JP5207430B2 - NOVEL ENZYME AND COMPOSITION CONTAINING THE SAME - Google Patents

Description

  The present invention relates to a uric acid oxidase whose action is not inhibited by halide ion, nitrate ion, or Tris buffer, and whose action is not inhibited by xanthine, a production method thereof, a composition containing the same, and uric acid measurement using the same. The present invention relates to a kit and a method for measuring uric acid in a sample using the kit.

  Gout is an inflammatory joint disease that is common in men after the middle age. This gouty arthritis occurs when blood levels of uric acid are elevated (hyperuricemia) and crystal formation of monosodium urate monohydrate occurs in the joint. In addition, uric acid is low in water solubility, and if it is secreted excessively (high uric aciduria), it causes kidney stones (uric acid stones). Severe hyperuricemia and gout are also associated with renal dysfunction (Non-Patent Documents 1 to 3). Therefore, measurement of blood and urine levels of uric acid is important for differential diagnosis of these diseases, and is one of the biochemical diagnostic items that are generally performed at present. An enzyme method using urate oxidase (Urate oxidase, uricase, Uricase, EC 1.7.3.3) is generally used as a reagent for measuring uric acid level (Patent Documents 1 to 3).

  Uric acid oxidase is an enzyme widely distributed in prokaryotes and eukaryotes, but it is thought that humans have lost urate oxidase as a result of mutations in evolution, resulting in hyperuricemia ( Non-Patent Documents 4 and 5). Therefore, urate oxidase is used to treat severe hyperuricemia in Europe (Non-patent Document 6).

  In addition, uric acid oxidase is used in place of hydrogen peroxide, sodium perborate, sodium percarbonate, sodium bromate, etc. as an oxidizing agent that does not cause hair or skin damage in the treatment of oxidative hair dyes and permanent waves. (Patent Document 4).

  Examples of the method for obtaining urate oxidase include Candida (Patent Document 5), Micrococcus, Brevibacterium (Patent Document 6), Streptomyces (Non-patent Document 7), Enterobacter (Patent Document 7). ), Methods using microorganisms belonging to the genus Bacillus (Patent Documents 8 and 9), and the like are known.

It is known that the enzymatic reaction of urate oxidase is currently represented by Formula 1 (Non-patent Document 8). The apparent action of urate oxidase is of formula 2 because the product 5-hydroxyisourea is immediately hydrolyzed to allantoin in several non-enzymatic reactions.
Uric acid + O ₂ + H ₂ O → 5-hydroxyisourea + H ₂ O ₂ (Formula 1)
Uric acid + O ₂ + 2H ₂ O → Allantoin + H ₂ O ₂ + CO ₂ (Formula 2)

  Since urate oxidase is an enzyme involved in purine nucleotide metabolism, conventional urate oxidase is strongly inhibited by xanthine, a substance in this metabolic pathway (Non-patent Document 9). Further, the enzymatic action of conventional urate oxidase is inhibited by KCl, NaCl, etc., and Non-Patent Document 10 reports that the enzyme reaction is inhibited by TrisHCl, which is a widely used buffer system. Due to the conventional urate oxidase inhibitors such as these, in the above-mentioned reagent for measuring uric acid level, therapeutic agent for hyperuricemia and oxidative hair dye or oxidative fixative composition for permanent wave, those raw materials are limited, The effect may be negatively affected. For example, in a conventional uric acid level measurement reagent using uric acid oxidase, if xanthine is present in the sample, a negative error occurs in the measured value. In addition, when the enzyme is stored in a liquid state, the stability of the enzyme generally increases in proportion to the salt concentration. Therefore, it is a common method to use a salt such as KCl or NaCl as a stabilizer. Naturally, a buffer solution is used to keep the pH of the solution constant, but when conventional urate oxidase is used, the use of salts or buffers thereof may be limited. Therefore, there has been a demand for urate oxidase that is not inhibited by halide ions, nitrate ions, or Tris buffer, and that is not inhibited by xanthine.

  Urate oxidase was once reported as a copper-containing enzyme (Non-patent Documents 11 and 12), but as a result of analysis such as crystal structure analysis of some bacteria and eukaryotic urate oxidase, the enzyme was found to be copper. It has come to be considered that the enzyme is not contained (Non-Patent Documents 10, 13, http://br.expasy.org/enzyme/1.7.3.3).

  On the other hand, multi-copper oxidase is laccase (EC 1.10.3.2), bilirubin oxidase (EC 1.3.3.5), ascorbate oxidase (EC 1.10.3.3), ceruloplasmin (EC 1 16.3.1), or a group of copper-containing oxidases such as phenoxazinone synthase, which removes electrons from a substrate and produces water by reducing molecular oxygen by four electrons It is an enzyme that catalyzes. The characteristics of multi-copper oxidase are as follows: 1) Four atoms of copper required for enzyme activity are fixed in a tetrahedral coordination structure, and three atoms of copper form a trinuclear copper cluster. 2) Four atoms of copper The amino acids related to the coordination of are highly conserved, and 3) there is absorption near 610 nm (Non-Patent Documents 13 to 15).

  A multi-copper oxidase composed of three domains among multi-copper oxidases is called a laccase-type multi-copper oxidase, and includes laccase, bilirubin oxidase, ascorbate oxidase, phenoxazinone synthase, etc. Patent Documents 13 to 15).

  Laccase is well known as an enzyme with wide substrate specificity, but it is usually known as an enzyme that oxidizes benzenediol and polyphenol (http://br.expasy.org/cgi-bin/nicezyme.pl? 1.10.3.2). Among its broad substrate specificities, laccase-type multi-copper oxidase with high substrate specificity is particularly called bilirubin oxidase, and laccase-type multi-copper oxidase with high substrate specificity to ascorbic acid is called ascorbate oxidase. . However, no laccase-type multi-copper oxidase having a high substrate specificity for uric acid has been reported so far.

Nephron, 1990, 56 (3): 317-321, Acute hyperuricemic nephropathy and renal failure after transplantation, Venkataseshan VS, Feingold R, Dikman S, Churg J. Transplantation proceedings, 1992 Aug; 24 (4), 1391-1392, Cyclosporine-induced hyperuricemia after renal transplant: clinical characteristics and mechanisms, Ahn KJ, Kim YS, Lee HC, Park K, Huh KB. Transplantation proceedings, 1992, Oct; 24 (5), 1773-1774, Hyperuricemia and gout in renal allograft recipients, Delaney V, Sumrani N, Daskalakis P, Hong JH, Sommer BG. Proceedings of the National Academy of Sciences of the United States of America, 1989, Dec; 86 (23): 9412-9416, Urate oxidase: primary structure and evolutionary implications, Wu XW, Lee CC, Muzny DM, Caskey CT. Journal of molecular evolution, Two independent mutational events in the loss of urate oxidase during hominoid evolution, 1992, Jan; 34 (1): 78-84, Wu XW, Muzny DM, Lee CC, Caskey CT. The Journal of pediatrics, 1982, Jan; 100 (1): 152-155, Urate-oxidase prophylaxis of uric acid-induced renal damage in childhood leukemia, Masera G, Jankovic M, Zurlo MG, Locasciulli A, Rossi MR, Uderzo C , Recchia M. Agric. Biol. Chem. 33, 1282, (1969) Biochemistry. 1997, Apr 22; 36 (16): 4731-4738, Kinetic mechanism and cofactor content of soybean root nodule urate oxidase.Kahn K, Tipton PA. The Journal of biological chemistry, 1953, Oct; 204 (2): 613-621, The inhibition of uricase by xanthine.VAN PILSUM JF. Biochemistry, 1997, Apr 22; 36 (16): 4731-4738, Kinetic mechanism and cofactor content of soybean root nodule urate oxidase, Kahn K, Tipton PA. The EMBO journal. 1983, 2 (12): 2333-2339, Nodulin-35: a subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules.Bergmann H, Preddie E, Verma DP. Biochimica et biophysica acta, 1991, Jan 29; 1076 (2): 203-208, Purification and molecular properties of urate oxidase from Chlamydomonas reinhardtii, Alamillo JM, Cardenas J, Pineda M. Nature structural biology, 1997, Nov; 4 (11): 947-952, Crystal structure of the protein drug urate oxidase-inhibitor complex at 2.05 A resolution, Colloc'h N, el Hajji M, Bachet B, L'Hermite G, Schiltz M, Prange T, Castro B, Mornon JP. Biochemistry, Vol. 77, No. 2, 148-153, Kunishige Kataoka Cellular and molecular life sciences, 2005, Sep; 62 (18): 2050-2066, Function and molecular evolution of multicopper blue proteins, Nakamura K, GO N. JP-A-6-70798 JP 2002-159299 A JP-A-9-70299 Japanese Patent Laid-Open No. 8-217652 Japanese Patent Publication No.42-5192 Japanese Patent Publication No. 44-14783 Japanese Patent Publication No. 60-19990 JP 61-280272 A Japanese Patent Laid-Open No. 2-53488

  In the present invention, the action is not inhibited by a conventionally known urate oxidase which is not inhibited by a urate oxidase inhibitor, that is, halide ion, nitrate ion, and Tris buffer, and xanthine does not inhibit the action. Providing a novel urate oxidase was an issue to be solved. Furthermore, the present invention relates to a microorganism that produces the urate oxidase, a polynucleotide encoding the urate oxidase, a recombinant vector containing the polynucleotide, a transformant having the recombinant vector, a method for producing the urate oxidase, and the uric acid An object to be solved is to provide a composition containing oxidase, a kit for measuring uric acid using the uric acid oxidase, and a method for measuring uric acid in a sample using the uric acid oxidase.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research. As a result, Lysobacter sp. T-15 strain was not inhibited by halide ions, nitrate ions, or Tris buffer, and xanthine was used. It has been found that a protein having urate oxidase activity whose action is not inhibited is produced, and that the protein is a novel enzyme completely different from known urate oxidase. Furthermore, the present inventors have established a method for measuring uric acid in a sample using the urate oxidase and a method for producing the urate oxidase, and have completed the present invention. That is, the present invention relates to the following.

<1> A urate oxidase derived from the genus Lysobacter having the following characteristics.
(1) Enzyme action: acts on uric acid in the presence of oxygen and water to produce 5-hydroxyisourea, hydrogen peroxide, and carbon dioxide.
(2) Inhibitory action: The action of (1) is not inhibited by halide ions, nitrate ions and / or Tris buffer. And xanthine does not inhibit the action of (1).
(3) Optimal pH: 6.3 to 7.8.
(4) Optimal temperature: 55 ° C. or higher.
(5) Range of temperature suitable for action: At least 30 to 60 ° C.
(6) Stability: Retains 98% or more of activity at 30 ° C. for 15 minutes in the absence of a stabilizer.
(7) Molecular weight: about 31000 and 32000 by SDS-PAGE or about 120800 ± 10000 by gel filtration.

<2> The urate oxidase according to <1>, wherein an absorption spectrum exists at 610 ± 20 nm.
<3> The urate oxidase according to <1> or <2>, wherein the urate oxidase is derived from Lysobacter sp. T-15 strain (Accession No. FERM P-21056).

<4> A urate oxidase comprising the amino acid sequence of any one of (a) and (b) below.
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) in the amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence in which one or several amino acids are deleted, substituted or added, and also has an amino acid sequence <5> laccase activity having urate oxidase activity, The urate oxidase according to any one of <1> to <4>.
<6> The urate oxidase according to any one of <1> to <5>, which also has bilirubin oxidase activity.

<7> A microorganism that produces the uric acid oxidase according to any one of <1> to <6>.
<8> Lysobacter sp. T-15 strain having a deposit number of FERM P-21056.

<9> A polynucleotide comprising the following base sequence (a) or (b):
(A) the base sequence represented by SEQ ID NO: 1;
(B) a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 1 and which encodes a protein having urate oxidase activity

<10> A recombinant vector comprising the polynucleotide according to <9>.
<11> A transformant having the recombinant vector according to <10>.
<12> The transformant according to <11> is cultured in a medium, the urate oxidase according to any one of <1> to <6> is produced and accumulated in the culture, and the uric acid is produced from the culture. A method for producing uric acid oxidase, comprising collecting oxidase.

<13> A composition containing the urate oxidase according to any one of <1> to <6>.
<14> A uric acid measurement kit comprising at least the uric acid oxidase according to any one of <1> to <6> or the composition according to <13>.
<15> In a sample containing one or more components selected from the group consisting of xanthine, nitrate ions, and halide ions, urate oxidase according to any one of <1> to <6>, <13> Or a method for measuring uric acid using the uric acid measurement kit according to <14>.

  According to the present invention, a novel urate oxidase that is not inhibited by a conventionally known inhibitor of urate oxidase, i.e., a halide ion, a nitrate ion, or a tris buffer solution, and a novel effect that is not inhibited by xanthine. Urate oxidase is provided. By using the urate oxidase of the present invention, uric acid in a sample can be measured.

The present invention will be specifically described below.
The sample that can be used in the present invention is not particularly limited as long as it contains uric acid, but in addition to seawater, natural water, beverages, waste liquid, research samples containing uric acid, biological samples such as plasma and serum And urine.

  The urate oxidase activity of the present invention is a known urate oxidase (EC 1.7) described in Enzyme Handbook (Asakura Shoten, 1984), Enzyme nomenclature detabase (http://ca.expasy.org/enzyme/) and the like. 3.3) refers to the catalytic action of the reaction that changes uric acid to its oxide.

  The laccase activity of the present invention refers to the catalytic action of a known laccase (EC 1.10.3.2) described in enzyme handbooks, Enzyme nomenclature detabase, etc., and includes syringaldazine and 2′2-azinobis- (3-ethylbenzothiazole). An example is the catalytic action of a reaction that changes -6-sulfonic acid (hereinafter sometimes referred to as ABTS) to its oxide.

  The bilirubin oxidase activity of the present invention refers to the catalytic action of known bilirubin oxidase (EC 1.3.3.5) described in the enzyme handbook, Enzyme nomenclature database, etc., and is a reaction of changing bilirubin to its oxide. Catalysis can be exemplified.

  In the present specification, the simple description of “bilirubin” includes all of direct bilirubin, indirect bilirubin, and total bilirubin.

  The protein concentration described in this specification was measured according to the method described in the instruction manual using a protein assay kit manufactured by Bio-Rad, and calculated using BSA (bovine serum albumin) as a standard.

  Unless otherwise specified, the reagents used in the experiments of the present invention can be easily obtained commercially, such as those manufactured by Wako Pure Chemical Industries, Ltd., Kokusan Kagaku Co., Ltd., and Sigma Aldrich. Recombinant DNA experimental enzyme reagents (such as restriction enzymes), vector DNA, and kits used in the experiments were purchased from Takara Bio Inc. unless otherwise specified.

The urate oxidase in the present invention (hereinafter sometimes referred to as the enzyme of the present invention) is not inhibited by at least halide ions, nitrate ions, and / or Tris buffer, and is not inhibited by xanthine. The origin is not particularly limited as long as it is a urate oxidase that is not inhibited by a conventionally known inhibitor of urate oxidase, but it is preferably at least Cl ⁻ , Br ⁻ , nitrate ion, and / or Tris buffer. Examples include varieties and mutants of microorganisms capable of producing uric acid oxidase that are not inhibited by known urate oxidase inhibitors, such as those that are not inhibited by xanthine, such as Lysobacter genus and mutants thereof, Optimally, Lysobacter sp. T-15 strain, and variants and mutants of Lysobacter sp. T-15 strain can be mentioned.

The mycological properties of Lysobacter sp. T-15 strain are as follows.
In the identification of this strain, the identification experiment was conducted in accordance with the classification and identification experiment method of microorganisms and Microbiological Methods (Vol. 3), and the experimental results were obtained from the results of Bergey's Manual of Systematic Bacteriology (Vol. 3), Bergey's Manual of Determinative. Identification was performed in comparison with Bacteriology, International Journal of Systematic Bacteriology and International Journal of Systematic Evolutionary Microbiology.

<Growth characteristics>
(1) Growth on top plate medium Grows on LB (Luria-Bertani) agar medium and TSB (Trypticase soy broth) agar medium, which are widely used as culture medium for bacteria, but diluted LB medium and TSB medium 10 times. 1/10 LB agar medium and 1/10 TSB agar medium, or R2A agar medium (Difco), NM-1 medium (International Journal of Systematic Bacteriology, Microlunatus phosphovorus gen. Nov., Sp. Nov., A new gram-positive polyphosphate-accumulating bacterium isolated from activated sludge, 1995, Vol.45, pp.17-22, Nakamura K, Hiraishi A, Yoshimi Y, Kawarasaki M, Matsuda K, Kamagata Y.) Grows better. It grows well under a culture temperature of 30 ° C, a culture pH of 7.0, and aerobic conditions.

(2) Growth in liquid medium Although it grows uniformly, the bacterial cell concentration shows a low value of 0.2 to 0.4 or less at OD (Optical Density) 600 nm. It grows well under a culture temperature of 30 ° C, a culture pH of 7.0, and aerobic conditions.

<Morphological features>
The colony has a circular shape (2 to 4 mm in diameter) with a smooth surface, rounded edges and viscosity. The color of the colony is yellow. It is also opaque and glossy. There is no gliding. The cell form is 0.3 to 0.5 μm × 4 to 7 μm long bacillus and has no motility. Gram staining test shows negative. Neisser staining test is positive.

<Catalase / oxidase test>
Both catalase and oxidase tests are positive.

<Characteristics of chemical classification index>
The GC content of DNA (GC mol% [HPLC]) is 67%, and the main isoprenoid quinone is ubiquinone-8 (UQ-8). The main fatty acid composition is as follows: iso-C15: 0 50%, iso-C16: 0 16%, C16: 1 10%, iso-C17: 1 5%, C16: 0 5%, anteiso- C15: 0 5%, iso-C11: 0-3OH 5%, iso- or anteiso-C11: 0 4%.

<Molecular phylogenetic identification based on 16S rRNA gene>
The almost full length (1471 bp) base sequence of the 16S rRNA gene of Lysobacter sp. T-15 was determined, and homology search was performed using the international base sequence database (GenBank / DDBJ / EMBL). Specifically, a BLAST (http://www.ncbi.nih.gov/BLAST/) search provided by NCBI in the United States was performed. The results are shown in Table 1.

  As a result of homology search by BLAST, it was revealed that Lysobacter sp. T-15 strain belongs to the Gammaproteobacteria class and is closely related to the genus Lysobacter or Thermomonas. In particular, the most related species that showed the highest homology (98%) to the 16S rRNA gene sequence of Lysobacter sp. Strain T-15 was Lysobacter brunescens.

  Next, a molecular phylogenetic tree based on the base sequence of the 16S rRNA gene was prepared in order to clarify the phylogenetic relationships of the Lysobacter sp. T-15 strain, the Lysobacter spp. Alignment of base sequences and creation of molecular phylogenetic trees by the neighborhood joining method were performed using the ARB program package (http://www.arb-home.de/). As a result, similar to the result of the homology search by BLAST, the closest relative of Lysobacter sp. T-15 strain was Lysobacter brunescens, and the homology value of 16S rRNA gene sequence was 97.5%. On the other hand, the homology between Lysobacter sp. Strain T-15 and other related species was relatively low at 93.3-95.9%. In addition, Lysobacter sp. T-15 strain formed the same cluster as Lysobacter brunescens on the molecular phylogenetic tree (see FIG. 1). Lysobacter sp. T-15 was similar in many respects to Lysobacter brunescens in terms of chemical classification indices such as the GC content of DNA, main isoprenoid quinone and fatty acid composition, and various physiological and biochemical properties. Therefore, Lysobacter sp. T-15 strain is considered to be a bacterial species belonging to the genus Lysobacter. However, as described below, the Lysobacter sp. T-15 strain exhibits properties that are different from the recent relative Lysobacter brunescens in several ways:

(1) Growth temperature: Lysobacter brunescens can grow at 50 ° C, but Lysobacter sp. T-15 strain cannot grow (2) Cell morphology: Lysobacter brunescens cells are very long, 7-70 μm, but Lysobacter sp. The -15 strain is a long bacillus of about 4-7 μm.
(3) Colony morphology: Lysobacter brunescens colonies spread thinly, but Lysobacter sp. T-15 strain forms a circular colony of about 2 to 4 mm.
(4) Glidability: Lysobacter brunescens has gliding properties, but Lysobacter sp. T-15 strain does not have gliding properties.
(5) Agar resolution: Lysobacter brunescens shows agar resolution, but not in Lysobacter sp. T-15.
(6) 16S rRNA gene sequence: The base sequences of 16S rRNA genes of Lysobacter brunescens and Lysobacter sp. T-15 strains differ by 2.5%.

  From the above morphological characteristics, physiological and biochemical properties, and the results of molecular phylogenetic analysis based on the 16S rRNA gene, Lysobacter sp. T-15 strain belongs to the genus Lysobacter. It is thought that it is a new microorganism that cannot. Therefore, this sample Lysobacter sp. T-15 strain was named Lysobacter sp. T-15 strain, and on October 6, 2006, the National Institute of Advanced Industrial Science and Technology (AIST) Deposited at 1-chome 1-address 1 center 6). The accession number is FERM P-21056.

<Enzyme action>
The catalytic reaction formula when uric acid of the enzyme of the present invention is used as a substrate is Formula 1. The product, 5-hydroxyisourea, is oxidized non-enzymatically to allantoin, so the apparent reaction equation is:

The catalytic reaction formula when the benzenediol of the enzyme of the present invention is used as a substrate is Formula 3.
4 benzenediol + O ₂ → 4 benzosemiquinone + 2H ₂ O (Formula 3)

The catalytic reaction formula when bilirubin of the enzyme of the present invention is used as a substrate is Formula 4. Depending on the conditions, the product biliverdin may be further converted into a colorless substance by bilirubin oxidase and / or air oxidation.
Birubin + O ₂ → bilivedin + H ₂ O (Formula 4)

<Urate oxidase activity measurement method 1>
1 ml of enzyme activity measurement solution consisting of 0.04 ml of 1M potassium phosphate buffer (pH 7.0 (25 ° C.)), 0.023 ml of 5 mM uric acid, and 0.937 ml of distilled water in a quartz cell having a layer length of 1 cm. After preheating at 37 ° C. for 2 minutes, 20 μl of the urate oxidase solution of the present invention diluted to an appropriate concentration with 40 mM potassium phosphate buffer (pH 7.0 (25 ° C.)) is added to start the enzyme reaction. The difference in absorbance of the substrate at 293 nm from 1 minute to 2 minutes after the start of the reaction is measured (As). The same operation is performed using distilled water as a blind test, and the absorbance difference is measured (Ab). 1 unit of enzyme activity (1 unit) is the amount of enzyme that changes 1 micromole of uric acid per minute at 37 ° C, and the millimolar extinction coefficient of uric acid in the above reaction solution is 12.6. Ask.

<Uric acid oxidase activity measurement method 2>
40 mM potassium phosphate buffer (pH 7.0 (25 ° C.)), 10 U / ml peroxidase, 1.5 mM 4-aminoantipyrine (4-AA), 0.04% N, N′-dimethylaniline, 1 ml of enzyme activity measurement solution consisting of 1 mM uric acid is pre-warmed in a quartz cell with a layer length of 1 cm for 2 minutes at 37 ° C., and then adjusted to an appropriate concentration with 40 mM potassium phosphate buffer (pH 7.0 (25 ° C.)). Enzyme reaction is started by adding 20 μl of diluted urate oxidase solution of the present invention. The difference in absorbance of the substrate at 565 nm from 1 minute to 2 minutes after the start of the reaction is measured (As). The same operation is performed using distilled water as a blind test, and the absorbance difference is measured (Ab). 1 unit of enzyme activity (1 unit) is the amount of enzyme that changes 1 micromole of uric acid per minute at 37 ° C, and the millimolar extinction coefficient of quinonediimine dye in the above reaction solution is 23.56. Ask for. Unless otherwise specified in this specification, the activity of urate oxidase was measured by urate oxidase activity measurement method 2.

<Laccase activity measurement method>
100 ml of sodium citrate buffer (pH 4.5 (25 ° C.)) 1 ml of enzyme activity measurement solution consisting of 5 mM ABTS was preheated in a quartz cell with a layer length of 1 cm for 2 minutes at 37 ° C., and then 10 mM phosphoric acid. The enzyme reaction is started by adding 20 μl of the enzyme solution of the present invention diluted to an appropriate concentration with potassium buffer (pH 7.0 (25 ° C.)). The difference in absorbance of the substrate at 420 nm from 1 minute to 2 minutes after the start of the reaction is measured (As). The same operation is performed using distilled water as a blind test, and the absorbance difference is measured (Ab). Enzyme activity is determined from As-Ab, where 1 unit of enzyme activity (1 unit) is the amount of enzyme that changes 1 micromole of ABTS per minute at 37 ° C., and the mmol extinction coefficient of 420 nm that increases in the reaction solution is 36. .

<Method for measuring bilirubin oxidase activity>
Enzyme solution of the present invention diluted to an appropriate concentration with 0.1 ml of 1 M potassium phosphate buffer (pH 6.0 (25 ° C.)) and 0.1 M potassium phosphate buffer (pH 6.0 (25 ° C.)) 1 ml of enzyme activity measurement solution consisting of 0.02 ml and distilled water 0.88 ml was preheated at 37 ° C. for 1.5 minutes in a quartz cell having a layer length of 1 cm, and then interference check A bilirubin of International Reagent Co., Ltd. as a substrate. 20 μl of F (component is bilirubin) adjusted to 0.9 mg NaCl to 20 mg / dl was added to start the enzyme reaction, and the difference in absorbance of the substrate at 450 nm from 1 minute to 2 minutes after the start of the reaction was determined. Measure (As). The same operation is performed using distilled water as a blind test, and the absorbance difference is measured (Ab). Enzyme activity is determined from As-Ab, where 1 unit of enzyme activity (1 unit) is the amount of enzyme that changes 1 micromole of bilirubin per minute at 37 ° C., and the millimolar extinction coefficient of bilirubin F in the reaction solution is 36. .

<Ascorbate oxidase activity measurement method>
1 ml of ascorbate oxidase activity measurement solution (90 mM sodium phosphate buffer (pH 5.5 (25 ° C.)) containing 0.5 mM L-ascorbic acid, 0.45 mM ethylenediaminetetraacetic acid) quartz cell with a layer length of 1 cm After preheating at 30 ° C. for 5 minutes, 0.1 ml of the enzyme solution of the present invention diluted to an appropriate concentration with 10 mM sodium phosphate buffer (pH 5.5 (25 ° C.)) containing 0.05% BSA To start an enzyme reaction, and the difference in absorbance of L-ascorbic acid at 245 nm from 1 minute to 5 minutes after the start of the reaction is measured (As). The same operation is performed using distilled water as a blind test, and the absorbance difference is measured (Ab). The enzyme activity is determined from the difference in absorbance (As-Ab) between the absorbance (As) using this enzyme solution and the absorbance (Ab) in the blind. 1 unit of enzyme activity (1 unit) is the amount of enzyme that changes 1 micromole of L-ascorbic acid per minute at 30 ° C., and the millimolar extinction coefficient of L-ascorbic acid in the above reaction solution is 10, As-Ab Determine enzyme activity.

<Inhibitory action 1>
NaCl, KCl, NaBr, Na ₂ SO ₄ , NaNO _3, or CH ₃ COONa was added to the enzyme activity measurement solution of the urate oxidase activity measurement method 2 at the concentrations shown in Table 2, and the urate oxidase of the present invention was added. The activity of Arthrobacter globiformis-derived urate oxidase (manufactured by Asahi Kasei Pharma Corporation) was measured. Table 2 shows the relative values when the additive-free case is taken as 100%. As is clear from Table 2, although the reaction of Arthrobacter globiformis-derived urate oxidase is inhibited in a concentration-dependent manner by NaCl, KCl, NaBr, and NaNO ₃ , the urate oxidase of the present invention inhibits the reaction under the same conditions. It has been found that it has features that are not.

<Inhibitory action 2>
10 mM NaCl, KCl, Na ₂ SO ₄ , and CH ₃ COONa are added to the enzyme activity measurement solution of the urate oxidase activity measurement method 2, and the urate oxidase of the present invention, Arthrobacter globiformis-derived urate oxidase, Candida sp. The activities of urate oxidase (Toyobo) and Bacillus sp.-derived urate oxidase (Toyobo) were measured. Table 3 shows the relative values when the additive-free case is taken as 100%. As is apparent from Table 3, the urate oxidase derived from Arthrobacter globiformis, Candida sp., And Bacillus sp. Was inhibited by NaCl and KCl, but the urate oxidase of the present invention reacted under the same conditions. Was found to have uninhibited characteristics.

<Inhibitory action 3>
Conventional urate oxidase has been reported to be strongly inhibited by xanthine (9). 1 mM xanthine was added to the enzyme activity measurement solution of the above-described method for measuring uric acid oxidase activity 2, and the activities of the urate oxidase of the present invention and the urate oxidase derived from Arthrobacter globiformis were measured. Table 4 shows the relative values when the additive-free case is taken as 100%. As is clear from Table 4, although the urate oxidase derived from Arthrobacter globiformis, derived from Candida sp., And Bacillus sp. Is inhibited by xanthine in a concentration-dependent manner, the urate oxidase of the present invention is not subjected to the same conditions. It was found that the reaction has the characteristic that it is not inhibited.

<Inhibitory action 4>
Non-Patent Document 10 reports that a conventional enzyme reaction of urate oxidase is inhibited by a TrisHCl buffer, which is a commonly used buffer. The concentration of potassium phosphate buffer (pH 7.0) in the enzyme activity measurement solution of the above-described method for measuring uric acid oxidase activity 2 was changed to the TrisHCl buffer (pH 7.0) having the concentrations shown in Table 5. The activities of urate oxidase and urate oxidase from Arthrobacter globiformis were measured. Table 5 shows the relative values when the additive-free case is taken as 100%. As is clear from Table 5, although the reaction of Arthrobacter globiformis urate oxidase is inhibited depending on the concentration of TrisHCl buffer, the urate oxidase of the present invention has the characteristic that the reaction is not inhibited under the same conditions. I understood.

<Inhibitory action 5>
The Ki value was calculated by a line Weber-Burk reciprocal plot of the inhibition reaction of NaCl between the urate oxidase of the present invention and the urate oxidase derived from Arthrobacter globiformis. FIG. 3 is a uline oxidase of the present invention, and FIG. 3 is a line Weber-Burk reciprocal plot of the inhibition reaction of Arthrobacter globiformis urate oxidase by NaCl. 2 and 3, white circle (◯) represents 0 mM, black circle (●) represents 50 mM, white triangle (Δ) represents 100 mM, black square (黒) represents 150 mM, and white square (□) represents 200 mM NaCl. FIG. 4 shows a quadratic plot of the line Weber-Burk reciprocal plot of the inhibition reaction in which the slopes of FIGS. 2 and 3 are plotted against the NaCl concentration. In FIG. 4, white circles (◯) indicate urate oxidase of the present invention, and black circles (●) indicate Arthrobacter globiformis-derived urate oxidase. From FIG. 4, it can be seen that the urate oxidase of the present invention is subjected to very weak inhibition by NaCl so as not to cause a practical problem, and Ki is about 200 mM. On the other hand, it was found that Arthrobacter globiformis-derived uric acid oxidase was strongly inhibited by NaCl to a practical level, and Ki was about 5.7 mM. In addition, it is clear from FIG. 2 that the inhibition form that the urate oxidase of the present invention receives by NaCl is an antagonistic form, and from FIG. 3, the inhibition form that Arthrobacter globiformis-derived urate oxidase receives by NaCl is a non-antagonistic form that is different from each other. It was. 2 and 3, V represents the reaction rate, and [I] represents the concentration of the inhibitor.

<Optimum pH (when uric acid is used as a substrate)>
The optimum pH for the uric acid of the enzyme of the present invention was determined. The measurement solution for measuring the optimum pH was obtained by the same method as in Urate Oxidase Activity Measurement Method 2 while changing the buffer solution. In FIG. 5, as the buffer solution, the pH 4.2 to pH 5.2 range is acetic acid-sodium hydroxide buffer, the pH 5.9 to pH 7.4 range is potassium phosphate buffer, pH 7.8 to pH 10.0. The range shows a relative value when the maximum activity with respect to uric acid when Tris-hydrochloric acid buffer is used is 100%. The relative activity of the enzyme of the present invention relative to uric acid was 85% or more between pH 6.3 and 7.8, and the optimum pH was near neutral.

<Optimum pH (when bilirubin is used as a substrate)>
The optimum pH for interference check A bilirubin C and bilirubin F of the international reagent of the enzyme of the present invention was determined. The measurement solution for measuring the optimum pH was 0.1 ml of 1M buffer solution, 0.02 ml of enzyme solution appropriately diluted with 0.1M potassium phosphate buffer solution (pH 6.0 (25 ° C.)), and Enzyme reaction rate was calculated | required by the same method as the said bilirubin oxidase activity measuring method using 1 ml of enzyme activity measuring solution which consists of 0.78 ml of distilled water. Changes in the millimolar extinction coefficient of bilirubin C and bilirubin F accompanying changes in pH were not considered. In FIG. 6, as the buffer solution, the pH range from 3.3 to 5.5 is acetic acid-sodium hydroxide buffer, the pH range from 4.8 to 6.1 is citric acid-sodium hydroxide buffer, pH 6.0 to pH 7. .4 range is potassium phosphate buffer, and pH 7.5 to pH 9.6 range is bilirubin C (black circle (●) in the figure) or bilirubin F (in the figure, when tris-hydrochloric acid buffer is used) The relative values with respect to the maximum activity with respect to white circles (◯) are 100%. The optimum pH for bilirubin C of the enzyme of the present invention was around 5, and the optimum pH for bilirubin F was around neutral.

<Optimum pH (when ABTS is used as a substrate)>
The optimum pH for ABTS of the enzyme of the present invention was determined. The measurement solution for measuring the optimum pH was obtained by the same method as the laccase activity measurement method, with the buffer solution changed. In FIG. 7, as the buffer, pH 3.2 to pH 4.2 ranges from acetic acid-sodium hydroxide buffer, pH 4.8 to pH 6.2 ranges from citrate-sodium hydroxide buffer, pH 6.3 to pH 6 The range of .9 shows a relative value when the maximum activity against ABTS is 100% when a potassium phosphate buffer is used. The optimum pH of the enzyme of the present invention for ABTS was around 4.

<Range of optimum temperature and working temperature>
The reaction rate of the urate oxidase of the present invention was measured by changing the reaction temperature from 25 to 58 degrees according to the activity measurement method of the urate oxidase activity measurement method 1 described above. The results are shown as Arrhenius plots in FIG. As is clear from FIG. 8, the optimum temperature of the urate oxidase of the present invention was at least 55 ° C. or higher, and the range of the action temperature was at least 30 ° C. to 60 ° C. Further, as is apparent from FIG. 8, the Arrhenius plot showed biphasic properties, and the activation energy was 62 kJ / mol between 25 and 36.5 ° C. and 37 kJ / mol between 39.5 and 58 ° C. . In the figure, T represents an absolute temperature.

<Stability>
The residual activity of uric acid oxidase after dissolving the urate oxidase of the present invention in 40 mM potassium phosphate buffer (pH 7.0 (25 ° C.)) to 0.03 mg / ml and heat-treating at each temperature for 15 minutes. It was measured according to the above-mentioned method 2 for measuring uric acid oxidase activity. The results are shown in FIG. As is apparent from FIG. 9, the urate oxidase of the present invention retains 98% or more of activity at 30 ° C. for 15 minutes in the absence of a stabilizer.

<Specific activity>
The reaction rate was measured using uric acid, bilirubin, ascorbic acid or ABTS of the enzyme of the present invention as a substrate, and the specific activity (μmol / mim / mg) was calculated and summarized in Table 6. For comparison, the specific activity (μmol / mim / mg) of laccase-type multi-copper oxidase using uric acid, bilirubin, ascorbic acid, and ABTS as substrates was also shown. In Table 6, (a) is Applied and Environmental Microbiology, 2006, 72, 972-975, (b) is The Journal of biological chemistry, 2002, 277, 18849-18859, (c) is Biochemistry, 1999, 38, 3034-3042, (d) is a value quoted from J. Microbiology, 2005, 43, 555-560, and * indicates Vmax. As is apparent from Table 6, the enzyme of the present invention has a urate oxidase activity, a laccase activity, and a bilirubin oxidase activity at the same time, and is a novel enzyme having completely different characteristics from the laccase-type multi-copper oxidase reported in the past. all right.

<Molecular weight>
When Lysobacter sp. T-15 was cultured and the urate oxidase of the present invention was purified, molecular weights of about 31000 and 32000 were measured (arrows in lane 1) as shown in the SDS-PAGE photograph of FIG. When the molecular weight was measured by pp, the molecular weight was estimated to be about 120800 ± 10000 as shown in FIG. When a transformant containing a recombinant plasmid containing the urate oxidase gene of the present invention encoding the amino acid sequence represented by SEQ ID NO: 2 is cultured and the urate oxidase of the present invention is purified, a photograph of the SDS-PAGE of FIG. The molecular weight was determined to be about 67,000 as indicated by (lane 2 arrow). The calculated value from the amino acid primary sequence was 64516. In the figure, white circles (◯) indicate enzyme activity (U / ml), and black circles (●) indicate molecular weight (kDa).

<Isoelectric point>
The isoelectric point of the urate oxidase of the present invention was about pH 5.2 ± 0.5 by electrophoresis using carrier ampholine.

<Apparent Km and Vmax>
Apparent Km and Vmax for uric acid of the enzyme of the present invention, interference check A bilirubin F, ascorbic acid and ABTS of International Reagent Co., Ltd. were measured. According to each enzyme activity measurement method described above, the concentration of each substrate in the enzyme activity measurement solution is set to 5 or more different concentrations between 1/10 to 10 times the Km values shown in Table 7. Each apparent Km value and Vmax value was calculated by a line Weber-Burk reciprocal plot. In Table 7, for comparison, Bacillus subtilis-derived CotA (Asahi Kasei Pharma Co., Ltd.), Arthrobacter globiformis-derived urate oxidase (Asahi Kasei Pharma Co., Ltd.), Trachderma tsunodae-derived bilirubin oxidase (Takara Shuzo Co., Ltd.), Acremonium sp. The apparent Km values and Vmax values of acid oxidase (manufactured by Asahi Kasei Pharma Co., Ltd.) and Trametes versicolor-derived laccase (manufactured by Sigma) were also shown. In the table, (a) is Applied and Environmental Microbiology, 2006, 72, 972-975, (b) is The Journal of biological chemistry, 2002, 277, 18849-18859, (c) is Biochemistry, 1999, 38, 3034. -3042, (d) is a value quoted from J. Microbiology, 2005, 43, 555-560, (d) is a value quoted from J. Microbiology, 2005, 43, 555-560, and ± indicates a slight reaction. ,-Indicates no reaction at all. As is apparent from Table 7, the enzyme of the present invention has uric acid oxidase activity, laccase activity, and bilirubin oxidase activity. In addition to uric acid oxidase reported in the past, laccase-type multi-copper oxidase, ie, bilirubin oxidase, ascorbic acid It was found to be a novel enzyme having completely different characteristics from oxidase and laccase.

<Primary array>
The N-terminal side and C-terminal side of the amino acid sequence of urate oxidase represented by amino acid numbers 1 to 661 of SEQ ID NO: 2 of the present invention may contain amino acid residues or polypeptide residues, and the amino acids Examples of the residue include a signal peptide or T7 tag, His tag, S tag, Trx tag, CBD tag, DsbA tag, GST tag, Nus tag and the like.

  Further, the amino acid sequence constituting the urate oxidase of the present invention is the amino acid sequence of SEQ ID NO: 2 that expresses the same effect as the enzyme activity expression by the polypeptide consisting of the amino acid sequence represented by amino acid numbers 1 to 661 of SEQ ID NO: 2. Also included are amino acid sequences consisting essentially of part of the amino acid sequences of Nos. 1 to 661 and equivalents obtained by mutating, deleting or adding a part of amino acid sequences not involved in the expression of enzyme activity.

  The DNA encoding the amino acid sequence represented by amino acid Nos. 1 to 661 of SEQ ID NO: 2 of the present invention has an amino acid sequence including amino acid residues or polypeptide residues on the N-terminal side and C-terminal side thereof. What is necessary is just DNA which consists of any one codon among corresponding series of codons.

  Furthermore, the DNA encoding the urate oxidase of the present invention exhibits the same effect as that of the enzyme activity expression by the polypeptide consisting of the amino acid sequence represented by amino acid numbers 1 to 661 of SEQ ID NO: 2, among amino acid numbers 1 to 661. DNA encoding a substantially amino acid sequence consisting of a part of the above, or a DNA encoding the equivalent amino acid sequence of a part of the amino acid sequence which is not involved in the expression of enzyme activity, which is mutated, deleted or added It may be.

  Microorganisms that are donors of DNA include the known urate oxidase inhibitors that are not inhibited by at least halide ions, nitrate ions, or Tris buffer, and are not inhibited by xanthine. The organism is not limited in any way as long as it produces uninhibited urate oxidase, and preferred examples include Lysobacter sp. T-15 and variants and mutants of Lysobacter sp. T-15.

  As a vector into which DNA encoding the urate oxidase of the present invention is incorporated, a vector constructed for genetic recombination from a phage or plasmid that can autonomously grow in the host microorganism is suitable. Examples of the phage vector include Escherichia When using a microorganism belonging to Kori as a host microorganism, λgt · λC, λgt · λB, etc. can be used. Moreover, as a plasmid vector, for example, when Escherichia coli is used as a host microorganism, pET vectors (Novagen) such as plasmids pET-3a, pET-11a, and pET-32a, or pBR322, pBR325, pACYC184, pUC12, pUC13 , PUC18, pUC19, pUC118, pIN I, BluescriptKS +, pWH1520, pUB110, pKH300PLK when Bacillus subtilis is the host, pIJ680, pIJ702, when using actinomycetes as the host, and yeast, especially Saccharomyces cerevisiae YRp7, pYC1, YEp13, etc. can be used. A vector fragment is prepared by cleaving such a vector with a restriction enzyme that produces the same end as the DNA end produced by the restriction enzyme used to cleave the DNA encoding the urate oxidase of the invention. A DNA fragment encoding an oxidase and a vector fragment can be combined with a DNA ligase enzyme according to a conventional method to incorporate the DNA encoding the urate oxidase of the present invention into the target vector.

  As the host microorganism for transferring the plasmid, it is sufficient that the recombinant DNA can be stably and autonomously propagated. For example, when the host microorganism is a microorganism belonging to Escherichia coli, Escherichia coli BL21, Escherichia coli BL21 (DE3), Escherichia coli BL21trxB, Escherichia coli Rosetta (DE3), Escherichia coli Rosetta, Escherichia coli Rosetta (DE3) pLysS, Escherichia coli Rosetta (DE3) pLac, Escherichia coli Luis et Kori Origami, Escherichia coli Origami, Escherichia coli Tuner, Escherichia coli DH1, Escherry Hia coli JM109, Escherichia coli W3110, Escherichia coli C600, etc. can be used. Further, when the microorganism host is a microorganism belonging to the genus Bacillus, Bacillus subtilis, Bacillus megaterium, etc., a microorganism belonging to actinomycetes, Streptomyces lividans TK24, etc., a microorganism belonging to Saccharomyces cerevisiae, Saccharomyces cerevisiae INVSC1 can be used.

  Further, the urate oxidase of the present invention may be mutated by a known gene manipulation means without damaging the property of catalyzing the original reaction. Such a mutant gene is derived from the gene of urate oxidase of the present invention. This means an artificially mutated gene created by genetic engineering techniques. This artificially mutated gene is a variety of genetically engineered methods such as the above-mentioned site-specific mutation method and replacement of specific DNA fragments of the target gene with artificially mutated DNA. Obtained using the method. It is possible to express the mutant urate oxidase of the present invention by inserting the artificially mutated urate oxidase gene of the present invention thus obtained into a vector and transferring it to a host microorganism, and a mutation having excellent properties It is also possible to produce the body's urate oxidase of the present invention.

  In addition, when the urate oxidase of the present invention is produced by the transformed microorganism, the transformed microorganism is cultured in a nutrient medium to produce the urate oxidase of the present invention in the cells or in the culture solution. The cultured cells are collected by means such as filtration or centrifugation, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, EDTA and / or an appropriate interface. Concentrate the aqueous solution of urate oxidase of the present invention by adding an activator or the like, or processing by adsorption chromatography such as ammonium sulfate fractionation, gel filtration, affinity chromatography, or ion exchange chromatography without concentrating, A good urate oxidase of the present invention can be obtained.

  Culture conditions for transformed microorganisms may be selected in consideration of their nutritional physiological properties. Usually, liquid culture is used in many cases, but industrially, deep aeration and agitation culture is advantageous. is there. As a nutrient source for the medium, those commonly used for culturing microorganisms can be widely used. The culture temperature can be appropriately changed within the range in which microorganisms grow and produce the urate oxidase of the present invention, but in the case of Escherichia coli, it is preferably about 10 to 45 ° C, more preferably about 20 to 30 ° C. The culture conditions vary somewhat depending on the conditions, but the culture should be terminated at an appropriate time in anticipation of the time when the urate oxidase of the present invention reaches the maximum yield. In the case of Escherichia coli, it is usually about 12 to 48 hours. . The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce the urate oxidase of the present invention, but in the case of Escherichia coli, the pH is preferably about 6 to 8.

  The urate oxidase of the present invention is mainly contained and accumulated in the microbial cells, and may be extracted from the microbial cells. To illustrate the extraction method, first, the culture is solid-liquid separated, and the obtained wet cells are dispersed in a solution such as phosphate buffer or Tris-HCl buffer, lysozyme treatment, ultrasonic treatment, French press treatment, A crude cell uric acid oxidase solution of the present invention or a uric acid oxidase inclusion body of the present invention is obtained by appropriately selecting and combining cell disruption means such as dynomill treatment.

  As a method for solubilizing the urate oxidase of the present invention in inclusion bodies, in vivo, a method of increasing the solubility by conjugating the E. coli chaperone gene, a method using a cold shock protein vector (Takara Bio Inc.), a transformant There is a method of expressing in the periplasmic region. In vitro, after the inclusion body is completely unfolded with a denaturing agent such as mercaptoethanol, urea, or guanidine, the denaturing agent can be removed by dialysis or dilution to be refolded. At this time, cycloamylose, cyclodextrin, surfactant, glutathione, arginine or the like may be used to increase the refolding efficiency.

  A purified enzyme is obtained from the crude enzyme solution of the present invention by means of isolation and purification of known proteins and enzymes. For example, the target enzyme is precipitated and recovered by applying a fractional precipitation method using an organic solvent such as acetone, methanol, ethanol, or a salting out method using ammonium sulfate, sodium chloride, or the like to the crude enzyme solution of the present invention. In addition, after performing dialysis and isoelectric precipitation of this precipitate as necessary, column chromatography using an ion exchanger, gel filter agent, adsorbent, etc. until a single band is shown by electrophoresis or the like Purify by etc. Moreover, when the purification degree of a target enzyme increases by combining these methods appropriately, it can carry out combining suitably.

  Enzymes obtained by these methods can be used as stabilizers in the form of liquid or lyophilized by methods such as ultrafiltration concentration and lyophilization with or without the addition of various salts, sugars, proteins, lipids, surfactants, etc. The solid urate oxidase of the present invention can be obtained, and may be freeze-dried as appropriate. In this case, saccharose, mannitol, sodium chloride, albumin, etc. may be added in an amount of about 0.5 to 10% as a stabilizer. Good.

  According to the present invention, it has a urate oxidase activity that is not inhibited by halide ions, nitrate ions, or Tris buffer, and is not inhibited by a conventionally known inhibitor of urate oxidase, such as xanthine. Proteins can be provided. As a result, in the composition containing the enzyme, there are no restrictions on the raw materials other than the enzyme, and it is expected that a composition containing the enzyme or a method for oxidizing uric acid in a sample using the enzyme can be provided. .

  The reagent and method for measuring uric acid using the uric acid oxidase of the present invention are a reagent and method for oxidizing uric acid in a sample by the action of the uric acid oxidase of the present invention.

  Although it will not specifically limit if it is a kit containing the enzyme of this invention as a kit containing the enzyme of this invention, For example, a uric acid measuring reagent is mentioned. These reagents and kits can be provided as liquid products, frozen products of liquid products, freeze-dried products of liquid products, or dried products of liquid products (by heat drying and / or air drying and / or vacuum drying, etc.). Liquid products, liquid frozen products, and liquid freeze-dried products are preferred, liquid products and liquid freeze-dried products are more preferred, and liquid products are most preferred. In another embodiment, a frozen liquid product may be preferable. As yet another aspect, lyophilization of a liquid product may be preferred.

  The present invention also includes a therapeutic agent for hyperuricemia containing the urate oxidase of the present invention as an active ingredient, use of the medicament for treating hyperuricemia, a method for treating hyperuricemia using the medicament, and the like. Range.

  Furthermore, the oxidizing agent containing the urate oxidase of the present invention as an active ingredient, the use of the oxidizing agent for oxidative hair coloring, and permanent wave are also within the scope of the present invention.

  The present invention will be described based on examples, but the scope of the present invention is not limited to the following examples. In the Examples, the gene manipulation technique described in accordance with the conventional method includes, for example, the method of Maniatis et al. (Maniatis, T., et al. Molecular Cloning. Cold Spring Harbor Laboratory 1982, 1989) and various commercially available enzymes. It can be carried out by following the procedure attached to the kits.

[Example 1]
<Culture>
Lysobacter sp. T-15 was cultured in the following medium; per liter ammonium sulfate 0.5 g, sodium glutamate 0.5 g, sodium succinate 0.5 g, sodium acetate 0.5 g, yeast extract 0.5 g, casamino Acid 0.5g, Sodium thiosulfate 0.5g, EDTA3Na 24.66mg, Iron sulfate heptahydrate 5.55mg, Magnesium sulfate heptahydrate 123.25mg, Calcium chloride dihydrate 14.7mg, Sodium chloride 117 mg, manganese sulfate tetrahydrate 0.558 mg, zinc sulfate heptahydrate 0.144 mg, cobalt nitrate heptahydrate 0.146 mg, copper sulfate pentahydrate 0.126 mg, sodium molybdate dihydrate 0 121 mg, boric acid 0.155 mg, nicotinic acid 1 mg, thiamine 1 mg, biotin 1 mg, paraamino Ikikosan 0.5 mg, Vitamin B12 0.01 mg, calcium pantothenate 0.5 mg, pyridoxine hydrochloride 0.5 mg, folic acid 0.5 mg. The medium was autoclaved (113 ° C., 20 minutes), inoculated with Lysobacter sp. T-15, and cultured aerobically at 30 ° C. for 24 hours. After completion of the culture, the culture was centrifuged at 7,000 rpm for 20 minutes to collect bacteria.

[Example 2]
<Extraction of DNA>
A part of the cells of Lysobacter sp. T-15 cultured in Example 1 was mixed with a 1 mg / ml lysozyme solution containing 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, and 15% sucrose at 37 ° C., 10 ° C. After the minute treatment, SDS was added to a final concentration of 0.25% to dissolve the cells. Further, an equal amount of a phenol / chloroform = 1: 1 mixed solution was added, stirred for 30 minutes, and then centrifuged at 12,000 rpm for 15 minutes to recover the aqueous layer. A 1/10 volume of 3M sodium acetate (pH 5.5) was mixed with the recovered aqueous layer, and then twice the amount of ethanol was gently overlaid, and the genomic DNA was wrapped around a glass rod and separated. The separated genomic DNA is dissolved in 20 ml of 10 mM Tris-hydrochloric acid (pH 8.0), 1 mM EDTA aqueous solution (TE buffer), 200 μl of 20 mg / ml RNase A is added, and the mixture is kept at 37 ° C. for 1 hour and mixed. RNA was degraded. Next, an equal amount of a phenol / chloroform mixed solution was added, and the mixture was treated in the same manner as described above to separate the aqueous layer. One-tenth volume of 3M sodium acetate (pH 5.5) and twice the volume of ethanol were added to the collected aqueous layer, and genomic DNA was separated once again by the method described above. This chromosome was dissolved in 50 ml of TE (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA (pH 8.0)), and 20 ml of a 1: 1 mixture of TE saturated phenol and chloroform was added. After suspending, the same centrifugation was repeated, and the upper layer was transferred again to another container. 2 ml of 3M sodium acetate buffer (pH 5.5) and 50 ml of ethanol are added to 20 ml of the separated upper layer, and after stirring, cooled at -70 ° C. for 5 minutes and then centrifuged (2,000 G, 4 ° C., 15 minutes). The precipitated chromosome was washed with 75% ethanol and dried under reduced pressure. By the above operation, about 1 mg of a Lysobacter sp. T-15 DNA preparation was obtained.

[Example 3]
<Method for obtaining cell extract>
The cells collected in Example 1 were suspended in 10 mM Tris-HCl buffer (pH 8.5), and the cells were crushed using an ultrasonic crusher, and then centrifuged (15,000 G, 5 minutes). 4 ° C.) and the supernatant was obtained to obtain a cell extract.

[Example 4]
<Method for purifying urate oxidase of the present invention>
The crude enzyme solution prepared in Example 3 was directly equilibrated with 10 mM Tris-HCl buffer (pH 8.5). It was made to adsorb | suck to FF (made by Amersham Pharmacia Biotech). After thorough washing with 10 mM Tris-HCl buffer (pH 8.5), elution was performed with a linear gradient using 10 mM Tris-HCl buffer (pH 8.5) containing 0 and 0.5 M potassium chloride. did. Phenyl sep. Was added to the active fraction to a final concentration of 20%, and equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing 20% ammonium sulfate. It was adsorbed on FF (manufactured by Amersham Pharmacia Biotech) and eluted with a linear gradient using 10 mM Tris-HCl buffer (pH 7.5) containing 20 and 0% ammonium sulfate. The active fraction was desalted with G-25 (Amersham Pharmacia Biotech) equilibrated with 10 mM Tris-HCl buffer (pH 8.5), and then 10 mM Tris-HCl buffer (pH 8.5). Q sep. It was adsorbed on HP (Amersham Pharmacia Biotech) and eluted with a linear gradient using 10 mM Tris-HCl buffer (pH 8.5) containing 0 and 0.5 M potassium chloride. The active fraction was desalted with G-25 equilibrated with 10 mM phosphate buffer pH 7.0, and equilibrated with 10 mM phosphate buffer pH 7.0 and 100 mM copper sulfate. It was adsorbed on FF (Amersham Pharmacia Biotech) and eluted with a linear gradient using 10 mM phosphate buffer pH 7.0 containing 0 and 1 M imidazole. The active fraction was desalted with G-25 equilibrated with 10 mM Tris-HCl buffer (pH 7.5) and then hydroxyapatite (BioRad) equilibrated with 10 mM Tris-HCl buffer (pH 7.5). And eluted with a linear gradient using 0 and 1 M phosphate buffer pH 7.5. The active fraction was subjected to gel filtration with Superdex 200 pp (manufactured by Amersham Pharmacia Biotech) equilibrated with 10 mM phosphate buffer pH 7.0 containing 0.15 M. FIG. 11 shows the result of this Superdex 200 pp column chromatography. The obtained active fractions were measured to have molecular weights of about 31000 and 32000 (arrows in lane 1) as shown in the SDS-PAGE photograph of FIG. The purification results of Example 5 are summarized in Table 8. The obtained enzyme solution was blue and the absorption spectrum was as shown in FIG.

[Example 5]
<Partial amino acid sequencing of the enzyme of the present invention>
1 mg of the enzyme of the present invention obtained in Example 4 dissolved in 1 M urea pH 8 was fragmented with aminopeptidase Lys-C or Asp-N (37 ° C., 17 hours). The fragments were separated and purified by reverse-phase column chromatography in acetonitrile-water system containing 0.1% trifluoroacetic acid according to a conventional method, and the partial amino acid sequence was determined by Edman degradation method.

[Example 6]
<Preparation of radioactive DNA probe>
Among the partial amino acid sequences of the enzyme of the present invention determined in Example 5, an oligonucleotide probe used for gene cloning was designed based on the sequence corresponding to amino acid numbers 298 to 307 of the amino acid sequence of SEQ ID NO: 2, and SEQ ID NO: The oligonucleotide having the base sequence represented by 3 was prepared by outsourcing synthesis to an external organization (Bex). 200 ng of the completed oligonucleotide was added to T4 polynucleotide kinase buffer (50 mM Tris-HCl buffer (pH 8.0), 10 mM magnesium chloride, 10 mM 2-mercaptoethanol), and [γ- ³² P] ATP in 740 kBq (kilobecrel). (Sold by NEN) In the presence of 8.5 u T4 polynucleotide kinase, the mixture was reacted at 37 ° C. for 30 minutes to incorporate radioisotope ³² P to obtain a radioactive oligonucleotide probe.

[Example 7]
<Assay of DNA fragment containing enzyme gene of the present invention>
The chromosomal DNA (10 μg) of Lysobacter sp. T-15 obtained in Example 2 was digested with various restriction enzymes, and 1.5% agarose gel (Agarose gel H14 manufactured by Takara Bio Inc., 40 mM Tris-acetate buffer (pH 7)). .4) Electrophoresis with 2 mM EDTA) at 150 V for 1.5 hours, Southern blotting was performed according to a conventional method, and DNA was transferred from an agarose gel to a nylon membrane (manufactured by PALL: Biodyne A).

  The membrane was air-dried, prehybridized according to the manual attached to the nylon membrane, and further hybridized using the radioactive probe prepared in Example 6 at 45 ° C. overnight. After hybridization, the membrane was washed with a washing solution at 55 ° C. (6 × SSC, 0.05% sodium pyrophosphate [1 × SSC: 0.15 M sodium chloride, 15 mM sodium citrate]: about 50 ml per 100 square cm of membrane) for 10 minutes. After washing, the membrane was naturally dried. This dried membrane was overlaid on an X-ray film (New RXO-H manufactured by Fuji Photo Film Co., Ltd.), and autoradiography was performed at -70 ° C. for 24 hours under light shielding.

  After completion of autoradiography, the film was developed, and the size of the positive band indicated by the chromosome cleaved by each restriction enzyme was observed. As a result, it was revealed that the gene of the enzyme of the present invention was contained on a DNA fragment of about 8 kb by cleavage with XhoI, and a gene library was prepared from the 8 kb fragment of chromosomal DNA cleaved with XhoI.

[Example 8]
<Generation of gene library>
Lysobacter sp. T-15 chromosomal DNA (10 μg) obtained in Example 2 was cleaved with restriction enzyme XhoI, and a DNA fragment of about 8 kb was isolated according to a conventional method. This DNA fragment was ligated with 1 μg of pUC119 cleaved with restriction enzyme SalI and dephosphorylated at alkaline phosphatase (hereinafter abbreviated as BAP) 1u with a DNA ligation kit (DNA Ligation Kit). Using this, Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.), which was made into competent cells in accordance with a conventional method, was transformed, and 50 μg / ml ampicillin-containing LB (bacttripton (manufactured by DIFCO) 10 g / l, yeast extract (Manufactured by DIFCO) 5 g / l, NaCl 10 g / l) It was cultured overnight on a 1.5% agar plate medium to obtain about 2,000 ampicillin-resistant colonies to obtain a gene library.

[Example 9]
<Screening of clones containing enzyme gene of the present invention>
The gene library obtained in Example 8 was replicated on a nylon membrane (manufactured by PALL: Biodyne A), and bacterial cell DNA was immobilized on this membrane according to the attached manual.

  The membrane immobilizing this DNA was air-dried, prehybridized according to the manual attached to the nylon membrane, and further hybridized using the radioactive probe prepared in Example 6 at 45 ° C. overnight. After hybridization, the membrane was washed with a washing solution at 55 ° C. (6 × SSC, 0.05% sodium pyrophosphate [1 × SSC: 0.15 M sodium chloride, 15 mM sodium citrate]: about 50 ml per 100 square cm of membrane) for 10 minutes. After washing, the membrane was naturally dried. This dried membrane was overlaid on an X-ray film (New RXO-H manufactured by Fuji Photo Film Co., Ltd.), and autoradiography was performed at -70 ° C. for 24 hours under light shielding. After completion of autoradiography, the film was developed and one colony showing a positive signal was confirmed.

[Example 10]
<Extraction of recombinant plasmid and determination of enzyme gene base sequence>
A colony showing a positive signal selected in Example 9 was inoculated into 1.5 ml of 50 μg / ml ampicillin-containing LB liquid medium and cultured with shaking at 37 ° C. for 16 hours, and then a plasmid was extracted according to a conventional method. When the base sequence of the chromosome fragment inserted into this plasmid was analyzed by requesting an external organization (BMR), the structural gene region encoding the partial amino acid sequence of the enzyme of the present invention determined in Example 5 was confirmed. . The base sequence of the enzyme gene of the present invention was determined by determining the structural gene region and the surrounding base sequence. The nucleotide sequence of the enzyme gene of the present invention and the encoded amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[Example 11]
<Preparation of plasmid for expression of recombinant enzyme>
A PCR primer (sense) of SEQ ID NO: 4 and a PCR primer (antisense) of SEQ ID NO: 5 were prepared by synthesis, and the DNA of SEQ ID NO: 1 was amplified by PCR. The composition of the PCR solution was 1 μl of KOD DNA polymerase, 5 μl of buffer attached to 10-fold concentrated KOD DNA polymerase, 2 μl of 1 mM magnesium chloride, 7.5 μl of 0.2 mM dNTP, 10 μg / ml Lysobacter sp. T-obtained in Example 2 It consists of 15 chromosomal DNA 10 μl, 10 pmol / μl sense primer 5 μl, antisense primer 5 μl, and distilled water 14.5 μl. The PCR conditions were (1) 98 ° C. for 15 seconds, (2) 65 ° C. for 20 seconds, and (3) 72 ° C. for 60 seconds. The amplified PCR product was cleaved with NdeI and BamHI and purified, and inserted into the NdeI and BamHI cleavage sites of pET-21a to construct a plasmid in which the urate oxidase gene of the present invention was linked. In addition, the PCR product was blunt-ended by a conventional method and inserted into the SmaI cleavage sites of pUC118 and pUC119 to construct a plasmid linked with the urate oxidase gene of the present invention. The plasmid constructed by pET-21a was transformed into Escherichia coli BL21 (DE3) by a conventional method. The plasmid constructed by pUC119 was transformed into Escherichia coli JM109 by a conventional method.

[Example 12]
<Culture of transformed Escherichia coli and preparation of cell extract>
Escherichia coli BL21 (DE3) or Escherichia coli JM109 introduced with the plasmid prepared in Example 11 was inoculated into LB medium containing 50 μg / ml ampicillin, cultured at 37 ° C., and the absorbance at 600 nm of the culture solution was 0. When the temperature reached .6, the culture temperature was lowered to 24 ° C., and 1 mM IPTG as a lac promoter inducer was added. Thereafter, the cells are further cultured at 24 ° C. for 4 hours, collected by centrifugation (15,000 G, 1 minute, 4 ° C.), suspended in 10 mM Tris-HCl buffer (pH 8.5), and subjected to an ultrasonic crusher. The cells were crushed and centrifuged (15,000 G, 5 minutes, 4 ° C.), and the supernatant was obtained to obtain a cell extract.

[Example 13]
<Method for purifying urate oxidase of the present invention from transformed Escherichia coli>
The crude enzyme solution prepared in Example 12 was directly equilibrated with 10 mM Tris-HCl buffer (pH 8.5). It was made to adsorb | suck to BB (made by Amersham Pharmacia Biotech). After thorough washing with 10 mM Tris-HCl buffer (pH 8.5), elution was performed with a linear gradient using 10 mM Tris-HCl buffer (pH 8.5) containing 0 and 0.5 M potassium chloride. did. Phenyl sep. Was added to the active fraction to a final concentration of 20%, and equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing 20% ammonium sulfate. It was adsorbed on FF and eluted with a linear gradient using 10 mM Tris-HCl buffer (pH 7.5) containing 20 and 0% ammonium sulfate. The active fraction was desalted with G-25 equilibrated with 10 mM Tris-HCl buffer (pH 8.5) and then equilibrated with 10 mM Tris-HCl buffer (pH 8.5). It was adsorbed on HP and eluted with a linear gradient using 10 mM Tris-HCl buffer (pH 8.5) containing 0 and 0.5 M potassium chloride. The active fraction was desalted with G-25 equilibrated with 10 mM Tris-HCl buffer (pH 7.5) and then adsorbed to hydroxyapatite equilibrated with 10 mM Tris-HCl buffer (pH 7.5). , And eluted with a linear gradient using 0 and 1 M phosphate buffer pH 7.5. The active fraction was gel-filtered with Superdex 200 pp equilibrated with 10 mM phosphate buffer pH 7.0 containing 0.15 M. The obtained active fraction was determined to have a molecular weight of about 67,000 as shown in the SDS-PAGE photograph of FIG. 10 (arrow in lane 2). The purification results of Example 13 are summarized in Table 9.

[Example 14]
<Quantification of uric acid using urate oxidase of the present invention>
1) Preparation of uric acid measurement reagent using uric acid oxidase of the present invention The following reagent kit was prepared as a uric acid measuring reagent using the uric acid oxidase of the present invention.

1-1) Reagent 1
50 mM phosphate buffer pH 7.0

1-2) Reagent 2
50 mM phosphate buffer pH 7.0
5 U / ml peroxidase (manufactured by Sigma)
0.03% 4-AA
0.03% DAOS (N-Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, sodium salt)
1 mU / ml Urate oxidase of the present invention prepared in Example 13

2) Preparation of calibration curve Prepare 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / dl uric acid standard solution for calibration curve using Suitrol N of Nissui Pharmaceutical Co., Ltd. The measurement was performed using a Hitachi 7080 automatic analyzer. The analysis parameters of the above-mentioned reagent of Hitachi 7080 automatic analyzer were 2-point end measurement, sample amount 4 μl, reagent 1 210 μl, reagent 2 70 μl, measurement main wavelength 600 nm, subwavelength 700 nm. As a result, as shown in FIG. 13, the amount of change in uric acid concentration and absorbance was plotted linearly with R ² = 0.9950, and uric acid could be quantified by measuring the absorbance change.

[Example 15]
<Preparation of serum uric acid measurement reagent using urate oxidase of the present invention, and measured value of specimen using the measurement reagent, and commercially available serum uric acid measurement reagent (Determiner LUA (manufactured by Kyowa Medex Co., Ltd.)) Comparison with measured values of the same sample used>

1) Preparation of reagent for measuring serum uric acid using urate oxidase of the present invention The reagent kit shown below was prepared as a reagent for measuring serum uric acid using the uric acid oxidase of the present invention.
1-1) Reagent 1
40 mM phosphate buffer pH 7.0
10 U / ml peroxidase 0.03% DAOS

1-2) Reagent 2
40 mM phosphate buffer pH 7.0
0.03% 4-AA
0.5 mM EDTA-2Na
10 U / ml Urate oxidase of the present invention prepared in Example 13

2) Measurement of serum uric acid Using a Hitachi 7080 automatic analyzer, 70 human serum samples were measured using the above reagents and the Determiner L UA kit. The analysis parameters of the above-mentioned reagent of Hitachi 7080 automatic analyzer were Rate-A measurement (20-24 points), sample amount 4 μl, reagent 1 210 μl, reagent 2 70 μl, measurement main wavelength 600 nm, and subwavelength 700 nm. The Determiner L UA kit was used according to the package insert. The measured value used the calibrator for Determiner LUA kit. As shown in FIG. 14 as a correlation diagram of the measurement results, the correlation equation is Y = 1.01X−0.55 and the correlation coefficient is R ² = 0.9922, indicating a good correlation. This shows that uric acid in human serum can be measured with high accuracy using the reagent for measuring serum uric acid using the urate oxidase of the present invention.

[Example 16]
<Quantification of bilirubin F and bilirubin C using the enzyme of the present invention>
1) Preparation of bilirubin F and bilirubin C measuring reagent using the enzyme of the present invention The reagent kit shown below was prepared as a bilirubin F and bilirubin C measuring reagent using the enzyme of the present invention.

1-1) Reagent 1
100 mM phosphate buffer pH 7.2
0.2% sodium cholate 0.5 mM EDTA-2Na

1-2) Reagent 2
100 mM phosphate buffer pH 7.2
0.02% TX-100
0.5 mM EDTA-2Na
4 U / ml enzyme of the present invention prepared in Example 13

2) Preparation of calibration curve 0, 0.5, 1, 2, 3, 4, 5, 6, 7, interference check A bilirubin F and bilirubin C of International Reagents Co., Ltd. diluted with 0.9% NaCl water Standard solutions for calibration curves of 8, 9, and 10 mg / dl were measured using a Hitachi 7080 automatic analyzer. The analysis parameters of the above-mentioned reagent of Hitachi 7080 automatic analyzer were 2-point end measurement, sample amount 10 μl, reagent 1 200 μl, reagent 2 50 μl, measurement main wavelength 450 nm, and subwavelength 546 nm. In the figure, black circles (●) indicate bilirubin C and white circles (◯) indicate bilirubin F. As a result, as shown in FIG. 15, the bilirubin F and bilirubin C concentrations and the amount of change in absorbance were plotted linearly at R ² = 0.99991 and R ² = 0.9996, respectively, and bilirubin was measured by measuring the absorbance change at a wavelength of 450 nm. Quantification of F and bilirubin C was possible.

[Example 17]
<Preparation of a reagent for measuring serum total bilirubin using the enzyme of the present invention, a measured value of a specimen using the reagent, and the same specimen using a commercially available reagent for measuring total bilirubin in serum (IAtro T-Bil kit) Comparison with measured values>
1) Preparation of reagent for measuring serum total bilirubin using enzyme of the present invention The following reagent kit was prepared as a reagent for measuring serum total bilirubin using the enzyme of the present invention.

1-1) Reagent 1
100 mM phosphate buffer pH 7.2
0.2% sodium cholate 0.5 mM EDTA-2Na

1-2) Reagent 2
100 mM phosphate buffer pH 7.2
0.02% TX-100
0.5 mM EDTA-2Na
4 U / ml enzyme of the present invention prepared in Example 13

2) Measurement of serum total bilirubin Using a Hitachi 7080 automatic analyzer, 70 human serum samples were measured using the above reagents and Iatro T-Bil kit (manufactured by Mitsubishi Chemical Iatron). The analysis parameters of the above-mentioned reagent of Hitachi 7080 automatic analyzer were 2-point end measurement (15-31 points), sample amount 10 μl, reagent 1 200 μl, reagent 2 50 μl, measurement main wavelength 450 nm, sub-wavelength 546 nm. The Iatro T-Bil kit was used according to the package insert. The measured value was converted into the total bilirubin concentration using a calibrator for Iatro T-Bil kit. As shown in FIG. 16 as a correlation diagram of the measurement results, the correlation equation was Y = 0.98X + 1.38, the correlation coefficient was R ² = 0.930, and a good correlation was shown. This shows that total bilirubin in human serum can be measured with high accuracy using the reagent for measuring total bilirubin in serum using the enzyme of the present invention.

  According to the present invention, urate oxidase that is not inhibited by halide ions, nitrate ions, or Tris buffer, and that is not inhibited by a conventionally known inhibitor of urate oxidase such as xanthine, and uric acid thereof An efficient method for producing oxidase is provided. Furthermore, a composition containing the urate oxidase and a method for measuring uric acid in a sample using the urate oxidase are provided.

FIG. 1 shows a molecular phylogenetic tree based on the 16S rRNA gene sequences of Lysobacter sp. T-15 strain and related species. The length of the compared gene sequences was 1466 bp, and a molecular phylogenetic tree was created by the neighborhood joining method. FIG. 2 shows a line Weber-Burk reciprocal plot of the inhibition reaction of the urate oxidase of the present invention with NaCl. FIG. 3 shows a line Weber-Burk reciprocal plot of the inhibition reaction of Arthrobacter globiformis-derived urate oxidase by NaCl. FIG. 4 shows a quadratic plot of a line Weber-Burk reciprocal plot of the inhibition reaction in which the slopes of FIGS. 9 and 10 are plotted against NaCl concentration. FIG. 5 shows the optimum pH when uric acid of the enzyme of the present invention is used as a substrate. FIG. 6 shows the optimum pH when bilirubin C and bilirubin F of the enzyme of the present invention are used as substrates. FIG. 7 shows the optimum pH when ABTS of the enzyme of the present invention is used as a substrate. FIG. 8 shows the relationship between the reaction rate and temperature of the urate oxidase of the present invention. FIG. 9 shows the thermal stability of the urate oxidase of the present invention. FIG. 10 shows the results of SDS-PAGE. Lane 1, molecular weight marker. Lane 2, the urate oxidase of the present invention obtained by culturing and purifying Lysobacter sp. T-15. Lane 3, urate oxidase of the present invention obtained by culturing and purifying a recombinant plasmid transformant containing the gene of urate oxidase of the present invention. FIG. 11 shows a chromatogram (X first axis) using Superdex 200 pp of the urate oxidase of the present invention obtained by culturing and purifying Lysobacter sp. T-15 and a calibration curve (X second axis) using molecular weight markers. . FIG. 12 shows the absorption spectrum of the urate oxidase of the present invention. FIG. 13 shows a calibration curve for measuring uric acid using the urate oxidase of the present invention. FIG. 14 shows measured values obtained by measuring 70 human serum samples using a serum uric acid measurement reagent using the urate oxidase of the present invention, and the same sample using a commercially available serum total bilirubin measurement reagent (Determiner L UA kit). The correlation with the measured value obtained by measuring is shown. FIG. 15 shows a calibration curve of bilirubin C and bilirubin F using the enzyme of the present invention. FIG. 16 shows the measurement values obtained by measuring 70 human serum samples using the serum total bilirubin measurement reagent using the enzyme of the present invention and the commercially available serum total bilirubin measurement reagent (IAtro T-Bil kit). The correlation with the measured value obtained by measuring the specimen is shown.

Claims (14)

A urate oxidase derived from the genus Lysobacter having the following characteristics.
(1) Enzyme action: acts on uric acid in the presence of oxygen and water to produce 5-hydroxyisourea, hydrogen peroxide, and carbon dioxide.
(2) Inhibitory action: The action of (1) is not inhibited by halide ions, nitrate ions and / or Tris buffer. And xanthine does not inhibit the action of (1).
(3) Optimal pH: 6.3 to 7.8.
(4) Optimal temperature: 55 ° C. or higher.
(5) Range of temperature suitable for action: At least 30 to 60 ° C.
(6) Stability: Retains 98% or more of activity at 30 ° C. for 15 minutes in the absence of a stabilizer.
(7) Molecular weight: about 31000 and 32000 by SDS-PAGE or about 120800 ± 10000 by gel filtration. The urate oxidase according to claim 1, wherein an absorption spectrum exists at 610 ± 20 nm. The urate oxidase according to claim 1 or 2, characterized in that it is derived from Lysobacter sp. T-15 strain (Accession No. FERM P-21056). A urate oxidase comprising the amino acid sequence of either (a) or (b) below.
(A) the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 and having urate oxidase activity The urate oxidase according to any one of claims 1 to 4, which also has laccase activity. The urate oxidase according to any one of claims 1 to 5, which also has bilirubin oxidase activity. Lysobacter sp. T-15 strain having accession number FERM P-21056. A polynucleotide comprising the following base sequence (a) or (b):
(A) the base sequence represented by SEQ ID NO: 1;
(B) a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 1 and which encodes a protein having urate oxidase activity A recombinant vector comprising the polynucleotide according to claim 8. A transformant comprising the recombinant vector according to claim 9. Culturing the transformant according to claim 10 in a medium, producing and accumulating the urate oxidase according to any one of claims 1 to 6 in the culture, and collecting the urate oxidase from the culture A method for producing urate oxidase, characterized in that The composition containing the urate oxidase of any one of Claim 1 to 6. A kit for measuring uric acid, comprising at least the uric acid oxidase according to any one of claims 1 to 6 or the composition according to claim 12. A sample containing one or more components selected from the group consisting of xanthine, nitrate ions, and halide ions, the urate oxidase according to any one of claims 1 to 6, the composition according to claim 12, or A method for measuring uric acid using the uric acid measurement kit according to claim 13.