JPH0212557B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing pyranose oxidase by fermentation. More specifically, the present invention involves culturing in a nutrient medium a microorganism belonging to the genus Coriolus, Daedaleopsis, Proirotus, or Gloeophylum that has the ability to produce pyranose oxidase, and producing and accumulating pyranose oxidase in the culture. The present invention relates to a method for producing pyranose oxidase by a fermentation method characterized by collecting pyranose oxidase. Pyranose oxidase (enzyme number 1.1.3.10) converts glucose to glucosone,
It is an enzyme that catalyzes the reactions that oxidize xylose to xylosone and sorbose to 5-ketoractose (Methods in Enzymol VolXLI, P170,
(1975), absorbs enzymes when oxidizing each substance,
Produces hydrogen peroxide. Conventionally, as a method for producing pyranose oxidase using a fermentation method, a method is known in which it is obtained by culturing microorganisms belonging to the genus Polyporus. [Method in
Enzymol volXLI, P170 (1975)]. The present inventors investigated various methods for producing pyranose oxidase by fermentation, and found that pyranose oxidase belongs to the genus Coriolus, Daedaleopsis, Proirotus, or Gloeophylum.
The present invention was completed by discovering that pyranose oxidase is produced in a culture by culturing a microorganism capable of producing oxidase in a culture medium. The present invention will be explained in detail below. The microorganisms used in the present invention include Coriolus spp.
Any strain may be used as long as it belongs to the Daedaleopsis layer, the genus Proirotus, or the genus Gloeophylum and has the ability to produce pyranose oxidase. Examples of suitable strains include Coriolus
Persicolor (Coriolus versicolor) IFO4937,
Daedaleopsis styracina (Daedaleopsis)
styracina) IFO4910, Gloeophyllum sepiarium (Gloeophyllum sepiarium) Z-41
(MRRL12506), Pleuroutus ost-reatus Z-64 (NRRL12507)
etc. can be mentioned. The mycological properties of these microorganisms are described in the following literature: Coriolus versicoloradaedaleopsis Styracinagroeophyllum sepiarium Seiya Ito "Japanese Journal of Mycology" Volume 2, No. 4 (1955) Yokendo Proirotus ostreatus: Rokuya Imazeki, Tsuguo Hongo ""Illustrated Encyclopedia of Japanese Fungi" (1957), Yukusha The nutrient medium used in the present invention may be either a synthetic medium or a natural medium, as long as it contains carbon sources, nitrogen sources, inorganic substances, and other micronutrients required by the strain used. is also available. Carbon sources include glucose, sucrose,
Carbohydrates such as dextrin, starch, glycerin, and molasses are used. Nitrogen sources include inorganic or organic nitrogen compounds such as amino acids such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, and glutamic acid; nitrogen-containing natural sources such as pentones; meat extracts, yeast extracts, malt extracts, and corn steep liquor. Things can be used. As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium sulfate, ferric chloride, etc. can be used. Culture is usually carried out by shaking culture or aerated agitation culture. The culture temperature is 20-35°C, preferably 25-30°C. It is desirable that the pH of the medium is in the range of 3.0 to 8.0, preferably 4.5 to 6.5. The culture period is completed in 1 to 7 days, usually 2 to 4 days. By culturing in the above method, in the culture,
Pyranose oxidase is mainly produced and accumulated in the bacterial body. Pyranose oxidase is collected from the culture, for example, as follows. After completion of the culture, the culture solution is filtered to obtain bacterial cells, which are then disrupted by an appropriate means, and a supernatant liquid is obtained from the disrupted fluid by centrifugation or the like. The supernatant liquid is treated by methods commonly used for enzyme purification, such as salting out, organic solvent precipitation, dialysis, ion exchange column chromatography, gel filtration, and lyophilization. Purified pyranose oxidase can be collected as described above. Pyranose in the present invention
Oxidase activity is measured by the following method. [Measurement method] 1.0 ml of 0.1M Tris-HCl buffer (PH7.0), coloring solution [4-aminoantipyri and phenol each]
Add peroxidase to 0.1M Tris-HCl buffer (pH 7.0) containing 10 mmol per 100 ml of the solution.
Containing 2000 units] 0.2ml, 1M glucose solution 0.1ml, pyranose oxidase solution 0.1
ml, 0.1mMRAD0.1ml, water 1.5ml total volume 3.0
ml of enzyme-containing solution was reacted at 37°C, and the increase in absorbance at 500 nm was automatically recorded with a recorder. The molecular extinction coefficient of the quinoneimine dye produced under these conditions is
5.3×10 ³ per minute at 37℃ and pH 7.0.
If the amount of enzyme that produces 1 μmol of hydrogen peroxide is 1 unit, the unit of pyranose oxidase is expressed as ΔA ₅₀₀ /5.3×3.0/0.1×enzyme dilution rate. ΔA ₅₀₀ : Increase in absorbance at 500 nm per unit time (1 minute) Next, the properties of the pyranose oxidase obtained in Example 1 are shown. (1) Stable pH range Dissolve the enzyme preparation in citrate buffer, Tris buffer, and borate buffer having various pH values shown in Figure 1 to a concentration of 100 mU/0.7 ml, and dilute these solutions at 50 mU/0.7 ml. It was kept at ℃ for 30 minutes. After cooling, it was neutralized and the residual activity was measured. From FIG. 1, it can be seen that the stable pH range of this enzyme is 5.0 to 7.4. (2) Optimum pH Dissolve the enzyme preparation in Tris buffer solutions with various pH values shown in Figure 2 to a concentration of 250 mU/3.7 ml, and use a cell for oxygen absorption measurement (manufactured by Water Supply Science Institute).
BIOXYGRAPH) and 1M glucose 50μ
was added to measure the amount of oxygen absorbed. From Figure 2, it can be seen that the optimum pH of this enzyme is around 6.2. (3) Optimal temperature A reaction solution with the same composition as the activity measurement method.
using 100 mU of pyranose oxidase,
The reaction was carried out for 5 minutes at each temperature shown in FIG. Third
The figure shows that the optimum temperature for this enzyme is 50°C. (4) Heat resistance Add this enzyme to 0.6ml of 0.2M Tris buffer pH6.0.
The solution was dissolved to a concentration of 100 mU/0.7 ml, and after heat treatment for 30 minutes at each temperature shown in Figure 4, the residual activity was measured. From FIG. 4, it can be seen that this enzyme has 90% residual activity even after treatment at 60°C for 30 minutes. (5) Substrate specificity A reaction solution with the same composition as the activity measurement method, containing 2.5 units of pyranose oxidase and various sugar solutions (100mM) shown in Table 1 as substrates for the reaction.
0.1ml was used. Activity was expressed as relative activity. As shown in Table 1, this enzyme had high specificity for glucose, and also acted on sorbose and xylose.

[Table] The substrate specificity for sorbose and xylose is extremely weak compared to the conventional pyranose oxidase obtained from microorganisms of the genus Polyporus (see Table 1 of BBA 167 501-510 (1968)). (6) Electron acceptor 1.4 ml of 50 mM Tris-HCl buffer pH 7.0, 0.1 ml of enzyme solution (250 mU), and 0.5 ml of various electron acceptor solutions having the concentrations shown in Table 3 were placed in a cell with a Thunberg tube. After removing the dissolved enzyme by aspiration, mix 0.5 ml of 0.1 M glucose solution in the side chamber. After mixing, the change in absorbance per minute from 30 seconds to 90 seconds was measured, and the activity of each electron acceptor was expressed as relative activity. From Table 2, the molecular enzyme of this enzyme is considered to be the optimal electron acceptor, and 2,6
-Diclophenol It can be seen that indophenol and cytochrome C can also be electron acceptors.

[Table] (7) Example of inhibition Determined by oxygen absorption measurement similar to the optimum pH measurement in (2). A reaction solution consisting of 3.4ml of 50mM Tris-HCl buffer (pH 7.0), 0.1ml of enzyme solution (280mU), and 0.2ml of inhibitor solution (18.5mM) was placed in a cell for oxygen absorption measurement, and 50μ of 1M glucose was added at 37°C. was added to carry out the reaction. The results are shown in Table 3. This enzyme contains Cu ²⁺ , Ag ⁺ , Ni ²⁺ ,
It can be seen that this is inhibited by PCMB.

[Table] (8) Isoelectric point The isoelectric point of this enzyme was determined to be 4.2 by the isoelectric focusing method using carrier ampholite with a pH of 3.5 to 10.0. (9) Molecular weight Determined by gel filtration method using Cephadex G-200. Egg albumin, bovine serum albumin, alcohol dehydrogenase, γ-globulin, and milk xanthine oxidase were used as standard proteins. As a result, the molecular weight of this enzyme was approximately 220,000.
was asked. (10) Km value: Lineweaver-Park (Lineweaver-Park) using 2.5 units of pyranose oxidase
Burk) blot for glucose
Km value was calculated. As a result, the Km value for glucose is
It was 0.83mM. This value is Km for glucose oxidase
It is about 1/24 to 1/24 compared to the value (10 to 20 mM). Next, measurement of the substrate of pyranose oxidase using pyranose oxidase obtained by the method of the present invention will be explained. Pyranose from microorganisms of the genus Coriolus, Daedaleopsis, Proirotus, or Gloeophyllum is used as a substrate for pyranose oxidase.
The substrate can be quantified by allowing oxidase to act in a suitable buffer, introducing the generated hydrogen peroxide into a dye system, and measuring absorption by the dye. As an example, the reaction formula of pyranose oxidase when glucose is the substrate is shown below. In addition to quantifying glucose in a sample containing glucose, it also measures compounds that participate in reactions in systems that can quantitatively produce glucose through reaction, decomposition, etc., and the activity of enzymes that catalyze reactions. It can be applied to For example, it can be applied to the determination of glucose derivatives such as maltose and methyl glucoside. These glucose derivatives are decomposed by acids or enzymes,
Since it is converted to glucose, these can be quantified. Specific examples of glucose derivative degrading enzymes include glucosidase, maltose, phosphorylase, amylase, and the like. For example, when maltose is treated with maltose phosphorylase, glucose and glucose-1-phosphate are produced, when maltose is treated with glucosidase, 2 moles of glucose are produced, and when glucosidase is treated with methyl-α-D-glucoside, glucose is produced. is produced, and when α-amylase and maltose phosphorylase act on starch, glucose and glucose-1-phosphate are produced. By quantifying the glucose produced in these reactions, it is possible to quantify the original compound and measure the activity of the enzyme used. When quantifying the substrate, prepare the sample containing the substrate and pyranose oxidase at a pH of approximately 7.0~
It can be reacted by adding it to 8.0 buffer solution. Pyranose oxidase can also be used in microencapsulation or immobilized on a carrier, if necessary. As the buffer, phosphate buffer, Tris-HCl buffer, etc. are used. Enzymes usually have a substrate of 0.1~
0.5 to 15 U is used per 1 μmol, and the reaction is carried out at a concentration of 3 ml with water. Any known means can be used to introduce the generated hydrogen peroxide to the dye system. For example, peroxidase and a color former are added to produce a pigment. Color formers include 4-aminoantipyrine and phenol, 4-aminoantipyrine and N,N-dimethylaniline, 3-methyl-2-benzothiazolinone hydrazone (hereinafter abbreviated as MBTH) and o-toluidine, MBTH and N,N -Dimethylaniline and the like are exemplified. The reaction is carried out in the presence of 2 to 10 U and 5 to 20 μmol of peroxidase and coloring agent in 3 ml of the reaction solution. The reaction is carried out at 30-50°C and is completed in 5-20 minutes. Measure the absorption in the visible region of the reaction solution due to the generated dye. Absorption is usually measured between 400 and 600 nm. The relationship between the content of the substrate and the absorbance is determined in advance as a calibration curve for a standard substance, and the content of the substrate can be determined from the absorbance obtained for the sample. As a method for quantifying the substrate, the oxidation reaction of the substrate and the reaction that leads the generated hydrogen peroxide to the dye can be performed separately, but it is preferable to use pyranose.
The objective can be easily achieved by adding oxidase and a composition for quantifying hydrogen peroxide together to a sample to obtain a colored reaction solution in a one-step reaction, and measuring the absorption in the visible region of this reaction solution. The present invention also includes a measuring composition and a kit using the same. This composition contains pyranose oxidase, peroxidase and color former from 0.5 to 15 U and from 2 to 10 U.
and 5 to 20 μmol, and the kit consists of a combination of this composition and 1 to 3 ml of buffer. The composition may include an enzyme or degrading agent capable of degrading the glucose derivative to glucose. For example, as a composition for measuring α-amylase, a composition containing 1 to 10 U of maltose phosphorylase in a composition containing starch is exemplified. Examples and reference examples of the present invention (determination of glucose using pyranose oxidase obtained in Example 1) are shown below. Example 1 Glucose 0.5g/dl, yeast extract 0.4g/dl,
Malt extract 1g/dl, ferric chloride 1mg/dl
Pour 30 ml of seed culture medium consisting of (pH 6.0) into a 300 ml flask and sterilize at 120°C for 15 minutes. One platinum loop of Coriolus versicolor IFO4937 is inoculated into this medium and cultured with shaking at 30°C for 5 days. 30 ml of the obtained seed culture solution (first type culture solution) was placed in a second culture flask. Seed culture medium having the same composition as above.
Inoculate 300ml and culture with shaking at 30°C for 4 days. The obtained seed culture solution (second type culture solution) 1.2 was inoculated into a fermentation medium 8 having the same composition as the seed culture medium placed in a 30-jar fermentor, and incubated at 30°C, 250 rpm, and aeration rate 1//min for 2 days. Perform main culture. Pyranose oxidase in the culture is 161 mU/ml of fermentation broth. Pyranose oxidase is collected from the culture solution as follows. The culture was aspirated to obtain about 1 kg of wet bacterial cells. After suspending the cells in 50 mM Tris-HCl buffer (pH 7.0) (all buffers used below are this buffer), the cells are shredded with a homogenizer. Supernatant obtained by centrifugation
After adjusting the pH to 7.0 by adding alkali to 1.8,
1 of HPA-75 (Mitsubishi Kasei) equilibrated with buffer solution
Pour into the column and wash with buffer. Next, wash with a buffer containing 0.1M ammonium sulfate, and elute the enzyme with a buffer containing 0.25M ammonium sulfate. Combine the active fractions and add ammonium sulfate.
Obtain the precipitate by centrifugation at 80% saturation. The precipitate is dissolved in 30 ml of buffer, applied to one column of Sepharose 4B, and eluted with the buffer. The active fractions are combined and the ammonium sulfate 80% saturated precipitate is collected by centrifugation. This was dissolved in 30 ml of buffer, applied to a 500 ml column of Sephadex G-100, and eluted with the buffer. The active fraction is lyophilized to obtain 815 mg of pyranose oxidase powder. The purification yield from the bacterial cell extract was 43%, and the specific activity was 9.6 U/mg protein. Example 2 In Example 1, the strain is replaced with Gloeofilm sepialium Z-41, and the strain is cultured for 6 days in a medium (two culture flasks) having the same medium composition as in Example 1. Pyranose oxidase in culture is
It is 365 mU/ml. Example 3 Culture is carried out in the same manner as in Example 1 except that the bacterial strain in Example 1 is replaced with Daedaleopsis tyracina IFO4910. Pyranose oxidase in the culture is 189 mU/ml. Example 4 Culture is carried out in the same manner as in Example 1 except that the bacterial strain in Example 1 is changed to Ploylotus ostreatus Z-64. Pyranose oxidase in the culture is 59 mU/ml. Reference example 0.2 ml of a solution containing glucose as a substrate at the concentration shown at each measurement point in Figure 5, 1.5 ml of 0.1 M potassium phosphate buffer pH 7.5, coloring solution [4-aminoantipyrine, phenol, 10 mmol each] include
0.1M potassium phosphate buffer (pH 7.5) containing 2000 units of peroxidase per 100ml of the solution] 0.2ml, 0.1mMFAD 0.1ml, and pyranose oxidase 1ml (6.7U) added for a total volume of 3.0ml. The reaction solution was reacted at 37°C for 10 minutes, and the absorbance at 500 nm was measured. The results are shown in Figure 5. A proportional relationship was observed between glucose concentration and absorbance.

[Brief explanation of drawings]

FIG. 1 shows the relationship between the relative activity of pyranose oxidase and the stable pH range. Figure 2 shows the relationship between relative activity of pyranose oxidase and optimum pH. FIG. 3 shows the relationship between relative activity of pyranose oxidase and optimum temperature. FIG. 4 shows the relationship between relative activity of pyranose oxidase and heat resistance. FIG. 5 shows the relationship between absorbance and glucose concentration.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Coriolus, Daedaleopsis, Ploylotus, or Gloeaphyllum that has the ability to produce pyranose oxidase is cultured in a nutrient medium, and pyranose oxidase is produced and accumulated in the culture, and the microorganism is collected. A method for producing pyranose oxidase using a fermentation method.