JP2008022767A - Stable reagent for determination of uric acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform accurate and easy determination of uric acid useful for clinical examination and various diagnoses by providing a uric acid determination reagent having extremely high stability, more particularly to improve the accuracy and easiness of determination and improve the easiness in the distribution and long-term storage of the reagent by providing a one-pack liquid reagent composition for the determination of uric acid storable at normal temperature (15-25°C by JIS Specification) or higher temperature not to mention of the storage in refrigerated state. <P>SOLUTION: The invention provides a liquid reagent composition for the determination of uric acid keeping the determination potency even after leaving at 60°C for 1 day by using a uricase having the optimum reaction temperature of ≤45°C and exhibiting residual activity of ≥80% after the treatment at 80°C for 5 min in the form of an aqueous solution. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a reagent composition for measuring uric acid that is more stable than before. More specifically, the present invention relates to a liquid uric acid measurement reagent composition that maintains its measurement ability even after being left at 60 ° C. for 1 day.

  Since humans do not have urate-degrading enzymes, uric acid is the end product of purine metabolism. Serum uric acid levels are elevated in diseases related to either gout and uric acid synthesis or excretion. Uric acid excess is also associated with renal dysfunction and ketoacidosis. Also, leukemias, lymphomas, erythrocytosis, and diseases that affect various nucleoprotein production and metabolism will lead to excess uric acid. Accordingly, there is a need for accurate and simple reagents and methods for measuring uric acid concentration in connection with these diseases.

  So far, various methods have been developed for measuring uric acid. Measurement methods utilizing chemical reactions were first developed, but these methods were driven out of the low specificity that is an essential problem of chemical reactions. A so-called enzymatic measurement method utilizing an enzymatic reaction was developed, and the specificity was dramatically improved by using uricase which oxidizes uric acid to allantoin. The uric acid concentration was measured by the change in absorbance before and after uricase treatment (Non-Patent Document 1).

  A method using catalase and aldehyde dehydrogenase has also been developed to measure hydrogen peroxide formed by the uricase reaction. Under the present circumstances, the production | generation of NADH by the reduction | restoration of NAD accompanying an enzyme reaction was utilized for the measurement of a uric acid concentration (nonpatent literature 2).

  A method using peroxidase to detect hydrogen peroxide formed by the uricase reaction has also been developed and is most popular today. In this method, peroxidase catalyzes a reaction in which hydrogen peroxide generated by the reaction of uricase performs oxidative coupling between 4-aminoantipyrine and a phenolic compound. Interference with bilirubin was reduced by the addition of potassium ferrocyanide (Non-patent Document 3).

  Due to the above situation, the uric acid measurement reagent has an enzyme as a main component. Therefore, the shelf life, ie stability, of the reagent is limited by the stability of the enzyme. In particular, regardless of which enzymatic measurement method is used, uricase, which is the main reaction enzyme, is important in the shelf life of the reagent.

  The stability of commercially available uric acid measurement reagents varies. For example, the stability time of the uric acid measurement reagent has been reported, and it is described that the uric acid measurement reagent set composed of two types of liquid reagents had stability for 3 months at 2 to 8 ° C. It cannot be said that the stability is satisfactory in the properties (Non-patent Document 3). On the other hand, the stability of a single liquid uric acid measurement reagent was poor. A single, ready-to-use liquid reagent can be measured quickly without taking reagent preparation time, and is accurate because there is no volume error in mixing.

  It is known that uricase is stabilized with a nonionic surfactant, serum albumin and / or basic amino acid in a lyophilized state (Patent Document 1).

  The uric acid measurement reagent containing uricase was stabilized by EDTA. The reagent had uricase, peroxidase, O-dianisidine, 0.03% bovine serum albumin, and 1 mM EDTA in a Tris buffer solution at pH 7.5. The stability of the refrigerated reagent was at a level of several weeks and was not sufficient (Non-patent Document 4).

JP-A-60-224499 M. Herman et al., "J. Biol. Chem.", 1947, 167, p429 R.M.White et al., “Clin. Chem.”, 1977, Vol. 23, p1538 P. Fossati et al., “Clin. Chem.”, 1980, Vol. 26, p227 J.H.Jr.Marymont et al., “Am.J.Clin.Pathol.”, 1980, Vol. 42, p630

  An object of the present invention is to provide a highly stable uric acid measurement reagent composition that makes accurate and simple uric acid measurement useful for clinical examinations and various diagnoses. More specifically, by providing a liquid uric acid measuring reagent composition having a single composition that can be stored and maintained at normal temperature (15 to 25 ° C. according to JIS standards) or at a temperature higher than refrigerated storage, The purpose is to improve accuracy and convenience during measurement, and also to improve convenience during circulation and long-term storage of reagents.

  As a result of intensive studies to achieve the above object, the present inventors have succeeded in obtaining a highly stable uricase by artificially modifying the amino acid sequence. And, it was found that such a uricase has both high temperature stability and an optimum reaction temperature close to a practical range (near 37 ° C.), a property that is not found in natural enzymes, and a liquid using the uricase. The present invention was completed by developing a uric acid measurement reagent.

That is, the present invention has the following configuration.
(1) A liquid uric acid measurement reagent composition that maintains its measurement ability even after being left at 60 ° C. for 1 day.
(2) The liquid uric acid measurement reagent composition according to (1), which contains uricase having a residual activity of 80% or more after treatment at 80 ° C. for 5 minutes in an aqueous solution.
(3) The liquid uric acid measurement reagent composition according to (1), which contains a uricase having an optimal reaction temperature of 45 ° C. or lower and a residual activity of 80% or more after treatment at 80 ° C. for 5 minutes in an aqueous solution.
(4) The uricase is a uricase variant comprising an amino acid sequence having 50% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing (2) or (3) The liquid uric acid measurement reagent composition as described.
(5) The liquid uric acid according to (4), wherein the uricase is a modified uricase comprising an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing Measurement reagent composition.
(6) The uricase is a modified uricase comprising an amino acid sequence in which one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing are deleted, substituted or added. (2) The liquid uric acid measurement reagent composition according to (2) or (3).
(7) The uricase is a uricase variant consisting of an amino acid sequence in which the amino acid at position 298 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or an amino acid sequence equivalent thereto is substituted with another amino acid. The liquid uric acid measurement reagent composition as described in (2) or (3),
(8) The uricase is a uricase variant in which the amino acid at position 298 in the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing or an amino acid at an equivalent position thereof is substituted with cysteine (7) The liquid uric acid measuring reagent composition according to claim).
(9) The liquid uric acid measurement reagent composition according to any one of (1) to (8), which is used in combination with a reagent for detecting hydrogen peroxide derived from oxidation of uric acid.

  According to the present invention, it is possible to improve the accuracy and simplicity of uric acid measurement. Furthermore, it becomes possible to supply a novel uric acid measurement reagent with improved ease of distribution and long-term storage.

  Hereinafter, the present invention will be described in detail.

  Examples of the sample for uric acid measurement in the present invention include biological samples such as serum, urine, and plasma, but are not limited thereto.

  The liquid uric acid measurement reagent composition of the present invention contains at least a uricase and a buffer such as phosphate, GOOD buffer, or Tris buffer. Furthermore, chelating reagents such as EDTA and O-dianisidine that capture ions that interfere with the enzyme reaction, ascorbic acid oxidase that eliminates ascorbic acid, which is a substance that interferes with the determination of hydrogen peroxide, Triton X-100 and NP-40 And various antibacterial agents such as streptomycin and sodium azide, preservatives and the like. These reagents may be a single reagent or may be composed of two or more kinds of reagents, but a simple single reagent is more preferable in order to take advantage of the present invention. In order to take advantage of the present invention, a liquid reagent that is easy to handle is preferred.

  As the buffer used in the present invention, any buffer having a sufficient buffer capacity in the pH range of 6.5 to 8.5 can be used. Buffers in this pH range include phosphate, tris, bis-trispropane, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), and 3- [N-tris (hydroxymethyl) methyl. Amino] -2-hydroxypropanesulfonic acid (TAPSO). The preferred buffer is phosphate because of its low cost and high stability. A preferred concentration range is 20-200 mM phosphate, pH 7-8.

  In the present invention, the reagent for detecting hydrogen peroxide derived from the oxidation of uric acid is not particularly limited as long as it is a reagent for detecting hydrogen peroxide derived from the oxidation of uric acid. And hydrogen peroxide coloring reagent. The peroxidase and hydrogen peroxide coloring reagent used are not limited in any way. Preferred indicators are those that are stable in solution and have low bilirubin interference.

  As the hydrogen peroxide coloring reagent, for example, 4-aminoantipyrine or 3-methyl-2-benzothiazoline hydrazone (MBTH) and phenol or a derivative thereof or aniline or a derivative thereof are used in combination. Examples of the phenol derivative include 2-chlorophenol, 4-chlorophenol, 1,2-dichlorophenol and the like. Examples of aniline derivatives include N, N-dimethylaniline, N, N-diethylaniline, N, N-diethyl-m-toluidine, N, N-dimethyl-m-anisidine, N-ethyl-N- (3-methylphenyl). ) -N′-acetylethylenediamine, N-ethyl-N- (β-hydroxyethyl) -m-toluidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N-ethyl- N-sulfopropyl-m-toluidine, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N -Ethyl-N-sulfopropyl-m-anisidine, N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine, N-ethyl -N- (2-hydroxy-N-sulfopropyl) -m-anisidine and the like. In addition, 10-X-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine, bis [8-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine, 1,4-bis (dimethylamino) diphenyl A leuco dye such as-(2,7-dihydroxy-4-naphthyl) methane may be used.

  In the present invention, preferred indicators for detecting hydrogen peroxide derived from oxidation of uric acid include benzidines, leuco dyes, 4-aminoantipyrine, phenols, naphthols and aniline derivatives. More preferred indicators are 4-aminoantipyrine and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS). The preferred concentration range is 0.05-10 mM for 4-aminoantipyrine and 0.05-10 mM for TOOS.

  The peroxidase used for detecting hydrogen peroxide derived from the oxidation of uric acid in the present invention is preferably a horseradish peroxidase because it is commercially available with high purity and low cost. The enzyme concentration must be high enough for a rapid and complete reaction, preferably 1,000-50,000 U / L.

  Ferrocyanide may be added to the reagent to minimize bilirubin interference. However, the presence of metal ions such as ferrocyanide can destabilize indicators and enzymes. The stability of the reagent of the present invention is high enough to allow the addition of ferrocyanide. The preferred concentration range of ferrocyanide is 1-400 μM, the maximum concentration is the concentration that inhibits enzyme activity.

  Inactive proteins may be added to further increase stability. Inactive proteins include serum albumins, globulins and fibrous proteins. A preferred protein is bovine serum albumin and a preferred concentration in wt / vol is 0.05-1%. Lower concentrations can be useful. Preferred inactive proteins are those that do not contain protease impurities that will cause enzymatic degradation.

  The uric acid concentration is measured using a specific volume of the sample and a specific volume of the reagent. Absorbance measurements are performed as soon as possible after mixing and before significant changes in absorbance due to uric acid metabolism occur to measure the sample blank. A first absorbance measurement after 0.5-5 seconds is appropriate. The second absorbance measurement is typically 3-5 minutes at 37 ° C. at a uric acid concentration of 20 mg / dL after the absorbance has become steady. Typically, the reagent is standardized with an aqueous or serum solution having a known uric acid concentration.

The uricase used in the present invention preferably has a residual activity of 80% or more after treatment in an aqueous solution at 80 ° C. for 5 minutes. More preferably, the optimum reaction temperature is 45 ° C. or lower, and the residual activity is 80% or higher after treatment in an aqueous solution at 80 ° C. for 5 minutes.
As a result of various studies, the present inventors have come to the conclusion that unless the uricase has such properties, it cannot be stored at room temperature and cannot be distributed at room temperature. In order to circulate at room temperature, depending on the season and region, it is necessary to prepare for leaving for one day to one week at a high temperature around 40 ° C. Thus, the requirement that the measurement ability is maintained even after being left at 60 ° C. for one day in the present invention is by no means an excessive requirement.
Such a uricase is not known in nature and must be prepared by artificially modifying the amino acid sequence.
In the present invention, uricase is preferably used at an enzyme concentration of 100 to 5,000 U / L in the uric acid measurement reagent composition.

  The term “used in combination” with the reagent for detecting hydrogen peroxide derived from the oxidation of uric acid in the present invention includes being used simultaneously with or before or after the reagent, and the reagent and the liquid uric acid measuring reagent. This includes providing the composition in a separate container, or including the reagent in a liquid uric acid measurement reagent composition and providing it in the same container.

  The method for modifying uricase used in the present invention comprises a deletion, substitution or addition of one or several amino acids in the amino acid sequence constituting the protein before modification having uricase activity, thereby comparing with the protein before modification. , To improve stability. In the present invention, “improvement of stability” means stability when the variant (a) and the parent protein (b) before modification are sufficiently purified and dissolved in an appropriate buffer. Is a state where (a)> (b). “Sufficiently purified” means a state in which no contaminating protein other than uricase is observed, for example, by SDS polyacrylamide gel electrophoresis. The “appropriate buffer” is not particularly limited as long as its type and concentration are selected so that it has a sufficient buffer capacity in the pH range where uricase acts, but for example, 50 mM phosphate buffer (pH 8.0), 50 mM A borate buffer (pH 8.0), a 50 mM Tris buffer (pH 8.0), or the like is selected. The buffer solution may further contain a surfactant, a salt, a chelating reagent, a preservative, etc., assuming a diagnostic use.

Examples of the protein having uricase activity to be used for the modification of uricase used in the present invention include uricase derived from microorganisms such as Bacillus, Candida, Enterobacter, Cellulomonas, Arthrobacter, etc. It is not particularly limited.
Specifically, for example, uricase derived from Bacillus sp. Strain TB-90 is exemplified, the amino acid sequence thereof is SEQ ID NO: 1 in the sequence listing, and the gene encoding the amino acid sequence is SEQ ID NO: 2, respectively. Indicated. These are all described in Japanese Patent No. 1966484. In SEQ ID NO: 1, the amino acid notation is numbered with methionine as 1.

  As long as the uricase used in the present invention retains uricase activity, the uricase is intermolecular or intramolecularly crosslinked, chemically modified with a sugar chain or other functional group, or provided with a histidine tag. Even if it is a thing, various fusion proteins, it does not become a problem in particular.

The variant of uricase used in the present invention preferably has 50% or more homology with the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing. More preferably, one or several amino acids located in the loop region located at the interface of the subunit or in the region interacting with it are deleted, substituted or added. (Here, the mode of modification may be two or more of deletion / substitution and addition)
Examples of such a modified site include the amino acid at position 298 in the amino acid sequence (SEQ ID NO: 1) encoding uricase derived from Bacillus sp. Strain TB-90. The amino acid at position 298 in SEQ ID NO: 1 is particularly preferably one substituted with cysteine.

  The above conversion position may be an equivalent position in the amino acid sequence of a protein having uricase activity derived from a source other than Bacillus sp. TB-90. Whether the positions are equivalent can be determined based on knowledge of the primary structure and the three-dimensional structure.

  The three-dimensional structure of uricase is not limited to those derived from the aforementioned genus Bacillus, but also for uricase from Aspergillus flavus and Arthrobacter globiformis (Protein Data Bank (http: //www.rcsborg)). /pdb/Welcome.do), etc., but there has been no description suggesting that the stability is improved by protein engineering.

For proteins with uricase activity derived from sources other than the above, the amino acid to be converted can be selected using information on the primary structure and the three-dimensional structure, and a modified body with improved stability can be obtained without undue investigation. Preferably, a uricase variant having 50% or more homology with the amino acid sequence described in SEQ ID NO: 1 of the sequence listing is mentioned, and more preferably, it is described in SEQ ID NO: 1 of the sequence listing. And a modified uricase having 80% or more homology with the amino acid sequence.
For example, uricase derived from Aspergillus flavus is known to have a low primary sequence homology of 26% with Bacillus sp. Strain TB-90 (J. Biochem. 119, 80-84, 1996), a highly stable uricase that has a high degree of similarity in three-dimensional structure and can be used in the present invention.
In addition, the homology of an amino acid sequence can be searched by homology search of two types of sequences using commercially available gene analysis software, such as GENETYX.

  Furthermore, the variant of uricase used in the present invention may also include a chimeric protein composed of a combination of fragments of wild-type uricase from several sources, so long as the activity against uric acid is essentially maintained.

The uricase variant is synthesized by a gene encoding the variant. A gene encoding a modified uricase can be obtained, for example, by modifying a DNA fragment containing a gene encoding a wild-type uricase obtained from various sources (origins) such as a microorganism. Specifically, for example, Bacillus sp. TB-90 strain, Aspergillus flavus, Arthrobacter globiformis, Candida utilis (Candida utilis) It can be prepared using a gene encoding a known uricase such as Cellulomonas flavigena).
The gene encoding the uricase variant is preferably a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence described in SEQ ID NO: 1 and encodes a protein having uricase activity It is. Here, the stringent condition refers to a condition in which nucleic acids having high homology, for example, hybridize at a temperature ranging from Tm of a perfectly matched hybrid to a temperature 15 ° C., preferably 10 ° C. lower than the Tm. Specifically, for example, hybridization is performed at 68 ° C. for 20 hours in a general hybridization buffer (for example, × 6 SSC, × 5 Denhardt, 0.1% SDS, 100 μg / ml salmon sperm DNA). The conditions for soybean. In the present invention, the base sequence encoding an amino acid sequence having 50% or more homology, preferably 80% or more homology with the amino acid sequence (SEQ ID NO: 2) encoded by the base sequence described in SEQ ID NO: 1, This is considered to correspond to a base sequence that hybridizes with the base sequence described in SEQ ID NO: 1 under the stringent conditions of

  Further, the gene encoding the uricase variant may include a modified codon usage to improve uricase expression.

  As a method for modifying a gene encoding uricase, a commonly used technique for modifying genetic information is used. That is, DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. Specific methods for converting bases in DNA include, for example, the use of a commercially available kit (Transformer Site-Directed Mutagenesis Kit; manufactured by Clonetech, QuickChange Site Directed Mutagenesis Kit; manufactured by Stratagene), or PCR. Use.

The prepared DNA having the genetic information of the modified uricase is transferred into the host microorganism in a state of being linked to the plasmid, and becomes a transformant that produces the modified protein.
When using a plasmid as a vector, for example, pBluescript, pUC18, etc. can be used when Escherichia coli is used as a host microorganism. Examples of host microorganisms that can be used include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5α. As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method of transferring the recombinant DNA in the presence of calcium ions can be employed. An electroporation method may be used. Furthermore, a commercially available competent cell (for example, competent high JM109; manufactured by Toyobo) may be used.

  Such genes may be extracted from these strains or chemically synthesized. Furthermore, it is possible to obtain a DNA fragment containing a uricase gene by using the PCR method.

  As a method for obtaining a gene encoding uricase before modification, for example, in the case of a uricase-producing bacterium having an unknown gene sequence, after separating and purifying the chromosome, the DNA is cleaved using sonication, restriction enzyme treatment, etc. A linear expression vector and the blunt end or sticky end of both DNAs are closed with DNA ligase or the like to construct a recombinant vector. After transferring the recombinant vector to a replicable host microorganism, screening can be performed using the expression of the vector marker and enzyme activity as an index to obtain a microorganism carrying a recombinant vector containing a gene encoding uricase. .

  If the gene sequence is known, after creating a primer that amplifies the gene encoded by uricase, the gene is obtained using the PCR method, and linked to an appropriate vector, A recombinant vector containing a gene encoding uricase can be obtained relatively easily.

  The host microorganism for transformation is not particularly limited as long as the recombinant vector is stable, can autonomously grow, and can express a foreign gene. Generally, Escherichia coli W3110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH5α, and the like can be used. The resulting microorganism, which is a transformant, can stably produce a large amount of uricase by being cultured in a nutrient medium. The selection as to whether or not the target recombinant vector is transferred to the host microorganism may be made by searching for a microorganism that expresses a drug resistance marker of the vector holding the target DNA.

  The base sequence of the uricase gene obtained by the above method is decoded by the dideoxy method described in Science, Vol. 214, 1205 (1981). The uricase amino acid sequence is deduced from the base sequence determined as described above.

  Thus, transfer from a recombinant vector carrying a uricase gene once selected to a recombinant vector that can be replicated in a microorganism capable of producing uricase can be carried out by using a restriction enzyme or PCR from the recombinant vector carrying the uricase gene. It can be easily carried out by recovering DNA, which is a uricase gene, by a method and binding it to other vector fragments. Moreover, the transformation of the microorganisms which can produce uricase by these vectors can use the competent cell method by electrolysis, the electroporation method, etc.

  For example, a microorganism, which is a transformant obtained as described above, can stably produce a large amount of a modified protein by being cultured in a nutrient medium. The culture form of the host microorganism, which is a transformant, may be selected in consideration of the nutritional physiological properties of the host, and in many cases, the culture is performed in liquid culture. Industrially, aeration and agitation culture is advantageous.

  As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

  The culture temperature can be appropriately changed within the range in which the fungus grows and produces a modified uricase. For example, when Escherichia coli is used as a host, it is preferably about 20 to 42 ° C. Although the culture time varies slightly depending on the conditions, the culture may be completed at an appropriate time when the uricase variant reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be changed as appropriate as long as the bacteria grow and produce a modified uricase, but is preferably in the range of about pH 6.0 to 9.0.

  Although the culture solution containing the microbial cells producing the uricase variant can be collected and used as it is, generally, according to a conventional method, if the uricase variant is present in the culture solution, filtration, centrifugation, etc. It is used after separating the uricase modified substance-containing solution and the microbial cells. In the case where the modified uricase is present in the microbial cells, the microbial cells are collected from the obtained culture by means such as filtration or centrifugation, and then the microbial cells are collected by a mechanical method or an enzymatic method such as lysozyme. In addition, if necessary, a chelating agent such as EDTA and a surfactant are added to solubilize the modified uricase and separated and collected as an aqueous solution.

  The uricase variant-containing solution obtained as described above is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. It can be precipitated by. Heat treatment and isoelectric point treatment are also effective purification means. Thereafter, a purified uricase variant can be obtained by performing gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

  For example, gel filtration using Sephadex gel (GE Healthcare Bioscience), column chromatography such as DEAE Sepharose CL-6B (GE Healthcare Bioscience), octyl Sepharose CL-6B (GE Healthcare Bioscience), etc. The purified enzyme preparation can be obtained by separation and purification. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

  Hereinafter, the present invention will be specifically described by way of examples. In the examples, the liquid uric acid measurement reagent was prepared with the following composition. The measurement conditions were as follows. The activity of uricase was measured as follows. The reagent used was purchased from Nacalai Tesque.

<Liquid uric acid measurement reagent>
Uricase 500U / L
Triton X-100 0.001% (W / V)
EDTA 0.5 mM
50 mM potassium phosphate buffer (pH 7.5)
<Uric acid measurement method>
In uric acid measurement, disappearance of uric acid due to uricase reaction was measured by change in absorbance.
Sample: 10-100 μM uric acid aqueous solution 3 ml of liquid uric acid measurement reagent is pre-warmed at 37 ° C. for 5 minutes, and then 0.1 ml of uric acid aqueous solution is added to start the reaction. After reacting accurately at 37 ° C. for 5 minutes, 0.2 ml of 20% (W / V) aqueous KOH solution is added to stop the reaction, and the absorbance at 290 nm is measured (ΔODtest). In the blind test, 0.1 ml of water was added in place of the aqueous uric acid solution, and the operation was performed in the same manner as described above to measure the absorbance (ΔOD blank). From the obtained absorbance, the amount of uric acid was calculated based on a calibration curve.
<Uricase activity measurement method>
The uricase activity was determined by measuring the change in absorbance of uric acid disappearance due to uricase reaction using uric acid as a substrate. After pre-warming 2.5 ml of 42 mM borate buffer (pH 8.0) containing 40 μM uric acid, 0.00083% (W / V) Triton X-100 and 0.83 mM EDTA at 37 ° C., Add 0.5 ml of uricase solution previously diluted with enzyme diluent (50 mM borate buffer (pH 8.0) containing 0.001% (W / V) Triton X-100 and 0.1 mM EDTA), Start the reaction. After reacting accurately at 37 ° C. for 5 minutes, 0.2 ml of 20% (W / V) aqueous KOH solution is added to stop the reaction, and the absorbance at 290 nm is measured (ΔODtest). In the blind test, 0.5 ml of an enzyme diluent was added instead of the enzyme solution, and the absorbance was measured by performing the same operation as above (ΔOD blank). From the obtained absorbance, the enzyme activity of uricase was calculated based on the following formula. The amount of enzyme that oxidizes 1 micromole of uric acid per minute under the above conditions is defined as 1 unit (U).
Formula activity value (U / ml) = {ΔOD / min (ΔODtest−ΔODblank) × 3.2 (ml) × dilution ratio} / {12.2 × 1.0 (cm) × 0.5 (ml)}
3.2 (ml): Total liquid volume
12.2: millimolar extinction coefficient when uric acid was measured under the above measurement conditions
1.0cm: Optical path length of the cell
0.5 (ml): Enzyme sample volume

Example 1 Preparation of Gene Encoding Uricase Variant Expression plasmid pKU1 (Patent No. 1966484) containing a uricase gene derived from Bacillus sp. TB-90, synthetic oligonucleotide described in SEQ ID NO: 3 in the sequence listing, and this Using a synthetic oligonucleotide complementary to the amino acid sequence, using the QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE), performing a mutation treatment operation according to the protocol, further determining the base sequence, A recombinant plasmid (pUOD-R298C) encoding a modified uricase in which the 298th arginine was replaced with cysteine was obtained.

Example 2 Production of Modified Uricase Variant Escherichia coli JM109 strain competent cell (manufactured by Toyobo) was obtained using the recombinant plasmid (pUOD-R298C) obtained in Example 1 to obtain the transformant. did. The obtained transformant was named Escherichia coli JM109 (pUOD-R298C).
500 ml of TB medium was dispensed into a 2 L Sakaguchi flask, autoclaved at 121 ° C. for 20 minutes, and after standing to cool, ampicillin and isopropyl-β-D-thiogalactoside, which were separately sterile filtered, each had a final concentration of 100 μl / ml and 0. Added to 1 mM. The medium was inoculated with 5 ml of a culture solution of Escherichia coli JM109 (pUOD-R298C) previously cultured in an LB medium containing 100 μl / ml ampicillin at 30 ° C. for 16 hours, followed by aeration-agitation culture at 37 ° C. for 24 hours. After completion of the culture, the cells are collected by centrifugation, suspended in 50 mM borate buffer (pH 8.0), crushed with a French press, further centrifuged, and the supernatant is used as a crude enzyme solution. Obtained. The obtained crude enzyme solution was subjected to denucleic acid removal with polyethyleneimine and ammonium sulfate fractionation, and after heat treatment at 55 ° C. for 1 hour, dialyzed against 50 mM borate buffer (pH 8.0). Furthermore, the purified enzyme preparation (R298C) was obtained by separating and purifying with DEAE Sepharose CL-6B (manufactured by GE Healthcare Bioscience) and octyl sepharose (manufactured by GE Healthcare Biosaoens). . The specimen obtained by this method was confirmed to be single by SDS polyacrylamide gel electrophoresis.

Example 3 Stability of uricase variant and reaction temperature evaluation The reaction temperature and temperature stability of the uricase obtained in Example 2 were evaluated. As a target, uricase derived from Bacillus sp. TB-90 strain (product code: UAO-211, manufactured by Toyobo Co., Ltd.) was used (comparative example uricase). UAO-211 is a commercially available uricase, and is an enzyme derived from a thermophilic Bacillus genus, and therefore has excellent stability among currently known uricases. As reaction temperature measurement conditions, uricase diluted with an enzyme diluent (50 mM borate buffer (pH 8.0) containing Triton X-100 of 0.001% (W / V) and 0.1 mM EDTA) was used. The activity was measured at each reaction temperature. The stability evaluation method at each temperature was performed by diluting uricase to 5 U / ml with 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and treating the enzyme activity after 5 minutes treatment at each temperature. Was performed by comparing with before treatment.
The results are shown in FIG. 1 and FIG. The optimal reaction temperature of the modified uricase was 40 ° C., and the residual activity after treatment at 80 ° C. for 5 minutes was about 90%. That is, it was confirmed that a uricase having an optimal reaction temperature of 45 ° C. or lower and having a residual activity of 80% or more was obtained by treatment at 80 ° C. for 5 minutes in an aqueous solution.
In contrast, the optimum reaction temperature of the comparative uricase was 50 ° C., and the residual activity after treatment at 80 ° C. for 5 minutes was about 0%.

Example 4 Stability Evaluation of Liquid Uric Acid Measurement Reagent Composition Next, using the uricase obtained in Example 2, a liquid uric acid measurement reagent (reagent of the present invention) was prepared. As a control, a liquid uric acid measurement reagent (comparative example reagent) using a uricase (product code: UAO-211, manufactured by Toyobo Co., Ltd.) derived from Bacillus sp. TB-90 strain was prepared.
The stability of the reagent of the present invention and the comparative example reagent was confirmed by storage at 60 ° C. As shown in FIG. 3, the reagent of the present invention maintained 50% or more (58%) of the initial measurement value even after being left at 60 ° C. for 1 day. On the other hand, the value of the comparative example reagent decreased to about 40% of the initial measured value after standing at 60 ° C. for 1 day.

  According to the present invention, the stability of the liquid uric acid measurement reagent is dramatically improved, and the accuracy and convenience of uric acid measurement can be improved. In addition, it is possible to supply a novel reagent for measuring uric acid with improved ease of distribution and long-term storage, which can contribute to the further spread of clinical tests based on preventive medicine, for example.

FIG. 1 shows the evaluation results of the reaction temperature of the modified uricase of the present invention and the comparative uricase. ●: Modified uricase of the present invention, ○: Comparative uricase FIG. 2 shows the temperature stability evaluation results of the uricase variant of the present invention and the comparative uricase. ●: Modified uricase of the present invention, ○: Comparative uricase FIG. 3 shows the stability evaluation results at 60 ° C. of the reagent of the present invention and the comparative reagent. ●: Reagent of the present invention, ○: Reagent of comparative example

Claims (9)

A liquid uric acid measurement reagent composition that maintains its measurement ability even after being left at 60 ° C. for 1 day. The liquid uric acid measuring reagent composition according to claim 1, comprising uricase having a residual activity of 80% or more after treatment at 80 ° C for 5 minutes in an aqueous solution. The liquid uric acid measuring reagent composition according to claim 1, which contains uricase having an optimal reaction temperature of 45 ° C or lower and a residual activity of 80% or more after treatment at 80 ° C for 5 minutes in an aqueous solution. The liquid uric acid measurement according to claim 2 or 3, wherein the uricase is a modified uricase consisting of an amino acid sequence having 50% or more homology with the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. Reagent composition. The liquid uric acid measurement reagent composition according to claim 4, wherein the uricase is a modified uricase comprising an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. object. The uricase is a uricase variant comprising an amino acid sequence in which one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing are deleted, substituted or added. 4. The liquid uric acid measurement reagent composition according to 2 or 3. The uricase is a uricase variant comprising an amino acid sequence in which the amino acid at position 298 in the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing or an equivalent position thereof is substituted with another amino acid. The liquid uric acid measurement reagent composition according to claim 2 or 3. The uricase is a variant of uricase comprising an amino acid sequence in which the amino acid at position 298 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing or an equivalent position thereof is substituted with cysteine. Item 8. The liquid uric acid measurement reagent composition according to Item 7. The liquid uric acid measuring reagent composition according to any one of claims 1 to 8, which is used in combination with a reagent for detecting hydrogen peroxide derived from oxidation of uric acid.

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