JP2004337165A - New enzyme for esterification of cholesterol and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology applicable for amelioration of state of the skin (prevention and amelioration of dry rough skin, prophylaxis, prevention, of melasma, wrinkle, etc.) or treatment of hyperlipidemia, etc., by developing a technology controlling amount of free fatty acids and cholesterol in the body. <P>SOLUTION: The new cholesterol esterifying enzymes catalyze cholesterol producing reaction (transfer of an acyl group), without mediating action of coenzyme, by acting free fatty acids and cholesterol or its derivatives as substrates, not acting on fatty acid-CoA complex. The enzyme has 40-45 kDa of molecular weight (gel filtration technique), 3.0-7.0 of acting pH, 4.0-5.0 of optimal pH, acting at 4-60°C, 20-50°C of optimal temperature. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel cholesterol esterification enzyme and its use. The present invention is used mainly in the technical fields of cosmetics, reagents, diagnostic agents, and drug development.

  Fatty acids and cholesterol present in living organisms (organs, blood, skin, etc.) are very important substances as energy sources for living organisms and constituents of biological membranes. Numerous studies have been made on these metabolisms, and the presence of esters with fatty acids (cholesterol esters) is also known as a cholesterol storage type.

Among them, Unna and Golodetz reported that studies on the skin show that the amount of cholesterol esters in the epidermis is particularly high in the stratum corneum (around 1910). In addition, reports on the activity of converting cholesterol and free fatty acids to cholesterol esters in the skin have been made by experiments using isotope-labeled oleic acid and cholesterol (see Non-Patent Document 1). This conversion reaction from free fatty acids and cholesterol to cholesterol ester was found to be activated by coenzyme A (CoA) and adenosine triphosphate (ATP) (see Non-Patent Document 2). It was concluded that this would be a transfer reaction via acyl-CoA, which was also suggested in the experiment of No. Similarly, bovine breast extracts also show the effect of the addition of CoA, ATP, Mg ²⁺ and the like (Askew et al., "Lipids", 6, 326-331, 1971).

  In fact, two acyltransferases, LCAT (lecithin-cholesterol acyltransferase) and ACAT (acyl-CoA-cholesterol acyltransferase), were found as transferases, and their respective cDNAs were cloned in 1986 (see Non-Patent Document 3). It was made in 1993 (see Non-Patent Document 4). While LCAT mainly exists in the blood and acts, ACAT exists in various tissues such as the foreskin, small intestine, and liver, and is involved in regulating the amount of cholesterol in the living body, and also in the absorption of cholesterol from the blood. Therefore, development of an inhibitor thereof is being promoted as a target of a therapeutic agent for hyperlipidemia (see Non-Patent Document 5).

  On the other hand, experiments using ACAT knockout mice showed that the amount of cholesterol ester was not so reduced, and that a considerable amount of ester was still synthesized, suggesting the presence of other esterifying enzymes other than ACAT. (See Non-Patent Document 6). Although there have been reports of ACAT isozymes since then (see Non-Patent Document 7), the existence of another enzyme could not be denied.

  Incidentally, it is known that the presence of excessive free fatty acids in the skin causes acne and rough skin, and a method for rapidly reducing free fatty acids is also required. The use of drugs that reduce the total amount of sebum, including free fatty acids and cholesterol, may be effective, but excessive amounts can cause dry skin and are not always desirable, especially for middle-aged and elderly people who have reduced sebum secretion . Although there is a method using sebum-adsorbing powder or powder, it is not always satisfactory, and it is effective to convert free fatty acids into harmless cholesterol esters.

  In addition, as a therapeutic agent for hyperlipidemia, an inhibitor for HMG-CoA reductase (= hydroxymethylglutaryl-CoA reductase), which is a rate-limiting enzyme for cholesterol synthesis, currently occupies the mainstream, but this alone is still insufficient. It is not a satisfactory situation, and there is a need to discover new key metabolic enzymes and to develop new therapeutic agents.

Freinkel and Aso, "J. Invert. Dermatol.", 52, 148-154, 1969. Freinkel and Aso, "Biochim. Biophys. Acta", 239, 98-102, 1971. McLean et al., "Proc. Natl. Acad. Sci. USA", 83, 2335-2339. Chang et al., "J. Biol. Chem.", 268, 20747-20755. Yoji Hirakawa and Hiroaki Shimokawa, "Therapeutic Agents for Hyperlipidemia", Pharmaceutical Journal of Japan, Vol. 118, pp. 389-395 (2001) Meiner et al., "Proc. Natl. Acad. Sci. USA", 93, 14041-14046, 1996. Anderson et al., "J. Biol. Chem.", 273, 26747-26754, 1998.

  The present invention has been made in order to solve the problems of the above-mentioned conventional technology, and has as its object to develop a technology for controlling the amounts of free fatty acids and cholesterol in a living body. Using this technology, it becomes possible to prevent and improve rough skin, prevent and prevent spots and wrinkles, and to apply it to drugs for treating hyperlipidemia.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies, and as a result, are enzymes that are different from LCAT and ACAT conventionally known, and directly react with cholesterol without converting free fatty acids into acyl-CoA. A novel enzyme that produces cholesterol ester was found, and the present invention was completed.

That is, the present invention relates to a novel cholesterol esterifying enzyme having the following properties.
(1) Action Directly acts on free fatty acids and cholesterol or its derivatives without mediating the action of coenzymes to catalyze the cholesterol ester formation (acyl transfer) reaction.
(2) Substrate specificity Free fatty acids and cholesterol or derivatives thereof act as substrates. It does not act on fatty acid-CoA complexes.
(3) Molecular weight The molecular weight by gel filtration is 40 kDa to 45 kDa, and the molecular weight by SDS-PAGE is 60 kDa to 70 kDa.
(4) Working pH and optimum working pH
The working pH is 3.0 to 7.0, and the optimum pH is 4.0 to 5.0.
(5) Working temperature and optimum temperature It works in a wide range of 4 to 60 ° C, but the optimum working temperature is found at 20 to 50 ° C.
(6) Thermal stability When heat-treated at each temperature for 10 minutes in an acetate buffer (pH 5.0), almost no inactivation occurs at 37 ° C or lower, and almost complete inactivation occurs at 80 ° C.
(7) Influence of activity EDTA (ethylenediaminetetraacetic acid) and DTT (dithiothreitol) do not affect the activity.
(8) Purification method Purification is performed by one of the following methods (i) and (ii).

  (I) A crude enzyme solution is obtained from an animal or human-derived tissue by a conventional method, and the enzyme solution dialyzed after fractionation with ammonium sulfate is adsorbed to (a) a cholesterol sepharose column and eluted with an acetate buffer to collect fractions. And (b) adsorbing the fraction to a heparin sepharose column and eluting with an acetate buffer (containing NaCl) to collect the fractions. May be performed first).

(Ii) A crude enzyme solution is obtained from an animal or human-derived tissue by a conventional method, and the enzyme solution dialyzed after fractionation with ammonium sulfate is adsorbed to (a) a cholesterol sepharose column and eluted with an acetate buffer to collect fractions. A step of (b) adsorbing the fraction on a heparin sepharose column and eluting with an acetate buffer (containing NaCl) to collect the fraction; (c) a step of chelating the fraction on a chelating Sepharose column (Cu is a ligand) And eluted with an acetate buffer (containing imidazole) to collect fractions. (D) The fraction is adsorbed to a phosphorylcholine-modified silica column (a sodium phosphate buffer as a mobile phase) and collected. (However, any of the steps (a) to (d) may be performed first).
(9) Peptide sequence The fraction obtained by the purification method described in (ii) above was subjected to SDS-PAGE, and a peptide fragment obtained from a band that appeared in the 50 to 70 kDa region was identified in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. , 65-78, 106-111, 238-242, 287-299, 289-299, 319-327, 328-340, 360-370, 426-437, 438-446 , 470-481 and 486-492.

  The present invention also relates to the above enzyme, which is derived from mouse or human.

  The present invention also relates to a method for producing a cholesterol ester, wherein the enzyme is added to a system containing a free fatty acid and cholesterol or a derivative thereof to produce a cholesterol ester without using a coenzyme.

  The present invention also relates to the use of a cholesterol esterification enzyme, which is used to reduce free fatty acids and / or cholesterol in a living body.

  The present invention also relates to a method for searching for a drug for reducing free fatty acids and / or cholesterol using the above enzyme.

  The present invention also relates to a skin improving method for reducing free fatty acids and / or cholesterol in a living body, preventing and preventing spots and wrinkles, and / or preventing and improving rough skin by using the above enzyme.

  The present invention also relates to a method for searching for a therapeutic agent for hyperlipidemia for reducing free fatty acids and / or cholesterol in a living body using the above enzyme.

  The present invention provides a novel cholesterol esterification (acyltransferase) enzyme. By using this enzyme, cholesterol ester can be produced at low cost, and free fatty acids and cholesterol in the body can be reduced. Furthermore, the enzyme is also useful for preventing and preventing skin wrinkles and wrinkles, for suppressing rough skin, and for developing therapeutic agents for hyperlipidemia and the like.

  Hereinafter, the present invention will be described in detail.

  The enzyme according to the present invention has an activity of catalyzing the production of cholesterol ester using free fatty acids and cholesterol or a derivative thereof as substrates without the action of coenzymes such as CoA and ATP. This enzyme does not use a fatty acid-CoA complex as a substrate.

  The origin of the enzyme is not particularly limited as long as it has the above-mentioned activity, but is preferably derived from animals, particularly mammals. For example, human, rat, cow, horse, pig, guinea pig, hamster, mouse and the like are preferable. Among them, mice and humans are more preferable. The organ to be used is not particularly limited as long as an enzyme having the above activity can be obtained. It may be obtained from animal-derived cultured cells, which may be a primary culture, or may use commercially available cells. When the present enzyme is used, it may be in a state of being extracted from a living body or purified by a suitable column chromatography or the like. Alternatively, it may be obtained by obtaining a gene encoding the present enzyme, incorporating the gene into an appropriate host such as yeast or animal cells, and expressing it.

  The free fatty acid used as a substrate of the present enzyme includes saturated and unsaturated fatty acids, such as myristic acid, oleic acid, stearic acid, palmitic acid, palmitoleic acid, linoleic acid, linolenic acid, and arachidonic acid. Among them, stearic acid, oleic acid, palmitic acid, and palmitoleic acid having 16 or 18 carbon atoms and having 0 or 1 double bond are preferred.

  Cholesterol or a derivative thereof used as a substrate of the present enzyme includes, in addition to cholesterol, 24 (R) -OH-cholesterol, 7α-OH-cholesterol, 25-OH-cholesterol, 27-OH-cholesterol, 7-ketocholesterol and the like. Is mentioned.

When it is difficult to track the reaction because of a large amount of endogenous fatty acids or cholesterol, it is preferable to use a substrate labeled with an isotope or a fluorescent substance. For example, fatty acids labeled with ¹⁴ C and cholesterol labeled with ³ H are commercially available and can be suitably used.

  The catalytic reaction by the present enzyme acts in the range of pH 3.0 to 7.0, more preferably pH 3.0 to 6.0, and the optimum pH is 4.0 to 5.0. The reaction temperature is 4 to 60 ° C, but the optimum temperature is 20 to 50 ° C. The catalytic reaction is performed by adding a free fatty acid and cholesterol as an ethanol solution in an appropriate buffer, shaking or standing for about 5 to 10 minutes, and then adding the enzyme solution of the present invention. The reaction time is usually about 1 to 60 minutes.

  The reaction product is not particularly limited as long as it can detect cholesterol ester, but gas chromatography, thin layer chromatography, high performance liquid chromatography and the like are usually used. In this case, the reaction solution may be extracted with an organic solvent, for example, a chloroform: methanol (2: 1) solution, and subjected to chromatography. The amount of the solvent used for the extraction is preferably about 4 to 10 times the reaction solution.

  The gel and the developing solvent for thin layer chromatography are not particularly limited as long as fatty acids, cholesterol or its derivatives, and cholesterol esters can be separated from each other. Generally, a silica gel is used as the gel, and commercially available products such as "Silica Gel 60" (Merck) and "Silica Gel 70" (Wako Pure Chemical Industries) can be used. As the developing solvent, a mixed solvent of hexane: ether: acetic acid = 8: 2: 0.1 (mass ratio), hexane: ether: acetic acid = 8: 3: 0.1 (mass ratio) and the like are suitably used. Coloring may be performed using iodine or the like.

  The molecular weight of this enzyme measured by the gelation method is 40 kDa to 45 kDa. A specific example of the measurement will be described in Example 7 described later. The molecular weight measured by the SDS-PAGE method is 60 kDa to 70 kDa. Specific examples of the measurement will be described later in Example 9 and later.

  The enzyme is preferably stored at pH 3 to 8.

  The activity of this enzyme is not affected by either EDTA (ethylenediaminetetraacetic acid) or DTT (dithiothreitol). The activity is not affected by metal ions. Specific examples of the effects of these activities will be described later in Example 5.

  A specific example of a method for purifying the present enzyme will be described in Example 4 described later.

  This enzyme can be applied to the production of cholesterol esters and is also considered to be useful for removing free fatty acids that are irritating to the skin. For example, application of the activity promoter of the present enzyme to the skin can be expected to prevent and improve rough skin. In addition, the present invention can be applied to reduction of cholesterol concentration in a living body, hyperlipidemia, and the like. In particular, since it is an enzyme different from known ACAT, it is expected that useful materials can be provided even to patients whose efficacy has been weak conventionally. Further, if a drug that enhances the expression of the present enzyme can be obtained by obtaining the gene of the present enzyme, the same effect can be expected.

  In other words, the use of this enzyme makes it possible to control the amount of free fatty acids and cholesterol in the living body without the need for expensive and unstable coenzymes such as CoA and ATP. (Screening) to develop an agent for preventing acne and rough skin caused by free fatty acids and cholesterol. For example, a solution of various plant extracts or chemically synthesized compounds is added to the present enzyme solution to carry out a reaction, and a drug that enhances the conversion reaction to cholesterol ester is screened. The extract or compound thus obtained can be blended as a skin external preparation.

  Furthermore, it could be used for inexpensive production of cholesterol esters. Cholesterol esters are known to have a moisturizing effect (for example, Nobuaki Hattori, "Fragrance Journal", extra edition, 185-190, 2000), and can be applied to the development of external preparations containing this. In addition, it will be possible to develop drugs such as hyperlipidemia drugs by searching (screening) for drugs using this enzyme.

[Example]
Hereinafter, the present invention will be described specifically with reference to examples. Note that the technical scope of the present invention is not limited by these examples and the like.

[Detection of free fatty acid-cholesterol esterase (Fatty acid: cholesterol acyltransferase; hereinafter referred to as "FCAT") in mouse skin extract]
The skin was excised from the back site of a hairless mouse (HR-1, Hoshino animal) with scissors. 0.5 g of skin was crushed with a glass homogenizer for 1 minute using 50 mM acetate buffer (pH 5.6) containing 2 ml of 1 mM EDTA. This was then transferred to a 15 ml tube and sonicated (Astrason sonifier). After centrifugation (9,000 rpm, 10 minutes), the supernatant was recovered, and ammonium sulfate was added to the supernatant so as to be 80% saturated. After leaving at 4 ° C. for 3 hours, the mixture was centrifuged to collect the precipitate.

  The precipitate was dissolved in 2 ml of 50 mM acetate buffer (pH 5.0) containing 1 mM EDTA, and dialyzed against 200 ml of the same buffer (Spectra / pore membrane, molecular weight cut off 12,000 to 14,000). ). After standing at 4 ° C. overnight, the solution was recovered and used as a crude enzyme solution.

  The following experiment was performed using this crude enzyme solution as a reaction solution.

  First, the reaction was carried out by changing the amount of the reaction solution and the reaction time, and whether or not the reaction was an enzyme reaction was examined. The reaction was performed as follows.

That is, 50 mM acetate buffer (pH 4.5), 1 mM EDTA, 20 μg cholesterol (2 μl ethanol solution), 0.5 mg / ml BSA (bovine serum albumin. Use free of fatty acid; Nacalai Tesque) and 200,000 cpm (10 μM) ) of [1- ¹⁴ C] oleate (1.2Myueru ethanol solution. mixed Perkin Elmer) solution (0.2 ml) was prepared and pre-incubated for 5 minutes at 37 ° C..

  The above crude enzyme solution (reaction solution) was added to this solution and reacted (37 ° C., pH 4.5). The reaction was carried out while varying the reaction time and the amount of the crude enzyme solution (the amount of the reaction solution). When the reaction time was changed, the reaction was performed for 5 to 30 minutes after adding 20 μl of the crude enzyme solution. When the amount of the reaction solution was changed, the amount of the crude enzyme solution was changed between 10 and 60 μl, and the reaction was carried out for 10 minutes. A 1.5 ml Eppendorf tube was used for the reaction.

  After the reaction, 0.5 ml of a chloroform: methanol (2: 1) solution was added and mixed well to extract a substrate and a product. After centrifugation, the supernatant was poured into another container and further extracted with 0.5 ml of a chloroform: methanol (2: 1) solution. The organic layer containing the chloroform: methanol (2: 1) solution was collected in a glass tube and left overnight to remove the solvent. This was mixed well with 10 μl of a chloroform: methanol (2: 1) solution, and spotted on an aluminum plate silica plate (Aldrich) by thin layer chromatography. As a developing solvent, a mixed solvent of hexane: ether: acetic acid (8: 2: 0.1) (mass ratio) was used.

  As controls, unlabeled cholesterol oleate and oleic acid were spotted simultaneously.

  After development, spots were colored by spraying with iodine for identification, each spot was cut out with scissors, placed in a glass container, 5 ml of a liquid scintillator was added, and the mixture was counted for 5 minutes by a scintillation counter (Beckman “LS6000TA”). The cholesterol ester conversion was calculated by the following equation.

  The conversion rate (%) of the sample to which no enzyme was added was subtracted to obtain a true conversion value. The results are shown in FIGS. 1a and 1b.

  FIG. 1a is a graph showing the relationship between the amount of a reaction solution of FCAT (ammonium sulfate / dialysis sample) derived from mouse skin and the cholesterol ester conversion rate (pH 4.5, 37 ° C., 10 minutes). The horizontal axis shows the reaction liquid volume (μl), and the vertical axis shows the cholesterol ester conversion rate (%). FIG. 1b is a graph showing the relationship between the reaction time of FCAT (ammonium sulfate / dialysis sample) derived from mouse skin and the cholesterol ester conversion rate (pH 4.5, 37 ° C.). The horizontal axis shows the reaction time (minutes), and the vertical axis shows the cholesterol ester conversion rate (%). As is clear from the results of FIG. 1a, the cholesterol ester conversion increased as the amount of the crude enzyme solution (the amount of the reaction solution) increased. Moreover, as is clear from the results of FIG. 1b, the cholesterol ester conversion increased as the reaction time increased.

  The crude enzyme solution was separately heat-denatured at 80 ° C. for 10 minutes and then reacted at 37 ° C. for 10 minutes, but the cholesterol ester conversion did not increase (<0.02%). It could be assumed that the active form would be a protein.

  Further, the temperature dependence of the reaction was examined using the above-mentioned mouse skin-derived FCAT (ammonium sulfate / dialysis sample) at a reaction time of 10 minutes and a pH of 4.5. As shown in FIG. It was found that ester conversion could be obtained. From this, it was more certain that this reaction was performed by an enzyme.

[Detection of FCAT in human stratum corneum extract]
The tanned stratum corneum was collected from the human back and upper arm. To 0.3 g of the stratum corneum, 1.0 ml of 50 mM acetate buffer (pH 5.6) containing 1 mM EDTA was added, and a glass homogenizer and sonication were performed in the same manner as in Example 1. After centrifugation, the supernatant was taken as a crude enzyme solution. Using 20 μl of the crude enzyme solution, the reaction was carried out at 37 ° C. for 10 minutes in the same manner as in Example 1 except that the pH was changed, and the cholesterol ester conversion was determined. FIG. 3 is a graph showing the pH dependence at that time. As is clear from FIG. 3, the activity became maximum at pH 4-5.

[Preparation of cholesterol Sepharose gel for FCAT purification]
A cholesterol sepharose gel was prepared according to the method of Fukuyama and Miyake et al. ("J. Biochem.", 85, 1183-1193, 1979). 5 ml of EAH Sepharose 4B gel (Amersham Bioscience) was washed with 400 ml of 0.5 M NaCl solution using a glass filter. Separately, 25 mg of cholesterol hemisuccinate (Sigma) was dissolved in 12.5 ml of dimethylformamide, and the pH was adjusted to 4.7 with HCl. 250 mg of WSC (water-soluble carbodiimide) dissolved in 0.75 ml of MilliQ water and the above gel were added thereto (in a 50 ml tube), and the mixture was shaken overnight. After the reaction, the gel was recovered by washing with 120 ml of a 50% dimethylformamide solution.

[Partial purification of FCAT in mouse skin extract]
From 2.0 g of mouse skin, a glass homogenizer and ultrasonic crushing were performed according to the method described in Example 1 to obtain 7.8 ml of a crude extract.

  Ammonium sulphate was added to this so as to be 30% saturated and left at 4 ° C. for 2 hours. Then, the supernatant (8.1 ml) was recovered by centrifugation. Ammonium sulfate was added to the supernatant so as to be 80% saturated, and left at 4 ° C. for 3 hours. After collecting the precipitate by centrifugation, it was dissolved in 4 ml of 50 mM acetate buffer (pH 5.0). This was dialyzed overnight (Spectra / pore membrane) against 500 ml of the same buffer.

  After recovering the enzyme solution from the dialysis tube, the enzyme solution was adsorbed to the cholesterol sepharose column prepared in Example 3 (preliminarily equilibrated with 20 mM acetate buffer, pH 5.0, gel 2 ml). After washing with 40 ml of the same buffer, elution was carried out with 12 ml of the same buffer containing 1 M NaCl.

  The fractions were collected and concentrated to 3 ml with "Sentriplus-10" (molecular weight cut off 10,000, Millipore). This was dialyzed overnight against 400 ml of 20 mM acetate buffer (pH 5.0) containing 5 mM NaCl and desalted.

  The solution after the above treatment was added to Heparin Sepharose CL-6B (gel 2 ml, Amersham Bioscience) already equilibrated with a dialysis buffer. After washing with 24 ml of equilibration buffer, the FCAT fraction was collected and concentrated with "Centriplus-10" (1.3 ml). The purification steps are shown in Table 1. The protein amount was converted by a calibration curve using BSA (bovine serum albumin) using a kit from Bio Rad.

  As is clear from the results shown in Table 1, FCAT was purified about 20-fold by three steps.

[Properties of partially purified FCAT from mouse skin]
The properties of FCAT partially purified according to Example 4 were examined. In one reaction, an enzyme (10 μl) having an activity of 0.83 (pmol / min) was used, and an average value of n = 2 was shown.

  First, the pH dependence was examined. FIG. 4 shows the results. In the figure, the vertical axis indicates relative activity (%), and the horizontal axis indicates pH. Performed with acetate buffer and Hepes buffer. The optimum pH was around 4-5, similar to FCAT derived from human stratum corneum. In addition, it was inactivated by heat (treatment at 80 ° C. for 10 minutes).

Subsequently, the effect of adding CoA and ATP was examined (pH 4.5, 37 ° C., 20 minutes). FIG. 5 shows the results. The addition amount of ATP was 2.5 mM and the addition amount of CoA was 0.25 mM. At the same time, 4 mM MgCl ₂ was added, but the cholesterol ester conversion rate was hardly changed by any addition. Since CoA, ATP and Mg are cofactors for acyl-CoA synthetase, it was considered that the enzyme sample did not have the activity of producing cholesterol ester via acyl-CoA.

  In addition, glibenclamide reported as an ACAT inhibitor (Ohgami et al., "Biochem. Biophys. Res. Commun.", 277, 417-422, 2000) was added to examine the presence or absence of inhibition (pH 4.5, 37). ° C, 20 minutes). FIG. 6 shows the results. As is clear from the figure, it was found that the addition of glibenclamide reduced the residual activity of partially purified FCAT derived from mouse skin.

  Further, the effects of addition of EDTA (ethylenediaminetetraacetic acid), DTT (dithiothreitol) and metal ions were examined (pH 4.5, 37 ° C., 20 minutes). FIG. 7 shows the results. As is clear from the figure, no inhibition by EDTA and DTT was observed, and there was almost no effect of adding metal ions. Therefore, it was presumed that metal ions did not participate in the activity. It has been reported that ACAT increases the activity by DTT (Biopharmaceutical Science Laboratory Course 3 “Lipid III”, Keizo Inoue, Keiichi Nakagawa, Ed., P162, Hirokawa Shoten, 2002), so FCAT is an enzyme different from ACAT. It was shown that there is.

Further, by using in place of oleic acid [1- ¹⁴ C] palmitic acid and [1- ¹⁴ C] linoleic acid (both Perkin Elmer), was examined substrate specificity (pH4.5,37 ℃, 20 Minutes). FIG. 8 shows the results. In mouse ACAT-1, it was reported that oleoyl-CoA>palmitoyl-CoA> linoleoyl-CoA (Cases et al., "J. Biol. Chem.", 273, 26755-26764, 1998). As is evident from the viewpoint of the fatty acid portion, in FCAT, oleic acid> palmitic acid> linolenic acid (polyvalent double bond), which is considered to have the same tendency.

[Measurement of ACAT activity using partially purified FCAT fraction derived from mouse skin]
ACAT activity was measured in order to directly confirm that ACAT was not contained in the partially purified FCAT fraction.

  As samples, the mouse skin-derived FCAT (ammonium sulfate / dialysis sample) obtained in Example 1 and the mouse skin-derived partially purified FCAT fraction obtained in Example 4 were used. As an ACAT sample (positive control), an extract from mouse liver (mouse liver 80% ammonium sulfate / dialysis sample) was used. The measurement was performed according to a conventional method (for example, Uelmen et al., "J. Biol. Chem.", 270, 26192-26201, 1995).

That, 100 mM Tris-HCl buffer ^{(pH7.5), [1- 14 C} ] oleoyl -CoA (80,000cpm, 8μM, Amersham Biosciences), 1mM EDTA, 20μg cholesterol, 0.5 mg / ml BSA (Free of fatty acid) and left at 37 ° C. for 5 minutes.

  Next, a test solution (enzyme solution) was added and reacted at 37 ° C. for 20 minutes, and then 0.5 ml of an organic solvent (chloroform: methanol = 2: 1) was added. Was extracted. To the aqueous layer after extraction, 0.5 ml of a solvent was further added, and extraction was performed again. The count of the aqueous layer was taken as the unreacted base mass (cpm). On the other hand, the organic layer was recovered, dried, dissolved in 10 μl of a chloroform: methanol (2: 1) solution, and spotted on a thin-layer silica gel. After drying, the mixture was developed with a mixed solution of hexane: ether: acetic acid = 8: 2: 0.1, spots corresponding to cholesterol ester were collected, and the count was measured to examine the conversion rate of each cholesterol ester. Table 2 shows the results.

  As is clear from the results in Table 2, the mouse liver 80% ammonium sulfate / dialysis sample (ACAT sample) shows ACAT activity (cholesterol ester conversion), whereas the mouse skin ammonium sulfate / dialysis sample and partially purified product have ACAT activity. The activity (cholesterol ester conversion) was found to be very low. From this, it can be determined that ACAT and FCAT are different enzymes.

[Measurement of molecular weight of FCAT derived from mouse skin]
The molecular weight of mouse FCAT was measured by a gel (agarose carrier gel) filtration method using a “Superose 12 column” (1.0 × 30 cm, Amersham Bioscience). As a buffer, a 20 mM acetate buffer (pH 5.0) containing 0.15 M NaCl was used at a flow rate of 0.5 ml / min. From the molecular weight marker and the elution position of the FCAT activity, the molecular weight was estimated to be 40 kDa to 45 kDa. This is a value smaller than the molecular weight of mouse ACAT, 64 kDa (Uelmen et al., "J. Biol. Chem.", 270, 26192-26201, 1995-estimated from cDNA).

[Detection of FCAT activity in extracts prepared from human adult breast-derived keratinocytes and human adult skin-derived fibroblasts]
The title culture cells were purchased from Kurabo Industries. Regarding keratinocytes, frozen cells (5 × 10 ⁵ cells) were suspended in 10 ml of HuMedia KG2 medium (Kurabo) and inoculated in one 75 cm ² flask. After 1, 3, and 5 days, the medium was changed. After 7 days, the cells were treated with a trypsin / EDTA solution, and the cells were collected by centrifugation (1.9 × 10 ⁶ cells). After washing twice with 10 ml of PBS (phosphate-buffered saline), the cells were suspended in 7 ml of PBS.

For fibroblasts, frozen cells were suspended in 10 ml of DMEM [containing 10% FBS (fetal bovine serum)] medium (Invitrogen) and seeded in one 75 cm ² flask. After 1, 3, and 5 days, the medium was replaced. After 7 days, the cells were treated with a trypsin / EDTA solution, and the cells were collected by centrifugation (3.6 × 10 ⁶ cells). This was also washed twice with PBS and suspended in 8 ml of PBS.

  Both were subjected to ultrasonic crushing, and this liquid was used as an extract as it was. The activity and the protein concentration were measured by the methods described in Examples 1 and 4, respectively. ACAT activity was also measured according to Example 6. Table 3 shows the results. As a comparison, the activity of the mouse skin extract is also shown.

  As is clear from the results in Table 3, ACAT was confirmed in both, while FCAT activity was confirmed only in the keratinocyte extract. Thus, it was shown that the mode of activity by tissues differs from that of ACAT.

[Identification of FCAT derived from mouse skin]
According to the method described in Example 4, 4.0 g of mouse skin was subjected to the steps of crude enzyme extract, ammonium sulfate / dialysis (30% saturation, 80% saturation), a cholesterol sepharose column, and a heparin farose column, followed by partial conversion of FCAT. Purified. FCAT was further purified and identified from this partially purified fraction.

First, a 0.1 M CuSO ₄ solution was added to prepare a chelating Sepharose FF column (Amersham Bioscience) using Cu as a ligand. Next, 2M NaCl was added to the partially purified enzyme to a concentration of 0.5M, and this was equilibrated with a 20 mM acetate buffer (pH 5.0) containing 0.5M NaCl, and a chelating Sepharose FF column (3 ml gel) was used. Was added. After washing with an acetate buffer containing 5 mM imidazole, FCAT was eluted with an acetate buffer containing 10 mM imidazole.

  This fraction was collected and concentrated to 210 μl using “Centriplus-30” and “Centricut Mini-30” (both Amicon). The samples in each of the purification steps up to this point were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using a gel (Tesco) with a 10% acrylamide concentration. FIG. 9 shows the results. For staining, a silver staining kit (Wako Pure Chemical Industries, Ltd.) was used.

  As shown in lane 5 in FIG. 9, there are still some bands, so it was decided to purify further. As a result of examining various columns, it was found that elution peaks were separated into several peaks by fractionation with a phosphorylcholine-modified silica column (4.6 × 250 mm, Shiseido) (see FIG. 10). It was decided to use it.

  This phosphorylcholine-modified silica column uses a chromatographic packing material in which phosphorylcholine groups are chemically bonded directly to the surface of a carrier (such as spherical porous silica gel) for the GFC column. Exhibits extremely low and high separation ability. The packing material for chromatography has been previously filed by the present applicant (Japanese Patent Application No. 2003-052305).

  For this phosphorylcholine-modified silica column (hereinafter also referred to as “PC column”), 76 μl of 210 μl of the enzyme fraction obtained by the above chelating Sepharose column was used. Using a 50 mM sodium phosphate buffer solution (pH 6.3) containing 0.5 M NaCl as a mobile phase, the above 76 μl portion was added to the sample in 13 portions, and the peak portion was collected.

  Each fraction was collected and subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) again (silver staining) (FIG. 11). In the figure, it was found that FCAT activity was present in fraction 1 (lane 3), and that the main protein of 65 kDa in fraction 2 (lane 4) did not have FCAT activity. Therefore, this fraction 1 (lane 3) was collected and concentrated and desalted using "Sentricut mini-30".

  As shown in Table 4, although the specific activity was considerably increased by the treatment with the PC silica column, it was found that there were still at least four bands from 70 kDa to 50 kDa as shown in lane 3 in FIG. Various columns were examined in order to further determine which band the FCAT protein corresponds to, but further separation was extremely difficult with an ion exchange column or a gel filtration column. Therefore, for protein identification, after the sample is electrophoresed, the protein band is cut out, digested with trypsin in a gel, extracted, analyzed by LC-MS, and the fragment is searched for and identified on a database. I decided.

  First, the samples [four bands of 70 kDa, 62 kDa, 55 kDa, and 50 kDa (lane 3 in FIG. 11) and a total of five 65 kDa contained in a fraction different from FCAT on the PC column] were carboxymethylated with iodoacetic acid. After that, it was loaded on an SDS acrylamide gel (10% acrylamide). The same gel and marker were used as described above. After the electrophoresis, the plate was washed three times with MilliQ water and stained with Simply Blue Stain (invitrogen). The excised band was washed with acetonitrile and digested with trypsin (Amersham Bioscience) in ammonium bicarbonate at 37 ° C. overnight. After the reaction was stopped by adding a formic acid solution, the mixture was mixed well, centrifuged, and the supernatant was recovered. To the remaining gel, a 0.1% formic acid solution containing 30% acetonitrile and then a 0.1% formic acid solution containing 60% acetonitrile were added and mixed to extract the peptide, and after centrifugation, the supernatant was collected. Analysis by LC-MS was performed.

  The sequences of the detected peptide fragments were searched in the NCBI mouse protein database and arranged in the order of the number of matching sequences, and the ones with the highest possibility were examined.

  As a result, it was found that the band of 70 kDa had the highest homology to the enzyme esterase 1, and the second and third positions in the search result were keratin. The 62 kDa band could not be identified due to the small amount of the detected peptide, and both the 55 kDa and 50 kDa bands were serine protease inhibitors. Albumin was the first candidate for the 65 kDa band. Only the 70 kDa band was a candidate for the enzyme, and the matching sequence exceeded 20%. It was considered that the 70 kDa band was extremely likely to be esterase 1.

  The amino acid sequence of esterase 1 is the sequence shown in SEQ ID NO: 1 in the sequence listing. Among the sequences of the peptide fragments obtained by the analysis of the 70 kDa band, the peptide fragments having a sequence that matches the amino acid sequence of esterase 1 (the amino acid sequence of SEQ ID NO: 1 in the sequence listing) are as follows.

    Fragment 1: a peptide fragment showing the amino acid sequence from amino acid position 65 (Phe) to position 78 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 2: Peptide fragment showing the amino acid sequence from amino acid position 106 (Glu) to position 111 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 3: A peptide fragment showing the amino acid sequence from amino acid position 238 (Asp) to position 242 (Arg) of SEQ ID NO: 1 in the sequence listing.

    Fragment 4: Peptide fragment showing the amino acid sequence from amino acid position 287 (Gln) to position 299 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 5: Peptide fragment showing the amino acid sequence from amino acid position 289 (Thr) to position 299 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 6: A peptide fragment showing the amino acid sequence of amino acid positions 319 (Ala) to 327 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 7: Peptide fragment showing the amino acid sequence from amino acid position 328 (Ser) to position 340 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 8: Peptide fragment showing the amino acid sequence from amino acid position 360 (Met) to amino acid position 370 (Arg) of SEQ ID NO: 1 in the sequence listing.

    Fragment 9: Peptide fragment showing the amino acid sequence from amino acid position 426 (Asp) to position 437 (Arg) of SEQ ID NO: 1 in the sequence listing.

    Fragment 10: A peptide fragment showing the amino acid sequence of amino acid positions 438 (Tyr) to 446 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 11: Peptide fragment showing the amino acid sequence from amino acid position 470 (Glu) to position 481 (Lys) of SEQ ID NO: 1 in the sequence listing.

    Fragment 12: Peptide fragment showing the amino acid sequence of amino acid number 486 (Phe) to 492 (Arg) of SEQ ID NO: 1 in the sequence listing.

The MH ⁺ (molecular weight at the time of cationization), the mass% of the peptide fragment in the total molecular weight, and the AA% (the number of amino acids of the peptide fragment in the total number of amino acids in the whole protein) of the fragments 1 to 12 are shown in Table 5 below As shown in FIG. In comparison with the 70 kDa band (homology), a candidate other than esterase 1 was keratin, and no enzyme including ACAT or LCAT appeared.

  Esterase 1 is known as the major mouse esterase (EC.3.1.1.1) and is a glycoprotein (65 kDa) consisting of 554 amino acids (eg, THFinlay et al., J. Biol. Chem., 257, 10914-10919). , 1982; SS Kadner, et al., J. Biol. Chem., 260, 15604-15609, 1985; TL Genetta et al., Biochem. Biophys. Res. Commun., 151, 1364-1370, 1988). It is known to be present in blood and liver, etc., and has been found to be widely involved in estrogen metabolism with wide substrate specificity. However, its presence in the skin is not known, and it is not known that it is involved in the ester formation of cholesterol and free fatty acids, which we have now found.

  On the other hand, cholesterol ester degradation in mice has been shown to be catalyzed by cholesterol esterase (EC.3.1.1.13). This cholesterol esterase, unlike esterase 1, is composed of 593 amino acids (K. Mackay et al., Gene, 165, 255-259 1995), mainly works in the pancreas, and is called bile salt-stimulated activated by bile. It has also been found that this is the same as an enzyme (lipase) called lipase (J. Nilsson et al., Eur. J. Biochem., 192, 543-550, 1990). The lipase is known to catalyze the synthesis of cholesterol ester from cholesterol and free fatty acids in vitro (E.M. Kyger et al., Biochemistry, 29, 3853-3858, 1990).

  In this study, the active fraction was not separated during the purification process of the crude enzyme extract from the skin. Therefore, esterase 1 that is highly homologous to FCAT, not cholesterol esterase, is the main factor in the production of cholesterol ester in the skin. It is thought that it works.

[Confirmation of degradation activity of synthetic substrate using purified fraction]
It has been reported that esterase 1 has the activity of decomposing a synthetic peptide substrate (SS Kandner et al., J. Bio. Chem., 260, 15604-15609, 1985). I checked. Since the commercial product of the β-alanine-nitrophenyl ester described in the above document was discontinued, a similar Boc-alanine-nitrophenyl ester (Sigma-Aldrich) was used.

  That is, in the presence of a 20 mM Tris-HCl buffer (pH 7.5), a 1/100 volume (final concentration: 0.3 mM) of a substrate solution dissolved in 50% dimethyl sulfoxide (DMSO) to a concentration of 30 mM was added. Incubated for 5 minutes. Thereafter, 20 μl of the present enzyme solution was added (3.0 ml in total), and the activity was measured at 25 ° C. The measuring device used was a JASCO V-560 spectrophotometer. The increase in absorbance accompanying release of the nitrophenyl group was observed over time (measuring wavelength: 405 nm). The results are shown in FIG. In the figure, the vertical axis indicates absorbance (Abs), and the horizontal axis indicates time (seconds). As a result, an increase in absorbance was confirmed by the addition of the purified enzyme (fraction 1 of the PC column; “lane 3” in FIG. 11) (graph indicated by “PC-1” in FIG. 12). On the other hand, in fraction 2 (PC column; “lane 4” in FIG. 11) having a small FCAT activity, the increase in absorbance was small (graph indicated by “PC-2” in FIG. 12).

[Effect of UV irradiation on mouse back on FCAT activity and cholesterol esterification ratio]
The FCAT activity of mice that induced wrinkle formation by ultraviolet (UVB) irradiation was measured and compared with those without UV irradiation. That is, the back of the SKH-1 hairless mouse (Charles River Japan) is irradiated with ultraviolet rays for 2 to 8 minutes per day for 10 weeks (ultraviolet irradiation apparatus: DERMARAY M-DMR-100, lamp model number: FL20S-E-30 / DMR). (Seven in parallel) and wrinkles were formed. After 8 weeks, the skin on the back of the mouse (5 animals) was excised and stored frozen. As a control (control group), UV-irradiated ones (4 individuals) under the same growth conditions were used.

  After 0.5 g of the preserved skin was excised and cut with scissors, 2.0 ml of 50 mM acetate buffer (pH 5.6) containing 1 mM EDTA was added, and crushed with a glass homogenizer. After transferring to a 15-ml tube and further sonication, the mixture was centrifuged (9000 rpm, 15 minutes), the supernatant was recovered, and the activity was measured. The reaction conditions were the same as in Example 1. As a result, the average value of FCAT specific activity in the group not irradiated with ultraviolet light was 38.5 (pmole / min · mg), and the average value of FCAT specific activity in the irradiation group was 32.4 (pmole / min · mg). From these results, it is considered that the FCAT activity is reduced by the irradiation of ultraviolet rays.

  Furthermore, the ratio of free cholesterol to esterified cholesterol in the skin was examined and quantified. First, 0.25 g of the skin was cut off, cut into pieces, and immersed in 2 ml of hexane overnight to extract cholesterol. After removing skin residues, hexane was removed by air drying in a 10 ml glass tube. After dissolving in 100 μl of ethanol, free cholesterol and total cholesterol were quantified using a Determiner LFC kit and a Determiner TC-555 kit, respectively (both manufactured by Kyowa Medex). The value obtained by removing free cholesterol from total cholesterol was defined as cholesterol ester.

  As a result, the ratio of cholesterol esterification in the unirradiated group was 56%, and the ratio in the irradiated group was 53%. It was revealed that the UV irradiation reduced the esterification ratio as well as the FCAT activity. There is a report that the irradiation of human back with ultraviolet rays reduces the esterification ratio of cholesterol in the skin (M. Gloor et al., Dermatologia, 154, 5-13, 1977). This has been associated with a decrease in FCAT activity and the esterification ratio of cholesterol in the skin as the skin condition worsens.

[Effect of aging and ultraviolet irradiation on esterase 1 expression level]
The skin of HR-1 mice aged 8 weeks, 20 weeks, 40 weeks, 60 weeks, and 87 weeks (2 individuals only for 20 weeks old, 3 others) was excised and RNA was prepared. Using this, the expression level of esterase 1 was analyzed using a microarray (Agilent). Table 6 shows the results. In Table 6, an 8-week-old sample was represented as 1.0.

  As is clear from the results shown in Table 6, the expression level of esterase 1 tended to decrease as the age of the week increased.

  On the other hand, cholesterol esterase, an enzyme involved in cholesterol metabolism in the liver and pancreas, was hardly expressed in the skin.

  In addition, the same experiment was performed in a blot model [F1 mouse, HR-1 (female) × Hr / De (male), Hoshino animal]. With respect to the three individuals, half of the back was irradiated with ultraviolet rays and the other half was not irradiated. The skin on each back was excised to prepare RNA, and the expression level of esterase 1 was analyzed by microarray. As a result, it was found that the expression level of esterase 1 was reduced to 0.82 in the irradiated part, where the unirradiated part was 1.0. This was consistent with the fact that FCAT activity in skin was reduced by ultraviolet irradiation as shown in Example 11, assuming that FCAT and esterase 1 were the same.

[Application effect of cholesterol ester]
The improvement by the skin barrier function by the cholesterol ester was observed using the amount of water evaporation (TEWL value) and the amount of retained water as indexes. Patch test bandage (Torii Pharmaceutical Co., Ltd.) in which 100 μl each of 1% by mass oleic acid, 1% by mass cholesterol, and 1% by mass cholesterol oleate (Wako Pure Chemical, ethanol solution) was impregnated into a round piece of cloth. Was adhered to one male panel forearm and occluded for 2 hours. After the continuation for 6 days, the measurement was performed on the next day using “TEWA meter TM210”. Table 7 shows the results.

As is clear from the results shown in Table 7, an increase in the TEWL value was suppressed with cholesterol oleate as compared with oleic acid and cholesterol.

  Cholesterol esters have a higher hydrophobicity than cholesterol and fatty acids, which are their materials, and have an effect of increasing skin barrier and suppressing water evaporation. From this fact, it is assumed that the barrier function can be exhibited by blending cholesterol ester itself into cosmetics, but the blending is extremely limited due to its high hydrophobicity. In particular, it is quite difficult to formulate a composition having a high water content such as a lotion. Therefore, it is very significant to increase the amount of cholesterol ester by increasing the activity of FCAT involved in the synthesis of cholesterol ester in the skin.

It is a graph which shows the relationship between the reaction liquid amount of FCAT (ammonium sulfate and dialysis sample) derived from mouse skin, and the cholesterol ester conversion rate. 4 is a graph showing the relationship between the reaction time of mouse skin-derived FCAT (ammonium sulfate / dialysis sample) and the cholesterol ester conversion rate. 4 is a graph showing the relationship between the reaction temperature of mouse skin-derived FCAT (ammonium sulfate / dialysis sample) and the cholesterol ester conversion rate. It is a graph which shows pH dependence of FCAT derived from human stratum corneum. It is a graph which shows the pH dependency of mouse skin-derived partially purified FCAT. It is a graph which shows the influence of the addition of CoA and ATP to partially purified FCAT derived from mouse skin. It is a graph which shows the effect of the addition of glibenclamide to mouse skin-derived partially purified FCAT. It is a graph which shows the effect of EDTA, DTT, and metal ion addition to mouse skin-derived partially refined FCAT. It is a graph which shows the substrate specificity of the free fatty acid of mouse skin-derived partially purified FCAT. It is a photograph (a photograph substituted for a drawing) which shows an electrophoretogram (SDS-PAGE) of a sample in each purification stage until fractionation by a chelating Sepharose column using a crude enzyme solution derived from mouse skin. 4 is a graph showing an elution pattern (measured at a wavelength of 210 nm) of a phosphorylcholine-modified silica column (PC column). It is a photograph (a photograph substituted for a drawing) which shows an electrophoretogram (SDS-PAGE) of a sample in each purification stage until fractionation by a phosphorylcholine (PC) silica column using a crude enzyme solution derived from mouse skin. It is a graph which shows Boc-alanine-nitrophenyl ester decomposition activity by the phosphorylcholine modified silica column (PC column) refinement | purification sample.

Claims (6)

A cholesterol esterifying enzyme having the following properties.
(1) Action Directly acts on free fatty acids and cholesterol or its derivatives without mediating the action of coenzymes to catalyze the cholesterol ester formation (acyl transfer) reaction.
(2) Substrate specificity Free fatty acids and cholesterol or derivatives thereof act as substrates. It does not act on fatty acid-CoA complexes.
(3) Molecular weight The molecular weight by gel filtration is 40 kDa to 45 kDa, and the molecular weight by SDS-PAGE is 60 kDa to 70 kDa.
(4) Working pH and optimum working pH
The working pH is 3.0 to 7.0, and the optimum pH is 4.0 to 5.0.
(5) Working temperature and optimum temperature It works in a wide range of 4 to 60 ° C, but the optimum working temperature is found at 20 to 50 ° C.
(6) Thermal stability When heat-treated at each temperature for 10 minutes in an acetate buffer (pH 5.0), almost no inactivation occurs at 37 ° C or lower, and almost complete inactivation occurs at 80 ° C.
(7) Influence of activity EDTA (ethylenediaminetetraacetic acid) and DTT (dithiothreitol) do not affect the activity.
(8) Purification method Purification is performed by one of the following methods (i) and (ii).
(I) A crude enzyme solution is obtained from an animal or human-derived tissue by a conventional method, and the enzyme solution dialyzed after fractionation with ammonium sulfate is adsorbed to (a) a cholesterol sepharose column and eluted with an acetate buffer to collect fractions. And (b) adsorbing the fraction to a heparin sepharose column and eluting with an acetate buffer (containing NaCl) to collect the fractions. May be performed first).
(Ii) A crude enzyme solution is obtained from an animal or human-derived tissue by a conventional method, and the enzyme solution dialyzed after fractionation with ammonium sulfate is adsorbed to (a) a cholesterol sepharose column and eluted with an acetate buffer to collect fractions. A step of (b) adsorbing the fraction on a heparin sepharose column and eluting with an acetate buffer (containing NaCl) to collect the fraction; (c) a step of chelating the fraction on a chelating Sepharose column (Cu is a ligand) And eluted with an acetate buffer (containing imidazole) to collect fractions. (D) The fraction is adsorbed to a phosphorylcholine-modified silica column (a sodium phosphate buffer as a mobile phase) and collected. (However, any of the steps (a) to (d) may be performed first).
(9) Peptide sequence The fraction obtained by the purification method described in (ii) above was subjected to SDS-PAGE, and a peptide fragment obtained from a band that appeared in the 50 to 70 kDa region was identified in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. , 65-78, 106-111, 238-242, 287-299, 289-299, 319-327, 328-340, 360-370, 426-437, 438-446 , 470-481 and 486-492.   The enzyme according to claim 1, which is derived from mouse or human.   A method for producing a cholesterol ester, comprising adding the enzyme according to claim 1 or 2 to a system containing a free fatty acid and cholesterol or a derivative thereof to produce a cholesterol ester without using a coenzyme such as CoA or ATP.   Use of a cholesterol esterification enzyme, wherein the enzyme according to claim 1 or 2 is used for reducing free fatty acids and / or cholesterol in a living body.   A method for improving skin, comprising using the enzyme according to claim 1 or 2 to reduce free fatty acids and / or cholesterol in a living body, thereby preventing and preventing skin wrinkles and wrinkles, and / or preventing and improving skin roughness. A method for searching for a therapeutic agent for hyperlipidemia for reducing free fatty acids and / or cholesterol in a living body using the enzyme according to claim 1 or 2.