JPS61204197A - Production of sterol fatty acid ester - Google Patents

Abstract

PURPOSE:A specific enzyme is used to effect the reaction between a cholesterol and fatty acid whereby the title compound which is used as a hydrophilic base material for medicines and cosmetics is obtained in high yield readily under mild reaction conditions. CONSTITUTION:The catalytic reaction between a compound bearing a steroid skeleton and hydroxyls in its molecule such as cholesterol and a fatty acid is carried out using a lipase or cholesterol esterase, usually in an aqueous medi um, preferably at 10-60 deg.C to give the objective compound. It is preferred to use 5,000-50,000 units of lipase or 500-50,000 units of cholesterol esterase per 1g. of the cholesterol. Further, the amount of the fatty acid used is 3-6 times the molar amount of the cholesterol.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new method for producing esters of sterols and fatty acids.

Conventional Techniques Esters of sterols and fatty acids have been used, for example, in cholesteric liquid crystals (see JP-A-52-24992) and hydrophilic base materials for pharmaceutical and cosmetic applications (JP-A-52-41215).
(see Japanese Patent Laid-Open No. 52-79030), etc., and are widely used in various fields.

Conventionally, such sterol fatty acid esters have been produced exclusively by organic synthesis methods. However, in general, organic synthesis methods employ harsh reaction conditions, and disadvantages such as side reactions cannot be avoided. 0
The hydroxyl group of alcohols is secondary, and because it is close to the steroid skeleton, its reactivity is lower than that of ordinary aliphatic secondary alcohols. When reacting to produce a fatty acid ester, it is necessary to carry out the reaction at high temperature for a long time using an acid catalyst, or to perform the esterification reaction after first converting the fatty acid into an acid anhydride, acid halide, etc. Furthermore, when a trimethylsterol having two methyl groups at the 4-position and a hydroxyl group at the 3-position of the steroid skeleton is used as a raw material, the compound undergoes further reaction due to the steric hindrance of the two methyl groups at the 4-position. The esterification reaction with fatty acids is difficult unless it is converted into an acid halide. However, sterol esters that are desired as useful for various uses are generally esters of long-chain fatty acids, and halides of long-chain fatty acids, which are used as raw materials for the synthesis of such esters, are generally difficult to obtain. It usually requires complicated steps and must be synthesized separately, and has the disadvantage of being very expensive. In addition, such fatty acid halides are generally unstable and easily decomposed by moisture, and moreover, there is a possibility that irritating and corrosive products may be produced as by-products during the reaction, and they also have the disadvantage of requiring special reaction equipment.

Problems to be Solved by the Invention The present inventors have developed a method of producing a desired sterol fatty acid ester in a milder manner without using organic synthesis, which requires the harsh reaction conditions described above and requires expensive and unstable reaction reagents. We have been conducting extensive research with the aim of providing a method that can be produced under favorable conditions and with high yields. As a result, we succeeded in developing a new production technology for sterol esters that meets the above objectives using a specific enzyme, and have now completed the present invention.

The present invention relates to a method for producing sterol fatty acid esters, which is characterized in that a compound having a steroid skeleton and a hydroxyl group in the molecule is brought into contact with a fatty acid using lipase or cholesterol esterase.

According to the method of the present invention, by utilizing enzymatic action, harsh reaction conditions such as those found in conventional organic synthesis methods are employed, raw materials are synthesized separately requiring complicated operations, and special The desired sterol fatty acid ester can be produced easily and with high yield under very mild conditions, usually at room temperature and pressure, without the need for a reaction device, etc., and separation and purification from the reaction system is also easy. I can do it. Furthermore, according to the method of the present invention, esters that have conventionally been difficult to produce in good yield due to poor reactivity can be easily produced in high yield.

In the method of the present invention, it is important to use lipase or cholesterol esterase as the enzyme.

Here, lipase is an enzyme that catalyzes a reaction that hydrolyzes triglyceride into glycerin and fatty acids in stages, and cholesterol esterase is an enzyme that hydrolyzes the ester bond between cholesterin and fatty acids. The present inventors discovered for the first time that the above-mentioned lipase and cholesterol esterase catalyze the ester synthesis reaction between various sterols and fatty acids, and using this reaction, sterol fatty acid esters can be easily produced in high yield and on an industrial scale. It was successfully synthesized by

The lipase and cholesterol esterase used in the present invention are not particularly limited in their origin, and may be derived from various microorganisms, animals, or plants. The originating microorganism of lipase is, for example, Achromobacter+orurgus.
, Achromobacter riboriticum (Achromobacter
Acter Iipolyticum), Chromobacterium viscosum
Corynebacterium acnes
s), Pseudomonas aeruginosa (p 5eu
Pseudomonas aeruginosa), Pseudomonas fluorescens
), Pseudomonas fly (Pseudos+o
nas fraai), Staphy OD'/Kas
aureus (Staphy+ococcusaure)
us), Aspergillus niger
lus n1aer), Candida cylindracea (Candida cylindracea)
), 7 Mycola langinosa (Humicora
Ianuainosa), Penicillium caseicolul
, Penicillium crustosum (penicilliu)
mcrustosum), Benicilliua+ cyclopi
um), Penicillium locufuotei (pen
icilliulrOQtleforti), Torulopsis erno
bii), Bacillus subtilles (Bacillus
us 5ubtills), Alcaligenes 5D (
AICaliOeneS Sp), Thermomyces Ibadanensis
anensis), etc.

The originating microorganism of cholesterol esterase is the genus Pseudomonas, such as Pseudomonas aeruginos.
a), Pseudomonas fluorescens
), Pseudomonas knob sp., Pseudomonas dismolitii, etc., Achromobacter species such as Achromobacter delicaturus
genus Fusarium, genus Nocardia, genus Psytomonas, Streptomyces occidentalis, and genus Candida, such as Candida volitei, Candida too and chalice, Candida intermedia, and Candida syrinthracia.

Most of the above-mentioned enzymes are commercially available as purified enzymes, and these commercially available products can be used as they are in the present invention, but there is no particular need to use II-manufactured commercially available products. It is also possible to use microorganisms themselves having the ability to produce enzymes, their culture fluids, crude enzyme solutions obtained by processing the culture fluids, compositions containing enzymes, etc.
It is also possible to use immobilized enzymes obtained by immobilizing each of the above-mentioned enzymes on a suitable carrier or the like according to a conventional method.

In the present invention, sterols as one of the raw materials synthesized using the lipase or cholesterol esterase refer to compounds having a steroid skeleton and a hydroxyl group in the molecule. Here, the steroid skeleton is a skeleton represented by the formula, and the hydroxyl group is generally directly bonded to the skeleton. Specific examples of the above-mentioned steroids used in the present invention include cholesterol,
7-dehydrocholesterol, β-cholestanol,
Cholesterol, radosterol, thymosterol, thymostenol, desmostyrol, brassicasterol, ergosterol, campesterol, β-sitosterol, γ-sitosterol, α-spinasterol,
Examples include stigmasterol, lanosterol, dihydro-lanosterol, agnosterol, dihydro-agnosterol, and isocholesterol obtained by separating them from wool wax as a mixture thereof.

In the present invention, the fatty acid used as the other raw material has 2 carbon atoms.
-32 saturated or unsaturated fatty acids, which may be linear or branched.

Examples of saturated fatty acids include acetic acid, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, montanic acid, and melisic acid. acids, saturated straight chain fatty acids with an even number of carbon atoms such as n-todoriacontanoic acid, and propionic acid, n-valeric acid, enanthic acid, pelargonic acid, hendecanoic acid, tridecanoic acid, pentadecanoic acid, heptadecanoic acid, nonadecane. Examples include saturated straight chain fatty acids having an odd number of carbon atoms, such as heneicosanoic acid, tricosanoic acid, bentafusanoic acid, and heptacosanoic acid.

Examples of saturated branched chain fatty acids include isobutyric acid, isocaproic acid, isocaprylic acid, isocapric acid, inlauric acid, 11-methyl-dodecanoic acid, isomyristic acid, 13-methyl-tetradecanoic acid, isopalmitic acid,
15-methyl-hexadecanoic acid, isostearic acid, 1
7-methyl-octadecanoic acid, isoarachidic acid, 19-
Methyl-eicosanoic acid, α-ethyl-hexanoic acid, α-
Hexyldecanoic acid, α-heptylundecanoic acid, 2-decyltetradecanoic acid, 2-undecyltetradecanoic acid,
Examples include 2-decylpentadecanoic acid, 2-undecylpentadecanoic acid, and Fine Oxocol 1801 (manufactured by Nissan Chemical Co., Ltd.) represented by the formula %.Furthermore, the above-mentioned saturated odd-branched fatty acids include, for example, 6- Methyl-octanoic acid, 8-methyl-
Decanoic acid, 10-methyl-dodecanoic acid, 12-methyl-
Tetradecanoic acid, 14-methyl-hexadecanoic acid, 16
-Antiiso-based where the terminal is an isobutyl group such as methyl-octadecanoic acid, 18-methyl-eicosanoic acid, 20-methyl-tocosanoic acid, 22-methyl-tetracosanoic acid, 24-methyl-hexacosanoic acid, 26-methyl-octacosanoic acid, etc. fatty acids are included.

Examples of unsaturated fatty acids include tuccinic acid, caproleic acid, Linderic acid, lauroleic acid, thuzuic acid, fisetoleic acid, myristoleic acid, palmitoleic acid,
Petroselic acid, oleic acid, elaidic acid, vaccenic acid, cadreic acid, cis-11-eicosenoic acid, cetoleic acid, erucic acid, ceracoleic acid, 17-hexacosenoic acid, 6.9.12.15-hexadecatetraene acid,
Linoleic acid, linuric acid, α-eleostearic acid, β-
Eleostearic acid, bunicic acid, 6,9゜12.15-
Octadecatetraenoic acid, parinaric acid, arachidonic acid, 5.8.11.14.17-nicosapencitric acid,
7,10.13,16゜19-docosapentaenoic acid, 4
, 7.10,13°16.19-docosahexaenoic acid and the like.

Further, the fatty acid used in the present invention may be an oxyfatty acid having a hydroxyl group in the molecule. The oxyfatty acids include, for example, α-hydroxylauric acid, α-hydroxymyristic acid, α-hydroxypalmitic acid, α-
Hydroxystearic acid, ω- and droxylauric acid,
α-Hydroxyarachidic acid, 9-hydroxy-12-octadecenoic acid, ricinoleic acid, α-hydroxybehenic acid, 9-hydroxy-trans-10,12-octadecadienoic acid, camolenic acid, ibrolylic acid, 9.10 Examples include -dihydroxystearic acid and 12-hydroxystearic acid.

Furthermore, the above-mentioned fatty acids include, for example, oxalic acid, malonic acid, succinic acid, geltaric acid, adipic acid, pimelic acid, seberic acid, azelaic acid, sebacic acid, D.

It may also be a polycarboxylic acid such as malic acid.

The above-mentioned fatty acids may be used alone in the reaction of the present invention, or two or more of the above-mentioned fatty acids belonging to the same group or different groups may be mixed and used in the present invention. When two or more types are used in combination, for example, lanolin fatty acids, which are a mixture of saturated linear and branched fatty acids and oxyfatty acids, can be advantageously used as the fatty acid raw material.

The reaction of the present invention proceeds by using the above sterols and fatty acids as substrates and bringing them into contact in the presence of lipase or cholesterol esterase. The above contact is
This can be carried out by simply mixing both substrates and an aqueous solution of the enzyme, and therefore the reaction system is usually aqueous, but other suitable organic solvents such as n-hexane, rhohebutane, n-
An organic solvent system using hydrocarbon solvents such as octane, isooctane, cyclohexane, n-decane, n-tridecane, n-tetradecane, n-hexadecane, polybutene, diisobutylene, liquid paraffin, squalane, squalene, and Prista. You can also do it. By using such an organic solvent, the physical properties of the reaction system may be improved and the above-mentioned contact may become better, and the resulting esters and the enzymes used can be separated and recovered from the reaction system more efficiently. It may be possible to do so. Further, the above-mentioned mixing is preferably carried out by stirring in order to make the reaction system more uniform. The reaction conditions may be any conditions as long as there is no inactivation of the enzyme used or this is suppressed to a minimum, and the optimum 11H and temperature conditions for the enzyme are usually employed. Generally, the above temperature is preferably in the range of about 10 to 60°C. The pH can be alkaline, neutral, or acidic depending on the enzyme used, and to adjust the pH, an appropriate acid or alkali, such as hydrochloric acid, sulfuric acid, sodium hydroxide, potassium hydroxide, etc. An appropriate buffer solution such as phosphoric acid am solution can also be added to the reaction system, if necessary.

In addition, in the reaction system, enzyme inhibitors such as "Tween 80" (manufactured by Seisei Atlas Co., Ltd.) and [Triton A suitable surfactant or the like may also be added. Further, known activators of the enzymes used, such as casein, albumin, calcium ions, bile acids and salts thereof, can also be added.

The ratio of the enzyme and both substrates to be present in the reaction system is appropriately selected depending on their types, reaction conditions, etc., and is not particularly limited. Usually, the ratio of the enzyme is per 10 sterol of the raw material, and that of the lipase is about 10,000 to 100,000 per 100,000 sterol. Unit, preferably 5000-5
For cholesterol esterase, it is preferably about 10,000 to 100,000 units, preferably about 500 to 50,000 units.

The ratio of both substrates to be used can be arbitrarily determined, and there is no particular restriction, but it is usually preferable to use fatty acids in an amount of about 1 to 20 times, preferably about 3 to 6 times, molar amount relative to sterol. The reaction can be carried out in an aqueous medium or an organic solvent system, but the reaction system contains at least about 0%
．． 1112, typically 1 or more water is preferably present. In general, in a water-based system, the ratio of water to sterol is about 5~
It is preferable to use 500 times the weight, so that the reaction proceeds advantageously. In addition, when using an organic solvent system, the amount of solvent used varies slightly depending on the type of solvent, but for example, in the case of isooctane, the ratio of isooctane/water is usually approximately
~100 weight ratio, preferably 0.01 to 10 weight ratio, and in this case also the total solvent (specified solvent + water)
The amount used is preferably about 5 to 500 times the weight of the sterol. Other organic solvents can also be used within substantially the same range as the above-mentioned isooctane.

The reaction of the present invention varies depending on the reaction conditions, the type of enzyme used, the amount used, the type of substrate, the ratio used, etc., but usually about 3
It ends in 0 minutes to 120 hours.

In the present invention, after the above reaction is completed, the target product is separated from the reaction system according to a conventional method and purified if necessary. For isolation and purification of the target product from the reaction system, for example, the reaction solution was extracted using a suitable solvent such as ether, unreacted fat WI raw material was removed by alkaline deacidification, and the solvent layer was dehydrated and dried. After that, a method for removing the solvent can be exemplified. The crude sterol fatty acid ester thus obtained can be produced by conventional methods such as column chromatography.

The sterol fatty acid ester thus obtained can be used in a wide variety of applications in which this type of sterol fatty acid ester has been conventionally used.

Experimental Examples and Examples will be given below to explain the present invention in more detail.

In addition, the enzyme amount on each side was expressed using international units determined by the method shown below.

Measurement of lipase activity> Polyvinyl alcohol solution (“Poval #117J”)
(manufactured by Kurashiki Rayon Co., Ltd.) 18g and [Poval #205J (
(manufactured by the same company) in 800°C of water and heated to 75-80°C.
After heating and stirring to completely dissolve, cool and add water to make 1000ml) 75-, 2 ml of olive oil
2. Emulsify 9 with a homogenizer! ! 1 sample enzyme solution was added to a mixture of the prepared olive oil emulsions 5 and O, IM Rin WI Enoki Shokuso 4-, and the mixture was reacted at 37°C for 20 minutes while stirring at 500 rpm with a magnetic stirrer. Add ethyl alcohol 40- to this and
．． Titrate the free fatty acids with 05M potassium hydroxide solution.

The amount of enzyme that releases 1 μmole equivalent of fatty acid per minute under these conditions is defined as 1 rB unit (tJ).

Measurement of activity of cholesterol esterase> One unit (1U) of cholesterol esterase is the activity of liberating 1 μmol of cholesterol per minute at 37°C using calf serum as a substrate. It is determined by oxidizing free cholesterol with cholesterol oxidase and colorimetrically measuring the generated hydrogen peroxide with peroxidase.

Reaction solution composition 00.2M phosphate buffer (1) 86.5) 0.6
+++GO peroxidase [manufactured by Sigma Chemical Co., type I [NO, P-8250: l 0.
3II1200.35% 4-aminoantipyrine aqueous solution 0.311Go0
．． 2w/w% phenol aqueous solution 0.311120 Cholesterol oxidase aqueous solution [manufactured by Toyo Jozo Co., Ltd., product NO, T-04]
1M phosphate buffer (pH 7, containing 0,0,05% w/v Triton X-100) to 10U/-]
0.6Peng0 Calf Serum [Grand Island Biorosica/L, manufactured by (USA)) 0.3ml
20 distilled water
0.3-For the sample enzyme solution, use IOIM phosphoric acid as the enzyme! It is used by dissolving it in a buffer solution (pH 7, 5, 0, containing 1% albumin) to a concentration of about 1 U/-. The above reaction solution 31
! Ill was placed in a colorimetric cell, incubated at 37°C for 10 minutes, added a 0.05-sample enzyme solution, mixed gently by inversion, and measured over time at 493 rv to determine the rate of increase in absorption (ΔAS/ minutes).

The same process is performed using a dilution buffer instead of the sample enzyme solution to determine the rate of increase (ΔAb/min).

The difference in the above absorption increase rate (ΔA/min - ΔAs - ΔAb> is 0
．． If it is less than 0.05, the concentration of the sample enzyme solution is increased and the operation is repeated until the concentration becomes 0.05 or more. Enzyme activity (U
/l1l(If) is calculated by the following formula.

Enzyme activity (U/Il + (1) - The synthesis rate of the target ester was calculated by the following method. After the reaction was completed, the reaction solution was made acidic, extracted four times with diethyl ether, washed with water, and dehydrated. After drying and distilling off the diethyl ether, a whole fat sample is obtained.A known amount of internal standard substance (n-todoriacontane) is added to this sample to prepare a constant liter sample.This sample is placed in a chroma rod (quartz rod). Silica gel welded, manufactured by Yatron, Chromarod Sn)
Charge, deploy, Iatro Scan TH-10
(manufactured by Yatron) and FID (flame ionization) detector to determine the amount of synthesized ester, and divide this by the amount of theoretically synthesized ester based on sterol calculated from the amount of reaction solution charged to obtain the synthesis rate. shall be.

In each experimental example, unless otherwise specified, the reaction solution was stirred and mixed in a 20 wax 300 Cl test tube shaking medium!
The test was carried out using 11l (manufactured by Iwashiya Bioscience Co., Ltd., RMR-3-20). In addition, in Experimental Examples 1 to 8, Candida cylinthrasia (Candida cylinthracea) was used as an enzyme.
racea)-derived lipase (“Lipase MYJ” manufactured by Meito Sangyo Co., Ltd.) was used. In other examples, each individual is shown.

Experimental Example 1 This example clarifies the relationship between the ester synthesis rate and the amount of substrate. Cholesterol is used as the sterol, and oleic acid as the fatty acid is varied in a fixed amount (0,1+11). Both were stirred and mixed at 37° C. for 18 hours to react in the presence of 0.5 mQ (500 U) of lipase at a constant concentration. The results are shown in Table 1 below.

Table 1 Test example Amount of oleic acid Ester synthesis rate No.
(g) (%)1 0.07
58.5 2 0.1 77.9 3 0.15 83.0 4 0.22 87.7 5 0.29 88.6 6 0.37 89.7 7 0.44 91.8 8 0.66 92 .0 From Table 1 above, it can be seen that by using about 1 to 6 times the weight of oleic acid per 1 weight of cholesterol, the desired oleic acid ester of cholesterol can be synthesized with a synthesis rate of about 80% or more. It also contains 2 to 6 times more oleic acid! It can be seen that when using (3 to 9 times the mole), the target ester can be synthesized quickly and with the highest synthesis rate.

Experimental Example 2 This example was conducted to investigate how the amount of water in the reaction system affects the synthesis rate of the target ester. The experiment consisted of 0.1Ω cholesterol and 0.1Ω oleic acid.
Using 22g, add 0.5U of lipase (500U) to it.
When acting, water 0.5-11.0nIi2.2.0
IQ, 4. This was carried out by adding Qml and 6.0- and reacting each. The results of determining the synthesis rate for each reaction are shown in Table 2 below.

Table 2 Test example Water amount Ester synthesis rate NO9 (carpet)
(%) 1 0 87.5 2 0.5 97.6 3 1.0 97.4 4 2.0 98.2 5 4.0 97.4 6 6.0 96.9 From Table 2 above, water It has been found that it is preferable that the amount of addition l be about 1 to 3 mQ (about 10 to 3 ome times that of cholesterol).

Experimental Example 3 In this example, the ester synthesis rate was determined when the reaction system was a Ti solvent system. The experiment was carried out in the same manner as in Experimental Example 2, except that in place of water, predetermined amounts of isooctane, n-octane, or n-hexane saturated with water were used. The results are shown in Table 3 below.

Table 3 2 Inoctane (1) 96.23
(2>96.74 〃
(4) 91.45 (6
) 83.46 (8)
78.87 #(1067,6゛extension11NO= medium Wl12
Gold” 1m knee 8 0-octane (1) 8
3.59 <2)
78.110 (4)
29.811
(6) 13.912
(8) 8.513
(10) 7.714 n-
Hexane (1) 76.415
(2) 68.816
(4) 29.617
(6) 11
．． 018 (8)
9.519 〃 (106
, 6 Table 3 shows that in the above system, isooctane causes the least amount of enzyme deactivation. The isooctane is 0.5 to
By adding 3WfJ, the synthesis rate of the desired ester could be significantly improved.

Experimental Example 4 In this example, the synthesis rate of the target ester was investigated in a system of water and isooctane. was added and the reaction was repeated under the same conditions. The results are shown in Table 4 below.

Table 4 Test No. Amount of water added “Gold” & “L”!翫”-1
0 (No additive') 73.0 2 1 92, 7 3 2 94, 6 4 4 96, 6 5 6 96, 8 6 7 96, 5 7 8 97.5 8 9 94, 9 9 10 95, 5 Experiment Example 5 This example shows the synthesis rate of the target ester when the amount of water is fixed at 2.0IQ and the amount of isooctane added is varied in a reaction in a mixed solvent system of water and in-octane, similar to Experimental Example 4. This study investigated changes in The conditions are the same as in Experimental Example 4. The results are shown in Table 5 below.

5 Table 11 and L Iso-octane addition amount Synthesis rate (%) 1 0
(No additives) 87.92 0, 5
94.93 1.0 95.1
4 1, 5 96.45 3,
0 94.06 4, 0
90.47 6,0 83.48
8,0 71.5 From Table 5, water 2IQ
and isooctane 0.5-3.0m (sterol 5-3
It can be seen that the combined use with 0x) significantly improves the synthesis rate.

The example in the experimental example compares the reaction in an organic solvent system and the reaction in an aqueous system when the fatty acid is solid. The conditions were the same as in Experimental Example 1. However, in the case of organic solvent-based reactions,
In addition, n-octane 2.0112 and water 7.51 were added to the reaction solution.
11 G was added, and in the case of an aqueous reaction, 2.01112 g of water was added. Valmitic acid and stearic acid were used as solid fatty acids. The results are shown in Table 6. For reference, Table 6 also shows the results when oleic acid, which is liquid at room temperature, was used.

Table 6 Palmitic acid 99.0 91.62 Stearic acid 98.5 51.63 Olein? m
96.5 91.6 From Table 6 above, it can be seen that especially in the case of stearic acid, the use of an organic solvent has a remarkable effect on improving the synthesis rate.

Experimental Example 7 In this example, cholesterol 0.1g, oleic acid 0.2g
Lipase solutions with various concentrations of 0.5!112 were added to a reaction solution system of 2a, n-octane 2.0- and water 8.0-, and the relationship between enzyme concentration and ester synthesis rate was determined. It is. The reaction conditions were the same as in Experimental Example 1: 37°C, 20ux3
00 cpm, 18 hours). The results are shown in Figure 1. FIG. 1 plots the ester synthesis rate (%) on the vertical axis and the enzyme unit (international unit, U) on the horizontal axis, and plots the synthesis rate when enzymes are used in each enzyme unit.

From Figure 1, approximately 5,000
It can be seen that if enzyme U is present, the reaction proceeds rapidly and the desired ester can be synthesized in high yield.

Experimental Example 8 This example differs from Experimental Example 7 in that approximately 30
When using a lipase with a volume (approximately 100 LJ) showing a synthesis rate of ~40%, the action time was further extended and the relationship between the reaction time and the ester synthesis rate was determined.

The results are shown in Table 7 below.

Table 7 Actual JIINo0 reaction time (hr,) Synthesis rate (%) 1
16 45.6 2 24 57, 7 3 40 65, 8 4 48 70, 0 5 64 74, 4 6 72 74, 9 7 96 78, 0 8 120 79.8 From Table 7, even when using 100 U of enzyme It can be seen that when the reaction time is extended to 120 hours, the synthesis rate of the target ester can be increased to about 80%.

Experimental Example 9 The procedure was carried out in the same manner as in Experimental Example 1, using the prescribed amounts of sterols (used 10.io), fatty acids, and enzymes shown in Table 8, and employing the reaction system (water and organic solvent) shown in Table 8. The target ester was synthesized and its synthesis rate was investigated. The results are also listed in Table 8.

However, the symbols for sterols, fatty acids, enzymes, and organic solvents in Table 8 indicate the following.

Costerols> A-1...Cholesterol A-2...β-Sitosterol A-3...
・・Stigmasterol A-4・・・・β-cholestanol A-5・・・・Ergosterol A-6・・・・Isocholesterol (fat)
Acid> B-1...Oleic acid 8-2...Valmitic acid B-3...Stearic acid B-4...Linoleic acid B-5. ...α-human O-xybalmitic acid 8-6
...Lanolin fatty acid distillate> E-1 ...Lipase MY (manufactured by Meito Sangyo Co., Ltd., derived from Candida cilintrasia) E-2 ...Lipase T-01 (Toyo Manufactured by Jozosha,
(derived from Chromobacterium viscosum) E-3...Lipase ``Amano JA (manufactured by Ohno Pharmaceutical Co., Ltd., derived from the Asberigillus genus) E-4...Lipase [Amano JP (manufactured by Ohno Pharmaceutical Co., Ltd., derived from the Asberigillus genus)
(derived from Pseudomonas genus) E-5...Cholesterol esterase T-1
8 (manufactured by Toyo Jozo Co., Ltd.) E-6... Cholesterol esterase (derived from Candida cilinthracia) Organic medicinal agent> S-1... n-octane S-2... Isooctane Table 8 Test Stereofatty acid Water Organic solvent Synthesis rate No., Enzyme (U) O- (anti-stereo
(store) (1110) (%) Ru -
1 E-1(5(X)) A-I B-1(3)
8 S-1(2>96.52 E-1(5
00) A-2B-1(3) 8 S-1(2)
95.53 E-1 (500) A-3B-1
(3) 8 8-1 (2) 69.04 E
-1 (500) A-4B-1 (3) 8 S-
1 (2) 92.85 E-1 (500) A
-5B-1(3) 8 S-1(2) 55
．． 06 E-1 (500) A-6B-1 (1,4
) OS-2(4,3>70.47 E-1(
500) A-I B-1(3) 8 3-1
(2) 96.58 E-1 (500) A-
I B-2(3) 8 S-1(2) 99.
09 E-1 (500) A-I B-3 (3)
8 S-1(2) 98.510 6-1(5
00) A-I B-4(3) 8 S-1(
2) 96.511 2-1 (500) A-I
B-5(3) 8 S-1(2) 35.01
2 2-2 (1000) A-I B-6 (3)
2 - (0) 55.013 E-2 (10
00) A-I B-1(3) 8 S-2(
2) 85.314 E-3 (1000) A-
I B-1 (3) 2 8-2 (0,5) 55
．． 215 6-4 (1000) A-I B-1 (
3) 8 8-2 (2) 52.416 E
-5 (500) A-I B-1 (3) 8
S-1 (2) 97.717 6-5 (500)
A-2B-1(3) 8 S-1(2) 88.
318 6-6 (500) A-I B-1 (3)
8 S-2(2) 95.0 Example 1 Cholesterol 10Q, olein! 122g, isooctane 50mG, Lipase MY (manufactured by Meito Sangyo Co., Ltd.) 5011
111 (50000LJ) was heated to 20Or
The reaction was allowed to proceed at 37° C. for 18 hours while stirring at a speed of 18 pm. Ether was added to this reaction solution, and the aqueous layer was removed while washing with an aqueous sodium bicarbonate solution. After repeating this process several times, the ether layer was dehydrated and dried over sodium 1a acid anhydride. Then, the ether was distilled off to obtain white translucent crude cholesterol oleate.

The purity of this crude cholesterol oleate is 96
．． 3%, and the yield is 16.0 (J (yield 95.0%)
Met.

6 g of the crude product obtained above was subjected to silica gel column chromatography (Wakogel C200, manufactured by Wako Pure Chemical Industries, Ltd., 160
(1) and eluted with 2000ml of benzene to obtain 5.3g of cholesterol oleate. The infrared absorption spectrum (IRE), melting point, color development on thin layer chromatography (TLC), and Rf value of this product completely matched those of the standard cholesterol oleate.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the amount of enzyme used and the synthesis rate of the target ester. (The above) Agent Patent attorney Eiji Saegusa ・i, ::H1
' -1 Procedural amendment (self-restraint August 9, 1985 1 Description of the case 1985 Patent Application No. 45128 2 Title of the invention Process for producing sterol fatty acid esters 3 Person making the amendment Relationship to the case Patent applicant Furukawa Oil Co., Ltd. 4 Agent Sawanotsuru Building (6521) 2-10 Hirano-cho, Higashi-ku, Osaka Patent attorney Eiji Saegusa 5 Date of amendment order Voluntary 6 Section 7 of “Detailed Description of the Invention” in the specification subject to amendment Amendment [Contents of the amendment as attached in the attached sheet]) The phrase "etc." in the third line from the bottom of No. 6 of the specification is corrected as follows. Achromobacter, Chroneptacterium, Corynebacterium, Pseudococcus, Staphylococcus, Aspergillus, Candida, Fumi-La, Penicillium, Torulopsis, Pacillus, Alkali Earth metals, various microorganisms belonging to the genus Thermomyces, etc.'' 2) In the fourth line of page 8 of the specification, the phrase ``synthesis reaction'' has been corrected to ``synthesis.'' 3) On page 9, line 2 of the specification, “Stigmasterol,
'' should be corrected to ``stigmasterol, etc., as trimethylsterol.'' 4) On page 9, line 6 of the specification, the words "roll, etc." are corrected to "roll, cycloartenol, etc." 5) In the third line from the bottom of detailed cylinder 13, correct the word ``Prista'' to ``Pristan.''6> The statement "suitable" on page 14, line 10 of the specification is corrected as follows. [Preferably, when using heat-stable lipase etc.
Higher temperatures can also be employed depending on the enzyme. ] 7) In the first line from the bottom of No. 14 of the specification, the phrase ``such as'' is corrected to read ``1st grade nonionic surfactant, etc.''. 8) In page 15, line 12 of the specification, the phrase "can be determined," is corrected to "can be determined, and any of them may be in excess." 9) In the 5th line from the bottom of page 20 of the specification, “manufactured by the company” and FI
D (Hydrogen) should be corrected to read "FID Hydrogen, manufactured by FID." 10) Delete "sterol-based" from the second line from the bottom of page 20 of the specification. 11) In the fourth line from the bottom of No. 21 of the specification, the word "lipase" is corrected to "1 lipase aqueous solution." 12) On page 23, line 8 of the specification, the word "reaction" is corrected to "reaction (conditions are the same as in Example 1)." 13) In Table 8 on page 33 of the Mingto letter, “Test No.
, 18J, the following "Test No. 19" and [Test No. 20j are added. r19 E-3 (1000) A-6B-1 (3)
8 S-2(2) 63.020 E-1(1
000) A-4B-3(3) 8 S-2(2)
99.2J14) In the first line of page 34 of the specification, the ``Example 1'' must be corrected as follows. "Experimental Example 10 For 0.1c+ of cholesterol, fatty acids (3 moles of sterol) and enzymes (1 mole of sterol) shown in Table 9 were
000U), isooctane 10.05M
Using a reaction system of phosphate buffer D)H7)=3m12/7mf2, the synthesis rate of the target ester was investigated over the time period shown in the same table. The results are also listed in Table 9. However, for the enzymes in Table 9, the symbol () is the same as in Table 8.
The symbols for fatty acids are the same as those in Table 8 or indicate the following: <Fatty acids> B-7...Propionic acid B-8...Capric acid B-9...Lignoceric acid B-10...Succinic acid B-11...Sebacic acid Table 9 Experimental Example 11 Cholesterol 0.1q, oleic acid 0.22 to l,') part MY1000U, i'#cutane 10.05M phosphate buffer (pH 7) = 2+nQ
A reaction system consisting of /8m12 was employed, and the reaction was carried out at 37°C to examine changes in the synthesis rate over time. The results are listed in Table 10. Table 10 Experimental Example 12 In this example, cholesterol 0.1C], oleic acid 0.1C]
22Ω, lipase MY500tJ and organic solution W10.0
5M phosphate buffer (pH 7, 0>=2m(?/8
A reaction system consisting of mG was reacted at 37°C for 6 hours, and changes in the cholesterol oleate synthesis rate depending on the type of organic solvent were investigated. The following organic solvents were used. S-2...Isooctane S-3...Cyclohexane S-4...n-hexadecane S-5...[Ivy Solvent 1016J (manufactured by Idemitsu Petrochemical Company, Ca: 63%, C9: 30%) S-6 is an isoparaffinic organic solvent mixture whose main components are Isopar El (manufactured by Exxon Chemical Co., Ltd., Ca: 25-35%, C9 5-60%. It is shown in Table 11 below. Test NO, Organic solvent Synthesis rate (%) 1 S-297,0 23-396,8 33-496,7 43-596,0 53-696,0 Example 1" 15) Details On page 34, lines 3-4 of the book, “Lipase...
(50,000 tJ) J is corrected to ``Lipase MY (manufactured by Octoso Sangyo Co., Ltd.) aqueous solution 200 mQ (50,000 U) J.'' 16) In the second line of page 35 of the specification, ``...matched.''
The statement is corrected as follows. [...A match was made. Example 2 Cholesterol 1q, isostearic acid (manufactured by Emery Industries >2.2cx, lipase MY0.33
3g (10000LI), isooctane 30mG and 0
．． 05M phosphate buffer 80ml (1)
The reaction was carried out at 37° C. for 45 hours while stirring at a speed of 0 Orpm. This reaction solution was sampled sequentially during the reaction to check the synthesis rate of the target ester, which was 28.5% in 3 hours, 85.9% in 23 hours, and 45% in 23 hours.
The time was 91.0%. 45 hours after the start of the reaction, the reaction was stopped, extracted with methanol aqueous solution, and the isooctane layer was distilled off from the extract to obtain 0.09 Q of unreacted cholesterol, 1.53 CI of unreacted isostearic acid, and the desired isostearic acid ester 1. .54 CI was obtained. The obtained isostearic acid ester gave a single spot on TLC and had a purity of 100% on Iatroscan analysis.
% was rare. (Above) Procedural Amendment (Voluntary) June 2, 1985 1 Indication of the case 1985 Patent Application No. 45128 2 Name of the invention Process for producing sterol fatty acid ester 3 Person who wrote the amendment, Pa case Relationship with Patent Applicant (. ゝ・, - Nikofugawa Oil Co., Ltd. 4 Agent 6 Sawanotsuru Building, 2-10 Hirano-cho, Higashi-ku, Osaka-shi Section 7 of ``Detailed Description of the Invention'' in the Specification Subject to Amendment) Contents of Amendment 2 As attached to Fube 5111, Contents of Amendment 1 On page 4 of the specification, lines 19-20, the phrase "triglyceride in stages" is corrected to "glyceride". 2 Page 6 of the specification In the bottom line, ``originating microorganisms'' should be corrected to ``Origins include various tissues of mammals, such as the pancreas, liver, brain, adrenal glands, green glands, ovaries, etc.''. 3 Akira Am Page 7 In line 9, delete [Psitomonas genus]. 4. In the bottom line of page 20 of the specification, correct the statement "shall be." as follows. "Shall be. However, Experimental Examples 10 to 13 The synthesis rates in Examples 2 and 3 were determined by the following method.That is, after the reaction was completed, the reaction solution was made into a water-organic solvent two-phase system, and the concentration of the organic solvent phase was adjusted appropriately. Then, charge about 20 to 40 μ9 as a fat buffer to the resin, develop under suitable conditions to separate the target ester and unreacted substrate (for example, hexane/ether/formic acid = 56/1410.3), and then dry for several minutes. Then, the Chromarod from which the developing solvent was removed was applied to an IA-1-Roscan T I-1-10 detector.
The peak area of the lipid component in the reaction solution was determined, and based on the area, the synthesis rate (%) of the target ester was calculated using the following formula. However, the first component means a small mole component of both substrates charged as reaction substrates. ” 5 “Origin)” on page 31, line 19 of the specification [Origin, 30U/mg)J
I am corrected. 6. In the first line of page 32 for the specification, "Origin)" is corrected to [Origin, 2801J/mg)J. 7. The phrase “origin)” on page 32, line 3 of the specification is corrected to “origin, 4U/l11(+)J.” 8 The phrase “origin)” on page 32, line 5 of the Book of Akira II is corrected as “origin, 4U/l11(+)J.”
The origin is corrected to 30tJ/m(1)J. 9. In the 7th line of page 32 of the specification, the phrase “manufactured by the company)” is corrected to “Made by the company, 105 Ll/+no) J.” 10 In the 9th line of page 32 of the specification, the phrase “origin)” is corrected to “Made by the company, 105 Ll/+no) J” Origin, 20LJ/mg protein, manufactured by Seikagaku Kogyo Co., Ltd.) J. 11. In the third line of page 35 of the specification, the phrase "in the drawings" is corrected as follows. [Experimental Example 13 Using the specified amounts of enzymes, sterols, and fatty acids shown in Table 12, isooctane 1 0.05M phosphate buffer (DI-17) = 3 mQ/8
-The reaction was carried out for a predetermined period of time. The results are also listed in Table 12 along with the reaction time. In addition, the abbreviations in Table 12 are as described above or indicate the following. <Enzymes> E-7... Cholesprolesterase (derived from Pseudomonas sp., 100 U/III (), manufactured by Funakoshi Pharmaceutical Co., Ltd.) E-8... Lipase OF (derived from Candida syrinthracia, 360 U/ma, Octacaccharide Industry) <Fatty acids> B-12... Isostearic acid (manufactured by Emery Industries) B-13... Rirun M (manufactured by Tokyo Kasei Kogyo) No. 1
2 Table I E-7(66,7) A-1(100) B-1(
147) 396.62 E-6(6,7) A-1(
100) B-12 (147) 2486.73 E-
7 (66,7) A-1 (100) B-12 (147
) 590.84 E-1 (1000) A-1 (10
0) B-13 (216> 1.593.25 E-6
(6,7> A-1 (100) B-6 (159) 4
872,86 E-6 (6,7) A-6 (100)
B-1 (146) 12292.17 E-8 (100
0) A-1 (100) B-1 (147) 1896
．． 5 Example 3 10 g of dihydrocholesterol, 11 g of oleic acid, lipase MY333w+. (10000U) and cyclohexane 5-
The reaction was stirred at 0.00 cpm for 64 hours. The synthesis rate 40 hours after the start of the reaction was 73.1%, and that after 64 hours was 83.1%. After the above reaction, the reaction mixture was extracted with an aqueous methanol solution to remove unreacted dihydrocholesterol and oleic acid, and cyclohexane was distilled off from the cyclohexane layer to obtain 12.40 g of the desired dihydrocholesterol oleate. of drawings” (or more)

Claims (1)

[Claims] (1) A method for producing styrene fatty acid ester, which comprises contacting and reacting a compound having a steroid skeleton and a hydroxyl group with a fatty acid using lipase or cholesterol esterase.