JPH03160992A - Carrier for immobilization of enzyme and production of immobilized enzyme - Google Patents

Abstract

PURPOSE:To obtain an immobilized enzyme having excellent ester synthesis and exchange activities and high durability by contacting an aqueous solution of a lipase with a carrier consisting of an ampholytic ion exchange resin having a specific cation exchange group and a specific anion exchange group. CONSTITUTION:An aqueous solution of a lipase is made to contact with a carrier for the immobilization of enzyme. The carrier used in the above process is composed of an ampholytic ion exchange resin having carboxyalkyl group or alkenyl group (alkyl or alkenyl is a 2-6C straight or branched-chain group which may have substituents) and/or phosphate group as cation exchange group and having primary amino group, secondary amino group, tertiary amino group and/or quaternary ammonium group as the anion exchange group. The carrier can be used in the form of powder, granule, fiber, sponge, etc., preferably as a porous material having large specific surface area.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a carrier for immobilizing an enzyme, and a method for producing an immobilized enzyme suitable for the synthesis and exchange reaction of esters of fats and oils and fatty acid derivatives.

x Sf Ruli (D Synthesis reaction is the synthesis of alcohol fatty acid esters from alcohol fatty acids, the synthesis of polyhydric alcohol fatty acid esters such as monoglycerides, polyglycerin fatty acid esters, and sugar esters, and the production method of fragrances such as geranyl butyrate. This is an important technology.

On the other hand, transesterification of oils and fats is an important technology along with hydrogenation for the modification of edible processed oils and fats such as margarine and shortening.

[Problems to be solved by conventional techniques and inventions] In recent years,
BACKGROUND ART Research and industrialization of combination and exchange reactions of fats and oils and esters using lipase as an enzyme are becoming active in various fields. This takes advantage of the fact that lipase reacts under mild conditions and has regioselectivity and alkyl selectivity. However, these reactions differ from the hydrolysis reactions inherent to lipase, and can proceed only in a system with limited moisture. On the other hand, in order to increase the esterification or exchange activity of Reverse 3~, water is required as an enzyme. For example, JP-A-55-71
In the low-moisture system reaction disclosed in Publication No. 797, a sufficient reaction rate cannot be obtained, and if more water than necessary is added to increase the reaction rate, the ester decomposition reaction proceeds preferentially. There is a point. Also, JP-A-60-19
As disclosed in Japanese Patent No. 495 and Japanese Patent Application Laid-Open No. 60-203196, there have been proposals for a method in which the reaction is divided into two stages: a step of decomposing a polyhydric system and a step of removing water. It cannot be said that the reaction rate of this reaction is sufficient compared to the usual rate of transesterification, and the process operation becomes unavoidably complicated.

In order to solve the above problems and use lipase efficiently, attempts have been made to immobilize lipase.

The expected advantages of lipase immobilization are as follows. In other words, (i> Conventionally, when lipase was used in the form of an aqueous solution, it was difficult to mix and disperse it uniformly in oil. However, by immobilizing lipase on the surface of an insoluble carrier, it can be easily mixed and dispersed in oil.) It becomes dispersible and can hold an appropriate amount of moisture in the carrier, making it easier to carry out esterification or exchange reactions in low moisture conditions.Gi) Reverse, which is expensive as a catalyst, can be easily recovered and reused; In the industrial implementation of a certain reaction or exchange reaction, it becomes easy to use a continuous reactor.

However, even with the above-mentioned advantages of immobilized enzymes, it has not been possible to simultaneously maintain the amount of water necessary for increasing the lipase synthesis activity and suppress hydrolysis, which is the reverse reaction. . For example, Journal of
American Oil Chemist's Soc
iety, Vol. 60, 291-294 (19
83) also pointed out that the hydrolysis reaction progresses when a small amount of water is added. Further, when a polyhydric alcohol such as glycerin is added instead of water, the hydrolysis reaction is suppressed to some extent, but the ester synthesis/exchange reaction becomes extremely slow. In addition, after comprehensively bonding a porous carrier and a super absorbent resin with chitosan in order to retain enzyme moisture,
In the method (Japanese Unexamined Patent Application Publication No. 59-213390) using a crushed carrier, a two-step reaction method (Japanese Unexamined Patent Application Publication No. 60-1988) is used to achieve both the ester synthesis/exchange reaction and the decomposition reaction of the immobilized enzyme. -203
No. 196] is adopted. However, such a method has disadvantages in that the operation is complicated and it is difficult to suppress diglyceride in the by-product bottom material.

As mentioned above, in ester synthesis and exchange reactions,
It is desired to develop an immobilized enzyme with higher esterification and exchange activity and excellent durability.

[Means to solve the problem]

As a result of previous research into factors that increase the esterification and transesterification activities of lipase, the present inventor found that
In the method disclosed in JP-A No. 60-25188, in which fats and oils are added to lipase and immobilized while causing a hydrolysis reaction to obtain a highly active immobilized enzyme, the coexistence of fatty acids, fatty acid derivatives, alcohols, or phospholipids is used. He discovered that the esterification and transesterification activities were increased by immobilization under
No. 153090). However, the enzymes immobilized in this manner also have problems such as a gradual decrease in activity when used at high temperatures of 50° C. or higher for a long period of time, and shedding of coexisting fatty acid derivatives.

Therefore, as a result of various studies in order to find a more durable immobilization method that strengthens the bond between the enzyme and the carrier, prevents the desorption of the coexisting fatty acid derivatives, and results in a more suitable carrier, an amphoteric one with a specific ion exchange group, Discovered an ion exchange resin and completed the present invention.

That is, the present invention provides that the cation exchange group is a carboxyalkyl group or an alkenyl group (the alkyl group or alkenyl group is a straight chain or branched chain having 2 to 6 carbon atoms, and the alkyl group or alkenyl group has a substituent). ), and/or a phosphoric acid group, in which the anion exchange group is one or more selected from the group consisting of a l-class amino group, a secondary amino group, a tertiary amino group, and a quaternary ammonium group. Provided is a carrier for immobilizing an enzyme, characterized in that the carrier is made of a certain amphoteric ion exchange resin, and a method for producing an immobilized enzyme, characterized in that the carrier for immobilization is brought into contact with an aqueous solution of a lipolytic enzyme. It is something.

The cation exchange group of the amphoteric ion exchange resin according to the present invention is a carboxyalkyl group or an alkenyl group (the alkyl group or alkenyl group is a straight chain or branched chain having 2 to 6 carbon atoms,
The alkyl group or alkenyl group may have a substituent), and/or the phosphoric acid group, and a suitable method for introducing a carboxyalkyl group or alkenyl group into the resin includes general formulas XR, COON (X is a halogen atom, R
1 is an alkylene group or alkenylene group having 2 to 6 carbon atoms)
l CH. A carboxylic acid represented by R2CHR3COOH (X is a halogen atom, R2.R3 is an alkylene group or an alkenylene group, and the sum of the carbon numbers of R2 and R3 is 0 to 4), or has an unsaturated bond Polycarboxylic acid or its acid anhydride has a hydroxyl group, a primary amino group,
There is a method in which a resin having a secondary amino group, an imino group, a sulfhydryl group, or a derivative thereof is reacted in the presence of an alkali compound or under heating.

Specific examples of suitable carboxylic acids used for introducing carboxyalkyl groups or alkenyl groups include monocarboxylic acids such as α-chloropropionic acid and β-chloropropionic acid; polycarboxylic acids such as maleic acid and itaconic acid; maleic anhydride. Examples include acid anhydrides of polycarboxylic acids such as itaconic anhydride and succinic anhydride.

In addition, suitable phosphoric acids used for introducing phosphoric acid groups include:
Examples include compounds having an alkyl phosphoric acid group in which the alkyl group has 0 to 3 carbon atoms.

Furthermore, a sulfonic acid group may be introduced using an alkylsulfonic acid group having 0 to 3 carbon atoms.

Further, as the anion exchange group of the amphoteric ion exchange resin according to the present invention, one or more types selected from the group of primary amino group, secondary amino group, tertiary amino group, or quaternary ammonium group. In addition, it is preferable that instead of a simple amino group, a meth9-tylglucamyl group, a polyalkylene polyamine group, a polyalkylene polyamine group having a substituent, or the like is partially introduced.

In the present invention, any commercially available anion exchange resin having the above-described anion exchange group can be suitably used for introducing the cation exchange group.

Furthermore, a titanium coupling agent having a primary or secondary amine group (for example, isopropyl (N-aminoethyl-aminoethyl) titanate) can be used to add an anion to a commercially available cation exchange resin having the above-mentioned cation exchange group. An amphoteric ion exchange resin can also be obtained by introducing an ion exchange group.

The carrier according to the present invention has various shapes such as powder, granule, fiber, and sponge, and any of them can be used. A porous material with a large specific surface area is preferable. Particularly from the viewpoint of process operation, it is preferable to use a porous resin having a particle size of 400 to 1000 μm and a pore size of 100 to 1500 μm.

Lipid degrading enzymes used in the present invention include lipases, phospholipases, cholesterol esterases, sphingomyelieses, and various esterases. Among these lipases, Rhizopus reacts only with the 1st and 3rd positions of glycerides and has excellent regioselectivity.
pus) genus, Aspergillus
), Mucor, Geotrichum with fatty acid specificity, Candida without specificity, Pseudomonas, Penicillium, Chromobacterium ) and animal lipases such as pancreatic lipase. Among these, as lipases whose synthetic activity is particularly likely to increase, lipases originating from the genus Rhizopus, Mucor, and Chromobacterium, which have strong active positions in medium-chain or higher alkyl groups, are more preferable. Examples of cholesterol esterases include those originating from microorganisms such as the genus Candida. Examples of phospholipases include phospholipase D derived from plants such as cabbage, peanuts, carrots, soybeans, and rapeseed, and moss plants; phospholipase D1 derived from microorganisms such as Streptomyces; phospholipase A1 derived from yeast; and phospholipase A2 derived from viper. Examples include.

Enzyme immobilization is carried out by using the aforementioned porous amphoteric ion exchange resin, preferably by introducing a hydrophobic group into this carrier, equilibrating it at a stable pH for the enzyme, and bringing it into contact with an aqueous enzyme solution to adsorb the enzyme. be exposed. The enzyme aqueous solution may be a solvent of monohydric alcohol or polyhydric alcohol having 1 to 6 carbon atoms, or a mixed solution of salts commonly used as enzyme treatment agents, such as sodium chloride and ammonium sulfate.

In the present invention, the immobilization temperature may be any temperature that does not cause deactivation of the lipolytic enzyme, and is preferably 0 to 60°C, preferably 25 to 40°C. The pH of the lipolytic enzyme aqueous solution is preferably within a range that does not cause denaturation of the lipolytic enzyme, preferably from 3 to 9. In particular, when using a lipase whose optimum pH is acidic, the pH is preferably 4 to 6 in order to obtain maximum activity. Further, the type of buffer used in the enzyme aqueous solution is not particularly limited, but general acetate buffer, phosphate buffer, Tris-HCl buffer, etc. can be used.

When immobilizing an enzyme according to the present invention, the concentration of the enzyme in the aqueous solution is not particularly specified, but from the viewpoint of immobilization efficiency, it is desirable that the concentration is lower than the solubility of the lipolytic enzyme and is sufficient. Furthermore, if necessary, the insoluble portion may be removed by centrifugation and the supernatant may be used. In addition, the usage ratio (weight ratio) of enzyme and immobilization carrier is 0 for enzyme and 0 for 1 part of immobilization carrier.
1 to 1 part is preferable, but is not particularly limited to this.

More preferably, in carrying out the present invention, the carrier before immobilization is crosslinked using a polyfunctional reagent, thereby further improving the durability of the immobilized enzyme in repeated use.

13 As the polyfunctional crosslinking reagent, polyaldehydes such as glyoxal, glutaraldehyde, malonaldehyde, and succinylaldehyde are preferred, and hexamethylene dithioisocyanate, N, N'-ethylene bismaleimide, etc. can also be used. Carbodiimides can also be used.

Further, after immobilization, it can be wrapped in PVA or the like to further improve stability.

In addition, before or at the same time as immobilization, the amphoteric ion exchange resin can be infused with one or more oil-soluble compounds selected from fatty acids, fatty acid derivatives, phospholipids, alcohols, ethers, carbonyl compounds, and alkyl halides. By adsorption treatment with a compound, a highly active and highly durable immobilized enzyme can be obtained. At this time, in order to prevent contamination with impurities, it is preferable to perform pretreatment, that is, dissolve these oil-soluble compounds in a volatile solvent, bring this solution into contact with an amphoteric ion exchange resin, filter it, and then dry it. The ratio of the oil-soluble compound to the immobilization carrier is suitably 0.001 to 1 part by weight of the oil-soluble compound-14 per 1 part by weight of the immobilization carrier, but is not limited thereto. An excessive amount of the oil-soluble compound is not adsorbed on the immobilization carrier, but is released into the solution and adsorbs the enzyme, resulting in a decrease in the yield of immobilization on the immobilization carrier, and is therefore not effective. Appropriate adsorption temperature is 0~
A temperature of 60°C, preferably 5 to 30°C is suitable. A suitable adsorption time is 5 minutes to 2 hours. The above temperatures and times are not limited to these.

Examples of fatty acids used in the amphoteric ion exchange resin treatment in the present invention include straight saturated saturated fatty acids, unsaturated fatty acids, and branched fatty acids having 4 to 24 carbon atoms. Preferred fatty acids include, for example, oleic acid, ricinoleic acid, and linoleic acid.

Suitable fatty acid derivatives used in the amphoteric ion exchange resin treatment in the present invention include monoglycerides, diglycerides,
and derivatives thereof, triglycerides, polyhydric alcohol fatty acid esters such as propylene glycol and polyglycerin, sugar esters such as 15 sucrose fatty acid ester, and sugar alcohol esters such as sorbitan fatty acid ester.

Examples of alcohols used in the amphoteric ion exchange resin treatment in the present invention include linear or branched aliphatic monohydric alcohols having 8 to 24 carbon atoms and polyhydric alcohols having 2 to 6 carbon atoms. In addition, phenolic compounds, sterols, terpene alcohols having 10 to 20 carbon atoms, and fat-soluble vitamins are also effective.

Examples of the ethers used in the amphoteric ion exchange resin treatment in the present invention include ethers having 10 to 18 carbon atoms, glyceryl ethers having 12 to 18 carbon atoms, and ethers having 10 to 18 carbon atoms.
-18 glyceride-like compounds such as glycidyl ether, polyoxy compounds, and silicon compounds of the alcohols mentioned above.

Examples of the carbonyl compound used in the amphoteric ion exchange resin treatment in the present invention include aliphatic aldehydes having 10 to 18 carbon atoms, aliphatic ketones, and the like.

(6) Examples of the alkyl halide used in the amphoteric ion exchange resin treatment in the present invention include alkyl halides having 8 to 18 carbon atoms.

It is preferable for the above-mentioned oil-soluble compounds to be liquid at room temperature in terms of process operation, but the invention is not limited thereto. Further, although these may be used alone, even more effects may be exhibited by appropriate combinations.

Examples of reactions of lipids using the immobilized enzyme obtained in the present invention include transesterification reactions using immobilized lipase. Examples of such transesterification reactions include acidolysis reactions using esters and fatty acids, and alcoholysis reactions using esters and alcohols. Examples include reactions, interesterification reactions between esters, and the like.

Examples of substrates for transesterification using the immobilized enzyme obtained in the present invention include vegetable oils such as soybean oil, olive oil, and palm oil, and animal fats and oils such as beef tallow, lard, and fish oil. Although these oils and fats may be used alone, it is preferable to use two or more types of oils or to carry out transesterification between oils and fats and higher fatty acids or between oils and fats and lower alcohol esters of higher fatty acids. When transesterifying a specific fat and oil with another fat, fatty acid, or a derivative thereof, the ratio of the two is 0.05 to 20 parts by weight, preferably 0.1 to 10 parts by weight of the other substance to 1 part by weight of the specific fat or oil. If it is not in parts by weight, it is difficult to obtain the effect of modifying fats and oils. Particularly preferred is transesterification of an oil or fat having a large number of oleic acid residues at the 2-position, such as palm oil, with stearic acid. In this reaction, the melting point of stearic acid is high and the viscosity of fats and oils is high, so in order to carry out the continuous transesterification reaction in a column reaction without a solvent, it is necessary to maintain the temperature of the reaction system at 60 to 90°C. The immobilized enzyme of the present invention is suitable for this purpose, and the resulting fats and oils are useful for chocolate.

Other examples of transesterification using the immobilized phospholipase obtained in the present invention include transesterification of natural phospholipids and various aliphatic alcohols, polyhydric alcohols, terpene alcohols, sugars, sugar alcohols, sterols, etc. Others, guanine, adenine,
Examples include transphosphatidylation with bases such as thymine and uracil.

Furthermore, examples of ester synthesis reactions using the immobilized enzyme obtained in the present invention include ordinary monohydric alcohols such as methanol, ethanol, propanol, and oleyl alcohol;
or polyhydric alcohols such as propylene glycol, glycerin, sorbitol and polyglycerin, terpene alcohols such as geranionopecitronellol and menthol, or sterols such as cholesterol;
Examples include esterification reactions with fatty acids having 2 to 24 carbon atoms.

Ester synthesis or reaction is carried out at 20°C to 90°C, more preferably at 3°C.
It is carried out at 0 to 80°C without a solvent or in an inert solvent such as a hydrocarbon or ether. Further, the amounts of alcohol and fatty acid are adjusted as appropriate depending on their valences and the intended product. For example, when the purpose is to synthesize diglycerides, about 2 moles of fatty acids are reacted with 1 mole of glycerin, and when the aim is to synthesize monoglycerides, about 1 mole of fatty acids is reacted with 1 mole of glycerin.

In these reactions such as transesterification, esterification, and transphosphatidylation, the amount of water in the reaction system, including the amount of water in the immobilized enzyme, should be 5% by weight or less, preferably 0.1%. It is preferable to keep it at 1% by weight.

The immobilized lipolytic enzyme obtained in the present invention can also be suitably used for hydrolysis reactions of fats and oils or various lipids by utilizing the inherent properties of the lipolytic enzyme.

〔Effect of the invention〕

When the immobilized lipolytic enzyme obtained by immobilization according to the method of the present invention is used, for example, in the transesterification of fats and oils or the esterification reaction of glycerides using the immobilized lipase, the durability is significantly improved, making it economical. This will further enhance the effectiveness of the project.

Furthermore, since the immobilized enzyme of the present invention has excellent heat resistance, the reaction can be carried out at a temperature of 50 to 80°C, so there is no need for a reaction solvent, and the reaction rate is increased. Great economic effects can be obtained in achieving this goal.

〔Example〕

The present invention will be explained in detail below with reference to Examples and Comparative Examples for transesterification and esterification, respectively.

Example 1 100 ml of ethanol, 8 g of sodium hydroxide, and 6 g of water were added to each Log of the commercially available anion exchange resin shown in Table 1. After stirring for 30 minutes, 10 g of β-monochloropropionic acid was added and the mixture was heated to room temperature. The mixture was allowed to react for 5 hours. After that, it was collected by filtration, washed with water, and then washed with 0.5M acetate buffer (pH 5).
) and finally equilibrate with 50mM acetate buffer (pl
After equilibration with 15) and drying under reduced pressure, each was used as a carrier. When 2 g of ricinoleic acid was adsorbed onto 5 g of this carrier in an acetate buffer (C), when it was used as a carrier as it was (B), and when it was treated with β-monochloropropionic acid (hereinafter sometimes abbreviated as CE). A study was conducted on the case (A) in which the original resin itself without any additives was used. 5 g of each carrier was mixed with 50-
was added and stirred at 30°C for 2 hours. The lipase aqueous solution is Riberze [Rhizapus japonicus (Rhizapus j
aponIcus), product name “Lipase Saiken 100” manufactured by Osaka Bacteria Research Institute Co., Ltd., 1
8000 Unit/g] 5 g at pH 5. 50 of 0
It was dissolved in 45mM acetate buffer. The resin was filtered from the suspension, washed with a buffer solution, and then dried under reduced pressure at room temperature to a moisture content of 5% to obtain immobilized lipase.

5 g of the immobilized lipase thus obtained was added to glycerin 2
3g (moisture content 0.8%, manufactured by Kao Corporation) and oleic acid (trade name "Lunac 0-L1, manufactured by Kao Corporation) 70
．． 5 g and an esterification reaction was carried out at 65° C. with stirring. A portion of the reaction liquid was taken out as a sample over time, and the acid value of the sample was measured according to the standard oil and fat analysis test method. The esterification rate was determined from the acid value of the sample using the following formula.

22 Here, AV. , A.V. are each AVt: acid value of sample after t hours A.V. = Acid value of mixed sample before reaction represents.

Furthermore, the enzyme adsorption rate was expressed as a percentage by subtracting the activity of the filtrate after immobilization from the activity of the enzyme stock solution. Table 1 shows the test results.
Shown below.

23 Table 1 Ester synthesis reaction using immobilized lipase 24 - Example 2 All conditions were the same as in Example 1 except that 10 g of α-chloropropionic acid was used in place of β-monochloropropionic acid. A carrier was prepared, and then lipase was immobilized under the same conditions to prepare immobilized lipase.

Using 5 g of the obtained immobilized lipase, the esterification reaction of Example 1 was carried out in the same manner as in Example 1, and the results are shown in Table 2.

25- Table 2 Ester synthesis reaction using immobilized lipase l) 2) Duolite is a product of Decoolite International Co., Ltd. Sumichelate is a product of Sumitomo Chemical ■Product DI
AION is manufactured by Mitsubishi Kagaku Kogyo ■Product treatment A: Use commercially available anion exchange resin as is (
Same as the comparative example listed in Table 1) Treatment B: Commercially available resin was treated with α-chloropropylene acid,
Amphoteric ion exchange resin into which an α-methylcarboxymethyl group was introduced as a cation exchange group was used as it was (Example of the present invention)-26 Example 3 Duolite A-568 used in Example 1
10 each of untreated (A), treated (B), and (C)
g was prepared, and the enzyme was immobilized in the same manner as in Example 1.

16 g of glycerin per 10 g of this immobilized enzyme. 2
g, 100 g of oleic acid was added, 40°C, degree of vacuum 3
The reaction was carried out for 6 hours at mmftg. Furthermore, the reaction was repeated five times by recovering the immobilized enzyme, and the enzyme adsorption rate of each reaction was measured. The results are shown in Table 3. From Table 3 (
It can be seen that the stability is significantly increased compared to the case where the resin in A) is immobilized as is.

Table 3 Stability of immobilized lipase zero (A), (B). (C); Same as treatment methods A-C in Table 1.

Example 4 Using 1 g of each of the immobilized lipases obtained in Example 3, 10 g of palm oil medium melting point (iodine value 32.5, diglyceride content 4.6%) and commercially available stearic acid (trade name)
10 g of Lunac S-90'' (stearic acid purity 93%, manufactured by Kao Corporation) was added, and the reaction was carried out at 60° C. for 5 hours.

After the reaction, the stearic acid content contained in the triglyceride was analyzed by gas chromatography, and the reaction rate was calculated with the equilibrium value expressed by the following formula as 100%.

Reaction rate (after t hours) = 100X (S.-S.)/(S,,-S.) In the above formula, St, So, S... are each SL one hour t
The stearic acid content in the oil and fat So - Stearic acid content in the raw material oil and fat before reaction S ~ -1,3 means the stearic acid content at random equilibrium.

The results are summarized in Table 4. In the case of (C), the reaction reached equilibrium within 5 hours, and the production of by-products was also smaller than in (^).

Example 5 Duolite A-568 used in Example 1
10g of was prepared, washed with 10% sodium hydroxide solution, filtered and dried. Add this resin to 40% acetone
- Then, 2 g of itaconic anhydride was added and the mixture was reacted with stirring for 1 hour. After the reaction, it was collected by filtration, equilibrated according to Example 1, and then dried under reduced pressure.

This resin was subjected to the same treatments (A), (B), and (C) as in Example 1, except that log of commercially available lipase [lipase originating from Rhizopus deremer (Talipase, manufactured by Tanabe Seiyaku Co., Ltd.)] was used to obtain immobilized lipase. , A transesterification reaction was carried out in the same manner as in Example 3. The results are also shown in Table 4.

Example 6 Strong cation exchange resin Duolite C-10 10g
After equilibrating with acetate buffer (pH 5.50mM),
The mixture was transferred to 50 liters of distilled water, and 2 g of isopropyltri(N-aminoethyl)titanate was added for binding. This resin was collected by filtration and added to an acetate buffer (pH 5.0.5).
After washing with 0mM), it was dried. 10g per 100ml of the same buffer
[Lipase preparation originating from Rhizopus javonicus, trade name "Riverase Saiken 100" manufactured by Osaka Bacteria Research Institute Co., Ltd.] is dissolved, and this treated resin is added thereto,
Stirring and immobilization were performed. After 120 minutes, this resin was collected by filtration and dried to obtain an immobilized enzyme.

The thus obtained immobilized enzyme (C) and Duolite
[:-10 was immobilized as it was (A) and was compared according to Example 4. The results are also listed in Table 4. Incidentally, the enzyme adsorption amount was almost the same at 78% and 80%.

30 Table 4 Transesterification reaction of immobilized lipase (reaction rate at 2nd hour) 0 (A) to (C) correspond to treatment methods A to C in Table 1, respectively.

Example 7 In Example 1, monochloroacetic acid was used instead of β-monochloropropionic acid, and a carboxymethylated resin (commercially available resin) was used under the same conditions (however, Duolite A-568 was used as the commercially available resin). (sometimes abbreviated) was obtained.

3 1- The CE resin and the CM resin were each treated with ricinoleic acid according to the method of Example 1, and then lipase was immobilized. 100 g of the obtained immobilized enzyme was packed into a column, and at 70°C, using the stearic acid and palm oil medium melting point used in Example 4 as substrates, stearic acid/palm oil medium melting point (weight ratio) = In 1.5, the reaction rate specified in Example 4 was passed at a flow rate to maintain the reaction rate of 90% or more, a continuous reaction was carried out, and the durability of the immobilized enzyme was investigated, and the results shown in Table 5 were obtained.

Table 5 As shown in Table 5, in terms of continuous productivity (durability), the lipase immobilized on the CE resin of the present invention has significantly improved durability compared to the lipase immobilized on the CM resin. It is clear that he did.

Claims (1)

[Scope of Claims] 1 The cation exchange group is a carboxyalkyl group or an alkenyl group (the alkyl group or alkenyl group is a straight chain or branched chain having 2 to 6 carbon atoms, and the alkyl group or alkenyl group has a substituent) ), and/or a phosphoric acid group, the anion exchange group being a primary amino group, a secondary amino group,
1 selected from the group of tertiary amino group and quaternary ammonium group
A carrier for immobilizing an enzyme, characterized in that it is made of one or more amphoteric ion exchange resins. 2. The carrier for enzyme immobilization according to claim 1, wherein the cation exchange group of the amphoteric ion exchange resin is a carboxyethyl group and/or an α-methylcarboxymethyl group. 3. A method for producing an immobilized enzyme, which comprises bringing an aqueous solution of a lipolytic enzyme into contact with the immobilization carrier according to claim 1. 4. The method for producing an immobilized enzyme according to claim 3, wherein the cation exchange group of the immobilization carrier is a carboxyethyl group and/or an α-methylcarboxymethyl group. 5. When immobilizing lipolytic enzymes, immobilization is carried out in the presence of one or more compounds selected from fatty acids, fatty acid derivatives, phospholipids, alcohols, ethers, carbonyl compounds, and alkyl halides. 5. The method for producing an immobilized enzyme according to claim 3 or 4, wherein the method comprises: 6. The method for producing an immobilized enzyme according to claim 3, 4 or 5, wherein the lipolytic enzyme is selected from lipase, phospholipase, and cholesterol esterase.