JP2000175698A - Pyranose oxidase-containing reagent for measuring pyranose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent for measurement of pyranose for diagnosis of diabetes mellitus, or the like, by including a pyranose oxidase having a specific molecular weight and antimicrobial activity obtained from a fraction precipitated by ammonium sulfate precipitation method from an aqueous extract solution of tricholoma matsutake. SOLUTION: This reagent for measurement of pyranose is obtained by including a pyranose oxidase capable of obtaining from a fraction precipitated by ammonium sulfate method from an aqueous extract solution of Tricholoma matsutake, having antimicrobial activity against at least rice sheath blight or rice blast, having about 210 kD molecular weight by gel permeation method, exhibiting presence of components having about 50 kD and about 15 kD molecular weights by SDS-PAGE method, capable of retaining the above antimicrobial activity by heating for 10 min at 60 deg.C in a neutral aqueous solution and deactivating the above antimicrobial activity by heating for 10 min at 80 deg.C in a neutral aqueous solution. The reagent is useful as a diagnostic agent for diabetes mellitus, or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a pyranose oxidase useful for the enzymatic determination of glucose and other aldopyranoses, more specifically to a reagent for measuring pyranose containing the above pyranose oxidase, and to the use of the above reagent. The present invention relates to a method for measuring pyranose and a method for diagnosing a disease associated with abnormal sugar metabolism such as diabetes.

[0002]

2. Description of the Related Art Pyranose: ox
ygen 2-oxidoreductase, EC 1.1.3.10) oxidizes the 2-position carbon of D-glucose and other aldopyranoses,
-An enzyme that catalyzes a reaction that produces a keto product and hydrogen peroxide.

[0003] This enzyme produces certain antibiotics (Koths et a
l. (1992) Carbohydr. Res. 232: 59-75) and industrial production of fructose (Shaked and Wolfe (1988) Methods En)
zymol137: 599-615). This enzyme is known for its practical value as a glucose biosensor. Enzymatic measurement of glucose in a sample can be performed by quantifying the hydrogen peroxide generated by the present enzyme by conjugating peroxidase, catalase, or the like, and then subjecting it to an appropriate coloring system. Specifically, it can be used for measuring glucose in foods and body fluids. In particular, it has already been applied as a blood glucose test method in clinical settings such as a diagnostic test for diabetes.

[0004] An advantage of the present enzyme as compared with conventionally used glucose oxidase is that it has a higher substrate affinity (Nishimura et al. (1996) J. Biotechnol.
52: 11-20) and also acts on α-D-glucose as well as β-D-glucose, eliminating the need for the addition of mutarotase (Taguchi et al. (1985) J. App.
l. Biochem. 7: 289). Furthermore, it has recently been found that this enzyme uses 1,5-anhydroglucitol, which has been attracting attention as a blood sugar control marker for diabetes, as a substrate (Yabuuchi et al. (1989) Clin. C.
hem. 35: 2039-2043), and is also expected to be useful as a diagnostic agent for diabetes.

Although the present enzyme has been purified from various basidiomycetes and its enzymatic properties have been analyzed, the gene encoding the present enzyme was isolated from Coriolus ver.
sico lor ) (Japanese Patent Application No. 7-124)
835).

[0006]

An object of the present invention is to provide a novel pyranose oxidase useful as a reagent for measuring saccharides, particularly glucose, for example, a blood glucose test agent, more specifically, a diagnostic agent for diabetes. And to identify a DNA sequence encoding the present enzyme protein so that the present enzyme can be obtained in a large amount, at low cost, and efficiently by applying a gene recombination technique. That is, one of the objects of the present invention is to identify a novel pyranose oxidase and provide a reagent for measuring pyranose. Another object of the present invention is to provide a method for measuring pyranose using the reagent for measuring pyranose containing pyranose oxidase according to the present invention, and a method for diagnosing a disease accompanied by abnormal metabolism of saccharides.

[0007]

Means for Solving the Problems As means for solving the above problems, the present inventor first inverts phytopathogenic bacteria.
Established an assay system for proteins that have antibacterial activity in vitro, extracted proteins from matsutake mushrooms, one of the edible mushrooms, combined an ion exchange column and a gel filtration column, and supplied each fraction to the above assay system. By experimentation, the antibacterial protein fraction was identified, and the protein was separated and purified. Next, the N-terminal amino acid sequence of this purified protein was determined, and a database search was performed on this sequence. As a result, Coriorus versico
lor ) pyranose oxidase. Then, when the pyranose oxidase activity of the purified protein was measured, a strong activity was actually detected, and the present invention was completed.

Furthermore, oligo DNA was synthesized based on the determined partial amino acid sequence, and a partial-length cDNA encoding the protein was obtained by RT-PCR. Next, a full-length cDNA encoding the protein was identified by screening a Matsutake cDNA library using this as a probe, and the entire nucleotide sequence was determined. Thus, the entire amino acid sequence of pyranose oxidase derived from Matsutake and the DNA sequence of the gene encoding the same were identified, and the present invention was completed.

According to a first aspect of the present invention, a gel obtained from an aqueous extract of matsutake by an ammonium sulfate precipitation method has at least an antibacterial activity against rice sheath blight or rice blast, and a gel. The molecular weight by filtration is about 210 kD, and the molecular weight by SDS-PAGE is about 50 kD.
And the presence of a component of about 15 kD.
A reagent for measuring pyranose, comprising pyranose oxidase, wherein the antibacterial activity is maintained by heating at 10 ° C for 10 minutes, and the antibacterial activity is deactivated by heating at 80 ° C for 10 minutes in a neutral aqueous solution. .

The pyranose oxidase in the present invention preferably has the following enzymatic properties: (1) Substrate specificity: D (+)-glucose, 1,5-anhydro-D-glucitol, L (-)- Sorbose,
Using D (+)-xylose, trehalose, D (+)-galactose and maltose as substrates, mannose, mannitol, D (-)-fructose, D-sorbitol, sucrose, D (-)-arabinose and N
-Acetyl-D-glucosamine is not used as a substrate; (2) optimum temperature: about 50 ° C. is the optimum temperature;
Enzyme activity of 80% or more is maintained in the range of 0 ° C. (3) Temperature stability: The residual activity after being kept at each temperature for 30 minutes is 95% or more and 55 to 6 at the temperature of 30 to 50 ° C.
About 50% at a temperature of 0 ° C, 30% at a temperature of 60 to 70 ° C
(4) optimum pH: pH 7.5 to 8.0 is the optimum pH, and the enzyme activity of 80% or more is maintained in the range of pH 6.5 to 9.0; (5) pH stability : 7.0 after stable incubation at 50 ° C. for 30 minutes at each pH where the remaining activity is 80% or more.
From 11.0;

Preferably, the about 50 kD component of the pyranose oxidase in the present invention is an N-terminal amino acid sequence represented by SEQ ID NO: 3 in the sequence listing: His Glu Ile Val His Tyr
ThrAsp Val Phe Ile Ala Gly Ser Gly Pro Ile Ser Xaa
Thr Tyr Ala Arg His IleIle Asp Asn Thr Ser Thr Th
r: and a component of about 15 kD has an N-terminal amino acid sequence represented by SEQ ID NO: 4 in the sequence listing: Arg Glu Trp Glu Ala Gly
Val Thr Asp Thr Tyr Gly Met Pro Gln Pro Thr Phe Hi
s Val Lys Arg Thr Asn Ala Asp Gly Asp Arg Asp Gln
Arg Met Met Asn Asp Met Thr Asn Val Ala Asn Met Le
u Gly Gly Tyr Leu Pro Gly Ser Tyr Pro Gln Phe Met
Ala Pro Gly Leu Val Leu His Ile Thr Has Gly Thr.

Preferably, the pyranose oxidase in the present invention is an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing; an amino acid sequence having one or more amino acid mutations in this sequence; or 50% or more, preferably Has an amino acid sequence having a homology of 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90%, and most preferably 95% or more.

According to a second aspect of the present invention, a polypeptide consisting of the partial amino acid sequence 1-25 of SEQ ID NO: 2 in the sequence listing: a polypeptide consisting of the partial amino acid sequence 26-435: from the partial amino acid sequence 436-564 Polypeptide: a polypeptide consisting of the partial amino acid sequence 1-435: and a polypeptide consisting of the partial amino acid sequence 26-564: and any one of the above amino acid sequences
A pyranose oxidase comprising a polypeptide having a plurality of amino acid mutations and a polypeptide having an amino acid sequence having 50% or more homology with any of the above amino acid sequences; Are provided.

In the present invention, pyranose is preferably D (+)-glucose, 1,5-anhydro-D-
It is selected from glucitol, L (-)-sorbose, D (+)-xylose, trehalose, D (+)-galactose or maltose, most preferably the pyranose is D (+)-glucose. The reagent for measuring pyranose of the present invention can be used, for example, as a diagnostic agent for a disease associated with abnormalities in sugar metabolism, and particularly as a diagnostic agent for diabetes.

According to a third aspect of the present invention, the pyranose measuring reagent of the present invention is reacted with pyranose in a sample,
There is provided a method for measuring pyranose in a sample, comprising reacting the produced hydrogen peroxide with an enzyme to develop color.

According to a fourth aspect of the present invention, the method comprises reacting the pyranose measuring reagent of the present invention with pyranose in a sample collected from a subject, and reacting the produced hydrogen peroxide with an enzyme to form a color. And a method for diagnosing a disease associated with abnormal sugar metabolism (eg, diabetes).

According to a fifth aspect of the present invention, there is provided a kit for analyzing pyranose or diagnosing a disease associated with abnormal metabolism of saccharides, comprising the reagent for measuring pyranose of the present invention.

[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, more detailed embodiments of the present invention will be described. Pyranose oxidase in the present invention,
It was obtained in the process of separating and purifying the antibacterial protein from Matsutake, but its origin and production method are not limited as long as it has the characteristics described in this specification, and naturally occurring proteins and genetic engineering techniques May be any of a protein expressed from recombinant DNA or a chemically synthesized protein.

The pyranose oxidase of the present invention typically has a 564 amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. However, some natural proteins have one or more amino acid mutations due to differences in varieties of the species producing them, differences in genes due to differences in ecotypes, or the presence of similar isozymes. It is well known that there are mutant proteins that have In addition,
As used herein, the term "amino acid mutation" means substitution, deletion, insertion and / or addition of one or more amino acids. The pyranose oxidase in the present invention has the amino acid sequence of SEQ ID NO: 2 based on the inference from the nucleotide sequence of the cloned gene, but is not limited to only the protein having the sequence. It is intended to include all homologous proteins as long as they have the properties described therein. The homology is at least 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more.

In general, when a substitution between amino acids having similar properties (for example, substitution between hydrophobic amino acids, substitution between hydrophilic amino acids, substitution between acidic amino acids or substitution between basic amino acids) is introduced. The resulting mutant protein often has the same properties as the original protein. Techniques for producing recombinant proteins having such desired mutations using genetic recombination techniques are well known to those skilled in the art, and such mutant proteins are also included in the scope of the present invention.

[0021] The pyranose oxidase of the present invention comprises
-A molecular weight of about 50 kD by PAGE (corresponding to a polypeptide consisting of the 26th to 435th amino acid sequence in the sequence of SEQ ID NO: 2) and 15 kD (from the 436th to 564th amino acid sequence) And a molecular weight of about 210 kD on a gel filtration column. However,
There is a possibility that the polypeptide consisting of the 1st to 25th amino acid sequences in SEQ ID NO: 2 may also be one subunit of this protein.

The pyranose oxidase in the present invention can be purified from matsutake fruit bodies by, for example, ammonium sulfate precipitation, ion exchange column chromatography or the like according to Examples described later. That is, the finely divided matsutake is extracted from the matsutake with a buffer solution, then filtered, and ammonium sulfate (ammonium sulfate) is added to the supernatant at a suitable concentration, for example,
By adding the mixture until 75% saturation and allowing it to stand, a precipitate containing the protein of the present invention is obtained. The precipitate is further dialyzed and then subjected to ion exchange chromatography.
A salt concentration gradient (eg, 50
(mM to 1M), and the fraction containing the desired protein may be collected.

Alternatively, the pyranose oxidase according to the present invention is a DNA characterized by being a sequence from the 101st to the 1792th of the DNA sequence of SEQ ID NO: 1 in the sequence listing encoding the Matsutake pyranose oxidase according to the present invention. A sequence, a DNA sequence that is the 176th to 1792th sequence, or a DNA sequence that is the 176th to 1405th sequence, or a DNA sequence that is the 1406th to 1792th sequence , Or both, introduced into an animal such as Escherichia coli, yeast, or silkworm larvae or certain animal cells such as Xenopus using an expression vector that can be amplified in each host,
It is thought that it can also be obtained by expression.

The expression vector may be one produced by producing the protein to be expressed as it is, one produced by adding a histidine tag at the N-terminus or C-terminus, or maltose-linked at the N-terminus or C-terminus. The protein or GST peptide is appropriately selected from those produced in a fused form.

The introduction (transformation (transfection)) of an expression vector into which a gene of the present invention has been incorporated into a host cell can be carried out by a conventionally known method. For example, bacteria ( E.
col i, in the case of Bacillus subtilis, etc.), for example Cohen
(Proc. Natl. Acad. Sci. USA., Vol. 69, No. 2)
110, 1972), the protoplast method (Mol. Gen.
Genet., 168, 111, 1979) and the competent method (Journal of Molecular Biology (J. Mol. Biol.), 56, 209, 19).
71 years), the yeast ( Saccharomyces cerevisiae ,
Pichia postoris ), for example, Hinne
n) et al. (Proc. Natl. Acad. USA., Vol. 75, No. 19)
27, 1978) or the lithium method (J. Bacteriol., 153, 163, 1983), in the case of animal cells, for example, the method of Graham (Virology, 52 Volume, p. 456, 197
3 years), in the case of insect cells, for example, the method of Summers et al. (Mol. Cell. Biol., Vol. 3, No. 2)
156 to 2165 (1983)).

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

[0027] Specific media and culture conditions used according to the host cell are shown below, but the present invention is not limited thereto. Host is bacteria, actinomycetes, yeast,
In the case of a filamentous fungus, for example, a liquid medium containing the above-mentioned nutrient source is suitable. Preferably, LB medium, M9 medium (Miller et al., Exp.
Mol. Genet., Cold Spring Harbor Laboratory, No. 43
1 page, 1972). In such a case, culturing is carried out usually with aeration and agitation, if necessary, with 14 to 43 times.
C. for about 3 to 24 hours. The host is Bacill
In the case of a genus us, if necessary, with aeration and stirring, usually 3
The reaction can be performed at 0 to 40 ° C. for about 16 to 96 hours.

When the host is yeast, the medium may be, for example, Burkholder minimal medium (Bostian, Proc.
Natl. Acad. ScI. USA., 77, 4505, 198.
0 years), and the pH is desirably 5 to 8. Culture is usually performed at about 20-35 ° C. for about 14-144.
It is performed for a while, and if necessary, aeration and stirring can be performed. When the host is an animal cell, for example, about 5-2
MEM medium containing 0% fetal bovine serum (Scie
122), p. 501, 1952), DM
EM medium (Virology, Vol. 8, No. 39
6, 1959), RPMI 1640 medium (J. Am. Me
d. Assoc., 199, 519, 1967), 1
99 times (Proc. Soc. Exp. Biol. Med., Vol. 73, No. 1)
Pp. 1950). Medium p
H is preferably about 6 to 8, and the culture is usually 30 to
The reaction is carried out at 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed. When the host is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. U.
SA., 82, 8404, 1985), and the pH is preferably about 5-8. The cultivation is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring can be performed.

The protein of the present invention can be obtained from the culture obtained by the above-described culture as follows. That is, when the protein of the present invention accumulates in host cells, the host cells are collected by an operation such as centrifugation or filtration, and the collected host cells are buffered (for example, at a concentration of 10 mM).
~ 100 mM Tris buffer, phosphate buffer, HE
Buffers such as PES buffer and MES buffer. The pH varies depending on the buffer used, but the pH is preferably in the range of 5.0 to 9.0), and then the cells are disrupted by a method suitable for the host cell to be used, and the contents of the host cell are obtained by centrifugation. . On the other hand, when the protein of the present invention is secreted out of the host cells, the culture medium is separated from the host cells by an operation such as centrifugation or filtration to obtain a culture filtrate. Host cell disruption fluid,
Alternatively, the culture filtrate can be used for purification and isolation of the protein of the present invention as it is or after performing ammonium sulfate precipitation and dialysis. The following methods can be mentioned as methods for purification and isolation. That is, when a tag such as 6 × histidine, GST, or maltose binding protein is attached to the protein, a method using affinity chromatography suitable for each tag generally used can be mentioned. On the other hand, when the protein of the present invention is produced without attaching such a tag, for example, a method described in detail in Examples below, that is, a method by ion exchange chromatography can be used. In addition, a method in which gel filtration, hydrophobic chromatography, isoelectric point chromatography, or the like is combined may also be used.

The protein of the present invention, that is, a protein having an amino acid sequence of SEQ ID NO: 3 in the sequence listing as a sequence on the N-terminal side and having a molecular weight of about 50 kD by SDS-PAGE, A protein having a molecular weight of about 15 kD by SDS-PAGE and a molecular weight of about 210 kD estimated by a gel filtration column, Has strong pyranose oxidase activity.

The pyranose oxidase activity of the present protein was 25.78 U / mg, and the previously reported Polyporus
pyranose oxidase from Obtusus (Janssen and R
uelius (1975) Methods Enzymol. 41: 170-173) or pyranose oxidase from Coriolus versicolor (Nishimura et al. (1996) J. Biotechnol. 52: 11-).
20 times stronger than 20), Phanerochaete chrysosporiu
m- derived pyranose oxidase (Artolozaga et al.
(1997) Appl. Microbiol. Biotechnol. 47: 508-514)
It has the same activity as.

Matsutake pyranose oxidase is
In addition to glucose, 1,5-anhydroglucitol was found to be a good substrate, and sorbose, xylose, trehalose, galactose, and maltose were also found to be substrates. Polyporus Compared with the pyranose oxidase obtusu s, matsutake pyranose oxidase activity about especially for 1,5-anhydroglucitol 1.6
It was twice as expensive.

The Km of matsutake pyranose oxidase for glucose was calculated to be 1.28 mM, and the Km for 1,5-anhydroglucitol was calculated to be 10.7 mM. This indicates that matsutake pyranose oxidase is particularly effective not only for glucose measurement but also for enzymatic measurement of 1,5-anhydroglucitol, which has recently attracted attention as a diagnostic marker for diabetes. Was done.

The optimum temperature of Matsutake pyranose oxidase was around 50 ° C., and had an enzyme activity of 80% or more in the range of 40 ° C. to 60 ° C. Temperature stability (30 minutes) is 50
To around ° C. It retains 95% or more of activity up to 50 ° C, but about 55-60 ° C, about half,
It showed 30-40% residual activity.

Optimum pH of Matsutake pyranose oxidase
Was around 7.5 to 8.0, and had an enzyme activity of 80% or more in the pH range of 6.5 to 9.0. Stable pH range (30 ℃ at 50 ℃)
Min) was pH 7.0-11.0. The residual activity of Matsutake pyranose oxidase after freezing at -20 ° C was 78% when the activity before freeze-thawing was defined as 100%. Matsutake pyranose oxidase, compared to Polyporus obtusus pyranose oxidase,
Km, temperature stability and pH stability show almost the same value, but 1,
It was excellent in action and Km for 5-anhydroglucitol and freeze-thaw stability.

The enzymatic properties described above include the polypeptide consisting of the 26th to 435th amino acid sequence and the 436th to 564th amino acid sequence of SEQ ID NO: 2 in the sequence listing. It is expected that the polypeptide is naturally retained even in a protein bound by a certain number. Since the pyranose oxidase derived from Matsutake of the present invention has a strong enzymatic activity, glucose can be measured with a smaller amount of enzyme as compared with conventional pyranose oxidase. Also, 1,
Since the Km for 5-anhydroglucitol is low and the activity is higher than conventional ones, it can be used particularly effectively as a diagnostic agent for diabetes.

About the Matsutake-derived protein and its gene of the present invention, BLAST in the database DDBJ was used.
When the homology search is carried out, the homology is found with Pyranose oxidase of Pleurotus ostreatus. When the total amino acid sequence of the protein and the total nucleotide sequence of the gene of the matsutake-derived protein and the pyranose oxidase of Kawatake mushroom are compared, they show 35% and 46% homology, respectively. In addition, since there is no other homologous sequence and no report has been reported on purification and isolation of pyranose oxidase from Matsutake, this protein is considered to be a novel pyranose oxidase.

If the amino acid sequence of this protein and the DNA sequence encoding it are disclosed by the present invention, the sequence or a part thereof can be used to carry out hybridization or PCR, which is a basic technique of genetic engineering. A gene encoding a protein having a similar biological activity can be isolated from other species. In such a case, the use of the protein having pyranose oxidase activity is also included in the scope of the present invention.

The reagent for measuring pyranose of the present invention contains pyranose oxidase as an essential component, and the other components are not particularly limited, and may optionally contain any additive commonly used in the art. . Examples of such additives include buffers (e.g., phosphate, acetate, carbonate, Tris hydrochloride, borate, citrate, etc.), solubilizers (e.g., Triton X-100, Noni
det P-40, etc.), stabilizers (eg, glycerin,
Bovine serum albumin, α- or β-cyclodextrin), and coenzyme FAD.

The reagent for measuring pyranose of the present invention can be used either in a dried form or in a dissolved form, and may be used after being impregnated in a thin-film carrier such as a sheet or an impregnable paper. .

As described above, according to the present invention, there is provided a reagent for measuring pyranose, which comprises pyranose oxidase having the characteristics described above in the present specification. When the reagent of the present invention is brought into contact with a sample containing pyranose, and the pyranose in the sample is reacted with pyranose oxidase, hydrogen peroxide is generated. This hydrogen peroxide is reacted with peroxidase or catalase, and in the case of peroxidase, the quinone dye produced by the condensation reaction of 4-aminoantipyrine with phenols is reacted with methanol in the case of catalase to formaldehyde. And this is subjected to a Hantzsch reaction using acetylacetone.
The amount of pyranose in the sample can be measured by using a lutidine derivative and quantifying the resulting yellow dye.

The sample can be selected from plasma, serum, urine, or other bodily fluids, and is not limited to such biological samples, but may be industrial or industrial, such as chemicals and foodstuffs. It can also be applied to samples from commercial products. The method for measuring pyranose by pyranose oxidase as described above is well known to those skilled in the art, and is described, for example, in Machida and Nakanishi (1984) Agric. Biol. Chem. 4
8: 2463-2470 or Seiichi Sasaki (1984) Clinical Examination MOOK18: 13-28.

An example of the method for measuring pyranose according to the present invention will be described below. This is a measurement method using a typical pyranose oxidase / peroxidase reaction.
Pyranose and hydrogen peroxide generated by pyranose oxidase are measured as a red quinone dye by a condensation reaction between 4-aminoantipyrine and phenol in the presence of peroxidase. First, 1 to 4
0 mU / ml, preferably 5-20 mU / ml of the pyranose measuring reagent of the present invention, 0.2-3 mM, preferably 0.4-1.6 mM 4-aminoantipyrine, 0.2
~ 12 mM, preferably 0.4-1.6 mM phenol, and 0.2-10 U / ml, preferably 1-2 U.
PH 6.5-1 containing peroxidase / ml of peroxidase
An appropriate buffer solution having a pH of 0, preferably 7.5 to 8.0 is added, and the reaction is carried out at a temperature of 35 to 60 ° C, preferably 45 to 55 ° C, for 3 to 20 minutes, preferably 5 to 10 minutes. Next, the degree of color development of the generated quinone dye is measured at 500 to 510 nm using a colorimeter or the like.
Finally, the pyranose concentration in the sample is calculated by comparing the absorbance value of a standard sample containing a known concentration of pyranose, which has been measured in advance, with the absorbance obtained using the sample.

According to the present invention, there is provided a kit for analyzing pyranose or for diagnosing a disease accompanied by abnormal sugar metabolism. The components of the kit include (1) a reagent for measuring pyranose of the present invention, (2) a buffer solution for reacting a sample with the reagent for measuring pyranose, and (3) an enzyme used for a color-forming reaction (for example, Peroxidase, catalase, etc.), and (4) a substrate for color reaction (for example, 4-aminoantipyrine and phenol).
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[0045]

EXAMPLES Example 1: Construction of assay system 1) Establishment of assay system Culture of pathogenic bacteria: rice blast (strain TUS-1, race 33)
7; obtained from the Tohoku Agricultural Experiment Station of the Ministry of Agriculture and Fisheries) was cultured on an automill medium (Difco, supplemented with 1% sucrose) to obtain conidia. The spore solution was added to 10% glycerol and stored at -80 ° C. The rice sheath blight fungus (strain JT872) is cultured in a 1/2 potato decoction medium (PD medium: Difco) for 2 days, and three hyphae masses of about 5 mm are gently polished with a Teflon homogenizer together with a 1/2 PD medium. The fragmented mycelium obtained by crushing was used as an inoculum.

The above inoculum was added to a 96-well microtiter plate (manufactured by Corning Incorporated) in an amount of 100 μl so that about 1,000 blast conidia of blast fungus and about 300 fragmented hyphae of the sheath rot fungus per well. The cells were added together with a 1 / 2PD medium, and cultured in a thermostat at 28 ° C. For the growth of the bacteria, the absorbance at 595 nm was measured using a microplate reader (Benchmark manufactured by Bio-Rad). The effect of salts and buffers on the growth of bacteria
And a fixed amount of phosphate buffer, Tris buffer, Hepes buffer, bovine serum albumin, dithiothreitol and the like.

2) Protein Extraction from Matsutake Mushrooms 20 g of Japanese matsutake was finely cut in advance with scissors, frozen using liquid nitrogen, and ground in a mortar until fine powder was obtained. Three times the volume of 50 mM Hepes buffer was added. Extraction was performed for an additional 30 minutes. The extract was filtered through Miracloth, and centrifuged at 10,000 × g for 20 minutes. Ammonium sulfate was added to the supernatant at 75% saturation, and the mixture was allowed to stand at 4 ° C. overnight. Again, sediment by centrifugation at 15,000Xg for 20 minutes, and precipitate 3 ml of 10 mM Hepes buffer, p
Dissolve in H7.5 and dialyze tubing (Spectra / Por1 MWCO 6-80
00, Spectrum Medical Industries) or 10 mM Hepes using benzoylated dialysis tubing (SIGMA).
Dialysis was performed against the buffer solution, pH 7.5, and insolubles were removed by centrifugation to obtain a matsutake protein sample. The protein concentration of the Matsutake protein sample was measured by the Bradford method using bovine serum albumin (BSA) as a standard protein.

Example 2: Purification of protein having antibacterial activity 1) Antibacterial activity of matsutake crude protein sample A matsutake protein sample was added to the above-mentioned culture system of blast fungus and sheath blight fungus immediately after the start of cultivation, and was added for 2 days. And the change in absorbance was measured over time to determine the presence or absence of antibacterial activity. A dilution series was prepared for protein samples, and the dilution end point for antibacterial activity was detected.
As a result, very high antibacterial activity against both the blast fungus and the sheath blight fungus was found (Table 1).

[0049]

[Table 1] Table 1: Antibacterial activity of the aqueous extract of Matsutake mushroom pH complete growth inhibitory concentration (μg / ml) at the time of extraction Blast fungus Sheath blight fungus Matsutake 7.5 0.07 <~ 0.7 <3.6

The complete growth inhibitory concentration against the blast fungus is 0.
It was estimated to be 7 μg or less of total extracted protein / ml, which revealed that the extract of Matsutake contained substances having extremely high antibacterial activity. When the concentration was high, complete suppression of germination was observed, and when the concentration was low, inhibition of mycelial growth was observed. The degree of inhibition of hyphal elongation was clearly concentration-dependent. In addition, the cells of the blast fungus had their cytoplasm separated from the cell wall, and had a plasma-separated appearance.

In order to confirm whether or not these results were due to proteins, heat treatment was first performed and the residual activity was examined. Next, the sample was fractionated with an ultrafiltration membrane in order to determine the approximate molecular weight. The antibacterial activity against blast fungus of Matsutake protein samples was investigated. The heat treatment is performed at 60 ° C, 80 ° C, and 100 ° C for 10 minutes, respectively, and the ultrafiltration membrane is Centricut Mini V-10 (cutoff molecular weight 10,000), V-20 (cutoff molecular weight 20,000).
(Both from Kurabo Co., Ltd.)
Assays were performed on fractions that did not pass through the membrane. As a result, the activity was maintained at 60 ° C (slightly deactivated),
At 100 ° C and 100 ° C, the activity almost completely disappeared. In addition, in the ultrafiltration membrane used, both activities were present only in the fraction not passing through the membrane (Table 2).

[0052]

[Table 2] Table 2: Effect of heat treatment on antibacterial properties and estimation of molecular weight Mushroom heat treatment (10 minutes) Ultrafiltration membrane 60 ° C 80 ° C 100 ° C V-10 V-20 passing fraction Non-passing fraction Passing fraction not Flow- through fraction Matsutake +---+-+ (+: active-: no activity)

The fact that deactivation by heat was observed and that the molecular weight was estimated to be at least 20,000 or more strongly suggested that proteinaceous substances exhibit antibacterial activity.

2) Purification by ion exchange column Next, a protein having antibacterial activity was purified. First, 3.6 mg / 10 ml of protein sample was added to the ion exchange column MonoQ.
The product was subjected to HR5 / 5 (Pharmacia) to partially purify the antibacterial protein. The flow rate is 1 ml / min and the basic buffer is 50 mM Mes.
pH 6.0, 50 mM NaCl, elution buffer 50 mM Mes pH 6.0, 1
Gradient (50 mM to 1 M with NaCl) using M NaCl
Took 40 minutes from 20 minutes after hitting the sample. Each fraction (1
ml) was used for an antibacterial assay against blast fungus and SDS-
The sample was subjected to PAGE electrophoresis. The antibacterial activity was determined by taking 5, 1, 0.2, and 0.04 μl from each fraction of MonoQ, and performing an assay against the fungus pathogen, +++ (inhibited to 0.2 μl, not inhibited by 0.04 μl), ++ (inhibited to 1 μl), + (Inhibited up to 5 μl),-
(No inhibition at 5 μl). As a result, when the protein was monitored by A280 and antibacterial activity against blast fungus, the ionic strength (NaCl
(Concentration) around 375 mM, an elution peak of a protein having antibacterial activity appeared. Thereafter, it was revealed that the antibacterial activity of the protein per unit eluate gradually decreased as the ionic strength increased (FIG. 1).

Next, 5 μl of each fraction was taken and an equal volume of 2 × SDS
After adding an electrophoresis buffer (Sambrook et al. 1989) and treating at 95 ° C. for 5 minutes, SDS-protein was added according to the method of Laemmli (1970).
PAGE electrophoresis was performed. The gel was subjected to silver staining II kit Wako (manufactured by Wako Pure Chemical Industries) using 15% PAGL (manufactured by ATTO) to detect proteins. To estimate the approximate molecular weight and amount of a protein, a molecular weight marker (LMW: Pharma
cia LKB, 94 kD, 67 kD, 43 kD, 3
0 kD, 20.1 kD, and 14.4 kD) were electrophoresed so that one band was 20 ng. Fig. 2 shows the relationship between the electrophoresis image and the antibacterial activity.
It was shown to. The numbers displayed above each lane are the same as the numbers of the fractions in FIG. 1, and the antimicrobial strength is also displayed in accordance with FIG. A close look at the protein bands that are thought to be linked to antimicrobial activity identified two bands at approximately 50 kD and approximately 15 kD as promising candidates (arrows in FIG. 2). Since the concentrations of these two bands and the degree of antibacterial activity were positively correlated, it was strongly suggested that these bands may be the main protein having antibacterial activity. Molecular weight marker (67
kD albumin), the amount of antibacterial protein was estimated using an imaging densitometer (model GS-670, manufactured by BIO-RAD), and the complete growth inhibitory concentration against blast fungus was calculated to be approximately 20 ng protein / ml antibacterial assay. It became a medium.

3) Estimation of molecular weight by gel filtration column Next, in order to estimate the approximate molecular weight of the matsutake antibacterial protein in a native state, it is necessary to estimate the approximate molecular weight of 50 kD and 15 kD.
To confirm whether only two bands are involved in antibacterial activity, and to estimate whether these two bands are subunits of one protein complex, the above MonoQ image was used. Minute # 39 for Centricut V-
20 and concentrated on a gel filtration column Superose6 HR 10/30 (Phar
macia). The flow rate was 0.5 ml / min, and the buffer used was the above basic buffer. First, the Gel filtration stand
After grasping the molecular weight of the protein and the approximate elution time with ard (manufactured by BIO-RAD), a MonoQ fraction having antibacterial activity was injected. As a result, when the protein was monitored by A280, a sharp peak having a peak at 210 kD and a relatively gentle peak having a peak at 22 kD appeared (FIG. 3). The antibacterial activity is concentrated around the peak of 210 kD and before and after it, and the total antibacterial activity almost coincides with the antibacterial activity of the added MonoQ fraction.Therefore, the antibacterial activity of Matsutake is approximately 210 kD by gel filtration. From a single protein having a molecular weight of In addition, each fraction (0.25 m
When 20 μl of 1) was subjected to silver staining after SDS-PAGE, bands of 50 kD and 15 kD observed in 2) were detected only at around 210 kD (FIG. 4). Antimicrobial activity (+++) of MonoQ fraction # 39 (5 μl) migrated adjacently, antimicrobial activity of fractions # 28- # 32 detected by Superose6 and their fractions What contributes to antibacterial properties from the minute band pattern is
Again, it was strongly suggested here that they were 50 kD and 15 kD. In a gel filtration column that separates proteins based on their molecular weight, both 50kD and 15kD bands form antimicrobial protein subunits because both bands have the same antibacterial activity. It has been suggested. The main band was not seen in other fractions including 22kD.

4) Decoding of Amino Acid Sequence of Matsutake Protein The above MonoQ fractions # 39 to # 44 were concentrated with Centricut V-20 (manufactured by Kurabo Industries) and subjected to SDS-PAGE electrophoresis. Tris is removed, transferred to a PVDF membrane (Millipore) using a buffer system containing no glycine, and Coomassie Brilliant Blue R-250 is transferred.
After destaining, was destained. Thereafter, 50 kD and 15 kD protein bands considered to be involved in antibacterial activity were cut out. The N-terminal amino acid sequence was determined by Edman degradation using a gas phase protein sequencer (HP G1005A Protein Sequencing System).

As a result, from the 50 kD protein, the following 32 amino acids (SEQ ID NO: 3) N-terminal-HEIVHYTDVFIAGSG
PISXTYARHIIDNTSTT-C-terminal, and the following 67 amino acids (SEQ ID NO: 4) N-terminal-RE from the 15 kD protein
WEAGVTDT YGMPQPTFHV KRTNADGDRD QRMMNDMTNV ANML
GGYLPG SYPQFMAPGL VLHITGT-C-terminus was determined.

These amino acid sequences are stored in the database DD.
When a homology search was performed on BJ by BLAST, it showed homology to pyranose oxidase of Coriorus versicolor . This enzyme produces hydrogen peroxide as a by-product in the process of oxidizing glucose to glucosone. This suggests that the antibacterial activity we detected may be directly attributable to hydrogen peroxide generated by the enzyme from glucose contained in the culture medium.

Example 3: Measurement of pyranose oxidase activity The pyranose oxidase activity of each fraction from the above-mentioned ion exchange column and gel filtration column was measured. Activity measurement
According to the method of Machida and Nakanishi (1984), the determination was performed by determining the amount of H ₂ O ₂ produced with the oxidation of glucose. That is, 4 μmol of Tris was contained in a total volume of 120 μl reaction solution.
-HCl buffer (pH 7.0), 0.08 μmol of 4-aminoantipyrine, 0.08 μmol of phenol, 0.16 U of peroxidase (Type II, manufactured by SIGMA), 4 μmol of glucose,
And an enzyme solution were added, incubated at 37 ° C. for 10 minutes, and H ₂ O ₂ production was performed by measuring the increase in absorbance at 500 nm.
One unit (1 U) of the enzyme activity was defined as the activity of producing 1 μmol of H ₂ O ₂ per minute from glucose at pH 7.0 and 37 ° C.

As a result, in the fractionation by MonoQ, pyranose oxidase activity started to be detected from a salt concentration of 350 mM (fraction No. 38), and a salt concentration of 400 mM (fraction No. 40).
Then, the activity became maximum (0.66 U / ml), and then the activity decreased. The strength of the pyranose oxidase activity in each fraction and the strength of the antibacterial activity in that fraction were in excellent agreement (FIG. 1). In the fractionation by Superose 6, the activity value was maximum at a molecular weight of about 210 kDa, which was also in good agreement with the strength of the antibacterial activity (FIG. 3).

From the above, it was considered that the strong antibacterial activity against the rice blast fungus was caused by the pyranose oxidase activity of Matsutake. The mechanism of action was presumed to be that glucose in the medium by pyranose oxidase and hydrogen peroxide generated during the oxidation of other aldopyranose would damage blast cells.

Example 4: Analysis of enzymatic properties of pyranose oxidase derived from Matsutake: Using the fractions Nos. 42 to 44 obtained by MonoQ column, specific activity and substrate specificity of Matsutake pyranose oxidase were determined. Properties, Km, optimal temperature, temperature stability, optimal p
Investigations were made on H, pH stability, and freeze-thaw resistance. Pyranose oxidase (manufactured by Takara Shuzo) of Polyporus obtusus , a kind of basidiomycete, was used as a control. The amount of enzyme in the reaction mixture was 20 ng (0.52 mg) of Matsutake pyranose oxidase.
mU), Pyranose oxidase from Polyporus obtusus is 80
ng (0.91 mU).

1) Specific activity A part of the fraction of fraction No. 42 on the MonoQ column and a molecular weight marker (LMW: manufactured by Pharmacia LKB) whose amount was adjusted so that one band was 1 μg were subjected to SDS -PAGE electrophoresis, staining with Coomassie Brilliant Blue (CBB) R-250, and destaining. 50 kD from the concentration of the marker band
The a and 15 kDa bands were quantified using an imaging densitometer (model GS-670, manufactured by BIO-RAD).
Next, the pyranose oxidase activity of fraction No. 42 was measured, and the specific activity was calculated. As a result, the specific activity of Matsutake pyranose oxidase was calculated to be 25.8 U / mg protein.

In the same manner, the amount of pyranose oxidase of commercially available Polyporus obtusus was quantified, and the specific activity was calculated to be 11.3 U / mg protein. This value was in good agreement with the activity of 11.7 U / mg protein described in the data sheet attached to the enzyme. From this, it became clear that the pyranose oxidase of Matsutake has a specific activity which is about twice as high as that of Polyporus obtusus .

2) Substrate specificity The activity was measured when a total of 14 sugars were used as substrates, including glucose. The concentration of each sugar was 33 mM, and the activity of each was shown as a relative value with that of glucose being 100 (Table 3). As a result, Matsutake pyranose oxidase was found to use 1,5-anhydroglucitol as a good substrate in addition to glucose, and also used sorbose, xylose, trehalose, galactose, and maltose as substrates. Compared with pyranose oxidase of Polyporus obtusus , matsutake pyranose oxidase was particularly active against 1,5-anhydroglucitol (about 1.6 times).

[0067]

Table 3: Table specific relative activity of Matsutake pyranose oxidase (%) Matsutake Polyporus obtusus substrate (33 mM) Pyranose oxidase Pyranose oxidase D (+)-glucose 100 100 1,5-anhydro-D-glucitol 52.4 32.6 L (-)-Sorbose 24.1 25 D (+)-xylose 12.9 14.5 trehalose 8.8 15.8 D (+)-galactose 3.2 0.5 maltose 3.1 4.4 mannose 0 2.9 mannitol 0 0 D (-)-fructose 0 0 D-sorbitol 0 0 sucrose 0 0 D (-)-arabinose 0 0 N-acetyl-D-glucosamine 0 0

3) Km Next, for glucose and 1,5-anhydroglucitol
The Km value was determined. The enzyme activity was measured for glucose at 9 points in the range of 0.04 mM to 100 mM and for 1,5-anhydroglucitol at 10 points in the range of 0.1 mM to 300 mM. Table 4 shows the results. The Km of Matsutake pyranose oxidase for glucose was calculated to be 1.28 mM, and the Km for 1,5-anhydroglucitol was calculated to be 10.7 mM. On the other hand , Polyporus obtusus
The Km for pyranose oxidase of glucose is 1.
Km for 18 mM, 1,5-anhydroglucitol is 15.1 m
M. Although 1,5-anhydroglucitol has recently attracted attention as a diagnostic marker for diabetes, matsutake pyranose oxidase has been used not only for glucose measurement but also for enzymatic measurement of 1,5-anhydroglucitol. Has also been shown to be particularly effective.

4) Optimum temperature and temperature stability Pyranose oxidase activity was measured in a temperature range of 30 ° C. to 70 ° C. (reaction at each temperature for 10 minutes). as a result,
The optimal temperature of Matsutake pyranose oxidase was found to be around 50 ° C. (FIG. 5). Further, the enzyme had an enzyme activity of 80% or more in the range of 40 ° C to 60 ° C.

Next, the temperature stability (temperature resistance) was investigated.
In a temperature range of 30 ° C. to 70 ° C., the enzyme is incubated at 0.1 M Tris-HCl pH 7.0 for 30 minutes at each temperature, and then reacted in a reaction solution containing a substrate or the like at 37 ° C. for 10 minutes to obtain a residual enzyme. Activity was measured. As a result, it was found that the resistance temperature of Matsutake pyranose oxidase was around 50 ° C. (FIG. 6). It showed a residual activity of 95% or more up to 50 ° C.
It showed about half the activity at 60 ° C. and 30-40% at higher temperatures. The optimum temperature of Pyranose oxidase of Polyporus obtusus was 55 ° C, and the temperature stability was 50 ° C.

5) Optimum pH and pH Stability Pyranose oxidase activity was measured in the range of pH 3.5 to 11 (reaction at 37 ° C. for 10 minutes). As a result, it was found that the optimal pH of Matsutake pyranose oxidase was around 7.5 to 8.0 (FIG. 7). In addition, it had an enzyme activity of 80% or more in the pH range of 6.5 to 9.0.

Next, the pH stability (pH tolerance) was examined.
After keeping the enzyme at 50 ° C. for 30 minutes at each pH in the range of pH 3.5 to 11, the enzyme was reacted at 37 ° C. for 10 minutes in a reaction solution containing a substrate and the like, and the residual enzyme activity was measured. As a result, it was found that the stable pH region (remaining activity of 80% or more) of Matsutake pyranose oxidase was pH 7.0 to 11.0 (FIG. 8). The optimal pH of Pyranose oxidase of Polyporus obtusus was 7.0 to 7.5, and the stable pH was 7.0 to 11.0.

The salt concentration of the buffer solution was 33 m when the optimum pH was measured.
M, 100 mM at the time of pH stability analysis, according to each pH,
Sodium acetate-acetic acid buffer (pH 3.5-5.5), scalpel-sodium hydroxide buffer (pH 5.5-7.0) Hepes-sodium hydroxide buffer (pH 7.0-8.0), Tris-hydrochloric acid buffer (pH
8.0-9.5), CAP-sodium hydroxide buffer (pH9.5
111.0).

6) Freezing and thawing resistance The enzyme was treated in glycerol-free 0.1 M Tris-HCl pH 7.0 at -20 ° C for 2 hours, frozen, and then the remaining activity after thawing was examined. As a result, the residual activity of matsutake pyranose oxidase and the pyranose oxidase of Polyporus obtusus was 100% less than the activity before freeze-thawing.
Were 78% and 44%, respectively. Table 4 summarizes the enzymatic properties of the above Matsutake pyranose oxidase. As a control, the experimental results of pyranose oxidase of Polyporus obtusus are also described.

[0075]

[Table 4] Table 4: Matsutake andPolyporus obtususEnzymological Properties of Pyranose Oxidase in Rice Enzyme Protein Specific ActivityKm (mM) (U / mg protein) Glucose 1,5-anhydroglucitol Matsutake pyranose oxidase 25.8 1.28 10.7Polyporus obtusus Pyranose oxidase 11.3 1.18 15.1  Optimum temperature Temperature resistance Optimum pH Stable pH Freezing resistance(℃) (℃) Matsutake pyranose oxidase 50 50 7.5-8.0 7.0-11.0 78%Polyporus obtusus Pyranose oxidase 55 50 7.0-7.5 7.0-11.0 44%

[0076] Matsutake pyranose oxidase, Poly
Compared to porus obtusus pyranose oxidase, Km for glucose, temperature stability, and pH stability show almost the same value, but Km for 1,5-anhydroglucitol
m, and freeze-thaw stability. The specific activity is high, about twice Polyporus obtusus, also described earlier Corio
rus versicolor pyranose oxidase (Nish
Compared with imura et al. (1996) J. Biotechnol. 52: 11-20), it was found that the specific activity was about twice as high.

Example 5: Isolation of cDNA 1) Design of degenerate primer Based on the amino acid sequence determined in 4) of Example 2, a primer in which all possible bases were mixed was synthesized (Tm). Is 50-54 ° C., and the number in parentheses indicates degeneracy). 2 from 50kD protein (decoding 33 amino acids)
One primer, MT50R1 (5'-caytwyacigaygtittyathgc
-3 '(96)) (SEQ ID NO: 5) and MT50F1 (5'-gtigaigt
rttrtcdatdatrtg-3 '(72)) (SEQ ID NO: 6) was converted from a 15 kD protein (67 amino acids) by three primers.
MT15R1 (5'-acigayacitayggiatgcc-3 '(4)) (SEQ ID NO: 7), MT15F-1 (5'-crttigtcatrtcrttcatca-3' (8))
(SEQ ID NO: 8) and MT15F-2 (5′-aaicciggigccatra
aytg-3 ′ (4)) (SEQ ID NO: 9) was synthesized (where i is inosine, y is c or t, r is a or g, w is a or t, h is c or a or t, d Represents g or a or t, respectively).

2) Construction of a cDNA library of matsutake fruit body. All nucleic acids were extracted from matsutake fruit body by SDS phenol method, and total RNA was recovered by precipitation with lithium chloride. From this, Matsutake mRNA was prepared using an mRNA purification kit (Pharmacia). About 5 g to 10 μg of mRNA was obtained from fruiting bodies. 5 μg of this was used for ZAP cDNA synthes
A test was performed with is kit (manufactured by Stratagene) to synthesize cDNA. A 1-5 kb cDNA was fractionated by a gel filtration column, ligated to Uni-ZAP XR vector (Stratagene), and packaged with GigapackIII (Stratagene). All procedures followed the instructions attached to the kit. The titer of the constructed Matsutake cDNA library was calculated to be about 1 million pfu.

3) Acquisition of probe by RT-PCR cD synthesized in 2) using the primer synthesized in 1)
PCR was performed using NA as a template, and an attempt was made to amplify a partial-length cDNA of Matsutake protein, which serves as a probe for screening a library. The reaction conditions were as follows. In a 50 μl reaction, 60 ng of cDNA, 10X Extaq
buffer 5 μl, dNTPs 4 μl, primer 10 pmoles / each sequence, Ex taq (Takara) + Taq START antibody (Clo
ntech) (1 μl each), using the program temp control system PC-700 (ASTEK)
One cycle at 4 ° C. for 3 minutes was performed, then 40 cycles of 94 ° C. for 1 minute, 50 ° C. for 1 minute and 72 ° C. for 2 minutes, and finally, 72 ° C. for 6 minutes. . As a result, 1.3 kb and 1.4 kb products were amplified in the primer combinations MT50R1-15F1 and MT50R1-15F2, respectively. Among these, a 1.3 kb fragment by a combination of 50R1-15F1 having higher amplification efficiency was gel-purified and cloned into a vector pCRII (Invitrogen). As a result of determining the nucleotide sequences of six clones having slightly different sizes, two clones were very similar (92 to 98% homology) to the two amino acid sequences determined in Example 2, 4). Gender) sequences were both included.
This proved that the obtained clone had a sequence encoding the purified protein. It was also suggested that the purified 50 kD and 15 kD antimicrobial proteins were produced in this order from one precursor protein containing both peptides.

4) Estimation of copy number by genomic Southern analysis Total DNA was extracted from Matsutake mushroom, 5 μg of DNA was digested with 6 kinds of restriction enzymes, fractionated by agarose gel electrophoresis, and then Zeta-probe blotting membrane ( (Manufactured by BIO-RAD). 3) Cloning R
Redi-prime labeling kit (Amarsh
Am product) and used as a probe for genomic Southern analysis. Hybridization,
The washing conditions were in accordance with the instructions attached to the membrane. As a result, one band was detected in EcoRI and XhoI digestion, two bands were detected in BamHI and PstI digestion, and three bands were detected in HindIII digestion. In addition, a smear band was detected on the polymer side in the SalI digestion (FIG. 9). From this, it became clear that the gene encoding the matsutake protein had at least one copy and at most about three copies in the matsutake genome. EcoR
Since the concentration of band I is high, this is considered to be doublet, and the Matsutake pyranose oxidase gene is
It was thought that there were probably two copies in the Matsutake genome.

5) Screening of full-length cDNA The clone obtained in 3) was excised from a vector, and this was used as a probe to screen a Matsutake cDNA library constructed in 2). According to the description of ZAP cDNA synthesis kit (manufactured by Stratagene) in a 14 × 10 cm square petri dish, about 15,000 pfu of phage
Plated with 1-blue MRF '. H Plaque
It was brought into contact with a ybond-N + nylon membrane filter (manufactured by Amersham) and subjected to an alkali treatment according to the instructions attached to the membrane to denature the DNA and immobilized on the membrane. Hybridization and washing conditions were performed under high stringency conditions according to the instructions attached to the Zeta-probe membrane. In the primary screening, about 40,000 positive clones were obtained from about 120,000 pfu of phage. Secondary screening and tertiary screening combined with plaque purification were performed on 16 of the clones, and ZAP cDNA synthesis was performed on all 16 clones.
In vivo exci according to the instructions of the kit (Stratagene)
sion. As a result, five (# 1, 4, 5, 11,
The clone 14) is a phagemid vector pBluescrip
It was recovered as cDNA incorporated into tSK. The length of these clones was 1.7-2.3 kb, and restriction enzyme analysis suggested that they were derived from very similar genes.

Example 6 Determination of Nucleotide Sequence Approximately 500 bp of the 5 ′ nucleotide sequence was determined for the above five cDNA clones. Genetyx ver.9.0 analysis software (Software Development
(Manufactured by the company). As a result, # 1 (2.0 kb) and 1
4 (1.9 kb) are from the same gene, suggesting that # 5 (2.3 kb) is different from this group. That is, # 5
The base sequence on the 5 ′ side of 50 has the 50 determined in Example 2, 4).
A sequence very similar to 31 amino acids of kD (of 31 amino acids
(Coincidence is 29 amino acids), but the translation initiation codon does not appear in the upstream region in the reading frame, the translation stop codon appears continuously, and the first translation initiation codon is the same as in Example 2. It first appeared after the DNA sequence encoding 31 amino acids determined in 4).

On the other hand, in clones # 1 and 14,
It contains a sequence very similar to the 31 amino acids determined in 4) of Example 2 (29 amino acids are identical in 31 amino acids), and the translation initiation codon ATG is located 25 amino acids upstream from the N-terminal of 31 amino acids in its reading frame. Existed. 27
The presence of the translation termination codon TAA upstream of bp (same reading frame) strongly suggested that this ATG was the translation initiation site. For # 4 (1.8 kb) and 11 (1.7 kb)
Although the length of the cDNA insert sequence was short and both clones retained a part of the 31 amino acids of 50 kD, grouping was difficult due to the absence of a region upstream of which there was a difference. .

In the N-terminal sequence of 50 kDa in 4) of Example 2, the only 19th amino acid that was not determined was:
From the cDNA sequence corresponding to that part, cysteine (Cy
s). Since cysteine could not be analyzed by Edman degradation, it would have been undetectable during amino acid sequencing.

Next, when the base sequence of about 500 bp on the 3 ′ side was compared for the five clones, the base sequences were almost identical for all the clones, and were determined in Example 2-4).
A sequence very similar to the 67 amino acids of kD (of 67 amino acids
(63 amino acids matched) was conserved in all five clones. In addition, there is a difference between clones in the poly A addition site, and when starting from that of # 1, # 4, 5, 1
1 was 10 bases upstream and # 14 was 17 bases downstream.

In the clones # 1 and # 14, and the clone # 5 having a different primary structure of the gene, 1)
N-terminal 32 amino acids of 50kDa determined in the above and N-terminal of 15kDa
The nucleotide sequence corresponding to 67 amino acids was all conserved. In particular, the N-terminal 67 amino acids of 15 kDa
It was conserved in all three cDNA clones. That is, the positions and types of minor amino acid sequence differences between the amino acid sequence determined directly from the purified protein by amino acid sequencing and the amino acid sequence predicted from the cDNA were the same in all clones. .

The reason why the amino acid sequence determined in 4) of Example 2 does not completely match the amino acid sequence encoded by the cDNA corresponding to the portion is that a clone derived from a similar isozyme gene is used. Was isolated from the matsutake sample used to purify the protein and determine the amino acid sequence, and
It is thought that there is a high possibility that this is caused by a subtle variety of matsutake mushroom samples used when A was purified and a cDNA library was constructed, or a difference in ecotype. However, the amino acid sequence encoded by the isolated cDNA clone is:
Very similar to the amino acids determined by amino acid sequencing (93-94%),
It goes without saying that a protein obtained by expression of DNA also has a high possibility of having the same activity and various enzymatic properties as the above-mentioned purified protein.

Based on the above results, it was considered that all the information of Matsutake pyranose oxidase was encoded by the # 1 and # 14 groups, and the entire nucleotide sequence of the larger # 1 clone was determined. First, a 2.0 kb clone was cloned into the vector pBlues using several internal restriction sites.
cript and subcloned into the ABI PRISM fluorescence sequencer (Model 310 Genetic
Analyzer, Perkin Elmer). In addition, the surrounding sequence including the restriction site was synthesized with a primer based on the internal base sequence and used for sequencing. As described above, the sequences of both strands of the DNA were decoded, and the entire base sequence was determined (FIG. 10). As a result, the cDNA encoding Matsutake pyranose oxidase consisted of 1946 bases in total and encoded 564 amino acids (SEQ ID NOS: 1 and 2 in the Sequence Listing). The molecular weight estimated from the amino acid sequence was calculated to be about 61.9 kD, and the isoelectric point was calculated to be 5.68. In addition, two putative glycosylation sites (amino acids 53 to 56 in SEQ ID NO: 2 and N in 287 to 290 in SEQ ID NO: 2) were present.

When a region corresponding to the N-terminal amino acid determined from the purified protein was searched, the 50 kD N-terminal 32
26 to 57 of the amino acid sequence of SEQ ID NO: 2 in the sequence listing
The 15th amino acid sequence has 67 amino acids at the N-terminal
The amino acids corresponded to the amino acids 436 to 502, respectively. In addition, of the amino acid sequence of SEQ ID NO: 2,
Calculating the molecular weight of the 435th amino acid sequence and the 436th to 564th amino acid sequences,
The values were 45.3 kD and 13.8 kD, which were very similar to the approximate molecular weight estimated from SDS-PAGE. This indicates that Matsutake pyranose oxidase is first translated as a precursor form in Matsutake cells, then the N-terminal leader sequence (25 amino acids) is cleaved in the appropriate intracellular organ, and cleavage between amino acids 435 and 436 occurs. The resulting two polypeptides, approximately 50 kD and 15 kD, were presumed to associate with each other.

From the above results, the cloned cDN
It was concluded that A was derived from the gene encoding Matsutake pyranose oxidase. Complete amino acid sequence of pyranose oxidase from Matsutake of the present invention and total DN thereof
When the homology search (BLAST) was performed on the sequence A against the database (DDBJ), the sequence A was also found to be homologous to the pyranose oxidase of Coriolus versicolor. Therefore, Genetyx ver.9.0 analysis software (Software Develo
The homology search was performed using the following method. as a result,
The amino acid sequence showed 35% overall homology and the DNA sequence showed 46% overall homology. In addition, since there was no homologous sequence other than pyranose oxidase of Kawatake mushroom, this protein was considered to be a novel pyranose oxidase.

[0091]

According to the present invention, the sequence of SEQ ID NO: 2 in the sequence listing, the entire sequence excluding the 1st to 25th sequences, or the polypeptide consisting of the 26th to 435th amino acid sequences By pyranose oxidase, which comprises a polypeptide consisting of the 436th to 564th amino acid sequences, including glucose, sorbose and xylose, or trehalose,
Sugars such as galactose and maltose can be detected with a smaller amount of enzyme compared to conventional pyranose oxidase. Further, since the pyranose oxidase of the present invention has high activity also on 1,5-anhydroglucitol, it can be expected to be used as a diagnostic agent for diabetes.

Further, according to the present invention, of the DNA sequence of SEQ ID NO: 1 in the sequence listing, the DNA sequence is the 101st to 1792th sequence, or the 176th to 1792th DNA sequence. DNA characterized by being a sequence
The sequence, or the DNA sequence from 176 to 1405, the DNA sequence from 1406 to 1792, or both, can be amplified into E. coli, yeast, insects, or certain animal cells in each host. It is expected that the protein can be obtained in large quantities and at low cost by introducing and expressing it using a suitable expression vector.

[0093]

[Sequence List] SEQUENCE LISTING <110> JAPAN TOBACCO INC. <120> An agent for the determination of pyranose comprising a pyranose oxidase <130> 981883 <160> 9

<210> 1 <211> 1946 <212> DNA <213> Tricholoma matsutake <400> 1 cttccttcga tttctaggtc tcatattatt tgatacatgc cgcggcatag gggcaaagtc 60 acaggccagg acataaggtc agtcttaccc acctactacc atg ccg ata cgt Arg ccc atat cgt ccg ata cgt 115g gaa aaa atc aac gac ctg ctg caa cgt tct caa ggg gat ctt 163 Ser Lys Glu Lys Ile Asn Asp Leu Leu Gln Arg Ser Gln Gly Asp Leu 10 15 20 act tcc tcg caa gac gaa att gta cat tac act gat gtt ttc att gct 211 Thr Ser Ser Gln Asp Glu Ile Val His Tyr Thr Asp Val Phe Ile Ala 25 30 35 ggc agt ggt ccc att gcc tgt act tac gcc cgc cac atc att gac aat 259 Gly Ser Gly Pro Ile Ala Cys Thr Tyr Ala Arg His Ile Ile Asp Asn 40 45 50 acc tca act aca aag gtc tac atg gcc gaa ata ggt tct caa gat aac 307 Thr Ser Thr Thr Lys Val Tyr Met Ala Glu Ile Gly Ser Gln Asp Asn 55 60 65 cct gtc atc gga gcc cat cac agg aac tcc ata aag ttt cag aaa gac 355 Pro Val Ile Gly Ala His His Arg Asn Ser Ile Lys Phe Gln Lys Asp 70 75 80 85 act gac aag ttt gtg aat atc atc aac ggt gcc ctc cag ccc att tcg 403 Thr Asp Lys Phe Val Asn Ile Ile Asn Gly Ala Leu Gln Pro Ile Ser 90 95 100 att tcg cca tcg gac acc tac cag ccc act ctc gct gta gca gcg tgg 451 Ile Ser Pro Ser Asp Thr Tyr Gln Pro Thr Leu Ala Val Ala Ala Trp 105 110 115 gcg ccg ccc atc gat cct gcc gaa ggc cag ctc gtg att atg gga cac 499 Ala Pro Pro Ile Asp Pro Ala Glu Gly Gln Leu Val Ile Met Gly His 120 125 130 aat ccg aat cag gag gcc ggc ctg aac ctt ccc ggt agc gct gtc acg 547 Asn Pro Asn Gln Glu Ala Gly Leu Asn Leu Pro Gly Ser Ala Val Thr 135 140 145 agg aca gtc ggg gga atg gcg acc cac tgg act tgc gcg tgt cct act 595 Ar Thr Val Gly Gly Met Ala Thr His Trp Thr Cys Ala Cys Pro Thr 150 155 160 165 cca cat gac gaa gag agg gtc aac aac cca gtt gac aag cag gag ttc 643 Pro His Asp Glu Glu Arg Val Asn Asn Pro Val Asp Lys Gln Glu Phe 170 175 180 gac gca ctg ctc gaa cgt gct aaa aca ttg ctc aac gtt cac agc gac 691 Asp Ala Leu Leu Glu Arg Ala Lys Thr Leu Leu Asn Val His Ser Asp 185 190 195 cag tat gac gat tct atc cgt cag gt t gtc aaa gag acc ctt cag 739 Gln Tyr Asp Asp Ser Ile Arg Gln Ile Val Val Lys Glu Thr Leu Gln 200 205 210 cag acc ctt gat gcg tcg cgg ggt gtg acc act ctc ccg ctg ggg gtg 787 Gln Thr Leu Asp Ala Ser Arg Gly Val Thr Thr Leu Pro Leu Gly Val 215 220 225 gag cgc cgc acg gac aat cct att tat gtc acc tgg acc ggt gcc gat 835 Glu Arg Arg Thr Asp Asn Pro Ile Tyr Val Thr Trp Thr Gly Ala Asp 230 235 240 245 acc gtc ctt ggt gat gtg ccg aag agt ccc cga ttc gtt ttg gtt aca 883 Thr Val Leu Gly Asp Val Pro Lys Ser Pro Arg Phe Val Leu Val Thr 250 255 260 gag acg aga gtg acg aag ttt att gtc agt gaa acc aat ccg acg cag 931 Glu Thr Arg Val Thr Lys Phe Ile Val Ser Glu Thr Asn Pro Thr Gln 265 270 275 gtt gtt gct gcg ttg cta cgt aac ttg aat aca agc aac gat gaa ctt 979 Val Val Ala Ala Leu Leu Arg Asn Leu Asn Thr Ser Asn Asp Glu Leu 280 285 290 gtc gtg gcc cag agt ttc gtc ata gct tgt gga gca gtc tgc aca ccg 1027 Val Val Ala Gln Ser Phe Val Ile Ala Cys Gly Ala Val Cys Thr Pro 295 300 305 caa atc ctg tgg aac a ac atc cgc cca cat gcg ctt ggt cgc tac 1075 Gln Ile Leu Trp Asn Ser Asn Ile Arg Pro His Ala Leu Gly Arg Tyr 310 315 320 325 ctc agc gaa cag tcc atg act ttt tgt cag att gtt ctc aag agg agc 1123 Le Glu Gln Ser Met Thr Phe Cys Gln Ile Val Leu Lys Arg Ser 330 335 340 ata gtc gat tcc atc gct act gac cct cgc ttc gct gcg aag gtt gag 1171 Ile Val Asp Ser Ile Ala Thr Asp Pro Arg Phe Ala Ala Lys Val Glu 345 350 355 gcg cac aag aag aag cac ccc gat gac gtg ctg ccg att cca ttc cac 1219 Ala His Lys Lys Lys His Pro Asp Asp Val Leu Pro Ile Pro Phe His 360 365 370 gag cct gaa cct caa gtg atg att ccg tac tcg gac ttc cct tgg 1267 Glu Pro Glu Pro Gln Val Met Ile Pro Tyr Thr Ser Asp Phe Pro Trp 375 380 385 cat gtt cag gtc cat cgc tat gca ttt ggt gat gtt gga ccc aag gcc 1315 His Val Gln Val His Arg Tyr Ala Phe Gly Asp Val Gly Pro Lys Ala 390 395 400 405 gac ccg cgt gtt gtc gtc gat ctg agg ttt ttc ggc aaa tca gat att 1363 Asp Pro Arg Val Val Val Asp Leu Arg Phe Phe Gly Lys Ser Asp Ile 410 415 420 gt c gaa gaa aat cga gtg act ttc ggt ccg aac cct aag cta cgc gac 1411 Val Glu Glu Asn Arg Val Thr Phe Gly Pro Asn Pro Lys Leu Arg Asp 425 430 435 tgg gaa gcg ggt gtt aca gac act tat gga atg cca aca ttc 1459 Trp Glu Ala Gly Val Thr Asp Thr Tyr Gly Met Pro Gln Pro Thr Phe 440 445 450 cat gtc aag cgg acc aac gcc gat gga gac cgt gac cag agg atg atg 1507 His Val Lys Arg Thr Asn Ala Asp Gly Asp Arg Asp Gln Arg Met Met 455 460 465 aat gat atg acc aac gtc gcg aac ata ctg ggc ggg tac ctt cct ggc 1555 Asn Asp Met Thr Asn Val Ala Asn Ile Leu Gly Gly Tyr Leu Pro Gly 470 475 475 480 485 tcc tcc cca atg gca cct ggt ctc gca cag cac atc acg gga 1603 Ser Tyr Pro Gln Phe Met Ala Pro Gly Leu Ala Gln His Ile Thr Gly 490 495 500 act act cgg atc ggg aca gat gat caa act tct gtt gct gat ccg aca 1651 Thr Thr Arg Ile Gly Thr Asp Asp Gln Thr Ser Val Ala Asp Pro Thr 505 510 515 tca aag gtt cat aac ttc gac aat ctg tgg gtc ggc ggg aat ggg tgc 1699 520 525 525 530 att cca gat gcg act gcc tgc aac ccg act cgt ac agc gtc gcg tat 1747 Ile Pro Asp Ala Thr Ala Cys Asn Pro Thr Arg Thr Ser Val Ala Tyr 535 540 545 gcg ctt aag ggt gct gag gct gta gtc agt tac ctt ggc gtc tcc tgaaa 1797 Ala Leu Lys Gly Ala Glu Ala Val Ser Tyr Leu Gly Val Ser 550 555 560 gattctcgtg tcttgagagc gattcaagtt tgaaaagctg tacttaatat atctaggtcc 1857 tgacgttcgc attttaacat gtacttgtat ccgattgtttg tcttatgtga tgattttcaaa 1917 catatttactaaaaa

<210> 2 <211> 564 <212> PRT <213> Tricholoma matsutake <400> 2 Met Pro Ile Arg Leu Ser Lys Glu Lys Ile Asn Asp Leu Leu Gln Arg 1 5 10 15 Ser Gln Gly Asp Leu Thr Ser Ser Gln Asp Glu Ile Val His Tyr Thr 20 25 30 Asp Val Phe Ile Ala Gly Ser Gly Pro Ile Ala Cys Thr Tyr Ala Arg 35 40 45 His Ile Ile Asp Asn Thr Ser Thr Thr Lys Val Tyr Met Ala Glu Ile 50 55 60 Gly Ser Gln Asp Asn Pro Val Ile Gly Ala His His Arg Asn Ser Ile 65 70 75 80 Lys Phe Gln Lys Asp Thr Asp Lys Phe Val Asn Ile Ile Asn Gly Ala 85 90 95 Leu Gln Pro Ile Ser Ile Ser Pro Ser Asp Thr Tyr Gln Pro Thr Leu 100 105 110 Ala Val Ala Ala Trp Ala Pro Pro Ile Asp Pro Ala Glu Gly Gln Leu 115 120 125 Val Ile Met Gly His Asn Pro Asn Gln Glu Ala Gly Leu Asn Leu Pro 130 135 140 Gly Ser Ala Val Thr Arg Thr Val Gly Gly Met Ala Thr His Trp Thr 145 150 155 160 Cys Ala Cys Pro Thr Pro His Asp Glu Glu Arg Val Asn Asn Pro Val 165 170 175 Asp Lys Gln Glu Phe Asp Ala Leu Leu Glu Arg Ala Lys Thr Leu Leu 180 185 190 Asn Val His Ser Asp Gln Tyr Asp Asp Ser Ile Arg Gln Ile Val Val 195 200 205 Lys Glu Thr Leu Gln Gln Thr Leu Asp Ala Ser Arg Gly Val Thr Thr 210 215 220 Leu Pro Leu Gly Val Glu Arg Arg Thr Asp Asn Pro Ile Tyr Val Thr 225 230 235 240 Trp Thr Gly Ala Asp Thr Val Leu Gly Asp Val Pro Lys Ser Pro Arg 245 250 255 Phe Val Leu Val Thr Glu Thr Arg Val Thr Lys Phe Ile Val Ser Glu 260 265 270 Thr Asn Pro Thr Gln Val Val Ala Ala Leu Leu Arg Asn Leu Asn Thr 275 280 285 Ser Asn Asp Glu Leu Val Val Ala Gln Ser Phe Val Ile Ala Cys Gly 290 295 300 Ala Val Cys Thr Pro Gln Ile Leu Trp Asn Ser Asn Ile Arg Pro His 305 310 315 320 Ala Leu Gly Arg Tyr Leu Ser Glu Gln Ser Met Thr Phe Cys Gln Ile 325 330 335 Val Leu Lys Arg Ser Ile Val Asp Ser Ile Ala Thr Asp Pro Arg Phe 340 345 350 Ala Ala Lys Val Glu Ala His Lys Lys Lys His Pro Asp Asp Val Leu 355 360 365 Pro Ile Pro Phe His Glu Pro Glu Pro Gln Val Met Ile Pro Tyr Thr 370 375 380 Ser Asp Phe Pro Trp His Val Gln Val His Arg Tyr Ala Phe Gly Asp 385 390 395 395 400 Val Gly Pro Lys Ala Asp Pro Arg Val Val Val Asp Leu Arg Phe Phe 405 410 415 Gly Lys Ser Asp Ile Val Glu Glu Asn Arg Val Thr Phe Gly Pro Asn 420 425 430 Pro Lys Leu Arg Asp Trp Glu Ala Gly Val Thr Asp Thr Tyr Gly Met 435 440 445 Pro Gln Pro Thr Phe His Val Lys Arg Thr Asn Ala Asp Gly Asp Arg 450 455 460 Asp Gln Arg Met Met Asn Asp Met Thr Asn Val Ala Asn Ile Leu Gly 465 470 475 480 480 Gly Tyr Leu Pro Gly Ser Tyr Pro Gln Phe Met Ala Pro Gly Leu Ala 485 490 495 Gln His Ile Thr Gly Thr Thr Arg Ile Gly Thr Asp Asp Gln Thr Ser 500 505 510 Val Ala Asp Pro Thr Ser Lys Val His Asn Phe Asp Asn Leu Trp Val 515 520 525 Gly Gly Asn Gly Cys Ile Pro Asp Ala Thr Ala Cys Asn Pro Thr Arg 530 535 540 Thr Ser Val Ala Tyr Ala Leu Lys Gly Ala Glu Ala Val Val Ser Tyr 545 550 555 560 560 Leu Gly Val Ser

<210> 3 <211> 32 <212> PRT <213> Tricholoma matsutake <220><221> UNSURE <222> 19 <400> 3 His Glu Ile Val His Tyr Thr Asp Val Phe Ile Ala Gly Ser Gly Pro 5 10 15 Ile Ser Xaa Thr Tyr Ala Arg His Ile Ile Asp Asn Thr Ser Thr Thr 20 25 30

<210> 4 <211> 67 <212> PRT <213> Tricholoma matsutake <400> 4 Arg Glu Trp Glu Ala Gly Val Thr Asp Thr Tyr Gly Met Pro Gln Pro 5 10 15 Thr Phe His Val Lys Arg Thr Asn Ala Asp Gly Asp Arg Asp Gln Arg 20 25 30 Met Met Asn Asp Met Thr Asn Val Ala Asn Met Leu Gly Gly Tyr Leu 35 40 45 Pro Gly Ser Tyr Pro Gln Phe Met Ala Pro Gly Leu Val Leu His Ile 50 55 60 Thr Gly Thr 65

<210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <222> 9 and 15 <223> n represents i (inosine) <400> 5 caytwyacng aygtnttyat hgc 23

<210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <222> 3 and 6 <223> n represents i (inosine) <400> 6 gtngangtrt trtcdatdat rtg 23

<210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <222> 3, 9 and 15 <223> n represents i (inosine) <400> 7 acngayacnt ayggnatgcc 20

<210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <222> 5 <223> n represents i (inosine) <400> 8 crttngtcat rtcrttcatc a 21

<210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <222> 3, 6 and 9 <223> n represents i (inosine) <400> 9 aanccnggng ccatraaytg 20

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a chart of separation of Matsutake protein by MonoQ column, and shows a relationship between antibacterial activity and pyranose oxidase activity.

FIG. 2 shows the relationship between the electrophoretic image of separation of Matsutake protein using a MonoQ column and the antibacterial property. The numbers on the lanes correspond to the fraction numbers in FIG. 1, and M indicates a molecular weight marker. The symbols (-, +, ++, +++) below the lanes indicate the strength of the antibacterial activity. The proteins having antibacterial activity (50 kD and 15 kD) are indicated by arrows.

FIG. 3 shows a chart of purification of Matsutake protein by Superose 6, antibacterial properties, and a relationship with pyranose oxidase activity. The arrow is Gel filtration sta
The elution positions of protein samples of each molecular weight of ndard (BIO-RAD) are shown.

FIG. 4 shows the relationship between the electrophoresis image of the purification of Matsutake protein by Superose 6 and the antibacterial property. The numbers on the lanes correspond to the fraction numbers in FIG.
Indicates the 39th fraction of monoQ (see FIG. 4), and M indicates a molecular weight marker. The symbols under the lane (-, +, ++, ++
+) Indicates the strength of the antibacterial activity. The proteins having antibacterial activity (50 kD and 15 kD) are indicated by arrows.

FIG. 5 shows the results of analyzing the optimal temperature of Matsutake pyranose oxidase.

FIG. 6 shows the analysis results of the temperature stability of Matsutake pyranose oxidase.

FIG. 7 shows the results of analyzing the optimal pH of Matsutake pyranose oxidase. ▲ indicates 100 mM sodium acetate-acetic acid buffer (pH 3.5 to 5.5), □ indicates 100 mM female-sodium hydroxide buffer (pH 5.5 to 7.0), and ◆ indicates 100 mM Hepes-sodium hydroxide buffer (pH 7.0 to 5.5). 8.0), △ indicates 100 mM Tris-HCl buffer (pH 8.0 to 9.5), and 示 す indicates 100 mM Caps-sodium hydroxide buffer (pH 9.5 to 11.0).

FIG. 8 shows Matsutake pyranose oxidase p.
The analysis result of H stability is shown. ▲ is 100 mM sodium acetate-
Acetate buffer (pH 3.5-5.5), □ is 100 mM female-sodium hydroxide buffer (pH 5.5-7.0), ◆ is 100 mM Hepes-sodium hydroxide buffer (pH 7.0-8.0), △ is 100 mM Tris-HCl buffer (pH 8.0 to 9.5) indicates 100 mM Caps-sodium hydroxide buffer (pH 9.5 to 11.0), respectively.

FIG. 9 shows the results of Southern blot analysis of the gene encoding Matsutake pyranose oxidase on the Matsutake genome. X is XhoI, S is SalI, P is P
stI, H indicates HindIII, E indicates EcoRI, and B indicates BamHI.

FIG. 10 shows a restriction map of a cDNA encoding Matsutake pyranose oxidase and a method for determining a nucleotide sequence. Bold lines indicate cDNA, AAAA ...
Is shown. Restriction enzyme sites on the cDNA are indicated by bold lines (EcoRI and XhoI are cloning sites in the vector).
Each arrow indicates the direction and length of the region where the base sequence has been determined.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1: 645) (C12N 15/09 ZNA C12R 1: 645) (72) Inventor Shozo Ota Iwata, Shizuoka Prefecture 700 Toga, Toyota-cho, Gunma, Japan Tobacco Inc. Genetics Breeding Research Institute (72) Inventor Satoru Sami 700, Higashihara, Toyota-cho, Iwata-gun, Shizuoka Japan Tobacco Inc. Genetics Breeding Laboratory F-term (reference) 2G045 AA25 AA34 BB50 BB51 CB20 CB21 CB30 DA30 DA31 DA36 FB01 FB11 GC12 4B024 AA11 AA20 BA08 CA02 DA01 HA11 HA19 4B050 CC01 DD05 LL03 4B063 QA01 QA13 QA18 QA19 QQ03 QQ16 QQ44 QQ68 QR32 QR02 QR02 QR02

Claims (18)

[Claims] 1. It can be obtained from a fraction precipitated by an ammonium sulfate precipitation method from an aqueous extract of Matsutake mushroom, has at least an antibacterial activity against rice sheath blight or rice blast, and has a molecular weight of about 210 kD by a gel filtration method. , SDS-
The PAGE method shows the presence of components of about 50 kD and about 15 kD. The above antibacterial activity is maintained by heating at 60 ° C. for 10 minutes in a neutral aqueous solution, and the above antibacterial activity is obtained by heating at 80 ° C. for 10 minutes in a neutral aqueous solution. A reagent for measuring pyranose, comprising pyranose oxidase, which is inactivated. 2. The reagent for measuring pyranose according to claim 1, wherein the pyranose oxidase has the following enzymatic properties; (1) substrate specificity: D (+)-glucose, 1,5-anhydro-D- Glucitol, L (-)-sorbose,
Using D (+)-xylose, trehalose, D (+)-galactose and maltose as substrates, mannose, mannitol, D (-)-fructose, D-sorbitol, sucrose, D (-)-arabinose and N
-Acetyl-D-glucosamine is not used as a substrate; (2) optimum temperature: about 50 ° C. is the optimum temperature;
Enzyme activity of 80% or more is maintained in the range of 0 ° C. (3) Temperature stability: The residual activity after being kept at each temperature for 30 minutes is 95% or more and 55 to 6 at the temperature of 30 to 50 ° C.
About 50% at a temperature of 0 ° C, 30% at a temperature of 60 to 70 ° C
(4) optimum pH: pH 7.5 to 8.0 is the optimum pH, and the enzyme activity of 80% or more is maintained in the range of pH 6.5 to 9.0; (5) pH stability : 7.0 after stable incubation at 50 ° C. for 30 minutes at each pH where the remaining activity is 80% or more.
From 11.0; 3. An approximately 50 kD component of pyranose oxidase has an N-terminal amino acid sequence represented by SEQ ID NO: 3 in the sequence listing: His Glu Ile Val His Tyr Thr Asp Val Phe Ile Al
a Gly Ser Gly Pro Ile Ser Xaa Thr Tyr Ala Arg His
Ile Ile Asp Asn Thr Ser Thr Thr: about 15k
The component of D is the N-terminal amino acid sequence of SEQ ID NO: 4 in the sequence listing: Arg Glu Trp Glu Ala Gly Val Thr Asp Thr Tyr
Gly MetPro Gln Pro Thr Phe His Val Lys Arg Thr Asn
Ala Asp Gly Asp Arg Asp GlnArg Met Met Asn Asp Me
t Thr Asn Val Ala Asn Met Leu Gly Gly Tyr Leu ProG
ly Ser Tyr Pro Gln Phe Met Ala Pro Gly Leu Val Leu
3. The reagent for measuring pyranose according to claim 1, which has His Ile Thr Gly Thr. 4. An amino acid sequence wherein the pyranose oxidase is represented by SEQ ID NO: 2 in the sequence listing; an amino acid sequence having one or more amino acid mutations in this sequence; or an amino acid having 50% or more homology with this sequence The pyranose measuring reagent according to any one of claims 1 to 3, which has a sequence. 5. The reagent for measuring pyranose according to claim 4, wherein the pyranose oxidase has an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 6. The reagent for measuring pyranose according to claim 4, wherein the pyranose oxidase has an amino acid sequence having 70% or more homology with the amino acid sequence described in SEQ ID NO: 2 in the sequence listing. 7. The reagent for measuring pyranose according to claim 4, wherein the pyranose oxidase has an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 8. The reagent for measuring pyranose according to claim 4, wherein the pyranose oxidase has an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 9. The reagent for measuring pyranose according to claim 4, wherein the pyranose oxidase has an amino acid sequence having 95% or more homology with the amino acid sequence described in SEQ ID NO: 2 in the sequence listing. 10. A polypeptide consisting of the partial amino acid sequence 1-25 of SEQ ID NO: 2 in the sequence listing: partial amino acid sequence 2
Polypeptide consisting of 6-435: partial amino acid sequence 4
Polypeptide consisting of 36-564: polypeptide consisting of partial amino acid sequence 1-435: and polypeptide consisting of partial amino acid sequence 26-564: and having one or more amino acid mutations in any of the above amino acid sequences The polypeptide and any one of the above amino acid sequences and 5
A polypeptide having an amino acid sequence having 0% or more homology; and a pyranose oxidase comprising a pyranose oxidase alone or in combination with any one of the polypeptides. 11. Pyranose is D (+)-glucose, 1,5-anhydro-D-glucitol, L (-)
-Sorbose, D (+)-xylose, trehalose,
The reagent for measuring pyranose according to any one of claims 1 to 10, wherein the reagent is selected from D (+)-galactose or maltose. 12. The reagent for measuring pyranose according to claim 11, wherein the pyranose is D (+)-glucose. 13. The reagent for measuring pyranose according to any one of claims 1 to 12, which is a diagnostic agent for a disease accompanied by abnormal sugar metabolism. 14. The reagent for measuring pyranose according to claim 13, which is a diagnostic agent for diabetes. 15. The method according to claim 1, wherein the pyranose measuring reagent according to claim 1 is reacted with pyranose in the sample, and the generated hydrogen peroxide is reacted with an enzyme to form a color. Method for measuring pyranose. 16. A metabolic abnormality of saccharide, which comprises reacting the reagent for pyranose measurement according to claim 13 with pyranose in a sample collected from a subject, and reacting the produced hydrogen peroxide with an enzyme to develop color. Method for diagnosing associated diseases. 17. The diagnostic method according to claim 16, wherein the disease associated with abnormal sugar metabolism is diabetes. 18. A kit for analyzing pyranose or diagnosing a disease associated with abnormal sugar metabolism, comprising the reagent according to any one of claims 1 to 14.