WO2007148451A1 - Method for stabilizing polyamine oxidase and method for improving substrate specificity of polyamine oxidase - Google Patents

Method for stabilizing polyamine oxidase and method for improving substrate specificity of polyamine oxidase Download PDF

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Publication number
WO2007148451A1
WO2007148451A1 PCT/JP2007/052732 JP2007052732W WO2007148451A1 WO 2007148451 A1 WO2007148451 A1 WO 2007148451A1 JP 2007052732 W JP2007052732 W JP 2007052732W WO 2007148451 A1 WO2007148451 A1 WO 2007148451A1
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Prior art keywords
polyamine
oxidase
polyamine oxidase
diacetyl
measuring
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PCT/JP2007/052732
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French (fr)
Japanese (ja)
Inventor
Mikio Bakke
Kazuhiko Shimoji
Naoki Kajiyama
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Kikkoman Corporation
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Priority to JP2008522315A priority Critical patent/JP5166259B2/en
Publication of WO2007148451A1 publication Critical patent/WO2007148451A1/en

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    • 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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)

Definitions

  • the present invention relates to a method for stabilizing polyamine oxidase, a method for improving substrate specificity, a method for measuring diacetyl polyamine using polyamine oxidase, and the like, which are used for clinical diagnosis and production of useful substances.
  • the present invention relates to a method for producing monoacetyl polyamine by reacting polyamine oxidase, a method for purifying polyamine oxidase, a stabilized polyamine oxidase preparation, a reagent for measuring diacetyl polyamine, and the like.
  • Polyamine is a general term for compounds having two or more amino groups in the same molecule.
  • four types of polyamines such as putrescine, cadaverine, spermidine, and spermine, and their monoacetyl and diacetyl groups.
  • urinary polyamine content has been measured for cancer diagnosis (e.g., non-patented (Ref. 1). Since most of the urine polyamines exist in monoacetyl form, it was conventionally measured as the total polyamine content that does not distinguish between free polyamines that hydrolyze acetyl groups and are acetylated with acetyl ethers. (For example, see Patent Document 1).
  • N 1 , N 12 diacetyl spermine (II) (hereinafter simply referred to as diacetyl spermine) and N 1 , N 8 — diacetyl spermidine (II 2) 2
  • II diacetyl spermine
  • N 1 , N 8 diacetyl spermidine
  • HPLC-enzyme method for example, see Non-Patent Document 2
  • HPLC-enzyme method see Non-Patent Document 2
  • various polyamines in the sample are separated by HPLC, and then the free polyamine produced by the action of acetylhydrolase in the HPLC detector is oxidized with polyamine oxidase, and the resulting hydrogen peroxide is electrochemically detected. It is the method of detecting using a container.
  • this method has a problem that a large number of samples cannot be processed in a short time.
  • Patent Documents 2, 3, and 4 Non-Patent Documents 3 and 4
  • the operation was complicated, and the measured value was inversely proportional to the sample amount of the sample.
  • Such polyamine oxidase is thought to be useful for the measurement of diacetyl spermine.
  • the stability of the enzyme and its specificity for diacetyl spermine should be further improved. Was considered necessary.
  • N 1 -acetyl spermidine is about 30%, 50%, 90% (pH 7.5), respectively. This shows the reactivity under phosphate buffer conditions), and there has been a problem of measuring polyamines other than diacetyl spermine.
  • polyamine oxidase is a diacetyl spermine (Chemical Formula 1) N 1 -acetyls permidine (4) that is useful as a raw material for pharmaceuticals and agricultural chemicals. It has been reported that it can be used in manufacturing methods (PCT / JP200 67310648). Although this method is simple and requires fewer steps compared to the conventionally known organic synthesis methods (for example, see Non-Patent Documents 6 and 7), the enzyme is unstable. It had the disadvantage that a large amount of enzyme was required. In addition, since the substrate specificity is insufficient, a part of the produced N 1 acetyl spermidine is converted to putrescine, resulting in a decrease in yield.
  • Patent Document 1 Japanese Patent Laid-Open No. 1 85080
  • Patent Document 2 WO 2004Z081569 Nonfret
  • Patent Document 3 Japanese Patent Laid-Open No. 2006-38594
  • Patent Document 4 Japanese Unexamined Patent Publication No. 2006-199655
  • Non-patent document 1 “Cancer ResearchJ, (USA), 1971, 31 ⁇ , p. 1555-1558
  • Non-patent document 2 “ Journal of BiochemistryJ, (Japan), 1995, 117 ⁇ , p. 107-112
  • Non-Patent Document 3 "Clinical Cancer Research”, (USA), 2005, 11 ⁇ , p. 2986-2990
  • Non-Patent Document 4 “Journal of BiochemistryJ, (Japan), 2006, 139 ⁇ , p. 315-322
  • Non-Patent Document 5 “Clinica Chimica ActaJ, (Landland), 2004, 340 ⁇ , ⁇ 219—22 7
  • Non-Patent Literature 6 journal of Chemical society, Perkm Transactions I ”(UK), 1988, p. 1905- 1911
  • Non-Patent Document 7 “Acta Chemica ScandinavicaJ, (Denmark), 1989, 43 ⁇ , p. 990-994
  • the subject of the present invention is a method for stabilizing polyamine oxidase, a method for improving substrate specificity, And a method for producing diacetyl polyamine by using polyamine oxidase and a method for producing monoacetyl polyamine using the same, a method for purifying polyamine oxidase, It is intended to provide a decontaminated polyamine oxidase preparation and a reagent for measuring diacetylpolyamine.
  • the present inventors have (1) the stability of polyamine oxidase is improved by the coexistence of an alcoholic compound and Z or a chelating reagent. (2) The substrate specificity of polyamine oxidase is improved by the coexistence of surfactants, and (3) the substrate of polyamine oxidase by allowing polyamine oxidase to act at a pH below 7.5. The inventors have found that the specificity is improved and have completed the present invention based on these findings.
  • the present invention relates to the following.
  • a method for stabilizing polyamine oxidase comprising coexisting an alcohol compound and Z or a chelating reagent with polyamine oxidase.
  • a method for improving the substrate specificity of polyamine oxidase characterized by allowing polyamine oxidase to act in the presence of a surfactant.
  • a method for improving the substrate specificity of polyamine oxidase characterized by allowing polyamine oxidase to act at a pH of less than 7.5.
  • a method for measuring diacetyl polyamine which comprises measuring a diacetyl polyamine by allowing a reagent containing polyamine oxidase to act on a sample.
  • a stabilized polyamine oxidase preparation comprising an alcoholic compound and Z or a chelating reagent and a polyamine oxidase.
  • the reagent for measuring diacetyl polyamine according to (13) above which contains an alcoholic compound and Z or a chelating reagent, and polyamine oxidase.
  • an alcoholic compound and Z or a chelating reagent are allowed to coexist.
  • the polyamine oxidase can be stabilized.
  • the substrate specificity of polyamine oxidase can be improved by coexisting a surfactant and making the condition less than Z or PH 7.5.
  • monoacetyl polyamine can be produced by using polyamine oxidase to diacetyl polyamine, or polyamine oxidase can be used.
  • Diacetyl polyamine can be measured by V.
  • FIG. 1 is a graph showing the effect of enzyme stability due to the coexistence of an alcohol compound and a chelating reagent.
  • FIG. 2 is a graph showing the thermal stability of a polyamine oxidase preparation.
  • FIG. 3 is a graph showing the relationship between the concentration and absorbance when a diacetyl spermine sample was measured using a reagent containing diacetyl spermine oxidase.
  • FIG. 4 is a graph showing the relationship between the concentration and the absorbance when a sample containing diacetyl spermine oxidase and diacetylspermine added to urine was measured.
  • polyamine oxidase used in the present invention is an enzyme that catalyzes the reaction of oxidizing diacetyl polyamine in the presence of oxygen to produce monoacetyl polyamine, aldehyde, and hydrogen peroxide.
  • any of polyamine oxidase produced by microorganisms, plants, animals, etc. is not particularly limited. Examples thereof include polyamine oxidase originating from yeast and the like, and preferably, enzymes derived from the genera Debaryomyces, Candida and Pichia are used.
  • diacetyl spermine oxidase is particularly preferable. Used frequently.
  • the di ⁇ cetyl spermine O Kishida over Ze described herein, N 1 - Asechirusu Perumin, N 1 - ⁇ cetyl spermidine, N 8 - active high for di ⁇ cetyl spermine than activity against ⁇ cetyl spermidine refers to polyamine O Kishida over Ze, For example, Debaryomyces' Nonsenyi, Denoriomyces' malama, Denoriomyces' Marams, Candida glabrata, Pichia minuta diacetyl spermine oxidase and the like.
  • diacetyl spermine oxidase having each of the physical properties described in the following (la) to (lg) or a combination thereof as appropriate (the physicochemical properties of the following enzymes are: (Including inevitable errors in measurement):
  • C—N bond is cleaved to form N 1 -acetyl spermidine, 3-acetamidopropanal and hydrogen peroxide.
  • N 1 -acetyl spermine, N 1 -acetyl spermidine, N 8 -acetyl spermidine is the substrate.
  • the enzyme of the present invention having a physical property having an activity of 90 or less, preferably 60 or less, more preferably 50 or less, and most preferably 40 or less. There are no restrictions on the pH conditions or the type and concentration of the buffer in this evaluation!
  • 200 mM acetate buffer pH 4.5 to 6.0
  • 200 mM potassium phosphate buffer ⁇ 6.0 to 8.0
  • 200 mM PIPES-NaOH pH 6.5 to 7.5
  • the enzyme of the present invention having physical properties of pH 6.5 to 8.5, preferably pH 7.0 to 8.0 as the optimum pH.
  • Stable pH 6.0-8.0
  • 200 mM acetate buffer pH 4.5 to 6.0
  • 200 mM potassium phosphate buffer ⁇ 6.0 to 8.0
  • 200 mM PIPES-NaOH pH 6.5 to 7.5
  • 200 mM Tris-hydrochloric acid buffer pH 7.5 to 9.5
  • the stable pH range include the enzyme of the present invention having physicochemical properties of pH 6.0 to 8.0, preferably pH 6.5 to 7.5.
  • the activity of the present enzyme is measured at various temperatures to determine the optimum temperature range of action.
  • the enzyme of the present invention having a physical property of 37 to 55 ° C. as the temperature range of action. (If) Thermal stability: 37 ° C or less
  • the enzyme according to the present invention having physicochemical properties of 37 ° C or lower can be cited as a range where the enzyme can stably exist.
  • the molecular weight of the enzyme of the present invention is not particularly limited, but examples of the molecular weight include the enzyme of the present invention having a molecular weight of about 92,000 (gel filtration method).
  • Diacetyl spermine oxidase having each of the physical properties described in (2a) to (2g) below or a combination thereof as appropriate (the physical properties of the following enzymes are Including inevitable errors):
  • N 1 -acetyl spermine, N 1 -acetyl spermidine, N 8 -acetyl spermidine is the substrate.
  • the enzyme of the present invention having a physical property having an activity of 90 or less, preferably 60 or less, and more preferably 50 or less when used as. There are no restrictions on the pH conditions or the type and concentration of the buffer in this evaluation.
  • 200 mM acetate buffer pH 4.5 to 6.0
  • 200 mM potassium phosphate buffer ⁇ 6.0 to 8.0
  • 200 mM PIPES-NaOH pH 6.5 to 7.5
  • the enzyme of the present invention having physical properties of pH 7.0 to 8.5, preferably pH 7.5 to 8.0 as the optimum pH.
  • Stable pH 5.5 to 9.5
  • 200 mM acetate buffer pH 4.5 to 6.0
  • 200 mM potassium phosphate buffer ⁇ 6.0 to 8.0
  • 200 mM PIPES-NaOH pH 6.5 to 7.5
  • 200 mM Tris-hydrochloric acid buffer pH 7.5 to 9.5
  • the stable pH range include the enzyme of the present invention having physicochemical properties of pH 5.5 to 9.5, preferably pH 6.5 to 9.0.
  • the activity of the present enzyme is measured at various temperatures to determine the optimum temperature range of action.
  • the enzyme of the present invention having a physical property of 45 to 55 ° C as the temperature range of action can be mentioned.
  • the enzyme according to the present invention having physicochemical properties of 37 ° C or lower can be mentioned as a range where the enzyme can stably exist.
  • the molecular weight of the enzyme of the present invention is not particularly limited, and examples of the molecular weight include the enzyme of the present invention having a molecular weight of about 90,000 (gel filtration method).
  • the first enzyme mentioned above is Debaryomyces malama HUT 7199, Debaryomyces maramus NBRC 0668 and Denoriomyces Hansenii Tsukuba 42 (FERM BP-10603)
  • the second enzyme mentioned above is Each one is produced by Brata Noda 162 and can be easily prepared from these cells by those skilled in the art.
  • the enzyme-encoding gene can be cloned and assembled in other hosts such as Escherichia coli and yeast. It can also be prepared by expression.
  • Debariomyces Hansenii Tsukuba 42 (FERM BP-10603) and Canida glabrata Noda 162 (FERM BP-10602) are strains obtained by the present inventors from soils in Ibaraki Prefecture and Chiba Prefecture, respectively. As of May 10, 2006, it was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the Budapest Treaty on the International Approval of Deposit of Microorganisms in Patent Procedures, each with deposit number FERM BP— 10603 And FERM BP-10602.
  • polyamine oxidase those using FAD or copper as a coenzyme are known, and although not particularly limited, polyamine oxidase using FAD as a coenzyme is desirable.
  • polyamine oxidases those produced by gene recombinants can be used.
  • the alcohol compound coexisted in order to improve the stability of polyamine oxidase is an organic compound having a hydroxyl group (OH), and those skilled in the art can use any compound having such a functional group.
  • Any compound known in the art can be used in the present invention.
  • an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group is preferably used because it is easily available. More specific Examples include aliphatic alcohols such as methanol, ethanol, 1 propanol, 2-propanol, ethylene glycol and glycerol, and sugars such as trehalose, xylitol, sucrose and glucose.
  • the above alcohol compounds can be used either individually or in combination.
  • the concentration of the alcohol compound in the solution coexisting with the polyamine oxidase may be within a range in which the stabilizing effect of the polyamine oxidase is exhibited and there is no inconvenience in handling the reagent containing the enzyme.
  • each compound can be added at a concentration at which an appropriate stability effect is recognized.
  • it is used at a concentration of 0.5 to 20%, more preferably 1 to 10% (hereinafter referred to as “volume%” unless otherwise specified).
  • the chelating reagent that coexists in order to improve the stability of polyamine oxidase is a compound that coordinates to a metal ion to give a chelating compound, and has such an action. Any compound known to those skilled in the art can be used in the present invention.
  • ethylenediamine-acetic acid (Ethylenediamine—N, N, ⁇ ,, ⁇ , —tetraacetic acid, (hereinafter referred to as “EDTA”)), 1, 2-Diaminopropane-N, N, N, N, Tetraacetic acid (hereinafter referred to as “Me—EDTA”), N— (2-Hydroxymethyl) ethy lenediamine-N, N, N, N, unitetraacetic acid (hereinafter referred to as “EDTA—OH”), trans— 1, 2-Diaminocyclohexane- N, N, N ', N'— tetraacetic acid (hereinafter referred to as “CyDTA”), 1, 3— Diamino— 2— hydroxypropane— N, N, N, N, -tetraacetic acid (Hereinafter referred to as “DTPA—OH”), O, ⁇ '— Bis (2—aminoethylenediamine—N
  • the chelating reagents may be used alone or in combination.
  • Ma Examples of the polyamine oxidase stabilized by the chelating reagent include, for example, polyamine oxidase originating from yeast and the like, and more specifically, Devariomyces' Hansenii, Denoriomyces. Malama, Denoriomyces. Marams, Candida diacetyl spermine oxidase derived from glabrata, and Candida 'bodily derived from Candida as described in FEBS Letters, (UK), 476 ,, p. 150—154 1 — acetyl spermidine oxidase and the like.
  • the concentration of the chelating reagent coexisting with polyamine oxidase in the solution the stability of the polyamine oxidase is exerted and the concentration of the chelating reagent is not particularly inconvenient in handling the reagent containing the enzyme.
  • concentration of the chelating reagent is not particularly inconvenient in handling the reagent containing the enzyme.
  • it is preferably used at a concentration of 0.01 to 50 mM, more preferably 1 to LOmM.
  • the polyamine oxidase can be stabilized by using the alcohol compound and the chelating reagent in combination.
  • the alcoholic compound and the chelating reagent can be used alone or in combination.
  • the alcohol compound and Z or a solid powder to which a chelating reagent, polyamine oxidase, and other reagents are added, or a solution thereof, can be allowed to coexist by appropriately mixing them.
  • the concentration of polyamine oxidase used in the stability method of the present invention is not particularly limited.
  • This enzyme solution contains coenzymes such as FAD, NAD, NADP, NADH, NADPH, peroxidase, 4-aminoantipyrine, phenol, N-ethyl N-sulfopyl pill-m-toluidine (TOOS), 10- (carboxymethyl) (Aminocarbols) — 3, 7 Hydrogen peroxide detection reagents such as bis (dimethylamino) phenothiazine sodium salt (DA—67), redox coloring such as 1 Methoxy PMS and WST—8 (formazan reagent) One or more of reagents, enzymes such as catalase, and various polyamines such as diacetyl spermine may be contained.
  • coenzymes such as FAD, NAD, NADP, NADH, NADPH, peroxidase, 4-aminoantipyrine, phenol, N-ethyl N-sulfopyl pill-m-toluidine (TOOS), 10-
  • the stability of an enzyme is greatly affected by the pH during storage, it is preferable to simultaneously use various buffers in the stable pH range.
  • the stability of the enzyme of the present invention The type of buffer used, its concentration, and pH are not particularly limited, but for example, it is set to a concentration having a buffer capacity between pH 6 and 10 and maintaining a necessary and sufficient buffer capacity. It is desirable that Examples of such buffers include general-purpose Tris buffers and phosphate buffers, organic acid buffers such as acetic acid, citrate, and oxalic acid, MES, BES, HEPES, and TES. Good buffer such as bicine and tricine, amino acid buffer such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, imidazole buffer and the like can also be used.
  • the concentration of the buffer solution for storing the enzyme is not particularly limited, but preferably 5
  • the alcohol compound of the present invention and Z or a chelating reagent are added to the buffer, they may be added directly or, for example, an aqueous solution thereof adjusted to pH 6 to 10, preferably pH 6 to 8, may be added. Good. If the pH is outside the target range by adding an alcoholic compound and Z or a chelating reagent, for example, acetic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, ammonia water, etc. It is preferable to adjust the pH so that it falls within the target range.
  • reagents if necessary, for example, various inorganic salts such as sodium chloride sodium, potassium salt sodium, sodium azide, polysaccharides such as dextran, urine serum albumin (BSA), glycerol Further, chemotherapeutic agents such as amino acids, surfactants, antibiotics and sulfa drugs may coexist. These reagents may be preliminarily added to the buffer solution.
  • the stability was evaluated by storing polyamine oxidase under various conditions in accordance with the storage conditions, transport conditions, and measurement conditions of the enzyme actually used, and measuring changes over time.
  • an acceleration test is generally used to perform evaluation in a short time. For example, a method of keeping polyamine oxidase at a constant high temperature and measuring its change over time can be mentioned.
  • polyamine oxidase particularly diacetyl spermine oxidase
  • This stable method is used to produce a stabilized polyamine oxidase formulation containing an alcoholic compound and a Z or chelating reagent and a polyamine oxidase. It can be done.
  • a PH6 ⁇ : LO buffer solution containing 1 to 10% of an alcohol compound and Z or a chelating reagent at a concentration of 1 to 50 mM is prepared, and polyamine oxidase is added to this buffer solution. Is added to make LOOOUZml.
  • a coenzyme such as FAD may be added to be 0.01-: LOmM.
  • peroxidase may be added at 10 to: LOOOUZml, TOOS at 0.05 to 5 mM, and 4-aminoantipyrine at 0.1 to 20 mM. This mixture can be stirred to produce a polyamine oxidase preparation.
  • this liquid preparation may be freeze-dried or spray-dried.
  • the polyamine oxidase preparation thus obtained is significantly stabilized as compared with the preparation produced by the conventional method, and is stabilized in a simple manner and at a low cost by the method of the present invention. Polyamine oxidase preparations can be produced.
  • a purified polyamine oxidase can be produced using this stability method.
  • a method for purifying an enzyme a method commonly used for enzyme purification can be used. For example, ammonium sulfate salting out method, organic solvent precipitation method, ion exchange chromatography method, gel filtration chromatography method, hydrophobic chromatography method, adsorption It is preferable to carry out an appropriate combination of chromatographic methods, electrophoresis methods and the like.
  • the polyamine oxidase is isolated and purified in a stable state by adding and coexisting an alcohol compound and Z or a chelating reagent. Can do. This makes it possible to produce the enzyme with a high recovery rate.
  • diacetyl spermine oxidase described in PCTZJP2006Z310648 is used for cell disruption with dynomill, ammonium sulfate precipitation, dialysis, anion exchange column chromatography, hydrophobic column chromatography, gel filtration and the like. Purify in combination, but with one or more of these steps, alcohol compound and Z or chelating reagent are added and coexisted in one step or multiple steps to stabilize diacetyl spermine oxidase. In this state, it can be isolated and purified.
  • N 1 -acetyl spermidine can be efficiently produced using this stable method.
  • Addition and coexistence of a compound and Z or a chelating reagent stabilizes diacetyl spermine oxidase, which makes it possible to carry out the reaction with a smaller amount of enzyme, which is industrially advantageous.
  • the above reaction is usually carried out in a buffer solution, but if necessary, a hydrogen peroxide-eliminating enzyme such as catalase or a preservative such as sodium azide may be added.
  • the buffer solution is not particularly limited as long as it is a normal buffer solution having a pH of 5 to 10, but examples thereof include general-purpose Tris buffer solution and phosphate buffer solution, and include acetic acid, citrate, and oxalic acid.
  • Organic acid buffer such as MES, BES, HEPES, TES, Good buffer such as bicine and tricine, Amino acid buffer such as glycine-NaOH, Boric acid buffer, Bis-Tris propane buffer, Imidazole buffer Etc.
  • the reaction time may be any time, but it is preferable to stop the reaction in 1 hour to 3 days, preferably 3 hours to 2 days.
  • “improving the substrate specificity of polyamine oxidase” means the ratio of the reactivity of polyamine oxidase to “diacetyl spermine” to “reactivity to other polyamines”, that is, “diacetyl”. Improve reactivity to spermine Z and reactivity to other polyamines!
  • “other polyamines” refer to, for example, N 1 -acetyl spermine, N 1 -acetyl spermidine, N 8 -acetyl spermidine, spermine, spermidine and the like.
  • Substrate specificity was evaluated by measuring the enzyme activity by changing the type of substrate added to the reaction solution used to measure the enzyme activity, and comparing the reactivity to diacetylspermine with the reactivity to other polyamines.
  • the "other polyamine” if example embodiment, N 1 - ⁇ cetyl spermine, N 1 - ⁇ cetyl spermidine, N 8 - Asechirusuperumi Jin, spermine, and a method of measuring the like spermidine and the like.
  • it is possible to compare the reactivity to diacetyl polyamine and the reactivity to one other polyamine eg N 1 -acetyl spermidine).
  • a surfactant is composed of a hydrophilic group and a lipophilic group (hydrophobic group), and is a substance that exhibits an activity of dissolving in water and lowering the surface tension of water. It can also be used in combination.
  • surfactants are Ryoon surfactants (anionic surfactants), cationic surfactants (cationic surfactants)
  • Nonionic surfactants non-ionic surfactants
  • amphoteric surfactants include quaternary amphoteric surfactants
  • A-on surfactant is an ion that is ionized in water to become an organic anion.
  • anionic surfactant is an ion that is ionized in water to become an organic anion.
  • the lipophilic group in the molecule of the surfactant is R, RCOONa, R SO Na, RSO Na, etc.
  • a cationic surfactant (cationic surfactant) is ionized in water to become an organic cation.
  • the lipophilic group in the surfactant molecule is R and the halogen is X, , R -NH X, [ ⁇ 4 ⁇ ] ⁇ (quaternary ammonia salt), (C ⁇ — N) RX (alkyl pyrimido
  • Nonionic surfactants are those whose hydrophilic groups are nonionic, and those often used as hydrophilic groups include, for example, oxyethylene groups (one CH CH
  • sorbitan sucrose ester
  • glycerin derivative monoglyceride etc.
  • the lipophilic group in the surfactant molecule is R, RO (CH 2 CH 2 O) H, RCOO (
  • Amphoteric surfactants are those having both an anion group and a cationic group in the molecule.
  • nonionic surfactants and amphoteric surfactants are particularly preferably used for improving the substrate specificity of polyamineoxidase. More specifically, as nonionic surfactants, for example, Triton X-100 [Polyoxyethy lene (10J Octylphenyl Ether), Triton X-405 [p-tertiary Octylphenoxy-Polyethoxy Ethanols], Tween 20 [Polyoxyethylene ( 20) Sorbitan Monolaurate], Tween 40 [Polyoxyethylene (20) Sorbitan Monopalmitate], T ween 80 [Polyoxyethylene (20) Sorbitan Monooleate], Tween 85 [Polyo xyethylene (20) Sorbitan Trioleate], Brij 35 [Polyoxyethylene (23) Lauryl Ether], Brij 58 [Polyoxyethylene (20) Cetyl Ether], Brij 78 [Polyoxyethylene (20) Stearyl Ether], Emanoregen 9
  • the above surfactants can be used alone or in combination.
  • the polyamine oxidase and the surfactant can be mixed together in the same reagent and used together.
  • the surfactant since the surfactant only needs to be present when the reaction with polyamine oxidase proceeds, for example, reagent A containing polyamine oxidase and reagent B containing the surfactant are separately provided. It is also possible to prepare them and mix them as appropriate so that the polyamine oxidase reaction can proceed in the presence of the surfactant.
  • the concentration of the surfactant when coexisting with polyamine oxidase is not particularly limited, and each compound can be added at a concentration at which an appropriate substrate specificity improving effect is recognized. It is preferably used at a concentration of 0.001 to 20%, more preferably 0.0 to 1 to 5%.
  • the type, concentration, and pH of the buffer used for improving the substrate specificity by the surfactant of the present invention are not particularly limited.
  • the buffer is relaxed between pH 5 and L0. It is desirable that the concentration is set so as to have an impulsive capacity and to maintain a necessary and sufficient buffer capacity.
  • buffers include general-purpose Tris buffers and phosphate buffers, organic acid buffers such as acetic acid, citrate, and oxalic acid, MES, BES, HEPES, Good buffers such as TES, bicine, and tricine, amino acid buffers such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, and imidazole buffer can also be used.
  • the concentration of the buffer is, for example, preferably 5 to 500 mM, more preferably 20 to: LOOmM.
  • a method of performing an enzyme reaction at a pH of less than 7.5 can also be mentioned. More preferable pH conditions include, for example, 6.0 or more and less than 7.5.
  • the above-mentioned surfactant that can improve the substrate specificity of polyamine oxidase can also be present.
  • the polyamine oxidase can be used in such a form that it is preferentially contained in a reagent having a pH of less than 7.5, more preferably pH 6.0 or more and less than 7.5.
  • the pH should be less than 7.5, more preferably pH 6.0 or more and less than 7.5. containing Okishidaze) was prepared separately respectively, a mixture of them, pH 7. less than 5, more preferably P H6. 0 or 7. as a condition of less than 5 is advanced polyamine O Kishida over peptidase reaction It ’s easy to do.
  • the concentration of polyamine oxidase used for improving the substrate specificity by carrying out the enzymatic reaction in the presence of the above-mentioned surfactant of the present invention and less than Z or PH 7.5 is not particularly limited.
  • This enzyme solution includes coenzymes such as FAD, NAD, NADP, NADH and NADPH, peroxidases, hydrogen peroxide detection reagents such as 4-aminoantipyrine, phenol, TOOS and DA-67, 1-Methoxy PMS and WST— 8 Oxidation-reduction coloring reagents such as (formazan reagent), enzymes such as catalase, and various polyamines such as diacetyl spermine! /, Or more than one! /, May! / .
  • coenzymes such as FAD, NAD, NADP, NADH and NADPH
  • peroxidases such as 4-aminoantipyrine, phenol, TOOS and DA-67
  • Oxidation-reduction coloring reagents such as (formazan reagent)
  • enzymes such as catalase
  • various polyamines such as diacetyl spermine! /, Or more than one!
  • chelating reagents and other reagents which are stabilizers as described above can be used together.
  • various inorganic salts such as sodium salt, potassium salt, sodium salt, polysaccharides such as dextran, ushi serum albumin (BSA), glycerol, amino acids, antibiotics, sulfa drugs, etc. These chemotherapeutic agents may coexist.
  • N 1 -acetyl spermidine can be efficiently produced by using the method for improving substrate specificity by carrying out an enzymatic reaction in the presence of the above-described surfactant and under Z or PH 7.5. it can.
  • N 1 -Acetylspermidine can be obtained by acting diacetylspermine oxidase on diacetylenoperm, but at this time the substrate has substrate specificity in the reaction system.
  • the product N 1 -acetyl spermidine is less susceptible to further conversion to putrescine and the recovery is improved, which is industrially advantageous.
  • the present invention includes a method for measuring diacetylpolyamine by allowing a reagent containing polyamine oxidase to act on a sample.
  • the sample to be measured is not particularly limited, and examples thereof include urine, serum, food and extracts thereof.
  • the concentration of diacetylpolyamine contained in the measurement sample is not particularly limited, and examples thereof include ImM or lower, and the sample can be diluted as appropriate.
  • the amount of polyamine oxidase to be used is not particularly limited, and for example, it may be added so that the final concentration is 0.05-1000 U / mU, preferably 0.2-100 U / ml.
  • the pH to be applied is not particularly limited.
  • the pH is 5 to 10, preferably 6 to 9, particularly preferably 6.0 to less than 7.5.
  • the pH adjustment method is not particularly limited.
  • a general-purpose Tris buffer or phosphate buffer can be used, an organic acid buffer such as acetic acid, citrate, or oxalic acid, MES, or the like.
  • Good buffers such as BES, HEPES, TES, bicine, and tricine, amino acid buffers such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, and imidazole buffer can be used.
  • an alcohol compound which is a stabilizer of polyamine oxidase, a chelating reagent, and a surfactant, which is a substrate specificity improver, may be present as appropriate.
  • the action time is, for example, 30 seconds to 120 minutes, preferably 1 to 30 minutes.
  • the working temperature is, for example, 20 to 45 ° C., and a temperature used for a normal enzyme reaction can be appropriately selected.
  • the polyamine oxidase solution added to the sample includes Tris, phosphate, acetic acid, citrate, oxalic acid, MES, BES, HEPES, TES, bicine, tricine, glycine, boric acid, Bis—Tris propane, Even if a buffer such as imidazole, a coenzyme such as FAD, an alcohol compound chelating reagent that is a stabilizer of polyamine oxidase, or a surfactant that is a substrate specificity improver of polyamine oxidase is added. Good.
  • various inorganic salts such as sodium chloride, potassium salt and sodium salt, polysaccharides such as dextran, BSA, glycerol, amino acids and the like may coexist.
  • This liquid preparation may be freeze-dried or spray-dried.
  • the product of the polyamine oxidase reaction may be measured by any method.
  • the generated hydrogen peroxide should be measured by an enzymatic measurement method using peroxidase and an appropriate coloring reagent, a luminescent reagent, or an electrical method using an enzyme electrode. Can do.
  • the aldehyde produced can be measured by combining aldehyde oxidase or aldehyde dehydrogenase with an appropriate color reagent.
  • detection reagents for reaction products using enzymes include tris, phosphoric acid, acetic acid, citrate, oxalic acid, MES, BE S, HEPES, TES, bicine, tricine, glycine, boric acid, Bis—Tris propane Buffers such as imidazole, coenzymes such as FAD, NAD, NADP, NADH, NADPH, hydrogen peroxide detection reagents such as peroxidase, 4-aminoantipyrine, phenol, TOOS, DA-67, 1-Methoxy PMS
  • redox coloring reagents such as WST-8 (formazan reagent) and enzymes such as catalase can be contained.
  • an alcohol compound that is a stabilizer of polyamine oxidase, a chelating reagent, and a surfactant that is a substrate property improving agent may be appropriately added.
  • various inorganic salts such as sodium chloride, sodium chloride potassium and azido sodium, polysaccharides such as dextran, BSA, glycerol, amino acids and the like may coexist. Freeze or spray dry this liquid formulation.
  • the polyamine oxidase reaction and its product can be detected simultaneously. It is preferable to add polyamine oxidase to the above-mentioned detection reagent for the reaction product in a range of 0.05-: L000 U / mU, preferably 0.2-: LOO U / ml. This liquid preparation may be freeze-dried or spray-dried.
  • the amount of the specimen used for the measurement of diacetyl polyamine, the addition amount of each reagent, and the ratio thereof are not particularly limited.
  • the present invention provides a reagent for measuring diacetyl polyamine used in the above measurement method.
  • the measurement reagent for diacetylpolyamine may be prepared as a liquid product, or a frozen product or a freeze-dried product of the liquid product.
  • Enzyme is diluted in A (50 mM potassium phosphate buffer (pH 7.0) containing ImM EDTA and 0.2% (w / v) urine serum albumin (BSA, Sigma)) or Diluent B [Diluted appropriately with 50 mM potassium phosphate buffer (pH 7.0) containing ImM EDTA and 5% (v / v) ethanol].
  • 0.05 ml of enzyme sample was added to the reaction solution and mixed. A total volume of 1.15 ml was reacted at 37 ° C for 3 minutes, and the change in absorbance at 555 nm of the reaction solution for 3 minutes from the start of the reaction was measured using a spectrophotometer.
  • the enzyme 1U of diacetyl spermine oxidase was defined as the amount of enzyme that produces 1 mol of hydrogen peroxide per minute when diacetyl spermine is used as a substrate under the above measurement conditions.
  • the millimolar molecular extinction coefficient under these conditions was 1.57 cm 2 Z mol.
  • This measurement method is just an example, and there is no limitation on the type, concentration, or pH of the reagent for activity measurement.
  • a phosphate buffer or the like can be used in place of the Tris-HC1 buffer.
  • the stability test of polyamine oxidase was performed by diluting enzyme samples with 50 mM phosphate buffer (PH 7.0) supplemented with various reagents, and comparing the residual activity after accelerated tests by storage at 30 ° C. went.
  • the residual activity of the enzyme solution after the acceleration test was expressed as a relative amount (%) when the amount of change in absorbance at the time of preparing the enzyme solution was taken as 100%.
  • a 50 mM phosphate buffer solution (pH 7.0) containing ImM or 5 mM chelating reagents was prepared.
  • a 50 mM phosphate buffer (pH 7.0) containing 10% (vZv) ethanol as an alcohol compound and 5 mM EDTA as Z or a chelating reagent was prepared.
  • a diacetylsperminoxidase solution 0.1 lU / ml derived from Debaryomyces' Nonsenii Tsukuba42 (FERM BP-10603) described in PCTZJP2006Z310648 was prepared.
  • the above enzyme solution 0.1 lU / ml was prepared with a 50 mM phosphate buffer solution (pH 7.0) containing no reagent.
  • the enzyme protein was removed by dialyzing against water using Centrivrep 10 (Amicon).
  • Cetylspermidine dihydrochloride was obtained (yield 76%). [0087] The above 82 mg of N 1 -acetyl spermidine dihydrochloride was recrystallized from ethanol to obtain 52 mg of N 1 -acetyl spermidine dihydrochloride (yield 47%). Melting points and 1 H, 13 C—NMR measurements agreed with literature known values (“Acta Chemica Scandinavica J, (Denmark), 19 89, 43 ⁇ , p. 990—994). In the production method of N 1 -acetyl spermidine described in JP2006 / 310648, 2 U / ml diacetyl spermine oxidase was used. N 1 -acetyl spermidine dihydrochloride was obtained.
  • Triton X-100 (Non-ionic, Wako Pure Chemical Industries) 13. 2
  • Triton X-405 (Nonionic, manufactured by Nacalai Tesque) 11. 1
  • Tween 80 (nonionic, manufactured by Wako Pure Chemical Industries, Ltd.) 11.8
  • Debariomyces origin Debaryomyces. Hansenii T s u k u b a 42 ERM BP— 10603) Diacetyl spermine oxidase derived from Candida: Candida glabrata No d a l 62 (FERM
  • a 0.2M phosphate buffer solution having a pH of 6.5 to 7.5 was prepared.
  • the polyamine oxidase activity measurement method described above was performed using the Debariomyces 'Nonseneni Tsukuba42 (FERM BP-10603) -derived diacetyl spermine oxidase solution and Candida' Grabrata Nodal62 ( FER M BP-10602) -derived diacetyl spermine oxidase was measured and compared for enzyme activity against various polyamines. The results are shown in Table 4. Thus, the effect of improving the substrate specificity was recognized by making the pH less than 7.5.
  • a 0.2M phosphate buffer solution D0l ⁇ oo C (oi (pH 6.5 to 7.5) containing 0.3% TritonX-100 was prepared, and the denomination described in PCTZJP2006Z310648 was performed using the polyamine oxidase activity measurement method described above. Enzyme activity against various polyamines was measured and compared using diacetyl spermine oxidase solution derived from Riomyces'Nonsenii Tsukuba42 (FERM BP-10603), and the results are shown in Table 5. Further improvement of substrate specificity was observed by adding calorie and adjusting the pH to less than 7.5, and did not react with putretin under any of the conditions.
  • N 1 A c S pm: N 1 — Acetylspentolemin, N 1 — Ac S pd: N 1 — Acetylspermidine, N 8 — A C S pd: N 8 — Acetyls penolemidine, S pm: Spenoremin , S pd: Spenoremidin
  • TritonX 100 0.04%
  • Diacetyl sperminoxidase (derived from Debaryomyces) 24UZmL Peroxidase (derived from horseradish rust) 15UZmL
  • Diacetyl sperminoxidase (derived from Debaryomyces) 24UZmL Peroxidase (derived from horseradish rust) 15UZmL
  • a stable polyamine oxidase preparation containing an alcohol compound and Z or a chelating reagent and a polyamine oxidase is produced by a simple method and at a low cost using the stable method of the present invention. Can do.
  • a polyamine oxidase preparation with improved substrate specificity containing a surfactant and polyamine oxidase can be produced by a simple method and at low cost.
  • a reagent for measuring diacetylpolyamine can be produced by a simple method and at a low cost.

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Abstract

It is intended to provide a method for stabilizing polyamine oxidase, a method for improving substrate specificity of polyamine oxidase etc. The polyamine oxidase is used for clinical diagnosis of cancer, production of a useful material such as monoacetylpolyamine or the like. The method for stabilizing polyamine oxidase by allowing an alcohol compound and/or a chelating reagent to coexist, the method for improving substrate specificity of polyamine oxidase by allowing a surfactant to coexist and/or setting the pH at lower than 7.5, a method for producing monoacetylpolyamine and a method for purifying polyamine oxidase using the methods.

Description

明 細 書  Specification
ポリアミンォキシダーゼの安定化方法と基質特異性の改良方法  Methods for stabilizing polyamine oxidase and improving substrate specificity
技術分野  Technical field
[0001] 本発明は、臨床診断や有用物質生産等に用いられるポリアミンォキシダーゼの安 定化方法、基質特異性の改良方法、ポリアミンォキシダーゼを用いるジァセチルポリ ァミンの測定方法、及び、それらを利用してポリアミンォキシダーゼを作用させてモノ ァセチルポリアミンを製造する方法及びポリアミンォキシダーゼを精製する方法、並 びに、安定ィ匕されたポリアミンォキシダーゼ製剤及びジァセチルポリアミン測定用試 薬等に関する。 背景技術  [0001] The present invention relates to a method for stabilizing polyamine oxidase, a method for improving substrate specificity, a method for measuring diacetyl polyamine using polyamine oxidase, and the like, which are used for clinical diagnosis and production of useful substances. The present invention relates to a method for producing monoacetyl polyamine by reacting polyamine oxidase, a method for purifying polyamine oxidase, a stabilized polyamine oxidase preparation, a reagent for measuring diacetyl polyamine, and the like. Background art
[0002] ポリアミンとは、同一分子内に 2個以上のアミノ基を有する化合物の総称であり、ヒト の体内には、プトレツシン、カダベリン、スペルミジン、スペルミンの 4種類のポリアミン とそれらのモノァセチル体及びジァセチル体が存在する。癌患者において、尿中ポリ ァミンの***量が増加することが 1971年に Russelによって報告されて以来、癌の診 断を目的として尿中のポリアミン含量の測定が行われてきた (例えば、非特許文献 1 参照)。尿中ポリアミンの大部分はモノアセチル体で存在するため、従来はァセチル 基を加水分解し、ァセチルポリアミンとァセチルイ匕されて ヽな 、遊離型のポリアミンを 区別しない総ポリアミン含量として測定されていた (例えば、特許文献 1参照)。  [0002] Polyamine is a general term for compounds having two or more amino groups in the same molecule. In the human body, four types of polyamines such as putrescine, cadaverine, spermidine, and spermine, and their monoacetyl and diacetyl groups. There is a body. Since Russel reported in 1971 that increased urinary polyamine excretion in cancer patients, urinary polyamine content has been measured for cancer diagnosis (e.g., non-patented (Ref. 1). Since most of the urine polyamines exist in monoacetyl form, it was conventionally measured as the total polyamine content that does not distinguish between free polyamines that hydrolyze acetyl groups and are acetylated with acetyl ethers. (For example, see Patent Document 1).
[0003] し力しながら、尿中総ポリアミンにぉ 、ては悪性腫瘍患者の中に偽陰性例が相当数 認められることが明らかになつている。また、悪性腫瘍以外にも、炎症性疾患、心筋 梗塞、肝硬変、創傷治癒過程など、種々の病態に関連して有意に上昇することが明 らかになり、腫瘍マーカーとしては実用的でないと考えられるようになった。  [0003] However, it has been clarified that a considerable number of false-negative cases are found in urine total polyamines and in malignant tumor patients. In addition to malignant tumors, it has been found that the level is significantly increased in relation to various pathological conditions such as inflammatory diseases, myocardial infarction, cirrhosis, and wound healing processes. It came to be able to.
[0004] 近年、 N1, N12—ジァセチルスペルミン（ィ匕 1) (以下、単にジァセチルスペルミンと 記載する）と N1, N8—ジァセチルスペルミジン（ィ匕 2)の 2種類のジァセチルポリアミン が尿中に***されていることが見出された (例えば、非特許文献 2参照)。健常者の 尿中においては、これらの成分は総ポリアミンのそれぞれ 0. 6%、 1. 4%を占めるに すぎないが、癌患者における増加の割合が総ポリアミンと比較して際立って高ぐ既 存の腫瘍マーカーよりも真陽性率が高いことから、新たな腫瘍マーカーとして注目を 集めている（例えば、非特許文献 3、 4参照)。 [0004] In recent years, N 1 , N 12 — diacetyl spermine (II) (hereinafter simply referred to as diacetyl spermine) and N 1 , N 8 — diacetyl spermidine (II 2) 2 It was found that a variety of diacetylpolyamines are excreted in urine (see, for example, Non-Patent Document 2). In healthy people's urine, these components account for only 0.6% and 1.4% of total polyamines respectively, but the rate of increase in cancer patients is significantly higher compared to total polyamines. It has attracted attention as a new tumor marker because it has a higher true positive rate than existing tumor markers (see, for example, Non-Patent Documents 3 and 4).
[0005] [化 1]
Figure imgf000004_0001
[0005] [Chemical 1]
Figure imgf000004_0001
[0006] [化 2]
Figure imgf000004_0002
[0006] [Chemical 2]
Figure imgf000004_0002
[0007] ジァセチルポリアミンを特異的に測定する方法としては、例えば、 HPLC—酵素法（ 例えば、非特許文献 2参照）が挙げられる。まず検体中の各種ポリアミンを HPLCに て分離した後、 HPLC検出器中にてァセチル加水分解酵素を作用させて生じるフリ 一ポリアミンをポリアミンォキシダーゼで酸ィ匕し、生じる過酸化水素を電気化学検出 器を用いて検出する方法である。し力しながら、本方法では多数の検体を短時間で 処理できない等の問題が生じていた。最近、ジァセチルスペルミンに対する特異性 が高い抗体が取得され、本抗体を用いる間接競合 ELISA法による測定法も開発さ れたが (例えば、特許文献 2、 3、 4、非特許文献 3、 4参照）、その操作は煩雑であり、 測定値は試料の検体量に反比例するものであった。 [0007] As a method for specifically measuring diacetyl polyamine, for example, HPLC-enzyme method (for example, see Non-Patent Document 2) can be mentioned. First, various polyamines in the sample are separated by HPLC, and then the free polyamine produced by the action of acetylhydrolase in the HPLC detector is oxidized with polyamine oxidase, and the resulting hydrogen peroxide is electrochemically detected. It is the method of detecting using a container. However, this method has a problem that a large number of samples cannot be processed in a short time. Recently, an antibody with high specificity to diacetylspermine was obtained, and an indirect competitive ELISA method using this antibody has been developed (for example, Patent Documents 2, 3, and 4, Non-Patent Documents 3 and 4). The operation was complicated, and the measured value was inversely proportional to the sample amount of the sample.
[0008] このような背景から、ジァセチルポリアミンに特異的に作用するポリアミンォキシダー ゼゃデヒドロゲナーゼを用いて、より簡便で直接的にジァセチルポリアミンを測定でき る方法が開発されれば非常に有用であると期待されるが (例えば、非特許文献 5参照 )、これまでにこのような試みはなかった。その理由として、これまでに知られているほ とんどのポリアミンォキシダーゼは、モノァセチルポリアミン（例えば、 N1—ァセチルス ペルミン、 N1—ァセチルスペルミジン、 N8—ァセチルスペルミジン、ァセチルプトレツ シン等）ゃァミノ基がァセチルイ匕されていないフリーのポリアミン (例えば、スペルミン、 スペルミジン、プトレツシン、カタべリン等）に高い反応性を示すものであり、ジァセチ ルポリアミンの測定には不向きであったためと考えられる。 [0008] Against this background, if a method for measuring diacetyl polyamine more easily and directly using polyamine oxidase dehydrogenase that specifically acts on diacetyl polyamine would be developed. Although it is expected to be very useful (see, for example, Non-Patent Document 5), no such attempt has been made so far. For this reason, most of the polyamine oxidases known so far are monoacetyl polyamines (eg, N 1 -acetylspermine, N 1 -acetyl spermidine, N 8 -acetyl spermidine, acetyl ptrettusin, etc. ) It is highly reactive with free polyamines whose amino groups are not acetylated (eg, spermine, spermidine, putretsin, catavelin, etc.). This is probably because it was not suitable for the measurement of rupolyamine.
[0009] し力しながら、最近になって、本発明者等によって、ジァセチルスペルミン (ィ匕 1)に 対して高い反応性を有し、（化 3)に示す反応を触媒するポリアミンォキシダーゼがデ ノ リオミセス、キャンディダ、ピキア等から単離された（PCTZJP2006Z310648参 照)。  [0009] However, recently, the present inventors have made polyamines having high reactivity with diacetyl spermine (I 匕 1) and catalyzing the reaction shown in (Chemical Formula 3). Xidase has been isolated from Denoriomyces, Candida, Pichia, etc. (see PCTZJP2006Z310648).
[0010] [化 3]
Figure imgf000005_0001
[0010] [Chemical 3]
Figure imgf000005_0001
^ AcHN^^N^^^N H2 + O^^^NHAc + H202 ^ AcHN ^^ N ^^^ NH 2 + O ^^^ NHAc + H 2 0 2
[0011] このようなポリアミンォキシダーゼはジァセチルスペルミンの測定に有用であると考 えられるが、実用化に向けては、酵素の安定性とジァセチルスペルミンに対する特異 性をさらに改善することが必要であると考えられた。 [0011] Such polyamine oxidase is thought to be useful for the measurement of diacetyl spermine. For practical application, however, the stability of the enzyme and its specificity for diacetyl spermine should be further improved. Was considered necessary.
[0012] まず、酵素の安定性について説明する。一般的に、酵素は液体に溶解した状態で は不安定であると 、う欠点を有して 、る。ポリアミンォキシダーゼにつ 、ても例外では なぐこの解決策として、種々の安定ィ匕方法が試みられてきたが有効な方法はなかつ た。  [0012] First, the stability of the enzyme will be described. In general, enzymes have the disadvantage of being unstable when dissolved in a liquid. As a solution for polyamine oxidase, which is no exception, various stable methods have been tried, but there has been no effective method.
[0013] 次に、酵素の基質特異性について説明する。本発明者等によって発見された上記 の酵素は、ジァセチルスペルミンに対する反応性を 100%とすると、例えば、 N1—ァ セチルスペルミジンにはそれぞれ約 30%、 50%、 90% (pH7. 5 リン酸緩衝液条件 下）の反応性を示すものであり、ジァセチルスペルミン以外のポリアミンも測定してしま うという問題が生じていた。 Next, the substrate specificity of the enzyme will be described. The above-mentioned enzyme discovered by the present inventors is assumed to have a reactivity to diacetyl spermine of 100%, for example, N 1 -acetyl spermidine is about 30%, 50%, 90% (pH 7.5), respectively. This shows the reactivity under phosphate buffer conditions), and there has been a problem of measuring polyamines other than diacetyl spermine.
[0014] また、ポリアミンォキシダーゼは、ジァセチルポリアミンの測定という用途以外にも、 ジァセチルスペルミン（化 1)力 医薬品や農薬の原料として有用な N1—ァセチルス ペルミジン (ィ匕 4)を製造する方法に利用できることが報告されて ヽる (PCT/JP200 67310648)。本法は、従来より知られていた有機合成法 (例えば、非特許文献 6、 7参照）と比較すると工程数が少なぐ簡便であるものの、酵素が不安定であるために 多量の酵素が必要となるという欠点を有していた。また、基質特異性が不十分である ために、生成する N1 ァセチルスペルミジンの一部がプトレツシンに変換されてしま い、収率が低下してしまうという欠点も有していた。 [0014] In addition to the use of diacetyl polyamine measurement, polyamine oxidase is a diacetyl spermine (Chemical Formula 1) N 1 -acetyls permidine (4) that is useful as a raw material for pharmaceuticals and agricultural chemicals. It has been reported that it can be used in manufacturing methods (PCT / JP200 67310648). Although this method is simple and requires fewer steps compared to the conventionally known organic synthesis methods (for example, see Non-Patent Documents 6 and 7), the enzyme is unstable. It had the disadvantage that a large amount of enzyme was required. In addition, since the substrate specificity is insufficient, a part of the produced N 1 acetyl spermidine is converted to putrescine, resulting in a decrease in yield.
[化 4]
Figure imgf000006_0001
[Chemical 4]
Figure imgf000006_0001
[0016] 特許文献 1 :特開平 1 85080号公報 Patent Document 1: Japanese Patent Laid-Open No. 1 85080
特許文献 2： WO 2004Z081569ノ ンフレット  Patent Document 2: WO 2004Z081569 Nonfret
特許文献 3：特開 2006 - 38594号公報  Patent Document 3: Japanese Patent Laid-Open No. 2006-38594
特許文献 4：特開 2006— 199655号公報  Patent Document 4: Japanese Unexamined Patent Publication No. 2006-199655
非特許文献 1：「Cancer ResearchJ , (米国）， 1971年， 31卷， p. 1555- 1558 非特許文献 2 :「Journal of BiochemistryJ , (日本）, 1995年， 117卷， p. 107 - 112  Non-patent document 1: “Cancer ResearchJ, (USA), 1971, 31 卷, p. 1555-1558 Non-patent document 2:“ Journal of BiochemistryJ, (Japan), 1995, 117 卷, p. 107-112
非特許文献 3 :「Clinical Cancer Research] , (米国）, 2005年， 11卷, p. 2986 - 2990  Non-Patent Document 3: "Clinical Cancer Research", (USA), 2005, 11 卷, p. 2986-2990
非特許文献 4:「Journal of BiochemistryJ , (日本）， 2006年， 139卷， p. 315 - 322  Non-Patent Document 4: “Journal of BiochemistryJ, (Japan), 2006, 139 卷, p. 315-322
非特許文献 5 :「Clinica Chimica ActaJ , (蘭国）， 2004年， 340卷， ρ219— 22 7  Non-Patent Document 5: “Clinica Chimica ActaJ, (Landland), 2004, 340 卷, ρ219—22 7
非特干文献 6： journal of Chemical society, Perkm Transactions I」 , ( 英国）， 1988年， p. 1905- 1911  Non-Patent Literature 6: journal of Chemical society, Perkm Transactions I ”(UK), 1988, p. 1905- 1911
非特許文献 7 :「Acta Chemica ScandinavicaJ , (デンマーク）， 1989年， 43卷， p. 990- 994  Non-Patent Document 7: “Acta Chemica ScandinavicaJ, (Denmark), 1989, 43 卷, p. 990-994
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0017] 本発明の課題は、ポリアミンォキシダーゼの安定ィ匕方法、基質特異性の改良方法、 及びポリアミンォキシダーゼを用いるジァセチルポリアミンの測定方法、及び、それら を利用してポリアミンォキシダーゼを作用させてモノァセチルポリアミンを製造する方 法及びポリアミンォキシダーゼを精製する方法、並びに、安定ィ匕されたポリアミンォキ シダーゼ製剤及びジァセチルポリアミン測定用試薬を提供することである。 The subject of the present invention is a method for stabilizing polyamine oxidase, a method for improving substrate specificity, And a method for producing diacetyl polyamine by using polyamine oxidase and a method for producing monoacetyl polyamine using the same, a method for purifying polyamine oxidase, It is intended to provide a decontaminated polyamine oxidase preparation and a reagent for measuring diacetylpolyamine.
課題を解決するための手段  Means for solving the problem
[0018] 本発明者らは、前記課題解決のために鋭意研究を重ねた結果、（1)アルコールィ匕 合物及び Z又はキレート試薬を共存させることにより、ポリアミンォキシダーゼの安定 性が向上すること、（2)界面活性剤を共存させることにより、ポリアミンォキシダーゼの 基質特異性が改良されること、（3)ポリアミンォキシダーゼを pH7. 5未満で作用させ ることにより、ポリアミンォキシダーゼの基質特異性が改良されることなどを見出し、こ れらの知見に基づき本発明を完成するに至った。  [0018] As a result of intensive studies to solve the above problems, the present inventors have (1) the stability of polyamine oxidase is improved by the coexistence of an alcoholic compound and Z or a chelating reagent. (2) The substrate specificity of polyamine oxidase is improved by the coexistence of surfactants, and (3) the substrate of polyamine oxidase by allowing polyamine oxidase to act at a pH below 7.5. The inventors have found that the specificity is improved and have completed the present invention based on these findings.
[0019] すなわち、本発明は、以下に関する。  That is, the present invention relates to the following.
(1)アルコールィ匕合物及び Z又はキレート試薬と、ポリアミンォキシダーゼを共存さ せることを特徴とする、ポリアミンォキシダーゼの安定ィ匕方法。  (1) A method for stabilizing polyamine oxidase, comprising coexisting an alcohol compound and Z or a chelating reagent with polyamine oxidase.
(2)ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、上記（1)に 記載のポリアミンォキシダーゼの安定ィ匕方法。  (2) The method for stabilizing polyamine oxidase according to (1) above, wherein the polyamine oxidase is diacetyl spermine oxidase.
(3)界面活性剤存在下にてポリアミンォキシダーゼを作用させることを特徴とする、ポ リアミンォキシダーゼの基質特異性の改良方法。  (3) A method for improving the substrate specificity of polyamine oxidase, characterized by allowing polyamine oxidase to act in the presence of a surfactant.
(4)ポリアミンォキシダーゼを pH7. 5未満で作用させることを特徴とする、ポリアミン ォキシダーゼの基質特異性の改良方法。  (4) A method for improving the substrate specificity of polyamine oxidase, characterized by allowing polyamine oxidase to act at a pH of less than 7.5.
(5)ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、上記（3)〜 (4)の 、ずれかに記載のポリアミンォキシダーゼの基質特異性の改良方法。  (5) The method for improving the substrate specificity of polyamine oxidase according to any one of (3) to (4) above, wherein the polyamine oxidase is diacetyl spermine oxidase.
(6)ポリアミンォキシダーゼを含有する試薬を試料に作用させて、ジァセチルポリアミ ンを測定することを特徴とする、ジァセチルポリアミンの測定方法。  (6) A method for measuring diacetyl polyamine, which comprises measuring a diacetyl polyamine by allowing a reagent containing polyamine oxidase to act on a sample.
(7)アルコールィ匕合物及び Z又はキレート試薬により安定ィ匕されたポリアミンォキシ ダーゼを用いることを特徴とする、上記（6)に記載の測定方法。  (7) The measurement method according to (6) above, wherein the polyamine oxidase stabilized with an alcohol compound and Z or a chelating reagent is used.
(8)界面活性剤により基質特異性が改良されたポリアミンォキシダーゼを用いること を特徴とする、上記（6)〜（7)の 、ずれかに記載の測定方法。 (9)ポリアミンォキシダーゼを pH7. 5未満で作用させることを特徴とする、上記（6)〜 (8)の 、ずれかに記載の測定方法。 (8) The measurement method according to any one of (6) to (7) above, wherein polyamine oxidase whose substrate specificity is improved by a surfactant is used. (9) The method according to any one of (6) to (8) above, wherein the polyamine oxidase is allowed to act at a pH of less than 7.5.
(10)ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、上記（6) 〜（9)の 、ずれかに記載のジァセチルポリアミンの測定方法。  (10) The method for measuring diacetyl polyamine according to any one of (6) to (9) above, wherein the polyamine oxidase is diacetyl spermine oxidase.
(11)アルコールィ匕合物及び Z又はキレート試薬と、ポリアミンォキシダーゼを含有す ることを特徴とする、安定化されたポリアミンォキシダーゼ製剤。  (11) A stabilized polyamine oxidase preparation comprising an alcoholic compound and Z or a chelating reagent and a polyamine oxidase.
(12)ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、上記 16に 記載の安定ィ匕されたポリアミンォキシダーゼ製剤。  (12) The stabilized polyamine oxidase preparation according to the above 16, wherein the polyamine oxidase is diacetyl spermine oxidase.
(13)ポリアミンォキシダーゼを含有することを特徴とする、ジァセチルポリアミンの測 定試薬。  (13) A reagent for measuring diacetyl polyamine, comprising polyamine oxidase.
(14)アルコールィ匕合物及び Z又はキレート試薬と、ポリアミンォキシダーゼを含有す ることを特徴とする上記（13)に記載のジァセチルポリアミンの測定試薬。  (14) The reagent for measuring diacetyl polyamine according to (13) above, which contains an alcoholic compound and Z or a chelating reagent, and polyamine oxidase.
(15)界面活性剤とポリアミンォキシダーゼを含有することを特徴とする上記（13)又 は（14)のいずれかに記載のジァセチルポリアミン測定試薬。  (15) The reagent for measuring diacetyl polyamine according to (13) or (14) above, which contains a surfactant and polyamine oxidase.
(16)ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、上記（13) 〜（15)のいずれかに記載のジァセチルポリアミン測定試薬。  (16) The diacetyl polyamine measuring reagent according to any one of (13) to (15), wherein the polyamine oxidase is diacetyl spermine oxidase.
(17)上記（1)又は（2)記載のポリアミンォキシダーゼの安定ィ匕方法、及び Z又は上 記（3)〜（5)の 、ずれか一項に記載のポリアミンォキシダーゼの基質特異性の改良 方法を利用することを特徴とする、ジァセチルポリアミンにポリアミンォキシダーゼを作 用させてモノァセチルポリアミンを製造する方法。  (17) The method for stabilizing polyamine oxidase according to (1) or (2) above, and the substrate specificity of the polyamine oxidase according to any one of Z or (3) to (5) above A method for producing monoacetyl polyamine by causing polyamine oxidase to act on diacetyl polyamine, which comprises using the improved method of 1.
(18)ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、上記（17) 記載のモノァセチルポリアミンの製造方法。  (18) The method for producing monoacetyl polyamine according to (17) above, wherein the polyamine oxidase is diacetyl spermine oxidase.
(19)上記（1)又は（2)記載のポリアミンォキシダーゼの安定ィ匕方法を利用することを 特徴とする、ポリアミンォキシダーゼの精製方法。  (19) A method for purifying polyamine oxidase, comprising using the method for stabilizing polyamine oxidase described in (1) or (2) above.
(20)ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、上記（19) 記載のポリアミンォキシダーゼの精製方法。  (20) The method for purifying polyamine oxidase according to the above (19), wherein the polyamine oxidase is diacetyl spermine oxidase.
発明の効果 The invention's effect
本発明の方法によれば、アルコールィ匕合物及び Z又はキレート試薬を共存させる ことにより、ポリアミンォキシダーゼを安定ィ匕することができる。また、界面活性剤を共 存させる及び Z又は PH7. 5未満の条件下とすることにより、ポリアミンォキシダーゼ の基質特異性を改良することができる。さらに、このような安定ィ匕方法及び Z又は基 質特異性の改良方法を用いて、ジァセチルポリアミンへポリアミンォキシダーゼを作 用させてモノァセチルポリアミンを製造したり、ポリアミンォキシダーゼを用 V、ることに よりジァセチルポリアミンを測定することができる。 According to the method of the present invention, an alcoholic compound and Z or a chelating reagent are allowed to coexist. Thus, the polyamine oxidase can be stabilized. In addition, the substrate specificity of polyamine oxidase can be improved by coexisting a surfactant and making the condition less than Z or PH 7.5. Furthermore, using such a stabilization method and a method for improving Z or substrate specificity, monoacetyl polyamine can be produced by using polyamine oxidase to diacetyl polyamine, or polyamine oxidase can be used. Diacetyl polyamine can be measured by V.
図面の簡単な説明  Brief Description of Drawings
[0021] [図 1]アルコールィ匕合物とキレート試薬を共存させることによる酵素の安定ィ匕効果を 示すグラフである。  [0021] FIG. 1 is a graph showing the effect of enzyme stability due to the coexistence of an alcohol compound and a chelating reagent.
[図 2]ポリアミンォキシダーゼ製剤の熱安定性を示すグラフである。  FIG. 2 is a graph showing the thermal stability of a polyamine oxidase preparation.
[図 3]ジァセチルスペルミンォキシダーゼを含有する試薬を用い、ジァセチルスペルミ ン試料を測定したときの濃度と吸光度の関係を示すグラフである。  FIG. 3 is a graph showing the relationship between the concentration and absorbance when a diacetyl spermine sample was measured using a reagent containing diacetyl spermine oxidase.
[図 4]ジァセチルスペルミンォキシダーゼを含有する試薬を用い、尿にジァセチルス ペルミンを添加した試料を測定したときの濃度と吸光度の関係を示すグラフである。 発明を実施するための最良の形態  FIG. 4 is a graph showing the relationship between the concentration and the absorbance when a sample containing diacetyl spermine oxidase and diacetylspermine added to urine was measured. BEST MODE FOR CARRYING OUT THE INVENTION
[0022] 以下、本発明を詳細に説明する。本発明に用いられる「ポリアミンォキシダーゼ」は 、ジァセチルポリアミンを酸素存在下で酸化して、モノァセチルポリアミン、アルデヒド 及び過酸化水素を生成する反応を触媒する酵素であり、如何なる起源のものでも用 いることができる。例えば、微生物、植物、あるいは動物などの生産するポリアミンォ キシダーゼ等、いずれであってもよぐ特に制限されない。例えば、酵母等を起源と するポリアミンォキシダーゼ等が挙げられ、好ましくはデバリオミセス属、キャンディダ 属、ピキア属由来の酵素等が用いられる。  [0022] Hereinafter, the present invention will be described in detail. The “polyamine oxidase” used in the present invention is an enzyme that catalyzes the reaction of oxidizing diacetyl polyamine in the presence of oxygen to produce monoacetyl polyamine, aldehyde, and hydrogen peroxide. But it can be used. For example, any of polyamine oxidase produced by microorganisms, plants, animals, etc. is not particularly limited. Examples thereof include polyamine oxidase originating from yeast and the like, and preferably, enzymes derived from the genera Debaryomyces, Candida and Pichia are used.
[0023] さらに具体的には、デバリオミセス'ハンセニイ（Debaryomyces hansenii)、デバリオ セス'マフマ (Debaryomyces maramaノ、ァノ リオ セス'マフムス (Debaryomyces mar amus)、キャンディダ'グラブラタ（Candida glabrata)及び「FEBS Letters] , (英国） , 2000年， 476卷， p. 150— 154に記載のキャンディダ 'ボイディ-ィ（Candida boid inii)由来のポリアミンォキシダーゼ等が好ましく用いられる。  [0023] More specifically, Debaryomyces hansenii, Debaryomyces marama, Anbariomyces mar amus, Candida glabrata and FEBS Letters , (UK), 2000, 476 卷, p. 150-154, polyamine oxidase derived from Candida boid inii is preferably used.
[0024] ポリアミンォキシダーゼの中でも、特にジァセチルスペルミンォキシダーゼは好まし く用いられる。ここで述べるジァセチルスペルミンォキシダーゼとは、 N1—ァセチルス ペルミン、 N1—ァセチルスペルミジン、 N8—ァセチルスペルミジンに対する活性より もジァセチルスペルミンに対する活性が高 、ポリアミンォキシダーゼを指し、例えば、 デバリオミセス'ノヽンセニイ、デノ リオミセス'マラマ、デノ リオミセス'マラムス、キャン デイダ ·グラブラタ、ピキア ·ミヌタ由来のジァセチルスペルミンォキシダーゼ等が挙げ られる。 [0024] Among polyamine oxidases, diacetyl spermine oxidase is particularly preferable. Used frequently. The di § cetyl spermine O Kishida over Ze described herein, N 1 - Asechirusu Perumin, N 1 - § cetyl spermidine, N 8 - active high for di § cetyl spermine than activity against § cetyl spermidine, refers to polyamine O Kishida over Ze, For example, Debaryomyces' Nonsenyi, Denoriomyces' malama, Denoriomyces' Marams, Candida glabrata, Pichia minuta diacetyl spermine oxidase and the like.
[0025] このようなジァセチルスペルミンォキシダーゼの好適例として、 PCTZJP2006Z3 [0025] As a preferred example of such diacetyl spermine oxidase, PCTZJP2006Z3
10648に記載されている以下の酵素を挙げることができる。 The following enzymes described in 10648 can be mentioned.
[0026] すなわち、以下の（la)〜（lg)に記載の理ィ匕学的性質をそれぞれ若しくはそれらを 適宜併せて有するジァセチルスペルミンォキシダーゼ（なお、下記の酵素の理化学 的性質は、測定上不可避的な誤差を含んでいる）： [0026] That is, diacetyl spermine oxidase having each of the physical properties described in the following (la) to (lg) or a combination thereof as appropriate (the physicochemical properties of the following enzymes are: (Including inevitable errors in measurement):
[0027] (la)作用：化 3に示したとおり、酸素存在下にてジァセチルスペルミンの 2級ァミンの[0027] (la) Action: As shown in Chemical Formula 3, the secondary amine of diacetyl spermine in the presence of oxygen
C— N結合を開裂して、 N1—ァセチルスペルミジン、 3—ァセトアミドプロパナール及 び過酸化水素を生成する。 C—N bond is cleaved to form N 1 -acetyl spermidine, 3-acetamidopropanal and hydrogen peroxide.
(lb)基質特異性：ジァセチルスペルミン > N1—ァセチルスペルミン， N1 -ァセチル スペルミジン， N8—ァセチルスペルミジン (lb) Substrate specificity: di § cetyl spermine> N 1 - § cetyl spermine, N 1 - Asechiru spermidine, N 8 - § cetyl spermidine
例えば、 pH7. 5の条件下において、ジァセチルスペルミンを基質として用いたとき の活性を 100としたとき、 N1—ァセチルスペルミン、 N1—ァセチルスペルミジン、 N8 —ァセチルスペルミジンを基質として用いたときの活性が 90以下、好ましくは 60以下 、さらに好ましくは 50以下、もっとも好ましくは 40以下である理ィ匕学的性質を有する 本発明酵素などが挙げられる。本評価における pHの条件や緩衝液の種類と濃度に つ!ヽてはなんら制限されな!ヽ。 For example, if the activity when diacetyl spermine is used as a substrate under the condition of pH 7.5 is 100, N 1 -acetyl spermine, N 1 -acetyl spermidine, N 8 -acetyl spermidine is the substrate. The enzyme of the present invention having a physical property having an activity of 90 or less, preferably 60 or less, more preferably 50 or less, and most preferably 40 or less. There are no restrictions on the pH conditions or the type and concentration of the buffer in this evaluation!
(lc)至適 pH : 6. 5〜8. 5  (lc) Optimum pH: 6.5 to 8.5
例えば、緩衝液として 200mM酢酸緩衝液 (pH4. 5〜6. 0)、 200mMリン酸カリ ゥム緩衝液（ρΗ6. 0〜8. 0)、 200mM PIPES -NaOH (pH6. 5〜7. 5)、 200m Mトリス—塩酸緩衝液 (pH7. 5〜9. 5)を用い、それぞれの pHにおいて、温度 37 °Cにて酵素反応を行い、至適 pHを求める。例えば、至適 pHとして pH6. 5〜8. 5、 好ましくは pH7. 0〜8. 0の理ィ匕学的性質を有する本発明酵素などが挙げられる。 (Id)安定 pH : 6. 0〜8. 0 For example, 200 mM acetate buffer (pH 4.5 to 6.0), 200 mM potassium phosphate buffer (ρΗ 6.0 to 8.0), 200 mM PIPES-NaOH (pH 6.5 to 7.5) Using 200 mM Tris-HCl buffer (pH 7.5 to 9.5), perform an enzymatic reaction at each temperature at a temperature of 37 ° C to obtain the optimum pH. For example, the enzyme of the present invention having physical properties of pH 6.5 to 8.5, preferably pH 7.0 to 8.0 as the optimum pH. (Id) Stable pH: 6.0-8.0
例えば、緩衝液として 200mM酢酸緩衝液 (pH4. 5〜6. 0)、 200mMリン酸カリ ゥム緩衝液（ρΗ6. 0〜8. 0)、 200mM PIPES -NaOH (pH6. 5〜7. 5)、 200m Mトリス—塩酸緩衝液（pH7. 5〜9. 5)を用い、それぞれの pHにおいて、 30°Cで 3 0分間処理した後、本発明酵素の残存活性を測定する。例えば、安定 pHの範囲とし て、 pH6. 0〜8. 0、好ましくは pH6. 5〜7. 5の理化学的性質を有する本発明酵素 などが挙げられる。  For example, 200 mM acetate buffer (pH 4.5 to 6.0), 200 mM potassium phosphate buffer (ρΗ 6.0 to 8.0), 200 mM PIPES-NaOH (pH 6.5 to 7.5) 200 mM Tris-hydrochloric acid buffer (pH 7.5 to 9.5) is used for 30 minutes at 30 ° C. at each pH, and then the residual activity of the enzyme of the present invention is measured. Examples of the stable pH range include the enzyme of the present invention having physicochemical properties of pH 6.0 to 8.0, preferably pH 6.5 to 7.5.
(le)至適温度： 37〜55°C (le) Optimal temperature: 37 ~ 55 ° C
例えば、後述の活性測定法における反応液と同一組成よりなる反応液を用い、種 々の温度にて本酵素の活性測定を行い作用適温の範囲を求める。例えば、作用適 温の範囲として 37〜55°Cの理ィ匕学的性質を有する本発明酵素などが挙げられる。 (If)熱安定性： 37°C以下  For example, using a reaction solution having the same composition as the reaction solution in the activity measurement method described later, the activity of the present enzyme is measured at various temperatures to determine the optimum temperature range of action. For example, the enzyme of the present invention having a physical property of 37 to 55 ° C. as the temperature range of action. (If) Thermal stability: 37 ° C or less
例えば、 200mMリン酸カリウム緩衝液 (pH7. 0)を用い、それぞれの温度におい て 30分間処理した後、本発明酵素の残存活性を測定する。このとき好ましくは 90% 以上の活性が残存している場合、その温度において酵素は安定であると言える。例 えば、酵素が安定に存在できる範囲として、 37°C以下の理化学的性質を有する本発 明酵素などが挙げられる。  For example, 200 mM potassium phosphate buffer (pH 7.0) is used for 30 minutes at each temperature, and then the residual activity of the enzyme of the present invention is measured. At this time, if 90% or more of the activity remains, it can be said that the enzyme is stable at that temperature. For example, the enzyme according to the present invention having physicochemical properties of 37 ° C or lower can be cited as a range where the enzyme can stably exist.
(lg)分子量:約 92, 000 (ゲルろ過法） (lg) Molecular weight: approx. 92,000 (gel filtration method)
分子量はゲルろ過法、 Native PAGE法、 SDS— PAGE法等を用いて測定する ことができる。本発明酵素の分子量については特に限定されないが、例えば、分子 量の一例として、分子量約 92, 000 (ゲルろ過法)の本発明酵素などが挙げられる。 以下の（2a)〜（2g)に記載の理ィ匕学的性質をそれぞれ若しくはそれらを適宜併せ て有するジァセチルスペルミンォキシダーゼ (なお、下記の酵素の理ィ匕学的性質は、 測定上不可避的な誤差を含んでいる）：  Molecular weight can be measured using gel filtration, Native PAGE, SDS-PAGE and the like. The molecular weight of the enzyme of the present invention is not particularly limited, but examples of the molecular weight include the enzyme of the present invention having a molecular weight of about 92,000 (gel filtration method). Diacetyl spermine oxidase having each of the physical properties described in (2a) to (2g) below or a combination thereof as appropriate (the physical properties of the following enzymes are Including inevitable errors):
(2a)作用：化 3に示したとおり、酸素存在下にてジァセチルスペルミンの 2級ァミンの C— N結合を開裂して、 N1—ァセチルスペルミジン、 3—ァセトアミドプロパナール及 び過酸化水素を生成する。 (2a) Action: As shown in Chemical Formula 3, the C—N bond of the secondary ammine of diacetyl spermine is cleaved in the presence of oxygen to produce N 1 -acetyl spermidine, 3-acetamidopropanal and And hydrogen peroxide.
(2b)基質特異性：ジァセチルスペルミン > N1—ァセチルスペルミン， N1 -ァセチル スペルミジン， N8—ァセチルスペルミジン (2b) Substrate specificity: diacetyl spermine> N 1 -acetyl spermine, N 1 -acetyl Spermidine, N 8 — Acetylspermidine
例えば、 pH7. 5の条件下において、ジァセチルスペルミンを基質として用いたとき の活性を 100としたとき、 N1—ァセチルスペルミン、 N1—ァセチルスペルミジン、 N8 —ァセチルスペルミジンを基質として用いたときの活性が 90以下、好ましくは 60以下 、さらに好ましくは 50以下である理ィ匕学的性質を有する本発明酵素などが挙げられ る。本評価における pHの条件や緩衝液の種類と濃度にっ ヽてはなんら制限されな い。 For example, if the activity when diacetyl spermine is used as a substrate under the condition of pH 7.5 is 100, N 1 -acetyl spermine, N 1 -acetyl spermidine, N 8 -acetyl spermidine is the substrate. And the like. The enzyme of the present invention having a physical property having an activity of 90 or less, preferably 60 or less, and more preferably 50 or less when used as. There are no restrictions on the pH conditions or the type and concentration of the buffer in this evaluation.
(2c)至適 pH : 7. 0〜8. 5  (2c) Optimum pH: 7.0 to 8.5
例えば、緩衝液として 200mM酢酸緩衝液 (pH4. 5〜6. 0)、 200mMリン酸カリ ゥム緩衝液（ρΗ6. 0〜8. 0)、 200mM PIPES -NaOH (pH6. 5〜7. 5)、 200m Mトリス—塩酸緩衝液 (pH7. 5〜9. 5)を用い、それぞれの pHにおいて、温度 37 °Cにて酵素反応を行い、至適 pHを求める。例えば、至適 pHとして pH7. 0〜8. 5、 好ましくは pH7. 5〜8. 0の理ィ匕学的性質を有する本発明酵素などが挙げられる。 (2d)安定 pH : 5. 5〜9. 5  For example, 200 mM acetate buffer (pH 4.5 to 6.0), 200 mM potassium phosphate buffer (ρΗ 6.0 to 8.0), 200 mM PIPES-NaOH (pH 6.5 to 7.5) Using 200 mM Tris-HCl buffer (pH 7.5 to 9.5), perform an enzymatic reaction at each temperature at a temperature of 37 ° C to obtain the optimum pH. For example, the enzyme of the present invention having physical properties of pH 7.0 to 8.5, preferably pH 7.5 to 8.0 as the optimum pH. (2d) Stable pH: 5.5 to 9.5
例えば、緩衝液として 200mM酢酸緩衝液 (pH4. 5〜6. 0)、 200mMリン酸カリ ゥム緩衝液（ρΗ6. 0〜8. 0)、 200mM PIPES -NaOH (pH6. 5〜7. 5)、 200m Mトリス—塩酸緩衝液（pH7. 5〜9. 5)を用い、それぞれの pHにおいて、 30°Cで 3 0分間処理した後、本発明酵素の残存活性を測定する。例えば、安定 pHの範囲とし て、 pH5. 5〜9. 5、好ましくは pH6. 5〜9. 0の理化学的性質を有する本発明酵素 などが挙げられる。  For example, 200 mM acetate buffer (pH 4.5 to 6.0), 200 mM potassium phosphate buffer (ρΗ 6.0 to 8.0), 200 mM PIPES-NaOH (pH 6.5 to 7.5) 200 mM Tris-hydrochloric acid buffer (pH 7.5 to 9.5) is used for 30 minutes at 30 ° C. at each pH, and then the residual activity of the enzyme of the present invention is measured. Examples of the stable pH range include the enzyme of the present invention having physicochemical properties of pH 5.5 to 9.5, preferably pH 6.5 to 9.0.
(2e)至適温度： 45〜55°C (2e) Optimal temperature: 45-55 ° C
例えば、後述の活性測定法における反応液と同一組成よりなる反応液を用い、種 々の温度にて本酵素の活性測定を行い作用適温の範囲を求める。例えば、作用適 温の範囲として 45〜55°Cの理ィ匕学的性質を有する本発明酵素などが挙げられる。 (2f)熱安定性： 37°C以下  For example, using a reaction solution having the same composition as the reaction solution in the activity measurement method described later, the activity of the present enzyme is measured at various temperatures to determine the optimum temperature range of action. For example, the enzyme of the present invention having a physical property of 45 to 55 ° C as the temperature range of action can be mentioned. (2f) Thermal stability: 37 ° C or less
例えば、 200mMリン酸カリウム緩衝液 (pH7. 0)を用い、それぞれの温度におい て 30分間処理した後、本発明酵素の残存活性を測定する。このとき好ましくは 90% 以上の活性が残存している場合、その温度において酵素は安定であると言える。例 えば、酵素が安定に存在できる範囲として、 37°C以下の理化学的性質を有する本発 明酵素などが挙げられる。 For example, 200 mM potassium phosphate buffer (pH 7.0) is used for 30 minutes at each temperature, and then the residual activity of the enzyme of the present invention is measured. At this time, if 90% or more of the activity remains, it can be said that the enzyme is stable at that temperature. Example For example, the enzyme according to the present invention having physicochemical properties of 37 ° C or lower can be mentioned as a range where the enzyme can stably exist.
(2g)分子量:約 90, 000 (ゲルろ過法）  (2g) Molecular weight: about 90,000 (gel filtration method)
分子量は、ゲルろ過法、 Native PAGE法、 SDS— PAGE法等を用いて測定する ことができる。本発明酵素の分子量については、特に限定されないが、例えば、分子 量の一例として、分子量約 90, 000 (ゲルろ過法)の本発明酵素などが挙げられる。  Molecular weight can be measured using gel filtration, Native PAGE, SDS-PAGE, and the like. The molecular weight of the enzyme of the present invention is not particularly limited, and examples of the molecular weight include the enzyme of the present invention having a molecular weight of about 90,000 (gel filtration method).
[0029] なお、上記の一番目に挙げた酵素はデバリオミセス マラマ HUT 7199、デバリ ォミセス マラムス NBRC 0668及びデノリオミセス ハンセニイ Tsukuba 42 ( FERM BP— 10603)、並びに、上記の二番目に挙げた酵素はキャンディダ グラ ブラタ Noda 162がそれぞれ生産するもので、当業者であれば、これらの菌体から 容易に調製することができるし、酵素をコードする遺伝子をクローユングして、大腸菌 や酵母等の他の宿主で組換え発現させて調製することもできる。 [0029] It should be noted that the first enzyme mentioned above is Debaryomyces malama HUT 7199, Debaryomyces maramus NBRC 0668 and Denoriomyces Hansenii Tsukuba 42 (FERM BP-10603), and the second enzyme mentioned above is Each one is produced by Brata Noda 162 and can be easily prepared from these cells by those skilled in the art. The enzyme-encoding gene can be cloned and assembled in other hosts such as Escherichia coli and yeast. It can also be prepared by expression.
[0030] デバリオミセス ハンセニイ Tsukuba 42 (FERM BP— 10603)及びキャンデ イダ グラブラタ Noda 162 (FERM BP— 10602)は、本発明者らが茨城県内 及び千葉県内の土壌よりそれぞれ分離して得た菌株であり、 2006年 5月 10日付け で、独立行政法人産業技術総合研究所 特許生物寄託センターに、特許手続上の 微生物の寄託の国際的承認に関するブダペスト条約に基づき寄託され、それぞれ、 受託番号 FERM BP— 10603、及び、 FERM BP— 10602が付与されている。  [0030] Debariomyces Hansenii Tsukuba 42 (FERM BP-10603) and Canida glabrata Noda 162 (FERM BP-10602) are strains obtained by the present inventors from soils in Ibaraki Prefecture and Chiba Prefecture, respectively. As of May 10, 2006, it was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the Budapest Treaty on the International Approval of Deposit of Microorganisms in Patent Procedures, each with deposit number FERM BP— 10603 And FERM BP-10602.
[0031] ポリアミンォキシダーゼには、 FAD又は銅を補酵素とするものが知られており、特に 限定はされな ヽが、 FADを補酵素として利用するポリアミンォキシダーゼが望ま ヽ 。これらのポリアミンォキシダーゼは、遺伝子組換え体によって製造されたものも用い ることがでさる。  [0031] As polyamine oxidase, those using FAD or copper as a coenzyme are known, and although not particularly limited, polyamine oxidase using FAD as a coenzyme is desirable. As these polyamine oxidases, those produced by gene recombinants can be used.
[0032] [ポリアミンォキシダーゼの安定性の向上]  [0032] [Improved stability of polyamine oxidase]
本発明にお 、て、ポリアミンォキシダーゼの安定性を向上させるために共存させる アルコール化合物とは、ヒドロキシル基 (OH)を有する有機化合物であり、そのような 官能基を有する化合物であれば当業者に公知の如何なる化合物でも本発明に用い ることができる。特に脂肪族炭化水素の 1個又は複数個の水素原子をヒドロキシル基 で置換した有機化合物は、入手が容易であることから好ましく用いられる。より具体的 には、例えば、メタノール、エタノール、 1 プロパノール、 2—プロパノール、エチレン グリコール及びグリセロール等の脂肪族アルコール、並びにトレハロース、キシリトー ル、スクロース及びグルコース等の糖類が挙げられる。本発明においては、上記アル コールィ匕合物は、それぞれ単独でも複数組み合わせても用いることができる。 In the present invention, the alcohol compound coexisted in order to improve the stability of polyamine oxidase is an organic compound having a hydroxyl group (OH), and those skilled in the art can use any compound having such a functional group. Any compound known in the art can be used in the present invention. In particular, an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group is preferably used because it is easily available. More specific Examples include aliphatic alcohols such as methanol, ethanol, 1 propanol, 2-propanol, ethylene glycol and glycerol, and sugars such as trehalose, xylitol, sucrose and glucose. In the present invention, the above alcohol compounds can be used either individually or in combination.
[0033] ポリアミンォキシダーゼと共存させる前記アルコール化合物の溶液中の濃度につい ては、ポリアミンォキシダーゼの安定ィ匕効果が発揮され、かつ酵素を含む試薬を取り 扱う上で不都合のない範囲内であれば特に限定されず、各化合物について適切な 安定ィ匕効果が認められる濃度で添加することができる。好ましくは 0. 5〜20%、より 好ましくは 1〜10% (以下、特に断りがない限り、「体積％」を意味する）の濃度で用い られる。 [0033] The concentration of the alcohol compound in the solution coexisting with the polyamine oxidase may be within a range in which the stabilizing effect of the polyamine oxidase is exhibited and there is no inconvenience in handling the reagent containing the enzyme. There is no particular limitation, and each compound can be added at a concentration at which an appropriate stability effect is recognized. Preferably, it is used at a concentration of 0.5 to 20%, more preferably 1 to 10% (hereinafter referred to as “volume%” unless otherwise specified).
[0034] 本発明において、ポリアミンォキシダーゼの安定性を向上させるために共存させる キレート試薬とは、金属イオンに配位しキレートイ匕合物を与えるような化合物であり、 そのような作用を有する化合物であれば当業者に公知の如何なる化合物でも本発明 に用いることができる。  [0034] In the present invention, the chelating reagent that coexists in order to improve the stability of polyamine oxidase is a compound that coordinates to a metal ion to give a chelating compound, and has such an action. Any compound known to those skilled in the art can be used in the present invention.
[0035] 例えば、エチレンジァミン 4酢酸（Ethylenediamine— N, N, Ν， , Ν，— tetraacet ic acid, (以下「EDTA」と称する））、 1, 2 - Diaminopropane - N, N, N，， N，一 t etraacetic acid (以下「Me— EDTA」と称する）、 N— ( 2 - Hydroxymethyl) ethy lenediamine-N, N, N，， N，一 tetraacetic acid (以下「EDTA— OH」と称する） 、 trans— 1, 2― Diaminocyclohexane― N, N, N' , N'— tetraacetic acid (以 下「CyDTA」と称する）、 1 , 3— Diamino— 2— hydroxypropane— N, N, N，， N， -tetraacetic acid (以下「DTPA— OH」と称する）、 O, Ο '— Bis (2— aminoethy 1) ethyleneglycol-N, N, N，， N，—tetraacetic acid (以下「EGTA」と称する）、 Triethylenetetramine— N, N, N' , , , — hexaacetic acid (以下 「TTHA」と称する）などのポリアミンカルボン酸系のキレート試薬や、クェン酸、リンゴ 酸、イソクェン酸などのォキシカルボン酸系のキレート試薬等が挙げられる。上記キレ ート試薬に配位する塩の有無、種類や数については何ら限定されるものではないが 、例えば、ナトリウム塩ィ匕合物が好ましく用いられる。  [0035] For example, ethylenediamine-acetic acid (Ethylenediamine—N, N, Ν,, Ν, —tetraacetic acid, (hereinafter referred to as “EDTA”)), 1, 2-Diaminopropane-N, N, N, N, Tetraacetic acid (hereinafter referred to as “Me—EDTA”), N— (2-Hydroxymethyl) ethy lenediamine-N, N, N, N, unitetraacetic acid (hereinafter referred to as “EDTA—OH”), trans— 1, 2-Diaminocyclohexane- N, N, N ', N'— tetraacetic acid (hereinafter referred to as “CyDTA”), 1, 3— Diamino— 2— hydroxypropane— N, N, N, N, -tetraacetic acid (Hereinafter referred to as “DTPA—OH”), O, Ο '— Bis (2—aminoethy 1) ethyleneglycol-N, N, N, N, —tetraacetic acid (hereinafter referred to as “EGTA”), Triethylenetetramine— N, N, N ',,, — Polyaminecarboxylic acid-based chelating reagents such as hexaacetic acid (hereinafter referred to as “TTHA”) and oxycarboxylic acid-based chelates such as citrate, malic acid, and isochenic acid Reagent, and the like. The presence, type and number of salts coordinated with the chelate reagent are not limited at all, but for example, sodium salt compounds are preferably used.
[0036] 上記キレート試薬は、それぞれ単独でも複数組み合わせても用いることができる。ま た、上記キレート試薬によって安定ィ匕させるポリアミンォキシダーゼとして、例えば、 酵母等を起源とするポリアミンォキシダーゼが挙げられ、さらに具体的には、デバリオ ミセス'ハンセニイ、デノ リオミセス.マラマ、デノ リオミセス.マラムス、キャンディダ.グ ラブラタ由来のジァセチルスペルミンォキシダーゼ、及び「FEBS Letters] , (英国 ) , 2000年， 476卷， p. 150— 154に記載のキャンディダ 'ボイディ-ィ由来の Ν1— ァセチルスペルミジンォキシダーゼ等が挙げられる。 [0036] The chelating reagents may be used alone or in combination. Ma Examples of the polyamine oxidase stabilized by the chelating reagent include, for example, polyamine oxidase originating from yeast and the like, and more specifically, Devariomyces' Hansenii, Denoriomyces. Malama, Denoriomyces. Marams, Candida diacetyl spermine oxidase derived from glabrata, and Candida 'bodily derived from Candida as described in FEBS Letters, (UK), 476 ，, p. 150—154 1 — acetyl spermidine oxidase and the like.
[0037] ポリアミンォキシダーゼと共存させる前記キレート試薬の溶液中の濃度については 、ポリアミンォキシダーゼの安定ィ匕効果が発揮され、かつ酵素を含む試薬を取り扱う 上で不都合のない範囲内であれば特に限定されないが、好ましくは 0. 01〜50mM 、より好ましくは 1〜： LOmMの濃度で用いられる。  [0037] Regarding the concentration of the chelating reagent coexisting with polyamine oxidase in the solution, the stability of the polyamine oxidase is exerted and the concentration of the chelating reagent is not particularly inconvenient in handling the reagent containing the enzyme. Although it is not limited, it is preferably used at a concentration of 0.01 to 50 mM, more preferably 1 to LOmM.
[0038] さらに、前記アルコール化合物と前記キレート試薬を併用することにより、ポリアミン ォキシダーゼを安定ィ匕させることができる。前記アルコールィ匕合物と前記キレート試 薬は、それぞれ単独でも複数組み合わせても用いることができる。  [0038] Furthermore, the polyamine oxidase can be stabilized by using the alcohol compound and the chelating reagent in combination. The alcoholic compound and the chelating reagent can be used alone or in combination.
[0039] このとき、必要により、その他の試薬が共存していてもよい。通常、アルコール化合 物及び Z又はキレート試薬、ポリアミンォキシダーゼ及び他の試薬等が添加された固 体粉末若しくはそれらの溶液をそれぞれ適宜混合することにより共存させることがで きる。  [0039] At this time, if necessary, other reagents may coexist. Usually, the alcohol compound and Z, or a solid powder to which a chelating reagent, polyamine oxidase, and other reagents are added, or a solution thereof, can be allowed to coexist by appropriately mixing them.
[0040] 本発明の安定ィ匕方法に用いられるポリアミンォキシダーゼの濃度は、特に制限され ないが、例えば、溶液の場合、 0. 05〜： LOOO U/mU好ましくは 0. 2〜： LOO U/ml である。この酵素溶液には、 FAD、 NAD、 NADP、 NADH、 NADPH等の補酵素 、ペルォキシダーゼ、 4—ァミノアンチピリン、フエノール、 N ェチル N—スルホプ 口ピル— m—トルイジン (TOOS)、 10— (カルボキシメチルァミノカルボ-ル）— 3, 7 ビス (ジメチルァミノ)フエノチアジンナトリウム塩 (DA— 67)等の過酸化水素検出 試薬、 1 Methoxy PMSや WST— 8 (ホルマザン試薬）等の酸化還元系発色試 薬、カタラーゼ等の酵素、ジァセチルスペルミン等の各種ポリアミン類のいずれか 1つ 以上を含有させることもできる。  [0040] The concentration of polyamine oxidase used in the stability method of the present invention is not particularly limited. For example, in the case of a solution, 0.05 to: LOOO U / mU, preferably 0.2 to: LOO U / ml. This enzyme solution contains coenzymes such as FAD, NAD, NADP, NADH, NADPH, peroxidase, 4-aminoantipyrine, phenol, N-ethyl N-sulfopyl pill-m-toluidine (TOOS), 10- (carboxymethyl) (Aminocarbols) — 3, 7 Hydrogen peroxide detection reagents such as bis (dimethylamino) phenothiazine sodium salt (DA—67), redox coloring such as 1 Methoxy PMS and WST—8 (formazan reagent) One or more of reagents, enzymes such as catalase, and various polyamines such as diacetyl spermine may be contained.
[0041] 一般に酵素は、保存時の pHによりその安定性が大きく影響を受けるため、安定な p H域の種々の緩衝液を同時に用いることが好ましい。本発明の酵素の安定ィ匕におい て用いられる緩衝液の種類及びその濃度、 pHは特に限定されるものではないが、例 えば、 pH6〜10の間で緩衝能を有し、かつ必要十分な緩衝能を保つ濃度に設定さ れていることが望ましい。この様な緩衝液として、例えば、汎用的なトリス緩衝液やリン 酸緩衝液を挙げることもできるし、酢酸、クェン酸、シユウ酸等の有機酸系緩衝液、 M ES、 BES、 HEPES、 TES、ビシン、トリシン等のグッドバッファー、グリシン— NaOH などのアミノ酸系緩衝液、ホウ酸緩衝液、 Bis-Tris propane緩衝液、イミダゾール 緩衝液などを使用することもできる。 [0041] In general, since the stability of an enzyme is greatly affected by the pH during storage, it is preferable to simultaneously use various buffers in the stable pH range. The stability of the enzyme of the present invention The type of buffer used, its concentration, and pH are not particularly limited, but for example, it is set to a concentration having a buffer capacity between pH 6 and 10 and maintaining a necessary and sufficient buffer capacity. It is desirable that Examples of such buffers include general-purpose Tris buffers and phosphate buffers, organic acid buffers such as acetic acid, citrate, and oxalic acid, MES, BES, HEPES, and TES. Good buffer such as bicine and tricine, amino acid buffer such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, imidazole buffer and the like can also be used.
[0042] 酵素を保存する際の緩衝液の濃度については特に限定されないが、好ましくは 5 [0042] The concentration of the buffer solution for storing the enzyme is not particularly limited, but preferably 5
〜500mM、さらに好ましくは 20〜100mMである。本発明のアルコール化合物及び Z又はキレート試薬を緩衝液に添加する場合は、直接添加するか、又は、例えば、 p H6〜10、好ましくは pH6〜8に調整したそれらの水溶液を添カ卩すればよい。アルコ 一ルイ匕合物及び Z又はキレート試薬を添加することにより、 PHが目的とする範囲か ら外れるときは、例えば、酢酸、塩酸、水酸化ナトリウム、水酸ィ匕カリウム、アンモニア 水等の添カ卩により pHが目的の範囲内におさまるように調整するのが好適である。 It is ˜500 mM, more preferably 20 to 100 mM. When the alcohol compound of the present invention and Z or a chelating reagent are added to the buffer, they may be added directly or, for example, an aqueous solution thereof adjusted to pH 6 to 10, preferably pH 6 to 8, may be added. Good. If the pH is outside the target range by adding an alcoholic compound and Z or a chelating reagent, for example, acetic acid, hydrochloric acid, sodium hydroxide, potassium hydroxide, ammonia water, etc. It is preferable to adjust the pH so that it falls within the target range.
[0043] さらに、その他の試薬として、必要により、例えば、塩ィ匕ナトリウム、塩ィ匕カリウム、ァ ジ化ナトリウム等の各種無機塩、デキストラン等の多糖類、ゥシ血清アルブミン (BSA )、グリセロール、アミノ酸、界面活性剤、抗生物質、サルファ剤等の化学療法剤等を 共存させてもよい。これらの試薬は、あら力じめ緩衝液に添カロしておいてもよい。  [0043] Further, as other reagents, if necessary, for example, various inorganic salts such as sodium chloride sodium, potassium salt sodium, sodium azide, polysaccharides such as dextran, urine serum albumin (BSA), glycerol Further, chemotherapeutic agents such as amino acids, surfactants, antibiotics and sulfa drugs may coexist. These reagents may be preliminarily added to the buffer solution.
[0044] 安定性の評価は、実際に用いる酵素の保存条件、輸送条件及び測定条件などに 即した種々の条件下に、ポリアミンォキシダーゼを保存、放置して、経時的にその変 化を測定することにより行なわれるが、一般に、短時間で評価を行うために、通常、加 速試験が用いられる。例えば、一定の高温下にポリアミンォキシダーゼを保温して、 経時的にその変化を測定する方法などが挙げられる。  [0044] The stability was evaluated by storing polyamine oxidase under various conditions in accordance with the storage conditions, transport conditions, and measurement conditions of the enzyme actually used, and measuring changes over time. In general, an acceleration test is generally used to perform evaluation in a short time. For example, a method of keeping polyamine oxidase at a constant high temperature and measuring its change over time can be mentioned.
[0045] [安定ィ匕されたポリアミンォキシダーゼ製剤]  [0045] [Stabilized polyamine oxidase preparation]
この様にして、アルコールィ匕合物及び Z又はキレート試薬を共存させることにより、 ポリアミンォキシダーゼ、特にジァセチルスペルミンォキシダーゼを安定ィ匕することが できる。この安定ィ匕方法を用いて、アルコールィ匕合物及び Z又はキレート試薬と、ポ リアミンォキシダーゼを含有する安定ィ匕されたポリアミンォキシダーゼ製剤を製造す ることがでさる。 In this way, polyamine oxidase, particularly diacetyl spermine oxidase, can be stabilized by the coexistence of an alcohol compound and Z or a chelating reagent. This stable method is used to produce a stabilized polyamine oxidase formulation containing an alcoholic compound and a Z or chelating reagent and a polyamine oxidase. It can be done.
[0046] 例えば、アルコール化合物を 1〜10%、及び Z又はキレート試薬を l〜50mMの 濃度で含有する、 PH6〜： LOの緩衝液を調製し、この緩衝液にポリアミンォキシダー ゼを濃度が 0. 05〜： LOOOUZmlとなるように添加する。次に、 FADなどの補酵素を 0. 01〜： LOmMとなるように添カ卩してもよい。さらに、必要により、例えば、ペルォキシ ダーゼを 10〜： LOOOUZml、TOOSを 0. 05〜5mM、 4ーァミノアンチピリンを 0. 1 〜20mMとなるように加えてもよい。この混合液を撹拌し、ポリアミンォキシダーゼ製 剤を製造することができる。  [0046] For example, a PH6 ~: LO buffer solution containing 1 to 10% of an alcohol compound and Z or a chelating reagent at a concentration of 1 to 50 mM is prepared, and polyamine oxidase is added to this buffer solution. Is added to make LOOOUZml. Next, a coenzyme such as FAD may be added to be 0.01-: LOmM. Further, if necessary, for example, peroxidase may be added at 10 to: LOOOUZml, TOOS at 0.05 to 5 mM, and 4-aminoantipyrine at 0.1 to 20 mM. This mixture can be stirred to produce a polyamine oxidase preparation.
[0047] さらに、必要により、この液状の製剤を凍結乾燥や噴霧乾燥してもよい。このように して得られたポリアミンォキシダーゼ製剤は、従来の方法により製造された製剤に比 ベて著しく安定化されており、本発明の方法により、簡単な方法でかつ安価に、安定 化されたポリアミンォキシダーゼ製剤を製造することができる。  [0047] Further, if necessary, this liquid preparation may be freeze-dried or spray-dried. The polyamine oxidase preparation thus obtained is significantly stabilized as compared with the preparation produced by the conventional method, and is stabilized in a simple manner and at a low cost by the method of the present invention. Polyamine oxidase preparations can be produced.
[0048] [ポリアミンォキシダーゼの精製方法]  [0048] [Method of purifying polyamine oxidase]
また、この安定ィ匕方法を用いて、精製したポリアミンォキシダーゼを製造することが できる。酵素の精製法としては、通常の酵素精製に用いられる方法が使用でき、例え ば、硫安塩析法、有機溶媒沈殿法、イオン交換クロマトグラフ法、ゲル濾過クロマトグ ラフ法、疎水クロマトグラフ法、吸着クロマトグラフ法、電気泳動法等を適宜組み合わ せて行うのが好ましい。例えば、これらの精製工程のいずれか 1工程若しくは複数の 工程において、アルコールィ匕合物及び Z又はキレート試薬を添加、共存させることに よってポリアミンォキシダーゼを安定ィ匕した状態で単離精製することができる。これに より酵素を高い回収率で製造することが可能となる。  In addition, a purified polyamine oxidase can be produced using this stability method. As a method for purifying an enzyme, a method commonly used for enzyme purification can be used. For example, ammonium sulfate salting out method, organic solvent precipitation method, ion exchange chromatography method, gel filtration chromatography method, hydrophobic chromatography method, adsorption It is preferable to carry out an appropriate combination of chromatographic methods, electrophoresis methods and the like. For example, in one or more of these purification steps, the polyamine oxidase is isolated and purified in a stable state by adding and coexisting an alcohol compound and Z or a chelating reagent. Can do. This makes it possible to produce the enzyme with a high recovery rate.
[0049] さらに具体的には、 PCTZJP2006Z310648に記載のジァセチルスペルミンォキ シダーゼは、ダイノーミルによる菌体破砕、硫安沈殿、透析、陰イオン交換カラムクロ マトグラフィー、疎水性カラムクロマトグラフィー、ゲルろ過等を組み合わせて精製す るが、これらの工程の ヽずれか 1工程若しくは複数の工程にぉ ヽてアルコール化合 物及び Z又はキレート試薬を添加、共存させることによりジァセチルスペルミンォキシ ダーゼを安定ィ匕した状態で単離精製することができる。  [0049] More specifically, diacetyl spermine oxidase described in PCTZJP2006Z310648 is used for cell disruption with dynomill, ammonium sulfate precipitation, dialysis, anion exchange column chromatography, hydrophobic column chromatography, gel filtration and the like. Purify in combination, but with one or more of these steps, alcohol compound and Z or chelating reagent are added and coexisted in one step or multiple steps to stabilize diacetyl spermine oxidase. In this state, it can be isolated and purified.
[0050] さらに、この安定ィ匕方法を用いて、 N1—ァセチルスペルミジンを効率よく製造するこ とができる。例えば、 PCTZJP2006Z310648〖こ記載のよう〖こ、ジァセチルスペルミ ンに対してジァセチルスペルミンォキシダーゼを作用させることにより、 N1 -ァセチル スペルミジンを得ることができる力 このとき反応系にアルコールィ匕合物及び Z又は キレート試薬を添加、共存させることによってジァセチルスペルミンォキシダーゼが安 定化されるため、より少量の酵素で反応を行うことが可能となり、産業上有利である。 [0050] Furthermore, N 1 -acetyl spermidine can be efficiently produced using this stable method. You can. For example, as described in PCTZJP2006Z310648, the ability to obtain N 1 -acetyl spermidine by acting diacetyl spermine oxidase on diacetyl spermine. Addition and coexistence of a compound and Z or a chelating reagent stabilizes diacetyl spermine oxidase, which makes it possible to carry out the reaction with a smaller amount of enzyme, which is industrially advantageous.
[0051] 上記の反応は、通常緩衝液中で行うが、必要に応じ、カタラーゼ等の過酸化水素 消去酵素やアジィ匕ナトリウム等の防腐剤を添加してもよい。緩衝液としては、 pH5〜l 0の通常の緩衝液であれば特に限定さないが、例えば、汎用的なトリス緩衝液やリン 酸緩衝液を挙げることもできるし、酢酸、クェン酸、シユウ酸等の有機酸系緩衝液、 M ES、 BES、 HEPES、 TES、ビシン、トリシン等のグッドバッファー、グリシン— NaOH などのアミノ酸系緩衝液、ホウ酸緩衝液、 Bis-Tris propane緩衝液、イミダゾール 緩衝液等を挙げることができる。反応時間はいかなる時間でもよいが、 1時間から 3日 間、好ましくは 3時間から 2日間程度で反応を停止させるのが好ましい。  [0051] The above reaction is usually carried out in a buffer solution, but if necessary, a hydrogen peroxide-eliminating enzyme such as catalase or a preservative such as sodium azide may be added. The buffer solution is not particularly limited as long as it is a normal buffer solution having a pH of 5 to 10, but examples thereof include general-purpose Tris buffer solution and phosphate buffer solution, and include acetic acid, citrate, and oxalic acid. Organic acid buffer such as MES, BES, HEPES, TES, Good buffer such as bicine and tricine, Amino acid buffer such as glycine-NaOH, Boric acid buffer, Bis-Tris propane buffer, Imidazole buffer Etc. The reaction time may be any time, but it is preferable to stop the reaction in 1 hour to 3 days, preferably 3 hours to 2 days.
[0052] [ポリアミンォキシダーゼの基質特異性の改良]  [0052] [Improvement of substrate specificity of polyamine oxidase]
本発明において、「ポリアミンォキシダーゼの基質特異性の改良」とは、ポリアミンォ キシダーゼの「ジァセチルスペルミンに対する反応性」の「その他のポリアミンに対す る反応性」に対する比、すなわち、「ジァセチルスペルミンに対する反応性 Zその他 のポリアミンに対する反応性」を向上させることを!、う。ここで 、う「その他のポリアミン」 とは、例えば、 N1—ァセチルスペルミン、 N1—ァセチルスペルミジン、 N8—ァセチル スペルミジン、スペルミン、スペルミジンなどを指す。 In the present invention, “improving the substrate specificity of polyamine oxidase” means the ratio of the reactivity of polyamine oxidase to “diacetyl spermine” to “reactivity to other polyamines”, that is, “diacetyl”. Improve reactivity to spermine Z and reactivity to other polyamines! Here, “other polyamines” refer to, for example, N 1 -acetyl spermine, N 1 -acetyl spermidine, N 8 -acetyl spermidine, spermine, spermidine and the like.
[0053] 基質特異性の評価は、酵素活性の測定に用いる反応液に添加する基質の種類を 変えて酵素活性を測定し、ジァセチルスペルミンに対する反応性とその他のポリアミ ンに対する反応性を比較することにより行う。ここでいう「その他のポリアミン」とは、例 えば、 N1—ァセチルスペルミン、 N1—ァセチルスペルミジン、 N8—ァセチルスペルミ ジン、スペルミン、スペルミジンなど測定する方法などが挙げられる。簡易的な評価を 行うために、ジァセチルポリアミンに対する反応性と 1種類のその他のポリアミン (例え ば、 N1—ァセチルスペルミジン）に対する反応性を比較して評価することもできる。 [0053] Substrate specificity was evaluated by measuring the enzyme activity by changing the type of substrate added to the reaction solution used to measure the enzyme activity, and comparing the reactivity to diacetylspermine with the reactivity to other polyamines. To do. The "other polyamine", if example embodiment, N 1 - § cetyl spermine, N 1 - § cetyl spermidine, N 8 - Asechirusuperumi Jin, spermine, and a method of measuring the like spermidine and the like. In order to make a simple evaluation, it is possible to compare the reactivity to diacetyl polyamine and the reactivity to one other polyamine (eg N 1 -acetyl spermidine).
[0054] 本発明にお 、て、ポリアミンォキシダーゼの基質特異性を改良するために使用する 界面活性剤とは、親水基と親油基 (疎水基)とで構成されており、水に溶けて水の表 面張力を低下させる活性を示す物質であり、そのような化合物であれば如何なるィ匕 合物でも用いることができる。一般的に、親水基の種類によって界面活性剤は、了二 オン界面活性剤 (陰イオン界面活性剤)、カチオン界面活性剤（陽イオン界面活性剤[0054] In the present invention, used to improve the substrate specificity of polyamine oxidase. A surfactant is composed of a hydrophilic group and a lipophilic group (hydrophobic group), and is a substance that exhibits an activity of dissolving in water and lowering the surface tension of water. It can also be used in combination. In general, depending on the type of hydrophilic group, surfactants are Ryoon surfactants (anionic surfactants), cationic surfactants (cationic surfactants)
)、非イオン性界面活性剤 (ノ-オン界面活性剤)、両性界面活性剤の 4種に分類さ れている。 ), Nonionic surfactants (non-ionic surfactants), and amphoteric surfactants.
[0055] ァ-オン界面活性剤（陰イオン界面活性剤）とは、水中で電離して有機陰イオンと なるものであり、例えば、界面活性剤の分子中の親油基を Rとすると、 RCOONa、 R SO Na、 RSO Naなどがある。  [0055] A-on surfactant (anionic surfactant) is an ion that is ionized in water to become an organic anion. For example, when the lipophilic group in the molecule of the surfactant is R, RCOONa, R SO Na, RSO Na, etc.
3 4  3 4
[0056] カチオン界面活性剤（陽イオン界面活性剤）とは、水中で電離して有機陽イオンと なるものであり、例えば界面活性剤の分子中の親油基を R、ハロゲンを Xとすると、 R -NH X、 [Κ 4Ν]Χ(第 4級アンモ-ゥム塩）、 (C Η— N)RX (アルキルピリ[0056] A cationic surfactant (cationic surfactant) is ionized in water to become an organic cation. For example, when the lipophilic group in the surfactant molecule is R and the halogen is X, , R -NH X, [Κ 4 Ν] Χ (quaternary ammonia salt), (C Η— N) RX (alkyl pyrimido
3 5 5 3 5 5
ジニゥム塩)などがある。  Zinium salt).
[0057] 非イオン性界面活性剤 (ノ-オン界面活性剤）とは、親水基が非イオン性のもので あり、親水基としてよく用いられるものとして、例えば、ォキシエチレン基（一 CH CH  [0057] Nonionic surfactants (non-ionic surfactants) are those whose hydrophilic groups are nonionic, and those often used as hydrophilic groups include, for example, oxyethylene groups (one CH CH
2 2 o— )、ソルビタン、ショ糖のエステル、グリセリン誘導体のモノグリセリドなどがあり、例 えば、界面活性剤の分子中の親油基を Rとすると、 RO (CH CH O) H、 RCOO (  2 2 o—), sorbitan, sucrose ester, glycerin derivative monoglyceride, etc. For example, if the lipophilic group in the surfactant molecule is R, RO (CH 2 CH 2 O) H, RCOO (
2 2 η  2 2 η
CH CH O) Hなどがある。  CH CH O) H and the like.
2 2 η  2 2 η
[0058] 両性界面活性剤とは、分子内にァニオン基とカチオン基の両方をあわせもつている ものである。  [0058] Amphoteric surfactants are those having both an anion group and a cationic group in the molecule.
[0059] 各種界面活性剤の中でも、特に非イオン性界面活性剤と両性界面活性剤はポリア ミンォキシダーゼの基質特異性を向上させるためにより好ましく用いられる。さらに具 体的には、非イオン性界面活性剤として、例えば、 Triton X— 100 [Polyoxyethy lene (10J Octylphenyl Ether]、 Triton X— 405 [p— tertiary Octylphenox y— Polyethoxy Ethanols]、 Tween 20 [Polyoxyethylene (20) Sorbitan Mo nolaurate]、 Tween 40 [Polyoxyethylene (20) Sorbitan Monopalmitate]、 T ween 80 [Polyoxyethylene (20) Sorbitan Monooleate]、 Tween 85 [Polyo xyethylene (20) Sorbitan Trioleate]、 Brij 35 [Polyoxyethylene (23) Lauryl Ether]、 Brij 58 [Polyoxyethylene (20) Cetyl Ether]、 Brij 78 [Polyoxyeth ylene (20) Stearyl Ether]、エマノレゲン 913 (Nonyl Phenol Ethoxylate) などが挙げられ、両性界面活性剤として、例えば、 3 ' - [ (3 - Cholamidopropyl) di methyl— ammonioj— 2— hydroxypropane— sulfonic acid (CHAPSO)など が挙げられる。 [0059] Among various surfactants, nonionic surfactants and amphoteric surfactants are particularly preferably used for improving the substrate specificity of polyamineoxidase. More specifically, as nonionic surfactants, for example, Triton X-100 [Polyoxyethy lene (10J Octylphenyl Ether), Triton X-405 [p-tertiary Octylphenoxy-Polyethoxy Ethanols], Tween 20 [Polyoxyethylene ( 20) Sorbitan Monolaurate], Tween 40 [Polyoxyethylene (20) Sorbitan Monopalmitate], T ween 80 [Polyoxyethylene (20) Sorbitan Monooleate], Tween 85 [Polyo xyethylene (20) Sorbitan Trioleate], Brij 35 [Polyoxyethylene (23) Lauryl Ether], Brij 58 [Polyoxyethylene (20) Cetyl Ether], Brij 78 [Polyoxyethylene (20) Stearyl Ether], Emanoregen 913 (Nonyl Phenol Ethoxylate) and the like, and amphoteric surfactants such as 3 ′-[ (3-Cholamidopropyl) dimethyl-ammonioj- 2-hydroxypropane-sulfonic acid (CHAPSO).
[0060] 上記界面活性剤は、それぞれ単独でも複数組み合わせても用いることができる。  [0060] The above surfactants can be used alone or in combination.
[0061] ポリアミンォキシダーゼと前記界面活性剤を同一の試薬内にあら力じめ混合し、共 存する形で用いることができる。若しくは、ポリアミンォキシダーゼによる反応が進行 するときに前記界面活性剤が存在していればよいので、例えば、ポリアミンォキシダ ーゼを含有する試薬 Aと前記界面活性剤を含有する試薬 Bをそれぞれ別途調製し、 これらを適宜混合して、前記界面活性剤存在下でポリアミンォキシダーゼ反応を進 行させることもできる。ポリアミンォキシダーゼと共存するときの前記界面活性剤の濃 度については特に限定されず、各化合物について適切な基質特異性改良効果が認 められる濃度で添加することができる。好ましくは 0. 001〜20%、より好ましくは 0. 0 1〜5%の濃度で用いられる。  [0061] The polyamine oxidase and the surfactant can be mixed together in the same reagent and used together. Alternatively, since the surfactant only needs to be present when the reaction with polyamine oxidase proceeds, for example, reagent A containing polyamine oxidase and reagent B containing the surfactant are separately provided. It is also possible to prepare them and mix them as appropriate so that the polyamine oxidase reaction can proceed in the presence of the surfactant. The concentration of the surfactant when coexisting with polyamine oxidase is not particularly limited, and each compound can be added at a concentration at which an appropriate substrate specificity improving effect is recognized. It is preferably used at a concentration of 0.001 to 20%, more preferably 0.0 to 1 to 5%.
[0062] 本発明の界面活性剤による基質特異性の改善にぉ ヽて用いられる緩衝液の種類 及びその濃度、 pHは特に限定されるものではないが、例えば、 pH5〜： L0の間で緩 衝能を有し、かつ必要十分な緩衝能を保つ濃度に設定されていることが望ましい。こ の様な緩衝液として、例えば、汎用的なトリス緩衝液やリン酸緩衝液を挙げることもで きるし、酢酸、クェン酸、シユウ酸等の有機酸系緩衝液、 MES、 BES、 HEPES、 TE S、ビシン、トリシン等のグッドバッファー、グリシン一 NaOHなどのアミノ酸系緩衝液、 ホウ酸緩衝液、 Bis-Tris propane緩衝液、イミダゾール緩衝液などを使用すること もできる。緩衝液の濃度については、例えば、好ましくは 5〜500mM、さらに好ましく は 20〜： LOOmMである。  [0062] The type, concentration, and pH of the buffer used for improving the substrate specificity by the surfactant of the present invention are not particularly limited. For example, the buffer is relaxed between pH 5 and L0. It is desirable that the concentration is set so as to have an impulsive capacity and to maintain a necessary and sufficient buffer capacity. Examples of such buffers include general-purpose Tris buffers and phosphate buffers, organic acid buffers such as acetic acid, citrate, and oxalic acid, MES, BES, HEPES, Good buffers such as TES, bicine, and tricine, amino acid buffers such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, and imidazole buffer can also be used. The concentration of the buffer is, for example, preferably 5 to 500 mM, more preferably 20 to: LOOmM.
[0063] また、ポリアミンォキシダーゼの基質特異性を改良するため、 pH7. 5未満にて酵素 反応を行う方法も挙げられる。より好ましい pH条件として、例えば、 6. 0以上 7. 5未 満が挙げられる。本発明においては、同様にポリアミンォキシダーゼの基質特異性を 改良する効果が得られる上記界面活性剤を共存させることもできる。 [0064] ポリアミンォキシダーゼは、 pH7. 5未満、より好ましくは pH6. 0以上 7. 5未満の試 薬内にあら力じめ含有される形で用いることができる。又は、ポリアミンォキシダーゼ による反応が進行するときに PH7. 5未満、より好ましくは pH6. 0以上 7. 5未満にな つていればよいので、例えば、 2種以上の試薬 (いずれカゝがポリアミンォキシダーゼを 含有する）をそれぞれ別途調製し、これらを適宜混合して、 pH7. 5未満、より好ましく は PH6. 0以上 7. 5未満の条件となるようにしてポリアミンォキシダーゼ反応を進行さ せることちでさる。 [0063] In addition, in order to improve the substrate specificity of polyamine oxidase, a method of performing an enzyme reaction at a pH of less than 7.5 can also be mentioned. More preferable pH conditions include, for example, 6.0 or more and less than 7.5. In the present invention, the above-mentioned surfactant that can improve the substrate specificity of polyamine oxidase can also be present. [0064] The polyamine oxidase can be used in such a form that it is preferentially contained in a reagent having a pH of less than 7.5, more preferably pH 6.0 or more and less than 7.5. Alternatively, when the reaction with polyamine oxidase proceeds, the pH should be less than 7.5, more preferably pH 6.0 or more and less than 7.5. containing Okishidaze) was prepared separately respectively, a mixture of them, pH 7. less than 5, more preferably P H6. 0 or 7. as a condition of less than 5 is advanced polyamine O Kishida over peptidase reaction It ’s easy to do.
[0065] 本発明の上記の界面活性剤存在下及び Z又は PH7. 5未満にて酵素反応を行うこ とによる基質特異性の改良に用いられるポリアミンォキシダーゼの濃度は、特に制限 されないが、例えば、溶液の場合、 0. 05〜： LOOO U/mU好ましくは 0. 2〜： LOO U Zmlである。この酵素溶液には、 FAD、 NAD、 NADP、 NADH、 NADPH等の補 酵素、ペルォキシダーゼ、 4—ァミノアンチピリン、フエノール、 TOOS、 DA— 67等の 過酸化水素検出試薬、 1 -Methoxy PMSや WST— 8 (ホルマザン試薬）等の酸 化還元系発色試薬、カタラーゼ等の酵素、ジァセチルスペルミン等の各種ポリアミン 類の!/、ずれか 1つ以上が存在して!/、てもよ!/、。  [0065] The concentration of polyamine oxidase used for improving the substrate specificity by carrying out the enzymatic reaction in the presence of the above-mentioned surfactant of the present invention and less than Z or PH 7.5 is not particularly limited. In the case of a solution, 0.05-: LOOO U / mU, preferably 0.2-: LOO U Zml. This enzyme solution includes coenzymes such as FAD, NAD, NADP, NADH and NADPH, peroxidases, hydrogen peroxide detection reagents such as 4-aminoantipyrine, phenol, TOOS and DA-67, 1-Methoxy PMS and WST— 8 Oxidation-reduction coloring reagents such as (formazan reagent), enzymes such as catalase, and various polyamines such as diacetyl spermine! /, Or more than one! /, May! / .
[0066] さらに、その他の試薬として、既に記載したような安定化剤であるアルコールィ匕合物 及び Z又はキレート試薬やその他の試薬を共存させて用いることもできる。また、必 要により、例えば、塩ィ匕ナトリウム、塩ィ匕カリウム、アジィ匕ナトリウム等の各種無機塩、 デキストラン等の多糖類、ゥシ血清アルブミン (BSA)、グリセロール、アミノ酸、抗生 物質、サルファ剤等の化学療法剤等を共存させてもよい。  [0066] Further, as other reagents, alcohol compounds and Z, chelating reagents and other reagents which are stabilizers as described above can be used together. If necessary, for example, various inorganic salts such as sodium salt, potassium salt, sodium salt, polysaccharides such as dextran, ushi serum albumin (BSA), glycerol, amino acids, antibiotics, sulfa drugs, etc. These chemotherapeutic agents may coexist.
[0067] さらに、上記の界面活性剤存在下及び Z又は PH7. 5未満にて酵素反応を行うこと による基質特異性の改良方法を用いて、 N1—ァセチルスペルミジンを効率よく製造 することができる。例えば、 PCTZJP2006Z310648〖こ記載のよう〖こ、ジァセチノレス ペルミンに対してジァセチルスペルミンォキシダーゼを作用させることにより、 N1—ァ セチルスペルミジンを得ることができるが、このとき反応系に基質特異性が向上して いるため、生成物である N1—ァセチルスペルミジンがさらなるプトレツシンへの変換を 受けにくくなり、回収率が向上するため、産業上有利である。 [0067] Further, N 1 -acetyl spermidine can be efficiently produced by using the method for improving substrate specificity by carrying out an enzymatic reaction in the presence of the above-described surfactant and under Z or PH 7.5. it can. For example, as described in PCTZJP2006Z310648, N 1 -Acetylspermidine can be obtained by acting diacetylspermine oxidase on diacetylenoperm, but at this time the substrate has substrate specificity in the reaction system. As a result, the product N 1 -acetyl spermidine is less susceptible to further conversion to putrescine and the recovery is improved, which is industrially advantageous.
[0068] [ジァセチルポリアミンの測定方法] さらに本発明は、ポリアミンォキシダーゼを含有する試薬を試料に作用させて、ジァ セチルポリアミンを測定する方法も含む。測定検体は特に限定されないが、例えば、 尿、血清、食品やその抽出物等が挙げられる。測定検体中に含まれるジァセチルポ リアミンの濃度についても特に限定されないが、例えば、 ImM以下が挙げられるし、 検体は適宜希釈することができる。用いるポリアミンォキシダーゼの量は、特に制限さ れないが、例えば、終濃度が 0. 05-1000 U/mU好ましくは 0. 2-100 U/ml となるように添加すればよい。作用させる pHは特に限定されるものではないが、例え ば、 pH5〜10、好ましくは pH6〜9、特に好ましくは pH6. 0以上 7. 5未満である。 p Hの調整法は、特に限定されず、例えば、汎用的なトリス緩衝液やリン酸緩衝液を挙 げることもできるし、酢酸、クェン酸、シユウ酸等の有機酸系緩衝液、 MES、 BES、 H EPES、 TES、ビシン、トリシン等のグッドバッファー、グリシン一 NaOHなどのアミノ酸 系緩衝液、ホウ酸緩衝液、 Bis-Tris propane緩衝液、イミダゾール緩衝液などを 使用することができる。この際、ポリアミンォキシダーゼの安定化剤であるアルコール 化合物ゃキレート試薬、基質特異性改良剤である界面活性剤が適宜存在して!/ヽても よい。作用時間は、例えば、 30秒〜 120分間、好ましくは 1〜30分間である。作用温 度は、例えば、 20〜45°Cであり、通常の酵素反応に用いられる温度を適宜選択する ことができる。 [0068] [Method for measuring diacetylpolyamine] Furthermore, the present invention includes a method for measuring diacetylpolyamine by allowing a reagent containing polyamine oxidase to act on a sample. The sample to be measured is not particularly limited, and examples thereof include urine, serum, food and extracts thereof. The concentration of diacetylpolyamine contained in the measurement sample is not particularly limited, and examples thereof include ImM or lower, and the sample can be diluted as appropriate. The amount of polyamine oxidase to be used is not particularly limited, and for example, it may be added so that the final concentration is 0.05-1000 U / mU, preferably 0.2-100 U / ml. The pH to be applied is not particularly limited. For example, the pH is 5 to 10, preferably 6 to 9, particularly preferably 6.0 to less than 7.5. The pH adjustment method is not particularly limited. For example, a general-purpose Tris buffer or phosphate buffer can be used, an organic acid buffer such as acetic acid, citrate, or oxalic acid, MES, or the like. Good buffers such as BES, HEPES, TES, bicine, and tricine, amino acid buffers such as glycine-NaOH, borate buffer, Bis-Tris propane buffer, and imidazole buffer can be used. In this case, an alcohol compound, which is a stabilizer of polyamine oxidase, a chelating reagent, and a surfactant, which is a substrate specificity improver, may be present as appropriate. The action time is, for example, 30 seconds to 120 minutes, preferably 1 to 30 minutes. The working temperature is, for example, 20 to 45 ° C., and a temperature used for a normal enzyme reaction can be appropriately selected.
[0069] 試料に添加するポリアミンォキシダーゼ溶液には、トリス、リン酸、酢酸、クェン酸、 シユウ酸、 MES、 BES、 HEPES、 TES、ビシン、トリシン、グリシン、ホウ酸、 Bis— Tr is propane,イミダゾール等の緩衝剤、 FAD等の補酵素、ポリアミンォキシダーゼ の安定化剤であるアルコールィ匕合物ゃキレート試薬、ポリアミンォキシダーゼの基質 特異性改良剤である界面活性剤が添加されていてもよい。また、必要により、塩ィ匕ナ トリウム、塩ィ匕カリウム、アジィ匕ナトリウム等の各種無機塩、デキストラン等の多糖類、 B SA、グリセロール、アミノ酸等を共存させてもよい。この液状の製剤を凍結乾燥や噴 霧乾燥してもよ ヽ。  [0069] The polyamine oxidase solution added to the sample includes Tris, phosphate, acetic acid, citrate, oxalic acid, MES, BES, HEPES, TES, bicine, tricine, glycine, boric acid, Bis—Tris propane, Even if a buffer such as imidazole, a coenzyme such as FAD, an alcohol compound chelating reagent that is a stabilizer of polyamine oxidase, or a surfactant that is a substrate specificity improver of polyamine oxidase is added. Good. If necessary, various inorganic salts such as sodium chloride, potassium salt and sodium salt, polysaccharides such as dextran, BSA, glycerol, amino acids and the like may coexist. This liquid preparation may be freeze-dried or spray-dried.
[0070] ポリアミンォキシダーゼの反応による生成物は、いかなる方法により測定してもよい 。例えば、生成する過酸化水素は、ペルォキシダーゼ及び適当な発色試薬、発光試 薬を用いる酵素的測定法、若しくは酵素電極を用いる電気的方法等で測定すること ができる。また、生成するアルデヒドをアルデヒドォキシダーゼやアルデヒドデヒドロゲ ナーゼと適当な発色試薬を組み合わせて測定することもできる。例えば、酵素を用い る反応生成物の検出試薬には、トリス、リン酸、酢酸、クェン酸、シユウ酸、 MES、 BE S、 HEPES、 TES、ビシン、トリシン、グリシン、ホウ酸、 Bis— Tris propane,イミダ ゾール等の緩衝剤、 FAD、 NAD、 NADP、 NADH、 NADPH等の補酵素、ペルォ キシダーゼ、 4ーァミノアンチピリン、フエノール、 TOOS、 DA— 67等の過酸化水素 検出試薬、 1 -Methoxy PMSや WST— 8 (ホルマザン試薬）等の酸化還元系発 色試薬、カタラーゼ等の酵素のいずれか 1つ以上を含有させることができる。これに、 ポリアミンォキシダーゼの安定化剤であるアルコールィ匕合物ゃキレート試薬、基質特 異性改良剤である界面活性剤を適宜添加してもよい。また、必要により、塩化ナトリウ ム、塩ィ匕カリウム、アジィ匕ナトリウム等の各種無機塩、デキストラン等の多糖類、 BSA 、グリセロール、アミノ酸等を共存させてもよい。この液状の製剤を凍結乾燥や噴霧乾 燥してちょい。 [0070] The product of the polyamine oxidase reaction may be measured by any method. For example, the generated hydrogen peroxide should be measured by an enzymatic measurement method using peroxidase and an appropriate coloring reagent, a luminescent reagent, or an electrical method using an enzyme electrode. Can do. In addition, the aldehyde produced can be measured by combining aldehyde oxidase or aldehyde dehydrogenase with an appropriate color reagent. For example, detection reagents for reaction products using enzymes include tris, phosphoric acid, acetic acid, citrate, oxalic acid, MES, BE S, HEPES, TES, bicine, tricine, glycine, boric acid, Bis—Tris propane Buffers such as imidazole, coenzymes such as FAD, NAD, NADP, NADH, NADPH, hydrogen peroxide detection reagents such as peroxidase, 4-aminoantipyrine, phenol, TOOS, DA-67, 1-Methoxy PMS One or more of redox coloring reagents such as WST-8 (formazan reagent) and enzymes such as catalase can be contained. To this, an alcohol compound that is a stabilizer of polyamine oxidase, a chelating reagent, and a surfactant that is a substrate property improving agent may be appropriately added. Further, if necessary, various inorganic salts such as sodium chloride, sodium chloride potassium and azido sodium, polysaccharides such as dextran, BSA, glycerol, amino acids and the like may coexist. Freeze or spray dry this liquid formulation.
[0071] ポリアミンォキシダーゼ反応の生成物の測定を行う際、ポリアミンォキシダーゼ反応 とその生成物の検出を同時に行うことも可能である。前述の反応生成物の検出試薬 に、ポリアミンォキシダーゼを 0. 05〜： L000 U/mU好ましくは 0. 2〜： LOO U/mlと なるように添加することが好ま 、。この液状の製剤を凍結乾燥や噴霧乾燥してもよ い。  [0071] When the product of the polyamine oxidase reaction is measured, the polyamine oxidase reaction and its product can be detected simultaneously. It is preferable to add polyamine oxidase to the above-mentioned detection reagent for the reaction product in a range of 0.05-: L000 U / mU, preferably 0.2-: LOO U / ml. This liquid preparation may be freeze-dried or spray-dried.
[0072] ジァセチルポリアミンの測定に用いる検体の量、上記の各試薬の添加量、比率に つ!ヽては特に限定されな 、。  [0072] The amount of the specimen used for the measurement of diacetyl polyamine, the addition amount of each reagent, and the ratio thereof are not particularly limited.
[0073] [ジァセチルポリアミンの測定試薬]  [0073] Diacetyl polyamine measurement reagent
さらに本発明では、上記測定方法に使用されるジァセチルポリアミンの測定試薬を 提供する。ジァセチルポリアミンの測定試薬は上述の各試薬を異なる容器に含むも のとして調製すればよぐ例えば、液状品、又は液状品の凍結物若しくは凍結乾燥品 として提供できる。あるいは、アルコールィ匕合物及び Z又はキレート試薬、及び Z又 は界面活性剤とポリアミンォキシダーゼを含有する測定試薬を調製することも可能で ある。  Furthermore, the present invention provides a reagent for measuring diacetyl polyamine used in the above measurement method. The measurement reagent for diacetylpolyamine may be prepared as a liquid product, or a frozen product or a freeze-dried product of the liquid product. Alternatively, it is also possible to prepare a measuring reagent containing an alcohol compound and Z or chelating reagent, and Z or a surfactant and polyamine oxidase.
[0074] [モノァセチルポリアミンを製造する方法] さらに、これまで記載したポリアミンォキシダーゼの安定ィ匕方法、及び Z又はポリア ミンォキシダーゼの基質特異性の改良方法を用いて、ジァセチルポリアミンへポリアミ ンォキシダーゼを作用させてモノァセチルポリアミンを製造することが可能である。 [0074] [Method for Producing Monoacetyl Polyamine] Furthermore, by using the method for stabilizing polyamine oxidase described above and the method for improving the substrate specificity of Z or polyamine oxidase, polyamine oxidase is allowed to act on diacetyl polyamine to produce monoacetyl polyamine. Is possible.
[0075] 以下、実験例及び実施例により、本発明をさらに具体的に説明する。本発明の技 術的範囲は、これらの例により何ら限定されない。  [0075] Hereinafter, the present invention will be described more specifically with reference to experimental examples and examples. The technical scope of the present invention is not limited by these examples.
実施例 1  Example 1
[0076] (ポリアミンォキシダーゼの活性測定法）  [0076] (Method of measuring polyamine oxidase activity)
0. 2M Tris— HC1緩衝液（pH7. 5) 0. 9ml、 7. OmM ポリアミン（例えば、ジァ セチルスペルミン）溶液 0. lml、 15mM TOOS溶液 0. 04ml, 150UZmlペル ォキシダーゼ（キッコーマン社製）溶液 0. 04ml, 1. 76% (w/v) 4—ァミノアンチ ピリン溶液 0. 02mlの混合液を 37°Cで 5分間インキュベートした。酵素は希釈液 A[ ImMの EDTA及び 0. 2% (w/v)のゥシ血清アルブミン（BSA、シグマ社製）を含 有する 50mMリン酸カリウム緩衝液 (pH7. 0) ]若しくは希釈液 B[ ImMの EDTA及 び 5% (v/v)のエタノールを含有する 50mMリン酸カリウム緩衝液 (pH7. 0) ]にて 適宜希釈した。酵素サンプル 0. 05mlを反応液に添加、混合した。全容 1. 15mlを 37°Cで 3分間反応させ、反応開始から 3分間の、反応溶液の 555nmにおける吸光 度の変化を、分光光度計を用いて測定した。ジァセチルスペルミンォキシダーゼの 酵素 1Uは、上記測定条件下において、ジァセチルスペルミンを基質とするときに 1分 間あたり 1 molの過酸ィ匕水素を生成する酵素量とした。なお、本条件におけるミリモ ル分子吸光係数を 1. 57cm2Z molとした。なお、本測定方法は一例を示したもの であり、活性測定の試薬の種類や濃度、 pHについては何ら制限するものではない。 例えば、 Tris— HC1緩衝液の代わりにリン酸緩衝液等を用いることもできる。 0.2 M Tris—HC1 buffer (pH 7.5) 0.9 ml, 7. OmM polyamine (eg diacetylspermine) solution 0.1 ml, 15 mM TOOS solution 0.04 ml, 150 UZml peroxidase (Kikkoman) solution 0.04 ml, 1. 76% (w / v) 4-Aminoantipyrine solution 0.02 ml of the mixture was incubated at 37 ° C for 5 minutes. Enzyme is diluted in A (50 mM potassium phosphate buffer (pH 7.0) containing ImM EDTA and 0.2% (w / v) urine serum albumin (BSA, Sigma)) or Diluent B [Diluted appropriately with 50 mM potassium phosphate buffer (pH 7.0) containing ImM EDTA and 5% (v / v) ethanol]. 0.05 ml of enzyme sample was added to the reaction solution and mixed. A total volume of 1.15 ml was reacted at 37 ° C for 3 minutes, and the change in absorbance at 555 nm of the reaction solution for 3 minutes from the start of the reaction was measured using a spectrophotometer. The enzyme 1U of diacetyl spermine oxidase was defined as the amount of enzyme that produces 1 mol of hydrogen peroxide per minute when diacetyl spermine is used as a substrate under the above measurement conditions. The millimolar molecular extinction coefficient under these conditions was 1.57 cm 2 Z mol. This measurement method is just an example, and there is no limitation on the type, concentration, or pH of the reagent for activity measurement. For example, a phosphate buffer or the like can be used in place of the Tris-HC1 buffer.
[0077] (ポリアミンォキシダーゼの安定性試験）  [0077] (Stability test of polyamine oxidase)
ポリアミンォキシダーゼの安定性試験は、各種試薬を添加した 50mMリン酸力リウ ム緩衝液 (PH7. 0)で酵素サンプルを希釈し、 30°C保存による加速試験後の残存活 性を比較して行った。加速試験を行った後の該酵素溶液の残存活性は、該酵素溶 液調製時における吸光度変化量を 100%としたときの相対量（％)として表した。  The stability test of polyamine oxidase was performed by diluting enzyme samples with 50 mM phosphate buffer (PH 7.0) supplemented with various reagents, and comparing the residual activity after accelerated tests by storage at 30 ° C. went. The residual activity of the enzyme solution after the acceleration test was expressed as a relative amount (%) when the amount of change in absorbance at the time of preparing the enzyme solution was taken as 100%.
[0078] (各種アルコール化合物の安定化効果の確認） 5%若しくは 10%の各種アルコールィ匕合物を含有する 50mMのリン酸緩衝液 (pH 7. 0)をそれぞれ調製した。これらの各種試薬を含有する緩衝液を用いて、 PCT/J P2006/310648に記載のデノ リオミセス'ノヽンセニイ Tsukuba42 (FERM BP 10603)由来ジァセチルスペルミンォキシダーゼ溶液 0. lUZmlをそれぞれ調製 した。なお、比較例として、アルコールィ匕合物を含有しない 50mMのリン酸緩衝液 (p H7. 0)で、上記酵素溶液 0. lUZmlを調製した。各酵素溶液について 30°C、 6時 間の加速試験を行い、その結果を表 1に示した。このように各種アルコール化合物に ついて安定ィ匕効果が認められた。特開平 5— 211869〖こ記載のよう〖こ、エタノール等 の有機溶媒の添カ卩は酵素を失活させやすいと考えるのが一般的であるが、今回、ェ タノール、メタノール、 1 プロパノール、 2—プロパノールのような有機溶媒も本酵素 の安定化剤として利用可能であることが示された。 [0078] (Confirmation of stabilizing effect of various alcohol compounds) A 50 mM phosphate buffer solution (pH 7.0) containing 5% or 10% of various alcoholic compounds was prepared. Using a buffer containing these various reagents, 0.1 μUZml of a diacetyl spermine oxidase solution derived from Denoriomyces ′ Nonsenii Tsukuba42 (FERM BP 10603) described in PCT / JP 2006/310648 was prepared. As a comparative example, the enzyme solution (0.1 UZml) was prepared with a 50 mM phosphate buffer (pH 7.0) containing no alcoholic compound. Each enzyme solution was subjected to an acceleration test at 30 ° C for 6 hours, and the results are shown in Table 1. Thus, a stable effect was observed for various alcohol compounds. As described in JP-A-5-211869, it is generally considered that adding an organic solvent such as ethanol is likely to inactivate the enzyme, but this time, ethanol, methanol, 1 propanol, 2 —It has been shown that organic solvents such as propanol can also be used as stabilizers for this enzyme.
[表 1] [table 1]
添加試薬 残存活性 (％) Additive reagent Residual activity (%)
メタノール 5% (ν/ν) 75  Methanol 5% (ν / ν) 75
メタノール 10% (ν/ν) 74  Methanol 10% (ν / ν) 74
エタノール 5% (ν/ν) 78  Ethanol 5% (ν / ν) 78
エタノール 10% (ν/ν) 92  Ethanol 10% (ν / ν) 92
エチレングリコール 5% (ν/ν) 69  Ethylene glycol 5% (ν / ν) 69
エチレングリコール 10% (ν/ν) 83  Ethylene glycol 10% (ν / ν) 83
1 -プロパノール 5% (ν/ν) 83  1-propanol 5% (ν / ν) 83
1 -プロパノール 10% (ν/ν) 69  1-propanol 10% (ν / ν) 69
2 -プロパノール 5% (ν/ν) 74  2-propanol 5% (ν / ν) 74
2-プロパノール 10% (ν/ν) 88  2-propanol 10% (ν / ν) 88
トレハロース 5% (w/v) 66  Trehalose 5% (w / v) 66
トレハロース 10% (w/v) 71  Trehalose 10% (w / v) 71
キシリトール 5% (w/v) 58  Xylitol 5% (w / v) 58
キシリ I ^一ル 10% (w/v) 62  Kisiri I ^ 1l 10% (w / v) 62
無添加 51 実施例 2 No additive 51 Example 2
[0080] (各種キレート試薬の安定化効果の確認）  [0080] (Confirmation of stabilizing effect of various chelating reagents)
ImM若しくは 5mMの各種キレート試薬を含有する 50mMのリン酸緩衝液（pH7. 0)をそれぞれ調製した。これらの各種試薬を含有する緩衝液を用いて、 PCT/JP2 006/310648に記載のデノ リオミセス'ノヽンセニイ Tsukuba42 (FERM BP— 1 0603)由来ジァセチルスペルミンォキシダーゼ溶液、キャンディダ'グラブラタ Nod a 162 (FERM BP— 10602)由来ジァセチルスペルミンォキシダーゼ溶液、及び「 FEBS LettersJ , (英国）， 2000年， 476卷， p. 150— 154に記載のキャンディダ' ボイディニイ NBRC 10574由来ポリアミンォキシダーゼ溶液を 0. lUZmlとして それぞれ調製した。  A 50 mM phosphate buffer solution (pH 7.0) containing ImM or 5 mM chelating reagents was prepared. Using a buffer containing these various reagents, a diacetyl spermine oxidase solution derived from Denoriomyces' Nonseneni Tsukuba42 (FERM BP-1 0603) described in PCT / JP2 006/310648, Candida glabrata Nod a 162 (FERM BP—10602) -derived diacetyl spermine oxidase solution and Candida boidinii NBRC 10574-derived polyamine oxidase described in “FEBS Letters J, (UK), 2000, 476 卷, p. 150-154” Solutions were prepared as 0.1 LUZml respectively.
[0081] なお、比較例として、キレート試薬を含有しな!、50mMのリン酸緩衝液 (pH7. 0)で 、上記各種酵素溶液 0. lUZmlを調製した。各酵素溶液について 30°C、 12時間の 加速試験を行い、その結果を表 2に示した。このように各種キレート試薬について、 3 種すベての酵素に対する安定ィヒ効果が認められた。なお、 EDTAの添加濃度が lm M及び 5mM添加のときは、安定ィ匕効果はほぼ同等であった。  As a comparative example, 0.1 UZml of the various enzyme solutions described above was prepared with a 50 mM phosphate buffer (pH 7.0) without containing a chelating reagent. Each enzyme solution was subjected to an accelerated test at 30 ° C. for 12 hours, and the results are shown in Table 2. In this way, various chelating reagents were found to have a stabilizing effect on all three enzymes. When the EDTA concentration was lmM or 5mM, the stability effect was almost the same.
[0082] [表 2] [0082] [Table 2]
残存活性 (％) Residual activity (%)
濃度  Concentration
添加試薬 (m ) デバリオミセス キャンディダ キャンディダ  Additive Reagent (m) Debaryomyces Candida Candida
ハンセニイ由来 グラブラタ由来 ボイディニイ由来 Hansenii origin Grabrata origin Boydinii origin
EDTA 1 52 86 76 EDTA 1 52 86 76
EDTA 5 53 88 77  EDTA 5 53 88 77
Me- EDTA 5 57 90 79 Me- EDTA 5 57 90 79
EDTA-OH 5 52 89 79EDTA-OH 5 52 89 79
CyDTA 5 59 89 77 CyDTA 5 59 89 77
DTPA-OH 5 60 89 80 DTPA-OH 5 60 89 80
EGTA 5 53 88 78 EGTA 5 53 88 78
TTHA 5 59 91 80 無添加 32 76 58  TTHA 5 59 91 80 No additive 32 76 58
実施例 3 Example 3
[0083] (アルコール化合物とキレート試薬の共存効果）  [0083] (Coexistence effect of alcohol compound and chelating reagent)
アルコール化合物である 10% (vZv)エタノール、及び Z又はキレート試薬である 5 mM EDTAを含有する、 50mMのリン酸緩衝液 (pH7. 0)をそれぞれ調製した。こ れらの各種試薬を含有する緩衝液を用いて、 PCTZJP2006Z310648に記載の デバリオミセス'ノヽンセニイ Tsukuba42 (FERM BP— 10603)由来ジァセチルス ペルミンォキシダーゼ溶液 0. lU/mlをそれぞれ調製した。なお、比較例として、試 薬を含有しない 50mMのリン酸緩衝液 (pH7. 0)で、上記酵素溶液 0. lU/mlを調 製した。各酵素溶液について 30°C、 6時間の加速試験を行い、加速試験後の酵素 活性を測定した。加速試験前の酵素活性と比較して、加速試験後の安定性を評価し 、その結果を図 1に示した。アルコール化合物であるエタノール、及びキレート試薬で ある EDTAを共存させることによりジァセチルスペルミンォキシダーゼがさらに安定に 保持された。  A 50 mM phosphate buffer (pH 7.0) containing 10% (vZv) ethanol as an alcohol compound and 5 mM EDTA as Z or a chelating reagent was prepared. Using buffers containing these various reagents, a diacetylsperminoxidase solution 0.1 lU / ml derived from Debaryomyces' Nonsenii Tsukuba42 (FERM BP-10603) described in PCTZJP2006Z310648 was prepared. As a comparative example, the above enzyme solution 0.1 lU / ml was prepared with a 50 mM phosphate buffer solution (pH 7.0) containing no reagent. Each enzyme solution was subjected to an acceleration test at 30 ° C for 6 hours, and the enzyme activity after the acceleration test was measured. Compared with the enzyme activity before the acceleration test, the stability after the acceleration test was evaluated, and the results are shown in FIG. By coexisting ethanol, which is an alcohol compound, and EDTA, which is a chelating reagent, diacetyl sperminoxidase was more stably retained.
実施例 4  Example 4
[0084] (安定ィ匕されたポリアミンォキシダーゼ製剤の製造） 10% (v/v) エタノール及び ImM EDTAを含有する 0. 2M リン酸緩衝液（pH 7. 0) 9ml、 15mM TOOS溶液 0. 4ml、 150U/ml ペルォキシダーゼ（キッコ 一マン社製）溶液 0. 4ml、 1. 76% (w/v) 4—ァミノアンチピリン溶液 0. 2ml、 0. 5U/ml ジァセチルスペルミンォキシダーゼ [デノ リオミセス'ノヽンセニイ Tsuk uba42 (FERM BP— 10603)由来、 PCTZJP2006Z310648記載のとおり] 0. 5mlを混合し、よく撹拌して、ポリアミンォキシダーゼ製剤 (溶液)を製造した。対照製 剤として、安定化剤を除いたポリアミンォキシダーゼ製剤を調製した。本発明製剤と 対照製剤について、 30°C、 24時間の***試験を行い、その結果を図 2に示した。対 照製剤は、酵素活性がほとんど失われているのに対して、本発明製剤は、酵素活性 が安定に保持されていた。 [0084] (Production of stabilized polyamine oxidase preparation) 0.2 ml phosphate buffer solution (pH 7.0) containing 10% (v / v) ethanol and ImM EDTA, 9 ml, 15 mM TOOS solution, 0.4 ml, 150 U / ml peroxidase solution (manufactured by Kikkoman) 0. 4 ml, 1.76% (w / v) 4-Aminoantipyrine solution 0.2 ml, 0.5 U / ml diacetyl spermine oxidase [Denoriomyces' nonseneni Tsuk uba42 (FERM BP— 10603) derived, PCTZJP2006Z310648 As described] 0.5 ml was mixed and stirred well to produce a polyamine oxidase formulation (solution). As a control product, a polyamine oxidase preparation without the stabilizer was prepared. The preparation of the present invention and the control preparation were subjected to an abuse test at 30 ° C. for 24 hours, and the results are shown in FIG. The control preparation almost lost the enzyme activity, whereas the preparation of the present invention stably maintained the enzyme activity.
実施例 5  Example 5
[0085] (安定化されたジァセチルスペルミンォキシダーゼを用いるモノァセチルポリアミン の製造）  [0085] (Production of monoacetyl polyamine using stabilized diacetyl spermine oxidase)
10% (vZv)エタノール、 ImM EDTA, 0. 5U/ml カタラーゼ（Sigma社製）及 び 7mM ジァセチルスペルミン · 2塩酸塩（150mg)を含有する 60mlの 20mM リン 酸緩衝液（PH7. 5)に、ジァセチルスペルミンォキシダーゼ [デノ リオミセス'ノヽンセ ニイ Tsukuba42 (FERM BP— 10603)由来、 PCTZJP2006Z310648に記 載のとおり]を lUZmlとなるように添カ卩した。 30°Cで 2日間インキュベートし、 TLCで N1 -ァセチルスペルミジンの生成を確認した（クロ口ホルム：メタノール：アンモニア水 ：水 = 2 :4 : 1 : 1、ニンヒドリン発色）。 60 ml of 20 mM phosphate buffer (PH 7.5) containing 10% (vZv) ethanol, ImM EDTA, 0.5 U / ml catalase (Sigma) and 7 mM diacetylspermine dihydrochloride (150 mg) Dicetylspermine oxidase [Denoriomyces' nonseneni Tsukuba42 (FERM BP-10603) as described in PCTZJP2006Z310648] was added to lUZml. Incubation was performed at 30 ° C for 2 days, and production of N 1 -acetyl spermidine was confirmed by TLC (black mouth form: methanol: aqueous ammonia: water = 2: 4: 1: 1, ninhydrin coloration).
[0086] セントリブレップ 10 (アミコン社製)を用いて水で透析し、酵素タンパクを除去した。 [0086] The enzyme protein was removed by dialyzing against water using Centrivrep 10 (Amicon).
透析外液を陽イオン交換榭脂 [DOWEX 50W—X4 (H+) ]カラム（lcm X 10cm) に通し、反応生成物を吸着させた。水で洗浄後、 1M アンモニアで溶出させた。溶 出液を減圧濃縮した後、クロ口ホルム:メタノール = 1 : 1の溶媒に溶解させて、シリカ ゲルカラムカラム（2cm X 15cm)に吸着させた。展開溶媒としてクロ口ホルム:メタノー ル（1： 1〜0： 1)を用いて溶出させ、 N1—ァセチルスペルミジン溶出画分に塩酸を添 加した後減圧濃縮し、 82mgの N1—ァセチルスペルミジン 2塩酸塩を得た (収率 76 %)。 [0087] 上記の 82mgの N1—ァセチルスペルミジン 2塩酸塩をエタノールで再結晶し、 52m gの N1—ァセチルスペルミジン 2塩酸塩を得た（収率 47%)。融点と1 H,13C— NMR の測定値は文献既知の値（「Acta Chemica ScandinavicaJ , (デンマーク）， 19 89年， 43卷， p. 990— 994)と一致した。力くして、 PCT/JP2006/310648に記 載の N1—ァセチルスペルミジンの製造方法では 2U/mlのジァセチルスペルミンォ キシダーゼを用いていたが、今回、安定化剤を用いることにより、半分の酵素量で同 量の N1—ァセチルスペルミジン 2塩酸塩を得ることができた。 The external dialysis solution was passed through a cation exchange resin [DOWEX 50W-X4 (H +)] column (lcm X 10 cm) to adsorb the reaction product. After washing with water, it was eluted with 1M ammonia. The eluate was concentrated under reduced pressure, dissolved in a solvent of chloroform-form: methanol = 1: 1, and adsorbed on a silica gel column column (2 cm × 15 cm). Elution was performed using chloroform-form: methanol (1: 1 to 0: 1) as a developing solvent, and hydrochloric acid was added to the N 1 -acetyl spermidine elution fraction, followed by concentration under reduced pressure to obtain 82 mg of N 1 -a. Cetylspermidine dihydrochloride was obtained (yield 76%). [0087] The above 82 mg of N 1 -acetyl spermidine dihydrochloride was recrystallized from ethanol to obtain 52 mg of N 1 -acetyl spermidine dihydrochloride (yield 47%). Melting points and 1 H, 13 C—NMR measurements agreed with literature known values (“Acta Chemica Scandinavica J, (Denmark), 19 89, 43 卷, p. 990—994). In the production method of N 1 -acetyl spermidine described in JP2006 / 310648, 2 U / ml diacetyl spermine oxidase was used. N 1 -acetyl spermidine dihydrochloride was obtained.
実施例 6  Example 6
[0088] (各種界面活性剤の基質特異性改良効果の確認）  [0088] (Confirmation of substrate specificity improvement effect of various surfactants)
0. 3%の各種界面活性剤を含有する 0. 2M リン酸緩衝液 (pH7. 5)をそれぞれ 調製した。これらの各種界面活性剤を含有する緩衝液を用いて、前記のポリアミンォ キシダーゼの活性測定法にて、 PCTZJP2006Z310648に記載のデバリオミセス' ハンセ-ィ Tsukuba42 (FERM BP— 10603)由来ジァセチルスペルミンォキシ ダーゼ溶液及びキャンディダ.グラブラタ Nodal62 (FERM BP— 10602)由来ジ ァセチルスペルミンォキシダーゼについて、ジァセチルスペルミン及び N1—ァセチ ルスペルミジンに対する酵素活性を測定、比較した。その結果を表 3に示した。このよ うに各種界面活性剤、特に非イオン性界面活性剤と両性界面活性剤を添加すること により、基質特異性の改善効果が認められた。 0.2 M phosphate buffer (pH 7.5) containing 0.3% of various surfactants was prepared. By using the buffer containing these various surfactants, the polyamine oxidase activity measurement method described above, the debaryomyces' Hansei Tsukuba42 (FERM BP-10603) -derived diacetyl spermine oxidase described in PCTZJP2006Z310648 Enzymatic activity against diacetyl spermine and N 1 -acetyl spermidine was measured and compared for diacetyl spermine oxidase derived from solution and Candida glabrata Nodal62 (FERM BP-10602). The results are shown in Table 3. Thus, by adding various surfactants, particularly nonionic surfactants and amphoteric surfactants, an effect of improving substrate specificity was observed.
[0089] [表 3] [0089] [Table 3]
N1—AcSpdに対する反応性（ ）※ Reactivity to N 1 —AcSpd () *
(DASpmに対する反応性を 100%とする) 界面活性剤  (Reactivity to DASpm is 100%) Surfactant
^リオミセス由来 キャンディダ由来 無添加 29.7 49.5 ^ Riomyces origin Candida origin No addition 29.7 49.5
Triton X-100 (非イオン性、和光純薬工業社製） 13. 2 Triton X-100 (Non-ionic, Wako Pure Chemical Industries) 13. 2
Triton X-405 (非イオン性、ナカライテスク社製） 11. 1  Triton X-405 (Nonionic, manufactured by Nacalai Tesque) 11. 1
Tween 20 (非イオン性、和光純薬工業社製） 7.4  Tween 20 (Non-ionic, Wako Pure Chemical Industries) 7.4
Tween 40 (非イオン性、和光純薬工業社製） 11.0  Tween 40 (Nonionic, Wako Pure Chemical Industries) 11.0
Tween 80 (非イオン性、和光純薬工業社製） 11.8  Tween 80 (nonionic, manufactured by Wako Pure Chemical Industries, Ltd.) 11.8
Tween 85 (非イオン性、和光純薬工業社製） 11.9  Tween 85 (nonionic, manufactured by Wako Pure Chemical Industries, Ltd.) 11.9
Brij 35 (非イオン性、和光純薬工業社製） 38.6  Brij 35 (Non-ionic, Wako Pure Chemical Industries) 38.6
Brij 58 (非イオン性、シグマ社製） 4.4  Brij 58 (Non-ionic, Sigma) 4.4
Brij 78 (非イオン性、シグマ社製） Ί.3  Brij 78 (Non-ionic, Sigma) Ί.3
ェマルゲン 913 (非イオン性、花王社製） 12.9  Emargen 913 (non-ionic, manufactured by Kao Corporation) 12.9
CHAPS0 (両性、同仁化学社製） 17.0  CHAPS0 (amphoteric, manufactured by Dojin Chemical) 17.0
※！^1— A c S p d ： N1—ァセチルスペルミジン *! ^ 1 — A c S pd: N 1 — Acetylspermidine
DAS pm：ジァセチルスペルミン  DAS pm: Diacetyl spermine
デバリオミセス由来：デバリオミセス . ハンセニイ T s u k u b a 42 ERM BP— 10603) 由来ジァセチルスペルミンォキシダ一ゼ キャンディダ由来：キャンディダ ·グラブラタ No d a l 62 (FERM  Debariomyces origin: Debaryomyces. Hansenii T s u k u b a 42 ERM BP— 10603) Diacetyl spermine oxidase derived from Candida: Candida glabrata No d a l 62 (FERM
P- 10602) 由来ジァセチルスペルミンォキシダーゼ  P-10602) Diacetyl spermine oxidase
実施例 7 Example 7
[0090] (pH条件の基質特異性改良効果の確認）  [0090] (Confirmation of substrate specificity improvement effect of pH condition)
pH6.5〜7.5の 0.2M リン酸緩衝液をそれぞれ調製した。これらの緩衝液を用 いて、前記のポリアミンォキシダーゼの活性測定法にて、 PCTZJP2006Z310648 に記載のデバリオミセス'ノヽンセニイ Tsukuba42(FERM BP— 10603)由来ジァ セチルスペルミンォキシダーゼ溶液及びキャンディダ 'グラブラタ Nodal62 (FER M BP— 10602)由来ジァセチルスペルミンォキシダーゼについて、各種ポリアミン に対する酵素活性を測定、比較した。その結果を表 4に示した。このように pHを 7.5 未満にすることにより、基質特異性の改善効果が認められた。  A 0.2M phosphate buffer solution having a pH of 6.5 to 7.5 was prepared. Using these buffers, the polyamine oxidase activity measurement method described above was performed using the Debariomyces 'Nonseneni Tsukuba42 (FERM BP-10603) -derived diacetyl spermine oxidase solution and Candida' Grabrata Nodal62 ( FER M BP-10602) -derived diacetyl spermine oxidase was measured and compared for enzyme activity against various polyamines. The results are shown in Table 4. Thus, the effect of improving the substrate specificity was recognized by making the pH less than 7.5.
[0091] [表 4] N1—AcSpdに対する反応性（ ）※ [0091] [Table 4] Reactivity to N 1 —AcSpd () *
酵素の由来 PH  Origin of enzyme PH
(DASpmに対する反応性を 100とする）  (Responsiveness to DASpm is 100)
7.5 7.5
デバリオミセス ハンセニイ  Debariomyces Hanseny
7.0  7.0
Tsukuba 42  Tsukuba 42
6.5  6.5
7.5  7.5
キャン丁イダ ワラブラタ  Cancho Ida Wallabrata
7.0  7.0
Noda 162  Noda 162
6.5  6.5
※！^1— Ac S p d ： N1—ァセチルスペルミジン *! ^ 1 — Ac S pd: N 1 — Acetylspermidine
DAS pm : ジァセチルスペルミン  DAS pm: Diacetyl spermine
実施例 8 Example 8
[0092] (界面活性剤の添加と pH条件を組み合わせた基質特異 M 」」 性改良効果の確認）  [0092] (Substrate-specific M combining the addition of surfactant and pH conditions)
0.3%の TritonX— 100を含有する 0.2M リン酸緩衝液D0l ω o o C (oi (pH6.5〜7.5)を調製 し、前記のポリアミンォキシダーゼの活性測定法にて、 PCTZJP2006Z310648に 記載のデノ リオミセス'ノヽンセニイ Tsukuba42(FERM BP— 10603)由来ジァセ チルスペルミンォキシダーゼ溶液にっ 、て、各種ポリアミンに対する酵素活性を測定 、比較した。その結果を表 5に示した。このように TritonX— 100を添カロし、かつ pHを 7.5未満とすることにより、さらなる基質特異性の改善効果が認められた。なお、いず れの条件においてもプトレツシンには反応しなかった。  A 0.2M phosphate buffer solution D0l ω oo C (oi (pH 6.5 to 7.5) containing 0.3% TritonX-100 was prepared, and the denomination described in PCTZJP2006Z310648 was performed using the polyamine oxidase activity measurement method described above. Enzyme activity against various polyamines was measured and compared using diacetyl spermine oxidase solution derived from Riomyces'Nonsenii Tsukuba42 (FERM BP-10603), and the results are shown in Table 5. Further improvement of substrate specificity was observed by adding calorie and adjusting the pH to less than 7.5, and did not react with putretin under any of the conditions.
[0093] [表 5] [0093] [Table 5]
各種ポリアミンに対する反応性※ Reactivity to various polyamines *
(DASpmに対する反応性を 100とする) μ  (Responsiveness to DASpm is 100) μ
Triton X— 100  Triton X— 100
DASpd N1 - N1— N8- DASpd N 1 - N 1 - N 8 -
Spm Spd AcSpm AcSpd AcSpd Spm Spd AcSpm AcSpd AcSpd
7.5 無添加 51.0 3.5 0 0 7.5 No additive 51.0 3.5 0 0
7.0 無添加 19.5 19.9 1.4 0 07.0 No additive 19.5 19.9 1.4 0 0
7.0 添加 18.6 7.8 7.8 1.0 0 07.0 Addition 18.6 7.8 7.8 1.0 0 0
6. 5 無添加 25.8 9.4 11.9 0.6 0 06.5 No additive 25.8 9.4 11.9 0.6 0 0
6. 5 添カロ 15.2 6. 1 7.0 0.5 0 0 6.5 Caro 15.2 6. 1 7.0 0.5 0 0
00  00
※ AS pm：ジァセチルスペルミン、 DAS p d :ジァセチルスペルミジン、 * AS pm: Diacetyl spermine, DAS p d: Diacetyl spermidine,
ω  ω
N1— A c S p m： N1—ァセチルスぺノレミン、 N1— Ac S p d ： N1—ァセチ ルスペルミジン、 N8— A c S p d ： N8—ァセチルスぺノレミジン、 S pm :ス ぺノレミン、 S p d :スぺノレミジン N 1 — A c S pm: N 1 — Acetylspentolemin, N 1 — Ac S pd: N 1 — Acetylspermidine, N 8 — A C S pd: N 8 — Acetyls penolemidine, S pm: Spenoremin , S pd: Spenoremidin
なお、 いずれの条件においてもプトレツシンには反応しなかった。 実施例 9  It did not react with putretin under any of the conditions. Example 9
[0094] (ポリアミンォキシダーゼを含有する試薬を用いる、ジァセチルポリアミンの測定方 法。）  [0094] (Method of measuring diacetyl polyamine using a reagent containing polyamine oxidase)
第 1試薬  First reagent
MES緩衝液（pH7.0) 50mM  MES buffer (pH 7.0) 50 mM
DA— 67 0.05mM  DA— 67 0.05mM
TritonX— 100 0.04%  TritonX— 100 0.04%
アジ化ナトリウム 0.09%  Sodium azide 0.09%
2纏  2 Summary
MES緩衝液（pH7.0) 50mM  MES buffer (pH 7.0) 50 mM
ジァセチルスペルミンォキシダーゼ（デバリオミセス由来） 24UZmL ペルォキシダーゼ（西洋ヮサビ由来） 15UZmL  Diacetyl sperminoxidase (derived from Debaryomyces) 24UZmL Peroxidase (derived from horseradish rust) 15UZmL
[0095] 生理食塩水で希釈調製したジァセチルスペルミン試料（125nM 500nM)各 10 μ Lに第 1試薬 80 Lを混和し、 37°Cで 5分間加温した。続いて第 2試薬 30 Lを添 加し、 5分間反応させた後の 658nmにおける吸光度を測定し、その結果を図 3に示 した。力べして、ジァセチルスペルミンォキシダーゼを用いた試薬でジァセチルスペル ミンを簡便かつ短時間に測定することができることがわ力 た。 [0095] 80 L of the first reagent was mixed with 10 μL of each diacetyl spermine sample (125 nM 500 nM) diluted with physiological saline, and heated at 37 ° C for 5 minutes. Subsequently, 30 L of the second reagent was added, the absorbance at 658 nm was measured after 5 minutes of reaction, and the results are shown in Fig. 3. did. In addition, it was proved that diacetyl spermine can be measured easily and in a short time with a reagent using diacetyl spermine oxidase.
実施例 10 Example 10
(ポリアミンォキシダーゼを含有する試薬を用いる、尿検体中のジァセチルポリアミン の測定方法。 ) 1纏  (Measurement method of diacetyl polyamine in urine samples using a reagent containing polyamine oxidase.) 1 Summary
TES緩衝液（pH7. 7) 50mM  TES buffer (pH 7.7) 50 mM
TOOS ImM  TOOS ImM
TritonX— 100 1. 5%  TritonX— 100 1.5%
KC1 70mM  KC1 70mM
カタラーゼ 300UZmL  Catalase 300UZmL
ァスコルビン酸ォキシダーゼ 3UZmL  Ascorbate oxidase 3UZmL
TES緩衝液（pH7. 7) 50mM TES buffer (pH 7.7) 50 mM
TritonX- 100 1. 5%  TritonX- 100 1.5%
KC1 70mM  KC1 70mM
4ーァミノアンチピリン 4mM  4-aminoantipyrine 4mM
ジァセチルスペルミンォキシダーゼ（デバリオミセス由来） 24UZmL ペルォキシダーゼ（西洋ヮサビ由来） 15UZmL  Diacetyl sperminoxidase (derived from Debaryomyces) 24UZmL Peroxidase (derived from horseradish rust) 15UZmL
アジ化ナトリウム 0. 09% 尿検体にジァセチルスペルミン（32nM〜500nM)を添加して調整した試料各 10 μ Lに、第 1試薬 80 Lを混和し、 37°Cで 5分間加温した。続いて第 2試薬 30 Lを 5分間反応させた後の 550nmにおける吸光度を測定し、その結果を図 4に示した。 力べして、ジァセチルスペルミンォキシダーゼを用いた試薬で尿試料中のジァセチ ルスペルミンを測定することができることがわかった。  Sodium azide 0.09% Each sample prepared by adding diacetyl spermine (32nM to 500nM) to a urine sample was mixed with 10μL of the first reagent and 80L of the first reagent, and heated at 37 ° C for 5 minutes. . Subsequently, the absorbance at 550 nm after the reaction with 30 L of the second reagent for 5 minutes was measured, and the result is shown in FIG. As a result, it was found that diacetyl spermine in urine samples can be measured with a reagent using diacetyl spermine oxidase.
産業上の利用可能性 本発明の安定ィ匕方法を用いて、アルコールィ匕合物及び Z又はキレート試薬とポリ アミンォキシダーゼを含有する安定ィ匕されたポリアミンォキシダーゼ製剤を、簡単な 方法でかつ安価に製造することができる。また、本発明の基質特異性の改良方法を 用いて、界面活性剤とポリアミンォキシダーゼを含有する基質特異性が改良されたポ リアミンォキシダーゼ製剤を、簡単な方法でかつ安価に製造することができる。さらに 、ジァセチルポリアミン測定用試薬を簡単な方法でかつ安価に製造することができる Industrial applicability A stable polyamine oxidase preparation containing an alcohol compound and Z or a chelating reagent and a polyamine oxidase is produced by a simple method and at a low cost using the stable method of the present invention. Can do. In addition, by using the method for improving substrate specificity of the present invention, a polyamine oxidase preparation with improved substrate specificity containing a surfactant and polyamine oxidase can be produced by a simple method and at low cost. Can do. Furthermore, a reagent for measuring diacetylpolyamine can be produced by a simple method and at a low cost.
Figure imgf000035_0001
Figure imgf000035_0001
g理官庁記入欄 -4 この用紙は国際出願とともに受理した  gScience Office entry field -4 This form was accepted along with the international application
(はい/いいえ） (Yes, No)
-4-1 権限のある職員 国際事務局記入 ft  -4-1 Authorized staff Completed by the International Bureau ft
0-5 この用紙が国際事務局に受理された日 0-5 Date when this form was received by the International Bureau
0-5-1 権限のある職員  0-5-1 Authorized staff
差替え用紙（規則 26) Replacement paper (Rule 26)

Claims

34 雜囲  34 Range
[I] アルコール化合物及び/又はギレ ト '薬と、ポリアミンォキシダ ゼを共存させる ことを特徴とする、ポリアミンォキシダ一 め安定ィ匕方法。  [I] A method of stabilizing a polyamine oxidase, comprising coexisting an alcohol compound and / or a drug and a polyamine oxidase.
[2] ポリアミンォキシダーゼがジァセチル ペルミンォキシダーゼである、請求項 1に記 載のポリアミンォキシダーゼの安定匕;^ 。  [2] The stability of the polyamine oxidase according to claim 1, wherein the polyamine oxidase is diacetyl perminoxidase; ^.
[3] 界面活性剤存在下にてポリアミン才キ. ダーゼを作用させることを特徴とする、ポリ アミンォキシダーゼの基質特異性 Φ改:良方法。 [3] Substrate specificity of polyamine oxidase, characterized by allowing polyamine oxidase to act in the presence of a surfactant.
〔4] ポリアミンォキシダ一せを pH7. 5未満 作用させることを特徴とする、ポリアミンォ キシダ一ゼの基質特異性の改良方法。  [4] A method for improving the substrate specificity of polyamine oxidase, characterized by allowing polyamine oxidase to act at a pH of less than 7.5.
[5] ポリアミンォキシダーゼがジァセ ルスペルミンォキシダーゼである、請求項 3〜4 のいずれかに記載のポリアミンォキシ ゼの.基質特異性の改良方法。  [5] The method for improving substrate specificity of polyamine oxidase according to any one of claims 3 to 4, wherein the polyamine oxidase is diacetylspermine oxidase.
[6] ポリアミンォキシダーゼを含有 る 試料に作用させて、ジァセチルポリアミン を測定することを特徴とする、ジァセチ リアミンの測定方法。 [6] A method for measuring diacetylamine, which comprises measuring a diacetylpolyamine by acting on a sample containing polyamine oxidase.
[7] アルコール化合物及ぴ 又は^".ト により安定化されたポリアミン才キシダ 一ゼを用 V、ることを特徴とする、鏞 錄 12載の測定方法。 [7] The measuring method according to 錄 錄 12, characterized by using a polyamine aged oxidase stabilized with an alcohol compound and / or ^ ".
[8] 界面活性剤により碁質時異性 ^^ た^リアミンォキシダーゼを用いることを 特徴とする、睛求項 6〜7のい ¾ 〖£ 測定方法。 . [8] A method for measuring the amount of claims 6 to 7 characterized in that isomerase is used in the course of surfactants. .
[9] ポリアミンォキシダーゼ pH7、 5未満？ きせることを特徴とする、請求項 6〜8 のいずれかに記載の測定方法。 [9] Polyamine oxidase pH 7, less than 5? The measuring method according to any one of claims 6 to 8, wherein the measuring method is characterized in that:
[10] ポリアミンォキシダ ゼがジァセチル ペルミンォキシダーゼである、請求項 6〜9 のいずれかに記載のジァセチルポリアミ の測定方法。 [10] The method for measuring diacetyl polyamido according to any one of claims 6 to 9, wherein the polyamine oxidase is diacetyl perminoxidase.
[II] アルコール化合物 ¾ぴ 又は ト戴^ ΐ、ポリアミンォキシダーゼを含有するこ とを特徴とする、安定ィ匕されたポリ ミ^キ ダーゼ製剤。  [II] A stabilized polymoxidase preparation characterized by containing an alcohol compound or a polyamine oxidase.
[12] ポリアミンォキシダーゼがジアセ^レスペルミンォキシダーゼである、請求項 11に 記載の安定化されたポリアミンオキシ^^ゼ製剤。  [12] The stabilized polyamine oxidase preparation according to claim 11, wherein the polyamine oxidase is diaceresperminoxidase.
[13] ポリアミンォキシダーゼを含有す と ¾き徼とする、ジァセチルポリアミンの測定試 薬。 [13] A reagent for measuring diacetyl polyamine, which contains polyamine oxidase.
[14] アルコール化合物及び/又はキレ ト試藥と、ポリアミンォキシダーゼを含有するこ  [14] Alcohol compound and / or chelate test and polyamine oxidase
差替え用紙 （規則 26) 3'5 Replacement paper (Rule 26) 3'5
とを特徴とする請求項 3に記載.のジァ ½チルポリアミンの測定試薬。 The reagent for measuring diatomylpolyamine according to claim 3, characterized in that:
[15] 界面活性剤とポリアミンォキシダ を含有することを特徴とする請求項 13又は 14 のレ、ずれかに記載のジァセチルポリアミ 測定試薬。 [15] The reagent for measuring diacetyl polyethylene according to [13] or [14], which contains a surfactant and a polyamine oxide.
[16] ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、請求項 13〜 1[16] The polyamine oxidase is diacetyl spermine oxidase.
5のいずれかに記載のジァセチルポ:リ ミ , ' 則定試薬。 5. Diacetylpo: Limi, 'Regular reagent according to any one of 5.
C17] 請求項 1又は 2言己載のポリアミシォキ タ^ゼの安定化方法、及び/又は請求項 3C17] Claim 1 or 2 Self-stabilizing polyamidase stabilization method and / or claim 3
〜5のいずれか一項に記載のポリ ミ ;^キシダ一ゼの基質特異性の改良方法を用Use the method for improving the substrate specificity of the polymer; ^ xidase as described in any one of
V、ることを特徴とする、ジァセチ ポリアミンへポリアミンォキシダーゼを作用させてモ ノアセチルポリアミンを製造する方法。 V. A process for producing monoacetylpolyamine by reacting diacetylpolyamine with polyamine oxidase.
[18] ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、請求項 17記 載のモノァセチルポリアミンの製瑋 ¾法。 [18] The process for producing monoacetyl polyamine according to claim 17, wherein the polyamine oxidase is diacetyl spermine oxidase.
[19] 請求項 1又は 2記載のポリアミンォキジダーゼの安定化方法を利用することを特徴と する、ポリアミンォキシダーゼの精製方法。 [19] A method for purifying polyamine oxidase, comprising using the method for stabilizing polyamine oxidase according to claim 1 or 2.
[20] ポリアミンォキシダーゼがジァセチルスペルミンォキシダーゼである、請求項 19記 載のポリアミンォキシダーゼの精製方法。 20. The method for purifying polyamine oxidase according to claim 19, wherein the polyamine oxidase is diacetyl spermine oxidase.
差替え用紙（規則 26) Replacement paper (Rule 26)
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