JPH0638778A - Production of fatty acid - Google Patents

Abstract

PURPOSE:To enable the use of a hydrolase for a long period and to produce a fatty acid in high purity, yield and productivity from a fatty acid lower alco hol ester used as a starting substance by using a specific process to use a hydrolase. CONSTITUTION:A fatty acid lower alcohol ester (e.g. methyl octanoate) and water are made to react with each other in a reactor 1 in the presence of a hydrolase (preferably lipase originated from the genus Rhizopus, esterase originated from the genus Pseudomonas, etc.) and, at the same time, the reaction mixture is separated into (A) a mixture of a lower alcohol and water and (B) an oil-water mixture composed of an oil layer containing unreacted fatty acid lower alcohol ester and fatty acid and a water layer using a membrane 3 impermeable to the hydrolase and selectively permeable to the water-layer in the reaction product. The oil-water mixture is returned to the reactor 1 and the procedures are repeated. The material of the membrane is preferably cellulose acetate, polysulfone, etc., having high affinity to water.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently producing a fatty acid using a fatty acid lower alcohol ester as a starting material and a hydrolase for a long period of time.

[0002]

BACKGROUND OF THE INVENTION Fatty acids are produced by hydrolyzing vegetable oils and animal oils under high pressure and high temperature. Also, a method for hydrolyzing a fatty acid lower alcohol ester has been studied for a long time (Japanese Patent Publication No. 3-24458). However, these methods have drawbacks that the energy cost is high because of the high temperature and high pressure reaction, the equipment becomes heavy, and the handling is difficult because a dangerous catalyst is used. On the other hand, much academic research has been conducted on the enzymatic hydrolysis of fatty acid lower alcohol esters (“Fats”, Vo.
42, No.2, P91), but no industrial studies have been conducted.
Further, in the hydrolysis reaction of the fatty acid lower alcohol ester, there is a chemical equilibrium, so that the decomposition rate of the fatty acid lower alcohol ester is very low, and it was necessary to use a large amount of water to improve the decomposition rate.

An object of the present invention is to provide a method for industrially producing a large amount of fatty acid from a fatty acid lower alcohol ester by enzymatic hydrolysis.

[0004]

The present inventors have completed the present invention as a result of intensive research to solve the above problems. That is, the present invention provides a method for producing a fatty acid characterized by repeating the following steps (1) to (3). (1) A step of reacting a fatty acid lower alcohol ester with water in the presence of a hydrolase. (2) A mixture of lower alcohol and water using a membrane capable of selectively permeating the aqueous layer side in the reaction product while allowing the reaction product to react with the reaction product in (1). And a step of separating the mixture into an oil layer and an aqueous layer. (3) A step of returning the mixture of the oil layer and the aqueous layer separated in (2) to the reaction system.

The fatty acid lower alcohol ester used in the production method of the present invention preferably has a fatty acid moiety corresponding to a fatty acid having 6 to 26 carbon atoms and an alcohol moiety corresponding to a monovalent lower alcohol having 1 to 4 carbon atoms. An ester, such as caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, palmitooleic acid, oleic acid, linoleic acid, Gondoinic acid, erucic acid and mixtures thereof, methanol, ethanol, propanol, isopropanol, normal butanol, secondary butanol, tertiary butanol,
Examples thereof include esters with isobutanol. Further, these fatty acid lower alcohol esters may be used alone or as a mixture of two or more kinds.

The hydrolase used in the production method of the present invention includes lipase and esterase.
These enzymes can be of microbial, animal or plant origin. Only one type of lipase or esterase can be used, or two or more types of lipase and esterase can be mixed and used. Preferably, as a lipase derived from a microorganism, those derived from the genus Rhizopus, those derived from the genus Chandida, those derived from the genus Geotrichum, and Mucor
Examples thereof include those derived from the genus, those derived from the genus Asprgillus, those derived from the genus Chromobacterium, and the like. Preferable examples of esterases include those derived from the genus Pseudomonas, those derived from the genus Chandida, those derived from the genus Penicillium, and the like. Examples of lipases and esterases derived from animals include those derived from organs such as humans, cows and pigs. Further, examples of plant-derived lipases and esterases include those derived from sunflower seeds, potatoes and the like. These hydrolases may be used in the form of powder, as an aqueous solution, or in the form of immobilized enzyme.

The water used in the production method of the present invention is preferably ion-exchanged water or distilled water. In order to obtain an effective reaction rate, an aqueous solution containing an alkali metal carboxylate, an alkaline earth metal carboxylate or a mixture thereof, an inorganic alkali metal salt, an inorganic alkaline earth metal salt or a mixture thereof is used. I don't mind. In the carboxylic acid alkali metal salt and the carboxylic acid alkaline earth metal salt, the carboxylic acid is a linear or branched aliphatic carboxylic acid having 2 to 8 carbon atoms, and examples thereof include fatty acids such as acetic acid, butyric acid, and propionic acid. Examples thereof include aromatic carboxylic acids such as group carboxylic acids and benzoic acid, and aliphatic carboxylic acids are preferable. Examples of the alkali metal include sodium and potassium, and examples of the alkaline earth metal include calcium and magnesium. Examples of the inorganic alkali metal salt or the inorganic alkaline earth metal salt include halides, carbonates and phosphates of the above metals. The amount of addition of these alkali metal salts of carboxylic acids, alkaline earth metal salts of carboxylic acids, inorganic alkali metal salts and inorganic alkaline earth metal salts is
It is preferable to add it in the range of 9.5. In the present invention, it is more preferable that pH is in the range of 5.0 to 7.0. If it deviates from this range, denaturation of the hydrolase due to pH change will occur, resulting in poor expression of hydrolase activity.

In the production method of the present invention, it is preferable to add glycerin when the fatty acid lower alcohol ester and water are reacted in the presence of a hydrolase. In this case, the mixture of the lower alcohol, water and glycerin and the mixture of the oil layer and the water layer are separated in the step (2).
Glycerin is used as a stabilizer for hydrolases, and is added to prevent denaturation of hydrolases by lower alcohols and heat denaturation of hydrolases caused by hydrolysis reaction of fatty acid lower alcohol esters. . The concentration of glycerin as a stabilizer is preferably 0.1 to 100% by weight based on the amount of water charged at the start of the reaction,
More preferably from 10 to the amount of water charged at the start of the reaction
80% by weight. If it is less than 0.1% by weight, the effect as a stabilizer of glycerin does not appear, and if it exceeds 100% by weight,
Fatty acid and glycerin produced by hydrolysis of fatty acid lower alcohol ester cause an esterification reaction by the catalytic action of the reverse reaction of hydrolase to form monoglyceride, diglyceride and triglyceride and a mixture thereof, which is caused by hydrolysis. The produced fatty acid disappears and the fatty acid cannot be efficiently produced.

The method for producing a fatty acid according to the present invention will be described with reference to FIG. The reactor 1 shown in FIG. 1 is charged with a fatty acid lower alcohol ester, and further, the reactor 1 is charged with 1 to 100 parts by weight of water per 1 part by weight of the above fatty acid lower alcohol ester. More preferably, the water content is 2 to 10 parts by weight with respect to 1 part by weight of the fatty acid lower alcohol ester. If the water content is less than 1 part by weight, the hydrolysis rate will be slow, and if it exceeds 100 parts by weight, the productivity will be poor. Then, a hydrolase is added. To enhance the stability of the hydrolase, glycerin is also charged in the reactor 1.

At the end of the above-mentioned preparation, the reactor 1
Is heated to the reaction temperature. The reaction temperature depends on the optimum temperature of the hydrolase used, but is preferably 5 to 90 ° C. More preferably, 30 to 70 ° C is adopted. If the temperature is lower than 5 ° C, the reaction rate is slow and the productivity is deteriorated. At the same time, the fatty acid lower alcohol ester and the produced fatty acid are coagulated, and the reaction system becomes a solid-liquid system, and the reactivity is extremely deteriorated. On the other hand, when the temperature exceeds 90 ° C, the heat deactivation of the hydrolase becomes large, and good reactivity cannot be obtained.

The hydrolase requires a concentration that allows the reaction to proceed sufficiently. Specifically, 1 to 1000 units are added to the reactor 1 with respect to 1 g of the fatty acid lower alcohol ester. More preferably, 10 to 500 units are added to 1 g of the fatty acid lower alcohol ester. If it is less than 1 unit, a sufficient reaction rate cannot be obtained, and if more than 1000 units are added, the reaction rate hardly increases. The term “one unit of enzyme unit” as used herein refers to the decomposing power of an enzyme that hydrolyzes a fatty acid lower alcohol ester to produce 1 μmol of fatty acid per minute.

Stirring during the reaction is performed at a stirring power requirement of 0.05.
It is preferred to provide ~ 2 kW / m ³ . More preferably, 0.
It is 1 to 1 kW / m ³ . The hydrolysis reaction of fatty acid lower alcohol ester with hydrolases proceeds at the water / oil interface, and the interfacial area is used for the term of substrate concentration in the usual enzyme reaction.
The reaction rate can be expressed by a Michaelis-Menten type formula. Therefore, the larger the interfacial area, the faster the reaction rate. Therefore,
If the power required for stirring is less than 0.05 kW / m ³ , the interfacial area required for the reaction cannot be secured and the reaction rate is low. On the other hand, if it exceeds 2 kW / m ³ , oil / water separation after the reaction becomes very difficult, and the yield of oil is significantly deteriorated.

In the present invention, the above reaction product is extracted from the reactor 1 to the outside of the reactor and fed to the membrane separation device 2.
In the membrane separation device 2, an oil layer (unreacted fatty acid lower alcohol ester and fatty acid) and an enzyme are not permeated, and an aqueous layer (water and lower alcohol. However, glycerin is also included when reacting by adding glycerin). A separation membrane 3 for permeation is attached to the reaction system, and only the aqueous layer is extracted to the outside of the reaction system, and the reaction product (including the enzyme) that has not permeated the separation membrane is returned to the reactor 1. The separation membrane 3 used in the present invention is not particularly limited as long as it has the above-mentioned performance, but it is a semi-permeable membrane such as a reverse osmosis membrane, an ultrafiltration membrane, a microfiltration membrane, an ion exchange membrane or a dialysis membrane. A permeable or porous membrane is used,
Although the material is not limited, those having a high affinity with water are desirable, and for example, cellulose acetate, polyacrylonitrile, polysulfone, polyamide, polyimide, polyvinyl alcohol, polyolefin, and ceramics are used. The form of the membrane is not particularly limited, and for example, a membrane such as a flat membrane, a hollow fiber membrane, a tubular membrane, and a spiral membrane can be used. As described above, a membrane that does not allow the enzyme to permeate, and can further permeate only the water layer, can be used in the present invention, but when the water layer permeates the membrane, the water in the water layer is lower than that of the lower alcohol. If it is a membrane that is easily permeated by water or a membrane that has a water permeability equivalent to that of lower alcohol, it is necessary to supply water to the reaction system in order to carry out the reaction efficiently. If a membrane that is easy to permeate is selected, it is not always necessary to add new water to the reaction system, and water may be added as necessary. Further, when a water amount equal to the amount of water removed by the membrane separator and the amount of water consumed in the hydrolysis reaction is added to the reaction system from the water supply tank 4, operation is performed while keeping the amount of water in the reaction system constant. can do. Water obtained by removing the lower alcohol from the mixture of the lower alcohol and water (which may include glycerin) separated by the membrane instead of adding the amount of water removed by the membrane separator (which may also include glycerin). May be returned to the reaction system. When water is supplied in this way, glycerin can be added if necessary. In order to allow the water layer in the reaction product to efficiently permeate through the membrane, a method of forming a pressure gradient between the feed liquid side and the permeate side is generally used. As a method of forming a pressure gradient, there are a method of pressurizing the supply liquid side, a method of depressurizing the permeate liquid side, and a method of using these methods simultaneously.
Further, if a water layer having a lower alcohol concentration lower than that of the supply liquid side is supplied to the permeate side, the lower alcohol can be efficiently permeated due to the concentration gradient. Further, there are a method of taking out the permeated liquid as a liquid and a method of taking out as a vapor (so-called pervaporation). Pressure applied to the feed side generally preferably 0.01~50kg / cm ^2, more preferably from 0.1 ~30kg / cm ^2. Pressure is 0.01kg / cm
^{When it is} less than ² , the permeation rate is low, and when it is more than 50 kg / cm ^2, there are problems such as durability of the membrane and heavy equipment of the device. The temperature for membrane separation is preferably 90 ° C. or lower, more preferably 70 ° C. or lower in consideration of the heat resistance of the membrane and the inactivation of the enzyme due to heat. However, when the immobilized enzyme is held in the reactor for the reaction and the enzyme is not present in the aqueous layer, the heat resistance of the membrane may be taken into consideration for membrane separation. By performing such an operation, the chemical equilibrium can be shifted to the production system side, the decomposition rate of the fatty acid lower alcohol ester can be increased, the fatty acid purity in the oil layer can be increased, and the fatty acid yield can be improved.

In the above operation, after reacting for a certain period of time, the whole amount of the reaction product is extracted, and in the membrane separation device, the operation of removing the aqueous layer is repeated, and after reacting for a certain period of time, a part of the reaction product is extracted and membrane separation is performed. It can be performed by a semi-batch operation in which the operation of removing the water layer is repeated in the apparatus, or a continuous operation of continuously withdrawing the reaction product from the reactor and removing the water layer in the membrane separation apparatus.

After the reaction is completed, an oil layer and a water layer which are composed of a mixture of fatty acid and unreacted fatty acid lower alcohol ester are separated by centrifugation or the like, and the obtained oil layer is separated into unreacted fatty acid lower alcohol ester and fatty acid by distillation or the like. The fatty acid can then be isolated.

[0016]

As described above, according to the method of the present invention,
Since the chemical equilibrium can be continuously, semi-batchly, or batchwise shifted to the production system side to improve the decomposition rate of the fatty acid lower alcohol ester, the fatty acid can be produced with high purity and high yield. . Further, it becomes possible to set up a manufacturing process with good productivity.

[0017]

The present invention is described in detail below with reference to examples, but the present invention is not limited to these.
In the examples,% is based on weight unless otherwise specified.

Example 1 In the reaction system shown in FIG. 1, the following reaction was carried out using an apparatus in which a vacuum pump was connected to the permeate side of the membrane separation apparatus. In a 1000 ml reactor 1, methyl octanoate (manufactured by Wako Pure Chemical Industries, Ltd.) 150 g, water 750 g, lipase OF powder (manufactured by Meito Sangyo Co., Ltd .: derived from Candida cylindracea) 9.4
1 g, 48000 U (activity 5.1 thousand U / g-lipase OF powder when using methyl octanoate as a substrate) was added,
The reaction was carried out at 37 ° C. During the reaction, the stirring speed of 400 rpm (the power required for stirring was about
0.2 kW / m ³ ) was given. After 3 hours from the start of the reaction, the reaction product was withdrawn from the reactor 1 at a rate of 100 ml / min and supplied to the membrane separation device 2. As the separation membrane 3 of the membrane separation device, a cellulose acetate membrane (manufactured by Daicel Chemical Industries, Ltd.) having an effective membrane area of 0.02 m ² was used. The permeate side of the membrane separator 2 was connected to a vacuum pump, and methanol and water were permeated through the membrane while reducing the pressure to 20 Torr and continuously withdrawn from the reaction system. on the other hand,
The reaction product that did not permeate the membrane was continuously returned to the reactor 1 by newly adding the amount of water removed by the membrane from the water supply tank 4. In this way, the reaction was carried out for 48 hours. After 24 hours and 48 hours, the purity of octanoic acid in the oil layer was 90% and 97%, respectively. Octanoic acid was quantified by gas chromatography (glass column φ 2.6 mm × 2 m, Uniport S 80-100 mesh with 15% loading of FFAP packing material, injection temperature)
250 ° C, initial temperature 100 ° C, final temperature 170
C, temperature rising rate 5 ° C / min, detector FID) was used for analysis.

Comparative Example 1 In a 1000 ml reactor, methyl octanoate (manufactured by Wako Pure Chemical Industries, Ltd.) 150 g, water 750 g, lipase OF powder (manufactured by Meito Sangyo Co., Ltd .: derived from Candida cylindracea) 9.41 g, 4800
0 U (activity when using methyl octanoate as substrate
5.1 thousand U / g-lipase OF powder) was added and reacted at 37 ° C. Stirring during the reaction was carried out using a 6 cm crescent blade type stirring blade, and the stirring speed was 400 rpm (required power of about 0.2 kW /
m ³ ) was given. In this way, the reaction was carried out for 48 hours. After 24 hours and 48 hours, the purity of octanoic acid in the oil layer is 58.
% And 60%. Octanoic acid was quantified by gas chromatography (glass column φ2.6mm × 2m, packing material FFAP 1
Uniport S 80-100mesh loaded with 5%, injection temperature 250 ℃, initial temperature 100 ℃, final temperature 1
It was analyzed by 70 ° C., heating rate 5 ° C./min, detector FID).

Example 2 In the reaction system shown in FIG. 1, the following reaction was carried out using an apparatus in which a vacuum pump was connected to the permeate side of the membrane separation apparatus. In a 1000 ml reactor 1, 100 g of methyl octoate (manufactured by Wako Pure Chemical Industries, Ltd.), 500 g of water, 250 of glycerin
g, lipase OF powder (manufactured by Meito Sangyo Co., Ltd .: Candida cy
derived from lindracea) 6.27 g, 32000 U (activity when using methyl octanoate as a substrate 5.1 1,000 U / g-lipase O
F powder) was added and reacted at 37 ° C. For stirring during the reaction, a stirring rotation speed of 400 rpm (required power of stirring: about 0.2 kW / m ³ ) was applied using a 6-cm crescent-shaped stirring blade. Reaction start 3
After a lapse of time, the reaction product was extracted from the reactor 1 at a rate of 100 ml / min and supplied to the membrane separation device 2. As the separation membrane 3 of the membrane separation device, a cellulose acetate membrane (manufactured by Daicel Chemical Industries, Ltd.) having an effective membrane area of 0.02 m ² was used. The permeate side of the membrane separator 2 was connected to a vacuum pump, and methanol and water were permeated through the membrane while the pressure was reduced to 20 Torr and continuously withdrawn from the reaction system. At this time, glycerin also permeated the membrane together with water and methanol and was extracted from the reaction system. On the other hand, for the reaction product that did not permeate the membrane, the amount of water removed by the membrane and glycerin were newly added from the water supply tank 4 and continuously returned to the reactor 1. After 24 hours of the reaction time, the whole amount of the reaction liquid in the reactor was extracted and oil / water separation was performed by centrifugation at 4000 rpm for 10 minutes. The oil layer was removed, and water and glycerin were newly added so that the weight of the remaining aqueous layer and the concentration of glycerin in the aqueous layer were the same as those at the time of initial charging, and the mixture was returned to the reactor 1 and 100 g of new methyl octanoate was added. Was added and the reaction was carried out under the same conditions as above. In this way, the reaction and centrifugation were repeated 5 times. Table 1 shows the octanoic acid purity of the oil layer at the end of each reaction. Octanoic acid was quantified by gas chromatography (glass column φ 2.6 mm × 2 m, loading FFAP 15%)
Uniport S 80-100 mesh, injection temperature 250
C., initial temperature 100.degree. C., final temperature 170.degree. C., heating rate 5.degree. C./min, detector FID).

[0021]

[Table 1]

[Brief description of drawings]

FIG. 1 is a schematic view of an apparatus used in the manufacturing method of the present invention.

[Explanation of Codes] 1 Reactor 2 Membrane Separation Device 3 Separation Membrane 4 Water Supply Tank

Claims (3)

[Claims] 1. A method for producing a fatty acid, which comprises repeating the following steps (1) to (3). (1) A step of reacting a fatty acid lower alcohol ester with water in the presence of a hydrolase. (2) A mixture of lower alcohol and water using a membrane capable of selectively permeating the aqueous layer side in the reaction product while allowing the reaction product to react with the reaction product in (1). And a step of separating the mixture into an oil layer and an aqueous layer. (3) A step of returning the mixture of the oil layer and the aqueous layer separated in (2) to the reaction system. 2. The method for producing a fatty acid according to claim 1, wherein water is supplied to the reaction system. 3. When the fatty acid lower alcohol ester and water are reacted in the presence of a hydrolase, the glycerin concentration in the aqueous layer is 0.1 to 100% by weight relative to the weight of water charged at the start of the reaction. The method for producing a fatty acid according to claim 1 or 2, wherein glycerin is added and reacted, and in the step (2), a mixture of a lower alcohol, water and glycerin and a mixture of an oil layer and an aqueous layer are separated.