JP5010291B2 - Method for producing new uricase - Google Patents

Description

  The present invention relates to a novel method for producing uricase. More specifically, the present invention relates to a method for producing a novel uricase derived from a microorganism belonging to the genus Cellulosimicrobium 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, the uricases obtained in Patent Documents 1 and 2 are both unstable at high temperatures (60 ° C. or higher). The uricase obtained in Patent Document 3 is derived from a thermophilic microorganism, but inactivation occurs when the temperature exceeds 50 ° C., and the activity is only maintained at about 50% around 60 ° C. (see FIG. 4). ). This can cause a reduction in the quality of the stored enzyme product in clinical diagnosis.

  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.

  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, it was found that uricase produced by bacteria (U-647 strain) belonging to Cellulosimicrobium hunkei isolated from nature (soil) is superior in heat resistance to known uricase, and the present invention It came to be completed.

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

  The uricase of the present invention does not inactivate even when heat-treated at 60 ° C. for 20 minutes and is excellent in heat resistance. Therefore, the storage stability of the enzyme product is improved, and the enzyme product can be stored for a long time in clinical diagnosis.

  Hereinafter, the present invention will be described in detail.

  The microorganism having the ability to produce uricase used in the present invention is not particularly limited as long as it is a genus of Cellulosimicrobium that produces uricase (hereinafter omitted in parentheses), but more preferably, cellulose. Cellulosimicrobium funkei (hereinafter omitted in parentheses), more preferably Cellulosimicrobium funkei U-647 FERM-P21098 (hereinafter referred to as "U-647 strain") It is a 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 cells or secreted into or out of the microbial cells.

  Here, the U-647 strain will be described in detail.

  The U-647 strain is a bacterial species isolated by the present inventors from soil in Gifu Prefecture by means of an accumulation culture of a medium using uric acid as the sole carbon source. Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the deposit number FERM-P21098.

  The U-647 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-647 strain showed high homology to 16S rDNA derived from Cellulosimicrobium, and the homology rate was 99.8. % Showed the highest homology to the 16S rDNA of the Cellulosimicrobium cellulans DSM 43879 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 cellulosimicrobium, and the reference strain is Cellulosimicrobium funkkei. ) High homology with 99.9% homology with 16S rDNA of ATCC BAA-886 strain. As a result of simple molecular phylogenetic analysis conducted by adding 16S rDNA of Cellulosimicrobium funkei to 16S rDNA of the top 30 strains of homology search against 16S rDNA of this strain and the bacterial reference strain database, It formed a phylogenetic branch almost identical to Um funky 16S rDNA and was shown to be very closely related. There is a one base difference between the 16S rDNAs of both, but the difference is due to the mixed bases, which is considered to be almost completely the same.

  This strain is a gram-positive gonococci having motility, and showed a “rod-coccus cycle” [rod-coccus cycle] whose morphology changes with the passage of culture time. The colony color on the Nutrient agar plate medium was yellow, and basal mycelium was formed after one week of culture. Catalase reaction was positive, oxidase reaction was negative, nitrate was reduced, gelatin was hydrolyzed, glucose and xylose were fermented, and mannitol was not fermented. In addition, casein was hydrolyzed, L-arabinose, glycerol and mannose were oxidized, and D-sorbitol was not oxidized. These properties are considered to be consistent with the properties of cellulosimicrobium funky and cellulosimicrobium cerance, which were suggested to be related in the 16S rDNA nucleotide sequence analysis. He supported Cellulosimicrobium funkei rather than Rosimicrobium cerance. From the results and various properties of the 16S rDNA base sequence analysis described above, this strain was determined to be Cellulosimicrobium funkei and named Cellulosimicrobium funkei U-647.

  This U-647 strain is 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-P21098. Therefore, the second of the present invention provides a Cellulosimicrobium funkei FERM-P21098 strain having uricase-producing ability.

(Method for producing uricase)
The method for producing uricase of the present invention includes a step (1) of culturing a microorganism belonging to the genus Cellulosimicrobium and having the ability to produce uricase, and a uricase from the culture obtained in the step (1). And (2) collecting the sucrose.

  Hereinafter, each step of the method for producing uricase using bacteria belonging to the genus Cellulosimicrobium will be described.

  (1) A step of culturing a microorganism belonging to the genus Cellulosimicrobium and capable of producing uricase.

  The medium used in the production method is not particularly limited as long as bacteria belonging to the genus Cellulosimicrobium grow, and contains moderately the carbon source, nitrogen source, inorganic substance, and other micronutrients required by the strain used. If so, either a synthetic medium or a natural medium can be used. The medium may be a solid or liquid medium.

  As a carbon source, any carbon source may be used as long as bacteria belonging to the genus Cellulosimicrobium 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 culturing bacteria belonging to the genus Cellulosimicrobium can be used. For example, ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, L- 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 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, those generally used for culturing bacteria belonging to the genus Cellulosimicrobium can be used. For example, sodium chloride, potassium chloride, calcium chloride, potassium phosphate, sodium phosphate And phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc, hydrochlorides, sulfates and acetates, such as magnesium sulfate and ammonium sulfate. 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.

  Cultivation of bacteria belonging to the genus Cellulosimicrobium is carried out under aerobic conditions, usually by shaking culture or normally 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 and accumulated in the culture solution and / or in the bacterial body of the bacterium belonging to the genus Cellulosimicrobium obtained as described above. Particularly preferred embodiments of the present invention When the U-647 strain is used, uricase produced in the cells of the U-647 strain is preferable because it is excellent in heat resistance, as described in detail in the following Examples.

  (2) A step of collecting uricase from the culture obtained in the step (1).

  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) A method for measuring the amount of hydrogen peroxide produced by reacting uric acid with uricase.

At this time, examples of the method 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 Cellulosimicrobium funkei U-647 strain Following composition: Tryptone (BD (Becton, Dickinson and Company)) 1% (w / v), yeast extract (BD (Becton) , Manufactured by Dickinson and Company) 0.1% (w / v), sodium chloride (manufactured by Kanto Chemical Co.) 0.5% (w / v), monopotassium phosphate (manufactured by Kanto Chemical Co.) 0.2% (W / v), uric acid (manufactured by Wako Pure Chemical Industries, Ltd.) 0.2% (w / v) was prepared, and then the pH was adjusted to 7.0 with NaOH and sterilized at 121 ° C. for 20 minutes. did.

  Next, 5 ml of the obtained culture solution was dispensed into a sterilized L-shaped test tube. Inoculate the U-647 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 is centrifuged, and 5 mL of phosphate buffer (10 mM, pH 7) and 3 g of glass beads are added to and mixed with the obtained bacterial cells. The cells were shaken for minutes to physically disrupt the cells. The supernatant of the crushed cells was collected by centrifugation, and the supernatant was used as an enzyme solution (total uricase activity 1.7 U).

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

(Evaluation Example 1) Method for measuring uricase activity The uricase activity was measured using the uricase peroxidase method, and oxidation of 4-aminoantipyrine and TODB (N, N-bis (4-sulfobutyl) -3-methylaniline) at 550 nm. The condensate quinone dye was measured with a spectrophotometer. More specifically, 120 μl of 0.83 mM uric acid solution, 5 μl of 75 mM TODB, 5 μl of 75 mM 4-aminoantipyrine, 5 μl of 80 U / ml peroxidase, and 65 μl of enzyme solution 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 enzyme solution obtained in Example 1 (2) was 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, the uricase obtained by the production method of the present invention was not inactivated even after heat treatment for 20 minutes at 60 ° C. or less, and maintained uricase activity of 95% or more at 60 ° C.

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

Claims (4)

A step (1) of culturing a microorganism belonging to the genus Cellulosimicrobium and capable of producing uricase;
Collecting uricase from the culture obtained in the step (1) (2);
A method for producing uricase, comprising:   The manufacturing method according to claim 1, wherein the uricase maintains an enzyme activity of 95% or more with respect to a heat treatment at 60 ° C for 20 minutes.   The production method according to claim 1 or 2, wherein the microorganism belonging to the genus Cellulosimicrobium is Cellulosimicrobium funkei U-647 FERM-P21098 strain.   Cellulosimicrobium funkei U-647 FERM-P21098 strain having uricase-producing ability.

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