JP5053648B2 - Method for producing new uricase - Google Patents

Description

  The present invention relates to a novel method for producing uricase. More particularly, the present invention relates to a method for producing a novel uricase derived from a microorganism belonging to the genus Brevibacterium having excellent heat resistance.

Uricase (EC 1.7.3.3) decomposes uric acid into allantoin, hydrogen peroxide, and carbon dioxide, ie, catalyzes the reaction of uric acid + O ₂ + 2H ₂ O → allantoin + CO ₂ + H ₂ O _2. It has an enzymatic degradation activity. This uricase is widely present in various animal tissues (eg, liver, kidney) and microbial tissues. Currently, uricase is used as an enzyme for measuring uric acid present in blood or urine for the purpose of clinical diagnosis of various diseases caused by uric acid accumulation in the human body.

Uricase production by microorganisms is widely performed (see, for example, Patent Documents 1 to 3). Patent Document 1 proposes a yeast belonging to the genus Torlopsis that produces a significant amount of uricase. Patent Document 2 discloses Micrococcus roseus, which produces a large amount of uricase, and it has been found that the uricase produced by the microorganism is close to the pH value of a physiological fluid. Patent Document 3 discloses that Bacillus sp. TB-90 strain produces uricase derived from a thermophilic microorganism having a wide action pH range.
Japanese Patent Laid-Open No. 56-124381 JP 57-115177 A JP 61-280272 A

  However, Torropsis uricoxidans TN1023 described in the above-mentioned Patent Document 1 (Mikken Kenki No. 55391) collects uricase from the culture and obtains the microbial cells from the culture by centrifugation or the like. Then, this bacterial cell was crushed by an appropriate means, and an enzyme purified solution was obtained from the crushed liquid (line 5 from the lower right column on page 3 to line 4 on the upper left column on page 4). Have been accumulated. Similarly, also in Patent Document 2, cells are collected by centrifugation in Example 1 and the enzyme is obtained from this cell paste (from the lower left column, line 6 to the upper right column, page 3). Is accumulated in the fungus body. Similarly, Patent Document 3 also describes that uricase is mainly produced in bacterial cells (2 to 1 line from the lower left column on page 4). That is, since all of the above Patent Documents 1 to 3 accumulate uricase in the microbial cell, in order to obtain a desired enzyme, the microbial cell must be physically disrupted and the enzyme must be purified from the crushed material. In addition, many purification steps are required, and the microbial cell disruption contains a large amount of cell components in addition to the desired uricase, so the purification step for obtaining pure uricase is very complicated / complex. There is a problem.

  Therefore, the present invention has been made in view of the above circumstances, and an object thereof is to provide a novel method for producing uricase having excellent heat resistance.

  Another object of the present invention is to provide a bacterium that produces and accumulates uricase excellent in heat resistance outside the cell.

  As a result of intensive studies in order to achieve the above-mentioned problems, the present inventors have extensively searched for microorganisms having a uricase-producing ability excellent in heat resistance. As a result, the uricase produced by the bacteria belonging to Brevibacterium (U-344 strain) isolated from the natural world (soil) is superior in heat resistance to the known uricase, and the bacterium is contained in the culture solution (bacteria). The uricase was produced and accumulated in vitro, and the uricase produced outside the fungus body was found to maintain an enzyme activity of 95% or more with respect to heat treatment at 50 ° C. for 20 minutes, thereby completing the present invention. .

  That is, the above object is a step (1) of culturing a microorganism belonging to the genus Brevibacterium and having the ability to produce uricase, and a step of collecting uricase from the culture obtained in the step (1) ( 2), and achieved by a method for producing uricase.

  The uricase of the present invention is excellent in heat resistance. In particular, among the uricases according to the present invention, uricase produced / accumulated outside the cells is hardly inactivated even when heat-treated at 50 ° C. for 20 minutes, and is excellent in heat resistance. Therefore, the enzyme product using the uricase of the present invention has good storage stability, and the enzyme product in clinical diagnosis can be stored for a long time.

  Hereinafter, the present invention will be described in detail.

  The present invention includes a step (1) of culturing a microorganism belonging to the genus Brevibacterium and having an ability to produce uricase (herein, also simply referred to as “uricase producing bacteria”), and the step (1). And a step (2) of collecting uricase from the culture obtained in (1) above, to provide a method for producing uricase.

  The uricase-producing bacterium used in the present invention is not particularly limited as long as it produces uricase having the above properties, but preferably belongs to the genus Brevibacterium (hereinafter omitted in parentheses), More preferably, it is Brevibacterium sp. U-344 FERM P-21097 strain (hereinafter referred to as “U-344 strain”). As long as the bacterium has the ability to produce uricase, the bacterium may exist in nature or may have been mutated by artificial genetic manipulation. The uricase produced by the bacterium may be accumulated in the microbial cell or secreted into or out of the microbial cell. However, considering simplification of the enzyme purification process, the uricase is preferably extracellular ( Produced and accumulated in the culture medium). Further, the uricase produced outside the cells can maintain an enzyme activity of 95% or more with respect to heat treatment at 50 ° C. for 20 minutes.

  Here, the U-344 strain will be described in detail.

  The U-344 strain is a bacterial species isolated by the present inventors from the soil in Gifu Prefecture by the enrichment culture of a medium using uric acid as the only carbon source. Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number FERM P-21097.

  The U-344 strain has the following (a) morphological properties, (b) culture properties, (c) physiological properties, and (d) other properties. In the following table, “+” indicates positive, “−” indicates negative, and “w” indicates that the reaction is weak.

  As a result of homology search against the bacterial reference strain database using BLAST, the 16S rDNA base sequence of U-344 strain showed high homology to 16S rDNA derived from Brevibacterium, with a homology rate of 98.6%. It showed the highest homology to 16S rDNA of Brevibacterium epidermidis NCDO2286 strain (currently NCIMB702286 strain). Also in the results of the homology search for GenBank / DDBJ / EMBL, the 16S rDNA of this strain shows high homology to the 16S rDNA derived from Brevibacterium, and the reference strain has a homology rate to the 16S rDNA of Brevibacterium epidermidis NCDO2286 strain. It showed 98.6% homology. In addition, as a result of a simple molecular phylogenetic analysis using 16S rDNA of U-344 strain and 16S rDNA of the top 30 homology search against the bacterial reference strain database, this strain was found in the cluster formed by Brevibacterium 16S rDNA. And formed a phylogenetic branch with Brevibacterium epidermidis 16S rDNA. However, the two 16S rDNAs are not completely the same, and when comparing the strains of this strain and Brevibacterium epidermidis and the various branches of Brevibacterium formed around it, the strain is found to be of Brevibacterium epidermidis. It is unlikely to be the same.

  This strain is a Gram-positive rod that has no motility, and exhibited a “rod-coccus cycle” whose morphology changes with the passage of time. The colony color on the Nutrient agar plate medium was orange, the catalase reaction was positive, and the oxidase reaction was negative. It exhibited pyrazine amidase and alkaline phosphatase activity and hydrolyzed gelatin but did not ferment sugars. In addition, it did not grow under anaerobic conditions and hydrolyzed casein. Although most of these properties are similar to those of Brevibacterium epidermidis, the color of orange is not considered because Brevibacterium epidermidis, which was suggested to be related in the results of 16S rDNA nucleotide sequence analysis, does not produce a specific pigment. This is different from Brevibacterium epidermidis in that it produces the above pigment.

  From the above 16S rDNA nucleotide sequence analysis results and various properties, this strain is included in Brevibacterium and is considered to be closely related to Brevibacterium epidermidis, but has the results of 16S rDNA nucleotide sequence analysis and pigment production ability Since a difference was recognized in this respect, this strain was judged to be a novel strain and named Brevibacterium sp. U-344. Accordingly, the second aspect of the present invention provides a Brevibacterium sp. U-344 FERM P-21097 strain capable of producing uricase. This U-344 strain was dated November 17, 2006, Tsukuba Center 1-1-1 Tsukuba City, Ibaraki Prefecture, Japan (Postal Code 305-8666) National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Deposited at the center as FERM P-21097.

(Method for producing uricase derived from Brevibacterium)
The uricase of the present invention can be produced from uricase-producing bacteria by commonly used culture means. Hereinafter, as one embodiment, a method for producing uricase using bacteria belonging to the genus Brevibacterium will be described.

  The medium used in the production method is not particularly limited as long as bacteria belonging to the genus Brevibacterium grow, and may contain carbon sources, nitrogen sources, inorganic substances, and other micronutrients required by the strain used. For example, either a synthetic medium or a natural medium can be used. The medium may be a solid or liquid medium.

  Any carbon source may be used as long as bacteria belonging to the genus Brevibacterium can grow well and produce a desired uricase. Specifically, sugars such as glucose, sucrose and galactose; alcohols such as ethanol, glycerol and sorbitol; organic acids such as citric acid, malic acid and succinic acid; starch or a fraction thereof, roasted dextrin, and processing Examples thereof include carbohydrates such as starch, starch derivatives, physically treated starch, and α-starch. Uric acid or the like may be used as a carbon source. Preferably, sugars such as glucose, sucrose and galactose, alcohols such as ethanol, glycerol and sorbitol, organic acids such as citric acid, malic acid and succinic acid, and uric acid are used. These carbon sources can be used alone or in the form of a mixture of two or more.

  As the nitrogen source, those generally used for culture of bacteria belonging to the genus Brevibacterium can be used, for example, ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, L-glutamic acid, etc. Inorganic or organic nitrogen compounds such as uric acid can be used. Furthermore, nitrogen-containing natural products such as peptone, polypeptone, meat extract, yeast extract, soybean hydrolysate, soybean powder, milk casein, casamino acid, corn steep liquor and the like may be used as the nitrogen source. Among these, amino acids such as ammonium chloride, ammonium sulfate, urea, and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid, nitrogen-containing natural products such as peptone, meat extract, and yeast extract are preferable. These nitrogen sources can be used alone or in the form of a mixture of two or more.

  As the inorganic substance, the same ones that are generally used for culture of bacteria belonging to the genus Brevibacterium can be used, for example, sodium chloride, potassium chloride, calcium chloride, potassium phosphate, sodium phosphate, magnesium sulfate, Phosphate, hydrochloride, sulfate, acetate, etc. such as magnesium sulfate, magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc are used. In addition, vitamins such as thiamine and biotin, and if necessary, nucleic acid-related substances such as adenine and uracil may be used. These inorganic substances can be used alone or in the form of a mixture of two or more.

  Bacteria belonging to the genus Brevibacterium are cultured under normal aerobic conditions, usually with shaking culture or with stirring culture. The culture conditions at that time are appropriately selected depending on the composition of the medium and the culture method, and are not particularly limited as long as the strain can grow and the uricase, which is the target enzyme, can be efficiently produced without being inactivated. Usually, the culture temperature is 25 to 45 ° C. The pH of the medium is in the range of 5.0 to 9.0, preferably 6.0 to 8.0. The culture time is usually 20 to 72 hours, preferably 24 to 48 hours. In the present invention, uricase is produced / accumulated in the culture solution and / or cells of bacteria belonging to the genus Brevibacterium obtained as described above, and is a particularly preferred embodiment of the present invention. When the U-344 strain is used, uricase produced in the culture solution of the U-344 strain is preferable because of excellent heat resistance, as described in detail in the following examples. In addition, the uricase produced in the culture solution as described above can be applied to a known purification method as described below without removing the culture solution, and also removes many impurities generated by disrupting the cells. Since a process is unnecessary, it is preferable also from the viewpoint of simplification of the purification process.

  The method for isolating the uricase of the present invention is described below.

  When the produced uricase is accumulated or secreted in the microbial cells, the microbial cells are recovered from the culture solution by centrifugation after completion of the culture, and then uricase is removed from the microbial cells by an appropriate means (for example, Triton X-100 etc.). After processing this extract by centrifugation, etc. to remove insoluble components, acid precipitation, organic solvent precipitation (for example, solvent precipitation with ethanol, acetone, etc.), salting out (for example, salting out with ammonium sulfate), dialysis, various types Enzyme purification methods commonly used by those skilled in the art, such as chromatography (eg, ion exchange chromatography, Sephadex chromatography, affinity chromatography, gel filtration chromatography, hydrophobic chromatography, etc.), ultrafiltration, lyophilization, electrophoresis, etc. The uricase with high purity can be obtained by purifying by the above. The degree of purification of the uricase can be confirmed by subjecting the obtained uricase to electrophoresis, and a highly purified uricase can be obtained by purifying the uricase until it becomes single electrophoretically. Alternatively, after completion of the culture, the cells are collected from the culture solution by filtration or centrifugation, and then the cells are collected by appropriate means (for example, ultrasonic, self-digestion, physical disruption using glass beads, etc.) To obtain a cell lysate, and a supernatant is obtained from the crushed material by centrifugation or the like. A highly purified uricase may be obtained from this supernatant by using the same purification method as described above.

  When the produced uricase is secreted outside the cells (in the culture), the cells are removed from the culture solution by filtration or centrifugation to obtain a filtrate or supernatant. A highly purified uricase can be obtained by directly applying the same purification method to the filtrate or supernatant thus obtained.

  As described above, the uricase obtained by the production method of the present invention can be used as an enzyme for measuring uric acid present in blood or urine for the purpose of clinical diagnosis of various diseases caused by uric acid accumulation in the human body. At this time, the uric acid quantification method using uricase includes the following methods.

(I) Method for measuring the amount of hydrogen peroxide produced by reacting uric acid and uricase At this time, examples of methods for measuring the amount of hydrogen peroxide include the following methods (a) to (c):
(A) Hydrogen peroxide is reacted with 4-aminoantipyrine and phenol or its derivative in the presence of peroxidase, and the dye produced in proportion to the amount of hydrogen peroxide is quantified from the absorbance (uricase in the enzyme colorimetric method).・ Perosidase method) method;
(I) a method in which hydrogen oxide and alcohol are reacted in the presence of catalase, and the resulting aldehyde is condensed with acetylacetone and ammonia to determine the amount of dye produced from the absorbance (uricase-catalase method in the enzyme colorimetric method); and (C) The reaction product aldehyde of the above (a) catalase is reacted with reduced nicotinamide adenine dinucleotide (NADH) in the presence of alcohol dehydrogenase to produce NAD, and the decreased NADH is quantified. (Ultraviolet absorption method)

  (Ii) A method of measuring oxygen consumed when reacting uric acid with uricase or generated carbon dioxide (electrode method).

  (Iii) A method for measuring a difference in ultraviolet absorption derived from uric acid before and after the reaction (ultraviolet absorption method).

  (IV) Chemical uric acid determination method (for example, phosphotungstic acid method) for uricase-treated and untreated samples.

  Any of the above methods can be applied to the uricase obtained by the production method of the present invention, but it can be preferably used in the method (i) (a).

  EXAMPLES The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited by the examples.

Example 1
(1) Culture of Brevibacterium sp. U-344 strain The following composition: Tryptone (BD (Becton, Dickinson and Company)) 1% (w / v), yeast extract (BD (Becton, Dickinson) and Company) 0.1% (w / v), sodium chloride (manufactured by Kanto Chemical) 0.5% (w / v), monopotassium phosphate (manufactured by Kanto Chemical) 0.2% (w / v) ), A culture solution composed of 0.2% (w / v) uric acid (manufactured by Wako Pure Chemical Industries, Ltd.), pH was adjusted to 7.0 with NaOH, and sterilized at 121 ° C. for 20 minutes.

  Next, 5 mL of the obtained culture solution was dispensed into a sterilized L-shaped test tube. Inoculate the U-344 strain previously cultured in a bouillon medium containing 1.5% agar into the culture solution of the above L-shaped test tube, and cultivate for 24 hours while stirring at 30 ° C at a speed of 120 rpm. went.

(2) Collection of enzyme The culture solution obtained in (1) above was centrifuged and the cells were removed to obtain an extracellular enzyme solution (total uricase activity 0.7 U).

  In addition, 5 mL of phosphate buffer (10 mM, pH 7) and 3 g of glass beads were added to the cells and mixed, and the cells were shaken for 3 minutes to physically disrupt the cells. The supernatant was recovered by centrifugation of this crushed cell body, and this supernatant was used as an intracellular enzyme solution (total uricase activity 1.8 U).

  In this example, the activity of uricase was measured by the following method.

(Evaluation Example 1) Method for measuring uricase activity The activity of uricase was measured by using uricase peroxidase method of 4-aminoantipyrine and TODB (N, N-bis (4-sulfobutyl) -3-methylaniline) at 550 nm. The oxidative condensate quinone dye was measured with a spectrophotometer. More specifically, 0.83 mM uric acid solution 120 μl, 75 mM TODB 5 μl, 75 mM 4-aminoantipyrine 5 μl, 80 U / ml peroxidase 5 μl, enzyme solution 65 μl were mixed and reacted at room temperature (25 ° C.) for 1 hour. Absorbance at 550 nm was measured. At this time, the unit of uricase was defined as “1 U (unit)” as the amount (titer) of the enzyme that decomposes 1 micromole of uric acid per minute under the above test conditions.

(Evaluation example 2) Temperature stability of enzyme As a sample, the extracellular enzyme solution and the intracellular enzyme solution obtained in Example 1 (2) were used.

  After maintaining the uricase preparation of the present invention at various temperatures (20 ° C., 30 ° C., 40 ° C., 50 ° C., 60 ° C.) for 20 minutes, the uricase activity was measured according to the method described in Evaluation Example 1 above. Relative residual activity at each temperature was calculated with the activity after holding at 20 ° C. for 20 minutes as 100, and the result is shown in FIG. From FIG. 1, both the intracellular uricase (in the figure, intracellular enzyme) and the extracellular uricase (in the figure, extracellular enzyme) obtained by the production method of the present invention were subjected to heat treatment at 40 ° C. for 20 minutes. However, the activity exceeding 80% is maintained, and both have excellent heat resistance. Also, from FIG. 1, the extracellular uricase of the present invention (extracellular enzyme in the figure) exhibits better heat resistance than the intracellular uricase of the present invention (intracellular enzyme in the figure). At 50 ° C. or lower, no deactivation was observed for 20 minutes of heat treatment, and the activity was maintained at almost 100%, and in particular, enzyme activity of 95% or more was maintained for 50 minutes at 50 ° C. .

It is a figure which shows the result of the evaluation example 2.

Claims (3)

A step (1) of culturing a microorganism belonging to the genus Brevibacterium and having the ability to produce extracellular uricase in the absence of a surfactant;
A step (2) of collecting uricase from the culture medium obtained in the step (1);
Only including,
The method for producing uricase, wherein the microorganism belonging to the genus Brevibacterium is Brevibacterium sp. U-344 FERM P-21097 strain .   The production method according to claim 1, wherein the uricase maintains an enzyme activity of 95% or more with respect to a heat treatment at 50 ° C for 20 minutes.   Brevibacterium sp. U-344 FERM P-21097 strain capable of producing extracellular uricase.

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