JPH0638753A - Immobilized lipase, its production and method for ester-interchanging fat and oil with the lipase - Google Patents

Abstract

PURPOSE:To provide the lipase immobilized on a carrier, excellent in the expression of the activity of the lipase, especially suitable for performing an ester interchange reaction in a system containing a fine amount of water, and capable of reducing the amount of the lipase used therein. CONSTITUTION:The immobilized lipase for the ester interchange reactions of fats and oils is characterized by immobilizing one kind or more of lipases selected from a group of lipases originated from the genuses Rhizopus, Mucor, Alcaligenes, and Candida on a carrier formed from a hydrophobic insoluble organic polymer, having fine pores of an average diameter of >=10nm, and having epoxy groups on its surface.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immobilized lipase suitable for transesterification of oils and fats, a method for producing the same, and a method for transesterification of oils and fats containing palm oil using the immobilized lipase.

[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 an immobilized lipase that can reduce the amount of lipase used. Another object of the present invention is to provide a method for efficiently producing the immobilized lipase. Another object of the present invention is to provide a method for transesterifying fats and oils containing palm oil using the immobilized lipase.

[0005]

According to the present invention, the above object can be solved by supporting a specific lipase on a specific carrier having an epoxy group on the surface of the carrier and formed of a macroporous adsorption resin. It was made based on the knowledge. That is, the present invention relates to a carrier formed of a hydrophobic insoluble organic polymer, which has pores having an average diameter of 10 nm or more and has an epoxy group on the surface of the carrier, Rhizopus and Mucor. The present invention provides an immobilized lipase for transesterification of fats and oils, which comprises one or more lipases selected from the group consisting of lipases derived from Alcaligenes and Candida. The present invention also provides a carrier formed from a hydrophobic insoluble organic polymer, which has pores having an average diameter of 10 nm or more and has an epoxy group on the surface of the carrier, and the specific lipase Provided is a method for producing an immobilized lipase, which comprises bringing an aqueous solution into contact with the carrier to support the lipase by a covalent bond, followed by drying.

[0006] The present invention further provides a method for transesterifying fats and oils, which comprises transesterifying fats and oils containing palm oil and fatty acids, or two or more fats and oils containing palm oil in the presence of the immobilized lipase. provide. In the present invention, as the hydrophobic insoluble organic polymer forming the carrier, a styrene-divinylbenzene copolymer, a methacrylic acid ester resin, an acrylic acid ester resin, polypropylene, nylon, a phenol resin or the like is used.
Among these, a copolymer of divinylbenzene and styrene and / or methacrylic acid ester is particularly preferable. Also,
The pore diameter of the resin is 10 nm or more, preferably 10 nm to 1,
000 nm. In the present invention, the epoxy functional group is located on the surface of the carrier formed of the hydrophobic insoluble organic polymer, so that the insoluble organic polymer constituting the carrier is
The epoxy functional groups may be attached via an alkyl or 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 carbon number of the alkyl chain following the epoxide is 1 to 20,
It is preferably 1 to 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, for example, by a copolymerization reaction of methacrylic acid glycid and divinylben, or the above-mentioned adsorption resin or a resin in which the functional group is generated by pretreating it, 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. Examples of such products are Levatit R259K and R260K from Bayer and FP40 from Organo.
00 etc. are available.

In the present invention, a carrier having an arbitrary particle size can be used, but in general, the carrier has a particle size of 90.
% Or more is preferably 50 to 1,000 .mu.m, and it is particularly preferable to use one having an average particle size of 300 to 600 .mu.m. The lipase immobilized on the carrier is selected from the group consisting of lipases derived from Rhizopus, Mucor, Alcaligenes and Candida. 1
Species or two or more types of lipases. In the present invention, by immobilizing a specific lipase in this manner, the desired highly active immobilized lipase was obtained for the first time. Of these, it is particularly preferable to use a lipase of the genus Mucor or the genus Rhizopus. The preferred transesterification activity of the immobilized enzyme is 150 per 1 g (dry weight) of the immobilized enzyme.
More than a unit.

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 during 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, palm olein 50 mM,
A hexane solution containing 50 mM myristic acid and 100 to 150 ppm of water was used as a substrate, and 20 to 20
One unit is defined as the activity of reducing 1 μmol of myristic acid per minute, which is calculated from the maximum reduction rate of myristic acid concentration when 0 mg (dry basis) of the immobilized enzyme was added and the reaction was carried out at 50 ° C.

In the present invention, the immobilized lipase can be used to efficiently transesterify fats and oils including palm oil. In particular, the water content in the reaction system is 50 to
It is suitable for carrying out the transesterification reaction in a slightly water system reduced to 2000 ppm, preferably 100 to 1000 ppm. 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 which does not reduce the activity of the immobilized enzyme is selected, and examples thereof include n-hexane and petroleum ether. The target fats and oils are fats and oils containing palm oil and fatty acids, or two or more fats and oils containing palm oil, and at least one or more fats and oils containing palm oil as a raw material for the transesterification reaction. Is required. Examples of oils and fats containing palm oil include processed oils and fats such as palm oil, fractionated oils thereof, hydrogenated oils, and various oils and fats obtained by mixing these. On the other hand, plant-derived oils and fats and fatty acids are preferable as raw materials to be combined with such oils and fats containing palm oil, but animal fats and oils, fish and shellfish oils and fatty acids thereof can also be used in addition to these.

[0011]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an immobilized lipase which has an extremely high lipase activity, is particularly suitable for carrying out a transesterification reaction in a slightly aqueous system, and can reduce the amount of lipase used. Therefore, the immobilized lipase of the present invention has extremely important industrial value. Further, when the immobilized lipase is used, the method of transesterifying oils and fats containing palm oil can be efficiently performed. Next, the present invention will be described with reference to examples.

[0012]

【Example】

Example 1 1 g (21 of lipase having lipase specificity of 1,3 position (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. Example 2 Porous resin having a base material of a copolymer obtained by introducing styrene into divinylbenzene and containing an epoxy group (Levatit R260K manufactured by Bayer, pore size 29 nm, average particle size 500)
Immobilized lipase (however, water content 5%) was prepared in the same manner as in Example 1 except that (.mu.m) was used.

Example 3 Immobilization was performed in the same manner as in 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. Comparative Examples 1 to 3 Weakly basic anion exchange resin containing phenol resin as a base material (Duolite A-56 manufactured by Rohm and Haas Co.)
8, particle size range 200 to 400 μm) [Comparative Example 1], adsorption resin having phenol resin as a base material (Duolite S-762 manufactured by Rohm and Haas Company, particle size range 200 to 4)
00 μm) [Comparative Example 2], and an adsorption resin (Diaion HP-40 manufactured by Mitsubishi Kasei Co., Ltd., average particle size 320 μ) [Comparative Example 3] using a styrene-divinylbenzene copolymer as a base material. Otherwise, the immobilized lipase was prepared in the same manner as in Example 1. Examples 1-3 and Comparative Examples 1-
The amounts of the immobilized lipase protein obtained in Example 3 and the hydrolysis activity of olive oil carried are shown in Table 1.

[0014]

[Table 1] Table-1 ─────────────────────────────────── Protein loading Hydrolysis activity (mg / g-dry Immobilized load (unit / g enzyme)-Immobilized concentration ──────────────────────────── ──────── Example 1 Levatit R259K 220 108300 1.4 Example 2 Levatit R260K 177 90100 1.5 Example 3 FP4000 102 84500 1.5 Comparative Example 1 Duolite A-568 244 26100 0.3 Comparative Example 2 Duolite S-762 167 88400 1.5 Comparative Example 3 Diaion HP-40 178 71900 1.2 ───────────────────────── ───────────

Example 4 Example 1 was added to 10 ml of a hexane solution (water content 100 ppm) containing 50 mM palm olein and 50 mM myristic acid.
The immobilized lipase (200 mg, dry matter standard) prepared in (4) was added, and the mixture was reacted at 50 ° C. with 100 strokes / min while shaking and stirring. 10 minutes, 20 minutes, 40 minutes after the start of the reaction,
The concentrations of myristic acid and oleic acid in the reaction solution after 60 minutes and 120 minutes were measured. The transesterification is determined by taking up myristic acid into triglyceride, and the maximum rate of myristic acid reduction is shown in Table 2 as transesterification activity (unit). Example 5 The transesterification reaction was performed in the same manner as in Example 4 except that the immobilized lipase prepared in Example 2 was used. The obtained transesterification activity is shown in Table 2.

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

[0017]

[Table 2] Table-2 ────────────────────────────────── Transesterification activity (unit / g carrier) -Immobilized enzyme) Expression efficiency ────────────────────────────────── Example 4 Levatit R259K 282.4 2 .61 × 10 ⁻³ Example 5 Levatit R260K 247.2 2.74 × 10 ⁻³ Example 6 FP4000 218.4 2.58 × 10 ⁻³ Comparative Example 4 Duolite A-568 11.2 0.43 × 10 ⁻³ Comparative Example 5 Duolite S-762 24.7 0.28 × 10 ⁻³ Comparative Example 6 Diaion HP-40 82.9 1.15 × 10 ⁻³ Comparative Example 7 Lipozyme IM60 145.3 --- −− ──────────────────────────────────

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 Example 1 except that it was used, and transesterification was performed in the same manner as in Example 4. Comparative Example 8 Resin Duolite S- of Comparative Example 2 as immobilized lipase
An immobilized lipase was prepared in the same manner as in Example 7 except that 762 was used, and transesterification was performed in the same manner as in Example 4. Table 3 shows the transesterification activity obtained in Example 7 and Comparative Example 8.

[Table 3] Table-3 ───────────────────────────────── Transesterification activity (unit / g carrier- Immobilized enzyme) Expression efficiency ───────────────────────────────── Example 7 Levatit R259K 203.4 5.21 × 10 ^-3 Comparative Example 8 Duolite S-762 10.4 0.80 × 10 ^-3 ───────────────────────────── ─────

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

Comparative Example 10 As lipase, lipase Sankyo (manufactured by Sankyo Co., Ltd., Aspergil )
( from lus niger ) 34,000 24,000 hydrolysis units / g
Immobilized lipase was prepared in the same manner as in Example 1 except that it was used, and transesterification was performed in the same manner as in Example 4. Table 4 shows the transesterification activities obtained in Examples 8 to 10 and Comparative Examples 9 to 10.

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

Example 11 and Comparative Example 11 The immobilized lipase obtained in Example 1 and the immobilized lipase obtained in Comparative Example 10 ( Aspergillus as a lipase)
Niger- derived one) was added to 100 g of 1: 1 mixed oil of palm oil: rapeseed oil (water content 180 ppm), and the transesterification reaction was performed while shaking and stirring at 50 ° C and 100 strokes / minute. went. After 2 hours, 6 hours, and 24 hours, the oil and fat portion was sampled to measure the transesterification rate (Example 11 and Comparative Example 11). Here, the transesterification rate is a value obtained by measuring the content of the triglyceride having 53 carbon atoms before the reaction and at the time of the substantial reaction equilibrium, and assuming that it is 0% before the reaction and 100% at the time of the substantial reaction equilibrium. Is. The results are shown in Table-5.

[Table 5] Table-5 ─────────────────────────────────── Transesterification rate (%) Lipase carrier 2 Hr 6 Hr 24 Hr ─────────────────────────────────── Example 11 Lipase F-AP15 Levatit R259K 46.5 83.0 99.8 Comparative Example 11 Lipase Sankyo Revatit R259K 8.5 19.0 47.3 ────────────────────────────────────

Example 12 The immobilized enzyme obtained in Example 1 was packed in a column having a volume of 100 ml, and a mixed oil of palm oil: rapeseed oil = 1: 1 was added to 65 parts.
Flowed at SV = 1 at ° C. After 400 hours, about 1 kg was sampled and the reaction rate was measured. Further, this transesterified oil and fat was wintered at 5 ° C., and the yield of the obtained liquid part was measured. Comparative Example 12 The immobilized enzyme obtained in Comparative Example 1 was packed in a column having a capacity of 100 ml, and the reaction and sampling were performed in the same manner as in Example 12 to measure the reaction rate and the yield of the liquid part at the time of wintering. Table 6 shows the reaction rates obtained in Example 12 and Comparative Example 12 and the liquid part yield at the time of wintering.

[Table 6] Table-6 ──────────────────────────────── Reaction rate Liquid fraction yield during wintering (% ) (%) ──────────────────────────────── Example 12 81 71 Comparative Example 12 42 54 54 ──── ────────────────────────────

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // (C12P 7/64 C12R 1: 845) (C12P 7/64 C12R 1: 785) (C12P 7 / 64 C12R 1:05) (C12P 7/64 C12R 1:72) (C12P 7/64 C12R 1:38) (C12P 7/64 C12R 1: 685) (72) Inventor Noboru Ando Kanagawa-ku, Yokohama-shi, Kanagawa 3-13 Moriyacho Chiyoda Kako Construction Co., Ltd. in Chiyoda Resarch Park (72) Inventor Takeshi Minami 3-13 Moriyacho Kanagawa-ku, Kanagawa-shi Kanagawa-ku Chiyoda Reconstruction Chikada Co., Ltd.

Claims (9)

[Claims] 1. A carrier formed from a hydrophobic insoluble organic polymer, having pores having an average diameter of 10 nm or more,
In addition, 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. Immobilized lipase for transesterification of oils and fats. 2. The immobilized lipase according to claim 1, wherein the insoluble organic polymer is a resin selected from styrene-divinylbenzene copolymer, methacrylic acid ester resin, polypropylene and nylon. 3. 90% or more of the particle size of the carrier is 50 to 1.0.
The immobilized lipase according to claim 1, which has a size of 00 μm. 4. The immobilized lipase according to claim 1, wherein the epoxy functional group is a 1,2 epoxide. 5. The immobilized lipase according to claim 1, wherein the water content is adjusted to 0.5 to 30% by drying under reduced pressure. 6. A carrier formed from a hydrophobic insoluble organic polymer, having pores having an average diameter of 10 nm or more,
And, a carrier having an epoxy group on the carrier surface is contacted with an aqueous solution of one or more lipases selected from the group consisting of lipases derived from Rhizopus, Mucor, Alcaligenes and Candida to form a covalent bond. A method for producing an immobilized lipase, which comprises supporting the lipase on a carrier and then drying. 7. The production method according to claim 6, wherein the drying is carried out under reduced pressure so that the water content becomes 0.5 to 30%. 8. A method for transesterification of fats and oils, which comprises transesterifying fats and oils containing palm oil and fatty acids, or two or more fats and oils containing palm oil in the presence of the immobilized lipase according to claim 1. 9. The transesterification method according to claim 8, wherein the fats and oils containing palm oil and the fatty acids are derived from plants.