JPS6019990B2 - Method for producing uricase by fermentation method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing uricase by fermentation.

More specifically, the present invention belongs to the genus Enterobacter;
Cultivating microorganisms with uricase production in a nutrient medium,
The present invention relates to a method for producing uricase, which comprises producing uricase in a culture and collecting the same. Uricase (ECI.7.3.3) is an enzyme that catalyzes the action of oxidizing uric acid to allantoin, and is used to quantify uric acid.

Conventionally, methods for producing uricase using fermentation methods include micrococcus genus, Brevibacterium genus (Special Publication No. 4411
4783), Candida genus (Hakama Kosho 42-5192
No.), Streptomyces genus (A turtleic.BioIChe
m. Vol. Many methods are known that use microorganisms belonging to the following groups, such as 33, 1282, 1969). The present inventors conducted extensive searches for microorganisms that have the ability to produce uricase, and as a result, they discovered that a strain belonging to the genus Enterobacter produces uricase in small amounts, and completed the present invention.

The present invention will be explained in more detail below.

According to the present invention, if a microorganism belonging to the genus Enterobacter and capable of producing uricase is cultured in a nutrient medium, uricase will be produced in the culture, and the microorganism is collected.

The microorganism used in the present invention belongs to the genus Enterobacter and has the ability to produce uricase.
Any strain may be used.

An example of a suitable strain is Enterobacter cloacae KY-3.
066 (Collection Research Institute No. 4077, NRRL B-11
155). For the mycological properties of Enterobacter cloacae, see Bergey's Man
ual of Detennative mother cter
It is described in Iolo Battle Volume 8, page 325. The nutrient base used in the present invention can be either a synthetic medium or a natural nutrient base, as long as it contains carbon sources, nitrogen sources, inorganic substances, and other essential nutrients required by the strain used. . As the carbon source, carbohydrates such as glucose, fractalk, sucrose, and molasses, nucleic acids such as guanine, hypoxanthine, and xanthine, and uric acid are used.

Ammonium chloride, ammonium sulfate,
Nucleic acids such as ammonium phosphate, guanine, hypoxanthine, and xanthine, amino acids such as glutamic acid,
Inorganic or organic nitrogen compounds such as uric acid can be used.

Further, as a nitrogen source, nitrogen-containing natural products such as veptone, meat extract, yeast extract, corn stave liquor, etc. can also be used. As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium sulfate, etc. can be used.

In the present invention, the amount of uricase produced can be increased by allowing uric acid to be present in the medium as an inducer of uricase. Uric acid may be present in the medium from the beginning or may be added to the medium during culture. When added during the cultivation, it is preferably added before the end of the logarithmic growth phase of the microorganism used. The uric acid concentration in the medium is 0. A value of d' or higher is preferable. Culture is usually carried out by Zhenliao culture or aerobic culture.

The culture temperature is 20 to 40°C, preferably 25 to 30 qo. The pH of the medium is 5.0-9. 7.0 to 9 for those familiar with the European Agency.
It is desirable that it be in the range of 0. The culture period is 1 to 5 days, usually 2 to 3 days. By culturing in this manner, uricase is produced and accumulated in the culture, mainly in the bacterial cells.

Uricase is collected from the culture as follows: o After the culture is completed, the bacterial cells are obtained from the culture by centrifugal separation, etc.
Then, the bacterial cells are crushed by an appropriate means, and a supernatant liquid is obtained from the crushed liquid by centrifugation or the like.

The supernatant liquid is treated with methods commonly used for enzyme purification, such as salting, organic solvent precipitation, dialysis, ion exchange cellulose chromatography, Sephadex column chromatography, ion exchange Sephadex column chromatography, and freeze drying. . Purified uricase can thus be obtained. The activity of uricase in the present invention is measured by the following method.

0.2M borate buffer containing 20 caps/d' of uric acid (
Add 0.5' of the enzyme solution whose activity is to be measured to the solution 2.0 (pH 9.0), and react for 10 minutes at 3700° C. in the presence of oxygen.

After the reaction, 0.1N hydrochloric acid (47.5%) was added, and the absorbance at 28 m was measured, and this was taken as the reaction value for 1 minute. For control, 0.1N hydrochloric acid is added to the solution containing uric acid immediately after adding the enzyme solution, and the absorbance is measured after 28 hours, which is used as a control value. The unit of urinary enzyme is displayed as the dilution rate of Mitsubishi Yume
J. death. Mahler, Cape Alytica I Bioch
emisist, 38, 65, 197).

The amount of enzyme that decomposes 1 rmole of uric acid in 1 minute is defined as 1 unit (U).

The preferred range of uric acid concentration in the medium will be shown below using Experimental Example 1.

Experimental Example 1 Uric acid (concentrations shown in Table 1), corn steep liquor 0. rudder/d', yeast extract 0. Acid/d', KH2P04
0.1 g/d' of Mizuchi (pH 7.4) 10' consisting of M04.7 & 00.0 chicks/d' was placed in a 70' capacity test tube and sterilized at 120° C. for one bond.

One platinum loop of Enterobacter cloacae KY3066 was inoculated into each medium, and shaken cultured at 30°C for 4 hours. After culturing, the culture was centrifuged (1000 pm, 18 minutes,
The following centrifugation is performed under these conditions. ) to obtain bacterial cells. This was mixed with 0.08 M Tris buffer (pH 8.0) 10
Suspended in the
, 10 minutes). The crushed solution is centrifuged to obtain a supernatant solution. Uricase activity is measured using the supernatant as an enzyme solution. The results are also shown in Table 1. Table 1 Uric acid concentration Uricase activity (U/d') 0
1.50.1
2.703
3.80.5
12.5○-7
17-〇10
27.91.5
28.520 3
1.33.0 29.
94.0 29.9
5.0 30.5 From the results of Experimental Example 1, the concentration of uric acid as an inducer of uricase in the medium is 0. It can be seen that a value of Satoshi no d' or higher is suitable.

Next, the properties of uricase obtained by the present invention will be shown.

The enzyme preparation obtained in Example 1 was used. '11
Stable pH range enzyme preparations were prepared at various pH levels (6.0, 7.0, 7.6, 8.
1, 8.0, 85, 9.0, 9.6, 10.0, 10.
5) in 0.09M Tris buffer or 0.09M Tris buffer with
Dissolve in 2M boric acid buffer at a concentration of 0.22U/' to obtain enzyme solutions of each pH.

These solutions are then kept at 45° C. for 3 hours, and the uricase activity of the treated solutions is measured.

The results are shown in Figure 1. From FIG. 1, it can be seen that the stability range of the present uricase is 7.5 to 10.0. '2'
A variety of ultra-dilute pH enzyme products (6.0, 7.0, 7.4
．． 7.8, 8.4, 8.1, 8.6, 9.0, 9.5,
10.1, 10.6, 11.0) in 0.08M Tris buffer or 0.2M borate buffer.
Uric acid of 0 cape/mole is dissolved to obtain a uric acid solution of each pH.

This solution contains 2.0 I and 0.3 U/secretion of uricase solution (0
．． 08M Tris buffer, pH 8.0) 0.5 [
is added, an enzymatic reaction is performed, and the enzymatic activity is measured. The results are shown in Figure 2. From FIG. 2, it can be seen that the best solution for this uricase is close to 9.9. The '3' optimal temperature enzyme preparation was dissolved in 0.2M boric acid buffer (pH 9.0) to a concentration of 0.23U/'.

Reaction temperature 20, 25, 30, 35 40, 45 50
, 55 6 and 0, uricase activity is measured in the same manner as the above-mentioned method for measuring uricase activity. The results are shown in Figure 3.

From FIG. 3, it can be seen that the optimum temperature for this uricase is 4030°C. [41 0.24U of substrate-specific enzyme preparation
0.2M boric acid buffer (pH 9)
．． 0). The amount of decrease in the substrate is measured in the same manner as the method for measuring uricase activity described above, except that the substrate shown in Table 2 is used. The maximum absorption wavelength of each substrate is used as the measurement wavelength for absorbance. The results are shown in Table 2. Table 2 Substrate Uricase activity (U/[) Uric acid
0.124 adenine
0 guanine 0 xanthine 0 hypoxanthine 0 theobromine 0 theophylline 0 The results show that the present uricase has substrate specificity for uric acid.

■ Heat resistance. The enzyme preparation was dissolved in 0.2M white acid buffer (pH 9.0) to a concentration of 0.23U/'.

This is 30,3540,45 50,55 60,65
The samples were treated at each temperature of 3° C. and the uricase activity was measured after cooling with tap water for 5 minutes. The results are shown in Figure 4. As shown in Figure 4, this uricase has a concentration of about 50% even after treatment at 65°C for 30 minutes.
% residual activity. Dissolve the 6' inhibitor enzyme preparation in 0.2 borate buffer (pH 9.0) to give 0.21 U/[.

Uricase activity is measured in the same manner as the method for measuring uricase activity described above, except that various enzyme activity inhibitors are added. As a result, it was found that the activity was inhibited by about 80% when 1.3 x 10-5M sodium p-chloromercurybenzoate was present in the reaction system. In addition, regarding inhibition of metal ions, CuS041.3×10-4M inhibited approximately 44%, C
It was found that oC〆21.3×10-4M inhibited the effect by about 54%. '71 Molecular Weight The molecular weight was measured using Sephadex G-200 equilibrated with 0.09 K Tris buffer (pH 8.0) containing 0.1M potassium chloride.

As standard proteins, cytochrome C, egg albumin, bovine serum albumin, and alcohol dehydrogenase were used. As a result, it was found that the molecular weight of this waricase was 105,000. ■ The isoelectric focusing point of the present IJ case was determined by the isoelectric focusing method (Proteins, Nucleic Acids, Enzymes, 12, 737, 1967).

Ampholine (Ampholi ship) (fence 3.5-5.
0) and the enzyme solution were mixed, packed into a 110' column, and after being energized for 2 hours, fractions were fractionated using a fraction collector. As a result of measuring the pH and uricase activity of each fraction, the equidistant point of IJ case was 4.
It turned out to be 6. Next, the present invention will be specifically explained using examples.

Example 1 Uric acid 0. Ship/d', Corn Steep Liquor 0.5g
/ yeast extract 0. drum/d', glucose 1.5g/d
', KH2P040.1g/M bag 04.7QOO.
Seed culture soil (pH 7.4) consisting of 0 Heron/10 shams and 7
Place in a 0-volume test tube and sterilize at 120°C for 15 minutes.

Enterobacter cloacae KY3066 was added to this medium.
Inoculate one platinum loop of and culture for 4 years at 3 and ○. The obtained seed culture solution (first type culture solution) 30% was inoculated into 300% of the same seed culture medium as described above placed in two Erlenmeyer flasks, and cultured for 4 hours at 300°C. 900 pieces of the obtained crab culture liquid (second type culture liquid) was inoculated into a fermentation tower with the same composition as the seed culture medium in 30 jars, and 30 ○, 30 ratio pm, and aeration amount. 1〆′〆/mi
Main culture is carried out for 4 hours at n. Uricase in culture is 0
．． 14U/'. Uricase is collected from the culture as follows.

The culture was centrifuged to obtain 57 dollars (silver grain amount) of bacterial cells. This bacterial cell was added to 0.08M Tris buffer (pH 8.0).
After suspending in
．． Mother chofen lm. (Switzerland) at 300 pm for about 1 hour. The obtained bacterial cell disruption solution is centrifuged to obtain 3 soybeans (containing 1820 U of uricase). Ammonium sulfate is added to the supernatant to make it 40% saturated with ammonium sulfate, and the resulting precipitate is removed by centrifugation. Further ammonium sulfate is added to the upper layer to make ammonium sulfate 70% saturated, and the resulting precipitate is obtained by centrifugation. This precipitate was dissolved in 0.08 M Tris buffer (pH 8.0) at 200 mL, using the same buffer for 10 minutes, and using Cellophantieup as a dialysis membrane.
Perform dialysis overnight at °C. Set the dialysis tube fluid 200 to 0.
．． 0.08M Tris buffer containing 1M NaC (
Charge a column packed with 1 DEAE-cellulose equilibrated with 0.2M NaC (pH 8.0), wash with about 2 sleeves of 0.08M Tris buffer (pH 8.0) containing 0.2M NaC, and then add 0.3M NaC. 0.09M Tris containing
Elute with buffer (pH 8.0) 2 times. The eluate is fractionated into two fractions, and the IJ case active fractions are combined. Ammonium sulfate is added to this to make it 70% saturated, and the resulting precipitate is obtained by centrifugal separation. This precipitate is dissolved in about 100% of 0.08M Tris buffer (PH 8.0), charged to a column packed with Sephadex G-150500M equilibrated with the same buffer, and eluted with the same buffer. Fractionate the eluate into two fractions and combine the uricase active fractions. This was mixed with 0.05M Tris containing 0.2M NaCZ.
Charge a column packed with DEAE-Sephadex A-50500 equilibrated with buffer (pH 80), wash with one sleeve of the same buffer containing 0.2M NaCZ, and then add 0.09M Tris containing 0.2-0.59M NaC. - Perform concentration gradient elution with one sleeve of buffer (pH 80). The eluate is fractionated into two fractions, and the IJ case active fractions are combined. Add this to 0.08M Tris buffer (Fune 80)
5. Using cellophane tube as a dialysis membrane, heat at 5℃.
Perform dialysis overnight. The dialysis fluid is freeze-dried to obtain uricase powder. The supernatant obtained by separating the disrupted bacterial cells by Liangxin separation contained 9 proteins with an activity yield of 10.5% and a specific activity of 1.9 U/. Example 2 Corn Steep Liquor 0. Beard/d', yeast extract 0
．． Mustache/KH2P040.1g/d [, MgS04・
A medium (pH 7.4) consisting of a 70% medium (pH 7.4) with a ratio of 0.7 to 0.0 is placed in a 70 volume test tube and sterilized at 120° C. for 15 minutes.

Add 1 part of Enterobacter cloacae KY3066 to this.
Platinum loops were inoculated and cultured in a shaking moat for 4 hours at 30 qC. 0.015 U of uricase was accumulated per culture 1.

[Brief explanation of drawings]

Figure 1 shows the stable pH range of the present uricase. Figure 2 shows the normal pH of the present uricase. Figure 3 shows the optimum temperature of this uricase. Figure 4 shows the heat resistance of the present uricase. 1 week of Z cooperation and 4 episodes

Claims (1)

[Claims] 1. A method for producing uricase, which comprises culturing a microorganism belonging to the genus Enterobacter and capable of producing uricase in a nutrient medium, producing uricase in the culture, and collecting the same.