JP2657886B2 - Immobilized enzyme and transesterification method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immobilized enzyme particularly suitable for transesterification of fats and oils, and a method for transesterifying fats and oils including palm oil using the same.

[0002]

2. Description of the Related Art Transesterification reactions include wax esters,
This 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. When lipase, a kind of lipolytic enzyme, is used as a catalyst for this transesterification reaction, transesterification can be performed under mild conditions, and the target product can be efficiently produced by its substrate specificity and positional specificity. Can be produced. Also, make the water in the system as small as possible,
In addition, it has been proposed to carry out a transesterification reaction in the presence of a lipase in an amount sufficient to express the activity of the enzyme.
However, lipase is water-soluble, and it is difficult to uniformly disperse it in a slightly water-based (oil-based) system. In order to solve such a problem, an immobilized lipase in which a lipase is carried on an insoluble carrier has been used. The use of the immobilized lipase has further obtained advantages such as easy separation of the product, repeated use of the lipase, and continuity of the reaction system.

[0003] However, despite having such advantages, an immobilized lipase that can withstand practical use has not been obtained. The method for preparing the immobilized lipase includes a porous chitosan molded product (JP-A-59-213390) and a macroporous anion exchange resin (JP-A-60-9898).
4), macroporous phenolic adsorption resin (JP-A-61-20268), animal bone (JP-A-64-8028)
6), a fired product of a foamable phenol resin (Japanese Patent Laid-Open No.
0678), a hydrophobic carrier having a pore diameter 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). Use has been 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 a reaction in a fine water system. Also, Japanese Patent Application Laid-Open No. 1-262
No. 795 discloses a method of immobilizing an enzyme on a chelate resin, but does not achieve a practically usable activity.

[0004]

DISCLOSURE OF THE INVENTION The present invention relates to a lipase immobilized on a carrier, which is excellent in the expression of these enzymatic activities, is particularly suitable for performing transesterification in a microaqueous system, and has a long life. It is intended to provide a long immobilized enzyme. Another object of the present invention is to provide a method for transesterifying fats and oils including palm oil using the immobilized lipase.

[0005]

SUMMARY OF THE INVENTION The present invention is directed to a macroporous resin which is a hydrophobic base material and has on its surface both a functional group capable of forming a covalent bond with an enzyme in an aqueous solution and an anion exchange group. It has been made based on the finding that by immobilizing the target enzyme on the formed specific carrier, an immobilized enzyme having high activity and long life can be obtained. That is, the present invention provides an immobilized enzyme, wherein lipase is immobilized on a resin carrier having a functional group capable of forming a covalent bond with an enzyme in an aqueous solution and a weakly basic anion exchange group. The present invention also provides a transesterification method comprising transesterifying fats and oils and fatty acids containing palm oil, or two or more fats and oils containing palm oil in the presence of the immobilized enzyme.

In the present invention, as the insoluble organic polymer forming the carrier, those based on divinylbenzene (DVB) copolymer, methacrylate, acrylate, polypropylene, nylon, phenol or the like are used. In particular, a divinylbenzene copolymer is preferably used. Further, the pore diameter of the resin is from 5 nm to
Thicknesses of 1000 nm, preferably 10 nm to 1000 nm are suitable. Examples of the functional group of the resin that forms a covalent bond with an enzyme in an aqueous solution include an epoxy group, a cyanide group, an aldehyde group, and a triazinyl group. Of these, an epoxy group is preferable, and a 1,2 epoxy group in which an oxygen atom is added to adjacent carbon is particularly preferable. Further, as the anion exchange group possessed by the resin, 1
Examples include a tertiary amino group, a secondary amino group, and a tertiary amino group, and a weakly basic tertiary amino group such as a diethylaminoethyl group (DEAE group) and a dimethylamino group are preferable.

In the present invention, the ratio of the above-mentioned functional group and anion exchange group in the resin carrier can be arbitrarily set, but the functional group forming a covalent bond to be copolymerized is 0.2 to 5.0.
mol / Kg, preferably 0.5 to 2.
It is 0 mol / Kg. In addition, 0.2 to 5.0 mol /
Kg is preferably contained, particularly preferably 0.5 to 2.0mo.
l / Kg. These functional groups and anion exchange groups can be introduced into the resin by a conventional method. For example, monomers having these functional groups or anion exchange groups were introduced by co-existing and copolymerizing during the polymerization of the resin, and the above-mentioned resin or a pre-treatment of the resin to form a reactive functional group. For example, it can be introduced by a general chemical bonding method such as an ester bond. The resin into which the specific functional group and the anion exchange group are introduced by such a method, for example,
Bayer's Levatit R260K is available and may be prepared as in Reference Example / or Example 3 below. In addition, Levatit R260K has an epoxy group and a secondary amino group, but the one described in Reference Example 1 having an epoxy group and a tertiary amino group is a more preferable carrier resin.

In the present invention, a carrier having an arbitrary particle size can be used.
% Or more is preferably used in a range of 50 to 1,000 μm, and particularly preferably one having an average particle size of 300 to 600 μm. As the lipase immobilized on the carrier, Mucor, Rhizopus, Aspergillus,
Alcaligenes, Geotricum, Candida,
Lipases derived from microorganisms such as Pseudomonas, Penicillium, Chromobacterium and the like can be mentioned. Among them, it is particularly preferable to use a lipase of the genus Mucor or Rhizopus.

[0009] The immobilized enzyme of the present invention can be produced, for example, by bringing a resin, which is a porous insoluble carrier, into contact with an enzyme solution to allow the carrier to carry lipase. Here, the enzyme solution is determined by the lipase to be used. For example, it is preferable to use 1 to 200 parts by weight of an aqueous solution containing 0.05 to 10% by weight of the enzyme 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 from 10 minutes to 40 hours, preferably from 1 hour to 24 hours. Temperature at the time of immobilization is 4
C. to 50.degree. C., preferably 5 to 25.degree. Further, if necessary, the enzyme solution can be prepared with a buffer solution. In this case, the adjusted pH is preferably around the optimum pH of the enzyme, and the optimum pH of the hydrolysis activity measured in the state of the free enzyme is adjusted.
It is preferable to adjust H, for example, to pH 5 to 9. The type of the buffer is not particularly limited, but an acetate buffer or a phosphate buffer can be used. The carrier carrying the enzyme is removed of residual liquid by filtration or the like, and washed with ion-exchanged water or the like as necessary. At this time, by using an aqueous solution containing a substance having an amino group such as a Tris-HCl buffer solution as the washing solution, it is possible to block the functional groups that form unreacted covalent bonds remaining on the carrier. It is preferable to dry the removed immobilized enzyme by a vacuum drying method or the like.
The content should be 0.5% to 30% by weight, preferably 5% to 10% by weight. When the water content after drying is less than 0.5% by weight, transesterification activity is not sufficiently exhibited, and when it exceeds 30% by weight, it causes deactivation and hydrolysis as a side reaction cannot be ignored. The ratio of the carrier and the enzyme used in the immobilization operation is 1 g of the carrier.
The protein in the enzyme is from 0.1 g to 10 g, preferably from 0.2 g to 5 g, based on (dry weight), but is not particularly limited to this.

By using the immobilized enzyme of the present invention, transesterification of fats and oils including palm oil can be efficiently performed.
In particular, the water content in the reaction system is 50 to 2000 ppm,
It is suitable for carrying out the transesterification reaction in a slightly water system preferably reduced to 100 to 1000 ppm. In the present invention, a suitable transesterification reaction includes a temperature of 30 to 7
The temperature is 0 ° C., and an organic solvent can be used if necessary. The organic solvent to be used is selected from those which do not decrease the activity of the immobilized enzyme, 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 2
It is essential that at least one or more kinds of fats and oils contain palm oil as raw materials for the transesterification reaction. Here, examples of fats and oils containing palm oil include palm oil, fractionated oil thereof, processed fats and oils such as hydrogenated oil and the like, and various fats and oils obtained by mixing them. On the other hand, as a raw material to be combined with such fats and oils containing palm oil, plant-derived fats and oils and fatty acids are suitable, but in addition, animal fats and oils, fish and shellfish fats and fatty acids can also be used.

[0011]

According to the present invention, it is possible to provide an immobilized enzyme which has excellent physical strength and chemical strength, is highly active, has a stable activity expression state of the carried enzyme, and has a long life. Therefore, the immobilized enzyme of the present invention is extremely important industrially. In addition, the transesterification of fats and oils including palm oil can be efficiently carried out by using this.
Next, the present invention will be described with reference to examples.

[0012]

【Example】

Reference Example 1 70% of divinylbenzene (DVB), 15% of glycidyl methacrylate and 15% of DEAE methacrylate were copolymerized by a usual method to obtain a resin carrier. This resin carrier had an average pore diameter of 12.3 nm and a pore volume of 0.5 cm ³ / g. Example 1 Lipase FAP-15 derived from Rhizopus sp. (150,000, manufactured by Amano Pharmaceutical Co., Ltd.) was added to 100 g of the resin carrier obtained in Reference Example 1.
u / g) at 1000C for 4 hours.
The mixture was immobilized while stirring. After filtration and washing, it was dried in a vacuum dryer for 3 hours. The obtained immobilized enzyme was 50 g and the water content was 5%. Example 2 In Example 1, the lipase was replaced with lipase LP derived from Chromobacterium Viscosum (100,000 u / Toyo Brewing Co., Ltd.).
g), an immobilized enzyme was prepared in place of Pseudomonus sp.-derived lipase CES (20,000 u / g, manufactured by Amano Pharmaceutical Co., Ltd.), and its transesterification activity was measured.

Example 3 One hundred grams of FE4612 (manufactured by Organo), which is a porous resin containing methacrylic acid as a base material and containing a primary amine, was previously added to 100 g.
Wt% glutaraldehyde (in 0.05M phosphate buffer)
200 ml was added, and the mixture was gently stirred at room temperature for 1 hour, washed with water, and then filtered to remove water, to thereby prepare an immobilized enzyme in the same manner as in Example 1. Comparative Example 1 Preparation was performed in the same manner as in Example 1 using Duolite S861, which is a hydrophobic resin having a carrier as a functional group and having neither a covalent bond group nor an anion exchange group. Comparative Example 2 Lipozyme (manufactured by Novo Nordisk Bioindustry) using a commercially available anion exchange resin as a carrier was used as the immobilized enzyme. Comparative Example 3 The procedure of Example 1 was repeated, except that Lewatit R259K, a hydrophobic resin having a functional group as a carrier and no anion exchange group, was used.

Example 4 The transesterification activity and life of the immobilized enzymes obtained in the above Examples and Comparative Examples were measured as follows. Transesterification activity of immobilized enzyme The transesterification activity was measured by the rate of incorporation of myristic acid into palm olein at 50 ° C. (μmol / min / g immobilized enzyme) and was converted to the unit volume of immobilized enzyme (unit: unit
/ ml immobilized enzyme). Lifetime of immobilized enzyme The immobilized enzyme was packed in a column, and a mixture of palm oil and rapeseed oil was allowed to flow at SV (space velocity) = 1 (liter / liter column · hr). From the change in the composition of the triglyceride of the reaction rate,
The amount of oil that can pass while maintaining a reaction rate of 70% was defined as the life of the immobilized enzyme. The results are shown in Table 1.

[0015]

[Table 1] Table-1 Example of immobilized transesterification activity long-lived enzyme (unit / ml immobilized enzyme) (1 feed oil / 1 immobilized enzyme) Example 1 93 1000 Example 2 68 800 65 800 Example 3 90 900 Comparative Example 1 85 400 Comparison Example 2 48 250 Comparative Example 3 83 500

Example 5 The immobilized enzyme obtained in Example 1 was packed in a column having a capacity of 100 ml, and a mixed oil of palm oil: rapeseed oil = 1: 1 was flowed at 50 ° C. and SV = 1. After 750 hours, about 1 kg was sampled and the reaction rate was measured. Further, this transesterified fat was wintered at 5 ° C., and the yield of the obtained liquid part was measured. Comparative Example 4 The immobilized enzyme obtained in Comparative Example 3 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 5 to measure the reaction rate and the yield of the liquid part during wintering. Table 2 shows the reaction rates obtained in Example 5 and Comparative Example 4 and the yield of the liquid part during wintering.

[0017]

Table 2 ────────────────────────────────── Reaction rate Liquid part yield at wintering (%) (%) 例 Example 5 80 71 Comparative Example 4 23 46 ──────────────────────────────────

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Noboru Ando 3--13 Moriyacho, Kanagawa-ku, Yokohama-shi, Kanagawa Prefecture Chiyoda Chemical Construction Co., Ltd. Chiyoda Research Park (72) Inventor Sachio Asaoka Moriyacho, Kanagawa-ku, Yokohama-shi, Kanagawa 3-13-13 Chiyoda Chemical Construction Co., Ltd. Chiyoda Research Park (72) Inventor Haruto Kobayashi 3-13 Moriyacho, Kanagawa-ku, Yokohama-shi, Kanagawa Chiyoda Chemical Construction Co., Ltd. Chiyoda Research Park

Claims (3)

(57) [Claims] 1. An immobilized enzyme comprising lipase immobilized on a resin carrier having a functional group capable of forming a covalent bond with the enzyme in an aqueous solution and a weakly basic anion exchange group. 2. The method according to claim 1, wherein the weakly basic anion exchange group is a tertiary amino acid.
2. The immobilized enzyme according to claim 1, wherein the functional group forming the group and the covalent bond is an epoxy group, and the resin carrier is porous. 3. A transesterification method comprising transesterifying an oil containing palm oil and a fatty acid or two or more fats and oils containing palm oil in the presence of the immobilized enzyme according to claim 1 or 2.