agent and coupler, peroxidase and fluorescent substrate, peroxidase and luminescent substrate, ferricyan ion and luminescent substrate in combination. As a method of detecting hydrogen peroxide, horseradish peroxidase (HRP) is used as a catalytic enzyme to oxidize various HRP substrates with hydrogen peroxide. Dyes, fluorescent substances and chemiluminescence generated as a result of oxidation reaction May be measured for absorbance, fluorescence measurement, and luminescence measurement, respectively.

  The saccharide-measuring reagent composition may be mixed together to form a single reagent. When components that interfere with each other are present, the components may be divided so as to form an appropriate combination. These may be prepared as a solution or powdery reagent, and may be prepared as a test paper or an analytical film by containing them in a suitable support such as filter paper or film. A deproteinizing agent such as perchloric acid or a standard reagent containing a certain amount of 1,5-anhydroglucitol may be attached. The amount of the enzyme in the saccharide measurement reagent composition is preferably about 0.5 to 20 units per sample. Examples of the specimen for quantifying 1,5-anhydroglucitol include cerebrospinal fluid, plasma, serum, urine and the like. In these specimens, saccharides such as glucose that can react with pyranose oxidase are present, so it is preferable to use a sample from which the saccharides have been removed.

  The saccharide measurement method of the present invention uses the pyranose oxidase of the present invention and is suitable for measurement of pyranose, particularly glucose and 1,5-anhydroglucitol, and the saccharide measurement reagent composition of the present invention. Is preferably used. Hereinafter, 1,5-anhydroglucitol will be described as an example of the saccharide to be measured. First, a sample obtained by treating a specimen with a saccharide removing agent, preferably a sample subjected to further deproteinization treatment, is preferably 0.5 to 10 units, more preferably 1 to 5 units of electron acceptor and pyranose oxidase per ml of the sample. A unit amount is added, preferably 4 to 50 ° C., particularly preferably 25 to 40 ° C., preferably 0.5 to 3 hours, particularly preferably 0.5 to 1 hour, and then hydrogen peroxide produced. The amount may be measured with a hydrogen peroxide detection reagent, and the amount of 1,5-anhydroglucitol may be determined from a separately prepared calibration curve.

A biosensor for measuring saccharides, preferably pyranose, particularly preferably glucose and 1,5-anhydroglucitol can be obtained using the pyranose oxidase of the present invention. An example of the biosensor of the present invention is shown below. An electrode system is formed on the insulating substrate by a method such as screen printing, and a reaction layer containing the hydrophilic polymer, the pyranose oxidase of the present invention, and an electron acceptor is formed on the electrode system. The biosensor can quantify the substrate concentration in the sample as follows. First, by dropping the sample solution onto the reaction layer of the biosensor, the reaction layer is dissolved, and an enzyme reaction proceeds between the substrate in the sample solution and the pyranose oxidase in the reaction layer. The electron acceptor is reduced. After the completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, whereby the substrate concentration in the sample solution is quantified from the oxidation current value obtained at this time.
When the enzyme used in the biosensor is a glycoprotein, it is desirable to remove the saccharide because the sugar chain part prevents the movement of electrons. In order to remove sugar chains from glycoproteins, for example, mold-derived glycoproteins (enzymes) are preferably pretreated with a sugar removal reagent such as glycosidase. However, when the pyranose oxidase of the present invention is expressed in a prokaryotic organism such as Escherichia coli, it is a protein that does not substantially contain a sugar chain, so that it can be easily used as a biosensor.

  Since the pyranose oxidase of the present invention is an oxidase, it can be used to produce a ketosaccharide under appropriate pH, temperature and aerobic conditions. For example, 3-deoxy-D-glucose can be converted to 3-deoxyglucosone and D-glucose can be converted to D-glucosone using pyranose oxidase.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

(Culture of pyranose oxidase producing bacteria)
Basidiomycetaus fungi 52 strain was cultured under the following conditions.
Medium composition Lactose: 4.0%, defatted soybean: 3.0%, CSL: 1.0%, glycine: 0.2%, adecanol: 0.1%, chloramphenicol: 50 mg / L (pH 6. 0)
◎ Culture conditions Dispense 10 mL of the above medium into 8 test tubes (diameter 2.2 cm × length 19.5 cm), sterilize and cool at 121 ° C. for 20 minutes, and then inoculate each strain with one platinum loop. The seed culture solution was prepared by shaking culture at 25 ° C. for 24 hours.
Next, 4 L of the above medium is filled in a 5 L jar, sterilized at 121 ° C. for 20 minutes and cooled, then the seed culture solution is added, and the aeration is 1 v / v / m, the rotation speed is 350 rpm, and the culture is performed at 25 ° C. for 130 hours. Went. After 95 hours from the start of the culture in which the pyranose oxidase activity was increasing, 3 g of wet cells were collected and used for preparation of mRNA.

(Acquisition of mRNA)
As shown below, mRNA was prepared based on the Total RNA Isolation method of Extract-A-Plant RNA Isolation Kit of CLONTEC.
3 g of the wet cells obtained above were washed with cold distilled water, dried, and frozen with liquid nitrogen to obtain freeze-dried cells. The cells were pulverized, 5 mL of Extraction Buffer and 5 mL of phenol were added, and the mixture was incubated at 80 ° C. The supernatant was recovered, 5 mL of phenol and 5 mL of chloroform / isoamyl alcohol were added and incubated, and the supernatant was recovered. Subsequently, 5 mL of 4M lithium chloride was added, and it frozen at -80 degreeC. This was dissolved and the precipitate was recovered. The recovered precipitate was added with 2 mL of 70% ethanol, dried, and then dissolved in 500 μL of DEPC-treated water. After DNase treatment, 500 μL of water-saturated phenol and 500 μL of chloroform / isoamyl alcohol were added to recover the supernatant. What was added with ethanol and precipitated was dissolved in 200 μL of DEPC-treated water. As a result, 454 μg of mRNA (about 4.5 μg / μL) was obtained from 3 g of wet cells.

(Preparation of primer)
With reference to the partial nucleotide sequence of the mRNA obtained above, a synthetic primer for RT-PCR was prepared in which restriction enzyme NdeI-EcoRI sites were designed upstream and downstream, respectively. The sequence represented by SEQ ID NO: 3 was used as a sense primer, and the sequence represented by SEQ ID NO: 4 was used as an antisense primer.

(RT-PCR)
RT-PCR was performed using TaKaRa RNA LA PCR Kit (AMV) Ver. 1.1 was used in the following procedure.
To 0.45 μg of mRNA, 8.5 μL of RNase-free distilled water and 1 μL of oligo dT adapter primer (0.125 μM) were added. Next, magnesium chloride 4 μL (5 mM), 10 × RNA PCR buffer 2 μL, dNTP-free distilled water 8.5 μL, RNase inhibitor 0.5 μL (1 U / μL), reverse transcriptase 1 μL (0.25 U / μL) And a thermal history of 70 ° C. for 10 minutes, 42 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 5 ° C. for 5 minutes. Next, 6 μL (2.5 mM) of magnesium chloride, 8 μL of 10 × LA PCR buffer (II), 63.5 μL of sterile distilled water, 1 μL of sense primer (0.2 μM), 1 μL of antisense primer (0.2 μM), LA Taq 0.5 μL (2.5 U / 100 μL) was added, and after heating at 94 ° C. for 2 minutes, a thermal history of 94 ° C. for 30 seconds, 55 ° C. for 30 minutes and 72 ° C. for 1 minute was performed for 28 cycles to obtain an amplification product. When this was subjected to 1.5% agarose gel electrophoresis, the expected RT-PCR product of about 1.9 kbp was amplified, and as a result of sequence analysis, the pyranose oxidase gene was confirmed (FIG. 1).

(Construction of expression plasmid)
pET21a (+) (5.4 kbp) was cleaved with EcoRI and NdeI, and the pyranose oxidase gene (1.9 kbp) obtained by linking NdeI on the upstream side and EcoRI site on the downstream side was ligated. A plasmid was constructed.

(Expression in E. coli)
The pET21a (+) incorporating the pyranose oxidase gene obtained above was introduced into E. coli BL21 (DE3), and cultured at 37 ° C. in an LB medium containing 50 μg / mL ampicillin. When the absorbance at 660 nm reached about 0.5, IPTG was added to 0.1 mM to induce pyranose oxidase expression. After 15 hours, the cells were collected by centrifugation, the cells were suspended in 20 mM phosphate buffer (KPB) (pH 7.0), and subjected to ultrasonic disruption to prepare a cell-free extract. Subsequently, pyranose oxidase activity was measured by the following method.
(Method for measuring pyranose oxidase activity)
In a mixture of 2.4 mL of water, 0.1 mL of 1M KPB (pH 7.0), 0.1 mL of peroxidase (100 U / mL), 0.1 mL of 4-aminoantipyrine (24.6 mM), 0.1 mL of phenol (420 mM) Then, 0.1 mL of the sample obtained above was added and incubated at 37 ° C. for 1 minute. Subsequently, 0.1 mL of D-glucose (1M) was added thereto, ΔOD at 500 nm was measured, and the activity was calculated by the initial velocity method using the following calculation formula.

  As a result, 0.2 U of pyranose oxidase activity was confirmed per 1 mL of the culture solution.

  The present invention can be used in technical fields such as sugar production and measurement of biological substances.

The result of having analyzed the product amplified by RT-PCR by agarose gel electrophoresis is shown.

Claims (19)

  A method for producing pyranose oxidase, comprising inserting a gene represented by SEQ ID NO: 1 into a microorganism belonging to the genus Escherichia to obtain a transformant, culturing the transformant, and collecting pyranose oxidase from the culture.   The pyranose oxidase manufactured by the manufacturing method of Claim 1.   The pyranose oxidase according to claim 2, comprising the amino acid sequence represented by SEQ ID NO: 2.   The pyranose oxidase according to claim 2, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid represented by SEQ ID NO: 2 and having pyranose oxidase activity.   Basidiomycetous fungi No. The pyranose oxidase according to any one of claims 2 to 4, which is derived from 52 strain (FERM P-10106).   A gene encoding the pyranose oxidase according to any one of claims 2 to 5.   The gene according to claim 6 represented by SEQ ID NO: 1.   A gene encoding a protein having 60% or more homology with the gene represented by SEQ ID NO: 1 and having pyranose oxidase activity.   A recombinant vector containing the gene according to any one of claims 6 to 8.   A transformant in which the gene according to any one of claims 6 to 8 is inserted and the gene is expressed.   11. The transformant according to claim 10, wherein the host is a microorganism belonging to the genus Escherichia, Bacillus, Saccharomyces, or Pichia.   A method for producing pyranose oxidase, comprising culturing the transformant according to claim 10 or 11 and collecting pyranose oxidase from the culture.   A method for measuring a saccharide, comprising using the pyranose oxidase according to any one of claims 2 to 5.   The method for measuring a saccharide according to claim 13, wherein the saccharide is glucose or 1,5-anhydroglucitol.   A saccharide-measuring reagent composition containing the pyranose oxidase according to any one of claims 2 to 5.   The saccharide measuring reagent composition according to claim 15, wherein the saccharide is glucose or 1,5-anhydroglucitol.   A biosensor for measuring sugars, wherein the pyranose oxidase according to any one of claims 2 to 5 is used.   The biosensor for saccharide measurement according to claim 17, wherein the saccharide is glucose or 1,5-anhydroglucitol. A method for producing a ketosaccharide, comprising using the pyranose oxidase according to any one of claims 2 to 5.

Images