JPH01218593A - Synthesis of fatty acid ester - Google Patents

Abstract

PURPOSE:To prolong activity duration time and to improve synthesis ratio per unit enzyme, by blending a specific enzyme with a substrate solution containing an alcohol component and an acid component and subjecting to contact reaction. CONSTITUTION:A lipase is selected from Pseudomonas, an ester bond comprising a secondary hydroxyl group is hydrolyzed and treated in an solution at 60 deg.C for 60minutes to give a thermostable lipase (A) having >=50% remaining relative hydrolyzing activity. Then, the component A is blended with an alcohol component such as cholesterol, an acid component such as oleic acid and a solvent such as isooctanoic acid, reacted and the solvent is removed to give a substrate reaction solution (B). Then, the component B and the component A are fed to a reacting part 8, N2 gas is introduced from a nozzle 6, the components A and B are mixed, brought into contact with each other and ester synthesis reaction is progressed. Then formed water produced with the reaction is removed from a gas outlet 9, the vaporized solvent is condensed by a condenser 1 and returned to the system. Then, the reaction is carried out for about 92 hours to collect a fatty acid ester in 99.9% synthesis ratio.

Description

DETAILED DESCRIPTION OF THE INVENTION (Objective of the Invention) The purpose of the present invention is to provide an improved method that enables industrially more advantageous implementation of the method for producing fatty acid esters using enzymes, which was previously developed by the present inventors. be.

(Prior art) For example, cholesteric liquid crystals (see JP-A-52-24992) and hydrophilic bases for pharmaceutical and cosmetic applications (JP-A-52-79)
(Refer to Publication No. 030) etc., and are widely used in various fields.

Furthermore, fatty acid esters of higher alcohols have long been used as hydrophilic bases for pharmaceutical and cosmetic applications.

Conventionally, such fatty acid esters have been produced mostly by organic synthesis methods, but in general organic synthesis methods employ harsh reaction conditions, and side reactions are unavoidable. It is necessary to isolate and purify the product. In particular, the hydroxyl group of sterols is secondary, and since this hydroxyl group is close to the steroid skeleton, the reactivity is lower than that of ordinary aliphatic secondary alcohols, and therefore, they have been sterolized.

The present inventors have demonstrated that desired esters can be advantageously produced in good quality and yield under milder conditions without using organic synthesis, which requires the harsh reaction conditions and expensive and unstable reaction reagents. As a result of intensive research with the aim of providing a method for efficient production, we first discovered that a specific enzyme catalyzes the synthesis of styrene esters, and based on this knowledge, invented a method for synthesizing fatty acid esters using enzymes. and filed a patent application. (JP 61-204197, JP 61-200997, JP 62-48391, JP 62-166895, JP 61-200997) However, these have the following problems. Improvements were needed for industrial implementation.

1. Heat-resistant conventional lipases have lower stability than general enzymes, and are particularly unstable in aqueous solutions. Therefore, if the amount of raw material substrate charged per enzyme is increased, it will be deactivated before a predetermined synthesis rate is reached, and it is necessary to use a large amount of lipase □ in order to obtain a sufficiently high synthesis rate. As a result, the production amount per enzyme was low, the production amount per batch was small, and non-production time such as preparing raw materials increased, resulting in low productivity.

2. The method previously applied by the present inventors, in which a stable emulsified dispersion is made from a substrate solution and an enzyme solution, and then allowed to stand still for reaction (Japanese Patent Application No. 61-200997), is a method that This method was excellent in that it did not require continuous stirring because the reaction occurred at the water/oil interface of the King Mulsion.
At the same time, the movement of water produced as esterification progresses is restricted, and water is essentially required, making it impossible to control the central water, making it difficult to obtain a sufficiently high synthesis rate, and making it difficult to use cheap dispersions. required a special emulsifying machine for its preparation.

3. The immobilization operation is complicated, it is difficult to granulate the carrier gel, and special equipment is required to scale up the immobilization.

4. When using a substrate that is solid or semi-solid at room temperature, a large amount of solvent is required to improve the fluidity of the enzyme, since the reaction temperature cannot be raised with enzymes that have poor heat resistance.

(Problems to be solved by the invention) Therefore, the inventors of the present invention proceeded with research and development to solve these problems of the conventional technology, and as a result, discovered the following knowledge and came to solve these problems. .

The enzyme selected from the lipases derived from the genus S. genus is stable and long-lived under the conditions of the present invention.

2. The above enzyme can catalyze ester synthesis of a sterically hindered secondary hydroxyl group close to the steroid 2 skeleton.

3. Continuous mixing gives movement to the water produced and the hydrophobicity of the substrate allows water to be effectively removed from the reaction field.

4. Unlike conventional lipases, which have the ability to synthesize sterol esters, the above enzyme exhibits a higher reaction rate in a non-aqueous system than in a water/oil two-phase system.

5. By carrying out continuous dehydration in a non-aqueous system, a large reaction rate can be obtained, and at the same time, the equilibrium of the reaction can be shifted and the process can proceed further to the synthesis side.

That is, the present invention uses an enzyme selected from lipases derived from the genus Pseudomonas that is heat-resistant and capable of hydrolyzing an ester bond consisting of a secondary water-X funic acid group to prepare a substrate solution containing an alcohol component and an acid component. This is a method for synthesizing fatty acid esters, which is characterized by continuous mixing and contact reaction with the above-mentioned enzymes.

Ester synthesis as used in the present invention refers to a reaction in which the product is a fatty acid ester, and also includes transesterification reactions such as so-called acidolysis and alcoholysis.

In the present invention, thermostable lipase is defined as 60°C in an aqueous solution.
The residual relative decomposition activity after 0 minute treatment is 60% or more. Lipases that can hydrolyze ester bonds consisting of secondary hydroxyl groups are selected from enzymes that can hydrolyze the β-position of glycerides, but there are various differences in their reactivity towards styrene hydroxyl groups. Although being able to hydrolyze the β-position of celite is a necessary condition, it is not a sufficient condition.

Its origin is, for example, Pseudomonas fluorescens biotype I (Pseudotsonus fl.
orescens boitype I, No. 10
21 Microtechnical Laboratory No. 5496), Pseudomonas mehitica lipolytica (Pseudo*onus m
Examples include ephitica+tpo+yttca, Kaikoken Funyori No. 1520).

These enzymes can be used in various forms; for example, purified enzyme powders or aqueous solutions thereof, microorganisms capable of producing enzymes or their dried products, culture solutions themselves, and crude enzyme solutions can be used.

In addition, these enzymes are treated with ion exchange resin (Dowex M
WA 1, DOWEX 66, etc.), adsorbent resins (Amberlite XAD-2, Amberlite XAD-8,
Couvemo Sove 101, etc.) etc. 4, top,! It can also be used by immobilizing it on a suitable carrier.

For immobilization, the enzyme solution may be shaken with the resin overnight, washed with buffer, and dried appropriately, or crosslinked with glutaraldehyde and washed.
(Japanese Patent Publication No. 59-20357 Reference M) When an immobilized enzyme is used in the reaction, the enzyme is further stabilized and the enzyme can be easily recovered from the reaction solution.

In addition, the alcohol components that are substrates include sterols such as cholesterol, β-cholestanol, ergosterol, campesterol, and β-sitostyol, lanosterol, dihydrolanosterol, and isoisomers obtained by separation and purification from wool wax as a mixture thereof. Cholesterol C', ``cols'', and esters thereof are used.

The alcohols may be saturated or unsaturated, linear or branched, and may be primary or secondary or higher,
A mixture of these may also be used.

The longer the carbon number, the greater the hydrophobicity, making it easier to form a hydrophobic interface, and the reaction equilibrium is on the synthesis side, so it is preferable, and a carbon number of 12 or more is particularly preferable.

Examples of the branched alcohol include those represented by the general formula and the general formula, and mixtures thereof, such as lanolin alcohol, and alcohol derived from it by fractionating various components.

Also, aliphatic-α, -β-diols, aliphatic-α, ω
- Diols etc. can also be exemplified.

Further, as branched chain synthetic alcohols, hexadecyl alcohol (Esso Standard), NGECOL 1BOA, 160B, and 181A.

20OA, 200C (all from Shin Nippon Chemical Co., Ltd.) Fine Oxocol 1800 (Nissan Chemical Co., Ltd.), Diadol 1
Examples include 8G (Mitsubishi Kasei) and octyldodecanol (Henkel).

In the present invention, the acid component used as the other raw material may be any of the following fatty acids, glycerin esters of fatty acids, and aliphatic alcohol esters having 1 to 32 carbon atoms.

The fatty acids include the following saturated odd or even straight chain fatty acids having 1 to 32 carbon atoms, branched chain fatty acids, unsaturated monoenes, dienes, triene fatty acids, and the alcohol components that constitute the fatty acid esters. , glycerin, and aliphatic alcohols with 1 to 32 carbon atoms.These fatty acid esters are once hydrolyzed by enzymes into their constituent components, and are thought to then participate in the reaction, and the transesterification rate is influenced by the equilibrium of the reaction. Ru.

The alcohols include primary and secondary linear or branched alcohols, among which those with up to 16 carbon atoms, especially aliphatic alcohols with 1 to 4 carbon atoms, have low hydrophobicity. This is preferable because the reaction equilibrium is on the decomposition side of the raw alcohol ester, and therefore the transesterification rate of the target ester becomes high.

The glycerin esters of the fatty acids mentioned above include monoesters,
Either diester or triester may be used, or a mixture thereof may be used.

Also includes natural or synthetic oils and fats (glycerides), natural or synthetic waxes, and the like. These natural oils and fats include, for example, linseed oil, olive oil, cacao butter, rice bran oil, soybean oil, camellia oil, rapeseed oil, palm oil, palm kernel oil, castor oil, cottonseed oil, wood wax, coconut oil, and mustard oil. , vegetable oils such as sunflower oil, animal oils such as beef tallow, milk fat, mutton tallow, cow leg oil, whale oil, cod liver oil, sardine oil, orange ratfish oil, and herring oil, and hydrogenated oils thereof.

Natural waxes such as whale wax, wart wax,
Examples include carnauba wax, candelilla wax, bran wax, shellac wax, beeswax, montan wax, wool wax, and cotton wax.

The above fatty acid esters may be used alone or in combination.
A mixture of two or more species can be used in the reaction of the present invention. (Unexamined Japanese Patent Publication No. 61-204197, Unexamined Japanese Patent Publication No. 61-61) When one of the substrates is a fatty acid ester such as glyceride or methyl oleate, water is required at the initial stage of the reaction to hydrolyze the raw fatty acid ester. If water is present in the system at the beginning of the reaction, the decomposition of the raw fatty acid ester will occur prior to the synthesis, so it can be used without problems by dehydrating it after the initial decomposition is complete.

In the present invention, the substrate used as a raw material is solid at room temperature,
If fluidity is poor as is, use a solvent that does not inhibit enzyme activity, such as hexane, heptane, octane,
Iso-octane (FOE), cyclohexane, decane, hexadecane, liquid paraffin, squalane, squalene, pristane, and mixed solvents thereof, such as Ivytrubene) 1016 (manufactured by Idemitsu Petrochemical Co., Ltd.), are preferred.

The substrate used in the present invention only needs to flow, and the present invention is suitable for implementation in the form of a slurry with an undissolved portion above the saturation concentration. When a slurry-like substrate is used, the substrate consumed by the reaction is , the undissolved substrate present in excess as a slurry in the reaction system is dissolved and replenished.

No substrate inhibition is observed even at concentrations as high as 85%.

Since the present invention uses a thermostable enzyme, the reaction temperature can be higher than when using a normal enzyme. Therefore, the solubility of the substrate increases and the reaction rate also increases. (At 37°C and 60°C, the reaction rate of so' c may be about twice as high.) However, if the reaction rate is too high, the active life may be shortened. In addition to the disadvantages, the reaction temperature must be determined in consideration of various conditions such as substrate solubility, active life, substrate ratio, substrate amount, reaction method, and energy cost.

Regarding the molar ratio of substrates, either one may be greater; the larger the ratio, the faster the reaction rate. However, if the molar ratio is too large, the burden in the refining process will increase.6 Since the acid component is usually cheaper, the acid component is set in excess.
The molar ratio is preferably in the range of 1 to 3.

When the substrate ratio (initial millimoles of alcohol component/synthesis unit of enzyme) is small, the synthesis rate is sufficiently fast, but the frequency of substrate exchange increases and non-productive time increases.

When the substrate ratio is large, it is thought that there is a large excess of substrate in the system, and the time to reach the turn-on synthesis rate of the enzyme-substrate complex slows down as the amount of substrate increases, but the number of substrate exchanges decreases. , the non-productive time during the active period is reduced.

In the case of ordinary lipases that are not thermostable, they are unstable in aqueous solutions, but thermostable enzymes are not only heat resistant but also have greater stability in aqueous solutions. Therefore, even if the substrate ratio is set high, there is little deactivation in the time it takes to reach a predetermined synthesis rate, so the synthesis rate becomes high, and the process can be carried out even with a sufficiently large substrate ratio.

The substrate ratio should be determined in such a way that the amount of ester synthesized per enzyme, per hour, and per unit of equipment is maximized, taking into consideration the production volume, time, and cost of one batch.

The substrate targeted by the present invention is basically a strongly hydrophobic substrate. Therefore, it is thought that a large amount of water is used.

This is because the equilibrium of the fatty acid ester synthesis reaction targeted by the present invention is unilaterally biased toward the synthesis side even without control to reduce the amount of water in the reaction system, and even in a water/oil two-phase system. That's the reason.

However, synthesis of fatty acid ester is a reversible reaction with decomposition, and removing water, which is one of the reaction products, from the reaction system is very effective in advancing the reaction further to the synthesis side. Therefore, the reaction system may be a two-phase system containing a large amount of water, as with conventional lipases capable of synthesizing sterol esters, but a non-aqueous system containing no water is more suitable from the viewpoint of reaction equilibrium. It was considered.

On the other hand, in terms of reaction rate, in the case of conventional lipases that are capable of synthesizing sterol esters and are not heat resistant, the reaction rate under non-aqueous i:mi conditions where water is limited - β... is water/oil It was known that in the case of a two-phase system, the value would be lower than ゛. (In the case of general ceride synthesis, if the water concentration is not sufficiently low, the apparent
The synthesis reaction hardly progresses.

Therefore, in order to obtain a sufficient reaction rate, it is necessary to take various measures to adjust the water concentration to an appropriate level. If so, it can be used as is, but when using the culture solution as it is, or in the form of a slightly purified crude enzyme solution, the reaction system should be mixed and stirred in an appropriate manner while being continuously mixed and stirred. Dehydration is preferable because it allows the reaction to proceed toward the synthesis side and at the same time increases the reaction rate.

″(ν・人－トトウロ） The presence of ester is preferable in terms of improving moisture retention, so a synthesis rate of about 90% is sufficient, but if a higher purity ester is desired, a further dehydration step is added. In particular, the synthesis rate can be easily increased.

Dehydration is easily accomplished by reducing the pressure of the reaction system or by blowing inert gas under continuous mixing and stirring. In either case, the solvent that is distilled off at the same time as water is condensed in a condenser, left to stand in a settling tank to separate the aqueous layer, and then removed by appropriate means such as using a reflux tube. It is preferable to return it to the reaction system.

There are no particular restrictions on the shape of the reactor used in this method, and the enzyme and liquid substrate can be mixed continuously.
Types in which these are in sufficient contact (effect) The substrates targeted by the present invention are basically highly hydrophobic substrates. Therefore, even in a water/oil two-phase system in which a large amount of water is used, water is excluded due to the hydrophobicity of the substrate, and it is thought that the actual reaction site is a hydrophobic mound.

Therefore, even in a water/oil two-phase system, the reaction equilibrium is unilaterally biased toward the synthesis side.

Synthesis of fatty acid ester by lipase is a reversible reaction with decomposition, and if the ice product is removed from the system, the reaction will inevitably proceed to the synthesis side.

In addition, unlike conventional enzymes capable of synthesizing styrene esters, the enzyme of the present invention has a reaction rate that is much higher in a non-aqueous system than in a water/oil two-phase system.

Moreover, the enzyme of the present invention is thermostable, so it is more stable and has a longer duration of activity under the conditions of the present invention, and can hydrolyze ester bonds consisting of secondary hydroxyl groups, so it It can act on various substrates, including sterically hindered alcohols such as

In addition, there is no substrate inhibition even at high substrate concentrations, and since higher reaction temperatures can be used, the amount of solvent used can be reduced, and the efficiency of the apparatus can be increased.

By using a highly concentrated substrate slurry, the dissolved substrate is consumed and reduced as the reaction progresses, but a commensurate amount of substrate is automatically dissolved and replenished into the reaction system.・The viscosity increases, but
The reaction always proceeds quickly.

In a non-aqueous system, the enzyme is not dissolved and exists as suspended particles, so after the reaction, it can be easily separated and recovered from the reaction solution by centrifugation or centrifugation. This recovered enzyme can be used as is for the next reaction.

The same applies to immobilized enzymes. When an immobilized enzyme is used, the disadvantage that it adheres to the wall of the container during the reaction, resulting in insufficient contact with the substrate, is overcome.

In addition, in the method of preparing a stable emulsified dispersion using a substrate solution and an enzyme solution, the movement of the generated water is restricted because the numerous water/oil interfaces in the stationary dispersion are used as reaction sites. is,
There was a problem that a sufficiently high synthesis rate could not be obtained. Rather than preparing a stable emulsified dispersion and using the water/oil interface as the reaction site, the enzyme and substrate solution are continuously mixed and contacted for reaction. - This gives movement to the water produced, and the hydrophobicity of the substrate can promote the removal of water from the reaction site. Continuous mixing is also important for dispersing enzymes and removing water from the system.

In the following examples, unless otherwise specified, the reaction was performed using an i.d.
20 mm x 300 cpm as a reaction vessel (hereinafter referred to as a reaction bottle), a screw glass bottle with an inner lid of 5 cm in height.
Shaking culture machine (RMR-9- manufactured by Iwashiya Biological Science Co., Ltd.)
20) in a thermostatic chamber at 37°C, and the following substrates were used.

Cholesterol (cho); Cholestemil USP, manufactured by Yoshikawa Oil Co., Ltd.; Oleic acid (OA); Extra Olein #80, manufactured by Nippon Oil & Fats Co., Ltd.; Pseudomonas fluorescens biotype I (No.
Lipase Bla powder derived from 1021.1m Koken Bokuyori No. 5495), 4.2EU/g 7000U/g Bib Crude enzyme solution, 0.78EU/g 1300U/m
1 Blc powder, 1024 EU/g 238000 U/g; Riba-ze Ml powder derived from Pseudomonas mehitii lipolytica (Feikoken Bacteria No. 520), 0.1
7EU/g 2160U/g The synthesis rate (A%) was determined for each component using TLC-FID (Iatroscan, Yatron Co., Ltd.).

A%=ARs″XI 00/ (ARs+ARc)k
Dashi, A%; area-based synthesis rate ARs; peak area of the produced ester ARc; peak area of unreacted alcohol component.Also, by measuring standard solutions of known concentrations in the same way, area-based synthesis rate (A%) - molar A conversion table with the standard synthesis rate (M%) was created. When A% was in the range of 5.5% to 93.0%, the following formula was used to obtain a close relationship. (Interphase coefficient = 0.996) M% = 0.763XA% + 5.31 Below, the synthesis rate is expressed in A% unless otherwise specified.

TLC-FID quantitative method Approximately 1 ml of the collected reaction solution is charged with isooctane. This is expanded in a suitable solvent system, such as hexane/ether/formic acid=56/1410.3, to separate each component. After development, after drying at 100° C. for several minutes to remove the developing solvent, the peak area of each lipid component in the reaction solution is determined using IatroScan TH-10.

The degrading activity of lipase was determined by the method of Yamada et al.
60-864.1962), using an olive oil emulsion with polyvinyl alcohol as an emulsifier as a substrate,
Measured at 6o''c with pi (=7.0), the amount of enzyme that releases 1M mole of fatty acids per minute was defined as 1 decomposition unit (U).

The synthetic activity of the enzyme was defined as follows. The potency to synthesize 18 mol of cholesterol oleate per minute under standard conditions was defined as 1 synthesis weight (EU).

Standard conditions Synthesis rate (A%) using 100 mg of cholesterol, 219 mg of oleic acid, and 3 ml of isooctane as the substrate solution.
Add an enzyme solution dissolved in O, 1M phosphate buffer so that the concentration is between 20% and 60%, and add it to a 20 mm x 30
0cpm shaking culture machine (RM made by Iwashiya Biological Science Co., Ltd.)
RS-20) at 37°C for 40 minutes.
However, in the case of low titer enzymes, the reaction was closed for a longer time and measured.

Example 1 Cholesterol (cho) 1.4816g, oleic acid (OA) 1.6018g, isooctane (ioc) 10
．． Table 1 Example 2 Fermentation*81a was added to 3 ml of 0.1M phosphate buffer p in each reaction bottle.
The reaction was carried out in the same manner as in Example 1 except that the mixture was dissolved in H=7.0 (PBX) and added to the reaction bottle. The second result is
Also listed in the table.

The results of Examples 1 and 2 clearly show that the non-aqueous system has a faster synthesis rate.

The synthetic unit of Bla is 4.2 EU/g, but when measured under non-aqueous conditions, it is 25. IEU/g, which was about 6 times that of the two-phase system.

As a result of continuing the reaction No. 1 of Example 2, the synthesis rate was 97.4% in 68 hours. It can be seen that even in a two-phase system, although the reaction rate is slow, the final synthesis rate can be sufficiently high.

□+1 Heat-stable lipase B near”c (1024EU/g) 6.3
mg to 1 ml of O,IM phosphate buffer pH=6.
In addition to 1 g of Dowex 66 dissolved in 0.0 (PB8) and washed with distilled water and PB6, add 1 g at 8'''C.
It was shaken at night and adsorbed. Then 160 μL of 2 at 8@C
A 5% glutaraldehyde solution was added, the mixture was shaken for 10 minutes, 0.2 ml of a 20% aqueous sodium hydrogen sulfite solution was added, and after 10 minutes of interlocking, the mixture was washed with PB6 and washed to prepare an immobilized enzyme.

The above immobilized enzyme was placed in a reaction bottle, and the molar ratio was OA/cho.
=3, ioc 10ml of substrate solution was added and reacted.

After the reaction, the reaction solution was removed using a Roca, and a new substrate solution was added to the recovered immobilized enzyme and the reaction was repeated. The results of the long-term repeated reactions carried out in this manner are shown in Table 3.

-:'l, 1. ,,l,+,Ko. Table-1---
・After this, the process was continued for the 43rd time (the synthesis rate was 94.6% when the reaction between preparations was carried out).The cumulative amount of synthesized ester up to the 43rd time was 18.091 g.
(0,0278 mol), 431 per synthetic unit
This means that O micromoles of ester have been synthesized.

From these results, the immobilization of the enzyme of the present invention does not require special equipment, is easy to scale up, has a long life, and is an economical synthesis method with a high synthesis amount of 4310 times per enzyme. I understand that.

Example 4 100 g of a reaction solution with a synthesis rate of 89.8% (initial raw material charging molar ratio = 1.5) obtained by separate reaction was desolventized and i
0.0 c 200 ml was placed in the reaction section shown in FIG. 1, and nitrogen gas was blown into it from the gas blowing nozzle (6) to cause a reaction. The enzyme and substrate were mixed and brought into contact by the circulating flow of the blown gas, and the reaction proceeded. Water produced during ester synthesis was removed from the gas outlet (9) as water vapor along with the blown gas. Most of the 10c vaporized together with water vapor was condensed in the condenser (1) and returned to the system. A corresponding amount of 10c contained in the nitrogen gas and removed from the system was replenished into the reaction system as appropriate.

The synthesis rate after 92 hours was 99.9%. From this result, it is possible to easily obtain a sufficiently high synthesis rate using the method of the present invention by using a thermostable enzyme and removing the water generated during continuous mixing even from about 90% of the reaction solution. Recognize.

Example 5 Same reaction solution as Example 4 (100 g of oil, ioc
200ml) was put into the reaction part (6) of the reaction apparatus shown in Figure 2, and the crude enzyme solution obtained by concentrating the culture solution Blb (
0.78 EU/ml) was added. Nozzle (1
) while blowing nitrogen gas into the reactor at 540 rpm using the stirrer (7), keeping the entire reactor at 45@C,
The upper part (2) of the condenser was connected to an aspirator, and the reaction was carried out under reduced pressure in the entire reaction system. Water and 10C are evaporated into steam by blowing nitrogen gas and reducing pressure, and condensation II
(3), it condenses and falls, and is separated into water and 10C in the stationary section (4). Foe above the liquid WA was automatically refluxed into the reaction system due to the difference in liquid level. The water accumulated in the stationary part was appropriately extracted from the extraction nozzle (5). The 0 synthesis rate after 20 hours of reaction in this manner was 9.9.8%.

Example 6 0.258 mmol of the alcohol component, 0.776 mmol of the acid component, and 3 ml of joc shown in Table 4 were placed in a reaction bottle as a substrate solution, and an enzyme solution prepared by dissolving 3.6 mg of Blc in a pH of 18 ml was added to carry out a reaction. . Table 4 shows the substrate groups, reaction times, and synthesis rates. The symbols used will be explained in Example 7.

Table 4 Example 7 0.258 mmol of the alcohol component shown in Table 5, 0.258 mmol of glyceride among the acid components, 0.516 mmol of methyl oleate, and 10C
・Pour 3 ml as a substrate solution into a reaction bottle, and add Blc −
A reaction was carried out by adding an enzyme solution dissolved in 3.6 mgt-PH18ml.

Table 5 shows the substrate groups, reaction times, and synthesis rates.

, Table 5 The synthesis rate was calculated using the formula.

The reaction formula is CO+X-+CX+CO+ch.

However, the symbols newly used in Table 4.5 are shown below.

SA; stearic acid isA; isostearic acid (manufactured by Emery) Cap, A
; Capric acid Pro, A; Propionic acid 0, alc; Oleyl alcohol) IPA; α-hydroxypalmitic acid S, alc; Stearyl alcohol Lau, a; (Yoshikawa Oil Co., Ltd. 81) CO; Cholesterol oleate C, Pro;
Cholesterol propionate ester 0, oil; Olive oil Ta1l; Beef tallow MO; Oleic acid methyl ester From the results of Example 6.7, the enzyme of the present invention can contain sterols, saturated or unsaturated higher alcohols, branched alcohols, mixtures thereof, and sterol esters. It can be seen that ester synthesis (direct ester synthesis, alcoholysis, acidolysis) can be performed using various substrates such as alcohol components and various fatty acids, glycerides, fatty acid esters, etc. as acid components.

Σgo-cho 1.4616g-olive oil 1.661g
, ioc 10ml was put into a reaction bottle as a substrate solution, and B
8.4 ml of ib was added and reacted. The synthesis rate of cholesterol oleate after 20 hours was 91.
8%, and the olive oil was completely decomposed. It can be seen that transesterification is possible using olive oil even at a large substrate ratio.

Example 9 Using the same substrate solution as in Example 1, Bla 300
m g into a reaction bottle and set the reaction temperature to 37°C or 60°C.
The reaction was carried out at 0C for 4 hours. The result was 44.
5% and 78.3% at 60@C. It can be seen that the reaction can be performed efficiently even at high reaction temperatures, and the reaction rate can be increased.

``Example 10 cho 1.4616g, OA 1.6016g,
Put 10ml of ioc into a reaction bottle as a substrate solution and add Bla
2150 mg was added and reacted for 200 hours. 20
The synthesis rate after hours was 98.0%. After the reaction solution was allowed to stand still, the supernatant was poured, the suspended enzyme was separated, and the supernatant was returned to the reaction bottle.

The same substrate solution as in the first reaction was put into the reaction bottle, and the reaction was carried out in the same manner. The second synthesis rate was 93.6%.

This result shows that in non-aqueous reactions, enzymes are suspended particles, which can be easily separated from the reaction solution using Roca, and their activity is preserved, making it possible to reuse them.

Example 11 1.4616 g of cho and 500 mg of OA 1.6016 reverse (Ml) were added and reacted for 116 hours. The 0 synthesis rate was 93.9%.

(Effect of the invention) From these examples, when a thermostable enzyme is used according to the method of the present invention, the duration of activity becomes longer, and per unit enzyme,
It is clear that the amount of synthesis per unit time and per unit device can be dramatically increased.

When used in a nonaqueous system, the enzyme of the present invention exhibits a higher reaction rate than in a water/oil two-phase system, and in addition, by continuously removing the water produced from the reaction system, the reaction equilibrium is further shifted toward the synthesis side. It is possible to proceed further and obtain a higher final synthesis rate in a shorter time.

Since it is a thermostable enzyme, the reaction temperature can be raised, thereby achieving a high reaction rate and reducing the amount of solvent used.

Furthermore, according to the present invention, not only direct ester synthesis but also transesterification can be carried out, and various substrates can be used as raw materials because it has a strong decomposition power for glycerides, fatty acid lower alcohol esters, etc.

This means that fatty acid esters derived from natural fats and oils that have not been thermally degraded by chemical decomposition can be easily obtained, and this has great economic significance.

The enzyme of the present invention has affinity for a wide range of substrates including branching compounds, and also has activity against secondary alcohols, so it can be used in various ester syntheses.

, -1 Furthermore, the immobilization is also 11, and since the structure is stabilized by immobilization, it has a longer life and does not require special equipment for scale-up.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of a reactor used in the method of the invention. 1. Condenser 2. Lid 3, straight body part 41 conical part δ, inner II 6. Gas blowing nozzle 7, gas distribution plate 88, reaction section 9, gas outlet FIG. 2 is a schematic diagram of a reactor used in the method of the present invention. 1. Gas blowing nozzle 2. Condenser upper part patent applicant Kozo Iizuka (m1 person), director of the Agency of Industrial Science and Technology Engraving of drawings (no changes to the contents) Figure 1 Figure 2 Procedure Supplementary stroke stationary type) 8 1 Indication of incident Patent Application No. 45068 of 1988 2 Name of the invention (114) Director of the Agency of Industrial Science and Technology Kozo Iizuka 4 Designated Agent 1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture Tomoo Suzuki Director of the Microbial Technology Laboratory, Agency of Industrial Science and Technology 5 Sub-Agent - 2 Yotsuno-cho, Higashi-ku, Osaka City No. 10 Sawanotsuru Building May 31, 1988 7 Target of amendment Contents of the amendment Engraving of the drawing (no change in content) As attached.

Claims (1)

[Claims] Using an enzyme selected from lipases derived from the genus Pseudomonas that is heat-resistant and capable of hydrolyzing ester bonds consisting of secondary hydroxyl groups, a substrate solution containing an alcohol component and an acid component is continuously mixed and contacted with the enzyme. A method for synthesizing fatty acid esters, characterized by synthesizing fatty acid esters.