JPH0710233B2 - Immobilized enzyme and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immobilized enzyme and a method for producing the same. More specifically, it relates to an immobilized lipase suitable for transesterification by lipase and an ester synthesis reaction, and a method for producing the same.

The transesterification reaction of oils and fats is an important technology along with hydrogenation for reforming edible oils and fats such as margarine and shortening.

The synthetic reaction of esters is an important technique as a method for synthesizing alcohol fatty acid ester from alcohol and fatty acid, synthesizing polyhydric alcohol fatty acid ester such as monoglyceride, polyglycerin fatty acid ester and sugar ester, and a method for producing fragrance such as geranyl butyrate.

[Conventional technology and its problems]

The transesterification and ester synthesis reactions of oils and fats and esters utilize the fact that lipase has characteristics such as reaction under mild conditions, regioselectivity and alkyl selectivity. However, these reactions are different from the lipase's original hydrolysis reaction and can proceed only in a system with limited water content.

On the other hand, a certain amount of water is required as an enzyme in order to increase the transesterification and ester synthesis activities of lipase. In the low-moisture-type transesterification reaction disclosed in JP-A-55-71797, a sufficient reaction rate cannot be obtained, and when water is added more than necessary to increase the reaction rate, ester decomposition reaction occurs. However, there is a problem in that Further, a proposal of a method in which the transesterification reaction disclosed in JP-A-60-19495 and JP-A-60-203196 is carried out in two steps, that is, a multi-moisture decomposition step and a synthesis step for removing water However, the latter synthetic reaction rate cannot be said to be sufficient as compared with the usual transesterification rate, and intricate process operation cannot be avoided.

Regarding the synthesis reaction of glycerides and esters by lipase, Iwai, Tsujisaka et al. (M.Iwai, Y. Tsujisak
a, J.Fukumoto, J.Gen.Appl.Microbiol., 10 , 13 (1964))
And known from Japanese Patent No. 831834, No. 1056738, etc., the equilibrium relationship between the hydrolysis action and the synthesis action is governed by the water content in the reaction system, the synthesis rate biased to the decomposition in the presence of water naturally naturally. Get smaller. As a means for solving this point, there is a proposal of a method disclosed in JP-A-57-8787, in which the reaction is carried out in a system as dry as possible and the water produced by the reaction is discharged to the outside of the system. However, the synthesis activity of the enzyme is extremely low under low water content, and the reduction of the synthesis rate and the long reaction time cannot be avoided.

For the purpose of solving the above problems and efficiently using lipase, attempts have been made to immobilize lipase.
The advantages expected by immobilization of lipase are as follows. Conventionally, it was difficult to mix and disperse uniformly in oil when using lipase in the form of an aqueous solution, but by immobilizing lipase on the surface of an insoluble carrier, it becomes possible to disperse easily in oil, and to the carrier. Since an appropriate amount of water can be retained, transesterification and synthesis reaction under low water becomes easy. Further, it is easy to recover and reuse lipase, which is still expensive as a catalyst, and it is easy to make the reaction device continuous even in the industrial implementation of transesterification or ester synthesis reaction.

However, even the immobilized enzyme having the above advantages has not been able to maintain both the amount of water necessary for increasing the lipase synthesis activity and the suppression of hydrolysis, which is a reverse reaction. For example, Journal of American Oil
Chemist's Soc., Volume 60, 291-294 (1983) has also been pointed out that the hydrolysis reaction proceeds when a small amount of water is given.

Further, when a polyhydric alcohol such as glycerin is added instead of water, the hydrolysis reaction is suppressed to some extent, but the transesterification and ester synthesis reactions are delayed.
Further, even in the esterification reaction, a method disclosed in JP-A-59-213390 discloses a method in which a porous carrier and a superabsorbent resin are entrapped and immobilized with chitosan for the purpose of retaining enzymatic moisture, and then a crushed carrier is used. It takes more than 20 hours and the synthesis rate is not sufficient.

As described above, the transesterification of oils and fats by lipase and the ester synthesis reaction have characteristic and advantageous points as compared with the above-mentioned chemical method, but on the other hand, there are many problems to be solved, It is necessary to solve these problems in order to implement.

In the transesterification reaction and the ester synthesis reaction as described above, it is desired to reliably control the water content of the immobilized enzyme or to develop an immobilized enzyme having a higher transesterification and ester synthesis activity.

Regarding the transesterification reaction, water control is also carried out in the two-step reaction described above (JP-A-60-203196), but it is complicated in terms of equipment and the first-stage decomposition is carried out. In the process, 1,2-diglyceride is selectively obtained in high yield, and the target triglyceride is selectively synthesized in the second step without transferring from 1,2-diglyceride to 1,3-diglyceride. It's difficult. In particular, it becomes more difficult to suppress the adverse effect of this transition as the temperature rises.

Regarding the ester synthesis reaction, most of the conventional methods use lipase as an aqueous solution,
The equilibrium relationship between decomposition and synthesis largely depends on decomposition,
The yield of the target ester is low. However, if the reaction is carried out with an immobilized enzyme, ester synthesis is performed even under lower water conditions, and the enzyme can be easily recovered, but even in this case, in order to obtain a reaction rate equivalent to that of a normal chemical method. It is desired to develop a more active immobilized enzyme.

In order to solve the above problems, various methods for increasing the ester synthesis activity and transesterification activity of immobilized lipase have been studied. However, to date
Nothing has been found other than the method disclosed in Japanese Patent No. 25188, in which fats and oils are added to the lipase to carry out a hydrolysis reaction and immobilization.

On the other hand, even in a system using immobilized lipase for hydrolysis, addition of a slight amount of Ca salt is also known including a stabilizing effect (J. Am. Oil Chemist's Soc., 61 , 776-781 ( 198
Four)). In addition, it is known that the addition of Ca salt, bile acid, various surfactants, etc. as a hydrolysis reaction system in the state of the aqueous enzyme solution has an activating effect. The fact is that little is known.

[Means for solving problems]

Therefore, as a result of intensive studies on the factors that increase the transesterification activity and ester synthesis activity of lipase, the present inventors have found that transesterification and ester synthesis activity are significantly increased when phospholipids are in contact with and bound to lipase. I found that I could see it. Based on this fact, the present inventors have completed the present invention by applying the lipase to which phospholipid is bound to various insoluble carriers.

There have been many reports on the relationship between lipases and phospholipids since Iwai et al. Reported at the Chemical Society of Japan in 1969. , Or only the method for stabilizing the fermentation production, and no report on activation in transesterification and ester synthesis reaction.

There is also a method reported by Noriaki Kadota et al. At the Japan Society for Agricultural Chemistry in April 1986, in which an enzyme is incorporated into a micelle by a phospholipid to cause a reaction. Further, as an application of this method, lipase is incorporated into a W / O (W means an aqueous phase, 0 means an oil phase) emulsion, and then this emulsion is subjected to W / O /
There is a report that a polyhydric alcohol fatty acid ester was produced by immobilizing it as a W emulsion. This method first creates a W / O emulsion, incorporates the enzyme into the emulsion, and then makes the emulsion W / O / W so that it does not break in the oil phase. In addition, since the emulsion-immobilized enzyme is physically unstable, a very complicated process of surrounding it with a photocrosslinkable resin is required.

On the other hand, according to the present invention, lipase may be bound to an insoluble carrier in a state where phospholipid is brought into contact with and bound in an aqueous phase, and it is not necessary to disperse it in an oil phase such as a W / O emulsion. Specifically, it suffices to add an insoluble carrier to a solution containing phospholipid and lipase, immobilize the lipase and phospholipid on the carrier, and then filter.

That is, the present invention relates to an immobilized enzyme suitable for transesterification and ester synthesis, which is obtained by immobilizing a lipase and a phospholipid in contact with and binding to an insoluble carrier.

Further, the present invention is an immobilized enzyme characterized by increasing the transesterification activity and ester synthesis activity of lipase by immobilizing lipase before or during immobilization of lipase on an insoluble carrier and immobilizing it. The present invention relates to a manufacturing method of.

The present invention is specifically as follows. That is, in immobilizing a lipase by adding an insoluble carrier to a lipase solution or a fermentation culture solution containing lipase, a phospholipid or a phospholipid is dispersed or dissolved in the lipase solution or the fermentation culture solution containing lipase before immobilization. The lipase and the phospholipid are brought into contact with and bound to each other by adding the above solution. Alternatively, a commercially available lipase may be dissolved in water or a buffer solution in which phospholipids are suspended or dissolved. Next, an insoluble carrier is added to the solution, and 1 minute to 20 hours, preferably
The solution is contacted for 30 minutes to 2 hours, then the insoluble carrier is filtered from the solution and washed with water. The immobilized lipase thus obtained is dried to obtain the immobilized lipase of the present invention. More preferably, a lipase solution or a fermentation broth containing lipase is dissolved in a phospholipid and an oil or fat or fatty acid alcohol solution (preferably an aliphatic 1 having 8 or less carbon atoms).
After adding (hydric alcohol) and stirring, the insoluble carrier is added thereto and contacted for 5 minutes to 20 hours, preferably 30 minutes to 2 hours, and then the insoluble carrier is filtered from the solution and washed with water. The immobilized lipase thus obtained is dried to obtain the immobilized lipase of the present invention.

As the lipase for immobilized lipase used in the present invention,
Rhizopus, Aspergillus, and Chromobacter with excellent regioselectivity
obacterium genus, Mucor genus, Pseudomonas genus, Penicillium
Genus, Geotrichum with fatty acid specificity
Examples include genus and lipases of microbial origin such as Candida that do not show specificity and animal lipases such as pancreatic lipase. Among these, Candida cylindracea, Rhizopus japonicus, and Mucor mehi (Mu) are particularly useful as lipases of microbial origin with high synthetic activity.
cor miehei), Chromobacterium viscous (Chromo)
More preferred is a lipase of bacterium viscoum) origin.

As the carrier used for immobilization, water, alcohol, various organic solvents, any insoluble carrier of oils and fats may be used, Celite, diatomaceous earth, kaolinite, silica gel, perlite, molecular sieves, porous glass, activated carbon,
Inorganic carriers such as calcium carbonate and organic polymers such as cellulose powder, polyvinyl alcohol, chitosan, ion exchange resins and adsorption resins do not affect the lipase activity and are physically and chemically stable from the operational point of view. Any material can be used. The carrier may have various shapes such as powder, fruit, fibrous, sponge, etc., and any of them may be used. 40 especially from the aspect of process operation
It is preferable to use a porous carrier having a particle size of 0 to 1000 μm and a pore size of 100 to 1500Å. In particular, examples of this type of immobilization carrier include macroporous phenol formaldehyde-based adsorption resin and ion exchange resin.

As the phospholipid used in the present invention, naturally occurring soybean lecithin, crude and / or purified mixed lecithin such as egg yolk lecithin may be used, and phosphatidylcholine obtained by fractionating these, phosphatidylserine, phosphatidylethanolamine. , Phosphatidylinositol,
Phosphatidyl glycerol, cardiolipin, phosphatidic acid and the like may be used alone or in combination. Further, synthetic phospholipids obtained by various synthetic methods and their derivatives can also be used. It is easy to handle a phospholipid having an alkyl group of a short chain or unsaturated as a phospholipid having a low melting point in operation, but the phospholipid is not limited thereto.

As a method for contacting the phospholipid with the lipase, the phospholipid may be added as it is to the lipase aqueous solution, but the phospholipid is once added to a solvent that does not affect the activity of the lipase in order to improve the dispersibility of the phospholipid in water. It is more effective to add after dispersing or dissolving. Examples of the solvent include aliphatic monohydric alcohols, chloroform, n-hexane, and the like, and aliphatic monohydric alcohols having 8 or less carbon atoms are preferable from the viewpoint of compatibility with water.

Further, when the lipase and the phospholipid are brought into contact with each other, coexistence of fatty acids, oils and fats, etc. as a substrate makes it possible to obtain an immobilized enzyme having higher activity. As the type of fatty acid, a straight chain having 2 to 24 carbon atoms and usually existing in nature, for example, saturated fatty acid such as palmitic acid and stearic acid, or unsaturated fatty acid such as oleic acid and linoleic acid may be used. it can. Examples of fats and oils include general vegetable fats and oils, animal fats and oils or processed fats and oils, or mixed fats and oils thereof. The same effect can be obtained by using diglyceride or monoglyceride obtained by deriving these oils and / or fatty acids and glycerin.

In the present invention, the temperature for immobilization may be a temperature that does not impair the activity of lipase, and is preferably 0 to 60 ° C, preferably 20 to 40 ° C. The pH of the lipase solution may be in the range that does not cause denaturation of the lipase, and is preferably in the range of pH 3-9. In particular, in the case of using a lipase whose optimum pH is acidic, the pH is preferably set to 4 to 6 in order to obtain the maximum activity. Further, the kind of the buffer solution used for the lipase solution is not particularly limited, but a common acetate buffer solution, phosphate buffer solution, Tris-HCl buffer solution or the like can be used.

In the immobilization method of the present invention, the concentration of lipase in the aqueous solution is not particularly specified, but from the viewpoint of immobilization efficiency, it is desirable that the concentration is not more than the solubility of the lipase and sufficient. If desired, the insoluble portion may be removed by centrifugation and the supernatant may be used. Further, it is preferable that the lipase and the phospholipid are contacted and bonded at a ratio of 0.01 to 1 part by weight of the phospholipid to 1 part by weight of the lipase. Further, the use ratio (weight ratio) of lipase and immobilization carrier is preferably 0.01 to 1 part by weight of lipase per 1 part by weight of immobilization carrier, but is not particularly limited thereto.

In the present invention, the carrier before immobilization and / or after the immobilization is completed, the carrier to which the lipase is bound obtained by separating the enzyme solution is crosslinked with a polyfunctional crosslinking agent, thereby repeating the immobilized enzyme. The durability in use can be improved. As the polyfunctional crosslinking agent, polyaldehydes such as glyoxal, glutaraldehyde, malonaldehyde, and succinylaldehyde are preferable, and hexamethylenedithioisocyanate and N, N′-ethylenebismaleimide can also be used. Further, the crosslinking with the polyfunctional crosslinking agent may be carried out at the same time during the above-mentioned immobilization operation.

〔The invention's effect〕

The method of the present invention is for sufficiently exhibiting the synthetic activity of lipase, and found that the transesterification activity and ester synthetic activity of lipase markedly increase due to the binding with phospholipid, and immobilization thereof was performed. It was obtained as a result of applying to.

Examples of transesterification reaction using immobilized lipase in the present invention include acidolysis reaction between ester and fatty acid, alcoholysis reaction between ester and alcohol,
There is an interesterification reaction by the ester comrades. Further, as an example of the ester synthesis reaction, ordinary methanol,
The esterification reaction of monohydric alcohols such as ethanol and propanol, polyhydric alcohols such as propylene glycol, glycerin, sorbitol and polyglycerin, or terpene alcohols such as geraniol, citronellol and menthol with fatty acids having 2 to 24 carbon atoms can give.

As an effect of the present invention, an immobilized lipase obtained by immobilizing a regioselective lipase by the method of the present invention has a remarkable activity, and an oil and fat containing a large amount of oleic acid at the 2-position of glyceride and a saturated fatty acid Reduction of diglyceride by-product and asymmetric conversion and consequent tri-saturated glyceride by-product for the purpose of producing symmetrical oils and fats having a structure similar to that of natural cocoa butter by the transesterification reaction of Is possible.

Further, in the synthesis of esters, in the conventional enzymatic method, the reaction reaches equilibrium due to the water produced as the reaction progresses, so that the esterification does not proceed. Therefore, it is attempted to further promote the esterification by dehydration operation such as reducing the pressure of the reaction system, but such operation cannot avoid the decrease of the ester synthesis activity of the enzyme. In such a case, when the immobilized lipase according to the method of the present invention is used, a sufficient ester synthesis activity is maintained even under low water conditions, so that a high esterification rate is achieved in a short time and the reaction is performed for a long time. This has the advantage that no deterioration in quality due to side reactions such as coloring due to oxidization and generation of off-flavor is observed.

As described above, it was discovered that contact and binding of phospholipids and lipase increase their transesterification and ester synthesis activities, making it possible to easily and inexpensively produce a highly active immobilized lipase in industrial practice. Became.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Commercially available lipase derived from Rhizopus japonicaus [trade name <
Lipase Saiken 100> Made by Osaka Bacterial Research Institute Co., Ltd.
Decomposition activity 35,000 Unit / g] 10 g was dissolved in 95 ml of 10 mM acetate buffer of pH 5.0. Separately, a liquid prepared by suspending 1 g of commercially available soybean lecithin (reagent, Wako Pure Chemical Industries, Ltd.) in 5 ml of ion-exchanged water was added to the above lipase solution and stirred for 1 hour to suspend the lipase and lecithin. A suspension was obtained. To this suspension of lipase and lecithin, a phenol-formaldehyde weak anion exchange resin [trade name <Duolit (Duolit
e) ES-568> made by Diamond Shamrock] 10 g (water content
10%) was added and the mixture was stirred at 30 ° C. for 2 hours. At this time, the pH of the mixed liquid was adjusted to 5.0. Next, the weak anion exchange resin was filtered off from the suspension, washed with water, and then water 5
The mixture was dried under reduced pressure at room temperature to obtain 10.5 g of immobilized lipase. At this time, the decomposition activity of the filtrate is 1,295 Unit / ml
As a result, 63% of the lipase initially used was immobilized on the immobilized lipase obtained from this result.

Using 1 g of the immobilized lipase obtained in this Example, 10 g of palm oil in the middle melting point (iodine value 32.5, glyceride content 4.6%)
Then, 10 g of commercially available stearic acid [Lunack S-90 (trade name), stearic acid purity 93%, manufactured by Kao Corporation] was added, and the reaction was carried out at 65 ° C. for 5 hours. After the reaction, Florisil (Floridin: 60-100 mesh, eluent hexane: ether = 2: 3) was used to separate the glyceride fraction by column chromatography to obtain methyl ester according to the standard oil and fat analysis test method, and stearic acid contained in the glyceride. The content was analyzed by gas chromatography. Equilibrium value shown by the following formula is 100%
Was calculated.

In the above formula, St: stearic acid content in fats and oils at time t So: stearic acid content in raw fats and oils before reaction S∞: 1,3 means stearic acid content at random equilibrium.

The results are shown in Table 1.

Comparative Example 1 10 g of the commercially available lipase used in Example 1 was dissolved in 100 ml of 10 mM acetate buffer of pH 5.0 and stirred for 1 hour to obtain a lipase solution. Add 10g of Duolite ES-568 to this lipase solution.
(Water content 10%) was added, and the mixture was stirred at 30 ° C. for 2 hours. At this time, the pH of the mixed solution was adjusted to 5. Next, the resin (Duolite ES-568) was filtered from the solution, washed with water, and then dried under reduced pressure at room temperature so that the water content became 5%.
g of immobilized lipase was obtained. Using this immobilized lipase, a transesterification reaction was performed in the same manner as in Example 1.

The results are shown in Table 1.

From the results shown in Table 1, the effect of increasing the transesterification activity of lipase by phospholipid treatment is recognized.

Example 2 10 g of the commercially available lipase used in Example 1 was dissolved in 95 ml of 10 mM acetate buffer having a pH of 5.0 and stirred for 1 hour to prepare a lipase solution. Separately, 1 g of soybean lecithin was dissolved in 5 ml of butanol to prepare a solution, which was added to the above lipase solution and stirred for 1 hour to obtain a mixture A (containing lipase, lecithin, butanol). 10 g of the weak anion exchange resin (Duolite ES-568) used in Example 1 was added to this mixture A.
(Water content 10%) was mixed and stirred at 30 ° C. for 2 hours. The pH at this time was adjusted to 5. The resin is then filtered off from the mixture,
After washing with water, vacuum drying was performed at room temperature so that the water content was 5% to obtain 11.2 g of immobilized lipase.

Using the immobilized lipase obtained here, a transesterification reaction was carried out in the same manner as in Example 1 (that is, 10 g of the melting point of palm oil used in Example 1 and 10 g of stearic acid were added, and the mixture was heated at 65 ° C. for 5 hours). went.

The results are shown in Table 2.

Comparative Example 2 10 g of the commercially available lipase used in Example 1 was dissolved in 95 ml of 10 mM acetate buffer having a pH of 5.0. Butanol was added to 5 ml of this lipase solution, and the mixture was stirred for 1 hour and then mixed with mixture B (lipase,
Containing butanol, but not lecithin). This mixture B was mixed with 10 g of the weak anion exchange resin (Duolite ES-568) used in Example 1 (water content 10%) and the mixture was mixed at 30 ° C. for 2 hours.
Stir for hours. The pH at this time was adjusted to 5.0. Next, the weak anion exchange resin was filtered off from the mixture, washed with water, and then dried under reduced pressure at room temperature so that the water content became 5%.
g of immobilized lipase was obtained.

Using the immobilized lipase obtained here, a transesterification reaction was carried out in the same manner as in Example 1.

The results are shown in Table 2.

Example 3 In this example, a choice of phospholipid solvent was made.

That is, in Example 2, the same operation as in the method of Example 2 was performed except that methanol, ethanol, propanol, pentanol, hexanol, heptanol, and octanol were used instead of butanol.

The results are shown in Table 2.

From the results shown in Table 2, it became clear that alcohol having 8 or less carbon atoms is effective as a dispersion solvent for phospholipids.

Example 4 In this example, a search for phospholipid types was performed.

That is, in Example 2, instead of commercially available soybean lecithin, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, as phospholipids,
The same operation as in Example 2 was performed except that phosphatidylserine (reagent, both manufactured by Sigma) was used.

A transesterification reaction was carried out in the same manner as in Example 1 using the immobilized enzyme obtained here. The results are shown in Table 3, and any phospholipid showed a sufficient effect.

Example 5 10 g of the commercially available lipase used in Example 1 was dissolved in 95 ml of 10 mM acetate buffer having a pH of 5.0. Separately, 1 g of phosphatidylcholine and 1 g of olive oil (Pharmacy olive oil, manufactured by Wako Pure Chemical Industries, Ltd.) were added to 5 ml of butanol and stirred to prepare a uniform mixture, which was added to the lipase solution and stirred for 1 hour. A mixture C (containing lipase, phosphatidylcholine, olive oil, butanol, etc.) was obtained. The mixture C
Weak anion exchange resin (Duolite) used in Example 1
ES-568) 10 g (water content 10%) was mixed and stirred at 30 ° C. for 2 hours. The pH during mixing was adjusted to 5.0. Next, the weak anion exchange resin was separated from the mixture by filtration, washed with water, and then dried under reduced pressure at room temperature so that the water content was 5% to obtain immobilized lipase.

Using the immobilized lipase obtained here, a transesterification reaction was carried out in the same manner as in Example 1.

The results are shown in Table 4.

Comparative Example 3 10 g of the commercially available lipase used in Example 1 was dissolved in 95 ml of 10 mM acetate buffer having a pH of 5.0. Separately, 1 g of olive oil used in Example 5 was added to 5 ml of butanol and stirred to prepare a uniform mixture, which was added to the above lipase solution and stirred for 1 hour to obtain a mixture D containing lipase, olive oil, butanol and the like. Obtained. The mixture D was mixed with 10 g of weak anion exchange resin (Duolite ES-568) used in Example 1 (water content 10%) and stirred at 30 ° C. for 2 hours. The pH during mixing was adjusted to 5.0. Next, the weak anion exchange resin was filtered off from this mixture, washed with water, and then dried under reduced pressure at room temperature so that the water content was 5% to obtain immobilized lipase.

Using the immobilized lipase obtained here, a transesterification reaction was carried out in the same manner as in Example 1.

The results are shown in Table 4.

Example 6 In Example 5, instead of olive oil, oleic acid diglyceride (reagent, manufactured by Sigma), oleic acid monoglyceride (trade name Exel O-95, manufactured by Kao Corporation), oleic acid (trade name Lunac O-LL, The same operation as in Example 5 was performed except that Kao Corporation) was used.

Using the immobilized lipase obtained here, a transesterification reaction was carried out in the same manner as in Example 1.

The results are shown in Table 4.

Results of Example 5, Example 6 and Comparative Example 3, and Example 4
The results of phosphatidylcholine alone are also collectively shown in Table 4. From these results, it was confirmed that the effect was increased by the coexistence of phospholipids and fats and oils which are substrates for lipase.

Example 7 In Example 5, weak anion exchange resin Duolite ES
-568 instead of Duolite ES-562, phenol formaldehyde adsorption resin Duolite S-861, Dowex M
WA-10 (Dow Chemical Company), Celite 5 as an inorganic carrier
Example 5 except that 45 (manufactured by Marine Building) was used respectively.
The same operation was performed.

Using the immobilized enzyme obtained here, the same transesterification reaction as in Example 1 was performed.

The results are shown in Table 5, but similar effects were observed not only with ion exchange resins but also with inorganic carriers.

Example 8 In Example 2, as a commercial lipase, Candida
Lipase of Syrindrass origin [Product name <Lipase OF>
The same method as in Example 2 was performed except that 0.97 g of 360,000 Unit / g manufactured by Meito Sangyo Co., Ltd. was used.

A transesterification reaction was carried out in the same manner as in Example 1 using the immobilized enzyme obtained here.

The results are shown in Table 6.

Example 9 In Example 2, the same procedure as in Example 2 was carried out except that 58 g of lipase derived from Rhizopus delemer [trade name <Taripase> manufactured by Tanabe Seiyaku Co., Ltd., 6000 Unit / g] was used as a commercial lipase. It was

A transesterification reaction was carried out in the same manner as in Example 1 using the immobilized enzyme obtained here.

The results are shown in Table 6.

Example 10 In Example 2, exactly the same as in Example 2 except that 5.8 g of lipase of Aspergillus niger origin [trade name <lipase AP6> Amano Pharmaceutical Co., Ltd., 60,000 Unit / g] was used as the commercial lipase. Made by way.

A transesterification reaction was carried out in the same manner as in Example 1 using the immobilized enzyme obtained here.

The results are shown in Table 6.

Example 11 In the same manner as in Example 2, except that 35 g of a commercial lipase of Mucor origin [trade name <Lipase AP10> Amano Pharmaceutical Co., Ltd., 10,000 Unit / g] was used as the commercial lipase. went.

A transesterification reaction was carried out in the same manner as in Example 1 using the immobilized enzyme obtained here.

The results are shown in Table 6.

Although the results of Examples 8 to 11 are collectively shown in Table 6, the same effect was observed using any of the microorganism-derived enzymes.

Example 12 In this example, the immobilized lipases obtained in Example 2 and Examples 8-11 were used for esterification.

That is, 1 g of each of the immobilized enzymes obtained in Example 2 or Examples 8 to 11 was treated with 6.3 g of octyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd.) and oleic acid (trade name: Lunac O-LL, manufactured by Kao Corporation). ) 13.7 g, and the ester synthesis (esterification) reaction was carried out at 40 ° C. with stirring. A part of the reaction solution was taken out as a sample with time, and the acid value of the sample was measured according to the standard oil and fat analysis test method. From the oxidation of the sample, the esterification rate was calculated by the following formula.

Here, AVt: acid value of sample after t hours AVo: acid value of mixed sample before reaction.

The results are shown in Table 7.

Comparative Example 4 The ester synthesis reaction was carried out in the same manner as in Example 12 using 35,000 units / g of the commercially available lipases used in Example 2 and Examples 8 to 11, respectively.

The results are shown in Table 7.

From the results shown in Table 7, it is clear that the immobilized enzyme of the present invention is also suitable for the ester synthesis reaction.

Example 13 Next, the durability of the immobilized enzyme obtained as described above was examined.

That is, 5 g of the immobilized enzyme prepared in Example 2 and Comparative Example 3 were each packed in a column with a jacket and kept warm with hot water at 70 ° C. A mixture of palm oil in the melting point and stearic acid in equal amounts was dissolved and continuously passed at a rate of 18 g / hr to examine the decrease in activity.

The half-life with respect to the initial reaction activity is about 50 hours when untreated (Comparative Example 3), whereas it is about 400 hours when the phospholipid-treated immobilized enzyme (Example 2) is used, which improves durability. . During this period, 1440 kg of reaction solution was treated with respect to 1 kg of immobilized enzyme.

Example 14 5 g of a 2% glutaraldehyde aqueous solution was added to 5 g of the immobilized enzyme obtained in Example 4, and a crosslinking reaction was carried out at room temperature for 1 hour.
Then, after removing the excess glutaraldehyde by decantation, it was washed twice with 20 ml of ion-exchanged water. After filtration, the product was dried at room temperature under reduced pressure so that the water content was 5% to obtain an immobilized enzyme crosslinked product. The immobilized enzyme obtained here is
The residual activity rate was 81%. Using this immobilized enzyme, the same continuous liquid passing test as in Example 13 was performed. The half-life for the initial reaction activity was about 630 hours, and stabilization by crosslinking was observed. During this period, 2300 kg of reaction solution was treated per 1 kg of immobilized enzyme.

Claims (10)

[Claims] 1. An immobilized enzyme suitable for transesterification and ester synthesis, which is immobilized on an insoluble carrier in a state where lipase and phospholipid are in contact with and bound to each other. 2. The immobilized enzyme according to claim 1, wherein the insoluble carrier is a macroporous resin and the immobilization is adsorption immobilization. 3. An immobilized enzyme characterized by increasing the transesterification activity and the ester synthesis activity of lipase by immobilizing lipase before or during immobilization of lipase on an insoluble carrier. Production method. 4. When a phospholipid is added to lipase and the lipase and the phospholipid are brought into contact with each other, an aliphatic monohydric alcohol having 8 or less carbon atoms is used to promote the binding between the phospholipid and the lipase. 4. A method for producing an immobilized enzyme according to claim 3. 5. The method for producing an immobilized enzyme according to claim 3 or 4, wherein a buffer solution is added to the lipase to dissolve the lipase. 6. A buffer for dissolving lipase, which has a pH of 3 to
The method for producing an immobilized enzyme according to claim 5, wherein the inactivation of lipase is suppressed by controlling the activity to 9. 7. An oil, a fatty acid, or a derivative thereof (diglyceride or monoglyceride) which serves as a substrate for lipase when immobilizing lipase and phospholipid on an insoluble carrier.
The method for producing an immobilized enzyme according to claim 3 or 4, wherein is added to protect the active site of lipase and suppress inactivation. 8. A method of treating an insoluble carrier with a polyfunctional crosslinking agent before, during, or after contacting with a lipase and a phospholipid, and then removing the remaining crosslinking agent. 8. The method for producing an immobilized enzyme according to any one of claims 3 to 7. 9. The method for producing an immobilized enzyme according to claim 3, wherein the immobilization is adsorption and immobilization on a macroporous resin. 10. The method for producing an immobilized enzyme according to claim 9, wherein the immobilization temperature is 0 to 60 ° C.