JP3720205B2 - Method for producing partial glyceride - Google Patents

Description

【００１８】
尚、上記例で反応終了油中の成分比は、サンプルをトリメチルシリル化してガスクロマトグラフィーで分析した結果である。
上記の実施例１と比較例１との対比より、本発明方法により固定化酵素の乾燥を行わずに反応を行った場合は、反応時間の短縮ができ、生成物の品質は損なわれないことが判明した。それぞれの酵素のエステル化の活性を比較したところ、反応基質で処理したものは150 ％（対乾燥品）の活性を発現したことになる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an esterification reaction of fatty acid and alcohol using an enzyme.
[0002]
[Prior art]
As an enzyme for reactions mainly intended for transesterification of fats and oils and esterification of fatty acids and alcohols, an immobilized enzyme in which a fat-and-oil degrading enzyme called lipase is immobilized on a carrier is used. In order to suppress hydrolysis, it is advantageous to perform these reactions at as low a water concentration as possible (1000 ppm or less). Therefore, it is compulsory to supply only a few percent of moisture to the carrier from the time of preparation of the immobilized enzyme. It is drying.
However, in the step of drying the immobilized enzyme, the adsorbed enzyme is likely to be deactivated, and in many cases, the maximum activity at the time of adsorption is not expressed when the actual activity is expressed.
Japanese Patent Application Laid-Open No. 60-134090 describes that after immobilization, the immobilized enzyme is dried under contact with a fatty acid derivative to increase the activity expression. However, this method is expensive for drying the immobilized enzyme. In addition to the need for equipment, the conditions for slow drying are complicated, making it impractical and efficient.
[0003]
[Problems to be solved by the invention]
Under the circumstances described above, it is desired to prepare an immobilized esterase for esterification that exhibits sufficient activity expression and further suppresses the desorption and inactivation of the enzyme to promote the esterification reaction.
[0004]
[Means for Solving the Problems]
As a result of the study by the present inventors to solve this problem, it is necessary to give a stable state to the enzyme adsorbed on the carrier. For this purpose, the reaction substrate and the enzyme are quickly combined after the enzyme is immobilized. It was found that contact is effective.
That is, the present invention is a method for producing a partial glyceride in which an oleolytic enzyme having positional specificity is adsorbed and immobilized on an immobilizing carrier and then directly contacted with a reaction substrate without being dried.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
In the case of an esterification reaction, the reaction is carried out while shifting the reaction under dehydration, so that it is possible to remove excess water remaining in the immobilized enzyme using the dehydration system while carrying out the reaction. In the first esterification reaction, in which the fat-degrading enzyme is adsorbed and immobilized on the immobilization carrier and then directly contacted with the reaction substrate without drying, it takes extra reaction time to remove this excess water. However, it takes a very short time compared to the drying time of the immobilized enzyme which has been conventionally performed. Furthermore, regarding the composition and quality of the product produced by this initial reaction, there is no difference from the product produced by the second and subsequent reactions. In this initial esterification reaction, the time required to reach the required activity-expressing moisture for the enzyme is approximately 1 hour, depending on the amount of immobilized enzyme used and the capacity of the dehydration system. With respect to the second and subsequent reactions, excessive water is removed in the initial reaction, so the time for removing the water required in the initial reaction is unnecessary.
[0006]
The carrier used in the present invention is preferably a porous anion exchange resin. The particle diameter of the resin is preferably 400 to 1000 μm, and the pore diameter is preferably 100 to 1500 mm. Examples of the material for the resin include phenol formaldehyde, polystyrene, acrylamide, and divinylbenzene. A phenol formaldehyde resin is particularly desirable. These pores give a large surface area to enzyme adsorption, and a larger adsorption amount can be obtained.
[0007]
In the present invention, it is preferable to treat the carrier with a fat-soluble fatty acid or a fat-soluble fatty acid derivative as a pretreatment for immobilization in order to obtain an adsorption state that exhibits high activity. As the fat-soluble fatty acid or fat-soluble fatty acid derivative to be used, those having 8 to 18 carbon atoms are desirable. Examples of the fatty acid include linear saturated fatty acids such as capric acid, lauric acid and myristic acid, unsaturated fatty acids such as oleic acid and linoleic acid, hydroxy fatty acids such as ricinoleic acid, and branched fatty acids such as isostearic acid. Examples of the fatty acid derivative include esters of a fatty acid having 8 to 18 carbon atoms and a compound having a hydroxyl group, monohydric alcohol esters, polyhydric alcohol esters, phospholipids, or derivatives in which ethylene oxide is further loaded on these esters. Is exemplified. Examples of monohydric alcohol esters include methyl esters and ethyl esters, and examples of polyhydric alcohol esters include monoglycerides, diglycerides, and derivatives thereof, polyglycerin fatty acid esters, sorbitan fatty acid esters, and sucrose fatty acid esters.
It is desirable in the process that these fatty acids and derivatives thereof are all liquid at room temperature. These may be used alone, but when combined, further effects are exhibited. It is considered that these derivatives are chemically or hydrolyzed with oleolytic enzymes in an aqueous medium to produce fatty acids.
As a method for contacting these fat-soluble fatty acids and derivatives thereof with the porous anion exchange resin, these may be added as they are in water or an organic solvent, but in order to improve dispersibility, the fat-soluble fatty acid or its The derivative may be once dispersed and dissolved, and then added to the porous anion exchange resin dispersed in water. Examples of the organic solvent at this time include chloroform, hexane, and ethanol. The ratio of the fat-soluble fatty acid and its derivative to the porous anion exchange resin is 0.01 to 1 part by weight, especially 0.05 to 0.5 part by weight, with respect to 1 part by weight (dry weight) of the porous anion resin. Is preferred. The contact temperature is 0 to 100 ° C, preferably 20 to 60 ° C. The contact time may be about 5 minutes to 5 hours.
[0008]
When preparing a partial glyceride such as monoglyceride or diglyceride, the oil-degrading enzyme used in the present invention has positional specificity, Rizopus genus, Aspergillus genus, Chromobacterium genus, mucor ( Examples include lipases derived from microorganisms such as Mucor, Pseudomonas, Geotrichum, Penicillium, and Candida, and animal lipases such as pancreatic lipase. In order to obtain triglycerides at a high esterification rate, a lipase having no position specificity (random type) is preferable, and Pseudomonas genus, Candida genus and the like are preferable for microbial origin.
[0009]
The temperature at which the immobilization is performed is 0 to 60 ° C., preferably 5 to 40 ° C. at which the enzyme is not deactivated, but can be selected depending on the characteristics of the enzyme. The pH of the enzyme solution may be in a range where no denaturation of the enzyme occurs and is preferably pH 3-9. This may be determined by the enzyme characteristics as well as the temperature. The buffer solution for maintaining these pHs is not particularly limited, but includes an acetate buffer solution, a phosphate buffer solution, a Tris-HCl buffer solution, and the like.
[0010]
The enzyme concentration in the enzyme solution at the time of immobilization is preferably not more than the solubility of the enzyme and sufficient concentration from the viewpoint of immobilization efficiency. If necessary, the insoluble part can be removed by centrifugation, and the supernatant can be used. The ratio of the immobilized carrier to the enzyme is preferably 0.05 to 10 parts by weight, particularly 0.1 to 5 parts by weight with respect to 1 part by weight of the immobilized carrier.
[0011]
The contact between the enzyme and the carrier treated as described above includes a method of dispersing the carrier in the enzyme solution and stirring, and a method of sealing the carrier in a packed tower such as a column and circulating the enzyme solution with a pump or the like. Any of these may be used. The immobilized enzyme adsorbed and immobilized is brought into contact with a reaction substrate in a state where water has been sufficiently removed by a physical method to perform an esterification reaction. At this time, the moisture in the immobilized enzyme varies depending on the type of carrier used, but is usually 20% by weight or more, preferably in the range of 40 to 60% by weight.
[0012]
Examples of the reaction substrate include fatty acids having 2 to 22 carbon atoms. These may be either saturated or unsaturated, and may contain a branch, a conjugated double bond, etc. in addition to a straight chain. Moreover, a structural isomer may be included and is not particularly limited. When preparing an ester, a partial glyceride, or a triglyceride having a single fatty acid composition, these fatty acids can be used alone or in combination of two or more. Furthermore, as the fatty acid, one obtained by completely or partially decomposing one or more kinds of vegetable oil / animal fat can be used. On the other hand, as alcohol, C1-C22 monohydric alcohol and bivalent or more alcohol are mentioned. The reaction substrate may be used in combination with the above fatty acid and alcohol so that the target esterified product can be prepared, and is not limited to the use of a specific one.
[0013]
The reaction method for esterification may be a known method such as a vacuum dehydration method, a glycerin dehydration method, or a dehydration reaction method using a dehydrating agent such as molecular sieve. The immobilized enzyme may be used in any of a stirring reactor, a packed column reactor, and a fluidized bed reactor.
[0014]
【The invention's effect】
According to the present invention, by providing a stable state to the enzyme on the immobilized enzyme and suppressing the deactivation of the enzyme by drying as much as possible, the maximum activity possessed by the adsorbed enzyme is drawn, and it can be extended over a long period. An efficient and stable esterification reaction can be performed.
[0015]
【Example】
Example 1
10 g of Duolite A-568 (Diamond Shamrock) was stirred for 1 hour in 100 cc of an N / 10 NaOH solution. After filtration, it was washed with 100 cc of ion-exchanged water, and the pH was equilibrated with 100 cc of 500 mM acetate buffer (pH 7). Thereafter, the pH was equilibrated twice for 2 hours with 100 cc of 50 mM acetate buffer (pH 7). Thereafter, filtration was performed to recover the carrier, and then ethanol substitution with 50 cc of ethanol was performed for 30 minutes. After filtration, 50 cc of ethanol containing 10 g of ricinoleic acid was added and ricinoleic acid was adsorbed on the carrier for 30 minutes. Thereafter, the carrier was recovered by filtration, and washed with 50 cc of 50 mM acetate buffer (pH 5) four times for 30 minutes, ethanol was removed, and the carrier was recovered by filtration. Thereafter, 10 g of a commercially available lipase (manufactured by Re-lipase Nagase Sangyo Co., Ltd.) was contacted with an enzyme solution dissolved in 90 cc of 50 mM acetate buffer (pH 7) for 5 hours for immobilization. After filtration, the immobilized enzyme was recovered, washed with 100 cc of 50 mM acetate buffer (pH 7), and unimmobilized enzyme and protein were washed. All the above operations were performed at 20 ° C. When the immobilization rate was determined from the difference between the residual activity of the enzyme solution after immobilization and the activity of the enzyme solution before immobilization, it was 98%.
Next, 100 g of fatty acid produced by decomposing soybean oil was added and stirred well, then 16 g of glycerin was added, and an esterification reaction was performed at 40 ° C. under reduced pressure (13 Pa or less). The DG (diglyceride) yield (diglyceride content + triglyceride content) in the reaction oil reached 63% after a reaction time of 4 hours. After this reaction, the reaction-finished oil was filtered off to recover the total amount of the immobilized enzyme, 100 g of soybean oil-decomposed fatty acid and 16 g of glycerin were added, and the reaction was repeated twice more. As a result, in these two repeated reactions, the DG yield in the reaction-finished oil was 62% after a reaction time of 2 hours. In the above repeated reaction, the composition of each component in the reaction oil at the time of reaction with a DG yield of about 62% is shown in Table 1.
[0016]
Comparative Example 1
The immobilized enzyme prepared in Example 1 was dried under reduced pressure (133 Pa or less) at 40 ° C. for 24 hours without adding the soybean oil-decomposing fatty acid shown in Example 1. The moisture content after drying was about 2%. Using 10 g of this immobilized enzyme, an esterification reaction was carried out in the same manner as in Example 1, and the reaction was repeated three times. As a result, in any reaction, the DG yield was 62 to 63% in 3 hours of reaction. Table 1 shows the composition of each component in the reaction oil when the reaction was performed with a DG yield of about 62%.
[0017]
[Table 1]

[0018]
In the above example, the component ratio in the reaction finished oil is the result of trimethylsilylation of the sample and analysis by gas chromatography.
From the comparison between Example 1 and Comparative Example 1 above, when the reaction is performed without drying the immobilized enzyme by the method of the present invention, the reaction time can be shortened and the quality of the product is not impaired. There was found. When the esterification activities of the respective enzymes were compared, those treated with the reaction substrate expressed 150% (vs. dry product).

Claims (5)

A method for producing a partial glyceride, in which an oil-degrading enzyme having positional specificity is adsorbed and immobilized on an immobilizing carrier and then directly contacted with a reaction substrate without drying, thereby carrying out an esterification reaction.   The method according to claim 1, wherein a porous anion exchange resin is used as the immobilizing carrier.   The method according to claim 1 or 2, wherein the carrier is treated with a fat-soluble fatty acid or a fat-soluble fatty acid derivative as a pretreatment for immobilization. Water in the immobilized enzyme after contact with the reaction substrate 40 ~ 60 The method according to any one of claims 1 to 3, wherein the method is% by weight. The method according to any one of claims 1 to 4, wherein the esterification reaction is repeated twice or more.