JPH0889242A - Xylitol oxidase, method for producing the same, and method for measuring sugar alcohol - Google Patents

Abstract

(57) [Summary] [Structure] A xylitol oxidase having the ability to oxidize xylitol in the presence of oxygen to produce hydrogen peroxide and D-xylose. The xylitol oxidase is
A microorganism belonging to the genus Streptomyces can be cultured and collected from the culture. [Effect] The xylitol oxidase of the present invention directly oxidizes xylitol and produces hydrogen peroxide for which a highly sensitive colorimetric detection method compatible with a general-purpose automatic biochemical measuring device has been established. It is extremely useful as a clinical diagnostic agent for.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to xylitol oxidase, a method for producing the same and a method for measuring sugar alcohol.

[0002]

2. Description of the Related Art Xylitol is an intermediate metabolite of the uronic acid pathway, which is a metabolic pathway of pentose, and is also present in human blood, and its concentration is known to increase in liver disease. In addition, the xylitol oral tolerance test is expected to be useful for diagnosing liver diseases as it is useful for determining liver function, particularly severity and preliminary liver function (Nippon Rinjo 47).
Special number 450-452 (1989)).

Conventionally, as a method for measuring xylitol, a method of decomposing and removing sugar with alkali, oxidizing the remaining xylitol with periodic acid, and then chemically coloring with xylomotropic acid to detect it (medical science) Ayumi 78 420 (1
971)), gas chromatography (Anal. Chem. 37 1602
(1965)), liquid chromatography (Anal. Sci. 2 165 (1
986)), the enzyme method (clinical pathology 23 381 (1975)), etc. are known.

[0004]

However, the color detection method using chromotropic acid is complicated and has poor specificity, and the gas chromatographic method requires complicated derivatization, and the liquid chromatographic method has a special method. It is not suitable for routine analysis because it requires a device and has a problem of electrode contamination, and both methods are problematic as clinical analysis methods for xylitol.

On the other hand, the enzymatic method uses L-xylulose reductase (EC 1.1.1.10) and D-xylulose reductase (EC 1.1.1.9) extracted from guinea pig liver, and xylitol is added in the presence of the coenzyme NADP. Oxidize,
This is a method of measuring the absorbance (340 nm) of reduced NADPH with a spectrophotometer. However, this method is not only easy to obtain the enzyme but also has a problem in the stability of the enzyme, and the absorption derived from the protein is the absorbance measurement. However, there is a problem that a complicated pretreatment operation such as deproteinization operation is required to interfere with the above.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a xylitol oxidase highly useful as a diagnostic agent for liver diseases and a method for producing the same. Another object of the present invention is to provide a method for measuring sugar alcohol using xylitol oxidase.

[0007]

That is, the first aspect of the present invention
The second gist resides in xylitol oxidase having the ability to oxidize xylitol in the presence of oxygen to generate hydrogen peroxide and D-xylose, and the second gist belongs to the genus Streptomyces, which is a xylitol oxidase. A method for producing xylitol oxidase, which comprises culturing a microorganism capable of producing in a nutrient medium, allowing xylitol oxidase to be produced and accumulated in the culture, and collecting the xylitol oxidase. The third gist is D-sorbitol. ,
A sample containing any of xylitol, D-galactitol, D-mannitol, and D-arabinitol is caused to act on the above-mentioned xylitol oxidase, the amount of hydrogen peroxide produced, D-glucose produced, D-xylose, D -Detecting the amount of any of galactose, D-mannose, D-arabinose or the amount of oxygen consumed, the corresponding sugar alcohol, D-sorbitol,
The method for measuring sugar alcohols comprises measuring any of xylitol, D-galactitol, D-mannitol, and D-arabinitol.

The present invention will be described in detail below. First, a method for producing xylitol oxidase according to the second aspect of the present invention will be described. The microorganism belonging to the genus Streptomyces used in the production method of the present invention and having xylitol oxidase-producing ability,
For example, Streptomycess
p.) IKD 472 (Deposit No., FERMP-14339, Institute of Biotechnology, Institute of Biotechnology, AIST).
The above strains were isolated from the soil by the present inventors, and their mycological properties are as follows.

1. Morphological properties As a result of observation after 2 weeks at 37 ° C, the aerial hyphae are simply branched and their tips are loop-shaped or spiral-shaped. No formation of sporangia or crustal filaments is observed. Also, sporangia and zoospores are not found. The spores have a cylindrical shape, the size thereof is 0.6 to 0.8 × 0.9 to 1.4 μm, and the surface of the spores has a smooth or rough surface. Also,
Spores are formed in a chain of 10 or more.

2. Growth in each medium Table 1 shows the growth state after 2 weeks at 37 ° C on each medium.

[Table 1]

3. Physiological properties

[Table 2]

4. Utilization of carbon source (on Pridham-Godlieve agar)

[Table 3]

5. Diaminopimelic acid in the cell wall: LL
-Diaminopimelic acid.

From the above-mentioned various properties, this strain has an L cell wall.
L-diaminopimelic acid, and the type of menaquinone is
MK-9 (H _6), is MK-9 (H _8). And
According to the method of the International Streptomyces Project (abbreviation ISP), the morphology of sporulating hyphae belongs to Section Spirals.
The surface of the spores has a smooth or rough surface, the color of mature hyphae is red color-series, and the spores produce melanin-like pigments and show purple pigments in the medium.

The color of the basic hyphae is brown or purplish brown. As the carbon source, L-arabinose, D-glucose, D-fructose, D-xylose, inositol, L-rhamnose, D-mannitol are used,
Do not use sucrose or raffinose.

[0016] Based on the above properties,
Buffeynan's Manual of Determinative, edited by Buzzey's Manual of Determinative
Bacteriology) 8th edition, 1974 ", and as a result, it was found that the above-mentioned" IKD 472 strain "belongs to the genus Streptomyces. Therefore, this bacterium is Streptomyces sp. IKD
It was named 472 and has been deposited and stored as "FERM P-14339" from May 27, 1994 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

For culturing the bacterium used in the present invention, a medium used for culturing ordinary actinomycetes can be used, and it appropriately contains carbon sources, nitrogen sources, inorganic substances and other micronutrients required by the bacterium used. Any synthetic medium and natural medium can be used as long as they are used.

As the carbon source, glucose, sucrose, dextrin, starch, glycerin, molasses, etc. can be used. As the nitrogen source, inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate and ammonium phosphate, amino acids such as D-alanine and L-glutamic acid, nitrogen containing such as peptone, meat extract, yeast extract, malt extract and corn steep licker Natural products can be used. As the inorganic substance, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate,
Magnesium sulfate, ferric chloride, etc. can be used.

The culture is usually carried out by shaking culture or aeration-agitation culture. The culture temperature is 10 to 60 ° C., preferably 20 to 50.
° C. The pH of the medium is in the range of 3 to 10, preferably 5 to 7. The culture period is 1 to 7 days, and the culture is usually completed in 2 to 4 days. By culturing by such a method, xylitol oxidase can be produced and accumulated in the culture, particularly in the bacterial cells.

As a method for obtaining xylitol oxidase from the culture, a method used in ordinary protein purification is used. That is, first, after culturing a bacterium, the culture solution is filtered to obtain a microbial cell, and then the microbial cell is crushed by an appropriate method, and a supernatant is obtained from the disrupted solution by centrifugation or the like. The xylitol oxidase contained in the supernatant can be purified by combining appropriate purification operations such as salting out, dialysis, ion exchange chromatography, hydrophobic adsorption chromatography, gel filtration and electrophoresis.

The enzymatic activity of the xylitol oxidase of the present invention can be measured by the following two methods.

[Activity measuring method 1] 1 mL of 100 mM potassium phosphate buffer (pH 7.5), 0.1 mL of 25 mM 4-aminoantipyrine, 0.1 mM of 420 mM phenol solution
L, 100 units / mL horseradish peroxidase 0.
1 mL, 100 mM xylitol 1 mL, water 0.65 mL 3
Place in a mL quartz cell, set in a spectrophotometer with a constant temperature cell holder, incubate at 37 ° C for 5 minutes, add 0.05 mL of enzyme solution, stir, and measure the absorbance change (ΔABS / min) at 500 nm for 5 minutes. To do. The enzyme activity is calculated by the following formula. In the formula, n represents a dilution ratio.

[0023]

[Equation 1]

[Activity measuring method 2] To 40 μL of 100 mM potassium phosphate buffer (pH 7.5), 40 μL of 100 mM xylitol and 35 μL of water, 5 μL of the enzyme solution was added and stirred, and after reacting at 40 ° C. for 40 minutes , At 100 ℃ 3
The reaction is stopped by heat treatment for a minute. The obtained reaction solution is centrifuged, deproteinized through a filter, and the product in the reaction solution is analyzed under the conditions of high performance liquid chromatography (HPLC) shown in Table 4. The enzyme activity is defined as the amount of enzyme that produces 1 μM D-xylose per minute.

[0025]

[Table 4]

Next, the xylitol oxidase according to the first aspect of the present invention will be described.

(1) Action In the presence of oxygen, it catalyzes the following oxidation reaction of xylitol and D-sorbitol to produce hydrogen peroxide, D-xylose and D-glucose.

[Formula 1] xylitol + O ₂ ── → D- xylose + H ₂ O ₂ D- sorbitol + O ₂ ── → D- glucose + H ₂ O ₂

(2) Substrate Specificity and Substrate Affinity In the enzyme activity measuring method described above, various other sugar alcohols (each having a final concentration of 33 mM) were used in place of D-sorbitol.
The relative reactivity (substrate specificity) when is used is as shown in Table 5. In addition, glycerin, ethanol, mesoerythritol, myoinositol, D-glucose, D
-Xylose, D-galactose, D-mannose, D
-For fructose, L-sorbose and L-fucose,
There was virtually no reaction. The substrate affinity (Km) and maximum reaction rate (Vmax) for xylitol, D-sorbitol and D-galactitol are as shown in Table 5.

[0029]

[Table 5]

Conventionally, as an oxidase which oxidizes D-sorbitol to produce hydrogen peroxide, cochlea (Helix aspersa) is used.
Derived mannitol oxidase (Int. J. Biochem. 18
(4) 337-344 (1986)) and the microbial Xanthomonas (Xanthomo
nas) -derived sorbitol oxidase (Japanese Patent Application Laid-Open No. 6-1
69764) is known. Table 6 shows the reactivity of these known enzymes with respect to the substrate.

[0031]

[Table 6]

Mannitol oxidase from the cochlea is
Unlike the enzyme of the present invention, mainly D-arabinitol and D-
It oxidizes mannitol and hardly oxidizes xylitol. On the other hand, xanthomonas-derived sorbitol oxidase is similar to the present enzyme in the property of oxidizing D-sorbitol, xylitol, D-mannitol, and D-arabinitol, but is reactive to D-mannitol (substrate specificity) and D-sorbitol. Clearly differs from the enzyme of the invention in its affinity for.

The enzyme of the present invention has a higher substrate affinity for xylitol than D-sorbitol (the smaller the Km value is, the higher the affinity is), and reacts strongly with xylitol at a low substrate concentration, so it is classified as xylitol oxidase. To be done. Further, the enzyme of the present invention is produced by a microorganism of the genus Streptomyces different from sorbitol oxidase derived from Xanthomonas.

(3) Optimal pH and pH stability (measured by activity measuring method 2) Citrate buffer solution, potassium phosphate buffer solution, Tris / hydrochloric acid buffer solution, glycine buffer solution having various pH values shown in FIG. In the presence of (each buffer was 33 mM), xylitol was oxidized with a purified enzyme at 40 ° C. for 40 minutes. After the reaction,
The D-xylose concentration in the reaction solution was quantified using liquid chromatography, and the pH showing the maximum reactivity was determined. From FIG. 1, it can be seen that the optimum pH of the enzyme of the present invention is around 7.5.

Further, the purified enzyme was dissolved in citrate buffer solution, potassium phosphate buffer solution, Tris / hydrochloric acid buffer solution, glycine buffer solution (50 mM each) having various pH values shown in FIG. After the treatment at 30 ° C. for 30 minutes, the residual enzyme activity was measured. From FIG. 2, it can be seen that the stable pH range of the enzyme of the present invention is 5.5 to 10.5.

(4) Optimum temperature and thermal stability (Activity measuring method 2
(Measurement with) A purified enzyme was used, xylitol was used as a substrate, and the reaction temperature was changed to carry out an oxidation reaction for 40 minutes. After the reaction, the concentration of D-xylose in the reaction solution was quantified using liquid chromatography, and the temperature at which the maximum enzyme activity was exhibited was measured. From FIG. 3, it can be seen that the optimum temperature of the enzyme of the present invention is around 55 ° C. The purified enzyme was added to 20 mM phosphate buffer (pH 7.
After dissolving in 5) and treating at each temperature for 30 minutes, the residual enzyme activity was measured. From FIG. 4, it can be seen that the enzyme of the present invention is stable up to 65 ° C.

(5) Inhibitor The enzyme activity of the purified enzyme was measured by adding various additives to the system of enzyme activity measuring method 2 so that the concentration was 1 mM. With respect to the control without the additive, the inhibitory effects as shown in Tables 7 and 8 were recognized by the additive. It can be seen that the enzyme of the present invention is strongly inhibited by cupric chloride, mercuric chloride and silver nitrate.

[0038]

[Table 7]

[0039]

[Table 8]

(6) Molecular weight The molecular weight of this enzyme was about 4 as a result of measurement by gel filtration.
It was 3,000. Also, sodium dodecyl sulfate-
As a result of measurement by polyacrylamide gel electrophoresis, it was about 43,000 and showed a single band.

(7) Absorption spectrum A large absorption maximum near protein at 274 nm and 342
It has two absorption maxima near nm and 451 nm (see Fig. 5). Therefore, the absorption spectrum of the purified enzyme of the present invention is
It can be seen that it exhibits a typical flavin enzyme spectral pattern.

Next, the substances produced in the reaction solution when xylitol is added to the purified enzyme of the present invention and reacted are described.

The system of the above-mentioned activity measuring method 1, that is,
Phenol and 4 in the presence of horseradish peroxidase
Since a red quinone dye is produced by an oxidative condensation reaction formula with -aminoantipyrine, it was found that hydrogen peroxide was generated by the reaction of the enzyme of the present invention and xylitol.

As a result of adding pyranose oxidase acting on D-xylose to the above reaction solution and further reacting it, the color development intensity was doubled.

Using the purified enzyme of the present invention in an excess amount, 20
Xylitol was oxidized in mM potassium phosphate buffer (pH 7.5). The molar balance between the product in the reaction solution and the reaction was analyzed by HPLC (condition of activity measurement method 2). By the reaction, xylitol with a retention time of 14.6 minutes disappeared and a new reaction product was generated. In addition, the retention times of this reaction product and D-xylose match,
It was 9.0 minutes. The molar balance of the reaction was also close to the theoretical value.

From the facts (1) to (3) above, it is understood that the enzyme of the present invention catalyzes the following reaction.

Embedded image Xylitol + O ₂ ---> D-xylose + H ₂ O ₂

Next, a substance produced in the reaction solution by adding D-sorbitol to the purified enzyme of the present invention and reacting it will be described.

Since a red quinone dye is produced in a system similar to the method for measuring the enzyme activity of xylitol oxidase, it can be seen that hydrogen peroxide is generated by the reaction of the enzyme of the present invention and D-sorbitol.

Using the purified enzyme of the present invention, in a 33 mM potassium phosphate buffer (pH 7.5) at 40 ° C. for 1 hour, D-
The sorbitol was reacted. Before and after this reaction,
As a result of analyzing the sugar in the reaction solution under the TLC analysis conditions shown in Table 9, the raw material D-sorbitol disappeared, and a spot of the reaction product appeared at the same Rf value of 0.35 as glucose. In addition, this reaction product showed a gray-blue color reaction similar to that of glucose, which is peculiar to aldose.

[0050]

[Table 9]

In the above reaction solution, glucokinase and A
After phosphorylation by the action of TP, glucose-
As a result of reacting hexaphosphate dehydrogenase with NADP, NADPH was produced which was almost equal to the concentration of D-sorbitol added before the reaction with the purified enzyme of the present invention. Since the glucokinase is specific to D-glucose and the glucose-6-phosphate dehydrogenase is specific to D-glucose-6-phosphate, it can be seen that the product of this reaction is D-glucose.

HPLC shown in the above-mentioned activity measurement method 2
The molar balance between the product and the reaction in the above reaction solution was analyzed under the conditions. By the reaction, D-sorbitol with a retention time of 21.8 minutes disappeared and a new reaction product was generated. Further, the retention times of this reaction product and D-glucose were in agreement and were 8.6 minutes. The molar balance of the reaction was also close to the theoretical value.

From the above results (1) to (4), it is understood that the enzyme of the present invention catalyzes the following reaction.

## STR00003 ## D-sorbitol + O ₂ --- → D-glucose + H ₂ O ₂

Next, the use of the enzyme (xylitol oxidase) of the present invention will be described. Xylitol oxidase catalyzes the oxidation reaction of sugar alcohols such as D-sorbitol, xylitol, D-galactitol, D-mannitol and D-arabinitol in the presence of oxygen, and hydrogen peroxide and D corresponding to these sugar alcohols are catalyzed. An enzyme that produces sugars such as glucose, D-xylose, D-galactose, D-mannose, D-arabinose.
Therefore, the enzyme of the present invention can be used for any purpose as long as the change due to such reaction can be used.

An example of directly utilizing the reaction product is a method for producing sugar from sugar alcohol. Also,
Examples of indirectly using the reaction product include a method for measuring sugar alcohol clinically important as a diagnostic agent, particularly xylitol as an index of liver disease, D-sorbitol as an index of diabetes, and an index of fungal infection. Examples include application to arabinitol. In addition, as an application to a diagnostic agent or a measuring reagent, not only a substrate that directly reacts with the enzyme of the present invention is detected but also a sugar alcohol indirectly reactive with the enzyme of the present invention is produced. It can also be applied to a method for measuring a precursor substrate of alcohol.

Next, a method for measuring sugar alcohol according to the third aspect of the present invention will be described. Sugar alcohol D-sorbitol, xylitol, D as a diagnostic agent and measuring reagent
-A method for measuring galactitol, D-mannitol and D-arabinitol will be described.

The xylitol oxidase of the present invention is D
-Sorbitol, xylitol, D-galactitol,
Since it catalyzes the oxidation reaction of sugar alcohols such as D-mannitol and D-arabinitol, xylitol oxidase is allowed to act on a sample containing these, the amount of hydrogen peroxide produced, and D- produced respectively from the corresponding sugar alcohol. The amount of glucose, D-xylose, D-galactose, D-mannose, D-arabinose or the like, or the amount of oxygen consumed by the oxidation of these sugar alcohols is detected, and the corresponding sugar alcohol, D-sorbitol. , Xylitol, D-galactitol, D-
Mannitol and D-arabinitol can be measured. Therefore, the xylitol oxidase of the present invention is
It is highly useful as a diagnostic agent and measurement reagent.

Samples containing sugar alcohols such as D-sorbitol, xylitol, D-galactitol, D-mannitol and D-arabinitol include serum, plasma, whole blood, red blood cells, urine, spinal fluid, tissue extract, Examples include food. As a method for detecting hydrogen peroxide produced, various known methods can be used, for example, a colorimetric method, a fluorescence method,
A chemiluminescence method, an electrode method and the like can be mentioned.

In the colorimetric method, a catalyst such as peroxidase is used, the substrate of peroxidase is oxidized and colored with hydrogen peroxide, and the color density is measured by a spectrophotometer. Substrates for peroxidase include 0-phenylenediamine, 5-aminosalicylic acid, 4-aminoantipyrine and phenol, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), bis [3-bis (4- Chlorophenyl) methyl-4-dimethyl-aminophenyl] amine and the like can be used. For example, the above-mentioned “method for detecting enzyme activity” can be exemplified.

In the fluorescence method, a catalyst such as peroxidase is used to oxidize the substrate of peroxidase with hydrogen peroxide to produce a fluorescent substance, and the fluorescence intensity is measured with a fluorometer. As a substrate for peroxidase, p-hydroxyphenylacetic acid, p-hydroxyphenylpropionic acid, etc. can be used.

In the chemiluminescence method, a catalyst such as peroxidase is used, a substrate of peroxidase is oxidized with hydrogen peroxide to emit light, and the luminescence intensity is measured with a luminometer.
Luminol compounds, lucigenin, compounds of allyl oxalates and the like can be used as the chemiluminescent substrate.

Further, as a method of detecting consumed oxygen,
Various known methods can be used, and examples include a method using an oxygen electrode.

When measuring sugar alcohol as a substrate, about p
The sample containing the substrate and xylitol oxidase may be added to the buffer solution of H 5.5 to 10.5 and reacted. The xylitol oxidase can be used as a lyophilized product, a microencapsulated product, or an insolubilized product fixed to a carrier, if necessary, in addition to the solution state. As the buffer solution, a phosphate buffer solution, a Tris / hydrochloric acid buffer solution, a glycine buffer solution, or the like is used. Enzyme is usually 1-500μ
It is used at a concentration of 0.5 to 50 units / ml for the M concentration of the substrate.

The methods for measuring sugar alcohols are classified into two methods, that is, the rate method and the end point method in terms of reaction rate. In the present invention, any of them can be adopted, and these measuring methods are appropriately selected depending on conditions such as the measuring system, reaction time, concentration of sugar alcohol as a substrate, and type of sample. It is also possible to prepare a reagent containing the above-mentioned reagent components necessary for detecting xylitol oxidase and the sugar alcohol as a substrate, and assemble it into a diagnostic agent as a sugar alcohol measurement kit.

[0065]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist thereof.

Example 1 (Production of xylitol oxidase by a Streptomyces bacterium) Polypeptone: 1%, yeast extract: 0.5%, MgSO _4.
7H ₂ O: 0.05%, KH ₂ PO ₄ : 0.1%, p
100 mL of H 6.0 medium was added to a 500 mL culture flask, and after sterilization cooling at 120 ° C. for 20 minutes, one platinum loop inoculation of Streptomyces genus (MICRO deposit number FERM P-14339) was carried out, and A seed culture was prepared by shaking culture at 30 ° C. for 30 hours.

1.6 L of a medium prepared by adding 0.01 V / V% of the antifoaming agent Adecanol KG-126 (manufactured by Asahi Denka Co., Ltd.) to the same medium composition as described above was put in a 2 L jar fermenter.
After sterilizing and cooling at 20 ° C. for 20 minutes, the above seed culture solution 16 was added thereto.
mL was inoculated and cultured at 35 ° C. for 27 hours under the conditions of an aeration rate of 1.6 L / min and a stirring speed of 300 rpm to obtain a preculture liquid.

Next, in a 200 L jar fermenter, 160 L of the medium having the same composition as above was put, and 120 ° C.
After sterilizing and cooling at 30 ° C. for 30 minutes, inoculate 1.6 L of the above pre-cultured solution at 35 ° C. for 21.5 hours at an aeration rate of 160 L / min and
The culture was performed under the conditions of a stirring speed of 00 rpm. After the culture was completed, the bacterial cells were collected by centrifugation with a Sharpless.

Half the amount of the obtained bacterial cells was added to a 20 mM potassium phosphate buffer solution (pH 7.
After suspending in 5) so that the total amount became 3 L, the microbial cells were crushed by a dyno mill grinder. The disrupted solution was centrifuged to remove the bacterial cell residue to obtain 3065 mL of a crude enzyme solution.

Ammonium sulfate was added to the above crude enzyme solution so as to be 40% saturated, and the mixture was allowed to stand overnight. Then, the deposited precipitate was removed by centrifugation, and the obtained supernatant liquid was saturated to 60%. Ammonium sulfate was added, the mixture was left standing again for 5 hours, and a precipitate was obtained by centrifugation. 20 containing 0.1 mM dithiothreitol
The above precipitate was dissolved in mM potassium phosphate buffer (pH 7.5), and then desalted with a buffer having the same composition using a dialysis membrane.

493 mL of the desalted solution was preliminarily diluted with 20 mM potassium phosphate buffer (pH 7.
5) DEAE-Cellulofine A-500 equilibrated in 5)
The enzyme was adsorbed through a column (diameter 6.0 cm × length 18 cm), washed with a buffer solution having the same composition, and then a 100 mM potassium phosphate buffer solution (pH 7.0) containing 0.1 mM dithiothreitol.
Elution was carried out in 5), and the active fractions of the enzyme were collected.

Ammonium sulphate was added to the above active fraction to adjust it to a 30% saturation concentration (pH 7.5), and then 20 mM containing 30% saturated ammonium sulphate and 0.1 mM dithiothreitol in advance.
Butyl Toyopearl 650M column (diameter 6.0 cm x length 14) equilibrated with potassium phosphate buffer (pH 7.5)
cm) to adsorb the enzyme, and after washing with a buffer solution of the same composition, a 20 mM potassium phosphate buffer solution (pH 7.5) containing 30% saturated ammonium sulfate and 0.1 mM dithiothreitol was added to 0.1 mM dithio. Elution was performed with a linear gradient to 20 mM potassium phosphate buffer (pH 7.5) containing threitol, and the active fractions of the enzyme were collected.

Next, ammonium sulfate was added to the above-mentioned active fraction so as to be 60% saturated, the mixture was allowed to stand overnight, and then a precipitate was obtained by centrifugation. This precipitate was added to a 20 mM potassium phosphate buffer solution (pH 7.
5) dissolved in 4.5 mL, and pre-equilibrated with a 20 mM potassium phosphate buffer (pH 7.5) containing 0.2 M NaCl and 0.1 mM dithiothreitol to obtain a Sephacryl S-100HR column (diameter). 1.5 cm x 110 cm long)
And eluted with the same buffer to collect active fractions.

Ammonium sulfate was added to the above-mentioned active fraction so as to be 60% saturated, the mixture was allowed to stand overnight, and a precipitate was obtained by centrifugation. This precipitate was dissolved in 20 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM dithiothreitol to make 10 mL, and subjected to preparative polyacrylamide gel electrophoresis. After the electrophoresis, the gel was immersed in an enzyme reaction solution to stain the enzyme activity band, which was cut out and ground,
The enzyme was extracted by immersing it in a 20 mM potassium phosphate buffer solution (pH 7.5) containing 1 mM dithiothreitol.

Ammonium sulfate was added to the above-mentioned active fraction so as to be 60% saturated, and the mixture was allowed to stand overnight, followed by centrifugation to obtain a deposited precipitate. This precipitate was dissolved in 20 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM dithiothreitol to make 2.0 mL, and 0.2 M NaCl and 0.1 mM were prepared in advance.
Sephacryl S-10 equilibrated with 20 mM potassium phosphate buffer (pH 7.5) containing dithiothreitol
Pass through a 0HR column (diameter 1.5 cm x length 110 cm),
Elution was performed with the same buffer, and active fractions were collected.

Ammonium sulfate was added to the above active fraction so as to be 60% saturated, the mixture was allowed to stand overnight, and then a precipitate was obtained by centrifugation. The above precipitate was dissolved in a 20 mM potassium phosphate buffer solution (pH 7.5) containing 0.1 mM dithiothreitol and then desalted with a buffer solution having the same composition using a dialysis membrane.

0.9 mL of the desalted solution was preliminarily added to a 20 mM potassium phosphate buffer solution containing 0.1 mM dithiothreitol (pH 7.
DE for high performance liquid chromatography equilibrated in 5)
The enzyme was adsorbed through an AE-5PW column (diameter 1.5 cm x length 4 cm), washed with a buffer solution of the same composition, and then 0.1 mM.
Dithiothreitol containing 20 mM potassium phosphate buffer (pH 7.5) to 0.1 mM dithiothreitol containing 1
Elution was carried out with a linear gradient to 00 mM potassium phosphate buffer (pH 7.5), and the active fractions of the enzyme were collected. The resulting enzyme showed a single band on SDS electrophoresis.

[0078]

[Table 10]

Example 2 (Measurement of xylitol) 25 μL of xitol solution was added to 475 μL of 0.1 M phosphate buffer (pH 7.5) (first reagent) containing 1.5 mM 4-aminoantipyrine, and the mixture was added at 37 ° C. Incubated for 5 minutes to incubate. Next, xylitol oxidase 10U / mL, horseradish peroxidase 25U / mL
After addition of 100 μL of 0.1 M phosphate buffer (pH 7.5) (second reagent) containing 7.5 mM of 3-hydroxy-2,4,6-triiodobenzoic acid and incubating at 37 ° C. for 5 minutes, the color was developed. Absorbance at 515 nm was measured with a spectrophotometer. The measurement result of the calibration curve is shown in FIG. The calibration curve is
The linearity was obtained from 1 to 50 mg / L, and the measurement of xylitol was possible.

Example 3 (Measurement of D-sorbitol) 25 μL of a xitol solution was added to 475 μL of a 0.1 M phosphate buffer (pH 7.5) (first reagent) containing 1.5 mM 4-aminoantipyrine. The cells were incubated at 37 ° C for 5 minutes to incubate. Next, xylitol oxidase 30U / mL, horseradish peroxidase 25U / mL
After addition of 100 μL of 0.1 M phosphate buffer (pH 7.5) (second reagent) containing 7.5 mM of 3-hydroxy-2,4,6-triiodobenzoic acid and incubating at 37 ° C. for 5 minutes, the color was developed. Absorbance at 515 nm was measured with a spectrophotometer. The measurement result of the calibration curve is shown in FIG. The calibration curve is
It became a straight line from 1 to 50 mg / L, and it was possible to measure D-sorbitol.

[0081]

According to the present invention described above, a novel xylitol oxidase and a method for producing the same are provided. The xylitol oxidase of the present invention directly oxidizes xylitol to form a general-purpose automatic biochemical measuring device. Since it produces hydrogen peroxide for which a highly sensitive colorimetric detection method has been established, it is extremely useful as a clinical diagnostic agent for liver diseases and the like.

[Brief description of drawings]

FIG. 1 Optimum pH of xylitol oxidase of the present invention
It is a graph which shows.

FIG. 2 is a graph showing the pH stability of the xylitol oxidase of the present invention.

FIG. 3 is a graph showing the optimum temperature of xylitol oxidase of the present invention.

FIG. 4 is a graph showing the temperature stability of xylitol oxidase of the present invention.

FIG. 5 is an absorption spectrum of the xylitol oxidase of the present invention.

FIG. 6 is a calibration curve for xylitol measurement.

FIG. 7 is a calibration curve for D-sorbitol measurement.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Minor Masuda Minoru Masuda 84-3-1 Nishiageo Daiichi housing complex 845-1 Oshigiya, Ageo City, Saitama Prefecture (72) Inventor Takashi Harada 3-15-11 Haneda, Ota-ku, Tokyo ( 72) Inventor Masahiko Yabuuchi 1-40-5 Kotakemachi, Nerima-ku, Tokyo

Claims (5)

[Claims] 1. Oxidizing xylitol in the presence of oxygen,
Xylitol oxidase having the ability to produce hydrogen peroxide, D-xylose. 2. The xylitol oxidase according to claim 1, which has a higher substrate affinity for xylitol than D-sorbitol and a Km value for xylitol of 1 mM or less. 3. The xylitol oxidase according to claim 2, which has the following physicochemical properties. (1) Action: In the presence of oxygen, oxidize xylitol and D-sorbitol to form hydrogen peroxide, D-xylose and D
-Produce glucose. (2) Substrate specificity: D-sorbitol, xylitol,
Acts strongly on D-galactitol, D-mannitol,
It also acts on D-arabinitol. (3) Optimum pH and pH stability: The optimum pH is around 7.5, and the stable pH range is 5.5 to 10.5. (4) Optimum temperature and thermal stability: The optimum temperature is around 55 ° C, and it is stable up to 65 ° C after 30 minutes of treatment under the condition of pH 7.5. (5) Inhibitors: Strongly inhibited by cupric chloride, mercuric chloride and silver nitrate. (6) Molecular weight: The molecular weight measured by gel filtration method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis method are both about 43,000. 4. A xylitol oxidase which is characterized in that a microorganism belonging to the genus Streptomyces and capable of producing xylitol oxidase is cultured in a nutrient medium to produce and accumulate xylitol oxidase in the culture, which is then collected. Manufacturing method. 5. D-sorbitol, xylitol, D-
A sample containing any of galactitol, D-mannitol and D-arabinitol is caused to act with the xylitol oxidase according to any one of claims 1 to 3,
Amount of hydrogen peroxide produced, D-glucose produced, D
-Xylose, D-galactose, D-mannose, D
-Detecting any amount of arabinose or the amount of oxygen consumed, and measuring any of the corresponding sugar alcohols, D-sorbitol, xylitol, D-galactitol, D-mannitol and D-arabinitol. A method for measuring sugar alcohol, which comprises: