JPS6243669B2 - - 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 relates to a method for producing uricase, which comprises culturing a microorganism belonging to the genus Torulopsis and capable of producing uricase in a nutrient medium, producing uricase in the culture, and collecting the microorganism. Uricase (EC1.7.3.3) is an enzyme that catalyzes the oxidative hydrolysis of uric acid to produce allantoin, hydrogen peroxide, and carbon dioxide. Uricase is used to measure uric acid in blood or urine. Conventionally, methods for producing uricase using fermentation methods include Micrococcus spp., Brevibacterium spp. Many methods are known that use microorganisms belonging to the genus Enterobacter (Japanese Unexamined Patent Publication No. 11296/1986), the genus Trichosporon (Japanese Unexamined Patent Publication No. 119086/1983), etc. . As a result of an extensive search for microorganisms capable of producing uricase, the present inventors found that a yeast belonging to the genus Torulopsis (referred to as strain TN-1023) isolated from the natural world (bird droppings) produced uricase in significant amounts. The present invention was completed based on this discovery. The mycological properties of this bacterium are as follows. 1 Growth status in each medium (1) YM liquid medium *1 After static culture at 25°C for 3 days, the growth of the bacterial cells was good, and the cells settled in a powder form, with no film formation. The cell morphology is oval, the size is (2-4) x (2.5-5) microns, and proliferation is due to multipolar budding. (2) YM agar medium *2 Growth is good at 25℃, and the surface of the colonies is smooth and buttery gray. The periphery of the colony is entire and rises in a platform-like manner. (3) Generation of giant colonies in YM gelatin medium *3 After culturing at 25℃ for 50 days, giant colonies with a diameter of 33 mm are generated. The periphery of the colony was wavy and descended into a platform, and was buttery and brownish cream in color. (4) All cells cultured on slides using potato extract agar medium are oval in shape and contain only vegetative cells, with no pseudohyphae, hyphae, spores, fission, etc. observed. *1 YM liquid medium composition: After dissolving 5g of peptone, 3g of yeast extract, 3g of malt extract and 10g of glucose in water,
Make the total volume to 1000ml with water. *2 YM agar medium composition: Dissolve 5 g of peptone, 3 g of yeast extract, 3 g of malt extract, 10 g of glucose, and 20 g of agar in water, then make up to 1000 ml with water. *3 YM gelatin medium composition: peptone 5g, yeast extract 3g, malt extract 3g, glucose 10g and gelatin 200%
Dissolve g in water and make up to 1000ml with water. 2. Formation of ascospores Using a medium such as gypsum/sodium acetate medium, Godlokova medium, improved Godlokova medium, glucose/peptone medium, Fewell medium, Crane medium, carrot piece medium, wort agar medium, vegetable juice agar medium, etc. The presence or absence of ascospore formation was examined, but no ascospores were observed. 3 Formation of extruded spores The presence or absence of extruded spore formation was examined using YM agar medium and wort agar medium, but no extruded spore formation was observed. 4 Physiological properties (1) Optimal growth conditions: PH5-6, temperature 23-33℃ (2) Growth range: PH2-7.5, temperature 11-39℃ (3) Nitrate assimilation: None (4) Fat decomposition : None (5) Urea decomposition: None (6) Gelatin liquefaction: None (7) Osmotic pressure resistance: NaCl growth limit concentration 21% (8) Carotenoid production: None (9) Significant organic acid production: None (10) Production of starch-like substances: None (11) Requirement for vitamins: Requires biotin for growth. (12) Degradability of glucosides: Yes (decomposes albutene) (13) Formation of esters: None 5 Fermentability of sugars Ferment D-glucose, D-galactose, sucrose, and trehalose. Roughinose's
Ferment 1/3 molecule. It does not ferment maltose, lactose, melibiose, meletitose, D-(+)-cellobiose, α-methylglucoside, etc. 6 Assimilation Assimilation: D-glucose, D-galactose, sucrose, maltose, raffinose, glycerol, ethanol, trehalose, D-
(+)-cellobiose, L-(-) sorbose,
L-(+)arabinose, D-xylose, D
-(+)-mannose, D-(-)-fructose, D-(+)-meletitose, α-methylglucoside, mannitol, D-(-)-sorbitol, dulcitol, sodium α-ketoglutarate, sodium citrate, Sodium succinate, sodium malate, DL-ammonium lactate (minor). Things that cannot be assimilated: lactose, melibiose, inositol, D-(-)-arabinose, L-rhamnose, soluble starch, inulin, dextrin, cellulose, lipose, meso-erythritol, sodium acetate, sodium formate. When the above-mentioned properties of this strain were compared with those of the known bacterial species described in Roder's "The East (1970)", it was found that Torulopsis candida, Torulopsis dattila, and Torulopsis salmanchi. Many similarities were found with Torulopsis salmanticensis and others. However, when comparing physiological properties, Torulopsis candida and
Between TN1023, glucose, galactose,
Differences can be seen in the fermentability of sucrose and raffinose, lactose, L-arabinose, L-rhamnose, soluble starch, and vitamin requirement. Furthermore, between Torulopsis datuchira and TN1023, there is a difference in the fermentability of galactose and raffinose, D-
arabinose, cellobiose, D-xylose,
There are differences in DL-lactic acid, succinic acid, citric acid, vitamin requirements, osmotic pressure resistance, etc., and the differences in properties between Torulopsis salmanticensis and TN1023 are the fermentability of maltose and raffinose, lactose, melibiose, It was recognized for its ability to assimilate soluble starch and glycerol, require vitamins, and resist osmotic pressure. Therefore, we determined that this bacterial strain (TN1023 strain) is a new species of the genus Torulopsis.
Torulopsis uricoxidans (Torulopsis)
uricoxidans) TN1023. This strain has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under the name Torulopsis uricoxidans TN1023, and as Fiber Science and Technology Research Institute No. 5391. The microorganism used in the present invention may be any strain as long as it belongs to the genus Torulopsis and has the ability to produce uricase. An example of a suitable strain is Torulopsis uricoxidans TN1023.
(Feikoken Bibori No. 5391). The culture medium used in the present invention includes a carbon source, a nitrogen source,
Both synthetic and natural media can be used as long as they contain appropriate amounts of inorganic substances and other micronutrients required by the strain used. Carbon sources include glucose, fructose,
Sugars such as sucrose and molasses, alcohols such as ethanol, glycerol, sorbitol, and mannitol, α-ketoglutaric acid, citric acid,
In addition to organic acids such as malic acid and succinic acid, uric acid is also used. Nitrogen sources include ammonium chloride, ammonium sulfate, ammonium phosphate, urea, etc.
Amino acids such as L-glutamic acid, or inorganic or organic nitrogen compounds such as uric acid can be used. Furthermore, nitrogen-containing natural products such as peptone, meat extract, yeast extract, and corn steep liquor can also be used as nitrogen sources. As an inorganic substance,
For example, sodium chloride, potassium chloride, potassium phosphate, sodium phosphate, magnesium sulfate,
Ammonium sulfate, iron sulfate, cobalt sulfate, manganese sulfate, cobalt chloride, and the like are used alone or in combination. Other vitamins such as biotin and thiamin, and if necessary, adenine,
Nucleic acid-related substances such as uracil and orotic acid are used alone or in combination. Culture is usually carried out by shaking culture or aerated agitation culture. Culture temperature is 15-38℃, preferably 23-33℃
Let's do it. The pH of the medium is 2.0-7.5, preferably 4.0-
Perform within the range of 9.0. By culturing for 1 to 4 days, uricase is produced and accumulated in the culture, mainly in the bacterial cells. Uricase is collected from the culture as follows. After completion of the culture, the bacterial cells are obtained from the culture by centrifugation or the like, and then the bacterial cells are disrupted by an appropriate means, and a supernatant liquid is obtained from the crushed liquid by centrifugation or the like. The supernatant solution is treated by methods commonly used for enzyme purification, such as salting out, organic solvent precipitation, dialysis, ion exchange cellulose chromatography, sepadex chromatography, affinity chromatography, ultrafiltration, and freeze drying. . Purified uricase can thus be obtained. In addition, the measurement of uricase activity is an indicator of uric acid.
Absorption at 283 nm (molecular extinction coefficient, 1.238×
The activity was calculated by measuring the degree of decrease in 10 ⁴ ) due to oxygen reaction. (Unexamined Japanese Patent Publication 1973-
(Refer to Publication No. 119086) Enzyme activity was measured for 1 minute (37℃,
PH8.5) and the amount of enzyme that breaks down 1 μmole of uric acid.
The unit is (U). Next, the properties of uricase using the enzyme preparation obtained in Example 2 will be shown. (1) Optimal PH Enzyme preparations were adjusted to various pH levels (5.8, 6.0, 6.5, 7.0,
7.5, 8.0, 8.5, 9.0, 9.2) M/20 borax.
Dissolve in M/10KH ₂ PO ₄ buffer to a concentration of 0.02U/ml. Enzyme activity was measured using 0.5 ml of these enzyme solutions according to the method described above. The buffer in the reaction solution has a pH shown in Table 1.
In place of the 8.5 buffer, various pH buffers used to dissolve the enzymes described above were used. PH
When the activity at 8.5 is set as 100, the relationship between the activities at each PH is shown in Figure 1.
Optimal pH was found nearby. (2) Stable PH range In this test, the following two types of buffers were used to check the stable PH depending on the PH range. In other words, in the range of PH6.0 to 9.8, M/20
Borax M/10KH ₂ PO ₄ buffer from PH5.0
In the range of 6.0, use a buffer of M/40 borax/M/40 succinic acid, dissolve the enzyme preparation in each pH buffer to give a uricase activity of 0.2 U/ml, and incubate at 25°C for 20 hours. After keeping, add M/20 borax to 1 ml of these enzyme solutions.
Add 9ml of 10KH ₂ PO ₄ buffer (PH8.5) to adjust the pH.
was set at around 8.5. 0.5ml of these enzyme samples
Measure the enzyme activity by the method described above using
The results expressed in terms of relative activity are shown in Figure 2. This enzyme was stable in the pH range of 7.0 to 9.8. (3) Optimal temperature Dissolve the enzyme preparation in M/20 borax/M/10KH ₂ PO ₄ (PH8.5) to a concentration of 0.02U/ml. Set the reaction temperature to 20, 25, 30, 35, 37, 40, 45, 50,
55, 60, 65, 70℃. The optimal reaction temperature was determined by changing only the reaction temperature using the method for measuring uricase activity described above. The results are shown in Figure 3, and it was found that the optimum temperature was in the range of 37°C to 40°C. (4) Stable temperature range Dissolve the enzyme preparation in M/20 borax/M/10KH ₂ PO ₄ buffer (PH8.5) to a concentration of 0.02U/ml, and heat it to each temperature shown in Figure 4 for 10 minutes. After holding, enzyme activity was measured using 0.5 ml of each sample according to the method described above. Highly active value
Figure 4 shows the relative activity at each temperature when it is set to 100. This enzyme showed no inactivation even after treatment for 30 minutes at temperatures up to 55℃, and at 65℃ and 10 minutes.
Approximately 40% activity remained even after treatment for several minutes. (5) Substrate specificity Dissolve the enzyme preparation in M/20 borax/M/10KH ₂ PO ₄ buffer (PH8.5) to a concentration of 0.02U/ml. The amount of substrate reduction is measured in the same manner as the method for measuring uricase activity described above, except that the substrate shown in Table 1 is used. The maximum absorption wavelength of each substrate is used as the measurement wavelength for absorbance.
Table 1 shows the relative activity for each substrate, taking the activity when using uric acid as a substrate as 100.

[Table] Table 1 shows that this uricase has substrate specificity for uric acid. (6) Michaelis constant Dissolve the enzyme preparation in M/20 borax/M/10KH ₂ PO ₄ buffer (PH 8.5) to a concentration of 0.02 U/ml. Prepare substrate solutions of various concentrations. The measurement is performed in the same manner as the method for measuring uricase activity described above except for the substrate concentration. As a result of reciprocal plotting of the initial substrate concentration [S] and the obtained enzyme activity value (U) according to the Lineweaver-Burk method, the Michaelis constant Km (uric acid) was 9.52×10 ⁻⁶ M. (7) Isoelectric point The isoelectric point was investigated using the electrofocusing method. A sample of 11.04U (0.96mg protein) was used. PH3.5~10.0 using 110ml column
Ampholine (manufactured by Carrier Ampholite LKB) in the range of 2°C on a molasses gradient,
Electrophoresis was performed at 900V for 40 hours. The fractionation was carried out at 2 g per fraction at a rate of 120 ml/hr. As a result of examining the relationship between the PH gradient and activity of the fraction, it was found that the isoelectric point (Ip) of this enzyme was 9.45. Examples of the present invention will be shown below. Example 1 Torulopsis uricoxidans TN1023 (Feikoken Bibori No. 5391) is used as a uricase-producing bacterium. Glucose 2g/dl, Sodium chloride 0.05g/
dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O 0.05g/
dl, (NH ₄ ) ₂ SO ₄ 0.6g/dl, Corn Steep・
30ml of medium (PH5.5) consisting of 2g/dl of liquor
Pour into a ml Erlenmeyer flask and sterilize at 120℃ for 15 minutes. Separately, dissolve 6 g of urea in water, prepare the volume with water to 100 ml, sterilize this prepared solution at 120°C for 3 minutes, and then add 1 ml of the solution to 30 ml of the above sterilized medium. The proportion of urea in this medium is 0.2g/
It is dl. After preparing the medium, the bacteria are inoculated. Inoculation was carried out by taking one platinum loopful from an agar medium cultured for two days in advance and transferring it to the above medium. Culture is 240
Carry out 24 hours at 30°C in a rotary shearer at revolutions per minute. Next, 90 g of glucose, 60 g of corn steep liquor, 1.5 g of KCl, 6 g of Na ₂ HPO ₄ 12H ₂ O,
KH ₂ PO ₄ 3g, MgSO ₄・7H ₂ O 1.5g,
This medium (PH 5.5) containing 18 g of (NH ₄ ) ₂ SO ₄ and 1.5 g of urea dissolved to a pH of 3.0 is placed in a 5-volume jar fermenter. After sterilizing this at 120° C. for 30 minutes and cooling, the jar fermenter is inoculated with the above flask solution. Main culture is aeration amount 3/
minutes, stirring at 500 revolutions/minute and temperature at 30°C for 30 hours. After culturing, centrifuge to obtain 220 g (wet weight) of bacterial cells. Add M/40 borax, M/20KH ₂ PO ₄ to this bacterial body.
Wash by adding buffer (PH8.5) 5, and obtain bacterial cells by centrifugation. After suspending the bacterial cells in the above-mentioned buffer 1, the bacterial cells were crushed in a Dyno Laboratory Mill KDL type (manufactured by Willy A., Bachofen Inc., Switzerland). Obtain a cell suspension. Centrifuge the cell suspension to obtain a supernatant. As a result of measuring the uricase activity of this supernatant, it was found that this supernatant 1.2 contained about 98.65 U of uricase. Ammonium sulfate is added to this supernatant to make it 30% saturated with ammonium sulfate, and the precipitate is removed by centrifugation to obtain a supernatant. Add ammonium sulfate to 60% ammonium sulfate.
% saturation and collect the precipitate by centrifugation. Dissolve this precipitate in 180ml of M/40 borax/M/20KH ₂ PO ₄ buffer (PH8.5) and add 20
using cellophane tube as a dialysis membrane, 5
Perform dialysis overnight at °C. M/ the fluid inside the dialysis tube.
Charge a column packed with DEAE-Cellulose 1 equilibrated with 40KH ₂ PO ₄ buffer (PH8.5) and add M/40 Borax/M/
Wash with 20KH ₂ PO ₄ buffer (PH8.5) 1.
By this operation, most of the proteins in the sample are adsorbed to DEAE-cellulose, but the present uricase is not adsorbed and most of the uricase is recovered in the washing solution. This washing solution was fractionated into 20g portions,
Collect the uricase elution fraction and make it 60% saturated with ammonium sulfate.
Collect the precipitate by centrifugation. This precipitate is mixed with M/40 borax, M/20KH _2PO borax (PH
8.5) Dissolve in 60ml of the same buffer (PH8.5) 10
Dialysis is performed overnight at 5°C using cellophane tube as a dialysis membrane. This dialysis fluid was mixed with M/40 borax, M/20KH ₂ PO ₄ buffer (PH
Charge the column packed with 500 ml of DEAE-cellulose equilibrated with 8.5), and add 500 ml of the same buffer.
Wash with water to elute uricase. 20g of eluate
Collect the uricase fraction. Ammonium sulfate is added to this to make it 60% saturated, and the resulting precipitate is obtained by centrifugation. This precipitate was mixed with M/40 borax.
Dissolve in 20 ml of M/20KH ₂ PO ₄ buffer (PH8.5), charge to a column packed with 600 ml of Sephadex G-200, and elute with the same buffer. Fractionate the eluate into 20 g portions, collect the uricase active fractions (240 ml), and filter them using an ultrafilter (Bio Engineering Co., Ltd., MC-2A model) using an ultrafiltration membrane diafilter F-50T (Bio Engineering Co., Ltd.). The mixture was concentrated to 20 ml to obtain 4 mg of uricase. The activity yield from the supernatant obtained by centrifuging the cell suspension was 26.9%, and the specific activity was 6.6 U/mg protein. Example 2 Citric acid 4g/dl, corn steep liquor 0.5
g/dl, meat extract (Kyokuto Ehrlitsu) 0.5g/
dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O 0.05g/
dl, FeSO ₄・7H ₂ O1mg/, CoSO ₄・7H ₂ O1mg/
dl, NaCl 0.1g/dl, (NH ₄ ) ₂ SO ₄ 0.6g/dl (PH is 3.50 with NaOH) 300ml
Place in an Erlenmeyer flask and sterilize at 120℃ for 15 minutes. Torulopsis uricoxidans after sterilization
Inoculate with TN1023 (Feikoken Bacteria No. 55391). Inoculation was carried out by inoculating the cells, which had been cultured for two days in advance and grown on an agar medium, into the above medium after suspending them in sterilized water. Cultivation is carried out using a rotary machine running at 240 revolutions/minute.
Perform in a sheaker at 30℃ for 24 hours. This culture solution was transferred to a 30-volume jar fermenter prepared as follows. This medium contains 1.2 kg of citric acid, 100 g of corn steep liquor, 100 g of meat extract (Kyokuto Ehrlich), 20 g of KH ₂ PO ₄ , MgSO _{4.7H 2} O10
g, _FeSO4・_7H2O20mg , _MnSO4・_7H2O20mg ,
_CoCl2・_6H2O20mg , NaCl20g, uric acid 40g at 15
It was dissolved so that Adjust the pH of the medium with NaOH.
After adjusting the temperature to 3.50, transfer to a 30-volume jar fermentor and sterilize by blowing steam at 120℃ for 5 minutes. After cooling to 30°C, add sterilized water to make the volume of the medium 20°C, and inoculate the above seeds. Cultivation is carried out for 28 hours at an aeration rate of 10/min, stirring at 300 rpm, and a temperature of 30°C. After culturing,
The culture solution was centrifuged to obtain 1465 g (wet weight) of bacterial cells. The cells are washed by adding 10 times of M/40 borax/M/20KH ₂ PO ₄ buffer (PH8.5), and centrifuged to obtain cells. After suspending the cells in the same buffer (PH8.5) 6, the cell suspension was prepared using a DynoLaboratory Mill KDL type (manufactured by Willy A, Bachofen Inc., Switzerland). Centrifuge to obtain the supernatant. When the uricase activity of this supernatant was measured, it was found that this supernatant contained 1460 U of uricase. Ammonium sulfate is added to this supernatant to make it 30% saturated, and the precipitate is removed by centrifugation to obtain a supernatant. Add ammonium sulfate to 60% ammonium sulfate.
% saturation and collect the precipitate by centrifugation. This precipitate is dissolved in 360 ml of M/40 borax/M/20 KH ₂ PO ₄ buffer (PH 8.5) and dialyzed overnight at 5° C. in the same buffer 30 using a cellophane tube as a dialysis membrane. The solution inside the dialysis tube is charged to a column packed with DEAE-cellulose 2 equilibrated with M/40 borax/M/20KH ₂ PO ₄ buffer (PH8.5), and washed with the same buffer 2.
With this procedure, most of the proteins in the sample are DEAE
Although it is adsorbed to cellulose, this uricase is not adsorbed and is recovered in the washing solution. This cleaning solution is 20
Fractionate into g units, collect the uricase elution fraction, and add ammonium sulfate.
Bring to 60% saturation and collect the precipitate by centrifugation.
This precipitate is dissolved in 120 ml of the above buffer, and dialyzed using cellophane tube in the same buffer 10. After dialysis was performed overnight at 5°C,
The dialyzed solution is charged to a column packed with DEAE-cellulose 1 equilibrated with M/40 borax/M/20 KH ₂ PO ₄ buffer (PH8.5), and washed with the same buffer 1 to elute uricase. Fractionate the eluate into 20 g portions and collect the uricase fraction.
Add ammonium sulfate to achieve 60% saturation, and obtain the resulting precipitate by centrifugation. This precipitate is dissolved in 40 ml of M/40 borax/M/20 KH ₂ PO ₄ buffer (PH 8.5), charged to a column packed with Sephadex G150 1.2, and eluted with the same buffer. The eluate was fractionated into 20 g portions, the uricase fraction was collected (420 ml), and the uricase fraction was collected using an ultrafilter MC-2A type and MC.
-4A type (Bio Engineering Co., Ltd.) and concentrated to 28 ml using an ultrafiltration membrane die filter F-50T (Bio Engineering Co., Ltd.). This enzyme solution was charged to a column packed with 220 ml of hydroxyapatite equilibrated with M/50K ₂ HPO ₄ (PH8.0), washed with 450 ml of the same buffer, and then washed with M/50K ₂ HPO ₄ . 5K ₂ HPO ₄・M/
Elute at 5KH ₂ PO ₄ buffer (PH8.0). Fractionate the eluate into 20g portions and separate the uricase fraction (200ml).
was collected and concentrated to 16 ml using the ultrafilter above to obtain 15.4 mg of uricase. The activity yield from the supernatant obtained by centrifuging the cell suspension was 12.1%.
The specific activity is 11.5U/mg protein. Example 3 Uric acid (concentration shown in Table 2), glucose 3.0g/
dl, corn stave liquor 1.0g/dl,
KCl0.05g/dl, KH ₂ PO ₄ 0.1g/dl, Na ₂ HPO ₄・
12H ₂ O 0.2g/dl, (NH ₄ ) ₂ SO ₄ 0.6g/dl, meat extract 1.0g/dl, yeast extract 0.1g/dl, urea*0.2
Pour 50 ml of a medium (PH 5.5) consisting of g/dl into a 500 ml shake flask and sterilize at 120°C for 15 minutes. [*At this time, prepare a solution of 10 g/100 ml of urea, sterilize it at 120°C for 3 minutes, and then add 1 ml of each to the sterilized medium (50 ml)]. One platinum loop of Torulopsis uricoxidans TN1023 (Feikoken Bacteria No. 5391) was inoculated into each medium and cultured with shaking at 28°C for 48 hours. After the cultivation is completed, the culture is centrifuged (8000 rpm for 15 minutes) to obtain bacterial cells. Same amount of M/40 borax as the culture solution
Wash using 20KH ₂ PO ₄ buffer (PH8.5) and centrifuge again to obtain bacterial cells. 1g of the obtained wet bacterial cells and 10 glass beads (0.25-0.50mmφ)
g was suspended in 5 ml of the above boric acid buffer, and ground using a pressure-resistant glass test tube while cooling the surrounding area with ice water and stirring the inside with a high-speed stirring blade. Centrifuge the triturate (10,000 rpm for 10 minutes) to obtain a supernatant. The uricase activity in the supernatant is measured to obtain the results shown in Table 2.

[Table] As can be seen from Table 2, by adding uric acid to the medium, especially by adding 0.05 g/dl or more, uricase was produced in a significant amount in the supernatant.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between the relative activity of the present uricase and the optimum pH. Figure 2 shows the relationship between the relative activity of the present uricase and the stable PH range. However, ●-●: M/40 borax, M/20KH ₂ PO ₄ buffer, Γ-Γ: M/40 borax, M/40 succinic acid buffer. FIG. 3 shows the relationship between the relative activity of the present uricase and the optimum temperature. FIG. 4 shows the relationship between the relative activity of the present uricase and the treatment temperature.

Claims (1)

[Claims] 1. A method for producing uricase, which comprises culturing a microorganism belonging to the genus Torulopsis and having uricase-producing ability in a nutrient medium, producing uricase in the culture, and collecting the same.