WO1999045106A1 - Cholesterol oxidase - Google Patents

Cholesterol oxidase Download PDF

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Publication number
WO1999045106A1
WO1999045106A1 PCT/JP1999/001022 JP9901022W WO9945106A1 WO 1999045106 A1 WO1999045106 A1 WO 1999045106A1 JP 9901022 W JP9901022 W JP 9901022W WO 9945106 A1 WO9945106 A1 WO 9945106A1
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Prior art keywords
enzyme
cholesterol
enzyme according
sterols
activity
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PCT/JP1999/001022
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French (fr)
Japanese (ja)
Inventor
Rikizo Aono
Toru Hamaya
Toshiaki Kono
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Meiji Seika Kaisha, Ltd.
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Publication of WO1999045106A1 publication Critical patent/WO1999045106A1/en

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • 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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03006—Cholesterol oxidase (1.1.3.6)

Definitions

  • the present invention relates to cholesterol oxidase which can be used for measuring cholesterol levels in body fluids and foods, for producing cholesterol derivatives, for insecticides, detergents and the like.
  • Cholesterol oxidase is an oxide that catalyzes the reaction between 3-hydroxysteroid and oxygen, producing the corresponding 3-oxosteroid and hydrogen peroxide.
  • thermostability is important for measuring the cholesterol concentration in body fluids. Therefore, search for new enzymes (Japanese Patent Laid-Open No. 6-169765, etc.) Attempts have been made to modify animals using protein engineering techniques (Japanese Patent Application Laid-Open No. Hei 8-242680). Also, the production of cholesterol derivatives requires organic solvent resistance.
  • the cholesterol nitridation reaction can be performed quickly at low substrate (cholesterol) concentrations. It is desirable that The development of roxidase was required.
  • the present inventors have now found cholesterol oxidase produced from Pseudomonas strain ST-200 and succeeded in isolating and purifying it.
  • This enzyme had the property of rapidly oxidizing the substrate at low substrate concentrations, acting over a wide pH range, being resistant to heat, and being strongly activated by organic solvents.
  • the present invention is based on this finding.
  • the enzyme according to the present invention is cholesterol oxidase produced by the ST-200 strain (FERM BP-6661).
  • the enzyme according to the invention has the following properties:
  • the enzyme according to the present invention is useful as a reagent for measuring cholesterol concentration, a composition for controlling pests and a bleaching agent.
  • FIG. 1 shows the pH dependence of the enzyme according to the invention.
  • ⁇ indicates 5 OmM acetic acid-sodium acetate buffer
  • ⁇ indicates 5 OmM monobasic potassium phosphate monosodium phosphate buffer
  • ⁇ Indicates that 50 mM Tris-monohydrochloride buffer was used
  • ⁇ indicates that 50 mM sodium carbonate-sodium bicarbonate buffer was used.
  • FIG. 2 shows the temperature dependence of the enzyme according to the invention.
  • FIG. 3 shows the pH stability of the enzyme according to the invention.
  • ⁇ indicates 5 O mM acetic acid-sodium acetate buffer
  • ⁇ indicates 5 O mM sodium phosphate monophosphate monosodium phosphate buffer
  • ⁇ indicates 50 mM Tris-HCl buffer
  • Okina indicates 50 mM sodium carbonate. This indicates that 50 mM sodium chloride-sodium hydroxide buffer was used.
  • FIG. 4 shows the temperature stability of the enzyme according to the invention.
  • the enzyme according to the invention is cholesterol oxidase which can act on and oxidize cholesterol. Specifically, it converts cholesterol to cholester-5-one-3-one and cholester-5-en-3-one to 6 yS-perhydroxycholest 1-4-en-3-one. Can be.
  • This reductase attaches an oxygen molecule (eg, oxygen present in the air) to the cholesterone 5-one 6-one 6; S-perhydroxycholesterone 4-one-one 3 -turn on.
  • free cholesterol oxidase reduces oxygen to produce hydrogen peroxide.
  • the reduced enzyme is oxidized to an acid enzyme.
  • This oxidized enzyme acts again on cholesterol and can oxidize it.
  • the enzyme according to the present invention includes both the oxidized enzyme and the reduced enzyme.
  • the enzyme according to the present invention has substrate specificity for 3 ⁇ -sterols.
  • “3 / 9-sterols” refers to sterols having a hydroxyl group in the 3rd position with an / S configuration, cholesterol,) 3-sitosterol, ⁇ -cholestanol, ⁇ -stigmasterol, predanenolone, ergo Includes sterols, dehydroepiandrosterone and epiandrosterone.
  • the enzyme according to the invention does not act on 3 ⁇ -hydroxysteroids, for example epicholesterol.
  • the activity of the enzyme according to the present invention is optimally at pH 5.0 to 8.5. It is stable at pH 4 to 11.
  • the cholesterol oxidase of the present invention has a maximum reaction rate (Vmax: zmole ⁇ min) in the presence of 0.3% surfactant Triton X—100 with respect to cholesterol.
  • the enzyme according to the present invention has an optimum temperature around 60 ° C and is stable at 4 to 55 ° C.
  • Naturally-derived cholesterol oxidase known to date has low thermostability.
  • the enzymes according to the invention have a wide temperature stability.
  • the cholesterol oxidase disclosed in the present invention may be an organic solvent having a logP of 2.1 or more and 4.5 or less, for example, benzene (2.1; representing logP ⁇ value; the same applies hereinafter), toluene (2.6), Paraxylene (3.1), propylbenzene (3.7), diphenylmethane (4.2), cyclooctane (4.5), etc. have the property of increasing the reaction rate. Therefore, the conversion of 3; 5-sterols can be carried out with high efficiency using the enzyme of the present invention under an organic solvent layer.
  • the logP ⁇ value is a common logarithm of a partition coefficient ⁇ value of an arbitrary substance between two phases of water and n-octanol, and represents a polarity of the substance.
  • the P nw value is calculated as (concentration in n-octanol phase) / (concentration in aqueous phase). Therefore, iogP n ji lower object quality indicates high polarity.
  • the values of logP nni defined in the present invention are calculated from the structures of various organic solvents. The calculation method is, for example, Chemical Review
  • Etc. can be used.
  • the enzyme according to the invention has a molecular weight of about 60 kDa as determined by SDS polyacrylamide gel electrophoresis.
  • the enzyme activity of the enzyme according to the present invention is inhibited by silver nitrate and mercury chloride when cholesterol is used as a substrate.
  • the enzyme according to the present invention can be produced by culturing Pseudomonas strain ST-200, and isolating and purifying from the culture.
  • the culture method is not particularly limited, and liquid culture or solid culture can be used.
  • As the medium use a medium containing an appropriate carbon source and nitrogen source and containing an appropriate amount of phosphate, inorganic ions, etc. as necessary. During the culture, it is preferable to appropriately adjust the conditions of stirring and aeration.
  • the cholesterol oxidase known to date is often produced in the cells of microorganisms, and its extraction requires the crushing process of the cells and requires a great deal of labor. Since the enzyme according to the present invention is produced outside the cells, it is advantageous in that it can be easily recovered.
  • a filtrate and a supernatant obtained by removing cells by filtration, centrifugation, etc. can be obtained and recovered by subjecting them to various types of chromatography.
  • solid culture after adding water to the medium, the cells are removed by filtration or centrifugation as in liquid culture to obtain a furnace solution or supernatant, which can be recovered by subjecting it to various types of chromatography.
  • Methods for purifying the cholesterol oxidase from the culture solution include fractional precipitation such as ammonium sulfate precipitation and solvent precipitation, ion exchange chromatography, hydrophobic chromatography, and chromatography such as gel filtration. . If necessary, desalting treatment such as dialysis may be performed.
  • fractional precipitation such as ammonium sulfate precipitation and solvent precipitation
  • ion exchange chromatography such as hydrophobic chromatography
  • chromatography such as gel filtration.
  • desalting treatment such as dialysis may be performed.
  • the type and order of chromatography are not particularly limited.
  • the enzyme according to the invention has cholesterol oxidase activity. Therefore, according to the present invention, there is provided a reagent for measuring the concentration of 3 ⁇ -sterols (particularly, cholesterol) containing the enzyme according to the present invention.
  • the concentration of 3-sterols can be measured by contacting a sample with the enzyme of the present invention and measuring the degree of oxidation of the substrate, ie, cholesterol oxidase activity.
  • the sample can be, for example, a sample separated from mammals or a sample separated from food. Cholesterol oxidase activity can be evaluated by measuring the amount of oxygen consumed by the oxygen electrode, or by measuring hydrogen peroxide generated during the enzymatic reaction.
  • a method for producing a sterol derivative is carried out by bringing the enzyme according to the present invention into contact with 3) 3-sterols, for example, under an overlay of an organic solvent, and recovering the oxidized 35-sterols.
  • the sterol derivatives produced include the oxidizing power of 3 ⁇ -sterols, for example, acids such as cholest-1-ene-3-one and 6-peroxycholest-4-1-ene-3-one. Dani cholesterol. Cholesterol oxidase destroys the midgut epithelium of insects and causes them to die (Purcell JP et al., Biochem. Biophy. Res. Comm., Vol.
  • compositions for controlling pests comprising the enzyme according to the present invention.
  • the term “pest-controlling composition” is used to include plant protection agents, pest control agents, pesticides, insect repellents, and the like.
  • the composition for controlling pests according to the present invention comprises, for example, adding an enzyme according to the present invention to a desired appropriate compounding agent (for example, a surfactant, a wetting agent, a solid diluent, a dispersant, an ultraviolet stabilizer, etc.), and adding water.
  • a desired appropriate compounding agent for example, a surfactant, a wetting agent, a solid diluent, a dispersant, an ultraviolet stabilizer, etc.
  • a desired appropriate compounding agent for example, a surfactant, a wetting agent, a solid diluent, a dispersant, an ultraviolet stabilizer, etc.
  • composition for controlling insect pests of the present invention can be used, if necessary and Z or desired, at the time of formulation or spraying, at the time of various insecticides and fungicides , A herbicide, a plant growth regulator, a synergist (including an activity enhancer contained in the culture supernatant), an attractant, a plant nutrient, a fertilizer, and the like.
  • pest control is carried out by adding a pest control composition diluted with a diluent (for example, water) to a plant that is or is expected to be damaged by the pest, or as it is.
  • a diluent for example, water
  • Cholesterol oxidase acts on substrates to generate hydrogen peroxide.
  • the enzyme system that produces hydrogen peroxide has a bleaching effect, and such an enzyme can be added to a detergent as a bleaching agent (WO89 / 09813, JP-A-3-50051). 0 0).
  • the detergent composition according to the present invention comprises the enzyme according to the present invention together with a surfactant, a builder, and other additives (for example, an optical brightener, a foaming agent, a foam inhibitor, a softener, a fragrance, etc.) May be contained, or the enzyme according to the present invention may be contained alone.
  • a surfactant for example, an optical brightener, a foaming agent, a foam inhibitor, a softener, a fragrance, etc.
  • the enzyme according to the present invention has a property that the reaction rate is high even in the presence of a low concentration of the substrate.
  • the enzyme of the present invention has thermostability higher than that of conventional natural cholesterol oxidase and thermostability equivalent to that of a genetically modified enzyme for the purpose of enhancing thermostability. Therefore, the present invention is advantageous for measuring the concentration of 3 ⁇ -sterols (particularly, low cholesterol concentration) in body fluids and foods.
  • the enzyme according to the present invention also has the property of having a high reaction rate under an organic solvent overlay. Enzymatic conversion of sterols is often carried out under an organic solvent overlay, and it is required that the conversion be labile under the organic solvent overlay.
  • the enzyme according to the present invention is advantageous in that sterols can be efficiently converted under an organic solvent layer.
  • Pseudomonas strain ST-200 (FERM BP-6661) in a 10 liter fermenter, 6 liter medium containing 1% tryptone (Difco), 0.5% yeast extract (Difco) and 1% sodium chloride And cultured at 30 ° C for 17 hours.
  • the stirring speed was 40 Orpm and the aeration was 12 liters per minute.
  • the obtained culture was centrifuged at 8, OOOxg for 15 minutes to obtain a supernatant. Subsequently, the precipitate was recovered by salting out with ammonium sulfate at 70% saturation (4 ° C, overnight) by centrifugation at 10,000 X g for 30 minutes.
  • the obtained precipitate was dissolved in 1 OmM Tris-HCl buffer (pH 8), and dialysis was repeated twice against the same buffer.
  • the dialyzed precipitate was applied to a DEAE cellulose DE 52 column, and eluted isocraticly with 10 mM Tris-hydrochloric acid buffer (pH 8). Add ammonium sulfate to the active fraction to a final concentration of 45% saturation and centrifuge (7, OOOxg, 15 minutes). After that, the supernatant was recovered. Subsequently, the active fraction was applied to a column packed with Ptiltoyopearl 650 S equilibrated with 1 OmM Tris-hydrochloride buffer (pH 8) containing 45% saturated ammonium sulfate, and the ammonium sulfate concentration was reduced to a linear gradient up to 0 M. Eluted. Since the active fraction was present in the 10% saturated to 0M ammonium sulfate concentration fraction, this was collected and salted out with the addition of 80% saturated ammonium sulfate.
  • the activity recovery of the obtained sample was 20%.
  • the specific activity was 15.2 U / mg, which was 36 times that before purification.
  • the molecular weight was 6 OkDa as measured by SDS polyacrylamide gel electrophoresis. It strongly oxidized cholesterol, ⁇ -sitosterol, / 3 / 3-cholesterol, etc., had no effect on epicholesterol, and was strongly inhibited by silver nitrate and mercuric chloride.
  • Test Example 1 Effect of ⁇ ⁇ and temperature on enzyme
  • Example 1 The ⁇ and temperature dependence and stability of the enzyme obtained in Example 1 were measured.
  • ⁇ ⁇ and temperature dependence were measured by measuring the amount of oxygen consumed when cholesterol was used as a substrate.
  • a solution containing 5 OmM phosphate buffer ( ⁇ 7.0), 64 mM sodium cholate, 0.34% surfactant Triton X-100, and 0.89 mM cholesterol was prepared according to the present invention. 3 ⁇ 4 was added and the amount of oxygen consumed was measured. The oxygen amount was measured using a DO meter (YSI Model53, Yellow Spring, Ohio, USA).
  • Cholesterol oxidase activity One unit was defined as the activity of oxidizing 1 micromole of cholesterol per minute.
  • pH dependence the activity at PH7 was set to 100%.
  • temperature dependence the activity at 60 ° C was set to 100%.
  • the pH and temperature stability was measured by measuring the residual activity (using cholesterol as a substrate) after 30 minutes at each pH and temperature at 30 ° (measurement of hydrogen peroxide generated during the enzymatic reaction at pH 7). Specifically, the method was performed as follows. In a solution containing the enzyme of the present invention at an appropriate concentration, a phosphate buffer (pH 7.0), a final concentration of 50 mM, a sodium cholate to a concentration of 64 mM, and a surfactant to a concentration of 0.34%, respectively.
  • a phosphate buffer pH 7.0
  • Triton X-100 Aminoantipyrine to 1.4 mM, phenol to 2 ImM, peroxidase from horseradish (Toyobo) to 5 units, cholesterol to 0.89 mM
  • the resulting solution (3 ml) was reacted at 30 ° C. for 5 minutes while measuring the absorbance at 500 nm.
  • Cholesterol oxidase activity One unit was defined as the activity of oxidizing 1 micole of cholesterol per minute.
  • the enzyme according to the present invention showed strong activity from pH 5.0 to 8.5 and was stable from pH 4.0 to 11.0. It showed strong activity at 50-60 ° C and was stable at 4-55 ° C.
  • Example 1 The substrate specificity of the enzyme obtained in Example 1 was examined. Enzyme activity was measured using various substrates at 30 at pH 7 according to Test Example 1. As a result, cholesterol shown as Table 1, ⁇ Shitosuteroru, YS cholestyramine evening to Sani ⁇ strongly Nord, 3 alpha-hydroxy sterols I de a is E peak cholesterol c Table 1 ST did not act on — Substrate specificity of cholesterol oxidase from 200 strains
  • Example 2 The effect of metal ions and the like on the activity of the enzyme obtained in Example 1 was examined.
  • the enzyme activity was measured at 30 ° (:, pH 7) according to Test Example 1 after adding various metal compounds to the reaction system so as to have an ImM concentration.
  • Table 2 Strong inhibition by silver nitrate and mercury chloride
  • the Michaelis constant (K ⁇ ) and maximum reaction rate (V) mill of the enzyme obtained in Example 1 were examined in the presence of 0.03% and 0.3% of the surfactant Triton X-100. .
  • the enzyme activity was measured at 30 ° C. and pH 7 according to Test Example 1 using various enzymes.
  • Table 3 Nocardia erythropolis, Pseudomonas sp., And Streptomyces sp. Streptomyces sp. Te rolls O Kishida over showed a small K and large Vmax compared to zero, the maximum of V n
  • Example 1 The effect of the organic solvent on the activity of the enzyme obtained in Example 1 was examined. At the time of activity measurement, 50% of the solution was overlaid with each organic solvent. The enzyme activity was measured at 30 ° C. and pH 7 according to Example 1. As a result, as shown in Table 4, the enzymes according to the present invention were obtained from Nocardiaerythropolis, Pseudomonas sp., Streptomyces sp., Brevibacterium sp. .) Compared to cholesterol oxidase, benzene, toluene, and phenol. The reaction rate was high under the laxylene, propylbenzene, diphenylmethane and cyclooctane layers.

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Abstract

A novel cholesterol oxidase having the following properties: acting on cholesterol and thus converting it into cholest-5-en-3-one; acting on 3β-sterols but not on 3α-hydroxysteroid; having an optical pH of from 5.0 to 8.5; and being stable within a pH range of from 4 to 11. This enzyme induces quick oxidation of the substrate even at a low substrate concentration, acts over a wide pH range, is resistant to heat and achieves a high reaction rate even in the presence of organic solvents. This enzyme is useful in reagents for measuring cholesterol concentration, insecticidal compositions and bleaching agents.

Description

明 細 書 コレステロールォキシダーゼ  Description Cholesterol oxidase
発明の背景 Background of the Invention
発明の分野  Field of the invention
本発明は、 体液及び食品中のコレステロール濃度の測定や、 コレステロール誘 導体の製造、 殺虫剤および洗剤等に使用することができるコレステロールォキシ ダーゼに関する。  The present invention relates to cholesterol oxidase which can be used for measuring cholesterol levels in body fluids and foods, for producing cholesterol derivatives, for insecticides, detergents and the like.
背景技術  Background art
コレステロールォキシダーゼは、 3 一ヒドロキシステロィドと酸素の間の反 応を触媒し、 相当する 3—ォキソステロイドと過酸化水素を生成する酸化 ¾で ある。 現在までに、 体液中のコレステロール濃度の測定 （特開平 6 - 1 6 9 7 6 5号等） 、 コレステロール誘導体の製造 （特開平 6 - 1 1 3 8 8 3号等） 、 殺虫 剤 （Purcell J. P. et al. , Biochem. Biophy. Res. Comm. , 196, 3, ppl406~14 13(1993)、 米国特許 5 5 5 8 8 6 2号等） 、 洗剤 (WO 8 9/ 0 9 8 1 3等） 等 に利用することを目的として開発 ·研究されている。  Cholesterol oxidase is an oxide that catalyzes the reaction between 3-hydroxysteroid and oxygen, producing the corresponding 3-oxosteroid and hydrogen peroxide. Up to now, measurement of cholesterol concentration in body fluids (JP-A-6-169765, etc.), production of cholesterol derivatives (JP-A-6-118388, etc.), insecticide (Purcell JP) et al., Biochem. Biophy. Res. Comm., 196, 3, ppl406-1413 (1993), U.S. Pat.No. 5,558,862, etc.), detergents (WO89 / 0981, 13 etc.) It is being developed and researched for the purpose of using it.
これらの酵素はストレブトマイセス （特開昭 6 2— 2 8 5 7 8 9号） 、 ブレビ パクテリゥム （特開平 4— 2 1 8 3 6 7号）、 ロドコッカス （特表平 3— 5 0 3 4 7 8号） 、 シユードモナス （特開平 6—1 8 9 7 5 4号） 等の種々の微生物に より生産されることカ知られている。  These enzymes are Streptomyces (Japanese Patent Application Laid-Open No. 62-287879), Brevi Pacterium (Japanese Patent Application Laid-Open No. 4-218369), Rhodococcus (Japanese Patent Application Laid-Open No. 3-503). No. 478) and Pseudomonas (Japanese Patent Laid-Open No. 6-189754) are known to be produced by various microorganisms.
求められる酵素特性は用途により異なるが、 例えば体液中のコレステロール濃 度測定のためには熱安定性が重要であり、 そのため新規酵素の探索 （特開平 6— 1 6 9 7 6 5号等） や、 タンパク工学的手法を利用した胜物の改変 （特開平 8 —2 4 2 8 6 0号） が試みられている。 また、 コレステロール誘導体の製造には 有機溶媒耐性が求められている。  The required enzyme properties vary depending on the application. For example, thermostability is important for measuring the cholesterol concentration in body fluids. Therefore, search for new enzymes (Japanese Patent Laid-Open No. 6-169765, etc.) Attempts have been made to modify animals using protein engineering techniques (Japanese Patent Application Laid-Open No. Hei 8-242680). Also, the production of cholesterol derivatives requires organic solvent resistance.
以上のような個々の目的にあわせた特性に加え、 体液や食品中のコレステロ一 ル濃度測定をはじめとする多くの用途において、 低基質 （コレステロール) 濃度 でコレステロ一ル酸ィ匕反応が速 、ことが望ましく、 この点に優れたコレステロ一 ルォキシダ一ゼの開発が求められていた。 In addition to the characteristics tailored to individual purposes as described above, in many applications including the measurement of cholesterol concentration in body fluids and foods, the cholesterol nitridation reaction can be performed quickly at low substrate (cholesterol) concentrations. It is desirable that The development of roxidase was required.
—方ヽ Applied and Environmental Microbiology, July 1994， pp2518 - 2523に はシユードモナス属に属する菌がコレステロ一ル酸化活性を有することが記載さ れている。  —Applied and Environmental Microbiology, July 1994, pp2518-2523, describe that bacteria belonging to the genus Pseudomonas have cholesterol oxidizing activity.
発明の概要  Summary of the Invention
今般本発明者らは、 シユードモナス ST— 200株から生産されるコレステロ 一ルォキシダ一ゼを見いだし、 これを単離 ·精製することに成功した。 この酵素 は低基質濃度で基質の酸化反応が早く、 幅広い pHで作用し、 熱に耐性であり、 さらに有機溶媒で強く活性ィヒされるという性質を有していた。 本発明はこの知見 に基づくものである。  The present inventors have now found cholesterol oxidase produced from Pseudomonas strain ST-200 and succeeded in isolating and purifying it. This enzyme had the property of rapidly oxidizing the substrate at low substrate concentrations, acting over a wide pH range, being resistant to heat, and being strongly activated by organic solvents. The present invention is based on this finding.
本発明による酵素は、 ST— 200菌株 （FERM BP— 6661) により 生産されるコレステロールォキシダーゼである。  The enzyme according to the present invention is cholesterol oxidase produced by the ST-200 strain (FERM BP-6661).
本発明による酵素は、 下記性質を有する：  The enzyme according to the invention has the following properties:
(1)作用： コレステロールに作用してこれをコレストー 5—ェンー 3—オンに 変換する；コレスト一 5—ェンー 3—オンに作用してこれを 6 ^—パーヒドロキ シコレスト一 4ェン一 3オンに変換する ；  (1) Action: It acts on cholesterol and converts it to cholesterol 5-ene-3-one; it acts on cholesterol 5-ene-3-one and turns it into 6 ^ -perhydroxylesterone 4-ene-3-on. Convert ;
(2)基質特異性： 3 /3—ステロール類に作用し、 3な一ヒドロキシステロイド には作用しない；  (2) Substrate specificity: 3 / 3-acts on sterols, not on 3 monohydroxysteroids;
(3)至適 pH： pH5. 0〜8. 5；  (3) Optimum pH: pH 5.0 to 8.5;
(4)安定 pH： pH4〜l 1。  (4) Stable pH: pH 4-l1.
本発明による酵素はコレステロール濃度測定用試薬、 害虫駆除用組成物および 漂白剤として有用である。  The enzyme according to the present invention is useful as a reagent for measuring cholesterol concentration, a composition for controlling pests and a bleaching agent.
微生物の寄託  Microbial deposit
Pseudomonas sp. 菌株 S T— 200は、 1998年 2月 4日付で工業技術院生 命工学工業技術研究所 (ΝΙ ΒΗ) (日本国茨城県つくば巿東 1丁目 1番 3号） に寄託された。 受託番号は、 FERM BP— 6661である。  Pseudomonas sp. Strain ST-200 was deposited on February 4, 1998 with the Institute of Life Science and Industrial Technology (ΝΙ ΒΗ), 1-3-1 Tsukuba East, Ibaraki, Japan. The accession number is FERM BP-6661.
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
図 1は、 本発明による酵素の pH依存性を示す。 〇は 5 OmM酢酸—酢酸ナト リウム緩衝液、 ▲は 5 OmMリン酸 1カリウム一リン酸 2ナトリウム緩衝液、 □ は 5 0 mMトリス一塩酸緩衝液、 ·は 5 0 mM炭酸ナトリウム—炭酸水素ナトリ ム緩衝液を使用したことを示す。 FIG. 1 shows the pH dependence of the enzyme according to the invention. 〇 indicates 5 OmM acetic acid-sodium acetate buffer, ▲ indicates 5 OmM monobasic potassium phosphate monosodium phosphate buffer, □ Indicates that 50 mM Tris-monohydrochloride buffer was used, and · indicates that 50 mM sodium carbonate-sodium bicarbonate buffer was used.
図 2は、 本発明による酵素の温度依存性を示す。  FIG. 2 shows the temperature dependence of the enzyme according to the invention.
図 3は、 本発明による酵素の p H安定性を示す。 〇は 5 O mM酢酸—酢酸ナ卜 リゥム緩衝液、 ▲は 5 O mMリン酸 1カリウム一リン酸 2ナトリゥム緩衝液、 □ は 5 0 mMトリス—塩酸緩衝液、 翁は 5 0 mM炭酸ナトリウム一炭酸水素ナトリ ム緩衝液、 國は 5 0 mM塩化ナトリウム—水酸化ナトリゥム緩衝液を使用したこ とを示す。  FIG. 3 shows the pH stability of the enzyme according to the invention. 〇 indicates 5 O mM acetic acid-sodium acetate buffer, ▲ indicates 5 O mM sodium phosphate monophosphate monosodium phosphate buffer, □ indicates 50 mM Tris-HCl buffer, and Okina indicates 50 mM sodium carbonate. This indicates that 50 mM sodium chloride-sodium hydroxide buffer was used.
図 4は、 本発明による酵素の温度安定性を示す。  FIG. 4 shows the temperature stability of the enzyme according to the invention.
発明の具体的説明  Detailed description of the invention
酵素の性質  Enzyme properties
本発明による酵素はコレステロールに作用してこれを酸化することができるコ レステロ一ルォキシダ一ゼである。 具体的には、 コレステロールをコレスト— 5 一ェン一 3—オンに変換し、 コレスト一 5—ェン一 3—オンを 6 yS—パーヒドロ キシコレスト一 4—ェン一 3—オンに変換することができる。  The enzyme according to the invention is cholesterol oxidase which can act on and oxidize cholesterol. Specifically, it converts cholesterol to cholester-5-one-3-one and cholester-5-en-3-one to 6 yS-perhydroxycholest 1-4-en-3-one. Can be.
コレステロールを酸化した酵素は、 補 ¾であるフラビンが還元された結果還 酵素となる。 この還元型酵素は、 酸素分子 （例えば、 空気中に存在する酸素） をコレスト一 5—ェン一 3—オンに付l口して 6 ;S—パーヒドロキシコレスト一 4 一ェン一 3—オンにする。 そして、 遊離型のコレステロールォキシダーゼが酸素 を還元して過酸化水素を生じさせる。 その結果、 還元型酵素は酸化され酸ィ 酵 素となる。 この酸化型酵素はコレステロールに再び作用して、 これを酸ィ匕するこ とができる。 本発明による酵素にはこの酸化型酵素および還元型酵素のいずれも が含まれるものとする。  The enzyme that oxidized cholesterol becomes a reductase as a result of the reduction of flavin, a supplement. This reductase attaches an oxygen molecule (eg, oxygen present in the air) to the cholesterone 5-one 6-one 6; S-perhydroxycholesterone 4-one-one 3 -turn on. Then, free cholesterol oxidase reduces oxygen to produce hydrogen peroxide. As a result, the reduced enzyme is oxidized to an acid enzyme. This oxidized enzyme acts again on cholesterol and can oxidize it. The enzyme according to the present invention includes both the oxidized enzyme and the reduced enzyme.
本発明による酵素は 3 ^—ステロール類に基質特異性を有する。 ここで、 「3 /9ーステロール類」 とは、 3位に /S配置の水酸基を持つステロールをいい、 コレ ステロール、 ）3—シトステロール、 ^ーコレスタノ一ル、 ^—スティグマステロ ール、 プレダネノロン、 エルゴステロール、 デヒドロェピアンドロステロンおよ びェピアンドロステロンを含む。 本発明による酵素は 3 α—ヒドロキシステロィ ド、 例えば、 ェピコレステロール、 には作用しない。 本発明による酵素の活性は、 p H 5. 0〜 8. 5が至適である。 また、 p H 4 〜 1 1において安定である。 The enzyme according to the present invention has substrate specificity for 3 ^ -sterols. Here, “3 / 9-sterols” refers to sterols having a hydroxyl group in the 3rd position with an / S configuration, cholesterol,) 3-sitosterol, ^ -cholestanol, ^ -stigmasterol, predanenolone, ergo Includes sterols, dehydroepiandrosterone and epiandrosterone. The enzyme according to the invention does not act on 3α-hydroxysteroids, for example epicholesterol. The activity of the enzyme according to the present invention is optimally at pH 5.0 to 8.5. It is stable at pH 4 to 11.
本発明のコレステロールォキシダーゼは、 コレステロールに対し、 0. 3 %界 面活性剤トライ トン X— 1 0 0存在下での最大反応速度 (Vmax: zmole · min The cholesterol oxidase of the present invention has a maximum reaction rate (Vmax: zmole · min) in the presence of 0.3% surfactant Triton X—100 with respect to cholesterol.
• mg— ½ とミハエリス定数 （K ： Μ) の比 V ΖΚ が 0 . 2 3、 0 . 0 3 πι max m • mg—the ratio of the ½ to the Michaelis constant (K: Μ) V ΖΚ is 0.23, 0.03 πι max m
%トライトン X— 1 0 0存在下での Vm。v /1 が 3. 2である。 Vm。v /Kmがこ max m max m のように大きな値であるコレステロールォキシダーゼは現在までに知られていな い。 % Triton X—V m in the presence of 100. v / 1 is 3.2. V m . Cholesterol oxidase in which v / K m is as large as max m max m is not known until now.
本発明による酵素は至適温度が 6 0 °C付近であり、 4〜 5 5 °Cで安定である。 現在までに知られている天然由来のコレステロールォキシダーゼは熱安定性が低 かつた。 これに対し本発明による酵素は幅広い温度安定性を有する。  The enzyme according to the present invention has an optimum temperature around 60 ° C and is stable at 4 to 55 ° C. Naturally-derived cholesterol oxidase known to date has low thermostability. In contrast, the enzymes according to the invention have a wide temperature stability.
本発明で開示するコレステロールォキシダーゼは、 logP が 2. 1以上、 4. 5以下の有機溶媒、 例えばベンゼン （2. 1 ； logP^値を表す。 以下同様） ， ト ルェン （2. 6 ) 、 パラキシレン （3. 1 ) 、 プロピルベンゼン （3. 7 ) 、 ジ フヱニルメタン （4. 2 ) 、 シクロオクタン （4. 5 ) 等で反応速度が上昇する 性質を有している。 従って、 本発明による酵素を用いて有機溶媒の重層下で 3；5 —ステロール類の変換を高効率で実施できる。 ここで、 logP ^値とは、 水と n-ォ クタノールとの二相間における任意の物質の分配係数 Ρηπι値の常用対数であり、 物質の極性を表す。 任意の物質において、 Pnw値は、 （n-ォクタノール相におけ る濃度） / (水相における濃度） として算出される。 従って、 iogPnjiが低い物 質は極性が高いことを示す。 本発明で定義する logPnniの数値は、 各種有機溶媒の 構造から算出されたものを使用する。 算出の方法は、 例えば Chemical ReviewThe cholesterol oxidase disclosed in the present invention may be an organic solvent having a logP of 2.1 or more and 4.5 or less, for example, benzene (2.1; representing logP ^ value; the same applies hereinafter), toluene (2.6), Paraxylene (3.1), propylbenzene (3.7), diphenylmethane (4.2), cyclooctane (4.5), etc. have the property of increasing the reaction rate. Therefore, the conversion of 3; 5-sterols can be carried out with high efficiency using the enzyme of the present invention under an organic solvent layer. Here, the logP ^ value is a common logarithm of a partition coefficient ηηπι value of an arbitrary substance between two phases of water and n-octanol, and represents a polarity of the substance. For any substance, the P nw value is calculated as (concentration in n-octanol phase) / (concentration in aqueous phase). Therefore, iogP n ji lower object quality indicates high polarity. The values of logP nni defined in the present invention are calculated from the structures of various organic solvents. The calculation method is, for example, Chemical Review
(Leo, A. J. ， Calculating log PQct from structure. 93 ； 1281-1306. )に記載 の方法や logP 計算プログラム C log P ver. 1. 0. 3 (Bio-Byte corp. , California) ow (Leo, AJ, Calculating log P Qct from structure. 93; 1281-1306.) And a logP calculation program C log P ver. 1.0.3 (Bio-Byte corp., California) ow
等を使用することができる。 Etc. can be used.
本発明による酵素は、 S D Sポリアクリルアミ ドゲル電気泳動による測定で約 6 O k D aの分子量を有する。  The enzyme according to the invention has a molecular weight of about 60 kDa as determined by SDS polyacrylamide gel electrophoresis.
本発明による酵素の酵素活性は、 コレステロールを基質とした場合、 硝酸銀お よび塩化水銀により阻害される。 本発明による酵素は、 シユードモナス菌株 S T— 2 0 0を培養し、 その培養物 から単離 ·精製することにより製造できる。 培養方法に特に制限はなく、 液体培 養や固形培養を利用できる。 培地としては適当な炭素源、 窒素源を含み必要に応 じてリン酸塩や無機イオン等を適量含有する培地を使用する。 培養時は撹拌や通 気の条件を適当に調整することが好ましい。 さらに、 シユードモナス菌株 S T—The enzyme activity of the enzyme according to the present invention is inhibited by silver nitrate and mercury chloride when cholesterol is used as a substrate. The enzyme according to the present invention can be produced by culturing Pseudomonas strain ST-200, and isolating and purifying from the culture. The culture method is not particularly limited, and liquid culture or solid culture can be used. As the medium, use a medium containing an appropriate carbon source and nitrogen source and containing an appropriate amount of phosphate, inorganic ions, etc. as necessary. During the culture, it is preferable to appropriately adjust the conditions of stirring and aeration. Furthermore, Pseudomonas strain ST—
2 0 0はシクロへキサンに耐性を有しているため、 培養時にシクロへキサンを重 層することによって、 コレステロールォキシダーゼの生産性を低下させることな く他の触物による汚染を防止することもできる。 Since 200 is resistant to cyclohexane, overlaying cyclohexane during culture prevents contamination by other tactile substances without reducing cholesterol oxidase productivity. Can also.
現在までに知られているコレステロールォキシダ一ゼは、 微生物の菌体内に生 産される場合が多く、 その抽出には菌体の破砕工程力必須であり、 多大の労力を 必要としていた。 本発明による酵素は菌体外に生産されるため、 安易に回収でき る点で有利である。 例えば、 液体培養の場合には、 濾過、 遠心分離等で菌体を除 去した炉液、 上澄み液を得、 各種クロマトグラフィーにかけることによって回収 できる。 固形培養では、 培地に加水した後、 液体培養と同様に濾過または、遠心 分離等で菌体を除去して炉液または上澄み液を得、 これを各種クロマトグラフィ 一にかけることによつて回収できる。  The cholesterol oxidase known to date is often produced in the cells of microorganisms, and its extraction requires the crushing process of the cells and requires a great deal of labor. Since the enzyme according to the present invention is produced outside the cells, it is advantageous in that it can be easily recovered. For example, in the case of liquid culture, a filtrate and a supernatant obtained by removing cells by filtration, centrifugation, etc., can be obtained and recovered by subjecting them to various types of chromatography. In solid culture, after adding water to the medium, the cells are removed by filtration or centrifugation as in liquid culture to obtain a furnace solution or supernatant, which can be recovered by subjecting it to various types of chromatography.
培養液から該コレステロールォキシダーゼを精製する方法としては、 硫安塩析 法や溶媒沈殿法等の分別沈殿、 ィォン交換ク口マトグラフィー、 疎水クロマトグ ラフィ一、 およびゲル濾過等のクロマトグラフィー等が挙げられる。 また、必要 に応じて透析などの脱塩処理を実施してもよい。 クロマトグラフィーの種類およ び順序は特に限定されるものではない。  Methods for purifying the cholesterol oxidase from the culture solution include fractional precipitation such as ammonium sulfate precipitation and solvent precipitation, ion exchange chromatography, hydrophobic chromatography, and chromatography such as gel filtration. . If necessary, desalting treatment such as dialysis may be performed. The type and order of chromatography are not particularly limited.
酵素の用途  Enzyme applications
本発明による酵素はコレステロールォキシダーゼ活性を有する。 従って、本発 明によれば、 本発明による酵素を含む 3 ^—ステロール類 （特に、 コレステロ一 ル） 濃度測定用試薬が提供される。 3 —ステロール類濃度の測定は試料と本発 明による酵素とを接触させ、 基質の酸化の程度、 すなわち、 コレステロールォキ シダ一ゼ活性を測定することにより行うことができる。 試料は、 例えば、 ほ乳類 から分離された試料や食品から分離された試料であることができる。 コレステロールォキシダーゼ活性は、 酸素電極により消費された酸素量を測定 することにより、 あるいは、 酵素反応に伴い生成する過酸化水素を測定すること により評価することができる。 The enzyme according to the invention has cholesterol oxidase activity. Therefore, according to the present invention, there is provided a reagent for measuring the concentration of 3 ^ -sterols (particularly, cholesterol) containing the enzyme according to the present invention. The concentration of 3-sterols can be measured by contacting a sample with the enzyme of the present invention and measuring the degree of oxidation of the substrate, ie, cholesterol oxidase activity. The sample can be, for example, a sample separated from mammals or a sample separated from food. Cholesterol oxidase activity can be evaluated by measuring the amount of oxygen consumed by the oxygen electrode, or by measuring hydrogen peroxide generated during the enzymatic reaction.
本発明によれば、 また、 ステロール誘導体の製造法が提供される。 ステロール 誘導体の製造は、 本発明による酵素と 3 )3—ステロール類とを、 例えば、 有機溶 媒の重層下で接触させ、 酸ィ匕された 3 5—ステロール類を回収することにより行 うことができる。 製造されるステロール誘導体としては、 3 ^—ステロール類の 酸化体力挙げられ、 例えば、 コレスト一 5—ェン一 3—オンや 6 —パーヒドロ キシコレスト一 4一ェン一 3—オンのような酸ィ匕コレステロールが挙げられる。 コレステロールォキシダ一ゼは昆虫の中腸上皮を破壊し、 昆虫を死に至らしめ る （Purcell J. P. et al.， Biochem. Biophy. Res. Comm. , Vol. 196, No. 3, pp 1406-1413(1993) 、 米国特許第 5 5 5 8 8 6 2号等） 。 従って、 本発明によれば、 本発明による酵素を含む害虫駆除用組成物が提供される。 ここで、 害虫駆除用組 成物とは、 植物保護剤、 害虫防除剤、 殺虫剤、 防虫剤等を含む意味で用いられる ものとする。  According to the present invention, there is also provided a method for producing a sterol derivative. The production of the sterol derivative is carried out by bringing the enzyme according to the present invention into contact with 3) 3-sterols, for example, under an overlay of an organic solvent, and recovering the oxidized 35-sterols. Can be. The sterol derivatives produced include the oxidizing power of 3 ^ -sterols, for example, acids such as cholest-1-ene-3-one and 6-peroxycholest-4-1-ene-3-one. Dani cholesterol. Cholesterol oxidase destroys the midgut epithelium of insects and causes them to die (Purcell JP et al., Biochem. Biophy. Res. Comm., Vol. 196, No. 3, pp 1406-1413 ( 1993), U.S. Patent No. 5,558,882, etc.). Therefore, according to the present invention, there is provided a composition for controlling pests comprising the enzyme according to the present invention. Here, the term “pest-controlling composition” is used to include plant protection agents, pest control agents, pesticides, insect repellents, and the like.
本発明による害虫駆除用組成物は、 例えば、 本発明による酵素を所望の適当な 配合剤 （例えば、 界面活性剤、 湿潤剤、 固体希釈剤、 分散剤および紫外線安定剤 等） を添加し、 水和剤、 粉剤、 フロアカレ剤等任意の剤型にして得ることができ また、 本発明の害虫駆除用組成物は、 必要によりおよび Zまたは所望により、 製剤時あるいは散布時に、 各種殺虫剤、 殺菌剤、 除草剤、 植物成長調節剤、 共力 剤 （培養上清中に含まれる活性増強物質等も含まれる） 、 誘引剤、 植物栄養剤、 肥料等を含んでいてもよい。  The composition for controlling pests according to the present invention comprises, for example, adding an enzyme according to the present invention to a desired appropriate compounding agent (for example, a surfactant, a wetting agent, a solid diluent, a dispersant, an ultraviolet stabilizer, etc.), and adding water. It can be obtained in any dosage form such as a Japanese wetting agent, a dusting agent, a floor curry agent, etc. In addition, the composition for controlling insect pests of the present invention can be used, if necessary and Z or desired, at the time of formulation or spraying, at the time of various insecticides and fungicides , A herbicide, a plant growth regulator, a synergist (including an activity enhancer contained in the culture supernatant), an attractant, a plant nutrient, a fertilizer, and the like.
害虫の駆除は、 一般に害虫の被害を受けている植物または被害を受けることが 予想される植物に、 希釈剤 （例えば、 水） で希釈した害虫駆除用組成物、 または 害虫駆除用組成物をそのままを散布することによつて実施することができる。 コレステロールォキシダーゼは基質に作用して過酸化水素を生じさせる。 過酸 ィ匕水素を生じさせる酵素系は漂白効果を有し、 このような酵素は漂白剤として洗 剤に添加できる （W0 8 9 / 0 9 8 1 3号、 特開平 3— 5 0 5 1 0 0号） 。 従つ て、 本発明によれば、 本発明による酵素を含む洗剤組成物が提供される。 In general, pest control is carried out by adding a pest control composition diluted with a diluent (for example, water) to a plant that is or is expected to be damaged by the pest, or as it is. By spraying. Cholesterol oxidase acts on substrates to generate hydrogen peroxide. The enzyme system that produces hydrogen peroxide has a bleaching effect, and such an enzyme can be added to a detergent as a bleaching agent (WO89 / 09813, JP-A-3-50051). 0 0). Follow Thus, according to the present invention, there is provided a detergent composition comprising the enzyme according to the present invention.
本発明による洗剤組成物は、 本発明による酵素を界面活性剤、 ビルダー、 およ びその他の添加剤 （例えば、 蛍光増白剤、 増泡剤、 抑泡剤、 柔軟化剤、 香料等） とともに含んでいてもよく、 あるいは本発明による酵素を単独で含んでいてもよ い。  The detergent composition according to the present invention comprises the enzyme according to the present invention together with a surfactant, a builder, and other additives (for example, an optical brightener, a foaming agent, a foam inhibitor, a softener, a fragrance, etc.) May be contained, or the enzyme according to the present invention may be contained alone.
本発明による酵素は、 低濃度の基質存在下でも反応速度が大きいという性質を 有する。 また、 本発明の酵素は、 従来の天然型コレステロールォキシダーゼ以上 の熱安定性、 および熱安定性を高める目的で遺伝子改変された遺伝子組み換え酵 素と同等の熱安定性を有する。 従って、 本発明による は体液や食品中の 3^3 ーステロール類濃度 （特に、 低濃度のコレステロール濃度） の測定に有利である。 本発明による酵素は、 また、 有機溶媒重層下において高い反応速度を有すると いう性質を有する。 ステロール類の酵素による変換は有機溶媒の重層下で行われ ること力く多く、 有機溶媒の重層下で変換隱カ活性であることが求められる。 本 発明による酵素は有機溶媒の重層下でステロール類の変換を高効率で実施できる 点で有利である。  The enzyme according to the present invention has a property that the reaction rate is high even in the presence of a low concentration of the substrate. In addition, the enzyme of the present invention has thermostability higher than that of conventional natural cholesterol oxidase and thermostability equivalent to that of a genetically modified enzyme for the purpose of enhancing thermostability. Therefore, the present invention is advantageous for measuring the concentration of 3 ^ -sterols (particularly, low cholesterol concentration) in body fluids and foods. The enzyme according to the present invention also has the property of having a high reaction rate under an organic solvent overlay. Enzymatic conversion of sterols is often carried out under an organic solvent overlay, and it is required that the conversion be labile under the organic solvent overlay. The enzyme according to the present invention is advantageous in that sterols can be efficiently converted under an organic solvent layer.
難例  Difficult case
本発明を下記例により説明するが、 本発明はこれに限定されるものではない。 実施例 1 酵素の精製  The present invention will be described with reference to the following examples, but the present invention is not limited thereto. Example 1 Purification of enzyme
シユードモナス菌株 ST— 200 (FERM BP— 6661) を 10リット ルのフアーメンターに、 1%トリプトン （Difco社） 、 0. 5%酵母エキス (Difco社） 及び 1%の塩化ナトリゥムを含む培地 6リッ トル中で 30°Cにて 17時間培養した。 攪拌速度は 40 Orpm とし、 通気は毎分 12リットルとした。 得られた培養液を 8， OOOxgで 15分間遠心分離をし、上清を得た。続 、 て飽和度 70%の硫安で塩析 （4°C、 一晩） 沈殿を 10， 000 X gで 30分遠 心分離することにより回収した。 得られた沈殿は 1 OmMトリスー塩酸緩衝液 (pH8) に溶解後、 同緩衝液に対し透析を 2回繰り返した。  Pseudomonas strain ST-200 (FERM BP-6661) in a 10 liter fermenter, 6 liter medium containing 1% tryptone (Difco), 0.5% yeast extract (Difco) and 1% sodium chloride And cultured at 30 ° C for 17 hours. The stirring speed was 40 Orpm and the aeration was 12 liters per minute. The obtained culture was centrifuged at 8, OOOxg for 15 minutes to obtain a supernatant. Subsequently, the precipitate was recovered by salting out with ammonium sulfate at 70% saturation (4 ° C, overnight) by centrifugation at 10,000 X g for 30 minutes. The obtained precipitate was dissolved in 1 OmM Tris-HCl buffer (pH 8), and dialysis was repeated twice against the same buffer.
次に、 透析にかけた沈殿を DEAEセルロース DE 52カラムにかけ、 10m M卜リス一塩酸緩衝液 （pH8) でイソクラティック溶出した。 活性画分に硫安 を終濃度 45%飽和となるよう添加し、 遠心分離 （7， OOOxg, 15分） し た後上清を回収した。 続いて、 45%飽和の硫安を含有する 1 OmMトリス—塩 酸緩衝液 （pH8) で平衡化したプチルトヨパール 650 Sを充填したカラムに 活性画分を供し、 硫安濃度を 0Mまで直線勾配で溶出した。 活性画分は 10%飽 和から 0Mの硫安濃度画分に存在したため、 これを集め、 飽和度 80%の硫安を 添加し塩析した。 Next, the dialyzed precipitate was applied to a DEAE cellulose DE 52 column, and eluted isocraticly with 10 mM Tris-hydrochloric acid buffer (pH 8). Add ammonium sulfate to the active fraction to a final concentration of 45% saturation and centrifuge (7, OOOxg, 15 minutes). After that, the supernatant was recovered. Subsequently, the active fraction was applied to a column packed with Ptiltoyopearl 650 S equilibrated with 1 OmM Tris-hydrochloride buffer (pH 8) containing 45% saturated ammonium sulfate, and the ammonium sulfate concentration was reduced to a linear gradient up to 0 M. Eluted. Since the active fraction was present in the 10% saturated to 0M ammonium sulfate concentration fraction, this was collected and salted out with the addition of 80% saturated ammonium sulfate.
沈殿を 1 OmMトリス一塩酸緩衝液 (pH8) に溶解した後、 同緩衝液に対し 透析を 2回繰り返した。 続いてセフアデックス G—100カラムを用いて、 10 mM卜リス—塩酸緩衝液 （pH8)、 5 OmM塩化ナトリウム及び 5mMコール 酸ナトリゥムを含む溶液で分画した。 活性画分を集め、 10 mMトリス一塩酸緩 衝液 (pH8) に対し透析し精製コレステロールォキシダーゼを得た。  After dissolving the precipitate in 1 OmM Tris-monohydrochloride buffer (pH 8), dialysis was repeated twice against the same buffer. Subsequently, using a Sephadex G-100 column, fractionation was performed using a solution containing 10 mM Tris-HCl buffer (pH 8), 50 mM sodium chloride and 5 mM sodium cholate. The active fractions were collected and dialyzed against 10 mM Tris-monohydrochloride buffer (pH 8) to obtain purified cholesterol oxidase.
得られたサンプルの活性回収率は 20%であった。 比活性は 15. 2U/mg であり、 これは精製前の 36倍であった。 また SDSポリアクリルアミ ドゲル電 気泳動により測定した結果、 分子量は 6 OkD aであった。 コレステロール、 β —シトステロール、 /3—コレス夕ノール等を強く酸化し、 ェピコレステロールに は作用せず、 硝酸銀や塩化水銀により強く阻害されるという特徴を有していた。 試験例 1 酵素への ρ Ηおよび温度の影響  The activity recovery of the obtained sample was 20%. The specific activity was 15.2 U / mg, which was 36 times that before purification. The molecular weight was 6 OkDa as measured by SDS polyacrylamide gel electrophoresis. It strongly oxidized cholesterol, β-sitosterol, / 3 / 3-cholesterol, etc., had no effect on epicholesterol, and was strongly inhibited by silver nitrate and mercuric chloride. Test Example 1 Effect of ρ Η and temperature on enzyme
実施例 1で得られた酵素の ρ Η及び温度依存性及び安定性を測定した。  The ρΗ and temperature dependence and stability of the enzyme obtained in Example 1 were measured.
Ρ Η及び温度依存性はコレステロールを基質としたときの酸素の消費量を測定 する方法で測定した。 具体的には 5 OmMのリン酸緩衝液 （ρΗ 7. 0)、 64 mMのコール酸ナトリウム、 0. 34%の界面活性剤トライトン X—100及び 0. 89mMのコレステロールを含む溶液に本発明による ¾を添加し、 消費さ れる酸素量を測定した。 酸素量の測定は、 DOメーター （YSI Model53, Yellow Spring, Ohio, USA ) を使用した。 コレステロールォキシダーゼ活性 1単位の定 義は、 1分間に 1マイクロモルのコレステロールを酸化させる活性とした。 pH 依存性については、 PH7のときの活性を 100%とした。 また、 温度依存性に ついては、 60°Cのときの活性を 100%とした。  Ρ Ρ and temperature dependence were measured by measuring the amount of oxygen consumed when cholesterol was used as a substrate. Specifically, a solution containing 5 OmM phosphate buffer (ρΗ7.0), 64 mM sodium cholate, 0.34% surfactant Triton X-100, and 0.89 mM cholesterol was prepared according to the present invention. ¾ was added and the amount of oxygen consumed was measured. The oxygen amount was measured using a DO meter (YSI Model53, Yellow Spring, Ohio, USA). Cholesterol oxidase activity One unit was defined as the activity of oxidizing 1 micromole of cholesterol per minute. Regarding pH dependence, the activity at PH7 was set to 100%. Regarding temperature dependence, the activity at 60 ° C was set to 100%.
また、 pH及び温度安定性は、 各 pH及び温度に 30分供した後の残存活性 (コレステロールを基質として使用） を 30° (、 p H 7にて酵素反応に伴い生成 する過酸化水素を測定する方法で実施した。 具体的には下記のようにして行つた。 適当濃度の本発明による酵素を含む溶液に、 終濃度がそれぞれ 50mMとなるよ うにリン酸緩衝液 （pH7. 0) 、 64mMとなるようにコール酸ナトリウム、 0. 34%となるように界面活性剤トライトン X— 100、 1. 4mMとなるよ うにァミノアンチピリン、 2 ImMとなるようにフエノール、 5単位となるよう にわさび由来ペルォキシダーゼ （東洋紡製） 、 0. 89mMとなるようにコレス テロールを加えた溶液 3m 1を、 500 nmの吸光度を測定しながら 30°Cで 5 分間反応させた。 コレステロールォキシダーゼ活性 1単位の定義は、 1分間に 1 マイク口モルのコレステロールを酸ィ匕させる活性とした。 The pH and temperature stability was measured by measuring the residual activity (using cholesterol as a substrate) after 30 minutes at each pH and temperature at 30 ° (measurement of hydrogen peroxide generated during the enzymatic reaction at pH 7). Specifically, the method was performed as follows. In a solution containing the enzyme of the present invention at an appropriate concentration, a phosphate buffer (pH 7.0), a final concentration of 50 mM, a sodium cholate to a concentration of 64 mM, and a surfactant to a concentration of 0.34%, respectively. Triton X-100, Aminoantipyrine to 1.4 mM, phenol to 2 ImM, peroxidase from horseradish (Toyobo) to 5 units, cholesterol to 0.89 mM The resulting solution (3 ml) was reacted at 30 ° C. for 5 minutes while measuring the absorbance at 500 nm. Cholesterol oxidase activity One unit was defined as the activity of oxidizing 1 micole of cholesterol per minute.
結果は、 図 1〜4に示される通りである。 本発明による酵素は pH 5. 0から 8. 5で強い活性を示し、 pH4. 0〜11. 0で安定であった。 また、 50〜 60 °Cで強い活性を示し、 4〜 55 °Cで安定であつた。  The results are as shown in FIGS. The enzyme according to the present invention showed strong activity from pH 5.0 to 8.5 and was stable from pH 4.0 to 11.0. It showed strong activity at 50-60 ° C and was stable at 4-55 ° C.
試験例 2 酵素の基質特異性 Test Example 2 Substrate specificity of enzyme
実施例 1で得られた酵素の基質特異性を調べた。 酵素活性の測定は各種基質を 用いて、 試験例 1に従い 30で、 pH7の条件下で行った。 その結果、 表 1に示 すようにコレステロール、 ^ーシトステロール、 yS—コレス夕ノールを強く酸ィ匕 し、 3 α—ヒドロキシステロィ ドであるェピコレステロールには作用しなかった c 表 1 ST— 200株由来コレステロールォキシダーゼの基質特異性 The substrate specificity of the enzyme obtained in Example 1 was examined. Enzyme activity was measured using various substrates at 30 at pH 7 according to Test Example 1. As a result, cholesterol shown as Table 1, ^ Shitosuteroru, YS cholestyramine evening to Sani匕strongly Nord, 3 alpha-hydroxy sterols I de a is E peak cholesterol c Table 1 ST did not act on — Substrate specificity of cholesterol oxidase from 200 strains
相対活性 Relative activity
(X) コレステロール （Cholest-5-en-3^- ol) 00 β—シトステロール (Sitost- 5-en - 3iS-ol) 84(X) Cholesterol (Cholest-5-en-3 ^ -ol) 00 β-Sitosterol (Sitost-5-en-3iS-ol) 84
^—コレスタノール （5- - Cholestan- 5-en - 3 β-ol 69 ースティグマステロール（Stigmast- 5-en- 3 β -ol) 59 プレグネノロン (3 /3 -Hydroxypregn-5-en-20-one) 32 エルゴステロール （Ergosta- 5, 7, 22-trien - 33-ol) 20 デヒドロェピアンドロステロン（33 -Hydroxyandrost-5-en-l 7-one) 16 ェピアンドロステロン（5 -Androstan-3 β -ol-17- one) 10 ェピコレステロール （Cholest- 5- en - 3び- ol) 0 コレステロールに対する酵素活性を 100とした 試験例 3 酵素活性への金属イオンの影響 ^ —Cholestanol (5--Cholestan-5-en-3 β-ol 69 -Stigmast-5-en-3 β-ol) 59 Pregnenolone (3/3 -Hydroxypregn-5-en-20-one ) 32 Ergosta- 5, 7, 22-trien-33-ol 20 Dehydroepiandrosterone (33-Hydroxyandrost-5-en-l 7-one) 16 Epiandrosterone (5-Androstan-3 β-ol-17-one) 10 Epicholesterol (Cholest-5-en-3-biol) 0 Enzyme activity on cholesterol was defined as 100 Test Example 3 Effect of metal ions on enzyme activity
実施例 1で得られた酵素の活性に対する金属イオン等の影響を調べた。 酵素活 性の測定は、 反応系に各種金属化合物を ImMの濃度になるように添加した後、 試 験例 1に従い 30° (：、 pH 7の条件下で行った。 その結果、 表 2に示すように硝 酸銀や塩化水銀により強く阻害された。 表 2 ST-200株由来コレステロールォキシダ一ゼに対する金属イオン等の  The effect of metal ions and the like on the activity of the enzyme obtained in Example 1 was examined. The enzyme activity was measured at 30 ° (:, pH 7) according to Test Example 1 after adding various metal compounds to the reaction system so as to have an ImM concentration. Table 2 Strong inhibition by silver nitrate and mercury chloride Table 2 Metal ions, etc., against cholesterol oxidase from ST-200 strain
Figure imgf000012_0001
Figure imgf000012_0001
添 物は全て ImMとした。  All additives were ImM.
添加物を加えないときの活性を 100とした 試験例 4 酵素反応速度論 The activity when no additives were added was set to 100 Test Example 4 Enzyme kinetics
実施例 1で得られた酵素のミハエリス定数 (K^) と最大反応速度 (V ) m ill を、 0. 0 3%及び0. 3%の界面活性剤トライトン X- 100 の存在下で調べた。 酵素活性の測定は、 各種酵素を用い、 試験例 1に従い 30 °C、 p H 7の条件下で 行った。 その結果、 表 3に示すようにノカルディアエリス口ポリス (Nocardia erythropolis) , シュ一ドモナス属 (Pseudomonas sp. ) ，ス卜レプ卜マイセ 禹 (Streptomyces sp. ) 'ブレビノ クテリゥム属 (Brevibacterium sp. のコレス テロールォキシダーゼに比べ小さな K および大きな Vmaxを示し、 最大の Vn The Michaelis constant (K ^) and maximum reaction rate (V) mill of the enzyme obtained in Example 1 were examined in the presence of 0.03% and 0.3% of the surfactant Triton X-100. . The enzyme activity was measured at 30 ° C. and pH 7 according to Test Example 1 using various enzymes. As a result, as shown in Table 3, Nocardia erythropolis, Pseudomonas sp., And Streptomyces sp. Streptomyces sp. Te rolls O Kishida over showed a small K and large Vmax compared to zero, the maximum of V n
max max
K を示した c C shows the K
表 3 コレステロールォキシダ一ゼのミハエリス定数 （Km) と最^応; « (Vmax) 由来 0, 03%トライトン X— 100'添加 0. 3%トライトン X— 100'添カロ Table 3 Michaelis constant (Km) and optimum of cholesterol oxidase; «(Vmax) origin, 0.3% Triton X-100% added calo with 0.3% Triton X-100%
Km Vm a x Vma /Km Km Vm a x Vm a x/Km (〃M) (jumole (〃M) (^mole  Km Vm a x Vma / Km Km Vm a x Vm a x / Km (〃M) (jumole (〃M) (^ mole
-1 -1  -1 -1
min mg b mm mg b  min mg b mm mg b
ST— 200 4. 04 13. 1 3. 2 52. 2 12. 1 0. 23 ノカルディァエリス口ポリス 5. 14 9. 8 1. 9 44. 0 6. 8 0. 15 シュ一ドモナス属 9. 41 11. 0 1. 1 76. 1 10. 3 0. 13 ストレプトマイセンス属 16 - 3 15. 8 0. 9 160 15. 3 0. 01 ブレビパ'クテリゥム属 63 . 3 12. 0 0. 2 925 11. 3 0. 01 ST—200 4.04 13.1 3.2 52.2 12.1 0.23 Nocardia Elis Mouth Police 5.14 9.8 1.9 44.0 6.8 0.15 Pseudomonas 9 41 11.0 1.1 76.1 10.3 0.13 Streptomyces 16-3 15.8 0.9 160 15.3 0.01 Brevipa's genus 63 .3 12.0 0.2 925 11.3 0.01
Km及び V maxは、 コレステロールが 0力、ら 1 mMの存在下で反応 を測定し、 Km and V max were measured in the presence of 0 mM cholesterol and 1 mM,
ラインウェーバー-パークプロットにより求めた。 It was determined by a line weber-park plot.
試験例 5 酵素活性への有機溶媒の影響 Test Example 5 Effect of organic solvent on enzyme activity
実施例 1で得られた酵素の活性に対する有機溶媒の影響を調べた。 活性測定時 に、 溶液の 5 0 %容の各有機溶媒を重層した。 酵素活性の測定は、 実施例 1に従 い 3 0 °C、 p H 7の条件下で行った。 その結果、 表 4に示すように本発明による 酵素は、 ノ力ルディァエリス口ポリス （Nocardiaerythropolis) ， シユードモナ ス属 CPseudomonas sp. ) , ス卜レプ卜マイセス属 (Streptomyces sp. ) ， ブレ ビバクテリウム属 （Brevibacteriuffl sp. ) のコレステロールォキシダーゼに比べ ベンゼン、 トルエン、 ノ、。ラキシレン、 プロピルベンゼン、 ジフヱニルメタンおよ びシク口オクタンの重層下で反応速度が大きかった。 The effect of the organic solvent on the activity of the enzyme obtained in Example 1 was examined. At the time of activity measurement, 50% of the solution was overlaid with each organic solvent. The enzyme activity was measured at 30 ° C. and pH 7 according to Example 1. As a result, as shown in Table 4, the enzymes according to the present invention were obtained from Nocardiaerythropolis, Pseudomonas sp., Streptomyces sp., Brevibacterium sp. .) Compared to cholesterol oxidase, benzene, toluene, and phenol. The reaction rate was high under the laxylene, propylbenzene, diphenylmethane and cyclooctane layers.
表 4 ST-200株由来コレステロールォキシダーゼに対する有機 の影 Table 4 Organic shadows on ST-200-derived cholesterol oxidase
有 機 溶 媒 1 o g P o 相 対 活 性  Organic solvent 1 o g P o Relative activity
ST-200 ノカルディア シユー ド ストレプト プレビバク エリス口ポリス モナス属 マイセス属 テリゥム属 無添カロ 1 1 1 クロ口ホフレム 1. 9 0. 2 3 0. 1 0. 1 ぐ 0. 1 ベンゼン 2. 1 3. 0 5 1. 3 0. 9 0. 5 トルエン 2. 6 3. 5 7 1. 4 1. 2 0. 5 パラキシレン 3. 1 3. 4 6 1. 5 0. 7 プロピルベンゼン 3. 7 3. 2 6 1. 3 0. 8 ジフヱニルメタン 4. 2 3. 1 6 1. 5 1. 0 0. 8 シクロオクタン 4. 5 1. 4 0. 8 0. 4 0. 2 活性測定は、 ρΗ7. 0、 30。Cで行った。  ST-200 Nocardia pseudo strepto Previbac Ellis mouth Polis Monas sp.Myces sp.Terium sp.Carbon free 1 1 1 Black mouth hofram 1.90.2 3 0.1 0.11.0.1 0.1 Benzene 2.1 3. 0 5 1.3 0.9.0.5 Toluene 2.6.3.5 7 1.4 1.2 0.5 Para-xylene 3.1.3.4 6.1.5 0.7 Propylbenzene 3.7.3 2 6 1.3 0.8 diphenyl methane 4.2. 3. 6 1. 5. 1. 0 0.8 Cyclooctane 4.5. 1. 0 0.8 0. 4 0.2 The activity measurement was ρΗ 7.0, 30. C went.

Claims

請 求 の 範 囲 The scope of the claims
1. ST— 200菌株 （F ERM B P— 6661) により生産されるコレ ステロールォキシダ一ゼ。 1. Cholesterol oxidase produced by ST-200 strain (FERM B P-6661).
2. 下記性質を有する酵素：  2. Enzymes with the following properties:
(1)作用：コレステロールに作用してこれをコレスト一 5—ェンー 3—オンに 変換する；コレスト一5—ェン一 3—オンに作用してこれを 6jS—パーヒドロキ シコレスト一 4一ェン一 3—オンに変換する ；  (1) Action: Acts on cholesterol and converts it to cholester-1-ene; on cholester-5-ene 3-one and acts on it to convert this to 6jS—perhydroxy cholesterol 4-one. 3—Convert to On;
(2)基質特異性： 3 —ステロール類に作用し、 3α—ヒドロキシステロィド には作用しない；  (2) Substrate specificity: acts on 3-sterols and does not act on 3α-hydroxysteroid;
(3)至適 ρΗ： ρΗ5. 0〜8. 5 ；  (3) Optimum ρΗ: ρΗ5.0 to 8.5;
(4)安定 ρΗ： ρΗ4〜11。  (4) Stable ρΗ: ρΗ4 ~ 11.
3. 下記性質を更に有する、 請求項 2に記載の酵素：  3. The enzyme of claim 2, further having the following properties:
(5)至適温度：約 60°C;  (5) Optimum temperature: about 60 ° C;
(6)安定温度： 4〜55°C;  (6) Stability temperature: 4 ~ 55 ° C;
(7)水と n-ォクタノールとの二相間における任意の物質の分配係数 Pnmの常用 対数値 （logPn ) が 2. 1以上、 4. 5以下の有機溶媒の存在下で反応速度が上 昇する； (7) The reaction rate increases in the presence of an organic solvent with a common logarithmic value (logP n ) of P nm of 2.1 or more and 4.5 or less for the partition coefficient of any substance between the two phases of water and n-octanol. Ascend;
(8) 量： SDS— PAGEによる測定で約 60kDa；  (8) Amount: about 60 kDa as measured by SDS-PAGE;
(9)阻害剤：コレステロールを基質とした場合の酵素活性が硝酸銀および塩化 水銀により阻害される。  (9) Inhibitor: Enzyme activity using cholesterol as substrate is inhibited by silver nitrate and mercury chloride.
4. コレステロールに対する、 0. 3%界面活性剤トライトン X— 100存在 下での最大反応速度 （ Vm。v ： ^mole- min"1 - mg"1) とミハエリス定数 （Km ：4. The maximum reaction rate (V m, v : ^ mole-min " 1 -mg" 1 ) and Michaelis constant (K m : cholesterol) in the presence of 0.3% surfactant Triton X-100
lciA ill 111 ) の比 ¥ /K 力 20以上であるか、 あるいは 0. 0 3%界面活性剤ト  lciA ill 111) ratio ¥ / K force 20 or more, or 0.03% surfactant
max m  max m
ライトン X-100存在下での VmaxZ Km力3.0以上である、 請求項 2に記載の酵素。 3. The enzyme according to claim 2, which has a VmaxZ Km force of 3.0 or more in the presence of Ryton X-100.
5. シクロへキサン耐性！^物により生産される、 請求項 2に記載の隱。 5. Cyclohexane resistant! 3. The cover according to claim 2, wherein the cover is produced by a product.
6. シユードモナス属のシクロへキサン耐性 物により生産される、請求 項 2に記載の酵素。 6. The enzyme according to claim 2, which is produced by a cyclohexane-resistant substance of the genus Pseudomonas.
7. ST— 200菌株 （FERM BP— 6661) により生産される請求 項 2に記載の酵素。 7. Claims produced by ST-200 strain (FERM BP-6661) Item 3. The enzyme according to Item 2.
8. 試料と請求項 1〜 7のいずれか一項に記載の酵素とを接触させ、 コレス テロールォキシダーゼ活性を測定することを含む、 3 yS—ステロール類の濃度の 測定法。  8. A method for measuring the concentration of 3 yS-sterols, comprising contacting a sample with the enzyme according to any one of claims 1 to 7, and measuring cholesterol oxidase activity.
9. 試料が、 ほ乳類から分離された試料または食品から分離された試料であ る、 請求項 8に記載の 3 )8—ステロ一ル類の濃度の測定法。  9. The method according to claim 8, wherein the sample is a sample separated from mammals or a sample separated from food.
1 0. 請求項 1〜 7の L、ずれか一項に記載の酵素を含む、 3 —ステロール 類濃度測定用試薬。  10. A reagent for measuring the concentration of 3-sterols, comprising the enzyme according to L of any one of claims 1 to 7.
1 1 . 請求項 1〜 7の L、ずれか一項に記載の酵素を含む、 害虫駆除用組成物。  11. A pest control composition comprising the enzyme according to any one of claims 1 to 7, L.
1 2. 請求項 1〜 7のいずれか一項に記載の酵素を含む、 洗剤組成物。  1 2. A detergent composition comprising the enzyme according to any one of claims 1 to 7.
1 3. 請求項 1〜7のいずれか一項に記載の酵素と 3 yS—ステロール類とを 接触させ、 ステロール誘導体を回収することを含む、 ステロール誘導体の製造法。  1 3. A method for producing a sterol derivative, comprising contacting the enzyme according to any one of claims 1 to 7 with 3yS-sterols to recover the sterol derivative.
1 4. 酵素と 3 yS—ステロール類とを有機溶媒の重層下で接触させる、請求 項 1 3に記載の方法。  14. The method according to claim 13, wherein the enzyme and the 3yS-sterol are contacted under an organic solvent layer.
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JP2007014329A (en) * 2005-06-08 2007-01-25 Kikkoman Corp Cholesterol oxidase stable in presence of surfactant

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