JP2012024048A - Deodorizer comprising unsaturated aldehyde blended with enzyme, and method of deodorization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To change a causative substance of body odor to another substance by utilizing an enzyme, and more concretely, to provide a method for eliminating the bad odor of the body and further changing it to an aroma.SOLUTION: It was found out that an enone-reductase, having enzyme activity for selectively reducing the α, β-unsaturated bond of an α, β-unsaturated ketone to form an α, β-saturated ketone, has an effect of eliminating the aging odor and also changing it to a good smell. By using the enone-reductase, it is possible to convert the generated substance becoming the cause of body odor to a substance having the aroma. That is, by using the enone-reductase, the body odor can be changed and also the once generated body odor can be fundamentally eliminated.

Description

The present invention relates to a method for deodorizing unsaturated aldehydes using a reaction for reducing carbon-carbon double bonds of unsaturated aldehydes. The present invention also relates to a composition for eliminating the odor of unsaturated aldehydes, which contains, as an active ingredient, a reducing catalyst that catalyzes a reaction of reducing carbon-carbon double bonds of unsaturated aldehydes. The invention further relates to the use of a reducing catalyst for catalyzing the reaction of reducing the carbon-carbon double bond of an unsaturated aldehyde for use in eliminating the odor of an unsaturated aldehyde.

Body odors can be roughly classified into "smell of each part of the body" such as halitosis, foot odor, axillary odor, and scalp odor, and "smell that combines odors emitted from the trunk". Regarding the former "smell of each part of the body", many research results have been reported so far, and the main components of the odor have been elucidated. Also, many excellent coping methods are presented.
For example, it is known that sweat odor, axillary odor, foot odor, and the like are produced by "sweat" that is the source of odor and "skin-resident bacteria" that produces odor. Sweat is secreted mainly by the sweat glands, which are composed of eccrine glands and apocrine glands. Sweat secreted by the eccrine glands distributed throughout the human body is called eccrine sweat. Most of them are composed of water, and also contain some sodium chloride, lactic acid, urea and the like. On the other hand, the apocrine gland is a sweat gland that accompanies hair, and is present only in specific parts such as the axilla, areola, and pubic area. The apocrine glands secreted from here are milk-like sweat containing proteins, lipids, fatty acids, cholesterol, glucose, ammonia, iron and the like.

These secreted sweats do not give off a strong odor by themselves. However, it is known that the bacteria resident on the skin surface transform it into an odor substance. As a result of analyzing the axillary odor and foot odor, lower fatty acids such as pelargonic acid and capric acid were specifically detected in the axillary odor. In addition, isovaleric acid is specifically detected in foot odor, which is regarded as the cause of body odor.

It has also been pointed out that there is a body odor (age odor) peculiar to middle-aged and elderly people that occurs with aging ("Fragrance Journal", September 1999, pp. 42-46 (Non-Patent Document 1). Japanese Patent Application Laid-Open No. 11-286428 (Patent Document 1)). For the generation of age-related odor, substances containing unsaturated groups such as 2-nonenal and 2-octenal, which are one of the volatile aldehydes generated from 9-hexadecenoic acid and the like, which increase in the sebum of middle-aged humans, are deeply involved. Known to be involved.

At present, various measures are taken as a means for preventing these body odors. They can be roughly classified according to the principle as follows.
(1) A masking or harmony of body odor using an aromatic component (fragrance, essential oil, etc.).
(2) A body odor component, or a body odor component that is physically adsorbed together with its precursor to suppress volatilization of the body odor component.
(3) A substance that suppresses the generation of body odor components from precursors due to oxidation reaction and bacterial activity.
The above method (1) does not suppress the generation of body odor components. In addition, a mixture of a problematic body odor and a fragrance due to an aroma component may lead to the generation of an undesired new odor. Therefore, it is hard to say that it is the solution to the fundamental problem.
As a typical example of the above method (2), for example, a method of suppressing volatilization of body odor components by utilizing the inclusion ability and adsorption ability of an inorganic porous material, cyclodextrin, or an organic porous material such as activated carbon There is. The method using cyclodextrin cannot be said to be a solution to the fundamental problem because the body odor component once clathrated may be released again due to the coexistence of another substance. On the other hand, the method using activated carbon is difficult to show immediate effect. In addition, it is difficult to maintain a sufficient amount of activated carbon in the form of powder on the skin, and it is not always effective because direct application to the living body is limited.
Typical examples of the above method (3) include an antioxidant substance that suppresses the oxidation of 9-hexadecenoic acid, which is a fatty acid that serves as a substrate of nonenal, an antibacterial substance that suppresses the decomposition of 9-hexadecenoic acid, and an enzyme inhibitor. Examples include methods using agents, trehalose, plant extracts and the like. This method may be effective in suppressing the generation of body odor. However, in order to use a sufficient amount of an antioxidant or an antibacterial substance to suppress the generation of body odor by itself, it is necessary to pay attention to irritation to the skin, for example.

As described above, all the known preventive measures against body odor are symptomatic. There is no known means for eliminating body odor itself by decomposing and removing the causative substance of body odor.

Japanese Patent Laid-Open No. 11-286428 Japanese Patent Laid-Open No. 11-286425 JP-A-2002-80336 JP 2002-247987 A JP-A-2003-33185 JP, 2010-57482, A JP 2001-302483 A

Fragrance Journal, September 1999, 42-46.

An object of the present invention is to provide a method of changing a causative substance of body odor to another substance using an enzyme, more specifically, a method of eliminating a bad odor of body odor and changing it to an aroma. In particular, it is an object of the present invention to provide a method for reducing the carbon-carbon double bond of an unsaturated aldehyde to eliminate the aging odor caused by the unsaturated aldehyde and change it to an aroma.

So far, the present inventors have repeatedly studied whether or not the structure of unsaturated aldehydes can be changed to eliminate the aging odor. Among them, the present inventors tried to react the enone reductase with a solution containing trans-2-nonenal. As a result, they have found that reduction of the carbon-carbon double bond of unsaturated aldehyde is effective in eliminating aging odor, and completed the present invention.

Specifically, the present inventors confirmed that the malodor caused by trans-2-nonenal disappears as a result of reacting enone reductase with a solution containing trans-2-nonenal. Furthermore, the present inventors have found that not only does the malodor disappear, but it also changes to an aroma with a refreshing sensation (smell of lemon lime). On the other hand, it was confirmed that when the alcohol dehydrogenase and the aldehyde dehydrogenase were reacted with the solution containing trans-2-nonenal, the malodor caused by trans-2-nonenal could not be eliminated.
Similar results were shown when enone reductase, alcohol dehydrogenase, and aldehyde dehydrogenase were reacted with a solution containing trans-2-hexenal and trans-2-octenal.
Furthermore, when the present inventors spray a solution containing enone reductase onto a gauze containing trans-2-nonenal or a shirt worn by a man for one day, the gauze or shirt eliminates a foul odor and changes into an aroma. I also confirmed that it is possible.
The present invention is based on such findings and relates to the following methods, compositions, uses and the like.
[1] A method for eliminating the odor of an unsaturated aldehyde, which comprises the step of reducing the carbon-carbon double bond of the unsaturated aldehyde.
[2] The step of reducing the carbon-carbon double bond of the unsaturated aldehyde is a step of contacting a reducing catalyst with the unsaturated aldehyde under conditions capable of reducing the carbon-carbon double bond, The method according to [1].
[3] The method according to [2], wherein the condition capable of reducing the carbon-carbon double bond is in the presence of an electron donor.
[4] The method according to [2], wherein the reduction catalyst is an enone reductase.
[5] The method according to [4], wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2,
(C) The amino acid sequence set forth in SEQ ID NO: 2, which comprises an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E) A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) and (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2, and which encodes a protein having the following physicochemical properties (A) and (E):
(A) Action Using reduced β-nicotinamide adenine dinucleotide phosphate as an electron donor, the carbon-carbon double bond of α,β-unsaturated ketone is reduced to generate the corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) It reduces the carbon-carbon double bond of an α,β-unsaturated ketone, but has substantially no ketone reducing activity.
(2) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(3) It does not substantially act on a substrate in which the two substituents from the ketone to the β-carbon are not hydrogen.
(4) The carbon-carbon double bond does not substantially act on the substrate present in the cyclic structure.
(C) Optimum pH
pH 6.5-7.0
(D) Optimum temperature 37-45°C
(E) Molecular weight sodium dodecylsulfate-polyacrylamide gel electrophoresis about 43,000. About 42,000 by gel filtration.
[6] The method according to [4], wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4,
(C) The amino acid sequence of SEQ ID NO: 4, which is composed of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E) A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) to (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, and which encodes a protein having the following physicochemical properties (A) to (E):
(A) Action The reduced β-nicotinamide adenine dinucleotide phosphate is used as an electron donor to reduce a carbon-carbon double bond of an α,β-unsaturated ketone to generate a corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(2) It reduces a carbon-carbon double bond of an unsaturated ketone, but has substantially no ketone reducing activity.
(3) 3-Methyl-4-(3-pyridyl)-3-buten-2-one is selectively reduced with α,β-unsaturated bonds to give 90% ee or more of (S)-3-methyl- Generates 4-(3-pyridyl)-3-butan-2-one.
(C) Molecular weight sodium dodecylsulfate-about 47,000 by polyacrylamide gel electrophoresis. About 92,000 by gel filtration.
(D) Working pH
pH 5.0-8.0
(E) Optimum temperature 37-45°C
[7] The method according to [3], wherein the electron donor is at least one coenzyme selected from the group consisting of (1) to (6) below:
(1) reduced nicotinamide adenine dinucleotide phosphate (NADPH),
(2) reduced nicotinamide adenine dinucleotide (NADH),
(3) reduced flavin mononucleotide (FMNH ₂ ),
(4) reduced flavin adenine dinucleotide (FADH ₂ ),
(5) Ubiquinone (UQ), and (6) Pyrroloquinoline quinone (PQQ).
[8] The method according to [1], wherein the unsaturated aldehyde is selected from the group consisting of 2-nonenal, 2-octenal, and 2-hexanal.
[9] A composition for eliminating an odor of an unsaturated aldehyde, which contains, as an active ingredient, a reduction catalyst that catalyzes a reaction of reducing a carbon-carbon double bond of an unsaturated aldehyde.
[10] The composition according to [9], which further contains an electron donor.
[11] The composition according to [9], wherein the reduction catalyst is an enone reductase.
[12] The composition according to [11], wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2,
(C) The amino acid sequence set forth in SEQ ID NO: 2, which comprises an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E) A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) and (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2, and which encodes a protein having the following physicochemical properties (A) and (E):
(A) Action Using reduced β-nicotinamide adenine dinucleotide phosphate as an electron donor, the carbon-carbon double bond of α,β-unsaturated ketone is reduced to generate the corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) It reduces the carbon-carbon double bond of an α,β-unsaturated ketone, but has substantially no ketone reducing activity.
(2) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(3) It does not substantially act on a substrate in which the two substituents from the ketone to the β-carbon are not hydrogen.
(4) The carbon-carbon double bond does not substantially act on the substrate present in the cyclic structure.
(C) Optimum pH
pH 6.5-7.0
(D) Optimum temperature 37-45°C
(E) Molecular weight sodium dodecylsulfate-polyacrylamide gel electrophoresis about 43,000. About 42,000 by gel filtration.
[13] The composition according to [11], wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4,
(C) The amino acid sequence of SEQ ID NO: 4, which is composed of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E): A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) to (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, and which encodes a protein having the following physicochemical properties (A) to (E):
(A) Action Using reduced β-nicotinamide adenine dinucleotide phosphate as an electron donor, the carbon-carbon double bond of α,β-unsaturated ketone is reduced to generate the corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(2) It reduces a carbon-carbon double bond of an unsaturated ketone, but has substantially no ketone reducing activity.
(3) 3-Methyl-4-(3-pyridyl)-3-buten-2-one is selectively reduced with α,β-unsaturated bonds to give 90% ee or more of (S)-3-methyl- Generates 4-(3-pyridyl)-3-butan-2-one.
(C) Molecular weight sodium dodecylsulfate-about 47,000 by polyacrylamide gel electrophoresis. About 92,000 by gel filtration.
(D) Working pH
pH 5.0-8.0
(E) Optimum temperature 37-45°C
[14] The composition according to [10], wherein the electron donor is at least one coenzyme selected from the group consisting of the following (1) to (6):
(1) reduced nicotinamide adenine dinucleotide phosphate (NADPH),
(2) reduced nicotinamide adenine dinucleotide (NADH),
(3) reduced flavin mononucleotide (FMNH ₂ ),
(4) reduced flavin adenine dinucleotide (FADH _2), and (5) ubiquinone (UQ), and (6) pyrroloquinoline quinone (PQQ).
[15] The composition according to [9], wherein the unsaturated aldehyde is selected from the group consisting of 2-nonenal, 2-octenal and 2-hexanal.
[16] A reduction catalyst which catalyzes a reaction for reducing a carbon-carbon double bond of an unsaturated aldehyde for use in eliminating the odor of an unsaturated aldehyde.

INDUSTRIAL APPLICABILITY The present invention provides a method for eliminating a causative substance of aging odor by reducing a carbon-carbon double bond of an unsaturated aldehyde, and a composition therefor. Conventionally, as a countermeasure against body odor, means such as masking, adsorption, and suppression of generation of a causative substance have been used. However, these methods are symptomatic means and were unable to completely eliminate the once-generated body odor.
On the other hand, in the method and composition of the present invention, the substance that causes the generated body odor can be changed to another substance. Therefore, by using the method and composition of the present invention, it is possible to fundamentally eliminate body odor once generated. Furthermore, in the method and composition of the present invention, the body odor can be changed from a bad odor to an aroma. Such an effect is not found in the known means for suppressing the aging odor.
Further, in the present invention, an enone reductase is used in a preferred embodiment. Enzymes, unlike antioxidants and antibacterial agents, can be used safely.

The present invention relates to a method for eliminating an odor of an unsaturated aldehyde, which comprises a step of reducing a carbon-carbon double bond of the unsaturated aldehyde.
In the present invention, the unsaturated aldehyde means an unsaturated hydrocarbon having an aldehyde group. The unsaturated aldehydes of the present invention often give off an odor that causes body odor. Examples of unsaturated aldehydes in the present invention include, but are not limited to, nonenal (eg, 2-nonenal), octenal (eg, 2-octenal), hexenal (eg, 2-hexenal), and the like.
The present invention is characterized by reducing a carbon-carbon double bond of an unsaturated aldehyde. Below, the reduction reaction of the carbon-carbon double bond of unsaturated aldehyde is illustrated. The following example is a reaction in which nonanal is produced by reducing the carbon-carbon double bond at the 2-position of 2-nonenal.

In the present invention, "eliminate an odor of an unsaturated aldehyde" means that the odor generated by the unsaturated aldehyde disappears. Therefore, "deodorizing" can also be expressed as "deodorize" or "cause an odor disappearing". In the present invention, it can be judged that the odor has disappeared when the odor generated due to the unsaturated aldehyde disappears as compared with that before the reduction of the carbon-carbon double bond.
Whether or not the odor of the unsaturated aldehyde has disappeared can be determined, for example, by directly smelling the odor. For example, 2-nonenal, which is one of unsaturated aldehydes, has both blue odor and greasy odor. Alternatively, it can be described as having a stink bug smell (the stink bug smell is derived from trans-2-hexenal). On the other hand, nonanal obtained by reducing C=C at the 2-position of 2-nonenal has a refreshing odor. From such a difference in odor, it is possible to determine whether or not the odor of the unsaturated aldehyde has disappeared.
Alternatively, an analyzer can be used to determine the presence or absence of the odor of unsaturated aldehydes. For example, when checking the presence or absence of unsaturated aldehyde contained in a shirt, gauze pad sewn on the shirt in advance is dipped in a solvent such as hexane to extract lipids. The presence or absence of unsaturated aldehydes in the sample can be confirmed by analyzing the sample collected in this manner using a device such as headspace gas chromatography or mass spectrum.
Alternatively, as described in Examples, the presence or absence of unsaturated aldehyde can be confirmed by derivatizing an aldehyde with dinitrophenylhydrazine (DNPH) by a conventional method and analyzing by GC.

In the present invention, "the step of reducing a carbon-carbon double bond of an unsaturated aldehyde" refers to, for example, "contacting a reduction catalyst with an unsaturated aldehyde under conditions capable of reducing a carbon-carbon double bond". Can be performed. The conditions under which the carbon-carbon double bond can be reduced include, for example, contacting a reducing catalyst with an unsaturated aldehyde in the presence of an electron donor. Examples of electron donors include, but are not limited to, the following.
・Reduced nicotinamide adenine dinucleotide phosphate (NADPH)
・Reduced nicotinamide adenine dinucleotide (NADH)
・Reduced flavin mononucleotide (FMNH ₂ )
And reduced flavin adenine dinucleotide (FADH ₂₎
・Ubiquinone (UQ)
・Pyrroloquinoline quinone (PQQ)

As a preferable reduction catalyst in the present invention, an enone reductase derived from Kleiberomyces lactis (KLER1, JP 2002-247987) can be mentioned. An example of such an enone reductase is an enzyme having physicochemical properties shown in the following (A1)-(E1).
(A1) Action
NADPH is used as an electron donor to reduce a carbon-carbon double bond of an α,β-unsaturated ketone to generate a corresponding saturated hydrocarbon.
(B1) Substrate specificity
(1) It reduces the carbon-carbon double bond of an α,β-unsaturated ketone, but has substantially no ketone reducing activity.
(2) As an electron donor, it has significantly higher activity against NADPH than NADH.
(3) It does not substantially act on a substrate in which the two substituents from the ketone to the β-carbon are not hydrogen.
(4) The carbon-carbon double bond does not substantially act on the substrate present in the cyclic structure.
(C1) Optimal pH
pH 6.5-7.0
(D1) Optimum temperature 37-45°C
(E1) Molecular weight Approximately 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS-PAGE). About 42,000 by gel filtration.
The nucleotide sequence of the enone reductase KLER1 derived from Kluyveromyces lactis is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO: 2.

Alternatively, as the enone reductase in the present invention, an enone reductase derived from Kleiberomyces lactis (KYE1 (KLER2), JP 2003-33185) having the physicochemical properties shown in (A2)-(E2) below. Is mentioned.
(A2) Action Using the reduced β-nicotinamide adenine dinucleotide phosphate as an electron donor, the carbon-carbon double bond of the α,β-unsaturated ketone is reduced to generate the corresponding saturated hydrocarbon.
(B2) Substrate specificity
(1) As an electron donor, it has significantly higher activity against NADPH than NADH.
(2) It reduces the carbon-carbon double bond of unsaturated ketones, but has virtually no ketone reducing activity.
(3) 3-Methyl-4-(3-pyridyl)-3-buten-2-one is selectively reduced with α,β-unsaturated bonds to give 90% ee or more of (S)-3-methyl- Generates 4-(3-pyridyl)-3-butan-2-one.
(C2) Molecular weight Sodium dodecyl sulfate-polyacrylamide gel electrophoresis about 47,000. About 92,000 by gel filtration.
(D2) Working pH
pH 5.0-8.0
(E2) Optimum temperature 37-45°C
The nucleotide sequence of the enone reductase KYE1 derived from Kluyveromyces lactis is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO: 4.

The phrase "the reactivity with NADPH is significantly higher than the reactivity with NADH" means an activity that is at least 2-fold or higher, preferably 3-fold or higher, and more preferably 5-fold or higher. The reactivity to NADPH and NADH can be compared, for example, as follows. That is, the same α,β-unsaturated ketone is used as a substrate, and both are used to generate a ketone. Reactivity can be compared by comparing the amount of NADPH or NADH consumed at this time.

Further, "the enone reductase does not substantially act on the substrate" or "the enone reductase has substantially no ketone reducing activity" means, specifically, the reducing activity of ethyl vinyl ketone for olefins. It means 1% or less. The selective reduction of the α,β-unsaturated bond means that the ketone is not substantially reduced under the condition for reducing the olefin. In the present invention, the ketone is not substantially reduced when the product in which the ketone moiety is reduced is, for example, 5% or less, usually 2% or less, and preferably 1% or less of the substrate. The activity of reducing the ketone moiety can be compared, for example, by allowing a test enzyme to act on 2-butanone and quantifying the amount of the reduction product. The amount of reduction products can also be known by using the decrease in NADPH as an index.

The enone reductase can be purified from the microorganism producing the enzyme by a conventional protein purification method. For example, after crushing the cells, protamine sulfate precipitation is performed, the centrifugation supernatant is salted out with ammonium sulfate, and further combined with anion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, etc. Can be purified by

In the present invention, the reducing activity for enone can be confirmed as follows. In the present invention, enone means α,β unsaturated ketone.
Method for measuring reducing activity for enone: 50 mM potassium phosphate buffer (pH 6.5), 0.2 mM NADPH, 20 mM ethyl vinyl ketone, and a reaction solution containing an enzyme at 30° C., and absorbance at 340 nm accompanying decrease in NADPH To measure the reduction of. 1 U can be the amount of enzyme that catalyzes the decrease of 1 μmol NADPH in 1 minute. The protein can be quantified by a dye binding method using a protein assay kit manufactured by Bio-Rad.

The enone reductase having the above-mentioned physicochemical properties can be purified, for example, from a culture of yeast of the genus Kleiberomyces. As a yeast of the genus Kliberomyces, Kluyveromyces lactis is particularly excellent in the ability to produce enone reductase. Kluyveromyces lactis that can be used to obtain enone reductase can be obtained from the Fermentation Research Institute as IFO0433, IFO 1012, IFO 1267, IFO 1673, IFO 1903, for example.

The above-mentioned microorganism is cultured in a general medium used for culturing fungi such as YM medium. After sufficiently proliferating, the microbial cells are collected and crushed in a buffer solution containing a reducing agent such as 2-mercaptoethanol or phenylmethaneflufonyl fluoride or a protease inhibitor to obtain a cell-free extract. Fractionation based on protein solubility (precipitation with organic solvents, salting out with ammonium sulfate, etc.), cation exchange, anion exchange, gel filtration, hydrophobic chromatography, chelates, dyes, antibodies, etc. from cell-free extracts It can be purified by an appropriate combination of affinity chromatography and the like. For example, it can be electrophoretically purified to a substantially single band through hydrophobic chromatography using phenyl-Sepharose, anion exchange chromatography using MonoQ, hydrophobic chromatography using phenyl-superose, and the like. ..
The enone reductase that can be purified from Kluyveromyces lactis has the physicochemical properties (A1)-(E1) or (A2)-(E2).

The enone reductases listed below can also be exemplified as the reductases that catalyze the reaction of reducing the carbon-carbon double bond of the unsaturated aldehyde in the present invention.
・Enone reductase from baker's yeast ・Enone reductase from tobacco ・Enone reductase from rat liver ・Saccharomyces ・Carlsbergensis and Saccharomyces ・Old yellow enzyme from Cerevisiae ・Clostridium ・Tyrobutyricum 2-enoate reductase ・Acryloyl-CoA reductase derived from Clostridium klaiberg, Saccharomyces cerevisiae-derived enzyme having enone reducing activity, and enone reductase derived from oyster mushroom extract

For example, a plurality of enone reductases have been purified from baker's yeast and reported. Kawai et al. of Kyoto University have purified enone reductase (YER-2) from baker's yeast and reported the enzyme chemistry (Proceedings of the 4th Biocatalyst Symposium p58 (2001)). This enzyme, like KLER2, also selectively reduces the α,β-unsaturated bond of 3-Methyl-4-(3-pyridyl)-3-buten-2-one to give (S)-3-methyl- It has been reported to produce 4-(3-pyridyl)-3-butan-2-one. Its molecular weight is 38,000 by gel filtration and 41,000 by SDS-PAGE. The optimum pH for the reaction is 7.5.
Wanner et al. reported the purification and properties of two enone reductases (EI and EII) from the same baker's yeast (Eur. J. Biochem. 255, 271-278 (1998)). EI is a heterodimer with a molecular weight of 75,000 in gel filtration and 37,000 and 34,000 by SDS-PAGE. EII is a heterodimer with a molecular weight in gel filtration of 130,000 and a molecular weight by SDS-PAGE of 56,000 and 64,000.

In plants, a large number of enone reductases (Verbenone reductase (aka p90, DDBJ accession number: amino acid sequence C0SUC5, base sequence AB488494), carvone reductase (alias, enone reductase-I, and Alias p44), enone reductase-II, p74) have been purified and their properties have been reported. The molecular weight of verbenone reductase (p90) in gel filtration is 90,000, and that by SDS-PAGE is 45,000. The optimum pH for the reduction reaction is 7.2.

Enone reductase-I (p44) has a molecular weight of 44,000 in gel filtration and a molecular weight of 22,000 by SDS-PAGE, and mainly utilizes NADH.
Enone reductase-II also uses a compound ((R)-pulegone) having no hydrogen at the β-position carbon of an α,β-unsaturated ketone as a substrate. The molecular weight in gel filtration is 132,000, and the molecular weight by SDS-PAGE is 22,000 and 45,000 (Phytochemistry 31, 2599-2603 (1992)).
p74 has a molecular weight of 74,000 by gel filtration and a molecular weight of 37,000 by SDS-PAGE.

In addition, from the plant species Euglena gracilis (gel filtration, molecular weight by SDS-PAGE 55,000;) and Astasia longa (Astasia longa; gel filtration, molecular weight by SDS-PAGE both 35,000 or 36,000) The enone reductase has been purified (Phytochemistry 49, 49-53 (1998)). All of these enzymes utilize NADH as a coenzyme.

In animals, enone reductase has been purified from rat liver (Arch. Biochem. Biophys. 282, 183-187 (1990)). This enzyme has a molecular weight of 39,500 by gel filtration and SDS-PAGE.

Old yellow enzyme (Old Yellow Enzyme, hereinafter abbreviated as OYE1) derived from Saccharomyces carlesbergensis and Saccharomyces cerevisiae has NADPH oxidative activity and not only oxygen as an electron acceptor but also It has been reported that cyclohexanone is produced by using Cyclohex-2-enone as an electron acceptor (J. Biol. Chem. 268, 6097-6106 (1993)). In addition, OYE2 and OYE3 having similar activity have been cloned from Saccharomyces cerevisiae. The OYE1 gene was cloned, its nucleotide sequence and predicted amino acid sequence were clarified, and it was revealed that it has high homology with the enone reductase KLER2 (homology search result using BLAST program, 71% identity, 84%similarity). It is estimated that this hydrogenation reaction of enone by OYE1 takes precedence over the Re-face for α-carbon and over the Si-face for β-carbon (Biochemistry 34, 4246-4256 ( 1995)). However, the optical purity of the ketone formed is not specifically shown, and it is clear whether the method for producing an optically active ketone using OYE1 satisfies the industrially required purity (at least 90%ee or higher). It hasn't been.

2-enoate reductase (EC1.3.1.31, DDBJ accession number: Y09960) derived from Clostridium tyrobutyricum is (E)-2-methyl-2-butenoic acid in a NADH-dependent manner. Is reduced to produce (R)-2-methylbutyric acid (J. Biotechnol. 6, 13-29 (1987)). In addition, this enzyme serves as a substrate when the carbonyl residue is a carboxylic acid, an aldehyde, or a keto acid, but no activity against a ketone body has been reported. Furthermore, the molecular weight is 800,000-940,000 in gel filtration.

Also, it has been reported that acryloyl-CoA reductase derived from Clostridium kluyveri has ethyl vinyl ketone reducing activity (Biol. Chem. Hoppe-Seyler 366, 953-961 ( 1985)). This enzyme utilizes reduced methyl viologen as a coenzyme, and its molecular weight is 28,400 by gel filtration and 14,200 by SDS-PAGE.

Further, the ORFs named YNN4, YL60, and YCZ2 derived from Saccharomyces cerevisiae are also known to have enone reducing activity. The KLER1 homology of the amino acid sequences of YNN4, YL60, and YCZ2 is 54%, 51%, 53% (identity), 69%, 68%, 69% (Positive). However, it has been confirmed that YNN4, YL60, and YCZ2 all have enone reducing activity.
The DDBJ accession numbers of YNN4, YL60 and YCZ2 are shown below.
YNN4: amino acid sequence P53912, base sequence Z46843
YL60: Amino acid sequence P54007, base sequence U22383
YCZ2: Amino acid sequence P25608, base sequence X59720

In addition, oyster mushroom-derived enone reductase can also be used as the reduction catalyst in the present invention. The aroma of mushrooms is known to be derived from volatile C8 compounds, which are linoleic acid metabolites. Since the enone reductase contained in oyster mushrooms is involved in the formation of volatile C8 compounds, it can be exemplified as a preferred embodiment of the reduction catalyst in the present invention.
The oyster mushroom-derived enone reductase can be isolated by a conventional method such as gel filtration chromatography, hydrophobic chromatography, affinity chromatography after streptomycin sulfate precipitation and methanol fractionation on a oyster mushroom crude enzyme solution ( The Agricultural Chemical Society of Japan 2004 Annual Conference Proceedings p116 Lecture No. 2B03p21, The Japanese Society of Agricultural Chemistry 2010 Annual Conference Proceedings p39 Lecture No. 2AIp09).

In the present invention, a polynucleotide encoding an enone reductase and a homolog thereof is known. In the present invention, the polynucleotide may be a naturally occurring polynucleotide such as DNA or RNA, or may be a polynucleotide containing an artificially synthesized nucleotide derivative.

The polynucleotide encoding the enone reductase in the present invention includes, for example, the nucleotide sequence shown in SEQ ID NO: 1 or 3. The nucleotide sequences shown in SEQ ID NOs: 1 and 3 respectively encode proteins containing the amino acid sequences shown in SEQ ID NOs: 2 and 4, and the proteins containing this amino acid sequence constitute a preferred embodiment of enone reductase. ..

The polynucleotide of the present invention includes any base sequence capable of encoding the amino acid sequence set forth in SEQ ID NO: 2 or 4. Since there are 1 to 6 codons corresponding to one amino acid, the DNAs encoding the proteins consisting of the amino acid sequences of SEQ ID NOs: 2 and 4 are the nucleotide sequences of SEQ ID NOs: 1 and 3, respectively. Not only the DNA having the following, but there are a plurality of DNAs that can be regarded as equivalent to the DNAs shown in SEQ ID NOs: 1, 3.

The polynucleotide of the present invention contains the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted, inserted and/or added, and has the physicochemical properties (A1). -Comprising a polynucleotide encoding a protein having (E1).
Alternatively, the polynucleotide of the present invention contains the amino acid sequence of SEQ ID NO: 4 in which one or more amino acids are deleted, substituted, inserted and/or added, and has the physicochemical properties (A2). )-(E2).
Those skilled in the art will appreciate that the method of introducing site-directed mutagenesis into the DNA shown in SEQ ID NO: 1 or 3 (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRLPress pp.200(1991) ), etc. can be used to introduce substitutions, deletions, insertions, and/or addition mutations as appropriate. Is.

The polynucleotide of the present invention is a polynucleotide that can hybridize with the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and has the physicochemical properties (A1)-(E1). It also includes polynucleotides that encode proteins.
Alternatively, the polynucleotide of the present invention is a polynucleotide that can hybridize with the DNA consisting of the nucleotide sequence of SEQ ID NO: 3 under stringent conditions and has the physicochemical properties (A2)-(E2). It also includes a polynucleotide encoding
The polynucleotide capable of hybridizing under stringent conditions means any one of at least 20, preferably at least 30, for example, 40, 60 or 100 consecutive sequences in SEQ ID NO: 1 or 3. Multiple selected DNA as a probe DNA, for example, using ECL direct nucleic acid labeling and detection system (manufactured by Amersham Pharmaica Biotech), in the conditions described in the manual (wash: 42 ℃, primary wash buffer containing 0.5x SSC), Refers to a hybridizing polynucleotide. Alternatively, the stringent hybridization conditions in the present invention may include, for example, 6M urea, 0.4% SDS, 0.1xSSC.

Polynucleotides that can hybridize with the DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3 under stringent conditions include those containing a base sequence similar to SEQ ID NO: 1 or 3. Such a polynucleotide is likely to encode a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4, respectively. Therefore, those skilled in the art can select a polynucleotide encoding a protein having an enone reductase activity from such polynucleotides based on the description in the present specification.

Furthermore, the polynucleotide of the present invention has at least 90%, preferably at least 95% or more (96%, 97%, 98%, 99%) identity (homology) with the amino acid sequence shown in SEQ ID NO: 2. And a polynucleotide encoding a protein having the physicochemical properties (A1)-(E1).
Alternatively, the polynucleotide of the present invention has at least 90%, preferably at least 95% or more (96%, 97%, 98%, 99%) identity (homology) with the amino acid sequence represented by SEQ ID NO: 4. And a polynucleotide encoding a protein having the physicochemical properties (A2)-(E2).
A protein identity search is performed by, for example, a database regarding the amino acid sequences of proteins such as SWISS-PROT and PIR, a database regarding DNA such as DNA Databank of JAPAN (DDBJ), EMBL and Gene-Bank, and a predicted amino acid sequence based on the DNA sequence. For example, on the Internet, using a FASTA program, a BLAST program, or the like for a database related to.
Alternatively, as a result of identity search using the BLAST program for DDBJ using the amino acid sequence of SEQ ID NO: 2, the highest identity among known proteins was Cochliobolus carbonum toxD. It was 36% (Identity) and 54% (Positives) of protein. In the present invention, the term “identity” means, for example, the value of Positive homology using the BLAST program.
As a result of an identity search using the BLAST program for SWISS-PROT using the amino acid sequence of SEQ ID NO: 4, the highest identity among known proteins was Saccharomyces carlesbergensis Old. Yellow Enzyme 1 (71% (Identity) and 84% (Positives) of OYE1).

A protein encoded by these polynucleotides and containing a mutation in the amino acid sequence is a homologue of the enone reductase according to the present invention as long as it has the physicochemical properties (A1)-(E1) or (A2)-(E2). included. The polynucleotide is useful for genetically engineering production of enone reductase. Alternatively, the polynucleotide can be used to genetically engineer a microorganism having an enone reductase activity useful for producing α,β-saturated ketones from α,β-unsaturated ketones.

The enone reductase according to the present invention includes a protein having the amino acid sequence of SEQ ID NO: 2 or 4 and having enone reductase activity, and a homolog thereof. The protein containing the amino acid sequence shown in SEQ ID NO: 2 or 4 constitutes a preferred embodiment of the enone reductase in the present invention.

In the present invention, the enone reductase homologue includes the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are deleted, substituted, inserted and/or added. A person skilled in the art would like to introduce a site-directed mutagenesis method into the DNA shown in SEQ ID NO: 1 or 3 (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983). ), Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp.200 (1991) ), etc., and appropriately introduce substitutions, deletions, insertions, and/or addition mutations. Thus, a DNA encoding a homolog of enone reductase can be obtained. By introducing a DNA encoding the enone reductase homolog into a host and expressing it, the enone reductase homolog described in SEQ ID NO: 2 or 4 can be obtained.

Furthermore, the homolog of enone reductase in the present invention means at least 90%, preferably 95% or more (96%, 97%, 98%, 99%) identity with the amino acid sequence shown in SEQ ID NO: 2 or 4. Refers to a protein having The protein identity search can be performed by the method described above.

The polynucleotide encoding the enone reductase in the present invention can be isolated by the following method, for example.

The polynucleotide of the present invention can also be isolated from other organisms by PCR cloning or hybridization based on the nucleotide sequence shown in SEQ ID NO: 1 or 3. The base sequence shown in SEQ ID NO: 1 or 3 is a gene isolated from Kleiberomyces lactis. A primer for PCR can be designed using the nucleotide sequence of SEQ ID NO: 1 or 3, and a polynucleotide encoding a protein having enone reductase activity can be obtained from a microorganism such as yeast of the genus Kleiberomyces. ..

Alternatively, the enone reductase having the physicochemical properties (A1)-(E1) or (A2)-(E2) can be isolated, and a polynucleotide can be obtained based on the structural characteristics. After purifying the enzyme, the N-terminal amino acid sequence is analyzed, further, after cleaving with an enzyme such as lysyl endopeptidase, V8 protease, etc., the peptide fragment is purified by reverse phase liquid chromatography and the amino acid sequence is analyzed by a protein sequencer. Multiple amino acid sequences can be determined.

When the partial amino acid sequence is clarified, the base sequence encoding it can be deduced. The present invention is designed by designing a primer for PCR based on the deduced nucleotide sequence or the nucleotide sequence shown in SEQ ID NO: 1 or 3, and performing PCR using the chromosomal DNA of an enzyme-producing strain or a cDNA library as a template. A portion of the polynucleotide in can be obtained.

Furthermore, using the obtained polynucleotide fragment as a probe, a restriction enzyme digestion product of the chromosomal DNA of the enzyme-producing strain was introduced into a phage, a plasmid, etc., and a library or cDNA library obtained by transforming E. coli was used. Then, the polynucleotide of the present invention can be obtained by colony hybridization, plaque hybridization, or the like.

Further, the nucleotide sequence of the polynucleotide fragment obtained by PCR is analyzed, and from the obtained sequence, a PCR primer for extending outside the known DNA is designed, and the chromosomal DNA of the enzyme-producing strain is treated with an appropriate restriction enzyme. After digestion with, by performing reverse PCR using DNA as a template by self-cyclization reaction (Genetics120, 621-623 (1988)), RACE method (Rapid Amplification of cDNA End, "PCR Experimental Manual" p25-33, It is also possible to obtain the polynucleotide of the present invention by, for example, HBJ Publishing Bureau.

The polynucleotide of the present invention can be obtained by synthesis in addition to genomic DNA or cDNA cloned by the above method.

By inserting the polynucleotide encoding the enone reductase thus isolated into a known expression vector, the enone reductase expression vector can be obtained. Then, the enone reductase of the present invention can be obtained from the recombinant by culturing the transformant transformed with this expression vector. Alternatively, the enone reductase can also be obtained from the recombinant by culturing a transformant in which the polynucleotide encoding the enone reductase according to the present invention is integrated into the genome.

In order to express the enone reductase in the present invention, the microorganism to be transformed is transformed with a recombinant vector containing a polynucleotide encoding a polypeptide having enone reductase activity and expresses the enone reductase activity. There is no particular limitation as long as it is a living organism. Examples of usable microorganisms include the following microorganisms.
Escherichia genus Bacillus genus Pseudomonas genus Serratia genus Brevibacterium genus Corynebacterium genus Corynebacterium genus Streptococcus genus Lactobacillus genus host vector systems The bacterium Rhodococcus genus Streptomyces genus Streptomyces genus actinomycete Saccharomyces genus Kluyveromyces genus Kluyveromyces genus Schizosaccharomyces genus Zygosaccharomyces spp. Yarrowia spp. Trichosporon spp. Rhodosporidium spp. Pichia spp. Candida spp. Yeast Neurospora spp. (Aspergillus) genus Cephalosporium genus Trichoderma (Trichoderma) genus and other molds have been developed host system

The procedure for producing a transformant and the construction of a recombinant vector suitable for a host can be performed according to the techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratories). In order to express an enone reductase gene in a microorganism, first, a polynucleotide encoding a polypeptide having an enone reductase activity is introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and its gene Information needs to be transcribed and translated. For that purpose, a promoter, which is a unit that controls transcription/translation, may be incorporated upstream of the 5′-side of the DNA chain, and more preferably, a terminator may be incorporated downstream of the 3′-side. As the promoter and terminator, it is necessary to use a promoter and terminator known to function in the microorganism used as the host. Regarding the vectors, promoters, terminators, etc. that can be used in these various microorganisms, "Basic Microbiology Course 8 Genetic Engineering/Kyoritsu Shuppan", especially regarding yeast, Adv. Biochem. Eng. 43, 75-102 (1990), Yeast 8, 423-488 (1992), and the like.

For example, in the genus Escherichia, in particular Escherichia coli (Escherichia coli), as a plasmid vector, pBR, pUC system plasmid can be used, lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp Fusion), a promoter derived from λ phage PL, PR and the like can be used. Moreover, as the terminator, a terminator derived from trpA, derived from phage, rrnB ribosomal RNA, or the like can be used. Among these, vector pSE420D (described in JP-A-2000-189170) obtained by partially modifying the multi-cloning site of commercially available pSE420 (manufactured by Invitrogen) can be preferably used.

In the genus Bacillus, pUB110 series plasmids, pC194 series plasmids and the like can be used as vectors, and they can also be integrated into the chromosome. In addition, apr (alkaline protease), npr (neutral protease), amy (α-amylase) and the like can be used as promoters and terminators.

In the genus Pseudomonas, host vector systems have been developed with Pseudomonas putida, Pseudomonas cepacia and the like. A broad host range vector (including genes necessary for autonomous replication derived from RSF1010, etc.) pKT240 based on the plasmid TOL plasmid involved in the decomposition of toluene compounds is available, and lipase (special Kaihei 5-284973) Genes can be used.

In the genus Brevibacterium, particularly Brevibacterium lactofermentum, a plasmid vector such as pAJ43 (Gene 39, 281 (1985)) can be used. As the promoter and terminator, the promoter and terminator used in E. coli can be used as they are.

For Corynebacterium, particularly Corynebacterium glutamicum, plasmid vectors such as pCS11 (JP-A-57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984)) can be used. Is.

In the genus Streptococcus, pHV1301 (FEMS Microbiol. Lett. 26, 239 (1985)), pGK1 (Appl. Environ. Microbiol. 50, 94 (1985)) and the like can be used as plasmid vectors.

In the genus Lactobacillus, pAMβ1 (J. Bacteriol. 137, 614 (1979)) developed for Streptococcus can be used, and the one used in E. coli as a promoter can be used. ..

In the genus Rhodococcus, a plasmid vector isolated from Rhodococcus rhodochrous can be used (J. Gen. Microbiol. 138, 1003 (1992)).

In the genus Streptomyces, a plasmid can be constructed according to the method described in Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual Cold Spring Harbor Laboratories (1985). In particular, in Streptomyces lividans, in pIJ486 (Mol. Gen. Genet. 203, 468-478, 1986), pKC1064 (Gene 103,97-99 (1991) ), pUWL-KS (Gene 165,149). -150 (1995)) can be used. In addition, the same plasmid can be used in Streptomyces virginiae (Actinomycetol. 11,46-53 (1997)).

In the genus Saccharomyces, especially Saccharomyces cerevisiae, YRp-type, YEp-type, YCp-type, and YIp-type plasmids can be used, and homologous recombination with multiple copies of ribosomal DNA in the chromosome is used. The integrated vector (such as EP 537456) is extremely useful because it can introduce a gene in multiple copies and can stably retain the gene. Also, ADH (alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK (phosphoglycerate kinase), ENO (enolase) Such promoters and terminators are available.

In the Kluyveromyces genus, especially Kluyveromyces lactis, it is involved in killer activity in a 2 μm plasmid derived from Saccharomyces cerevisiae, pKD1 plasmid (J. Bacteriol. 145, 382-390(1981)). A pGKl1-derived plasmid, a KARS-based plasmid for autonomous growth gene in the genus Kleiberomyces, a vector plasmid (EP 537456, etc.) that can be integrated into the chromosome by homologous recombination with ribosomal DNA, etc. can be used. In addition, promoters and terminators derived from ADH, PGK, etc. can be used.

In the genus Schizosaccharomyces, plasmid vectors containing ARS (gene involved in autonomous replication) from Schizosaccharomyces pombe and auxotrophic selection markers from Saccharomyces cerevisiae are available ( Mol. Cell. Biol. 6, 80 (1986)). In addition, the ADH promoter derived from Schizosaccharomyces pombe can be used (EMBO J. 6, 729 (1987)). In particular, pAUR224 is commercially available from Takara Shuzo and is easily available.

In the genus Zygosaccharomyces, plasmid vectors derived from pSB3 (Nucleic Acids Res. 13, 4267 (1985)) derived from Zygosaccharomyces rouxii are available, and the PHO5 promoter derived from Saccharomyces cerevisiae. Alternatively, a promoter of GAP-Zr (glyceraldehyde-3-phosphate dehydrogenase) derived from T. saccharomyces rouxii (Agri. Biol. Chem. 54, 2521 (1990)) and the like can be used.

In the genus Pichia, a host vector system has been developed that utilizes genes (PARS1, PARS2) involved in autonomous replication from Pichia such as Pichia pastoris (Mol. Cell. Biol. 5 , 3376 (1985)), and strong promoters such as AOX, which can be induced by high-concentration culture and methanol (Nucleic AcidsRes. 15, 3859 (1987)). In addition, a host-vector system has been developed in Pichia angusta (formerly Hansenula polymorpha). As the vector, genes (HARS1, HARS2) involved in autonomous replication derived from Pichia angusta can be used, but since they are relatively unstable, multicopy integration into the chromosome is effective (Yeast 7, 431- 443 (1991)). In addition, promoters such as AOX (alcohol oxidase) and FDH (formate dehydrogenase), which are induced by methanol, can be used.

In the genus Candida, in Candida maltosa (Candida maltosa), Candida albicans (Candida albicans), Candida tropicalis (Candida tropicalis), Candida utilis (Candida utilis), etc. Is being developed. In Candida maltosa, ARS derived from Candida maltosa has been cloned (Agri. Biol. Chem. 51, 51, 1587 (1987)), and a vector utilizing this has been developed. In Candida utilis, a strong promoter has been developed for a chromosome integrated type vector (Japanese Patent Laid-Open No. 08-173170).

In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most well-studied among molds, and their integration into plasmids and chromosomes is available. Promoters derived from extraprotease and amylase are available (Trends in Biotechnology 7, 283-287 (1989)).

In the genus Trichoderma, a host vector system utilizing Trichoderma reesei has been developed, and a promoter derived from extracellular cellulase gene and the like can be used (Biotechnology 7, 596-603 (1989)).

In addition to microorganisms, various host-vector systems have been developed in plants and animals, and especially in insects using silkworms (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants. A system for expressing a heterologous protein in a large amount has been developed and can be preferably used.

Further, the transformant expressing the enone reductase obtained by the above method can be used for the production of the enzyme and for eliminating the odor of unsaturated aldehyde by reducing the carbon-carbon double bond of unsaturated aldehyde.

The α,β-unsaturated ketone in the present invention is not limited. For example, the compound represented by the following general formula I can be represented as an α,β-unsaturated ketone.

General formula I

In the formula, R1 represents hydrogen, an alkyl group, an allyl group, an alkenyl group, an aralkyl group, or an alkoxy group (however, the alkyl group, the allyl group, the alkenyl group, the aralkyl group, and the alkoxy group may be substituted). , R2, and R3 represent hydrogen or an optionally substituted short chain alkyl group.

Examples of the alkyl group, allyl group, alkenyl group, aralkyl group, and alkoxy group for R1 include those having 1 to 8 carbon atoms. Further, these substituents may be substituted with halogen, nitro group, amino group, alkoxy group or the like. On the other hand, examples of the short-chain alkyl group for R2-R3 include methyl, ethyl, butyl, and propyl. These short chain alkyl groups may also be substituted with halogen, nitro group, amino group, alkoxy group or the like. In addition, R2-R3 can be the substituents independently or simultaneously.

Specifically, compounds having the following substituents as R1-R4 are desirable as the substrate compound in the present invention.
R1=H, methyl group, ethyl group, phenyl group, 3-pyridyl group, 2-pyridyl group, 3-nitrophenyl group, 3-methoxyphenyl group, and 4-methoxyphenyl group
R2=H, methyl group, and ethyl group
R3=H, methyl group, and ethyl group

More specifically, methyl vinyl ketone, ethyl vinyl ketone, 3-penten-2-one, 3-methyl-3-penten-2-one, 2-hexenone, 2-methyl-2-hexenone, 3-methyl- 4-(3-pyridyl)-3-buten-2-one, 3-methyl-4-phenyl-3-buten-2-one, 3-methyl-4-(3-nitrophenyl)-3-butene-2 -ON or the like is preferably used.

In the present invention, the contact between the reducing catalyst and the unsaturated aldehyde can be carried out, for example, on the surface of skin or clothing where the adhesion of the unsaturated aldehyde is suspected. For example, the composition containing the reducing catalyst of the present invention can be contacted with the surface of skin or clothing. Specific embodiments of the composition containing the reduction catalyst are described later.

The reducing catalyst in the present invention is, as described below, a liquid form such as an aqueous solution, lotion, spray liquid, suspension and emulsion, a solid form such as powder, granules and blocks, and a semi-form such as cream and paste. It may be in a solid form, a gel form or the like. When the reducing catalyst of the present invention is used in these forms, the contact can be carried out by directly applying it to a site such as skin where the odor generated due to unsaturated aldehyde is desired to be eliminated. Alternatively, it can be performed by spraying clothes such as a shirt.

The amount of the enone reductase used in the present invention can be selected within a range capable of eliminating the odor generated by the unsaturated aldehyde. For example, a shirt 10 cm ² per unsaturated aldehyde impregnated, or shirt 10 cm ² per wearing 1 day, 1 unit 0.001 unit, preferably 0.1 units from 0.01 units, more preferably the amount of enzyme 0.05 units from 0.02 units However, the invention is not limited to these.
It is difficult to express the enzyme extract by weight because the mass of the enzyme changes depending on the degree of purification, but an amount of, for example, 2 μg to 1 mg, preferably 10 μg to 200 μg can be used.
The amount of enzyme used can be adjusted according to the nature, area, and condition of the contact target.

In the method for eliminating unsaturated aldehyde of the present invention, an electron donor may be brought into contact with the reducing catalyst. The contact amount of the electron donor can also be appropriately adjusted depending on the property, area, and condition of the contact target. The contact amount of the electron donor can be, for example, 1 μg to 500 μg, preferably 5 μg to 100 μg, per 10 cm ² shirt impregnated with unsaturated aldehyde, or 10 cm ² shirt worn for one day, but is not limited thereto. ..

The mixing ratio of the reduction catalyst and the electron donor may be 2:1, but the present invention is not limited thereto. In the method for eliminating unsaturated aldehyde of the present invention, not only the mixing ratio of the reducing catalyst and the electron donor, but also the amount of the electron donor is important.

The enone reductase in the present invention is not limited to the purified enzyme, and includes partially purified enzyme, microbial cells containing enone reductase, and processed products thereof. The term "treated product" as used in the present invention is a generic term for cells, purified enzymes, partially purified enzymes and the like immobilized by various methods.

In the present invention, the contact between the reducing catalyst and the unsaturated aldehyde can be carried out, for example, in an aqueous solution. The enzymatic reaction with the enone reductase in the present invention can be carried out, for example, under the following conditions.
-Reaction temperature: 4-55°C, preferably 15-45°C
PH: 4-9, preferably 5.0-8.0, more preferably pH 6.0-7.0
・Substrate concentration: 0.01-90%, preferably 0.1-20%

If necessary, an organic solvent such as glycerin for stabilizing the enzyme and ethanol as a preservative may be added to the aqueous solution. The concentration of these organic solvents can be preferably about 1 to 10%, more preferably about 5% (for example, 3%, 4%, 5%, 6%, 7%), but is not limited thereto.
Furthermore, a surfactant can be added to the reaction solution. As the surfactant, 0.1 to 5.0% by weight of Triton X-100, Tween 60 or the like is used.
Further, a moisturizing agent, antibacterial agent, bacteriostatic agent, bactericidal agent, preservative in the reaction solution as long as the activity of the reduction catalyst that catalyzes the reaction of reducing the carbon-carbon double bond of unsaturated aldehyde is not hindered , Antioxidants, UV-screening agents, UV-absorbing agents, antiperspirants, components showing pharmacological effects, emulsifiers, texture-improving agents, powder raw materials, refreshing agents, propellants, pH adjusting agents, flavoring agents, aromatic agents, Colorants, proteins, amino acids, fibers, lipids, salts and the like can also be added. Specific examples of these are described later.

The present invention also relates to a composition for eliminating the odor of unsaturated aldehydes, which contains, as an active ingredient, a reducing catalyst that catalyzes a reaction of reducing carbon-carbon double bonds of unsaturated aldehydes. Examples of the reduction catalyst that catalyzes the reaction of reducing the carbon-carbon double bond of the unsaturated aldehyde include those mentioned above.
The composition of the present invention may further include an electron donor. The above-mentioned thing can be used for an electron donor.
The amount of the reducing catalyst and the electron donor used, the compounding ratio and the like are as described above.

The composition of the present invention may contain a known substance having an unsaturated aldehyde generation suppressing activity. Examples of the substance having an unsaturated aldehyde generation inhibitory activity include antioxidants capable of blocking the oxidation process that produces unsaturated aldehydes.
Specifically, for example, carotenoids such as α-carotene, β-carotene, γ-carotene, lycopene, cryptoxanthin, zeaxanthin, isozeaxanthin, rhodoxanthin, capsanthin, and crocetin,
Furans such as 2,5-dimethylfuran, 2-methylfuran, 2,5-diphenylfuran and 1,3-diphenylisobenzofuran,
Vitamin Es such as α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol,
Amino acids such as histidine, tryptophan, methionine, alanine, and alkyl esters of alanine,
1,4-diadicyclooctane, dibutylhydroxytoluene (BHT), gallic acid esters,
Examples thereof include, but are not limited to, ascorbic acid, tannins and flavonoids.

In addition, an extract of a crude drug having an antioxidant effect can be used as the above antioxidant. Specifically, for example, sardine extract, sagebrush extract, Acacia catechu extract, sage extract, tea extract, rosemary extract, fennel extract, thyme extract, nutmeg extract, pepper extract, turmeric extract Examples thereof include, but are not limited to, herbal medicine extracts such as vanilla extract, paprika extract, yokuinin extract, psycho extract, wood melon extract, suho extract, curen extract, and john lab extract.

When this antioxidant is added to the composition of the present invention, the amount of the antioxidant is generally 0.001% by weight or more and 10.0% by weight or less, preferably 0.01% by weight or more and 1.0% by weight or less. It can be a range.

Also, a substance that inhibits lipoxygenase can be incorporated into the composition of the present invention. Lipoxygenase is an in vivo enzyme that produces lipid peroxides (eg, linoleic acid hydroperoxide) that cause oxidation of palmitoleic acid to palmitoleic acid HPO. Therefore, a substance that inhibits the action of lipoxygenase can suppress the generation of age-related odor.

Examples of the substance that inhibits the action of lipoxygenase include tranexamic acid and β-carotene. When β-carotene is used as lipoxygenase, it is preferable to select an antioxidant other than β-carotene as the above-mentioned antioxidant.

When this lipoxygenase inhibitor is added to the composition of the present invention, it is generally 0.0001% by weight or more and 10.0% by weight or less, preferably 0.005% by weight or more and 5.0% by weight or less of the composition. It can be blended within the following range.

In addition, the antimicrobial fragrance dihydrofarnesol can also be included in the compositions of the present invention to mask or harm age-related odors.

Further, the composition of the present invention, further to the extent that does not hinder the activity of the reduction catalyst that catalyzes the reaction of reducing carbon-carbon double bond of unsaturated aldehyde, further moisturizing agent, antibacterial agent, bacteriostatic agent, bactericide, Preservatives, preservatives, antioxidants, ultraviolet shielding agents, ultraviolet absorbers, antiperspirants, components showing pharmacological effects, emulsifiers, surfactants, touch improvers, powder raw materials, cooling agents, propellants, pH A regulator, a flavoring agent, a fragrance, a coloring agent, a protein, an amino acid, a fiber, a lipid, a salt and the like can be added.

As the moisturizer, for example,
Polyhydric alcohols such as propylene glycol, glycerin, 1,3-butylene glycol, erythritol, dipropylene glycol, xylitol, sorbitol, polyethylene glycol, hexylene glycol, diglycerin and maltitol,
Sugars such as maltose, D-mannitol, starch syrup, glucose, fructose, lactose, glucosamine and cyclodextrin,
Polysaccharide derivatives such as carboxymethyl chitin,
Amino acids such as sodium pyrrolidonecarboxylate and salts thereof,
Lactic acid and sodium lactate, sodium chondroitin sulfate, sodium hyaluronate, organic acids such as sodium adenosine phosphate, and salts thereof can be mentioned.
The content of the above components depends on the kind of the components to be used and the field of application and usage form of the composition of the present invention, but is usually in the range of about 0.1% to about 50% based on the total weight. Is.

Examples of the antibacterial agent, bacteriostatic agent, bactericidal agent, preservative or preservative include, for example,
Organic acids such as benzoic acid and its salts, sorbic acid and its salts, dehydroacetic acid and its salts,
Benzoic acid ester derivatives such as methyl paraoxybenzoate and ethyl paraoxybenzoate,
Phenols such as phenol, cresol, chlorcresol, isopropylmethylphenol, trichlorohydroxydiphenyl ether,
Quaternary ammonium salts such as benzalkonium chloride, benzenetonium chloride and cetylpyrimidinium chloride,
Chlorhexidine gluconate, chlorhexidine derivatives such as chlorhexidine hydrochloride,
Diphenylurea derivatives such as trichlorocarbanilide, halocarbane,
Examples include dihydrofarnesol.
The content of the above components depends on the type of the components to be used and the field of application of the composition of the present invention and the usage form, but is usually 0.001% to 5%, preferably 0.01% based on the total weight. The range of 1% to 1% is preferable.

As the antioxidant, for example,
Ascorbic acid and its salts, ascorbic acid derivatives such as ascorbic acid stearic acid ester and sugar transfer ascorbic acid,
Examples thereof include citric acid and salts thereof, tocopherol acetate, dibutylhydroxytoluene, tocopherol, palmitic acid and salts thereof, ascorbyl palmitate, sodium pyrosulfite, butylhydroxyanisole, propyl gallate, and tea catechin.
The content of the above components depends on the kind of the components to be used and the field of application and usage of the composition of the present invention, but is usually about 0.001% to about 10%, preferably about 10% by total weight. A range of 0.01% to about 5% is preferred.

Examples of the ultraviolet shielding agent include
Inorganic substances with UV shielding ability such as titanium oxide, talc, kaolin, etc.
Examples thereof include organic substances having an ultraviolet shielding ability such as 5-chlorouracil, guanine and cytosine.

Further, as the ultraviolet absorber, for example,
Examples include ethyl paraaminobenzoate, ethylhexyl paradimethylaminobenzoate, cinoxate, ethylhexyl paramethoxycinnamate, 2-(2-hydroxy-5-methylphenyl)benzotriazole, oxybenzozone, urocanic acid, ethyl urocanate. ..
Furthermore, the following ultraviolet absorbers can be preferably used in the present invention.
Para-aminobenzoic acid type UV absorbers such as para-aminobenzoic acid,
Anthranilic acid type UV absorbers such as methyl anthranilate,
Salicylic acid-based UV absorbers such as octyl salicylate, phenyl salicylate, and homomenthyl salicylate,
Isopropyl paramethoxycinnamate, octyl paramethoxycinnamate, 2-ethylhexyl paramethoxycinnamate, glyceryl mono-2-ethylhexanoate diparamethoxycinnamate, [4-bis(trimethylsiloxy)methylsilyl-3 -Methylbutyl]-3,4,5-trimethoxycinnamic acid ester or other cinnamic acid-based UV absorber,
Benzophenone-based UV absorption such as 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, and sodium 2-hydroxy-4-methoxybenzophenone-5-sulfonate Agent,
UV absorbers such as 2-phenyl-5-methylbenzoxazole and 4-tert-butyl-4′-methoxydibenzoylmethane.
The content of the above components depends on the type of the components to be used and the field of application of the composition of the present invention, and is generally in the range of about 0.1% to about 50% based on the total weight. is there.

As an antiperspirant, for example,
Aluminum chloride, aluminum oxychloride, basic aluminum bromide, aluminum sulfate, chlorhydroxyaluminum, chlorohydroxyaluminum zirconium, allantoin chlorohydroxyaluminum, aluminum potassium sulfate (alum), zinc sulfate, aluminum phenolsulfonate, phenolsulfonate, base Examples thereof include zinc aluminum lactate, zinc oxide, and zinc paraphenolsulfonate.
Furthermore, astringent compounds such as a complex of the above compound and an amino acid, preferably glycine, can be mentioned.
The content of the components as described above depends on the type of the component used and the field of application and usage form of the composition of the present invention, but is usually preferably in the range of 0.01% to 40% based on the total weight.

As a component showing a pharmacological effect that can be used in the present invention, it is desirable to show a component that exhibits effects such as an anti-inflammatory action, a melanin production inhibiting action, and a wound healing promotion when applied to the skin of a living body. For example, vitamins, hormones, plant extracts, animal extracts, photosensitizers, etc. can be added to the composition of the present invention.
As vitamins,
Vitamin A including retinol,
Vitamin Bs such as thiamine, pantothenic acid and their derivatives,
Vitamin Cs such as ascorbic acid and its derivatives,
Vitamin Es including tocopherol and its derivatives,
Vitamin Ps including flavonoids such as rutin, hesperidin, and naringin, and derivatives thereof, and the like can be mentioned.
Examples of hormones include estrogen and pregnenerone.
Examples of the plant extract include chamomile extract and licorice extract.
Animal extracts include placenta extracts and the like.
As a photosensitizer,
Photosensitizer No. 101 (Chemical name: 2,2'[3'-[2-(3-heptyl-4-methyl-4-thiazoline-2-ylidene)ethylidene]propenylene]bis(3-heptyl-4-methylthia Zolium iodide), common name: Platonin),
Photosensitizer 201 (chemical name: 3-heptyl-2-[(3-heptyl-4-methyl-2(3H)-thiazolidene)methyl]-4-methylthiazolium iodide, trivial name: pionine),
Photosensitizer No. 301 (chemical name: 2-[2-[(5-bromo-2-pyridinyl)amino]ethenyl]-1-ethyl-6-methylpyridinium iodide, trivial name: tacanal),
Photosensitizer No. 401 (Chemical name: 3,4-dimethyl-2-[2-(phenylamino)ethenyl]oxazolium iodide, trivial name: Luminex)
And so on.
The content of the components as described above is usually 0.0001% to 5% based on the total weight, although it depends on the type of the components used and the field of application and usage of the composition of the present invention.

Examples of the emulsifier or surfactant include, for example,
Esters such as sorbitan fatty acid ester, polyoxyethylene fatty acid ester, lipophilic glyceryl monostearate, self-emulsifying glyceryl monostearate, trimethylolpropane trioctanoate, polyglycerin fatty acid ester, and sucrose fatty acid ester,
Ethers such as polyoxyethylene alkyl ethers,
Examples thereof include phospholipids such as lecithin.
The content of the above-mentioned components depends on the kind of the components used and the field of application of the composition of the present invention, and the form of utilization, but is preferably in the range of 0.01% to 20% in total.

As the texture-improving agent, a chain and/or cyclic silicone oil having a viscosity represented by a kinematic viscosity at 25° C. of 100 mm 2 /sec or less is usually desirable. Examples thereof include methylcyclopolysiloxane, dimethylpolysiloxane, hexamethyldisiloxane, octamethyltrisiloxane, methylphenylpolysiloxane, and organohydrogenpolysiloxane.
The content of the above components depends on the type of the components used and the field of application and usage of the composition of the present invention, but is usually preferably in the range of 0.01% to 50% based on the total weight.

As the thickener, for example,
Guar gum, quince seed, tragacanth, pectin, xanthan gum, chitin, chondroitin sulfate, dextran sulfate, sodium alginate, cationized cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, carboxymethyl starch, propylene glycol alginate, collagen, keratin, casein, albumin, gelatin, Water-soluble polymers such as hydroxypropyl trimethyl ammonium chloride ether, polyvinyl alcohol, polyvinyl pyrrolidone, sodium polyacrylate, polyvinyl methyl ether, carboxy vinyl polymer,
Electrolytes such as sodium chloride, potassium chloride, sodium sulfate,
Examples include various oil components.
The use of the thickener as described above is effective in improving or adjusting the feeling of use when the composition of the present invention is directly applied to the skin for use. The content of the above components depends on the type of the components to be used and the field of application and usage of the composition of the present invention, but is usually preferably in the range of 0.01% to 30% based on the total weight.

The powdery raw material means a powdery component which is substantially insoluble in water or an organic solvent and is generally acceptable for use in the field of external preparation for skin. As the powder raw material, for example,
Synthetic resin powder such as nylon (polyamide) powder, polyurethane powder, polyethylene powder, polystyrene powder, cross-linked polystyrene powder, cross-linked silicone powder,
Silicic anhydride, magnesium oxide, magnesia silica (composition containing silicic anhydride and magnesium oxide), montmorillonite, hydroxyapatite, magnesium silicate, mica, carbon black, sericite, zeolite, aluminum oxide, zirconium oxide, calcium carbonate , Magnesium carbonate, barium sulfate, titanium mica (mica powder coated with titanium oxide film), calcium oxide, barium silicate, calcium silicate, pearl powder, white, red, brown, yellow, Powdered black, purple, green or blue inorganic pigment,
Powdered products of various organic pigments,
Powdered metal pigments,
Examples include natural powder such as silk powder and wool powder.
The above powder raw materials can be used in the composition of the present invention, for example, as a colorant, a base, an excipient, a carrier, a coating agent, a brightening agent and the like. The content of the above components depends on the type of the components used and the field of application and usage of the composition of the present invention, but is usually preferably in the range of 0.01% to 50% based on the total weight.

Examples of the cooling agent include menthol, camphor, peppermint water, peppermint oil, cinnamon oil, fennel oil, bergamot oil, borneol and the like.
The content of the above components depends on the type of the components to be used and the field of application of the composition of the present invention and the usage form, but is usually 0.001% to 5%, preferably 0.01% based on the total weight. The range of 1% to 1% is preferable.
When the above-described components are used in direct or indirect contact with a living body, it is desirable to use a preparation that can be used externally or internally to the living body.

The desired properties and functions can be imparted to the composition of the present invention by blending the above components into the composition of the present invention according to the purpose. For example, to give just one example, a composition in which an antiperspirant, a bactericidal agent, a texture-improving agent, and a powder material are mixed with a reducing catalyst that catalyzes a reaction of reducing a carbon-carbon double bond of an unsaturated aldehyde, When applied to the skin of a living body, it has the effect of eliminating the odor caused by unsaturated aldehydes, and suppresses sweating as in the case of ordinary antiperspirant or deodorant products, giving a refreshing feeling of use.

For example, the composition of the present invention can be used as a skin external preparation. In that case, an ultraviolet absorber or a humectant can be added to the composition of the present invention. Examples of the ultraviolet absorber and the moisturizer include those mentioned above.

Furthermore, when the composition of the present invention is used as an external preparation for skin, it may contain the following substances.
・Vitamins such as inosit, pyridoxine hydrochloride, benzyl nicotinate, nicotinic acid amide, vitamin D2 (ergocalciferol) and biotin; hormones such as estradiol and ethinyl estradiol,
・ Anti-inflammatory agents such as allantoin, azulene, glycyrrhetinic acid,
・Whitening agents such as arbutin
・Astringents such as zinc oxide and tannic acid
・L-menthol, refreshing agent such as camphor,
・Sulfur, lysozyme chloride, etc.
Further, among the above-mentioned antioxidants, antioxidants other than the crude drug extract can be blended in the composition of the present invention as a skin external preparation.

It should be noted that the other components listed above are not limited to the other components that can be incorporated into the composition of the present invention. Further, the above-listed medicinal components may be blended alone in the composition of the present invention as a skin external preparation, and two or more kinds of the above components may be blended in an appropriate combination according to the purpose. is there.

The composition of the present invention is used as a skin external preparation for pharmaceuticals, quasi drugs (ointments, dentifrices, etc.) and cosmetics [basic cosmetics such as face washes, emulsions, creams, gels, essences (cosmetics), packs and masks. , Foundation, lipstick and other makeup cosmetics, oral cosmetics, fragrance cosmetics, hair cosmetics, body cosmetics, etc.].
Alternatively, the composition of the present invention may be in the form of liquid such as aqueous solution, lotion, spray, suspension and emulsion, solid such as powder, granule and block, semi-solid such as cream and paste, gel and the like. Can be If necessary, it can be enclosed in an appropriate container such as a bottle, bag, can, spray can, spray container, box, or pack.

In addition, the dosage form is a wide range of agents such as aqueous solution, solubilized system, emulsified system, powder system, oil liquid system, gel system, ointment system, aerosol system, water-oil 2-layer system, water-oil-powder 3-layer system, etc. Can take a mold. When the composition of the present invention is used as an external preparation for skin, known base components and the like depending on these desired forms and dosage forms may be blended and used within a range that does not impair the intended effect of the composition. it can.

That is, it is possible to add an oil component such as a liquid oil, a liquid or solid oil, a solid oil and wax to the composition of the present invention. In addition, ester oil, hydrocarbon oil, and silicone can be added to the composition of the present invention.

Further, a surfactant such as an anionic surfactant, a cationic surfactant, an amphoteric surfactant, a lipophilic nonionic surfactant, a hydrophilic nonionic surfactant, etc. should be added to the composition of the present invention. You can Then, lower alcohols, sterols, plant-based polymers, microbial-based polymers, starch-based polymers, animal-based polymers, and cellulosic polymers can be added to the composition of the present invention.

Further, a sequestering agent, a neutralizing agent, a pH adjusting agent and the like can be added to the composition of the present invention. Here, the above-mentioned base components and the like are examples, and the base components that can be added to the composition of the present invention are not limited to these base components and the like.

These base components can be appropriately combined and blended in the composition of the present invention according to the formulation according to the desired form.

The composition of the present invention can also be used as a means for eliminating the aging odor adhering to clothes and the like, and a means for preventing the aging odor generated in the body from adhering to clothes. Specifically, it can be used in the form of, for example, a laundry finishing agent for clothes or a coating agent for clothes.

When used in such an embodiment, a fixing agent or the like for fixing the composition of the present invention to an object such as clothes can be incorporated into the composition depending on the purpose. For example, the composition of the present invention can be used as a liquid agent or a spray agent.

The composition of the present invention can also be used as a form contained in an article that can contact a living body. The article that can come into contact with a living body is not particularly limited as long as it can be used directly or indirectly by coming into contact with a living body of a human or an animal.
For example, woven fabrics, non-woven fabrics, sponges, porous synthetic resins, cotton, absorbent cotton, etc. formed into shapes that can be used in the fields of cosmetics, daily necessities, hygiene, medical supplies, nursing care, exercise, etc. Is mentioned. Further, it can be used for the following articles.
Daily necessities such as tissue paper, wet tissue, paper towels, towels, towels, towels, handkerchiefs, mats for foot wipes, cushions, door covers, etc.
Laundry detergent for laundry such as clothes, bedding, daily necessities, laundry products such as laundry finishing agents, softening agents, and gluing agents,
Clothing such as underwear, socks, shirts, pants, and skirts, including disposable items molded from paper, etc.
Bed linen gloves such as sheets, duvet covers, pillowcases, blankets, and towels,
Personal items such as hats, mufflers, headbands, hair bands, and shoes,
Bandages, eye patches, gauze, adhesive bandages with pads, compresses, compress base fabrics, cotton swabs, masks, diapers, disposable diapers, sanitary products such as sanitary products, medical or nursing care products,
Sports shoes, training wear, supporters, bodice, helmet pads, gloves, mitts and other sports equipment,
Cleaning items such as rags, cloths and mops for cleaning floors, handrails, furniture, etc.
Aesthetic supplies,
Massage supplies,
Lotion as a body odor care product, cream, gel, external composition for skin such as sky pollen,
Compositions for scalp and hair such as shampoo, dry shampoo, conditioner, hair tonic, hair liquid, hair mousse, hair cream, pomade, hair restorer and hair restorer,
Oral compositions such as mouthwash, toothpaste,
Pet products for pets such as seafood bait, aquarium purifier, bath liquid for pet animals, etc. The above-exemplified articles containing the composition of the present invention, enone reductase per total weight, as a total thereof in terms of anhydrides. Normally about 0.00001% to about 30% by weight, preferably about 0.0001% to about 10% by weight.
The above-mentioned article eliminates the body odor generated by the person who uses the composition as compared with the article not containing the composition of the present invention, and the body odor caused by the secretion from the living body attached to the article or It has the function of eliminating body odor.

The composition of the present invention is effective for eliminating body odor of non-human animals including mammals, birds and seafood in addition to humans. Examples of animals other than humans include pet animals, ornamental animals and domestic animals. More specifically, the composition of the present invention is effective for the following animals.
・Meat such as dogs, cats, foxes, raccoons,
・Rodents such as squirrels, hamsters, mice, guinea pigs, and rabbits,
・Non-human primates, including lemur, aye, tarsier, gibbon,
・Couple, sheep, goat, boar, hoof, and other hoofed animals,
・Horsepods including horses (above mammals),
・Birds such as chickens, turkeys, squash, quail, parakeets, bunchos, canaries, pine trees, swan, pigeons,
-Seafood such as tropical fish, goldfish, carp, Thailand, shrimp, crab, etc. Therefore, the composition of the present invention is not only used by an individual on a daily basis, but, for example, barber/beauty industry, public bath industry including hot springs, It can also be advantageously used in the aesthetics industry, massage industry, cleaning industry, livestock/fishery industry, the industry of handling pets or ornamental animals including mammals/birds/seafood, the medical field, and the nursing field.

The present invention also provides an odor eliminating agent for unsaturated aldehydes, which contains, as an active ingredient, a reduction catalyst that catalyzes a reaction of reducing carbon-carbon double bonds of unsaturated aldehydes. In a preferred embodiment, the odor eliminating agent of the present invention can further contain an electron donor.

In addition, the present invention relates to the use of a reduction catalyst which catalyzes a reaction for reducing a carbon-carbon double bond of an unsaturated aldehyde, in the production of an odor eliminating agent for an unsaturated aldehyde.
Alternatively, the present invention relates to the use of a reducing catalyst, which catalyzes a reaction for reducing a carbon-carbon double bond of an unsaturated aldehyde, in eliminating the odor of an unsaturated aldehyde.
In addition, the present invention relates to a method for producing an odor eliminating agent, which comprises a step of combining a reducing catalyst which catalyzes a reaction of reducing a carbon-carbon double bond of an unsaturated aldehyde and a pharmaceutically acceptable carrier.
The invention further relates to the use of a reducing catalyst which catalyzes a reaction for reducing a carbon-carbon double bond of an unsaturated aldehyde, in the manufacture of a composition for eliminating an odor of an unsaturated aldehyde.
All prior art documents cited in the present specification are incorporated herein by reference.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.
[Example 1] Deodorant of trans-2-nonenal Alcohol dehydrogenase derived from yeast (Sigma-Aldrich), aldehyde dehydrogenase derived from yeast (Sigma-Aldrich), Enone reductase-1 derived from yeast (KLER1, JP 2002-247987), 1 U of yeast-derived enone reductase-2 (KYE1, JP2003-033185), 10 mM of coenzyme, 2 mM of trans-2-nonenal, and a reaction solution was prepared. The enzymatic reaction was carried out at 15°C for 15 minutes. Note that 1 U of each enzyme was the amount of enzyme that converted 1 μmol of the reference substance in 1 minute. The reference substances for each enzyme are ethanol for alcohol dehydrogenase, acetaldehyde for aldehyde dehydrogenase, and ethyl vinyl ketone for enone reductase.
After the reaction, a change in odor was evaluated in a sensory test. The results are shown in Table 1. Not only did the enone reductase not smell bad, but it turned into a refreshing aroma.

[Example 2] Deodorization of unsaturated aldehyde by enone reductase In the same manner as in Example 1, trans-2-hexenal and trans-2-octenal were also subjected to sensory evaluation. Enzymes used for evaluation were yeast-derived enone reductase-1 (KLER1, JP 2002-247987), yeast-derived enone reductase-2 (KYE1, JP 2003-033185), and coenzyme NADPH at 10 mM. The enzyme reaction was carried out at 30° C. for 15 minutes at pH 6.5 with each unsaturated aldehyde at 2 mM.
After the reaction, a change in odor was evaluated in a sensory test. The results are shown in Table 2. The enone reductase exhibited a deodorizing effect on any unsaturated aldehyde, and changed to a refreshing odor as in Example 1.

Further, the unsaturated aldehyde and its conversion product remaining in the reaction solution were analyzed. Aldehydes were derivatized with dinitrophenylhydrazine (DNPH) by a conventional method and analyzed by GC. The analysis conditions are as follows. That is, using DB-Wax (J&W, 0.25 mm×30 m), the column temperature was kept at 50° C. for 5 minutes, and then the temperature was raised to 200° C. at 5° C./min. A hydrogen flame ionization detector (FID) was used for detection. As a result, in each case, the unsaturated aldehyde was converted into the corresponding n-aldehyde with a yield of 100%.

[Example 3] Deodorization of unsaturated aldehyde attached to clothes
10 μg of trans-2-nonenal was dissolved in 1 mL of water, and the aqueous solution was absorbed into 10 cm×10 cm gauze. Next, 0.5 U of yeast-derived enone reductase-1 (KLER1, JP 2002-247987) and yeast-derived enone reductase-2 (KYE1, JP 2003-033185), 0.55 mg of NADPH was added to pH 6.5. The deodorizing enzyme solution was prepared by dissolving the solution in a potassium phosphate buffer solution prepared as described above and adjusting the final solution volume to 10 mL. Here, 1 U was the amount of enzyme that reduced the carbon-carbon double bond of trans-2-nonenal at 1 μmol/min. A 1 mL portion of this deodorizing enzyme solution was sprayed on trans-2-nonenal-containing gauze and allowed to stand at room temperature for 5 minutes, and a change in odor was evaluated in a sensory test. As a result, in any of the enone reductases, the bad odor of trans-2-nonenal disappeared, and conversely, it changed to a good odor with a refreshing feeling.

[Example 4] Deodorization of body odor attached to clothes
Two 57-year-old men's shirts that were worn for one day were cut into 10 cm x 10 cm pieces, and were named shirt A and shirt B. Shirt A was sprayed with 1 mL of the deodorizing enzyme solution prepared in Example 3, and shirt B (control) was sprayed with 1 mL of the enzyme-free solution. As a result, Shirt A did not smell much better than the control.

The present invention relates to a method for deodorizing unsaturated aldehydes using enone reductase. By the method of the present invention, it is possible to eliminate a body odor by decomposing and removing a substance which is a source of body odor (aging odor) generated with aging.

Claims (16)

A method for eliminating the odor of an unsaturated aldehyde, comprising the step of reducing the carbon-carbon double bond of the unsaturated aldehyde. The step of reducing the carbon-carbon double bond of the unsaturated aldehyde is a step of contacting the reducing catalyst with the unsaturated aldehyde under conditions capable of reducing the carbon-carbon double bond. The method described in. The method according to claim 2, wherein the condition capable of reducing the carbon-carbon double bond is in the presence of an electron donor. The method according to claim 2, wherein the reduction catalyst is an enone reductase. The method according to claim 4, wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2,
(C) The amino acid sequence set forth in SEQ ID NO: 2, which comprises an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E): A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) and (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2, and which encodes a protein having the following physicochemical properties (A) and (E):
(A) Action Using reduced β-nicotinamide adenine dinucleotide phosphate as an electron donor, the carbon-carbon double bond of α,β-unsaturated ketone is reduced to generate the corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) It reduces the carbon-carbon double bond of an α,β-unsaturated ketone, but has substantially no ketone reducing activity.
(2) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(3) It does not substantially act on a substrate in which the two substituents from the ketone to the β-carbon are not hydrogen.
(4) The carbon-carbon double bond does not substantially act on the substrate present in the cyclic structure.
(C) Optimum pH
pH 6.5-7.0
(D) Optimum temperature 37-45°C
(E) Molecular weight sodium dodecylsulfate-polyacrylamide gel electrophoresis about 43,000. About 42,000 by gel filtration. The method according to claim 4, wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4,
(C) The amino acid sequence of SEQ ID NO: 4, which is composed of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E): A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) to (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, and which encodes a protein having the following physicochemical properties (A) to (E):
(A) Action The reduced β-nicotinamide adenine dinucleotide phosphate is used as an electron donor to reduce a carbon-carbon double bond of an α,β-unsaturated ketone to generate a corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(2) It reduces a carbon-carbon double bond of an unsaturated ketone, but has substantially no ketone reducing activity.
(3) 3-Methyl-4-(3-pyridyl)-3-buten-2-one is selectively reduced with α,β-unsaturated bonds to give 90% ee or more of (S)-3-methyl- Generates 4-(3-pyridyl)-3-butan-2-one.
(C) Molecular weight sodium dodecylsulfate-about 47,000 by polyacrylamide gel electrophoresis. About 92,000 by gel filtration.
(D) Working pH
pH 5.0-8.0
(E) Optimum temperature 37-45°C The method according to claim 3, wherein the electron donor is at least one coenzyme selected from the group consisting of the following (1) to (6):
(1) reduced nicotinamide adenine dinucleotide phosphate (NADPH),
(2) reduced nicotinamide adenine dinucleotide (NADH),
(3) reduced flavin mononucleotide (FMNH ₂ ),
(4) reduced flavin adenine dinucleotide (FADH ₂ ),
(5) Ubiquinone (UQ), and (6) Pyrroloquinoline quinone (PQQ). The method of claim 1, wherein the unsaturated aldehyde is selected from the group consisting of 2-nonenal, 2-octenal, 2-hexanal. A composition for eliminating an odor of an unsaturated aldehyde, which comprises, as an active ingredient, a reduction catalyst that catalyzes a reaction of reducing a carbon-carbon double bond of an unsaturated aldehyde. The composition according to claim 9, further comprising an electron donor. The composition according to claim 9, wherein the reduction catalyst is an enone reductase. The composition according to claim 11, wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2,
(C) The amino acid sequence set forth in SEQ ID NO: 2, which comprises an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E): A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) and (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2, and which encodes a protein having the following physicochemical properties (A) and (E):
(A) Action Using reduced β-nicotinamide adenine dinucleotide phosphate as an electron donor, the carbon-carbon double bond of α,β-unsaturated ketone is reduced to generate the corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) It reduces the carbon-carbon double bond of an α,β-unsaturated ketone, but has substantially no ketone reducing activity.
(2) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(3) It does not substantially act on a substrate in which the two substituents from the ketone to the β-carbon are not hydrogen.
(4) The carbon-carbon double bond does not substantially act on the substrate present in the cyclic structure.
(C) Optimum pH
pH 6.5-7.0
(D) Optimum temperature 37-45°C
(E) Molecular weight sodium dodecylsulfate-polyacrylamide gel electrophoresis about 43,000. About 42,000 by gel filtration. The composition according to claim 11, wherein the enone reductase is a protein encoded by a polynucleotide selected from the group consisting of (a) to (e) below.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3,
(B) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4,
(C) The amino acid sequence of SEQ ID NO: 4, which is composed of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and/or added, and has the following physicochemical properties (A) to (E): A polynucleotide encoding a protein having
(D) a polynucleotide which hybridizes with the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 under highly stringent conditions and which encodes a protein having the following physicochemical properties (A) to (E):
(E) a polynucleotide which comprises an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 4, and which encodes a protein having the following physicochemical properties (A) to (E):
(A) Action The reduced β-nicotinamide adenine dinucleotide phosphate is used as an electron donor to reduce a carbon-carbon double bond of an α,β-unsaturated ketone to generate a corresponding saturated hydrocarbon.
(B) Substrate specificity
(1) As an electron donor, it has a significantly higher activity for reduced β-nicotinamide adenine dinucleotide phosphate than for reduced β-nicotinamide adenine dinucleotide.
(2) It reduces a carbon-carbon double bond of an unsaturated ketone, but has substantially no ketone reducing activity.
(3) 3-Methyl-4-(3-pyridyl)-3-buten-2-one is selectively reduced with α,β-unsaturated bonds to give 90% ee or more of (S)-3-methyl- Generates 4-(3-pyridyl)-3-butan-2-one.
(C) Molecular weight sodium dodecylsulfate-about 47,000 by polyacrylamide gel electrophoresis. About 92,000 by gel filtration.
(D) Working pH
pH 5.0-8.0
(E) Optimum temperature 37-45°C The composition according to claim 10, wherein the electron donor is at least one coenzyme selected from the group consisting of (1) to (6) below:
(1) reduced nicotinamide adenine dinucleotide phosphate (NADPH),
(2) reduced nicotinamide adenine dinucleotide (NADH),
(3) reduced flavin mononucleotide (FMNH ₂ ),
(4) reduced flavin adenine dinucleotide (FADH _2), and (5) ubiquinone (UQ), and (6) pyrroloquinoline quinone (PQQ). 10. The composition according to claim 9, wherein the unsaturated aldehyde is selected from the group consisting of 2-nonenal, 2-octenal, 2-hexanal. A reduction catalyst that catalyzes a reaction for reducing a carbon-carbon double bond of an unsaturated aldehyde for use in eliminating the odor of an unsaturated aldehyde.