JP3241712B2 - Cholesterol oxidase - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to cholesterol oxidase which can be used for measurement of cholesterol concentration in body fluids and foods, production of cholesterol derivatives, insecticides, detergents and the like.

BACKGROUND ART Cholesterol oxidase is an oxidase that catalyzes the reaction between 3β-hydroxysteroid and oxygen to produce the corresponding 3-oxosteroid and hydrogen peroxide.
Up to the present, measurement of cholesterol concentration in body fluids (JP-A-6-169765, etc.), production of cholesterol derivatives (JP-A-6-113883, etc.), insecticide (Purc
cell. P. et al. , Biochem. Bi
ophy. Res. Comm. , 196, 3, pp14
06-1413 (1993), U.S. Pat.
No., etc.), detergents (WO89 / 09813, etc.) and the like.

These enzymes are known as Streptomyces (JP-A-62-2).
No. 85789), Brevibacterium (JP-A-4-21)
8367), Rhodococcus (Tokuhyohei 3-503478)
) And Pseudomonas (JP-A-6-189754).

The required enzyme properties vary depending on the application. For example, thermostability is important for measuring the cholesterol concentration in body fluids.
765) and modification of microorganisms using protein engineering techniques (Japanese Patent Application Laid-Open No. 8-242860). In addition, the production of cholesterol derivatives requires organic solvent resistance.

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, it is desirable that the cholesterol oxidation reaction be rapid at low substrate (cholesterol) concentrations. There was a need for the development of superior cholesterol oxidase.

On the other hand, Applied and Environment
ntal Microbiology, July 19
94, pp2518-2523 describes that bacteria belonging to the genus Pseudomonas have cholesterol oxidizing activity.

SUMMARY OF THE INVENTION The present inventors have now found cholesterol oxidase produced from Pseudomonas strain ST-200,
This was successfully isolated and purified. This enzyme rapidly oxidizes the substrate at low substrate concentrations and acts over a wide pH range.
It had the property of being resistant to heat and being strongly activated by an organic solvent. The present invention is based on this finding.

The enzyme according to the invention is a ST-200 strain (FERM).
BP-6661).

The enzyme according to the present invention has the following properties: (1) action: acts on cholesterol to convert it to cholest-5-en-3-one; Is converted to 6β-perhydroxycholest-4en-3one; (2) substrate specificity: acts on 3β-sterols and 3α
(3) optimal pH: pH 5.0 to 8.5; (4) stable pH: pH 4 to 11.

The enzyme according to the present invention is a reagent for measuring cholesterol concentration,
Useful as pest control compositions and bleaches.

Deposit of microorganism Pseudomonas sp. Strain ST-200
Was deposited with the National Institute of Advanced Industrial Science and Technology (NIBH) on February 4, 1998 (1-1-3 Higashi, Tsukuba, Ibaraki, Japan). Accession number is FERM BP
-6661.

DETAILED DESCRIPTION OF THE INVENTION Enzyme Properties The enzymes according to the present invention are cholesterol oxidases that can act on and oxidize cholesterol. Specifically, cholesterol can be converted to cholest-5-en-3-one and cholest-5-en-3-one can be converted to 6β-perhydroxycholest-4-en-3-one. .

The enzyme that oxidized cholesterol becomes a reduced enzyme as a result of the reduction of flavin, a coenzyme. This reduced enzyme adds an oxygen molecule (eg, oxygen present in the air) to cholest-5-en-3-one to give 6β-perhydroxycholest-4-en-3-one. And
Free cholesterol oxidase reduces oxygen to produce hydrogen peroxide. As a result, the reduced enzyme is oxidized to an oxidized 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 enzymes according to the invention have substrate specificity for 3β-sterols. Here, “3β-sterols” refers to sterols having a β-positioned hydroxyl group at the 3-position, such as cholesterol, β-sitosterol, β-cholestanol, and β-cholesterol.
Including stigmasterol, pregnenolone, ergosterol, dehydroepiandrosterone and epiandrosterone. The enzymes according to the invention do 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 4-11.

The cholesterol oxidase of the present invention is based on 0.3% surfactant Triton X-100 based on cholesterol.
Reaction rate in the presence (V _max : μmole mi
n ^-1 mg ^-1 ) and the ratio V _max / K _{m of} Michaelis constant (K _m : μM) are 0.23 and 0.03% Triton X
V _max / K _m in the presence of −100 is 3.2. V
cholesterol oxidase _{max /} K _m is a large value in this way is not known to date.

The enzyme according to the present invention has an optimum temperature around 60 ° C.
Stable at ~ 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.

The cholesterol oxidase disclosed in the present invention has l
an organic solvent having an log P _ow of 2.1 or more and 4.5 or less, for example, benzene (2.1; representing a log P _ow value; the same applies hereinafter), toluene (2.6), para-xylene (3.1),
Propylbenzene (3.7), diphenylmethane (4.
2) It has the property that the reaction rate is increased by cyclooctane (4.5) or the like. Therefore, conversion of 3β-sterols can be carried out with high efficiency using the enzyme according to the present invention under an organic solvent layer. Here, the log P _ow value means water and n
_-Common logarithm of the partition coefficient _Pow value of any substance between the two phases with octanol, representing the polarity of the substance. For any substance, the P _ow value is calculated as (concentration in n-octanol phase) / (concentration in aqueous phase). Therefore, a substance having a low log _Pow value indicates a high polarity. As the numerical value of _logPow defined in the present invention, those calculated from the structures of various organic solvents are used. The calculation method is, for example, Chemical Review (L
eo, A .; J. , Calculating logP
_oct from structure. 93; 1281
-1306. The method and logP _ow calculation program C log P ver described). 1.0.3 (Bio-B
yte corp. , California) 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 mercuric chloride when cholesterol is used as a substrate.

Production of Enzyme The enzyme according to the present invention comprises Pseudomonas strain ST-20.
0 is cultured and isolated and purified from the culture. The culture method is not particularly limited, and liquid culture or solid culture can be used. As the medium, a medium containing an appropriate carbon source and nitrogen source and containing appropriate amounts of phosphate, inorganic ions, and the like as necessary is used. During the culture, it is preferable to adjust the conditions of stirring and aeration appropriately. Furthermore, since Pseudomonas strain ST-200 has resistance to cyclohexane, it is possible to prevent contamination by other microorganisms without lowering the productivity of cholesterol oxidase by overlaying cyclohexane during culture.

The cholesterol oxidase known to date is often produced in the cells of microorganisms, and the extraction thereof requires a step of crushing 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 furnace liquid and a supernatant obtained by removing cells by filtration, centrifugation and the like can be obtained and recovered by subjecting them to various types of chromatography. In solid culture, after adding water to a medium, the cells are removed by filtration or centrifugation as in liquid culture to obtain a furnace solution or a supernatant, which can be recovered by subjecting it to various types of chromatography.

As a method for purifying the cholesterol oxidase from the culture solution, fractional precipitation such as ammonium sulfate precipitation or solvent precipitation,
Examples include chromatography such as ion exchange chromatography, hydrophobic chromatography, and gel filtration. In addition, desalting treatment such as dialysis may be performed as necessary. The type and order of the chromatography are not particularly limited.

Use of the enzyme The enzyme according to the present invention has cholesterol oxidase activity. Therefore, according to the present invention, there is provided a reagent for measuring the concentration of 3β-sterols (particularly, cholesterol) containing the enzyme according to the present invention. The concentration of 3β-sterols can be measured by contacting a sample with the enzyme of the present invention and measuring the degree of oxidation of the substrate, that is, 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.

According to the present invention, there is also provided a method for producing a sterol derivative. The production of the sterol derivative can be carried out by bringing the enzyme according to the present invention into contact with 3β-sterols, for example, under an organic solvent layer, and collecting the oxidized 3β-sterols. Examples of the sterol derivative to be produced include oxidized forms of 3β-sterols, for example, cholest-5-en-3-one and 6β-sterol.
Oxidized cholesterol such as perhydroxycholest-4-en-3-one.

Cholesterol oxidase destroys the midgut epithelium of insects and causes them to die (Purcell JP.
et al. , Biochem. Biophy. Re
s. Comm. , Vol. 196, No. 3, pp 1
406-1413 (1993); U.S. Pat.
No. 62). Thus, according to the present invention, there is provided a composition for controlling pests comprising the enzyme according to the present invention. Here, the pesticidal composition is a plant protectant, a pesticide, an insecticide,
It shall be used to include insect repellents and the like.

The composition for controlling pests according to the present invention includes, for example, an enzyme according to the present invention and a desired suitable compounding agent (for example, a surfactant,
Wetting agents, solid diluents, dispersants, ultraviolet stabilizers, etc.) can be added to obtain any dosage form such as wettable powders, powders, and flowables.

In addition, the composition for controlling pests of the present invention may be used, if necessary and / or desired, at the time of preparation or spraying, at the time of various insecticides, fungicides, herbicides, plant growth regulators, synergists (in the culture supernatant). Included activity enhancers, etc.), attractants, plant nutrients, fertilizers and the like.

In general, pest control is carried out by adding a pest control composition or a pest control composition diluted with a diluent (eg, water) to a plant that is or is expected to be damaged by the pest. It can be carried out by spraying.

Cholesterol oxidase acts on substrates to generate hydrogen peroxide. Enzyme systems that generate hydrogen peroxide have a bleaching effect, and such enzymes can be added to detergents as bleaching agents (WO 89/09813, JP-A-3-505).
No. 100). Thus, according to the present invention there is provided a detergent composition comprising the enzyme according to the present invention.

The detergent composition according to the invention comprises the enzyme according to the invention together with surfactants, builders and other additives (for example, optical brighteners, foaming agents, foam inhibitors, softeners, perfumes, etc.). Or may comprise the enzyme according to the invention alone.

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. Further, 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 which has been genetically modified for the purpose of enhancing thermostability. Therefore, the enzyme according to the present invention is advantageous for measuring the concentration of 3β-sterols (particularly, low cholesterol concentration) in body fluids and foods.

The enzyme according to the invention also has the property of having a high reaction rate under an organic solvent overlay. In many cases, the conversion of sterols by an enzyme is performed under an overlay of an organic solvent, and it is required that the converting enzyme is active under the overlay of the organic solvent. The enzyme according to the present invention is advantageous in that sterols can be efficiently converted under an organic solvent layer.

Examples The present invention will be described with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Purification of enzyme Pseudomonas strain ST-200 (FERM BP-
6661) in a 10 liter fermenter, 1% tryptone (Difco), 0.5% yeast extract (Di
fco) and 6% of a medium containing 1% sodium chloride at 30 ° C. for 17 hours. Stirring speed is 40
0 rpm and aeration at 12 liters per minute.

The obtained culture was centrifuged at 8,000 × g for 15 minutes to obtain a supernatant. Subsequently, the precipitate was salted out (4 ° C., overnight) with ammonium sulfate having a saturation of 70%, and the precipitate was collected by centrifugation at 10,000 × g for 30 minutes. The obtained precipitate was dissolved in a 10 mM Tris-HCl buffer (pH 8), and dialysis was repeated twice against the same buffer.

Next, the dialyzed precipitate was subjected to DEAE cellulose DE5.
Apply to 2 columns, 10 mM Tris-HCl buffer (pH
Eluted in isocratic in 8). Ammonium sulfate was added to the active fraction to a final concentration of 45% saturation and centrifuged (700,000).
(0 × g, 15 minutes), and the supernatant was recovered. Then, 45
The active fraction was applied to a column packed with butyltoyopearl 650S equilibrated with 10 mM Tris-HCl buffer (pH 8) containing% saturated ammonium sulfate, and eluted with a linear gradient of ammonium sulfate up to 0M. The active fraction was present in the 10% saturated to 0M ammonium sulfate concentration fraction, and was collected to a saturation degree of 80%.
Of ammonium sulfate was added for salting out.

After dissolving the precipitate in 10 mM Tris-HCl buffer (pH 8), dialysis was repeated twice against the same buffer. Subsequently, fractionation was performed using a solution containing 10 mM Tris-HCl buffer (pH 8), 50 mM sodium chloride and 5 mM sodium cholate using a Sephadex G-100 column.
The active fractions were collected and 10 mM Tris-HCl buffer (pH
8) was dialyzed to obtain purified cholesterol oxidase.

The activity recovery of the obtained sample was 20%. The specific activity was 15.2 U / mg, which was 36 times that before purification. As a result of measurement by SDS polyacrylamide gel electrophoresis, the molecular weight was 60 kDa. Strongly oxidizes cholesterol, β-sitosterol, β-cholestanol, etc., does not act on epicholesterol,
It had the characteristic of being strongly inhibited by silver nitrate and mercury chloride.

Test Example 1 Effect of pH and Temperature on Enzyme The pH, temperature dependence and stability of the enzyme obtained in Example 1 were measured.

The pH and temperature dependence were measured by a method of measuring the amount of oxygen consumed when cholesterol was used as a substrate. Specifically, the enzyme according to the present invention is added to a solution containing 50 mM phosphate buffer (pH 7.0), 64 mM sodium cholate, 0.34% surfactant Triton X-100 and 0.89 mM cholesterol. Then, the amount of consumed oxygen was measured. The measurement of the amount of oxygen is performed using a DO meter (YSIM
ode153, Yellow Spring, Ohi
o, USA). One unit of cholesterol oxidase activity was defined as the activity of oxidizing 1 micromole of cholesterol per minute. Regarding pH dependency, the activity at pH 7 was set to 100%. Regarding temperature dependency, the activity at 60 ° C. was set to 100%.

The pH and temperature stability is 30
The remaining activity (using cholesterol as a substrate) after the dispensing was carried out at 30 ° C. and pH 7 by a method of measuring hydrogen peroxide generated during the enzymatic reaction. Specifically, the procedure was performed as follows.

Phosphate buffer (pH 7.0) is added to the solution containing the enzyme of the present invention at an appropriate concentration so that the final concentration is 50 mM each.
0), sodium cholate to a concentration of 64 mM;
Surfactant Triton X-100 to be 34%,
Aminoantipyrine, 21 mM to be 1.4 mM
5 ml of a solution containing phenol, 5 units of wasabi-derived peroxidase (manufactured by Toyobo Co., Ltd.) and 0.89 mM of cholesterol, and
The reaction was carried out at 30 ° C. for 5 minutes while measuring the absorbance at m.
One unit of cholesterol oxidase activity was defined as the activity of oxidizing 1 micromole of cholesterol per minute.

The results are as shown in FIGS. The enzyme according to the present invention shows strong activity at pH 5.0 to 8.5,
It was stable at 4.0 to 11.0. Also, 50-60 ° C
Showed strong activity and was stable at 4-55 ° C.

Test Example 2 Substrate specificity of enzyme The substrate specificity of the enzyme obtained in Example 1 was examined. The enzyme activity was measured using various substrates according to Test Example 1.
The reaction was carried out at a temperature of 7 ° C. and a pH of 7. As a result, as shown in Table 1, cholesterol, β-sitosterol and β-cholestanol were strongly oxidized and did not act on epicholesterol which is a 3α-hydroxysteroid.

Test Example 3 Effect of Metal Ions on Enzyme Activity The effects of metal ions and the like on the activity of the enzyme obtained in Example 1 were examined. The enzyme activity was measured at 30 ° C. and pH 7 according to Test Example 1 after adding various metal compounds to the reaction system to a concentration of 1 mM. as a result,
As shown in Table 2, it was strongly inhibited by silver nitrate and mercury chloride.

Test Example 4 Enzyme Reaction Kinetics The Michaelis constant (K _m ) and the maximum reaction rate (V _max ) of the enzyme obtained in Example 1 were 0.03% and 0.3%.
In the presence of the surfactant Triton X-100.
The enzyme activity was measured using various enzymes according to Test Example 1.
The test was performed at 0 ° C. and pH 7. As a result, as shown in Table 3, Nocardia erythropolis (Nocardia)
erythropolis, Pseudomonas (Ps
eudomonas sp. ), Streptomyces sp. And Brevibacterium sp. Cholesterol oxidase showed a small K _m and a large V _max, and showed a maximum V _max / K _m .

Test Example 5 Effect of Organic Solvent on Enzyme Activity 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% volume of each organic solvent was overlaid on the solution. Enzyme activity was measured in 30
The reaction was carried out at a temperature of 7 ° C. and a pH of 7. As a result, as shown in Table 4, the enzymes according to the present invention were obtained from Nocardiaerythropolis, Pseudomonas sp. And Streptomyces ssp.
p. ), Brevibacte
rium sp. The reaction rate was higher under the superposition of benzene, toluene, valaxylene, propylbenzene, diphenylmethane and cyclooctane as compared to cholesterol oxidase of (1).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the pH dependence of the enzyme according to the invention. ○ indicates 50 mM acetic acid-sodium acetate buffer, は indicates 50 mM 1 potassium phosphate-disodium phosphate buffer, and □ indicates 50 mM acetic acid-sodium acetate buffer.
mM Tris-HCl buffer, ● indicates that a 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 50 mM acetic acid-sodium acetate buffer, は indicates 50 mM 1 potassium phosphate-disodium phosphate buffer, and □ indicates 50 mM acetic acid-sodium acetate buffer.
mM Tris-HCl buffer, ● indicates the use of 50 mM sodium carbonate-sodium bicarbonate buffer, and Δ indicates the use of 50 mM sodium chloride-sodium hydroxide buffer.

 FIG. 4 shows the temperature stability of the enzyme according to the invention.

Continuation of front page (56) References Appl. Environ. Microbiol. , Vol. 60 [7] (1994) p. 2518-2523 Appl. Microbiol. Biotechnol. , Vol. 31 [5/6] (1989) p. 542-546 Biochem. Biophys. Acta. , Vol. 999 [3] (1989) p. 233-238 Gen. Microbiol. , Vol. 137 [5] (1989) p. 1213-1214 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9/04 A01N 63/00 C11D 3/386 BIOSIS (DIALOG) WPI (DIALOG)

Claims (13)

(57) [Claims] 1. An enzyme having the following properties: (1) action: acting on cholesterol to convert it to cholest-5-en-3-one; acting on cholest-5-en-3-one Is converted to 6β-perhydroxycholest-4-en-3-one; (2) substrate specificity: acting on 3β-sterols, 3α
(3) optimal pH: pH 5.0 to 8.5; (4) stable pH: pH 4 to 11. 2. The enzyme according to claim 1, which further has the following properties: (5) optimum temperature: about 60 ° C .; (6) stable temperature: 4 to 55 ° C .; (7) water and n-octanol. The reaction rate increases in the presence of an organic solvent having a common logarithmic value (logP _ow ) of the partition coefficient P _ow of any substance between the two phases of 2.1 to 4.5; (8) molecular weight: SDS- Approximately 60k measured by PAGE
Da; (9) Inhibitor: Enzyme activity when cholesterol is used as a substrate is inhibited by silver nitrate and mercury chloride. 3. The maximum reaction rate (V _max : μmole · min ⁻¹ · mg ⁻¹ ) and Michaelis constant (K _m : μM) of cholesterol in the presence of 0.3% surfactant Triton X-100. or the ratio V _max / K _m is 0.20 or more, or 0.03% surfactant TRITON X-100 Vmax / Km in the presence of 3.0 or more, enzymes according to claim 1. 4. The enzyme according to claim 1, which is produced by a cyclohexane-resistant microorganism. 5. The enzyme according to claim 1, which is produced by a cyclohexane-resistant microorganism of the genus Pseudomonas. 6. The ST-200 strain (FERM BP-6)
661). The enzyme of claim 1 produced according to (661). 7. A method for measuring the concentration of 3β-sterols, comprising a step of contacting a sample with the enzyme according to any one of claims 1 to 6 and measuring cholesterol oxidase activity. 8. The method according to claim 7, wherein the sample is a sample separated from a mammal or a sample separated from a food.
A method for measuring the concentration of β-sterols. 9. A reagent for measuring the concentration of 3β-sterols, comprising the enzyme according to any one of claims 1 to 6. 10. A pest control composition comprising the enzyme according to any one of claims 1 to 6. 11. A detergent composition comprising the enzyme according to any one of claims 1 to 6. 12. A method for producing a sterol derivative, comprising a step of bringing the enzyme according to any one of claims 1 to 6 into contact with 3β-sterols to recover the sterol derivative. 13. The method according to claim 12, wherein the enzyme and the 3β-sterol are contacted under an organic solvent layer.