JPH05137574A - Method for reactivating immobilized lipase - Google Patents

Abstract

PURPOSE:To reactivate an immobilized lipase having deteriorated activity by its use for reaction such as modification or synthesis of esters so as to enable maintenance of high activity for a long period by treating the immobilized lipase used once or more for reaction with a nonpolar solvent and a polar solvent. CONSTITUTION:A lipase derived from Rhizopus delemar is dissolved and dispersed in ion exchange water and stirred with an anion exchange resin to provide a slurry form, which is then dried to prepare an immobilized lipase. The resultant immobilized lipase is filled in a reaction vessel and subjected to ester synthetic reaction of isobutyric acid, etc., with n-octanol, etc., and subsequently used for reaction to produce n-octyl isobutyrate. The immobilized lipase, used for the reaction and deteriorated in activity is then treated with a nonpolar solvent such as n-hexane and a polar solvent such as acetone to reactivate the immobilized lipase having the deteriorated activity. Thereby, the immobilized lipase is kept in a state of high activity for a long period.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for reactivating immobilized lipase for use in modification and synthesis of esters.

[0002]

2. Description of the Related Art In recent years, ester modification and synthesis reactions have been studied by utilizing the chemical action of lipase. The modified and synthesized esters thus obtained are used for foods, cosmetics, toiletries and pharmaceuticals. , Agricultural, feed, fisheries, ink coatings, resins, fuel oil, metal processing, electricity, electronics, machinery, etc.

Lipase, which is one of the methods for modifying or synthesizing the above-mentioned esters used in a wide range of industrial fields, is usually immobilized mainly for the purpose of reuse. In general, the activity of immobilized lipase, like other enzymes, is lower than that of native lipase itself. Possible causes for this are that the conformation of the enzyme protein changes due to the immobilization procedure itself, that the active center is blocked, that the frequency of contact with the substrate during the reaction is reduced, etc. Examples include heat denaturation and coexistence of activity inhibitors. For this reason, many proposals have been made regarding the activation method when adjusting the immobilized lipase.

For example, it has been reported that lecithin, chitosan, water-soluble chitin, etc. are effective as activators of lipase (JP-A-3-130079). Also,
As a method for reactivating the immobilized lipase, once hydrolyzed, the recovered enzyme agent is washed with a non-polar solvent such as hexane, dried, and then impregnated with water or a divalent or trivalent lower alcohol. Another method has been proposed (Japanese Patent Laid-Open No. 61-149084). This method is also effective for immobilized lipase used once or more for transesterification.

[0005]

[Problems to be Solved by the Invention] When using immobilized lipase, not only the technical development for preparing immobilized lipase having higher activity but also the use of immobilized lipase which has been used more than once is reduced. The technological development to be activated is also an extremely important technical development subject from the viewpoint of practical production cost of useful substances. However, there are still few reports regarding this, and the above-mentioned Japanese Patent Laid-Open No. 61-1490.
Also in Japanese Patent Publication No. 84, not only it takes a long time to dry the used immobilized lipase, but also the amount of water to be infiltrated needs to be set strictly, for example, 0.015 g needs to be prepared for 1.87 g of the lipase preparation. However, there are many practical problems, such as complicated and precise operations required.

[0006] That is, a method for reactivating the immobilized lipase in a simple and short time has not been proposed yet, and the development of such a technique includes the modification of oil and fat in the oil and fat industry. It is considered to be one of the technologies that make a significant contribution as an efficient practical means in the modification and synthesis of esters that are usefully used in various industries.

In view of such circumstances, the present inventors have aimed to develop a technique for simply and quickly reactivating an immobilized lipase whose activity has been reduced by using it once or more in the modification and synthesis of esters. Through intensive studies, the present invention has been completed.

[0008]

That is, in the present invention, in the modification or synthesis of esters, the immobilized lipase used in the reaction once or more is treated with a non-polar solvent and a polar solvent. Is the reactivation method of.

The immobilized lipase used in the present invention may be prepared by a conventional method, and lipase of animal or plant origin or microbial origin, for example, lipase derived from pig pancreas or castor seed,
Aspergillus niger, Aspergil oryzae, Rhizopus
Rhizopus delemar, Rhizopus arrhizus, Mucor
miehei), Mucor javanicus
), Candida cylindrac
ea), Geotrichumcandi
dum), Penicillium Cyclopium (Penicillium cy
Microbial lipase such as clopium) is adsorbed on adsorbents such as activated carbon, diatomaceous earth, celite, zeolite, silica gel, glass, animal bones, chitin, chitosan, hydroxyapatite, cellulose, cation exchange resin, anion. Covalently bonded to ion exchange resin, both ion exchange resins, polyether and polysulfone polymer materials,
Alginic acid, polyacrylic acid, and gums that are also thickeners may be incorporated and immobilized, and the like.

Next, the esters in the present invention are carboxylic acids and alcohols which are ester-bonded to each other regardless of natural products and synthetic products, and are monovalent to polyvalent, linear or pendant. It refers to esters of these carboxylic acids and alcohols with or without chain, hydroxyl groups, unsaturated bonds and cyclic structures.

As the natural products, general animal and plant oils and fats, that is, soybean oil, rapeseed oil, safflower oil,
Sunflower oil, sesame oil, cottonseed oil, palm oil, palm kernel oil,
Palm oil, kapok oil, camellia oil, almond oil, cacao butter, shea butter, butter oil, linseed oil, castor oil, egg oil, beef tallow, lard, fish oil, etc., as well as these fractionated oils and fats, part or all of hydrogen Including added oils and fats, transesterified oils and fats,
Furthermore, algae and microbial oils and fats can also be used.

In the synthetic ester, a carboxylic acid having an alkyl group residual chain having 1 to 25 carbon atoms is preferable, and a fatty acid having an alkyl group residual chain having 5 to 21 carbon atoms is further preferable. Preferably, examples of these are:
Caproic acid, capryl, acid 2-ethylhexanoic acid, nonanoic acid, capric acid, lauric acid, dodecadicarboxylic acid,
Myristic acid, pentadecanoic acid, palmitic acid, palmitooleic acid, isopalmitic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, ricinoleic acid, 12-hydroxystearic acid, linoleic acid, α-
Linolenic acid, γ-linolenic acid, erucic acid, behenic acid,
Examples include arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, succinic acid, citric acid, malic acid and tartaric acid. On the other hand, alcohols have 1 to 1 carbon atoms
0 is preferable, and examples thereof include methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, 2-ethylhexanol, myristyl alcohol, stearyl alcohol, oleyl alcohol, 2-hexyldecanol, isostearyl alcohol, and 2-octyldodeca. Noor, Eicosanol,
Monohydric alcohols such as octacosanol, ethylene glycol, propylene glycol, butylene glycol,
Hexylene glycol, etc., and their polymers, their dihydric alcohols such as partial ethers or esters, and their derivatives, and trihydric or higher polyols such as glycerin, trimethylolpropane, erythritol, pentaerythritol, and their polymers, and their derivatives. , Saccharides such as glucose, sucrose and xylose, amino alcohols and sterols. The present invention is directed to, but not limited to, mono- to polyvalent esters of carboxylic acid and alcohol as described above.

In the present invention, the lipase activity is reduced by using the immobilized lipase as described above once or more when the ester is modified, that is, hydrolyzed or transesterified, or synthesized. It is characterized by efficiently increasing the activity of immobilized lipase.
INDUSTRIAL APPLICABILITY Among the treatments of esters, the present invention exhibits a remarkable effect particularly on transesterification reaction. The transesterification reaction means an intramolecular and / or intermolecular transesterification reaction of a single or a plurality of types of esters, an acidolysis reaction using an ester and a carboxylic acid, and an alcoholysis reaction using an ester and an alcohol, for example, in the oil and fat industry. Reactions using fats and oils, fats and oils and fatty acids and / or lower alcohol esters of fatty acids, and fats and oils and glycerin are suitable for the present invention.

As the non-polar solvent used in the present invention, hydrocarbon solvents such as pentane, hexane, heptane, isooctane and petroleum benzine, ether solvents such as petroleum ether, dimethyl ether, diethyl ether, methyl ethyl ether and isopropyl ether, Methyl acetate,
Examples include ester solvents such as ethyl acetate, isopropyl acetate, propyl ester of diethylene glycol monomethyl ether, and acetic acid ester of propylene glycol monoethyl ether, but hexane, petroleum benzine, petroleum ether, diethyl ether, ethyl acetate and the like are preferable in operation. .. As the polar solvent, acetone,
A ketone solvent such as methyl ethyl ketone, diethyl ketone, or methyl isobutyl ketone, or an alcohol solvent such as isopropyl alcohol, butyl alcohol, or cyclohexanol can be used, and acetone is particularly preferable.

The immobilized lipase is treated by combining such a non-polar solvent and a polar solvent. Among them, the combination of n-hexane and acetone is effective. The non-polar solvent and the polar solvent are preferably used in an amount of 1 to 50 times by weight, usually 3 to 30 times by weight, of the immobilized lipase. If the amount of solvent is less than 1 times by weight, the factor inhibiting the lipase activity of the immobilized lipase cannot be eliminated, and if it is more than 50 times by weight, the lipase protein itself is denatured and the inactivation is promoted. As the combination of the non-polar solvent and the polar solvent, the immobilized lipases may be treated individually or in any order, and may be used as a mixture, and the ratio thereof is not particularly limited.

In the present invention, the immobilized lipase used in the reaction more than once is treated with such a solvent. The treatment method may be as follows. That is, the immobilized lipase is recovered from the reaction system by precipitation, filtration, centrifugation, etc., and if necessary, the adhering raw materials, reaction intermediates, products, etc. are roughly removed in advance to remove the above-mentioned substances. It is immersed in a solvent or simply washed, and the remaining solvent is distilled off by a conventional method such as air drying, reduced pressure or freeze drying. With such a simple operation, the lipase activity of the immobilized lipase is reactivated, so that it can be subjected to the usual modification or synthesis reaction of esters again.

[0017]

【Example】

Reference example As an immobilized lipase, an anion-exchange resin with high specificity at the 1- and 3-positions of glycerides
LIPOZYME with immobilized lipase derived from miehei)
300 g (Novo) was packed in a 1 liter column,
360 g of palm oil and rapeseed oil (4: 6) at 60 ° C
The liquid was passed at a flow rate of / hr to carry out the transesterification reaction. At this time, the water content of the oil was 300 ppm, and the water content in LIPOZYME was 15% (weight ratio). When the transesterification rate dropped to 51% on the 15th day, LIPOZYM
E was recovered.

The transesterification activity and transesterification rate of this immobilized lipase were determined as follows. Transesterification activity: Triolein and palmitic acid were stirred with immobilized lipase in n-hexane, and the amount of palmitic acid incorporated in the product triglyceride was determined from GLC (gas chromatography) analysis values. The relative activity of the enzyme capable of taking up palmitic acid was evaluated. Transesterification rate: Calculated from the ratio of the actual reaction component to the component when the 1,3-position of the glyceride was completely transesterified.

Example 1 Each of the LIPOZYME obtained in the reference example was added to the LIPOZYME in an amount of 5 times the weight (weight ratio; the same applies hereinafter) of n.
-Washing with a mixed solvent of hexane and acetone, filling in the column again, and performing the transesterification reaction in the same manner as in Reference Example, the transesterification reaction rate was recovered to 92%. Further, the transesterification activity at this time was restored to 70% of the initial activity as shown in Table 1.

[0020]

[Table 1]

Comparative Example 1 As a comparative example of Example 1, LIPOZYM obtained in the reference example
E was washed only with a 5-fold amount of n-hexane, and its transesterification activity was measured. As a result, it was 30% of the initial activity.

Comparative Example 2 As a comparative example of Example 1, the LIPOZYM obtained in the reference example
E was washed only with 5-fold amount of acetone, and the transesterification activity thereof was measured. The result was 45% of the initial value.

Example 2 Five-fold amounts of n-hexane and acetone were added to n-hexane and LIPOZYME obtained in the reference example, respectively.
Then, the product was washed with acetone in that order, and the column was packed again to carry out a transesterification reaction. This operation was repeated 3 times, and the change with time of the transesterification rate at that time is shown in FIG. From the figure, it was found that LIPOZYME whose transesterification activity was reduced by the reactivation treatment of the present invention can be repeatedly used.

Example 3 The LIPOZYME obtained in the reference example was immersed in 3 times the amount of petroleum benzine and then 5 times the amount of methyl isobutyl ketone for washing, and the solvent was distilled off under reduced pressure. When 5% by weight of solvent-treated LIPOZYME was used to conduct the transesterification reaction in the same manner as in Example 1 in a batch reaction vessel equipped with a stirrer, the transesterification rate on the 4th day was reactivated to 87%. It was

Example 4 0.5 g of lipase (manufactured by Seikagaku Corporation) derived from Rhizopus delemar was added to 30 g of ion-exchanged water.
It was dissolved / dispersed in ml, stirred with 30 g of anion exchange resin to form a slurry, and dried to prepare immobilized lipase. 10 g of the immobilized lipase was added to a glass reaction vessel equipped with a stirrer and charged with 100 g of raw materials at a ratio of 1 mol of isobutyric acid and 5 mol of n-octanol, and the temperature was 40 ° C.
The ester synthesis reaction was carried out with gentle stirring. The production rate of the target n-octylisobutyric acid ester in the reaction product was analyzed by thin layer chromatography (TLC) and gas chromatography (GLC).
It was 85% in the reaction at 0 hours. The immobilized lipase was filtered off from the reaction product, and 20 times the amount of n-lipase was added to the immobilized lipase.
After washing with hexane and then with 5 times the amount of acetone to distill off the residual solvent, the synthesis reaction was carried out again using the same new raw material under the same conditions as above.
The synthesis rate of octyl isobutyric acid ester was 82%.

Comparative Example 3 As a comparative example of Example 4, the ester synthesis reaction of Example 4 was carried out once, and the immobilized lipase filtered from the reaction product was used as it was for the second synthesis as in Example 4. After the reaction, the synthesis rate of the target ester after 300 hours was 6
It was 8%.

Example 5 Using the immobilized lipase prepared in Example 4 instead of the LIPOZYME of Reference Example, transesterification was carried out under the same conditions as in Reference Example. On day 10, the transesterification activity dropped to 43% of the initial activity, at which time the immobilized lipase was filtered from the reaction and collected. Then, when the immobilized lipase was washed with a solvent and subjected to a transesterification reaction again in the same manner as in Example 1, the transesterification activity was restored to 75% of the initial level.

Example 6 Candida cylindracea
An immobilized lipase was prepared by using the lipase (manufactured by Meito Sangyo Co., Ltd.) derived from the method and operation of Example 4, and instead of 1 mol of oleic acid and 5 mol of n-decanol of Example 4, When 1 mol of oleic acid and 1 mol of glycerin were used to perform a glyceride synthesis reaction at 40 ° C., the esterification rate of oleic acid in the produced glyceride after 5 days was 85%. The immobilized lipase recovered from the reaction product was washed with 5 times each amount of petroleum ether and then with n-butyl alcohol, the residual solvent was distilled off under reduced pressure, and then a synthetic reaction of a new raw material was carried out under the same conditions as above. Do this operation 10
Repeated times, the esterification rate of oleic acid in glyceride was as follows: 2nd time: 83%, 5th time: 80%, 10%
Round: 75%. On the other hand, in the same operation as above, the glyceride yield in the case where the immobilized lipase was not treated with a solvent was as follows: second time: 70%, fifth time: 65%, tenth time: 41
%Met.

[0029]

Industrial Applicability By using the method of the present invention, the activity of the immobilized lipase used for modifying or synthesizing the ester can be reactivated and can be repeatedly used for a longer time. Furthermore, this method is very simple and easy to carry out on an industrial scale.

[Brief description of drawings]

FIG. 1 is a diagram showing the effect of reactivating the immobilized lipase in Example 2, in which the horizontal axis represents reaction time (days) and the vertical axis represents transesterification rate (%).

Claims (3)

[Claims] 1. Reactivation of immobilized lipase, which comprises treating the immobilized lipase used in the reaction more than once with a non-polar solvent and a polar solvent in the modification or synthesis of esters using immobilized lipase. Law. 2. The method for reactivating immobilized lipase according to claim 1, wherein the immobilized lipase is used for transesterification. 3. The immobilized lipase reactivation method according to claim 1, wherein the non-polar solvent and the polar solvent are n-hexane and acetone, respectively.