JP2000270886A - Transesterification - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quantitatively carry out a transesterification reaction useful for producing a fatty acid ester etc., without using a solvent by transesterifying an ester with an alcohol by using an esterase in an aqueous system. SOLUTION: An ester (e.g. oils and fats, etc.), is transesterified with an alcohol in an aqueous system containing 1-20 wt.% of water in the reaction system by using an esterase (e.g. lipase) so as to carry out the transesterification between the ester and the alcohol by a method for producing an automobile fuel (biodiesel fuel) from naturally occurring oils and fats of animal and plant and microorganism, especially waste oil instead of fossil fuel from the viewpoint of environment problem. Conventionally in a transesterification reaction, the reaction is carried out by eliminating water as much as possible but it is found that a transesterification reaction is performed even in a system sufficiently containing water and yet approximately quantitatively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for transesterification with an esterase. More specifically, the present invention relates to a method for performing transesterification in a water-containing system.

[0002]

2. Description of the Related Art In recent years, from the viewpoint of environmental problems, instead of fossil fuels, naturally occurring animal and plant and microorganism fats and oils have been used.
Attempts have been made to produce automotive fuels (so-called biodiesel fuels). In particular, the production of biodiesel fuel from waste oil is considered to greatly contribute to resource recycling and solving environmental problems, because waste oil causes environmental problems when it is discarded.

[0003] The production of biodiesel fuel from such natural fats and oils (waste oil) includes a chemical method and a biological method. The chemical method is a high-temperature, energy-consuming reaction, and has process problems such as necessity of a neutralization process. Therefore, a biological method that consumes less energy at room temperature is being studied.

The biological method is a method in which fats and oils are exclusively dissolved in a solvent (for example, hexane) and reacted with lipase in the presence of alcohol. The lipase that uses a solvent is an enzyme that hydrolyzes fats and oils into fatty acids and triglycerides in the presence of water. In the transesterification (transesterification) reaction between fats and oils and alcohol, the hydrolysis reaction proceeds due to the presence of water. However, it is considered that the transesterification reaction hardly proceeds. Therefore, in the transesterification reaction (transesterification reaction), a system in which water does not exist,
Alternatively, a trace water system is considered to be essential, and a method using a solvent is recommended. Accordingly, biological methods also have process problems such as recovery of the solvent and prevention of explosion.

[0005] In order to solve this problem, studies have been made on a system that does not use a solvent. For example, JAOCS 73
Pp. 1191-1195 (1996), fatty acid esters are obtained using branched alcohols, all of which are expensive alcohols, and inexpensive industrial alcohols such as methanol and ethanol have high reaction rates. The example shown is not reported.

On the other hand, when using fats and oils and waste oils of animals, plants and microorganisms and waste oils, it is considered that the water contained in the fats and oils or waste oils causes the ester decomposition reaction to proceed.

As described above, in order to efficiently use biodiesel fuel from fats and oils or waste oils, transesterification is carried out in a solventless system using an inexpensive alcohol such as methanol while considering the influence of moisture. Although it is necessary, at present, both the problem of the effect of water and the problem of using inexpensive alcohols in a solventless system remain unsolved.

[0008]

Accordingly, in order to efficiently use biodiesel fuel from fats and oils or waste oil, a solvent-free system using an inexpensive alcohol such as methanol, taking into account the effect of moisture. There is a need for a method of performing an exchange reaction.

[0009]

As a result of studying to solve the above problems, the present inventors overturned the conventional belief that the transesterification reaction hardly proceeds in the presence of water. But the transesterification proceeds,
The inventors have found that the transesterification reaction can be carried out without using a solvent and using an inexpensive alcohol, thereby completing the present invention.
According to the present invention, a transesterification reaction in a water-containing system, which has not been considered before, is performed, and the synthesis of a fatty acid ester (biodiesel fuel) from oils and fats containing water such as animals and plants and microbial oils or waste oil is efficiently performed. .

[0010] That is, the present invention relates to a transesterification method characterized in that a transesterification reaction between an ester and an alcohol is carried out in a water-containing system using an esterase.

In a preferred embodiment, the water-containing system is a system containing 1 to 20% by weight of water in a reaction system.

In a preferred embodiment, the reaction is performed in a solventless system.

[0013] In a preferred embodiment, the esterase is a lipase and the ester is a fatty acid ester.

[0014] The present invention also relates to an oil and fat, and an alcohol,
The present invention relates to a method for producing a fatty acid ester, comprising a step of reacting in the presence of water and lipase.

In a preferred embodiment, the water is contained in the reaction system in an amount of 1 to 20% by weight.

In a preferred embodiment, the reaction is performed in a solventless system.

In a preferred embodiment, the fat or oil is waste oil.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The reaction system used in the present invention contains esterase, alcohol, water and ester.

The most significant feature of the present invention is that water is present in the reaction system. Conventionally, ester hydrolysis proceeds in the presence of water, and the present invention is epoch-making in view of the fact that water is prevented from being present as much as possible.
Water is contained in the reaction system in an amount of 0.3% by weight or more, preferably 1 to 20% by weight, more preferably 1 to 10% by weight, and most preferably 5 to 8% by weight. The term “water-containing system” refers to a case where 0.3% by weight or more is contained in the reaction system.

In the transesterification method of the present invention, typically, these fats and oils are mixed with an alcohol and an aqueous enzyme solution is added thereto, or water is added to a reaction mixture of the fats and oils and the enzyme in a dry state. It is performed by an appropriate method such as addition. It is considered that the reaction forms an emulsion between the oil and fat and the enzyme, and proceeds at the interface. Therefore, it is considered that the progress of the reaction is controlled by the size of the emulsion, the concentration of the enzyme, and the like. The amount of water contained in the reaction system also
The optimum amount of water may be determined in consideration of such points. Thus, water may be included in excess of the preferred 20% by weight.

The above reaction system may contain a solvent (for example, hexane or the like). The case where no solvent is contained is referred to as a solvent-free system. The term "solvent-free system" means that a solvent for dissolving fats and oils is not contained, and the alcohol used for the transesterification reaction is not the solvent referred to in the present invention.

In the present invention, esterase refers to an enzyme that hydrolyzes an ester, and includes carboxylesterase, peptidase and the like. A typical example of a carboxylesterase is lipase. Lipase refers to an enzyme that has the ability to act on glyceride to break down into glycerin or partial glycerides and fatty acids.

Hereinafter, the present invention will be described using lipase as an example, but it goes without saying that the present invention can be applied to other esterases.

The origin of the lipase does not matter. Regardless of the shape of the enzyme, it may be a powder or may be immobilized. In addition, a case where a microorganism that produces lipase or an immobilized microorganism obtained by immobilizing the microorganism is directly used as an enzyme agent is also included. Among these, the immobilized lipase is most preferable from the viewpoints of rapid mass transfer during the reaction and reusability.

The lipase may be 1,3-specific or non-specific. From the viewpoint of the production of the fatty acid ester, the non-specific one is preferred. As lipase,
For example, the genus Rhizomucor, Muco (Muco
r) genus, Aspergillus genus, Rhizopus (Rhiz
opus), fungi belonging to the genus Penicillium (Penicilium), etc., genus Candida, yeast belonging to the genus Pichia, genus Pseudomonas (Pseudomonas), bacteria belonging to the genus Serratia (Seratia), pig pancreas And other lipases derived from animals.

Commercially available lipase is also used. Hereinafter, examples will be given, but the present invention is not limited thereto. As a lipase derived from a filamentous fungus, lipase A-10FG (trade name)
(From Rhizopus japonicas: Nagase Seikagaku Corporation), trade name Lipase F (from Rhizopus oryzae:
Amano Pharmaceutical Co., Ltd.) and trade name Lipase M (derived from Mucor Javanicus: Amano Pharmaceutical Co., Ltd.).

[0027] Bacterial lipases include trade name SM
Enzymes (from Serratia marcesens: Nagase Seikagaku Corporation), trade name lipase AH (from Pseudomonas cepacia: Amano Pharmaceutical Co., Ltd.), trade name lipase P: (Nagase Seikagaku Corporation) and the like. Can be

Examples of lipases derived from yeast include lipase L (trade name, derived from Candida lipolytica: Amano Pharmaceutical Co., Ltd.), lipase-OF (derived from Candida rugosa, Meito Sangyo Co., Ltd.) and the like.

[0029] Methods for immobilizing these lipases on carriers are known. Examples of the carrier include an ion exchange resin, a ceramic carrier, glass beads, activated carbon, and the like. In consideration of durability, affinity with lipase, and the like, ion exchange resins, ceramic carriers, and the like are most preferable. Examples of the immobilization method include an entrapment method, a cross-linking method, a physical adsorption method, an ion adsorption method, and a hydrophobic bonding method, and the cross-linking method and the hydrophobic bonding method are most preferable.

Commercially available immobilized lipases, for example, Novozyme 435 and Lipozyme IM60 (both from Novo Nordisk) are also suitably used in the present invention.

Examples of the microorganism include a lipase-producing filamentous fungus, bacteria, yeast, and the like. As filamentous fungi, Aspergillus (Aspergillus) genus, Galactomyces (Galactom
yces), Geotricum, Mucole
r), Phycomyces, Rhizomucor (Rhi
zomucor), Penicillium, Rhizopus
(Rhizopus) and the like.

The bacteria include Pseudomonas
As), bacteria belonging to the genus Alkaligenes, and the like.

As the yeast, Candida
Genus, Cryptococcus, Pichia
a) Genus, Rhodotorula, Yarrouia (Yarr
owia).

These microorganisms are examples, and it goes without saying that the microorganism of the present invention is not limited to these exemplified microorganisms.

The above microorganism may be used after being immobilized.
Immobilization is performed by a method appropriately used by those skilled in the art. An inclusive method, a physical adsorption method and the like can be mentioned, but a physical adsorption method using a porous carrier is preferable because of easy production.
In particular, microorganisms having cohesiveness (eg, bacteria, yeast, etc.)
Alternatively, if microorganisms having a floc-like or film-like morphology (for example, so-called molds) are used, an immobilized microorganism can be obtained simply by culturing with a porous carrier. When a microorganism is used directly as an enzymatic agent, treatment with acetone, alcohol, or the like brings the enzyme and the substrate into efficient contact with each other, which is a preferable state for increasing the reaction rate.

The ester is not particularly limited, but typical esters include fatty acid esters, particularly oils and fats. As fats and oils, glycerides, fats and oils containing a large amount of triglycerides are preferable, and vegetable fats and oils, animal fats and oils, fish oils, fats and oils produced by microorganisms, mixed fats and oils thereof,
Alternatively, these waste oils are used. Vegetable oils and fats include soybean oil, rapeseed oil, palm oil, olive oil and the like. Animal fats and oils include beef tallow, lard, whale oil, sheep fat and the like. Examples of fish oil include sardine oil, tuna oil, squid oil and the like. The fats and oils produced by microorganisms include Mortierella and Schizochytrium (S
chizochytrium) and the like.

The waste oil refers to, for example, fats and oils used for applications such as food production, such as tempura waste oil. Waste oil contains hydrogenated or peroxidized fats and oils when exposed to high temperatures, which can also be feedstocks for biodiesel fuels.

Examples of the alcohol include straight-chain alcohols such as methanol, ethanol, propanol, and butanol.
Examples include branched alcohols such as isopropanol, isobutanol and 2-butanol, and polyhydric alcohols such as glycerol. In the production of biodiesel fuel, it is preferable to use inexpensive alcohols such as methanol and ethanol.

The transesterification between the fat and the alcohol is generally carried out at 5 ° C. to 80 ° C., preferably at 15 ° C.
Carried out at 50 ° C., more preferably at 25 ° C. to 45 ° C.,
These may be determined by the lipase to be used. For example, in the case of a thermostable lipase, the reaction proceeds at a relatively high temperature.

Under the above reaction conditions, the transesterification proceeds in the presence of an ester, an alcohol, an esterase and water. When using fats and oils, fats and alcohols,
The reaction in the presence of water and lipase produces fatty acid esters and glycerin. This reaction may proceed irreversibly. The resulting fatty acid ester is
It is separated and recovered from glycerin or unreacted glyceride by a separation operation such as standing, centrifugation, membrane separation, molecular distillation, precision distillation and the like.

[0041]

The present invention will be described below with reference to examples.
The present invention is not limited to this embodiment.

Example 1 Commercially available enzymes described in Table 1
0 g was dissolved in 5 ml of distilled water to prepare an enzyme solution.

In a 20 ml glass container with a cap, add 0.
5 ml enzyme solution, 4.83 g olive oil, and 17
0 mg of methanol was mixed, shaken and stirred at 25 ° C. Sixteen hours after the start of the reaction, the resulting fatty acid methyl ester was quantified by gas chromatography.

The gas chromatography conditions were as follows:
It was as follows. Column; DB-5 (J & W Scientific, 10mx25m
m) Initial column temperature; 150 ° C (0.5 min) Heating rate; 10 ° C / min Final temperature; 300 ° C (3 min) Injector temperature; 245 ° C Detector temperature; 320 ° C Carrier gas; Helium (2.5 cm / Min) Spirit ratio; 1/100

Table 1 shows the results. The reaction rate in Table 1 is represented by the molar ratio of the resulting fatty acid methyl ester to the added fat and oil (olive oil). From the molar ratio of the added fat and oil to methanol, the reaction rate of 33.3% is theoretically Above, it means that 100% transesterification proceeded. The reason why methanol was added in an amount of 1/3 mol based on the fatty acid was that inhibition of lipase activity by methanol was considered.

[0046]

[Table 1]

The results in Table 1 show that the transesterification proceeded even when the water content was about 9% by weight.
In particular, lipase A-10FG, lipase F, lipase-OF, lipase P, lipase QL, lipase L, lipase M, lipase AH, lipase PS, and SM are catalyzed by the formation of fatty acid methyl esters despite the presence of water. The transesterification proceeded in high yield without being performed.

(Example 2) Transesterification in a water-containing system was performed using the filamentous fungi and yeasts shown in Table 2. First,
A pH 6.0 medium containing 1.0% yeast extract, 3.0% peptone, and 1.0% olive oil.
The yeast was cultured at 37 ° C. for 1 day at 2 ° C. for 2 days. 25 ml of each culture was freeze-dried to prepare an enzyme preparation, and the obtained enzyme preparation was dissolved in 500 μl of distilled water to prepare an enzyme solution.

50 in a 20 ml glass container with a cap
0 μl enzyme solution, 4.83 g olive oil, 170 m
g of methanol was added, and the mixture was shaken and stirred at 25 ° C.
Sixteen hours after the start of the reaction, the formation of fatty acid methyl esters was examined by thin-layer chromatography (TLC) using silica gel 60 (manufactured by Merck). TLC
Is a developing solution: hexane / ethyl acetate / acetic acid = 90: 1
0: 1, and after development, methanol / sulfuric acid = 50/50
Was sprayed and heated to develop a color.

Table 2 shows the results. In Table 2, + indicates a reaction rate of 2 to 10%, ++ indicates a reaction rate of 10 to 18%, ++
+ Indicates a reaction rate of 18 to 25%, and +++ indicates a reaction rate of 25%.
It means above.

[0051]

[Table 2]

It was shown that the transesterification proceeded even in the presence of about 9% by weight of water.

Example 3 Lipase F (Amano Pharmaceutical Co., Ltd.)
2.9 g) was dissolved in 10 ml of distilled water. Its 3m
1 is used as the enzyme solution, while 3 ml of it is used as 0.1 ml.
It was mixed with 8 g of Celite 545 and immobilized. Soybean oil 2
A transesterification reaction was performed using 8.95 g, 1.05 g of methanol and 3 ml of the enzyme solution or the immobilized enzyme. The results are shown in FIG.

In FIG. 1, ■ represents lipase F not immobilized, and ● represents lipase F immobilized.
After the reaction for one day, the transesterification reaction proceeded irreversibly with both the non-immobilized enzyme and the immobilized enzyme, and fatty acid methyl esters were produced almost quantitatively. Further, 1.05 g of methanol was added (first addition).
However, since the reaction proceeded almost quantitatively, 1.05 g of methanol was further added (second addition). The reaction further proceeded by this addition, and the fatty acid methyl ester was obtained almost quantitatively.

This result indicates that the knowledge of the present invention is a finding that a 100% transesterification reaction (ester synthesis reaction) proceeds in the presence of water, which has never been considered before.

(Example 4) Using lipase F, the influence of the water concentration on the reaction system was examined. An immobilized enzyme was prepared in the same manner as in Example 3, and 28.95 g of soybean oil and 1.05 g of methanol were mixed to perform a transesterification reaction. Note that 3.0 ml of distilled water was added to the reaction solution,
2.4 ml, 1.8 ml, 1.2 ml, 0.3 ml and 0.1 ml were added. Table 3 shows the conversion rate 24 hours after the start of the reaction.

[0057]

[Table 3]

From Table 3, it can be seen that a high conversion was achieved when the water content exceeded about 6% by weight, but when the water content was less than 0.3% by weight,
The conversion was not very high.

Example 5 Polypeptone 70 g / l, N
aNO ₃ 1 g / l, MgSO ₄ .7H ₂ O 0.5 g /
medium containing olive oil and 20 g / l of olive oil (pH
5.6) 100 ml and 100 porous carriers of 6 mm square polyurethane foam (manufactured by Bridgestone Corporation, Model HR-50) were put into a 500 ml flask, and the mixture was placed in a 500 ml flask.
Oryzae (Rhizopus oryzae) IFO 4697 was inoculated and cultured with shaking at 37 ° C. for 90 hours to immobilize the cells on a porous carrier to obtain immobilized cells. The immobilized cells were collected, washed twice with acetone, and then dried under vacuum to prepare immobilized dried cells having lipase activity.

In a 20 ml glass container with a cap, 9.65 g of soybean oil, 0.35 g of methanol, and 50 immobilized dried cells prepared above were mixed. 1 ml of distilled water was added to the mixture, and the mixture was shaken and stirred at 30 ° C. to react. On the first day and the second day after the start of the reaction, 0.35 g of methanol was added successively so that the soybean oil and the methanol finally became almost equimolar. Table 4 shows the results.

[0061]

[Table 4]

This result indicates that efficient reaction can be achieved even when lipase-containing microbial cells are directly used as an enzyme preparation.

[0063]

In the past, in the transesterification reaction, the reaction was carried out while excluding water as much as possible. However, it has been discovered that the transesterification reaction can be carried out even in a system containing sufficient water and that it can be performed almost quantitatively. Was done. In the presence of water, the acyl transfer reaction of esterases, especially lipases, proceeds almost 100%.

[Brief description of the drawings]

FIG. 1 is a diagram showing that a transesterification reaction proceeds in the presence of water.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B064 AD64 CA02 CA05 CA06 CA21 CB26 CC03 CD06 CD24 DA16 4H013 AA01 4H059 AA04 BA12 BA30 BB02 BB03 BC03 BC13 BC48 CA36 CA94 EA17 EA40

Claims (8)

[Claims] 1. A transesterification method, wherein the transesterification reaction between an ester and an alcohol is carried out in an aqueous system using an esterase. 2. The method according to claim 1, wherein the water-containing system is 1 to 20 in the reaction system.
2. The method according to claim 1, wherein the system contains water by weight. 3. The method according to claim 1, wherein the reaction is performed in a solvent-free system. 4. The method according to claim 1, wherein the esterase is a lipase and the ester is a fat or oil. 5. A method for producing a fatty acid ester, comprising a step of reacting an oil and fat with an alcohol in the presence of water and lipase. 6. The method according to claim 5, wherein the water is contained in the reaction system in an amount of 1 to 20% by weight. 7. The method according to claim 5, wherein the reaction is performed in a solvent-free system. 8. The method according to claim 5, wherein the fat is waste oil.