WO2015008637A1 - Gène xanthine oxydase et séquence d'acides aminés codant pour ce gène - Google Patents

Gène xanthine oxydase et séquence d'acides aminés codant pour ce gène Download PDF

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WO2015008637A1
WO2015008637A1 PCT/JP2014/067892 JP2014067892W WO2015008637A1 WO 2015008637 A1 WO2015008637 A1 WO 2015008637A1 JP 2014067892 W JP2014067892 W JP 2014067892W WO 2015008637 A1 WO2015008637 A1 WO 2015008637A1
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amino acid
seq
xanthine oxidase
acid sequence
sequence shown
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悠 歌島
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東洋紡株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y117/00—Oxidoreductases acting on CH or CH2 groups (1.17)
    • C12Y117/03—Oxidoreductases acting on CH or CH2 groups (1.17) with oxygen as acceptor (1.17.3)
    • C12Y117/03002—Xanthine oxidase (1.17.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0093—Oxidoreductases (1.) acting on CH or CH2 groups (1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

  • the present invention relates to a xanthine oxidase (hereinafter also abbreviated as XTO) enzyme gene obtained for heterologous protein expression in a specific host.
  • the present invention also includes two subunit proteins constituting xanthine oxidase, a nucleotide sequence encoding the protein, an accessory protein that plays an important role in efficiently expressing xanthine oxidase, and a nucleotide sequence encoding the protein.
  • the present invention relates to a method for efficiently recombinantly producing a stable quality xanthine oxidase using an approximate gene.
  • the present invention relates to a novel thermostable xanthine oxidase gene and a method for producing the same.
  • the xanthine oxidase of the present invention is an enzyme that catalyzes a reaction of oxidizing hypoxanthine and xanthine to produce hydrogen peroxide, and is characterized by excellent thermal stability.
  • the xanthine oxidase of the present invention is used to measure inorganic phosphorus, which is an indicator of nephritis / thyroid disorders, adenosine deaminase, which is an indicator of immune diseases, and the like.
  • xanthine oxidase is known to exist in milk.
  • milk-derived xanthine oxidase is allowed to act on hypoxanthine and xanthine-containing samples and the hydrogen peroxide produced is converted to a dye, it is known that the dye is decomposed in a short period of time and cannot be quantified based on hydrogen peroxide. It has been. The reason is due to the production ratio of superoxide radicals and hydrogen peroxide produced when milk-derived xanthine oxidase decomposes xanthine. In the case of milk-derived xanthine oxidase, it is combined with hydrogen peroxide at a high ratio of about 50%.
  • Non-patent Document 3 It is considered that superoxide radicals are produced (Non-patent Document 3). It is considered that superoxide radicals produced together with hydrogen peroxide oxidize organic dyes and cause dye discoloration. Thus, milk-derived xanthine oxidase is not suitable for measurement using dyes.
  • Non-patent document 1 Enzymes obtained from microorganisms belonging to the genus Pseudomonas, Escherichia, Arthrobacter, Nocardia, etc. are derived from microorganisms.
  • Non-patent document 2 Enzymes obtained from Enterobacter cloacae.
  • xanthine oxidase produced by a microorganism belonging to the genus Arthrobacter described in Patent Document 1 is excellent in thermal stability. Furthermore, it is substrate specific for hypoxanthine and xanthine.
  • xanthine oxidase which catalyzes a reaction (see the following reaction formula) that oxidizes hypoxanthine to xanthine and further oxidizes xanthine to uric acid, and produces hydrogen peroxide using an enzyme as an electron acceptor
  • a heat-resistant xanthine oxidase characterized in that it has a residual activity of at least 70% after treatment for 30 minutes. Reaction formula: hypoxanthine + O 2 ⁇ xanthine + H 2 O 2 Xanthine + O 2 ⁇ urea + H 2 O 2
  • Patent Document 1 when the quantitative property of the substrate was measured in a color developing system, it was shown that the xanthine oxidase derived from milk was 80%, whereas xanthine oxidase derived from Arthrobacter was 100%. Therefore, it can be said that Arthrobacter-derived xanthine oxidase has a low production amount of superoxide radicals and is advantageous also in the measurement of a coloring system.
  • the present inventor newly found a problem that the specific activity of the obtained enzyme may be reduced when xanthine oxidase is produced and purified by a wild-type microorganism belonging to the genus Arthrobacter.
  • the object of the present invention is to solve this problem and obtain a stable quality xanthine oxidase.
  • the present inventor found that the specific activity is reduced because protease produced by the microorganism belonging to the genus Arthrobacter appears in the disruption solution by crushing the cells at the early stage of the purification step and comes into contact with xanthine oxidase. I found out that it was for disassembly. Therefore, the present inventor performed cloning of a gene encoding xanthine oxidase derived from Arthrobacter, which has not been performed so far, to clarify the entire sequence thereof, and by gene recombination using the gene, This problem was solved by providing a method for producing xanthine oxidase by a host with low production.
  • xanthine oxidase can be efficiently produced by co-expressing an accessory protein considered to be important for coordinating molybdenum to xanthine oxidase. Furthermore, it discovered that the enzyme characteristic of the obtained xanthine oxidase is equivalent to the wild type xanthine oxidase derived from Arthrobacter.
  • the present invention has been completed based on these findings and comprises the following items 1 to 11.
  • Item 1 A xanthine oxidase comprising the polypeptide of any one of (a) to (c) below: (A) a combination of polypeptides composed of a subunit consisting of the amino acid sequence shown in SEQ ID NO: 1 and a subunit consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) in the amino acid sequence shown in SEQ ID NO: 1 , 0, 1 or several amino acid residue substitutions, deletions, insertions, additions and / or inverted subunits, and the amino acid sequence shown in SEQ ID NO: 2, 0, 1 or several
  • a combination of polypeptides having xanthine oxidase activity comprising a subunit consisting of an amino acid sequence substituted, deleted, inserted, added and / or inverted.
  • A A combination of DNA encoding the amino acid sequence shown in SEQ ID NO: 1 and DNA encoding the amino acid sequence shown in SEQ ID NO: 2
  • B DNA consisting of the base sequence shown in SEQ ID NO: 4, and SEQ ID NO: 5
  • C identity between the DNA consisting of the base sequence whose identity to the base sequence shown in SEQ ID NO: 4 is 80% or more and the base sequence shown in SEQ ID NO: 5
  • a combination of DNAs that encode a combination of polypeptides having xanthine oxidase activity (excluding the combination of (B) above).
  • Item 6 The vector according to Item 3, further comprising the DNA according to Item 4.
  • Item 7. A transformant comprising the vector according to item 5 or item 6.
  • Item 8 A method for producing a polypeptide having xanthine oxidase activity, comprising culturing the transformant according to item 7, and collecting xanthine oxidase from the obtained culture.
  • Item 9 Item 10.
  • a polypeptide produced by the method according to Item 8 and having xanthine oxidase activity Item 10.
  • a method for measuring a biological component comprising a step of allowing the polypeptide having xanthine oxidase activity according to Item 1 or Item 9 to act on hypoxanthine and / or xanthine.
  • Item 11 Item 11.
  • a biological component measurement reagent comprising the polypeptide having xanthine oxidase activity according to Item 1 or Item 9.
  • stable quality xanthine oxidase can be produced at a high level.
  • xanthine oxidase and DNA encoding xanthine oxidase (1-1) Xanthine Oxidase
  • One aspect of the xanthine oxidase of the present invention comprises (a) a subunit consisting of the amino acid sequence shown in SEQ ID NO: 1 and a subunit consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide sequence represented by SEQ ID NO: 1 is the ⁇ subunit sequence of mature xanthine oxidase
  • the polypeptide sequence represented by SEQ ID NO: 2 is the ⁇ subunit sequence.
  • Another aspect of the xanthine oxidase of the present invention is (b) an amino acid substituted, deleted, inserted, added and / or inverted in 0, 1 or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1.
  • a subunit consisting of a sequence, and a subunit consisting of an amino acid sequence in which substitution, deletion, insertion, addition and / or inversion of 0, 1 or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 A combination of polypeptides that are configured and have xanthine oxidase activity (except for the combination of (a) above).
  • Another aspect of the xanthine oxidase of the present invention is the (c) a subunit consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2. It is a combination of polypeptides having a xanthine oxidase activity (excluding the combination of (a) above), which is composed of a subunit consisting of an amino acid sequence having an identity of 80% or more.
  • the subunit of SEQ ID NO: 1 is identical to the amino acid sequence shown in SEQ ID NO: 1 as long as it retains xanthine oxidase activity when combined with another subunit. It is a subunit consisting of an amino acid sequence having a sex of 80% or more.
  • the identity with the amino acid sequence shown in SEQ ID NO: 1 is 85% or more, more preferably 88% or more, still more preferably 90% or more, still more preferably 93% or more, and still more preferably 95%. Above, especially preferably 98% or more, most preferably 99% or more. The same can be said for the subunit of SEQ ID NO: 2.
  • the xanthine oxidase of (b) or (c) is a known technique such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, position-directed mutagenesis method or random mutagenesis method. It can be produced by modifying using the method, or can be obtained by other methods such as ultraviolet irradiation. From the viewpoint of maintaining the activity of xanthine oxidase, it is preferable that the above-described modification is present at a site that does not affect the active site or substrate binding site of xanthine oxidase.
  • the xanthine oxidase of (b) or (c) includes naturally occurring variants (for example, single nucleotide polymorphisms) such as cases based on individual differences of microorganisms holding xanthine oxidase, differences in species or genera.
  • ⁇ Identity of amino acid sequence> Various methods are known as methods for calculating the identity of amino acid sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). In this document, the National Center for Biotechnology Information (NCBI) identity algorithm BLAST (Basic local search tool) http: // www. ncbi. nlm. nih. By using default (initial setting) parameters in gov / BLAST /, amino acid sequence identity is calculated.
  • NCBI National Center for Biotechnology Information
  • DNA encoding the xanthine oxidase of the present invention is (A) a combination of DNA encoding the amino acid sequence shown in SEQ ID NO: 1 and DNA encoding the amino acid sequence shown in SEQ ID NO: 2.
  • DNA encoding a protein refers to DNA from which the protein is obtained when it is expressed, that is, DNA having a base sequence corresponding to the amino acid sequence of the protein. Therefore, DNA that differs depending on codon degeneracy is also included.
  • Another embodiment of the DNA encoding xanthine oxidase of the present invention is (B) a combination of DNA consisting of the base sequence shown in SEQ ID NO: 4 and DNA consisting of the base sequence shown in SEQ ID NO: 5.
  • the base sequences shown in SEQ ID NOs: 4 and 5 are base sequences encoding the ⁇ subunit and ⁇ subunit of mature xanthine oxidase, respectively.
  • Another embodiment of the DNA encoding the xanthine oxidase of the present invention is (C) a DNA comprising a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 4 and a base shown in SEQ ID NO: 5.
  • a combination of DNAs (excluding the combination of (B) above) that is composed of a DNA consisting of a base sequence having identity to the sequence of 80% or more and that encodes a combination of polypeptides having xanthine oxidase activity .
  • a DNA comprising a base sequence having an identity of 80% or more with the base sequence shown in SEQ ID NO: 4 has a subunit having an amino acid sequence encoded by it, As long as it has xanthine oxidase activity when combined with another subunit, the identity with the base sequence shown in SEQ ID NO: 4 is 80% or more, preferably 85% or more, more preferably 88% or more, still more preferably It is 90% or more, more preferably 93% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.
  • the same can be said for DNA consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 5.
  • ⁇ Identity of base sequence> Various methods are known for calculating the identity of base sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
  • NBI National Biotechnology Information Center
  • identity algorithm Advanced BLAST 2.1 by using blastn as a program and setting various parameters to default values, a search is performed for nucleotide sequence identity values. (%) Is calculated.
  • Another embodiment of the DNA encoding the xanthine oxidase of the present invention is (D) a DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 4, and SEQ ID NO: 5
  • a DNA combination that encodes a combination of polypeptides having xanthine oxidase activity (provided above (B )).
  • stringent conditions generally refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art and are described, for example, in Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).
  • stringent conditions refers to the following conditions. 50% formamide, 5 ⁇ SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0) as a hybridization solution, 1 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / mL denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)) is used.
  • DNA that hybridizes under such conditions may include those in which a stop codon has occurred in the middle or those that have lost activity due to mutations in the active center, but these are incorporated into commercially available active expression vectors. It can be easily removed by expressing it in a suitable host and measuring the enzyme activity by a known method.
  • Another embodiment of the DNA encoding the xanthine oxidase of the present invention is (F) substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1. And a subunit comprising an amino acid sequence obtained by substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2.
  • the DNA encoding the xanthine oxidase of (C) to (F) can be obtained by modifying the DNA of (A) or (B) using a commercially available kit or PCR method.
  • the activity of the protein encoded by the obtained gene can be confirmed by the activity measurement method described later.
  • the DNA of the present invention is DNA that is present in an isolated state.
  • isolated DNA refers to a state separated from other components such as nucleic acids and proteins that coexist in the natural state.
  • the isolated DNA may contain some other nucleic acid components such as a nucleic acid sequence adjacent in the natural state (for example, a promoter region sequence and a terminator sequence).
  • an “isolated” state in the case of chromosomal DNA is preferably substantially free of other DNA components that coexist in the natural state.
  • the “isolated” state in the case of DNA prepared by genetic engineering techniques such as cDNA molecules is preferably substantially free of cell components, culture medium, and the like.
  • the “isolated” state in the case of DNA prepared by chemical synthesis is preferably substantially free of precursors (raw materials) such as dNTPs, chemical substances used in the synthesis process, and the like.
  • precursors raw materials
  • dNTPs chemical substances used in the synthesis process
  • DNA DNA in an isolated state.
  • the DNA of the present invention also includes DNA complementary to the DNA described herein (cDNA).
  • the DNA of the present invention can be produced and obtained by chemical DNA synthesis based on the sequence information disclosed in this specification or the attached sequence listing, for example, known standard genetic engineering techniques, molecules It can be easily prepared by using a biological technique, a biochemical technique or the like.
  • Accessory protein and DNA encoding accessory protein Another aspect of the present invention is a protein comprising any of the following polypeptides (d) to (f).
  • D a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3
  • a polypeptide comprising an amino acid sequence a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 3
  • DNA encoding accessory protein is any one of the following DNAs (G) to (L).
  • G DNA encoding the amino acid sequence shown in SEQ ID NO: 3
  • H DNA consisting of the base sequence represented by SEQ ID NO: 6
  • I DNA consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 6
  • J DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 6
  • K DNA consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted, added and / or inverted in the nucleotide sequence shown in SEQ ID NO: 6
  • L DNA encoding a polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted, added and / or inverted in the amino acid sequence shown in SEQ ID NO: 1
  • amino acid sequence represented by SEQ ID NO: 3 is the sequence of the accessory protein.
  • the base sequence represented by SEQ ID NO: 6 is a base sequence encoding an accessory protein and is derived from Arthrobacter luteus.
  • Vector (3-1) Vector obtained by incorporating DNA encoding xanthine oxidase
  • a vector obtained by incorporating DNA encoding the above xanthine oxidase, and a vector obtained by introducing this vector into a microorganism A transformant characterized by is also provided.
  • the vector of the present invention is a vector in which a DNA encoding the xanthine oxidase of the present invention described above is incorporated.
  • the “vector” is a nucleic acid molecule (carrier) capable of transporting a nucleic acid molecule inserted therein into a target such as a cell, and can replicate the DNA of the present invention in an appropriate host cell.
  • the type and structure are not particularly limited as long as it can be expressed. That is, the vector of the present invention is an expression vector.
  • an appropriate vector is selected in consideration of the type of host cell.
  • the vector include a plasmid vector, a cosmid vector, a phage vector, a virus vector (such as an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, and a herpes virus vector).
  • a virus vector such as an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, and a herpes virus vector.
  • E. coli vector include pBlueScript, pBR322, pUC19, pGEM-T, pCR-Blunt, pTA2, and pET.
  • the vector DNA includes an auxotrophic marker, a drug resistance marker, an expression promoter DNA sequence, and an expression terminator DNA sequence, and more preferably includes an ampicillin resistance gene derived from pBlueScript and a lactose promoter. It is also possible to use a vector suitable for the case where a host microorganism described later is used as a host, or a vector suitable for self-clo
  • Escherichia coli When Escherichia coli is used as a host, for example, M13 phage or a modified product thereof, ⁇ phage or a modified product thereof, pBR322 or a modified product thereof (pB325, pAT153, pUC8, etc.) can be used.
  • yeast When yeast is used as a host, pYepSec1, pMFa, pYES2, etc. can be used.
  • insect cells When insect cells are used as hosts, for example, pAc and pVL can be used.
  • mammalian cells When mammalian cells are used as hosts, for example, pCDM8 and pMT2PC can be used, but the present invention is not limited thereto. .
  • the vector vector for co-expressing the accessory protein may further incorporate DNA encoding the accessory protein described above.
  • cofactor synthesis and coordination of cofactors to enzyme proteins can be activated, and an increase in production as an active enzyme can be expected.
  • the present invention relates to a transformant in which the DNA of the present invention is introduced into a host cell.
  • the means for introducing the DNA of the present invention into the host is not particularly limited.
  • the DNA is introduced into the host in a state of being incorporated into the vector described above.
  • the host cell is not particularly limited as long as it can express the DNA of the present invention and produce xanthine oxidase.
  • prokaryotic cells such as Escherichia coli and Bacillus subtilis
  • eukaryotic cells such as yeast, mold, insect cells, plant culture cells, and mammalian cells can be used.
  • it is a microorganism classified as a prokaryote.
  • microorganisms belonging to the genus Actinomyces and Escherichia coli are suitable.
  • examples include the genus Escherichia, the genus Bacillus, the genus Brevibacillus, the genus Corynebacterium, and the like, each of which is an Escherichia coli (Escherichia). C600, Escherichia coli HB101, Escherichia coli DH5 ⁇ , Bacillus subtilis, Brevibacillus choshinensis, Corynebacterium glutamicum, etc.
  • Examples of the vector include pBR322, pUC19, and pBluescript.
  • the host is yeast, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Candida, the Pichia genus, the genus Cryptococcus, and the genus Rhodococcus Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utris, Pichia pastoris, Pichia pastoris, and Pichia pastoris. It is.
  • the vector include pAUR101, pAUR224, and pYE32.
  • examples include Aspergillus genus, Trichoderma genus, Colletotrichum genus, etc., and Aspergillus oryzae and gers gers. ), Trichoderma reesei, Colletotrichum hiemiris, and the like.
  • a microorganism belonging to the genus Arthrobacter from which the xanthine oxidase of the present invention has been isolated that is, in the transformant, exogenous DNA is usually present in the host cell, but a transformant obtained by so-called self-cloning using a microorganism from which the DNA is derived as a host is also a preferred embodiment.
  • the combination of the expression vector and the host microorganism used in the present invention is not particularly limited, but an auxotrophic marker gene or drug resistance marker gene derived from the host into which the gene is incorporated and an expression promoter DNA sequence from the host into which the gene is incorporated And a combination of an expression vector containing a terminator DNA sequence derived from a host into which the gene is incorporated and an auxotrophic mutant host or a drug-sensitive host, and most preferably an ampicillin resistance gene derived from pBlueScript, a lactose promoter, and E. coli The combination of is mentioned.
  • the method for transferring the recombinant expression vector into the cells of the host microorganism is not particularly limited. Preferably, it is prepared by transfection or transformation using the expression vector shown above. Transformation may be transient or stable. Transfection and transformation can be performed by methods such as electroporation.
  • xanthine oxidase culturing the transformant described above, a manufacturing method of xanthine oxidase comprising the step of collecting the protein with from xanthine oxidase enzymatic activity obtained culture is also provided.
  • the culture form of the transformant may be appropriately selected in consideration of the nutritional physiological properties of the host. Usually, liquid culture is used in many cases, but industrially, aeration and agitation culture is advantageous.
  • the nitrogen source used for the culture may be any nitrogen compound that can be used by the host microorganism except for a special N source such as a deletion of a specific amino acid component.
  • a special N source such as a deletion of a specific amino acid component.
  • These are mainly organic nitrogen sources, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline decomposition product and the like are used.
  • yeast extract and soybean protein are preferable, but the present invention is not limited to this, and the transformant can also be cultured by using casein polypeptone, fermented koji extract, malt extract, or the like.
  • nutrient sources commonly used for culturing microorganisms are widely used.
  • the carbon source any carbon compound that can be assimilated may be used.
  • glucose, sucrose, lactose, maltose, xylose, molasses, pyruvic acid and the like are used.
  • phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, molybdenum and other salts, 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 bacteria grow and produce xanthine oxidase, but in the case of E. coli, it is usually about 20 to 37 ° C.
  • the culture time varies slightly depending on the conditions, the culture may be terminated at an appropriate time in consideration of the time when the xanthine oxidase reaches the maximum yield, and is usually about 24 to 120 hours.
  • the pH of the medium can be appropriately changed within the range in which the bacteria grow and produce xanthine oxidase, but is usually about pH 3.0 to 9.0.
  • the xanthine oxidase of the present invention can be collected and used as it is by collecting a culture solution containing the cells obtained by culturing the above transformant. Thus, it can be used after separating the xanthine oxidase-containing solution and the bacterial cells.
  • xanthine oxidase may be purified from the xanthine oxidase-containing solution thus obtained and used.
  • Purification methods include, for example, vacuum concentration, membrane concentration, salting out treatment such as ammonium sulfate and sodium sulfate, fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc., heating treatment or isoelectric point treatment, adsorbent
  • treatments such as gel filtration with a gel filtration agent, adsorption chromatography, ion exchange chromatography, hydrophobic interaction chromatography and the like.
  • the xanthine oxidase obtained by the above production method is also an embodiment of the present invention.
  • the xanthine oxidase of the present invention produced by the above-described method of the present invention has the following properties (i) to (iv), and has higher quality than the conventional one.
  • Action Shows xanthine oxidase enzyme activity in the presence of mediator and hydrogen peroxide.
  • Molecular weight SDS 105 kDa and 31 kDa heterodimer
  • iii Stable pH range: pH 6.0 to 9.0.
  • Thermal stability has a residual activity of 90% or more after heat treatment at 50 ° C. for 10 minutes.
  • the xanthine oxidase having the enzyme chemical characteristics as described above can be suitably used for applications such as clinical examinations.
  • the various enzyme chemical properties described above can be examined by using known methods for specifying various enzyme properties, for example, the methods described in the following examples.
  • Various properties of the enzyme can be examined to some extent in the culture medium of the transformant producing the xanthine oxidase of the present invention and in the middle of the purification process, and more specifically, can be examined using the purified enzyme.
  • the purified enzyme refers to an enzyme that has been separated to a state that does not substantially contain components other than the enzyme, particularly proteins other than the enzyme (contaminating protein). Specifically, for example, it refers to an enzyme having a content of contaminating protein of less than about 20%, preferably less than about 10%, more preferably less than about 5%, and even more preferably less than about 1% based on weight. .
  • xanthine oxidase activity is measured under the following conditions.
  • Activity measurement method I ⁇ Reaction reagent> The following Tris-HCl buffer solution (8.0 ml), xanthine solution (1.0 ml) and potassium oxonate solution (1.0 ml) are mixed to obtain a reaction reagent. 100 mM Tris-HCl buffer pH 7.5 -10 mM xanthine solution-1 mM potassium oxonate solution ⁇ Measurement conditions> Pre-warm 3 ml of reaction reagent at 37 ° C. for 5 minutes.
  • one unit (U) in XTO activity is defined as the amount of enzyme that produces 1 micromole of uric acid per minute in the presence of xanthine at a concentration of 0.48 mM.
  • Activity value calculation formula XTO activity (U / ml) ⁇ ( ⁇ OD TEST ⁇ OD BLANK ) ⁇ 3.1 ⁇ dilution factor ⁇ / ⁇ 12.5 ⁇ 0.1 ⁇ 1.0 ⁇
  • reaction reagent Mix the following Tris-HCl buffer 33.27 ml, xanthine solution 2.0 ml, potassium oxonate solution 2.0 ml, horseradish peroxidase solution 0.5 ml, 4-aminoantipyrine solution 1.26 ml, and ADPS solution 0.97 ml. Use as a reaction reagent.
  • XTO activity is determined according to the following formula (I).
  • one unit (U) in XTO activity is defined as the amount of enzyme that produces 1 micromole of uric acid per minute in the presence of xanthine at a concentration of 0.48 mM.
  • Bio component measurement method and biological component measurement reagent Another aspect of the present invention is a biological component measurement method including the step of causing the xanthine oxidase of the present invention to act on hypoxanthine and / or xanthine.
  • another embodiment of the present invention is a reagent for measuring a biological component containing the xanthine oxidase of the present invention.
  • the biological component measurement method to which the present invention is applied is not particularly limited as long as it includes a step of allowing xanthine oxidase to act on hypoxanthine.
  • the biological component measurement method is an enzymatic method, particularly an oxidase-peroxidase- A method using a color former, that is, a method utilizing a colorimetric determination by reacting a substance to be measured in a specimen with an enzyme to generate hydrogen peroxide and reacting it with a color former in the presence of peroxidase.
  • Biological component measurement methods using this principle have already been established in the art. Therefore, the knowledge can be applied to the present invention to measure the amount or concentration of biological components in various samples, and the mode is not particularly limited. For example, methods for measuring biological components such as adenosine deaminase, guanase, inorganic phosphorus, and various nucleic acids can be exemplified.
  • purine nucleoside phosphorylase When measuring adenosine deaminase, purine nucleoside phosphorylase is allowed to act on inosine produced by the reaction of adenosine deaminase using adenosine as a substrate to change it to hypoxanthine.
  • the generated hydrogen peroxide can be quantified by a peroxidase-color former system. Since xanthine oxidase further generates hydrogen peroxide in the process of converting xanthine to uric acid, it may be quantified by a peroxidase-color former system.
  • xanthine oxidase when guanase is measured, xanthine oxidase can be allowed to act on xanthine produced by the reaction of guanase using guanine as a substrate.
  • inorganic phosphorus when measuring inorganic phosphorus, it can measure by paying attention to the point with consumption of inorganic phosphorus in the reaction of purine nucleoside phosphorylase which uses inosine as a substrate.
  • nucleic acids such as AMP, IMP, and adenosine can be quantified using a peroxidase-chromogenic system by designing an appropriate coupling reaction.
  • Means for carrying out the above method include a method using a liquid reagent (or kit) configured to be applicable to a general-purpose automatic analyzer (for example, Hitachi 7170 type automatic analyzer), a means such as lyophilization.
  • a method using a reagent (or kit) composed of a combination of the produced dry preparation and a solution a method using a kit or a sensor called a so-called dry system in which an enzyme is supported on an appropriate carrier, etc.
  • a form can be illustrated.
  • the analysis is performed by an automatic analyzer using a liquid reagent in which the reagent is divided into two (hereinafter also referred to as a two-reagent liquid reagent).
  • the first type of reagent (hereinafter also referred to as the first reagent or R1) is first added to the sample and allowed to react for a certain time, and then the second type of reagent (hereinafter also referred to as the first reagent or R1).
  • the target component can be quantified by adding and reacting, and measuring the change in absorbance during this period.
  • Example 1 N-terminal amino acid analysis
  • Arthrobacter luteus which is a xanthine oxidase producing bacterium
  • N-terminal amino acid analysis was performed from purified xanthine oxidase (XTO211 manufactured by Toyobo Co., Ltd.). Purified xanthine oxidase was dissolved in ion exchange water at a concentration of 1 mg / ml, subjected to SDS-PAGE, and the protein was fractionated by molecular weight. The results are shown in FIG.
  • bands were detected at locations of about 105 kDa, about 90 kDa, about 68 kDa, and about 31 kDa.
  • the band present at 68 kDa is predicted to be bovine serum albumin (BSA), which is used as a protective agent for proteins. Therefore, the protein was transferred from the SDS-PAGE gel to the nylon membrane, and the band was about 105 kDa, about A 90 kDa band of about 31 kDa was cut out and subjected to N-terminal amino acid analysis.
  • the detected amino acids were as follows.
  • Example 2 Acquisition of xanthine oxidase gene
  • Arthrobacter luteus which is a producer of xanthine oxidase
  • genome analysis of Arthrobacter luteus was performed. First, Arthrobacter luteus was cultured in LB medium, and the cells were collected, and then genomic DNA was purified. Genomic DNA was purified using MagExtractor Plant Genome manufactured by Toyobo Co., Ltd., and the purification method was in accordance with the instructions attached to the kit. The purified genomic DNA was analyzed by GS-FLX at Operon Biotechnology. The results of genome analysis are shown in FIG.
  • the first gene was presumed to encode XdhC, an accessory protein of xanthine oxidase, and showed high identity with XdhC derived from other microorganisms.
  • the second and third genes are presumed to encode the respective xanthine oxidase ⁇ subunit (XdhA) and ⁇ subunit (XdhB), and the N-terminal sequence of the gene product is the analyzed 105 kDa protein and 31 kDa. Each showed high identity with the N-terminal amino acid of the protein.
  • the 90 kDa protein was the product of partial degradation of the 105 kDa ⁇ subunit protein. It was thought that.
  • Example 3 In order to confirm that the DNA sequence obtained and expressed for xanthine oxidase gene encodes xanthine oxidase, the xanthine oxidase gene was cloned using the following primer set and introduced into the E. coli expression vector pBSKN at the NdeI and EcoRI sites.
  • XdhA_F NdeI
  • XdhB_R EcoRI
  • aaaagaatTCAGCGGGGTgGGGGTGGGCGCC The prepared xanthine oxidase expression vector pBSKN-XTO was transformed into E.
  • Xanthine oxidase activity was detected by the method shown in Activity measurement method I. As a result, 35 U / L xanthine oxidase activity was detected in E. coli.
  • E. coli JM109 strain into which pBSKN as an expression vector was introduced was cultured in the same manner and xanthine oxidase activity was measured, but no activity was detected.
  • Example 4 Co-expression of an accessory protein that modifies xanthine oxidase It was considered that co-expression of XdhC, an accessory protein, was effective for efficient expression of xanthine oxidase, and an attempt was made to co-express the XdhC gene.
  • XdhC_F (NdeI) aaaacATATGCTCCACATCGTCGACCGGCT XdhB_R (EcoRI) aaaagaatTCAGCGGGGTgGGGGTGGGCGCC
  • TB + IM medium TB medium + 1 mM IPTG, +0.01 mM ammonium molybdate
  • Xanthine oxidase activity was detected by the method shown in Activity measurement method I.
  • Activity measurement method I As a result, 68 U / L xanthine oxidase activity was detected in E. coli.
  • Example 5 FIG. Acquisition of E. coli recombinant xanthine oxidase
  • the enzyme was purified by disrupting the cells, anion chromatography, or hydrophobic chromatography. Enzyme characteristics were evaluated using the obtained enzyme solution.
  • Example 6 Characterization of E. coli recombinant xanthine oxidase (1) Comparison of hydrogen peroxide purification ratio The obtained enzyme solution and xanthine oxidase enzyme solution produced by wild strain of Arthrobacter luteus, and milk-derived xanthine oxidase enzyme solution as a control The enzyme activity was measured by activity measurement method 1 and activity measurement method II. The results are shown in Table 1.
  • the hydrogen peroxide production rate of the recombinant XTO was 91%, which was almost the same as that of the wild strain-derived XTO.
  • milk-derived XTO was 52%.
  • xanthine oxidase having the same characteristics and stable quality as wild-type xanthine oxidase derived from Arthrobacter can be efficiently recombinantly produced by microorganisms. Therefore, the present invention is extremely useful for producing xanthine oxidase used in clinical tests and the like.

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Abstract

La présente invention vise à fournir une nouvelle enzyme pour la mesure de la glycémie, ladite enzyme possédant des propriétés exceptionnelles de substrat et d'affinité de substrat, et est donc appropriée pour utilisation dans un capteur d'auto-surveillance de la glycémie. À cet effet, l'invention concerne une xanthine oxydase comportant une combinaison de polypeptides constituée d'une sous-unité comprenant une séquences d'acides aminés indiquée dans SEQ ID NO:1 et une séquences d'acides aminés illustrée dans SEQ ID NO:2.
PCT/JP2014/067892 2013-07-16 2014-07-04 Gène xanthine oxydase et séquence d'acides aminés codant pour ce gène WO2015008637A1 (fr)

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CN107119059A (zh) * 2017-04-20 2017-09-01 武汉轻工大学 可用于临床检测的黄嘌呤氧化酶、其编码基因及其应用
KR101899426B1 (ko) 2017-05-25 2018-09-17 (주)큐브바이오 종양 진단을 위한 퓨린 대사체 하이포잔틴 및 잔틴 농도 분석용 효소 조성물
CN109735511A (zh) * 2018-09-06 2019-05-10 西安文理学院 一种临床检测用黄嘌呤氧化酶的制备方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107119059A (zh) * 2017-04-20 2017-09-01 武汉轻工大学 可用于临床检测的黄嘌呤氧化酶、其编码基因及其应用
KR101899426B1 (ko) 2017-05-25 2018-09-17 (주)큐브바이오 종양 진단을 위한 퓨린 대사체 하이포잔틴 및 잔틴 농도 분석용 효소 조성물
WO2018216864A1 (fr) * 2017-05-25 2018-11-29 (주)큐브바이오 Composition enzymatique pour l'analyse de la concentration de métabolites de purine, d'hypoxanthine et de xanthine pour le diagnostic de tumeur
CN108929895A (zh) * 2017-05-25 2018-12-04 高普生物有限公司 作为用于肿瘤诊断的嘌呤代谢物的次黄嘌呤及黄嘌呤浓度分析用酶组合物
CN109735511A (zh) * 2018-09-06 2019-05-10 西安文理学院 一种临床检测用黄嘌呤氧化酶的制备方法
CN109735511B (zh) * 2018-09-06 2022-03-29 西安文理学院 一种临床检测用黄嘌呤氧化酶的制备方法

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