JPS6020995B2 - 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 relates to a method for producing uricase, which comprises culturing a microorganism belonging to the genus Trichosborone and having a uricase production facility in a nutrient medium, producing uricase in the culture, and collecting the microorganism. Uricase (EC17 paper) is an enzyme that catalyzes the action of oxidatively hydrolyzing uric acid to produce allantoin, hydrogen peroxide, and carbon dioxide gas. This enzyme is used as a diagnostic reagent to measure the uric acid content in blood or urine, and is also attracting attention as a therapeutic agent for gout, arthritis, etc. caused by excessive accumulation of uric acid in the body. Conventionally, methods for producing IJ case by fermentation include Micrococcus spp., Brevibacterium spp.
Genus Candida (Japanese Patent Publication No. 42-5192), Genus Streptomyces (Ag ratio. Biol. Chem. Vol.
．． A number of methods are known that use microorganisms belonging to the genus Microorganisms, 1969) and others.

In addition, Japanese Patent Application No. 11296/1985, written by the same inventor as the present inventor, discloses a method for producing uricase using a microorganism belonging to the genus Enterobacter.

As a result of extensive searches for microorganisms capable of producing uricase, the present inventors discovered that a strain belonging to the genus Trichosporon can economically produce uricase in small amounts, and have completed the present invention.

The present invention will be explained in more detail below.

According to the present invention, when a microorganism belonging to the genus Trichosporon and having the ability to produce uricase is cultured in a nutrient medium, uricase is produced in the culture, and the microorganism is collected.

The microorganism used in the present invention may be any strain as long as it belongs to the genus Trichosporon and has the ability to produce uricase.

A suitable strain is Trichosporon cutanium (Tr
iCho Sporon CutaMum) KY5575 (
IF.

0714) Trichosporon heteromorphum (Tric
hosporonheteromorphum) ATC
Examples include C20001.

The nutrient tower used in the present invention can be either a synthetic medium or a natural medium, as long as it contains adequate amounts of carbon sources, nitrogen sources, inorganic substances, and other essential nutrients required by the strain used.

As carbon sources, carbohydrates such as glucose, fructose/sucrose, and honeydew, as well as uric acid, are used.

Ammonium chloride, ammonium sulfate,
In addition to ammonium phosphate, urea, etc., amino acids such as glutamic acid, or inorganic or organic nitrogen compounds such as uric acid can be used.

Furthermore, nitrogen-containing natural products such as veptone, meat extract, yeast extract, and corn stave liquor can also be used as nitrogen sources. In addition, nucleic acid-related substances such as guanine, adenine, hypoxanthin, xanthine, uracil, cytosine, orotic acid, inositol, thiamine, riboflavin, pyridoxine, nicotinic acid, para-aminobenzoic acid,
Vitamins such as calcium pantothenate and biotin are used alone or in combination.

As the inorganic substance, salts such as common salt, potassium chloride, phosphate, magnesium, calcium, potassium, iron, manganese, zinc, steel, molybdenum, cobalt, and boric acid are used as necessary.

In addition, organic or inorganic substances that promote the growth of this bacterium and the production of uricase can be appropriately added. in general,
Uricase produced by microorganisms is an inducible enzyme, and uricase will not be produced if uric acid, xanthine, or hypoxanthine is not present in the culture medium or induction reactor. In this respect, this bacterium produces a chopstick amount of uricase without adding uric acid or the like to the medium when the medium contains corn, stew, liquor, etc. as a natural nutrient source. On the other hand, the presence of uric acid in the medium clearly increases the production of uricase compared to the case where uric acid is not present. However, in this case as well, the effect is observed at a much lower uric acid concentration than in the case of bacteria described in Japanese Patent Application No. 11296/1983. Therefore, it is possible to produce uricase more economically than with conventional strains.

When using uric acid, uric acid may be present in the medium from the beginning or may be added during the culture.

A concentration of uric acid in the medium of 5 females/d' is sufficient. The culture is
Usually, this is done by wainawa culture or aerated rope azusa culture. The culture temperature is preferably 20 to 40°C, preferably around 30°C, and the pH of the culture medium is preferably in the range of 3.0 to 7.0, preferably 4.0 to 6.0.

The culture period is 1 to 5 days, usually 2 to 4 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.

After the cultivation is completed, the bacterial cells are obtained from the culture by centrifugation, etc., and then the bacterial cells are disrupted by an appropriate means, and the supernatant liquid is obtained from the crushed liquid by centrifugal separation, etc. The supernatant is treated by methods commonly used for enzyme purification, such as salt folding, organic solvent precipitation, dialysis, ion-exchange cellulose chromatography, 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.

The principle of the measurement method described below is to calculate the activity by measuring the degree of decrease in the absorption of uric acid at 28 kalpas (molecular extinction coefficient, 12 spots x 1 peak) due to the enzymatic reaction. It is described in. In order to further improve measurement accuracy, the present inventors modified this method to measure activity. 1 Wonhington t
electrical sheet, 1733 (1
964) 2 J. L. Nbkler. AMI. BiMh
em. , 38, 65 (1970), that is, it has the composition shown in Table 1 below.

The absorbance of each reaction solution is measured using Reaction Solutions 1, 0 and each control solution, and the absorbance difference due to the decrease in uric acid in the reaction solution is measured using Equation 1, which will be described later, and then the activity is calculated using Equation 2. Table 1 However, for convenience of explanation below, in Table 1, uric acid, 0
．． Composition S, consisting of 2M boric acid buffer (pH 8.0) and water, 0.2M boric acid buffer (pH 8.0)
0) and water is referred to as composition S2.

Next, a method for measuring the absorbance of each reaction solution using an actual enzyme reaction and calculating the absorbance difference and thus the activity will be explained below.
Measurement of absorbance of reaction solution 1: Add enzyme solution to composition S, and react at 370°C for 1q minutes.

After the reaction, IN hydrochloric acid. is added and the absorbance at 28 m is measured. Let this measured value be A. Measurement of absorbance of control solution for reaction solution 1: After adding IN hydrochloric acid to composition S, enzyme solution is added, and the absorbance on fabric 2 is measured.

Let this measured value be B. Absorbance measurement of reaction solution 0: Add enzyme solution to composition S2 and react at 370 for 10 minutes.

After the reaction, IN hydrochloric acid is added and the absorbance at 28 ml is measured. Let this measured value be C. Absorbance measurement of control solution of reaction solution 0: After adding IN hydrochloric acid to composition S2, enzyme solution was added,
Measure the absorbance in leg blood.

Let this measured value be D. From the measured values A, B, C, and D above, the absorbance difference (△E) due to the decrease in uric acid is calculated by the following formula.
1}.

That is, △E=(B-A)-(01C)...{11The purpose of measuring the absorbance of reaction solution B and its control solution is to measure the change in absorbance due to enzyme protein, etc. under the reaction conditions. This is an attempt to correct this and improve the accuracy of quantitative determination (reduction of uric acid). Oxygen activity (U/autumn) △E min x 103 x 3 x dilution factor
．． ．． ．． (2) 1.238 x 104 x 0.5 In the above formula, the enzyme activity is defined as the amount of enzyme that decomposes uric acid in moZe between each dressing as 1 unit (U).

In the above formula, △E: Absorbance difference due to decrease in uric acid 3: Reaction liquid volume (secretion) dilution factor: Enzyme dilution ratio 0.5
: Volume of enzyme liquid used (ship).

Therefore, when expressing the activity per 100 and (d'), the value calculated by the above formula is multiplied by 100. The preferred range of uric acid concentration in the medium is shown below using experimental examples.

Experimental example 1 Uric acid (concentration shown in Table 2), glucose 5.0 tano d'
, Corn Steve Liquor 20 evening/d [, KC So.
05 evening/d', Na2HR04, 12th 20 0.2 evening/d', KH2P04 0.1 evening/d', M is Q, 7th 20 0.05 evening/d', (NH4)2SQ O. A medium (pH 5.
5) Put 50' of 500' into a flask and add 12
Sterilize at 0:00 for 18 minutes.

[*At this time, prepare a solution of 10/100 urea,
After sterilizing at 120 qo for 3 minutes, add 1 part of each to the above sterilized medium (50')]. Trichosporon in each medium
One platinum loop of Cutanium moo174 was inoculated, and cultured at 30:00 for 6-striped horses. After the cultivation is completed, the culture is centrifuged (800 pm for 15 minutes) to obtain bacterial cells. The cells are washed using the same amount of 0.08M boric acid buffer as the culture solution, and centrifuged again to obtain bacterial cells.

The obtained green bacterial cells and glass beads (0.25 to 0.5
The suspension was suspended in the above-mentioned 5M boric acid buffer, and the tube was ground using a pressure-resistant glass test tube while the surrounding area was cooled with ice water and the inside was stirred with a high-speed fly blade. This fly is equipped with a universal homogenizer (
A handmade polyurethane blade (2 廇岬◇) was attached to the tip of the homogenizer shaft of the homogenizer (manufactured by Nippon Seiki KK), and the rotation speed was set at 2000 to 300 pm. The lysate was centrifuged (1000 pml 0 min) to obtain the supernatant fluid. The supernatant was used as an enzyme solution, which was diluted with the buffer shown above to an appropriate enzyme concentration and used for measuring enzyme activity.
The results are shown in Table 2. As shown in Table 2, Experimental Example 1, the production of IJ case by this bacterium is such that in the above-mentioned medium, even without the addition of uric acid, a chopstick amount of uricase is produced, and by further adding uric acid, the production of IJ case is suppressed. An increase was observed.

However, it was found that sufficient effects were obtained when the concentration of uric acid added was 0.005 m/d'. Next, the properties of uricase using the enzyme preparation obtained in Example 2 will be shown.

'11 to pH Enzyme preparations were prepared at various pH levels (6.0, 6.5, 7.0, 7.
2,7.5,7. & 8.0, 8.2, 8.5, 9.0
,9. 9.8) in 0.2M boric acid buffer.
Dissolve to a concentration of 15 U/'.

Enzyme activity was measured using 0.5 of these enzyme solutions according to the method described above. The buffer in the reaction solution is the first
The pH 8.0 buffer shown in the table was replaced with the various bait buffers used to dissolve the enzymes described above. pH
When the activity at pH 8.0 is set as 100, the relationship between the activities in each state is shown in Figure 1. The optimum pH was found near the river. ■ Stable pH range Stable pH in this test
The following two types of buffers were used depending on the pH range as pH-maintaining buffers for investigating H.

That is, in the range of pH 60 to 9.8, the buffer is 0.029M borax - 0.1M boric acid - 0.02 aC, and in the range of pH 5.0 to 6.0, it is 0.029M boric acid -
Using a 0.025 1 M succinic acid buffer, the enzyme preparation was dissolved in each pH buffer to give a uricase activity of 15 U/', kept at 25 qo for 2 hours, and then dissolved in one of these enzyme solutions. 0.1M borax - 0.4
M boric acid-0.1M NaC buffer (pH 8.0
)9 was added to adjust the pH to around 8.0. Enzyme activity was measured using 0.5 mm of these enzyme samples by the method described above, and the results expressed as relative activity are shown in FIG. This enzyme was most stable between pH 7.6 and 9.3. [3
} Dissolve the optimal temperature enzyme preparation in 0.2M borate buffer (pH 8.0) to a concentration of 0.15U/'.

Reaction temperature 20, 25, 30, 35, 37, 40, 45
, 50, 55, 60, 65 and 70qo. Using the method for measuring uricase activity described above, only the reaction temperature was changed to find the lowest reaction temperature. The results are shown in Figure 3.
The optimum temperature was found to be in the range of 370 to 4020 degrees Celsius. (2) Dissolve the substrate-specific enzyme preparation in 0.2M borate buffer (pH 8.0) at a concentration of 0.15U/secretion.

The above-mentioned IJ except that those shown in Table 3 are used as substrates.
The amount of substrate reduction is measured in the same manner as the method used to measure case activity. The maximum absorption wavelength of each substrate is used as the measurement wavelength for absorbance. The results are shown in Table 3. The results in Table 3 indicate that the present uricase has substrate specificity for uric acid.

(51 Dissolve the Michaelis constant enzyme preparation in 0.2M borate buffer (pH 8.0) to a concentration of 0.15U/'.

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. The initial substrate concentration [S] and the obtained enzyme activity value [U] are calculated using Linewea.
As a result of reciprocal plotting according to the method of ver-Burk, the Michaelis constant km (uric acid) is 8.93 × 10-
It turned out to be 6M.

Examples are shown below. Example 1 Using Trichosporon cutanium mCO174 as a uricase producing strain Glucose 5/d and Corn Steve Liquor 3/d
Also 〇0.2 Tano, KH2P040.1 evening/d', M on 04.7th 200.05 evening/d', (N day) 040.
A medium (p
H5.5) Put 30' of 30 ribs into an Erlenmeyer flask,
Sterilize at 120°C for 18 minutes.

Separately prepare a 6/100% solution of urea, sterilize it at 120°C for 3 minutes, and then add 1% to the above sterilized medium.

The rate of urea in this medium is 0.2/d.
After preparing the medium, the bacteria are inoculated.

Inoculation was carried out by taking one platinum loop from an agar plate that had been cultured for two days in advance and transferring it to the above medium. Culture at 240 revolutions/
Incubate on a rotary shaker for 42 hours at 30°C. This culture solution was transferred to a 5-capacity jar fermentor prepared as follows. This medium contains 150 glucose,
Corn Steve Liquor 60 evenings, KC evening 1.5 evenings, N
a2HP04/12 also 06 evening, K mother P043 evening, MgS
On the evening of April 7th, 201.5 L (NA) evening, on the evening of 0418, a medium (pH 5
．． Place 5) in a 5-volume jar and fermenter at 120°C.
Sterilize for 20 minutes. In addition, dissolve 60% of urea to a concentration of 100% and sterilize it at 120% for 3 minutes. After sterilizing the main tower area, this urea solution is added, and the above seed culture solution 30' is transferred. Cultivation in a 5-capacity jar fermenter was carried out for 4 hours at an aeration rate of 3 degrees/minute, a rotation rate of 500 revolutions/minute, and a temperature of 30°C.

After culturing, centrifugation is performed to obtain bacterial mass (wet weight). The cells are washed with 5 ml of 0.05M boric acid buffer (pH 8.0), and the cells are obtained by centrifugation. After suspending the bacterial cells in 1 volume of 0.09M boric acid buffer (pH 8.0), they were suspended in a Dyno Laboratory Mill (Dyno Laboratory Mill) KDL type (Wmy A, Bac.
(manufactured by Hofen Inc, Switzer Sagind) to obtain a crushed bacterial cell solution. Centrifuge the cell suspension to obtain a supernatant solution. The uricase activity in this superfluous fluid was measured and converted to the production amount per ld' of the culture fluid, giving a value of 60.57 U/d'. (
Contains 1.2 U of total activity and 5 U of uricase).
Ammonium sulfate was added to this supernatant to make ammonium sulfate 30% saturated, the precipitate was removed by centrifugal D separation to obtain a supernatant, further ammonium sulfate was added to make ammonium sulfate 60% saturated, and the precipitate was removed by centrifugation. collect. This precipitate was mixed with 0.09M boric acid buffer (P
H8.0), and dialysis was performed overnight at 5°C using 200ml of the same buffer and using a cellophane tube as a dialysis membrane. The fluid in the dialysis tube was charged to a column packed with DEAE-cellulose 1, which had been equilibrated with 0.09 M boric acid buffer (pH 8.0), and after washing with 1 sleeve of 0.09 M boric acid buffer (pH 80), sodium chloride was added. Buffer containing 0.2 M sodium chloride (pH 80) without the above buffer (pH 8.0) 1.5 m
1.5 Perform sleeve concentration phantom elution. The eluate is fractionated into 20 fractions, the uricase eluted fractions are collected and saturated with ammonium sulfate to 60%, and the precipitate is collected by centrifugation. This precipitate was 0.
Dissolve in 08M boric acid buffer (pH 80) of about 100% and perform dialysis overnight at 5°C using the same buffer (solution 8.0) 10Z using a cellophane tube as a dialysis membrane. This dialyzed fluid was mixed with 0.09M borate buffer (p
DEAE-Sephadex A- equilibrated with H8.0)
After washing with the same buffer IZ, the same buffer 1.2 hours (without NaCZ, pH 8.0) and the same buffer 1.2 hours (without NaCZ, pH 8.0) and 0.2 hours of the same buffer (pH 8.0) were charged. Elution is performed using a concentration gradient (pH 8.0). The eluate is fractionated 20 times each and the uricase active fraction is collected. Add ammonium sulfate to this to make it 60% saturated,
The resulting precipitate is obtained by centrifugation. This precipitate is 5
Sephadex G- dissolved in 0.0' of 0.09K borate buffer (PH8.0) and equilibrated with the same buffer.
Charge a column packed with 200500 and elute with the same buffer. The eluate was fractionated 20 times each, and the uricase active fractions were collected and lyophilized to obtain uricase powder. The activity yield from the supernatant obtained by centrifuging the cell suspension was 10.7%, and the specific activity was 9.7 U/imitation protein. Example 2 Glucose 5.0 t/d', corn stave 1'car 2.0 t/d[, KC so 0.05 t/d', NaHP
〇4-12LOO. 1 Kuno d', MgS04・740
0.05 evening/d and (NH4) ○40.5 evening/d',
A medium (pH 5.50) consisting of uric acid at 0.005 m/d and urea at 0.3 m/d' (pH 5.50) was placed in a 500 Z customer flask and sterilized for 15 minutes at 120 m/d.

Urea was prepared separately in a solution of 10/100 °C and heated at 120 °C.
After sterilization for 3 minutes at o, add one secretion of each to the above medium (50 ml). The proportion of urea in each medium is 0.3/d'. One platinum loop of the bacterial strains shown in Table 4 grown on agar plate was inoculated into each medium. The culture time was set to muscle time, and the culture was carried out as described below. Culture temperature is 30qo, Shinkaku is 1
The test was carried out using a reciprocating box shaker with a main return rate of 4/min.

After the culture was completed, the bacterial cells were collected and ground to obtain an enzyme solution (supernatant) according to the method described above. The uricase activity in these enzyme solutions was measured by the method described above, and the results shown in Table 4 were obtained. This result indicates that many strains belonging to the genus Trichosporon have uricase productivity similar to that of the genus Trichosporon. Table 4 A simple explanation of the production diagram of ulj case by Trichosporon sp. strain. Figure 1 shows the relationship between the relative activity of this uricase and pH.

Figure 2 shows the relative activity and stable pH range of the present uricase. FIG. 3 shows the relationship between the relative activity of the present uricase and temperature. Elephant' Ranta Z1 Genta 3 Powder

Claims (1)

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