JP3509124B2 - Method for transesterification of fats and oils using immobilized lipase - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for transesterifying oils and fats containing no palm oil using an immobilized lipase suitable for transesterification of oils and fats.

[0002]

2. Description of the Related Art The transesterification reaction is a wax ester,
It is an important technique as a method for producing various fatty acid esters, sugar esters, steroids, etc., or a method for modifying vegetable oils and animal oils. As a catalyst for this transesterification reaction, it is possible to carry out the transesterification reaction under mild conditions by using lipase, which is a kind of fat and oil-degrading enzyme, and also to efficiently target the target product due to its substrate specificity and regiospecificity. Can be produced. Also, reduce the water content in the system as much as possible,
Moreover, it has been proposed to carry out the transesterification reaction in the presence of a sufficient amount of lipase so that the activity of the enzyme is expressed.
However, lipase is water-soluble, and it is difficult to uniformly disperse it in a fine water system (oil system). In order to solve such a problem, immobilized lipase in which lipase is carried on an insoluble carrier is used. Further, by employing the immobilized lipase, it is possible to further facilitate the separation of the product, the repeated use of the lipase, and the continuous reaction system.

However, in spite of such advantages, an immobilized lipase that can be put to practical use has not been obtained. As a method for preparing the immobilized lipase, a porous chitosan molded product (JP-A-59-213390) and a macroporous anion exchange resin (JP-A-60-9898) are used.
4), macroporous phenolic adsorption resin (JP-A-61-2268), animal bone (JP-A-64-8028)
6), a fired product of a foamable phenol resin (Japanese Patent Laid-Open No. 2-10
0678), a hydrophobic carrier having a pore size of 50 nm or more (JP-A-2-138986), a cation exchange resin (JP-A-3-64185), and a macroporous acrylic adsorption resin (JP-A-3-501922) as a carrier. The one to be used is proposed. However, when these carriers are used, sufficient lipase activity cannot be obtained. or,
JP-A-60-137290 discloses a method of immobilizing an enzyme on a polysaccharide carrier using an aldehyde group obtained by oxidizing a hydroxyl group of a polysaccharide. However, since the carrier is hydrophilic, it is not suitable for reaction in a slightly water system. In addition, JP-A-1-262
In 795, a method of immobilizing an enzyme on a chelate resin is shown, but it has not reached the activity enough for practical use.

[0004]

The present invention relates to a lipase immobilized on a carrier, which is excellent in expression of lipase activity,
It is particularly suitable for conducting transesterification in a slightly water system,
Another object of the present invention is to provide a method for transesterifying fats and oils containing no palm oil using an immobilized lipase that can reduce the amount of lipase used.

[0005]

The present invention uses an immobilized lipase in which a specific lipase is carried on a specific carrier having an epoxy group on the surface of the carrier and formed of a macroporous type adsorption resin. Therefore, it was made based on the finding that the above-mentioned object can be solved. That is, the immobilized lipase used in the present invention is a carrier formed from a hydrophobic insoluble organic polymer, which has pores with an average diameter of 10 nm or more and has an epoxy group on the carrier surface. 1 selected from the group consisting of lipases derived from the genera Rhizopus, Rhizopus, Mucor, Alcaligenes and Candida.
It is an immobilized lipase for transesterification of oils and fats in which one or more lipases are immobilized. Such an immobilized lipase is a carrier formed of a hydrophobic insoluble organic polymer, has a mean diameter of 10 nm or more, and has an epoxy group on the carrier surface. It can be produced by bringing an aqueous solution into contact with the carrier to covalently support the lipase on the carrier, and then drying. Thus, the present invention provides a method for transesterification of fats and oils that does not contain palm oil and fatty acids or two or more fats and oils that do not contain palm oil in the presence of the immobilized lipase. To do.

In the immobilized lipase used in the present invention,
Examples of the hydrophobic insoluble organic polymer forming the carrier include styrene-divinylbenzene copolymer, methacrylic acid ester resin, acrylic acid ester resin, polypropylene,
Nylon, phenol resin, etc. are used. Among these, a copolymer of divinylbenzene and styrene and / or methacrylic acid ester is particularly preferable. The pore diameter of the resin is 10 nm or more, preferably 10 nm to 1,000.
nm. In the immobilized lipase of the present invention, the epoxy functional group is attached to the insoluble organic polymer constituting the carrier so that the epoxy functional group is located on the surface of the carrier formed of the hydrophobic insoluble organic polymer. Alternatively, it is preferable to bond it via an aryl chain. Here, the epoxy group may be of any type, but preferably 1,2 epoxide in which oxygen atoms are added to adjacent carbons is used. The number of carbon atoms in the alkyl chain following the epoxide is 1
-20, preferably 1-10. It is also possible to use an aryl chain instead of an alkyl chain. in this case,
The number of benzene nuclei is 1 to 10, preferably 1 to 3.
The resin into which these functional groups have been introduced is obtained by, for example, a copolymerization reaction of methacrylic acid glycid and divinylbenzene, or the above-mentioned adsorption resin or a resin obtained by pretreating the adsorbent resin to produce a functional group, an ester bond, etc. Although it can be prepared by introducing an epoxy group by the general chemical bonding method described in 1., it is also possible to use a commercially available resin in which an epoxy functional group has been introduced in advance. As such, Levatit R259K and R260K manufactured by Bayer, and FP4000 manufactured by Organo are available.

In the immobilized lipase of the present invention, a carrier having an arbitrary particle size can be used, but it is generally preferred that 90% or more of the carrier particle size is 50 to 1,000 μm. However, especially the average particle size is 300 to 60
It is preferable to use one having a thickness of 0 μm. The lipase immobilized on the carrier is one or more lipases selected from the group consisting of lipases derived from Rhizopus, Mucor, Alcaligenes and Candida. In the present invention, a highly active immobilized lipase was obtained for the first time by immobilizing a specific lipase in this manner. Of these, it is particularly preferable to use a lipase of the genus Mucor or the genus Rhizopus. The transesterification activity of the preferred immobilized enzyme is 150 units or more per 1 g (dry weight) of the immobilized enzyme.

Next, a method for producing immobilized lipase will be described. In the present invention, a lipase is supported on a carrier by bringing an enzyme solution into contact with a porous insoluble carrier composed of a hydrophobic matrix into which an epoxy group has been introduced. Here, although the lipase solution depends on the lipase used, it is preferable to use 1 to 200 parts by weight of an aqueous solution containing 0.05 to 10% by weight of lipase per 1 part by weight of the carrier (dry weight). At this time, it is preferable to stir gently. The time required for immobilization is 10 minutes to 40 hours, preferably 4 hours to 24 hours. The temperature at the time of immobilization is 4
C. to 50.degree. C., preferably 5.degree. C. to 25.degree. Further, the enzyme solution can be prepared with a buffer solution if necessary. In this case, the adjusted pH is preferably around the optimum pH of the enzyme, and when lipase is used, it is preferably adjusted to the optimum pH of the hydrolysis activity measured in the free enzyme state, for example, pH 5-9. The type of buffer solution is not particularly limited, but an acetate buffer solution or a phosphate buffer solution can be used. The carrier carrying the enzyme is filtered to remove the residual liquid, and washed with ion-exchanged water or the like if necessary. At this time, it is possible to block the unreacted epoxy groups remaining on the carrier by using an aqueous solution containing a substance having an amino group such as Tris-hydrochloric acid buffer solution as the washing solution. The removed immobilized enzyme is preferably dried by a vacuum drying method or the like, and the water content after drying is 0.5% by weight to 30% by weight, preferably 5% by weight to
It is preferable to set it to 10% by weight. The water content after drying
If it is less than 0.5% by weight, the transesterification activity is not sufficiently expressed, and if it exceeds 30% by weight, it causes deactivation and hydrolysis as a side reaction cannot be ignored.
The ratio of carrier to enzyme used in the immobilization procedure is 0.1 g of protein in the enzyme per 1 g (dry weight) of carrier.
To 10 g, preferably 0.2 to 5 g, but is not particularly limited thereto.

Since the enzyme used for immobilization often contains a large amount of contaminating proteins, during the immobilization, lipase, which is a target protein, is selectively carried from these contaminants to some extent, and Effective expression of transesterification activity of the carried enzyme is an important factor for obtaining a highly active immobilized enzyme. Each of these can be evaluated by the degree of enrichment and the expression efficiency defined below. There are various methods for measuring the hydrolysis activity. Here, 1 part by weight of olive oil (Pharmacopoeia) and a 2% PVA aqueous solution (Kuraray's Poval 117: 18.5 g / 1, Poval 205:
1.5 g / 1) 1.5 parts by weight of emulsified solution was used as a substrate, and 5 ml of this emulsified solution was added to McKilven's buffer solution (pH).
7) One unit is defined as the activity of increasing 1 μmol of free fatty acid per minute calculated from the amount of free fatty acid generated by hydrolysis when 4 ml and 1 ml of enzyme solution are added and reacted at 37 ° C. for 60 minutes. There are various methods for measuring the transesterification activity. Here, a hexane solution containing 50 mM of triolein, 50 mM of palmitic acid, and 100 to 150 ppm of water is used as a substrate, and the substrate is 20 to 200
One unit is the activity of reducing 1 μmol of palmitic acid per minute, which is calculated from the maximum reduction rate of the concentration of palmitic acid in the case of adding mg (dry basis) of the immobilized enzyme and reacting at 50 ° C.

In the present invention, the immobilized lipase can be used to efficiently transesterify fats and oils that do not contain palm oil. Especially, the water content in the reaction system should be 5
0 to 2000 ppm, preferably 100 to 1000 pp
It is suitable for conducting a transesterification reaction in a fine water system reduced to m. In the present invention, a suitable transesterification reaction is at a temperature of 30 to 70 ° C, and an organic solvent can be used if necessary. As the organic solvent to be used, one that does not reduce the activity of the immobilized enzyme is selected, for example, n-
Examples include hexane and petroleum ether. Further, as the target fats and oils, plant-derived fats and oils containing no palm oil and fatty acids are preferable, but in addition, animal fats and oils, fish and shellfish fats and fatty acids thereof can also be targeted.
The palm oil includes palm oil, fractionated oil thereof, processed oils and fats such as hydrogenated oil, and various oils and fats mixed with them. Various transesterification reactions can be carried out as a method for producing wax esters, various fatty acid esters, sugar esters, steroids, etc., or as a method for modifying vegetable oils and animal oils.

[0011]

INDUSTRIAL APPLICABILITY According to the present invention, a fat and oil containing no palm oil, which has an extremely high lipase activity and is particularly suitable for conducting a transesterification reaction in a slightly water system, and which can reduce the amount of lipase used, is obtained. The transesterification method of a class can be efficiently performed. Next, the present invention will be described with reference to examples.

[0012]

【Example】

Reference Example 1 1 g (21 of lipase having 1,3 position specificity (Amano Pharmaceutical Co., Ltd. Lipase F-AP15, derived from Rhizopus oryzae ))
A hydrolyzable unit / g) was dissolved in 50 ml of ion-exchanged water to prepare an enzyme solution, and a porous resin containing an epoxy group as a base material of a copolymer prepared by introducing styrene into divinylbenzene (manufactured by Bayer) Levatit R259K
Pore diameter: 17 nm, average particle size: 500 μm) 5 g (water content: about 60%) was added, and the mixture was gently shaken and stirred at 5 ° C. for 4 hours to allow the lipase to be covalently bound to the resin. After removing the residual liquid by filtration, the water content is about 10% by vacuum drying.
Immobilized lipase was prepared. Reference Example 2 Porous resin having a copolymer of divinylbenzene and styrene as a base material and containing an epoxy group (Levacit R260K manufactured by Bayer, pore size 29 nm, average particle size 500)
Immobilized lipase (however, the water content was 5%) was prepared in the same manner as in Reference Example 1 except that μm) was used.

Reference Example 3 Immobilization was carried out in the same manner as in Reference Example 1 except that a porous resin containing polyacrylic acid as a base material and containing an epoxy group (FP4000 manufactured by Organo, pore size 55 nm, average particle size 100 μm) was used. A modified lipase was prepared. Reference Comparative Examples 1 to 3 Weakly basic anion exchange resin having phenol resin as a base material (Duolite A-56 manufactured by Rohm and Haas Co.)
8, particle size range 200 to 400 μm) [Reference Comparative Example 1],
Adsorption resin with phenol resin as base material (Duolite S-762 manufactured by Rohm and Haas Co., particle size range 200
400 μm) [Reference Comparative Example 2], an adsorption resin having a styrene-divinylbenzene copolymer as a base material (Mitsubishi Kasei Kogyo Co., Ltd. Diaion HP-40, average particle size 320 μ)
An immobilized lipase was prepared in the same manner as in Reference Example 1 except that [Reference Comparative Example 3] was used. Table 1 shows the amounts of the immobilized lipase obtained in Reference Examples 1 to 3 and Reference Comparative Examples 1 to 3 for the hydrolytic activity of protein and olive oil.
Shown in.

[0014]

[Table 1] Table-1 ───────────────────────────────────                               Protein loading Hydrolytic activity                               (mg / g-dry immobilized amount supported (unit / g                   Enzyme) -immobilized enzyme) Concentration ─────────────────────────────────── Reference Example 1 Revachit R259K 220 108300 1.4 Reference example 2 Revachit R260K 177 90100 1.5 Reference example 3 FP4000 102 84500 1.5 Reference Comparative Example 1 Duolite A-568 244 26100 0.3 Reference Comparative Example 2 Duolite S-762 167 88400 1.5 Reference Comparative Example 3 Diaion HP-40 178 71900 1.2 ───────────────────────────────────

Reference Example 4 200 mg of immobilized lipase prepared in Example 1 (on a dry matter basis) in 10 ml of a hexane solution (water content 100 ppm) containing triolein (50 mM) and palmitic acid 50 mM
Was added, and the reaction was carried out at 50 ° C. and 100 strokes / min while shaking and stirring. 10 minutes, 20 minutes, 40 after starting the reaction
After 60 minutes, 60 minutes, and 120 minutes, the concentrations of palmitic acid and other fatty acids in the reaction solution were measured. The transesterification is determined by incorporating palmitic acid into triglyceride, and the maximum rate of decrease of palmitic acid is shown in Table 2 as transesterification activity (unit). Reference Example 5 The transesterification reaction was performed in the same manner as in Reference Example 4 except that the immobilized lipase prepared in Reference Example 2 was used. The obtained transesterification activity is shown in Table 2.

Reference Example 6 The transesterification reaction was carried out in the same manner as in Reference Example 4 except that the immobilized lipase prepared in Reference Example 3 was used. The obtained transesterification activity is shown in Table 2. Reference Comparative Examples 4 to 6 Transesterification was performed in the same manner as in Reference Example 4 except that the immobilized lipase prepared in Reference Comparative Examples 1 to 3 was used. Reference Comparative Example 7 Commercially available immobilized lipase (Novo Nordisk
A transesterification reaction was carried out in the same manner as in Reference Example 4 using Lipozyme IM60 manufactured by Bioindustry. Table 2 shows the transesterification activities obtained in Reference Examples 4 to 6 and Reference Comparative Examples 4 to 7.

[0017]

[Table 2] Table-2 ─────────────────────────────────── Transesterification activity (unit / g charge) Body-immobilized enzyme) Expression efficiency ─────────────────────────────────── Reference Example 4 Levatit R259K 282. 4 2.61 × 10 ⁻³ Reference Example 5 Revat R260K 247.2 2.74 × 10 ⁻³ Reference Example 6 FP4000 218.4 2.58 × 10 ⁻³ Reference Comparative Example 4 Duolite A-568 11.2 0 .43 × 10 ⁻³ Reference Comparative Example 5 Duolite S-762 24.7 0.28 × 10 ⁻³ Reference Comparative Example 6 Diaion HP-40 82.9 1.15 × 10 ⁻³ Reference Comparative Example 7 Lipozyme IM60 145.3 −−−−− ────────────────────────────────────

Reference Example 7 As lipase, lipase A ( Rhiz manufactured by Nagase & Co., Ltd.)
(from opus japonicus ) 1g of 90,000 hydrolysis units / g
Immobilized lipase was prepared in the same manner as in Reference Example 1 except that it was used, and transesterification was performed in the same manner as in Reference Example 4. Reference Comparative Example 8 An immobilized lipase was prepared in the same manner as in Reference Example 7 except that the resin Duolite S-762 of Reference Comparative Example 2 was used as the immobilized lipase, and the transesterification reaction was performed in the same manner as in Reference Example 4. It was Table 3 shows the transesterification activities obtained in Reference Example 7 and Reference Comparative Example 8.

[Table 3] Table-3 ─────────────────────────────────── Transesterification activity (unit / g charge) Body-immobilized enzyme) Expression efficiency ─────────────────────────────────── Reference Example 7 Levatit R259K 203. 4 5.21 × 10 ^-3 Reference Comparative Example 8 Duolite S-762 10.4 0.80 × 10 ^-3 ───────────────────────── ─────────────

Reference Example 8 Lipase M (manufactured by Amano Pharmaceutical Co., Ltd., Mucor as lipase
(from javanicus ) 3g of 15,000 hydrolysis units / g
Immobilized lipase was prepared in the same manner as in Reference Example 1 except that it was used, and transesterification was performed in the same manner as in Reference Example 4. Reference Example 9 As lipase, lipase PL (manufactured by Meito Sangyo Co., Ltd., Alca
ligenes sp )) Immobilized lipase was prepared in the same manner as in Reference Example 1 except that 1 g of 90,000 hydrolysis units / g was used, and transesterification was performed in the same manner as in Reference Example 4. Reference Example 10 As lipase, lipase OF (manufactured by Meito Sangyo Co., Ltd., Cand
Immobilized lipase was prepared in the same manner as in Reference Example 1 except that 1 g of 220,000 hydrolysis units / g ( from ida cylindracea ) was used, and transesterification was performed in the same manner as in Reference Example 4.

Reference Comparative Example 9 Lipase CES (manufactured by Amano Pharmaceutical Co., Ltd., Ps as lipase
( from eudomonas sp ) 34,000 hydrolysis units / g 3
Immobilized lipase was prepared in the same manner as in Reference Example 1 except that g was used, and transesterification was performed in the same manner as in Reference Example 4. Reference Comparative Example 10 Lipase Sankyo (manufactured by Sankyo Co., Ltd., Aspergil ) as lipase
( from lus niger ) 34,000 24,000 hydrolysis units / g
Immobilized lipase was prepared in the same manner as in Reference Example 1 except that it was used, and transesterification was performed in the same manner as in Reference Example 4. The transesterification activities obtained in Reference Examples 8 to 10 and Reference Comparative Examples 9 to 10 are shown in Table 4.

[0021]

[Table 4] Table-4 ────────────────────────────────── Transesterification activity (unit / g enzyme- Immobilized enzyme) Expression efficiency ────────────────────────────────── Reference Example 8 Lipase M 190.1 4. 53 × 10 ⁻³ Reference Example 9 Lipase PL 215.5 5.26 × 10 ⁻³ Reference Example 10 Lipase OF 228.3 1.62 × 10 ⁻³ Reference Comparative Example 9 Lipase CES 45.2 0.51 × 10 ^{− 3} Reference Comparative Example 10 Lipase Sankyo 29.8 0.89 × 10 ^-3 ────────────────────────────────── ─

Example 1 and Comparative Example 1 Immobilized lipase obtained in Reference Example 1 and Reference Comparative Example 1
Immobilized lipase obtained with 0 ( Aspergil as lipase
lus niger to use) each 1g those derived from, high oleic sunflower oil (83% oleic acid content): the rapeseed oil 1:
1 Addition to 100 g of mixed oil (water content 180 ppm), the transesterification reaction was performed while shaking and stirring at 50 ° C. and 100 strokes / min. 2 hours, 6 hours and 2
After 4 hours, the oil and fat portion was sampled and the transesterification rate was measured (Example 1 and Comparative Example 1). Here, the transesterification rate means that the carbon number is 53 before the reaction and at the time of the substantial reaction equilibrium.
It is a value obtained by measuring the triglyceride content of and measuring 0% before the reaction and 100% at the time of substantial reaction equilibrium.
The results are shown in Table-5.

[0023]

[Table 5] Table-5 ─────────────────────────────────── Transesterification rate (%) Lipase carrier 2 Hr 6 Hr 24 Hr ─────────────────────────────────── Example 1 Lipase F-AP15 Levacit R259K 49.8 85.5 99.0 Comparative Example 1 Lipase Sankyo Revatit R259K 9.5 20.1 49.6 ─────────────────────────────────── Example 2 The immobilized enzyme obtained in Reference Example 1 was packed in a column, and a mixture of high oleic sunflower oil (oleic acid content 83%): stearic acid (purity 98%) = 1: 1 was added at 65 ° C. to give SV = 1.
I washed away. After 400 hours, sampling was performed to measure the triglyceride composition, and the reaction rate was calculated from the change in the triglyceride composition before and after the reaction. Free fatty acids were removed from this reaction oil by distillation to 5% or less. Next, hexane was used for two-stage fractionation at 18 ° C. and 5 ° C., and then the middle melting point portion was purified to obtain a cocoa substitute fat. The results are shown in Table-6. Comparative Example 2 The reaction rate was measured and the intermediate melting point was obtained in the same manner as in Example 2 except that the immobilized enzyme obtained in Reference Comparative Example 1 was used.
The results are shown in Table-6.

[Table 6] Table-6 ─────────────────────────────                   Reaction rate Mid-melting point yield                   (%) (%) ─────────────────────────────   Example 2 83 30   Comparative Example 2 42 18 ───────────────────────────── ─

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12P 7/64 C12R 1: 845 C12R 1: 845) 1: 785 (C12P 7/64 C12R 1:05 C12R 1: 785) 1 : 72 (C12P 7/64 C12R 1:05) (C12P 7/64 C12R 1:72) (72) Inventor Takeshi Minami 3-13 Moriyacho, Kanagawa-ku, Kanagawa Prefecture Chiyoda Research Park Co., Ltd. (56) References JP-A-63-17692 (JP, A) Tokuhyo 3-501922 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 1/00-41 / 00 Z C12N 11/00

Claims (6)

(57) [Claims] 1. A carrier formed from a hydrophobic insoluble organic polymer, having pores having an average diameter of 10 nm or more,
Also, the transesterification of oils and fats in which one or more lipases selected from the group consisting of lipases derived from Rhizopus, Mucor, Alcaligenes and Candida are immobilized on a carrier having an epoxy group on the surface of the carrier A method for transesterifying fats and oils, which comprises transesterifying fats and oils containing no palm oil and fatty acids or two or more fats and oils containing no palm oil in the presence of immobilized lipase. 2. The insoluble organic polymer of immobilized lipase comprises:
The transesterification method according to claim 1, which is a resin selected from a styrene-divinylbenzene copolymer, a methacrylic acid ester resin, polypropylene and nylon. 3. 90% of the particle size of the carrier of immobilized lipase
The transesterification method according to claim 1, wherein the above is 50 to 1,000 µm. 4. The epoxy functional group of the immobilized lipase is
The transesterification method according to claim 1, which is 1,2 epoxide. 5. The transesterification method according to claim 1, wherein the immobilized lipase has a water content adjusted to 0.5 to 30% by drying under reduced pressure. 6. The transesterification method according to claim 1, wherein the fats and oils containing no palm oil and the fatty acids are derived from plants.