JPH0314426B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to pyranose oxidase and a method for producing the same. More specifically, the present invention relates to a pyranose oxidase that acts not only on β-D-glucose but also on α-D-glucose, and a method for producing the same using a basidiomycete belonging to the genus Polyporus. When the pyranose oxidase of the present invention acts on α-D-glucose and β-D-glucose in the presence of oxygen, it produces D-glucozone and hydrogen peroxide. Therefore, by measuring the amount of hydrogen peroxide produced or the amount of oxygen decreased by a known method, it is possible to quickly measure the amount of glucose in a biological sample or food. [Prior Art] Pyranose oxidase, which oxidizes D-glucose to produce D-glucozone and hydrogen peroxide, has been discovered in the basidiomycete Polyporus obtusus (Biochimica e Biophysica).
Biochimica et Biophysica Acta
Vol167, p493, p501 (1968)]. However, although this enzyme acts on β-D-glucose, α-D-glucose cannot be a substrate. Recently, Tokukai Sho
No. 58-43785 also reports that microorganisms belonging to the genus Coriolus, Daedaleopsis, Ploylotus, or Gloeofilm produce pyranose oxidase that is highly specific for D-glucose. [Problems to be Solved by the Invention] Glucose oxidase has been frequently used as a method for specifically quantifying D-glucose in biological samples or foods. However, glucose oxidase acts only on β-D-glucose. D in biological samples or food
- The existing form of glucose is 36.5% as α-D-glucose and 63.5% as β-D-glucose. For this reason, when quantifying D-glucose using glucose oxidase, rapid quantification is not possible unless mutarotase is added. In view of the above-mentioned current situation, the present inventors have conducted extensive studies on pyranose oxidase produced by basidiomycetes, and have found that Polyporus obtusus, which is known as a pyranose oxidase-producing bacterium, converts α-D-glucose into α-D-glucose in culture. The present invention was completed based on the discovery that a previously unknown pyranose oxidase, which also acts as a pyranose oxidase, can be produced in large quantities. Therefore, an object of the present invention is to provide a method for industrially producing the above enzyme at low cost by culturing pyranose oxidase and Polyporus obtusus that can rapidly quantify D-glucose. [Means for Solving the Problems] To summarize the present invention, the present invention relates to pyranose oxidase and a method for producing the same, and the first invention of the present invention is characterized by having the following physical and chemical properties. Concerning pyranose oxidase. (1) Action: In the presence of oxygen, the second
Oxidizes carbon to produce D-glucozone and hydrogen peroxide. (2) Substrate specificity: The effect is highest on D-glucose, but L-sorbose, D-xylose, D-maltose, D-galactose, 2-
It also acts on deoxy-D-glucose, gluconate δ-lactone, etc. Furthermore, when acting on D-glucose, it oxidizes not only β-D-glucose but also α-D-glucose. (3) Optimal PH and PH stability: The optimal PH is around 5.0, and the pH is stable between 5.0 and 9.0 when treated at 37°C for 60 minutes. (4) Optimal temperature and thermal stability: The optimal temperature is around 55°C, and it is stable up to 60°C when treated at PH6.0 for 10 minutes. (5) Electron acceptor: Molecular oxygen is the best electron acceptor, but phenazine methosulfate and 2,6-dichlorophenol indophenol are also electron acceptors. (6) Molecular weight: The molecular weight measured by gel filtration method is approximately
195,000 Sodium Dodecyl Sulfate (SDS) −
Three subunits of approximately 40,000, one subunit of approximately 30,000, and one subunit of approximately 28,000 as determined by polyacrylamide gel electrophoresis.
It is composed of individuals. (7) Coenzyme: One molecule of the coenzyme flavin adenine dinucleotide (FAD) is covalently bonded to each of approximately 40,000 subunits, and three molecules to each enzyme molecule. (8) Isoelectric point: 6.95 (9) Inhibitor: Hg ²⁺ , Fe ²⁺ , Ag ⁺ , phenylmethylsulfonyl fluoride, sodium sulfide, p
- Inhibited by chloromercribenzoic acid, o-phenanthroline, etc. (10) Km value: 1.23×10 ^-3 M (D-glucose) In addition, the second invention of the present invention is to culture a strain having the ability to produce pyranose oxidase belonging to the genus Polyporus in a medium, and to obtain the present invention from the culture. The invention is characterized in that the pyranose oxidase of the first invention is collected. The basidiomycetes used in the present invention include strains belonging to the genus Polyporus, such as Polyporus obtusus.
There is ATCC26733. To explain the method of the present invention in more detail, the nutrient source added to the culture medium may be one that can be used by the strain used to produce pyranose oxidase.
Examples of carbon sources that can be used include glucose, starch, sucrose, maltose, lactose, glycerol, dextrin, and fats and oils; examples of nitrogen sources include yeast extract, peptone, defatted soybean, and
Corn steep liquor, meat extract, etc. are suitable. In addition, inorganic and metal salts such as phosphates, potassium salts, and magnesium salts may be added, as well as vitamins and growth-promoting factors. When culturing basidiomycetes, the production amount of pyranose oxidase varies greatly depending on the culture conditions, but generally the culture temperature is 20-35℃ and the pH of the medium is 4-40℃.
8 is good, and the production of pyranose oxidase reaches its maximum after 2 to 10 days of aerated agitation culture. It goes without saying that culture conditions should be set to maximize the production of pyranose oxidase depending on the strain used, medium composition, etc. Pyranose oxidase produced by the strain of the present invention is mainly located within the bacterial body. Purification of pyranose oxidase from bacterial cells is carried out as follows. The cells obtained by straining the culture solution are suspended in a buffer solution of pH 4 to 10 and stirred at 10 to 40°C for 2 to 48 hours to extract pyranose oxidase from the cells. For example, 10g of bacterial cells are 0.1M, 0.5M, 1M and
Suspend in 100ml of each 2M phosphate buffer (PH7.0),
After stirring and extracting at 20℃ or 30℃ for 20 hours,
Table 1 shows the pyranose oxidase activity in the supernatant obtained by centrifugation. As a control, 10 g of bacterial cells and 100 g of 0.1M phosphate buffer (PH7.0) were added.
The activity in the supernatant obtained by homogenizing ml using emulsion (manufactured by Teraoka Co., Ltd.) is also shown.

[Table] Add a precipitant, such as ammonium sulfate, to the obtained extract for 20 to 20 minutes.
By adding 80w/v% or organic solvents such as alcohol, acetone, etc.
By adding 80v/v%, it is separated as a precipitate. The obtained precipitate is desalted by ultrafiltration, dialysis, or Sephadex treatment to obtain a crude enzyme solution. To purify the obtained crude enzyme solution, the crude enzyme solution is adsorbed onto a column of DEAE-Sepharose (CL-6B) buffered in advance with 0.01M borate buffer (PH9.0), and the adsorbed matter is reduced to 0.03%. After washing with M borate buffer (PH9.0), wash with 0.1M borate buffer (PH9.0).
9.0) and collect the active fraction. Next, this active fraction was concentrated and desalted by ultrafiltration, and then adsorbed on a column of CM-Sepharose (CL-6B) buffered with 0.01M phosphate buffer (PH6.0) to reduce the adsorbate to 0.03M.
After washing with phosphate buffer (PH6.0), elute with 0.1M phosphate buffer (PH6.0) and collect the active fraction. This active fraction is filtered through distilled water and then freeze-dried to obtain purified enzyme powder. This enzyme powder is electrophoretically homogeneous on polyacrylamide gel discs. The enzymatic and physicochemical properties of the pyranose oxidase of the present invention are as follows. (1) Action: When this enzyme acts on D-glucose in the presence of oxygen, it oxidizes the secondary carbon of D-glucose to produce D-glucozone and hydrogen peroxide.
The reaction formula is as follows. D-glucose + O ₂ → D-glucozone + H ₂ O ₂ (2) Substrate specificity: When various sugar solutions (concentration in reaction solution: 1.67 mM) were used as substrates for this enzyme, it was investigated that this enzyme It had high specificity against L-sorbose, D-xylose, etc. (Table 2).

[Table] This enzyme also contains α-D-glucose and β-D
- Comparison of reaction rates when acting on glucose. The reaction system consists of 0.1ml of 100mM α-D-glucose or β-D-glucose solution, 1.5ml of pine kilvaine buffer (PH6.0), 0.1ml of 24.6mM 4-aminoantipyrine solution, 0.1ml of 0.42M phenol solution, and peroxidase derived from horseradish. Solution (240 units/ml) 0.1ml, water 1.0ml and this enzyme solution (30 units/ml) 0.1ml, reaction solution volume 3.0
The reaction was carried out in a cube at 37°C, and the change in absorption at 500 nm due to the produced quinoneimine dye was measured over time. The results are shown in FIG. In FIG. 1, the solid line is for β-D-glucose and the dotted line is for α-D-glucose, the horizontal axis represents reaction time (seconds), and the vertical axis represents absorbance. As is clear from Figure 1, this enzyme is β-D-
Although slightly lower than the activity towards glucose, α
-D-glucose was also found to have a sufficient effect. Incidentally, a similar experiment was conducted using glucose oxidase, which is known not to act on α-D-glucose. The results are shown in FIG. The solid line in FIG. 2 is for β-D-glucose, and the dotted line is for α-D-glucose.
The horizontal axis shows the reaction time (seconds), and the vertical axis shows the absorbance. Using 0.1 ml of glucose oxidase solution (300 units/ml manufactured by Toyobo Co., Ltd.), we tracked the absorption change at 500 nm and found that it appeared to have a slight effect on α-D-glucose, but this was not due to α-D-glucose.
It is due to the non-enzymatic conversion of D-glucose to β-D-glucose. (3) Optimal PH and PH stability: As shown by the curve in Figure 3, the optimal PH of this enzyme has high activity around PH5.0.
Figure 4 shows the PH stability when this enzyme was treated at 37°C for 60 minutes at each PH. Fourth
As is clear from the figure, this enzyme is stable between PH5.0 and 9.0. (4) Optimal temperature and thermostability: The optimal temperature of this enzyme is around 55°C, as shown by the curve in Figure 5.
Figure 6 shows the thermal stability of this enzyme when it was treated at pH 6.0 for 10 minutes at each temperature.
This enzyme was stable up to 60℃. (5) Electron acceptor: When we investigated the electron acceptor of this enzyme, we found that molecular oxygen is the best electron acceptor for this enzyme, but phenazine methosulfate and 2,6- Dichlorophenol indophenol was also used as an electron acceptor (Table 3).

[Table] (6) Molecular weight: The molecular weight of this enzyme is approximately 195,000 when measured by gel filtration using Cephacryl S-300 (manufactured by Pharmacia).
It was hot. Furthermore, when measured by (SDS)-polyacrylamide gel electrophoresis, it was composed of three subunits of about 40,000, one subunit of about 30,000, and one subunit of about 28,000. (7) Coenzyme: Purified enzyme solution 0.1% concentration (0.1M phosphate buffer PH
When the absorption spectrum of 7.0) was measured, the absorption spectrum of the flavoenzyme shown in FIG. 7 was obtained. This enzyme was heat-treated or trichloroacetic acid-treated, and the supernatant obtained by centrifugation was subjected to thin-layer chromatography (developing solvent: n-butanol:acetic acid:water = 4:1:5). It was suggested that the dye does not move from the origin and is covalently bonded to the enzyme protein. Therefore, when this enzyme was treated with pronase and then treated with phosphodiesterase, the production of adenosine-5'-1 phosphate (AMP) was observed, which revealed that FAD was a coenzyme of this enzyme. F.A.D.
Judging from the molecular extinction coefficient at 450nm,
Each molecule of this enzyme contained 3 molecules of FAD. In addition, the gel obtained by (SDS)-polyacrylamide gel electrophoresis of this enzyme was
% acetic acid solution, it was confirmed that FAD was present in only 40,000 of the three subunits. (8) Isoelectric point: The isoelectric point of this enzyme was determined to be 6.95 by focusing electrophoresis using Pharmalite (PH3-10, manufactured by Pharmacia). (9) Effects of inhibitors, metal ions, and metal chelating agents: This enzyme contains Hg ²⁺ , Fe ²⁺ , Ag ²⁺ , PMSF (phenylmethylsulfonyl fluoride), sodium sulfide, and PCMB (p-chloromercuribenzoin). acid), o-phenanthroline, etc. (Table 4).

〔Example〕

The method for producing pyranose oxidase according to the present invention will be shown below with examples, but the present invention is not limited to the following examples. In addition,%
is w/v% unless otherwise specified. Example 1 Glucose 2%, Ebios 0.5% and Agar 1.5
% (Ebios medium) was inoculated with Polyporus obtusus ATCC26733 and left to stand for one week at 25°C to obtain a seed culture. glucose 2
%, yeast extract 0.3%, peptone 1%,
Dispense 100 ml of a medium with a composition of KH ₂ PO ₄ 0.3% and MgSO ₄ 7H ₂ O 0.1% into a 500 ml Erlenmeyer flask,
After sterilizing at 120℃ for 20 minutes, cooling, scraping and inoculating with the above inoculum, and sterilizing at 100% per minute at 25℃ for 8 days.
Culture was performed with shaking by rotation. After the culture was completed, the culture solution was filtered to obtain bacterial cells. The bacterial cells were dissolved in 1M acetate buffer (PH
5.0) Place in an Erlenmeyer flask together with 50 ml and heat at 25℃ for 20 minutes.
Extraction of pyranose oxidase was performed while stirring for hours. A supernatant liquid was obtained from this extract by centrifugation. The total activity of this supernatant was 220 units. Example 2 Polyporus obtusus ATCC26733 cultured in the Ebios medium of Example 1 was treated with 2% glucose,
Yeast extract 0.3%, peptone 1%, KH ₂ PO ₄ 0.3%
and 100 ml of a medium containing 0.1% MgSO ₄ .7H ₂ O was inoculated into a sterilized (120° C., 20 minutes) 500 ml Erlenmeyer flask, and cultured at 25° C. for 6 days to prepare a seed culture. Glucose 2%, yeast extract 0.1%, peptone 2%, KH ₂ PO ₄ 0.3%, MgSO ₄ 7H ₂ O 0.1%
and antifoaming agent (CB-442 manufactured by NOF Corporation) 0.02%
A medium 20 having a composition of (v/v) was placed in a 30 volume jar fermenter and sterilized at 120°C for 20 minutes.
After cooling, 100 ml of the above seed culture solution was inoculated and cultured at 27° C. for 7 days at an aeration rate of 20 per minute and a stirring speed of 300 revolutions per minute. After the culture was completed, bacterial cells were obtained by straining. 1.50 kg of these bacterial cells and 10 ml of 1M phosphate buffer (PH7.0) were placed in a stirring tank, and pyranose oxidase was extracted while stirring at 30°C for 24 hours. Remove bacterial cell residue from the extract by filtration,
A supernatant was obtained. The pyranose oxidase activity of this supernatant 11 was 5.40 units/ml. Ammonium sulfate was added to this supernatant liquid 11 to make it 70% saturated, and after standing for a day and night, the obtained ammonium sulfate precipitate was
Dissolved in 500ml of 0.01M borate buffer (PH9.0). This crude enzyme solution was concentrated by ultrafiltration, and after desalting,
The adsorbed material was adsorbed onto a column (diameter 5.0 cm x length 10 cm) of DEAE-Sepharose (CL-6B) buffered in advance with 0.01M borate buffer (PH9.0), and the adsorbed material was buffered with 0.03M borate buffer (PH9.0). After washing with .0), 0.1M
The active fraction was collected by elution with borate buffer (PH9.0). Next, this active fraction was concentrated by ultrafiltration, desalted, and then column of CM-Sepharose (CL-6B) buffered with 0.01M phosphate buffer (PH6.0) (5.0cm diameter x 4cm length). ) to absorb the adsorbed material.
After washing with 0.03M phosphate buffer (PH6.0), the active fraction was collected by elution with 0.1M phosphate buffer (PH6.0). This active fraction was dialyzed against distilled water and then lyophilized to obtain 2.25 g of purified enzyme powder. The specific activity of this powder was 13.7 units/mg. This enzyme powder was uniform in polyacrylamide gel disc electrophoresis. The above purification steps are shown in Table 5.

〔Effect of the invention〕

The present invention provides pyranose oxidase, which is extremely useful as a clinical diagnostic reagent, and a manufacturing method suitable for its industrial production.

[Brief explanation of the drawing]

FIG. 1 shows α-D-glucose and β-D-glucose of pyranose oxidase obtained according to the present invention.
FIG. 2 is a graph showing the specificity of the reaction to glucose; FIG. 2 is a graph showing the specificity of the reaction of known glucose oxidase to α-D-glucose and β-D-glucose;
FIG. 3 is a graph showing the relationship between PH and activity of the pyranose oxidase obtained according to the present invention, and FIG. FIG. 5 is a graph showing the relationship between temperature and activity of the enzyme according to the present invention, and FIG. 6 is a graph showing the relationship between temperature and activity after 10 minutes of treatment at each temperature at PH6.0. FIG. 7 is a graph showing the absorption spectrum of the enzyme according to the present invention (enzyme concentration 0.1%, pH 7.0).

Claims (1)

[Scope of Claims] Pyranose oxidase having the following physical and chemical properties. (1) Action: In the presence of oxygen, the second
Oxidizes carbon to produce D-glucozone and hydrogen peroxide. (2) Substrate specificity: The effect is highest on D-glucose, but L-sorbose, D-xylose, D-maltose, D-galactose, 2-
It also acts on deoxy-D-glucose, gluconate δ-lactone, etc. Furthermore, when acting on D-glucose, it oxidizes not only β-D-glucose but also α-D-glucose. (3) Optimal PH and PH stability: The optimal PH is around 5.0, and the pH is stable between 5.0 and 9.0 when treated at 37°C for 60 minutes. (4) Optimal temperature and thermal stability: The optimal temperature is around 55°C, and it is stable up to 60°C when treated at PH6.0 for 10 minutes. (5) Electron acceptor: Molecular oxygen is the best electron acceptor, but phenazine methosulfate and 2,6-dichlorophenol indophenol are also electron acceptors. (6) Molecular weight: The molecular weight measured by gel filtration method is approximately
195,000 Sodium Dodecyl Sulfate (SDS) −
Three subunits of approximately 40,000, one subunit of approximately 30,000, and one subunit of approximately 28,000 as determined by polyacrylamide gel electrophoresis.
It is composed of individuals. (7) Coenzyme: One molecule of the coenzyme flavin adenine dinucleotide (FAD) is covalently bonded to each of approximately 40,000 subunits, and three molecules to each enzyme molecule. (8) Isoelectric point: 6.95 (9) Inhibitor: Hg ²⁺ , Fe ²⁺ , Ag ⁺ , phenylmethylsulfonyl fluoride, sodium sulfide, p
- Inhibited by chloromercribenzoic acid, o-phenanthroline, etc. (10) Km value: 1.23 x 10 ^-3 M (D-glucose) 2 A strain having the ability to produce pyranose oxidase belonging to the genus Polyporus was cultured in a medium, and the culture was used to produce pyranose having the following physical and chemical properties.
A method for producing pyranose oxidase, which comprises collecting oxidase. (1) Action: In the presence of oxygen, the second
Oxidizes carbon to produce D-glucozone and hydrogen peroxide. (2) Substrate specificity: The effect is highest on D-glucose, but L-sorbose, D-xylose, D-maltose, D-galactose, 2-
It also acts on deoxy-D-glucose, gluconate δ-lactone, etc. Furthermore, when acting on D-glucose, it oxidizes not only β-D-glucose but also α-D-glucose. (3) Optimal PH and PH stability: The optimal PH is around 5.0, and the pH is stable between 5.0 and 9.0 when treated at 37°C for 60 minutes. (4) Optimal temperature and thermal stability: The optimal temperature is around 55°C, and it is stable up to 60°C when treated at PH6.0 for 10 minutes. (5) Electron acceptor: Molecular oxygen is the best electron acceptor, but phenazine methosulfate and 2,6-dichlorophenol indophenol are also electron acceptors. (6) Molecular weight: The molecular weight measured by gel filtration method is approximately
195,000 Sodium Dodecyl Sulfate (SDS) −
Three subunits of approximately 40,000, one subunit of approximately 30,000, and one subunit of approximately 28,000 as determined by polyacrylamide gel electrophoresis.
It is composed of individuals. (7) Coenzyme: One molecule of the coenzyme flavin adenine dinucleotide (FAD) is covalently bonded to each of approximately 40,000 subunits, and three molecules to each enzyme molecule. (8) Isoelectric point: 6.95 (9) Inhibitor: Hg ²⁺ , Fe ²⁺ , Ag ⁺ , phenylmethylsulfonyl fluoride, sodium sulfide, p
- Inhibited by chloromercribenzoic acid, o-phenanthroline, etc. (10) Km value: 1.23×10 ^-3 M (D-glucose) 3 When collecting pyranose oxidase from the culture, the cultured bacteria obtained from the culture should be
3. The method according to claim 2, comprising the step of extracting pyranose oxidase by suspending it in a buffer solution of 10 to 10%. 4 The basidiomycete belonging to the genus Polyporus is Polyporus.
4. The method according to claim 2 or 3, wherein the method is obstusus.