JPH0412716B2 - - Google Patents

Description

[Detailed description of the invention]

B. Object of the Invention Industrial Application Field The present invention relates to a method for ester synthesis using lipase, and more specifically, the synthesis of triglyceride from fatty acids and monoglyceride and/or diglyceride using lipase immobilized on a porous carrier having macroscopic pores. Concerning how to synthesize. Conventional technology The main component of edible oils and fats is triglyceride (TG).
In addition, it contains free fatty acids (FFA), monoglycerides (MG), and diglycerides (DG), which are hydrolysis products of TG. These products are produced by hydrolysis of the main component TG by enzymes contained in the raw materials or external factors during the oil and fat extraction operation from the collection of raw materials. Among these hydrolysis products, FFA and MG can be removed in the purification step. For example, it is possible to separate and remove a significant proportion of FFA and MG from TG by alkyl purification, distillation, and deacidification. On the other hand, DG has physical and chemical properties such as boiling point.
Since it is similar to TG, it is difficult to separate it from each other, and a method for separating DG has not been established. The presence of DG in TG forms a eutectic mixture,
Problems arise such as a decrease in SFI (solid fat content index), interference with crystal nucleation, and difficulty in tempering operations in the thiokolate production process. If DG is contained in this way, its uses will be limited, so a method is needed to effectively reduce the DG content in fats and oils. It is widely known that lipase acts on fats and oils to cause hydrolysis, ester synthesis, and transesterification reactions. Among these, hydrolysis and synthesis of fatty acid esters are reversible reactions as shown in the following formula. DG + FFA ← → TG + H ₂ O (1) From equation (1), it is clear that the reaction proceeds to the hydrolysis side in a system with a lot of water, and to the TG synthesis side in a system with a little water. It acts as a catalyst that promotes decomposition or synthesis reactions. JP-A-59-179091, JP-A-59-183691 and JP-A-60-98984 disclose lipase immobilized on a carrier having macro pores, but all of these carriers are ion exchange resins. It is a carrier that has a functional group such as a derivatized polysaccharide or a derivatized polysaccharide, and the functional group of the carrier and lipase are covalently bonded to each other by a crosslinking agent such as polyaldehyde to form a strong bond. Although these methods can be applied to water-rich systems such as hydrolysis and transesterification,
This is not necessarily considered a good solution when applied to systems with little water. That is, approximately 50% water is required for hydrolysis, and 1 to 2% water is required for transesterification. Under this moisture condition, lipase, which is a water-soluble enzyme, leaks into water, which is in suspension with oil and fat, which is a substrate, so it is necessary to firmly immobilize it with a carrier. Although it is essential to use a crosslinking agent as a means for this purpose, this not only complicates the enzyme preparation process, but also inhibits the expression of synthetic activity and is feared to reduce the activity. JP-A No. 57-8787 relates to an esterification method in which an enzyme with transesterification activity acts on a substrate in a low moisture system. An example using stearic acid methyl ester as a substrate has been shown, but because the physical properties of the carrier surface are not taken into consideration, lipase is not effectively immobilized and activity expression is poor. It takes a long time, several days. Problems to be Solved by the Invention The present invention provides an efficient method for improving the properties of fats and oils and increasing the TG yield by reacting DG and/or MG with FFA to produce TG. The purpose is to (b) Means for solving the structural problems of the invention The method for ester synthesis using lipase of the present invention includes:
Immobilized lipase obtained by immobilizing lipase on a porous carrier having macropores with an average pore diameter of 800 Å or more by physical adsorption is
It is characterized in that triglycerides are synthesized from fatty acids and monoglycerides and/or diglycerides under conditions of 200 ppm or less. Enzymes used in the present invention include Rhizopus, Aspergillus, Penicillum,
Examples include lipases derived from the genus Candida, Pseudomonas, Mucor, and Geotricum. Since lipase is insoluble in oil and fat, the larger the surface area (specific surface area) per unit weight of the carrier, the better in order to immobilize it on the surface of the carrier and bring it into sufficient contact with the substrate. In order to increase the specific surface area, it is necessary to have many pores on the surface of the carrier. However, the diameter of the pores (pore diameter) requires the following conditions. Molecular weight 30,000-5
The size of a lipase molecule is said to be 30 to 50 Å. A distance is required in which this molecule is fixed, and the substrate and reaction products FFA, MG, DG, TG, and water molecules can freely enter and exit, and the electrical properties of these substrates are not affected.
In order for TG molecules to sufficiently diffuse within the pores, TG
It is estimated that pores of several hundred angstroms in diameter would be required for hydrophobic and hydrophilic substances with different surface electrical properties to freely pass through without affecting each other. The results of the study were obtained. After further investigation based on these study results, we found that the carrier should be a porous carrier with as few micro pores as possible, such as tens to hundreds of Å, and macro pores of several thousand Å or more. I found something favorable. A porous carrier having such macroscopic pores needs to have pores with an average pore diameter of 800 Å or more, preferably 1000 Å or more. However, if the average pore diameter is too high, the specific surface area will decrease and the expression of lipase activity will tend to deteriorate, so the average pore diameter is preferably 500,000 Å or less. Note that the average pore diameter is expressed as the pore diameter when the cumulative pore volume by mercury intrusion method reaches 50% of the total pore volume. The carrier used in the present invention may be any material as long as it has the above-mentioned physical properties.Inorganic natural products include clay minerals such as zeolite, diatomaceous earth, perlite, and kaolin, and inorganic synthetic carriers include pores on the surface of the carrier. Synthetic polymer compounds such as silica, alumina, and zeolite prepared with Any carrier that satisfies the above-mentioned conditions, such as a composite of these, can be used. In the present invention, an immobilized lipase obtained by immobilizing lipase on the above-mentioned carrier by a physical adsorption method is used. The substrate for the ester synthesis of the present invention may be any substrate as long as it contains at least one of monoglycerides and diglycerides in addition to fatty acids. Obtained by transesterification of fats and oils
In addition to TG, it can also be applied to substances containing by-products such as MG, DG, and FFA. It can also be purified in advance and used as MG.
Fatty acids may be added to the DG-containing material after FFA has been removed. In order to make the reaction of equation (1) more favorable to the TG synthesis side, the water concentration in the reaction system should be 200 ppm or less,
Preferably it is 150 ppm or less. The reaction temperature may be 20 to 75°C, depending on the lipase used. Test Example 1 0.3 g of commercially available lipase derived from the genus Pseudomonas and 0.3 g of lecithin as a surfactant are dissolved in 10 g of water to make an enzyme aqueous solution, and this enzyme aqueous solution is applied to Celite with an average pore diameter of 97200 Å and a specific surface area of 0.80 m ² /g.
It was added to 10 g of carrier No. 535 (manufactured by Wako Pure Chemical Industries, Ltd.) and uniformly impregnated. After drying at room temperature under vacuum conditions of 0.5 mmHg for 18 hours, the temperature was raised to 40°C and drying was carried out for 6 hours to prepare immobilized lipase containing 3% lipase based on the carrier. The water content of this immobilized enzyme was about 1%. In addition to the above, three types of immobilization carriers, Celite No. 545 (manufactured by Wako Pure Chemical Industries, Ltd.) and Celite No. 640 (manufactured by Tokyo Mining Trading Co., Ltd.) shown in Table 1, were used as immobilization carriers, and 3% lipase was added by the method described above. An immobilized lipase containing the following was prepared. The reaction was performed using TG dehydrated to a water concentration of 60 ppm:
To 30 g of crude palm olein oil consisting of 87.6%, DG: 7.2%, MG: 0.3%, and FFA 4.8%, 3 g of each of the immobilized lipases prepared by the above method was added, and the mixture was heated at 60°C under a 0.6 mmHg vacuum condition. The reaction was carried out in a batch manner in which the reaction was carried out for 24 hours. After 24 hours of reaction, the reaction oil was collected and the TG content was measured by gas chromatography. The results are shown in Table 1. The specific surface area of the immobilization carrier was determined by the BET method.
Pore volume was measured by mercury intrusion method. In addition, the pore diameter when the cumulative pore volume by mercury intrusion method reached 50% of the total pore volume was defined as the average pore diameter. As is clear from the results in Table 1, when comparing the three types of carriers, celite with an average pore diameter of 97,200 Å
The immobilized lipase prepared using Celite No. 535 and Celite No. 545 with an average pore diameter of 159,000 Å has an average pore diameter of
It was observed that the TG increase ΔTG, that is, the TG synthesis activity, was significantly better than that of the immobilized lipase prepared using 570 Å Celite No. 640. Test Example 2 Various carriers were impregnated with an enzyme aqueous solution in the same manner as in Test Example 1, using a commercially available lipase derived from the genus Pseudomonas at a concentration of 3% based on the carrier. The immobilized lipase was prepared by drying for 6 hours. 10% immobilized lipase was added to 30 g of crude palm olein oil used in Test Example 1, and
Similarly, ester synthesis was carried out for 24 hours under vacuum conditions of 60°C and 0.6 mmHg while removing generated water. The carrier for immobilized lipase used in this test example was
Natural inorganic substances similar to celite include diatomaceous earth (manufactured by Wako Pure Chemical Industries, Ltd.), perlite, and kaolin; synthetic silica with controlled pores includes SMB-2035 (manufactured by JGC Chemical);
SPG, MPG (manufactured by Ise Chemicals), ion exchange resin WK-10 (manufactured by Mitsubishi Chemical) as a synthetic polymer, styrene/
Test examples in which lipase was immobilized on a total of 11 types of immobilization carriers: microspheres with pores prepared from divinylbenzene copolymer SGP-70C (manufactured by Soken Chemical) and polymethyl methacrylate MP-1400 (manufactured by Soken Chemical). 1
The reaction was carried out in the same manner as above at 60°C for 24 hours, the reaction oil was collected, and the changes in TG composition were measured and are shown in Table 1.

[Table] Also, regarding the data of Test Example 1 and Test Example 2,
The relationship between the average pore diameter of the carrier (shown on the horizontal axis) and the TG increase amount ΔTG after 24-hour reaction (shown on the vertical axis) is
Shown in the figure. From Table 1 and Figure 1, it can be seen that carriers with average pore diameters of 600 Å or more, and further 800 Å or more, exhibit good TG synthesis activity, with TG concentrations of 92% or more and 94% or more, respectively. This clearly shows that the activity expression factor of immobilized lipase is strongly influenced by the pore size on the surface of the immobilized carrier, and the expression of activity is better when a carrier with an average pore diameter of 600 Å or more is used. It is recognized that there is. Test Example 3 Dawetsukes MWA-1 (manufactured by Dow Chemical Company:
20-50 mesh, average pore diameter 600 Å with tertiary amine as an exchange group, specific surface area 43 m ² /g, pore volume
Weakly basic anion exchange resin (0.926cc/g)
10g of M/15 Matsuklebain buffer (PH5.0)
An enzyme solution in which 0.3 g of lipase (derived from Pseudomonas genus as in Test Example 1) was dissolved in 10 ml was added, and the entire solution was shaken at 10°C. Next, add 0.8 ml of 25% glutaraldehyde solution to 10 ml of Muzzle Vein buffer prepared separately, shake for 10 minutes, and then add this to the mixture of enzyme solution and Dowex MWA-1 mentioned above to infuse lipase into the ion exchange resin. Covalently bonded. Finally, 2 ml of 20% sodium hydrogen sulfite was added to remove excess glutaraldehyde, and then washed with matsuklebain buffer and distilled water to wipe off water adhering to the ion exchange resin. This was dried under reduced pressure under the same conditions as Test Example 1 to prepare a lipase enzyme agent covalently bonded to a carrier (comparative example). On the other hand, 0.3 g of lipase was dissolved in Dawetzke's MWA-1 in water coexisting with 0.3 g of lecithin, and Test Example 1 was prepared.
An adsorbed and immobilized lipase enzyme agent (Example) was prepared in the same manner as described above. In addition, dowex was applied in the same manner as in Test Example 1 except that lecithin was not used.
A lipase enzyme agent (Example) adsorbed and immobilized on MWA-1 was prepared. The ester synthesis reaction was carried out under the same conditions as in Test Example 1, and the TG concentration in the palm olein reaction oil was measured. The results are shown in Table 2.

[Table] Lipase enzyme preparations prepared by adsorption method exhibit good activity, whereas lipase enzyme preparations immobilized by covalent bonding method have approximately
It was observed that only 20-30% of TG synthesis activity was expressed. C. Effects of the invention The TG content in fats and oils can be improved and the properties of fats and oils can be easily modified. The burden on the oil and fat refining process can be reduced, and an improvement in refining yield can be expected.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between the average pore diameter of the carrier and the increase in TG after 24 hours.

Claims (1)

[Scope of Claims] 1. Immobilized lipase obtained by immobilizing lipase on a porous carrier having macroscopic pores with an average pore diameter of 800 Å or more by a physical adsorption method, and acting on the immobilized lipase at a water concentration of 200 ppm or less. A method for ester synthesis using lipase, characterized in that triglyceride is synthesized from fatty acid and monoglyceride and/or diglyceride. 2. The ester synthesis method according to claim 1, wherein the porous carrier having macropores has an average pore diameter of 800 to 500,000 Å.