JPS62115283A - Immobilized lipase and production thereof - Google Patents

Abstract

PURPOSE:An immobilized enzyme, obtained by entrapping lipase or a biological tissual material containing the lipase in a resin capable of swelling in a fat or oil, glycerol ester of a fatty acid or fatty acid without dissolving therein and having improved diffusivity of a substrate or product in the resin and strength. CONSTITUTION:An immobilized lipase obtained by respectively dissolving or dispersing 100pts.wt. resin capable of swelling in a fat or oil, glycerol ester of a fatty acid or the fatty acid without dissolving therein, preferably polybutadiene, high-styrene rubber or chloroprene rubber and 1-100pts.wt. lipase or biological tissual material containing lipase in a volatile organic solvent to prepare a resin solution containing the suspensed enzyme and removing the solvent. A volatile solvent, e.g. tetrahydrofuran, chloroform or toluene, vaporizing at <=70 deg.C under ordinary or reduced pressure is preferred for the volatile organic solvent.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to an immobilized lipase and a method for producing the same. To,
The present invention relates to an immobilized lipase containing lipase or a dried body tissue containing lipase, and a method for producing the same.

[Conventional technology and problems]

Conventional methods for hydrolyzing fats and oils include saponification, toisochel, high-pressure decomposition, and enzymatic decomposition using lipase. Among them, the high-pressure decomposition method is mainly adopted as an industrially advantageous method. This method uses a high-pressure, high-tower reaction tank to decompose fats and oils with water vapor at a high temperature and pressure of 50 atm at 250°C, and is suitable for the continuous large-scale decomposition of a single fat and oil. It's a method. However, this method has drawbacks such as being an energy-consuming reaction, making it difficult to switch between multiple types of fats and oils, and being unsuitable for decomposing small amounts of fats and oils.

In addition, in saponification decomposition method and toisochel decomposition method,
Because the reaction process requires considerable high-temperature treatment, side reactions are likely to occur, and during the decomposition of fats and oils, the fatty acids are markedly colored and produce a bad odor, so it has the disadvantage that a light-colored product cannot be obtained unless repeated distillation is carried out. are doing.

In order to eliminate the above drawbacks, hydrolysis of fats and oils using free lipase was carried out (Japanese Patent Publications No. 46-16508 and Japanese Patent Publication No. 46-16509) These enzymatic decomposition methods are capable of hydrolyzing fats and oils at low temperatures. It is an energy-saving reaction that can decompose , and has the advantage that the fatty acids and glycerin produced are not colored or altered.However,
When using free lipase, protein derived from the lipase is mixed into the backwater after the reaction, and a step for removing it is necessary. Furthermore, since it is very difficult to reuse free lipase, there are drawbacks such as the need to use large amounts of expensive lipase.

To overcome these drawbacks of using free lipase, immobilization of lipase has been proposed. For example, a method of introducing hydrophobic groups onto the surface of a hydrophilic gel and adsorbing and immobilizing lipase (Japanese Patent Publication No. 51-27477), and a method of adsorbing or covalently bonding lipase to a macroporous hydrophobic carrier. Method of immobilization (Unexamined Japanese Patent Publication No. 5
9-28483) etc. have been proposed. However, when producing immobilized lipase using carrier binding methods such as adsorption and covalent bonding, in order to make it highly active, the immobilization carrier must be made finer and the surface area of the carrier must be increased. However, when considering industrial processes,
It is difficult to operate. Furthermore, when an adsorption method is used as an immobilization method, enzyme desorption is significant, and when a covalent bond method is used, the enzyme molecule itself undergoes a structural change during immobilization, resulting in a decrease in enzyme activity. There are significant problems.

In order to solve these problems related to the immobilization of lipase by adsorption or covalent bonding, a method has been proposed in which lipase is comprehensively immobilized on polyacrylamide and this is used for the production of fatty acids (Unexamined Japanese Patent Publication No. Showa 5
0-129789). However, this method uses polyacrylamide, a hydrophilic resin, for the reaction using fats and oils as a substrate, so it does not solve the problem of the difficulty of the substrate diffusing in the resin, which is a drawback of the entrapping immobilization method. . In addition, in consideration of this point, a method has been proposed in which lipase is comprehensively immobilized using a hydrophobic UV curable resin (Japanese Patent Publication No. 15877/1987 II). In this case, by making the resin hydrophobic, it is possible to improve the diffusivity of substrates and products within the resin, but although the resulting resin has a crosslinked structure, it lacks mechanical strength, resulting in poor reuse stability. There are problems such as a low molecular weight and a restriction on the form of the immobilized enzyme.

[Means for solving problems]

As a result of intensive studies to solve the above-mentioned problems, the present inventors found that fats and oils, fatty acid diesters or monoesters of glycerin, or resins that swell with but do not dissolve in fatty acids can be used as carriers for immobilizing lipase. The present invention has been completed based on the discovery that by using this method, an immobilized lipase can be obtained which has excellent dispersibility of the substrate and product in the resin and mechanical strength, as well as excellent storage stability and reuse stability.

That is, the present invention provides lipase or a lipase-containing biological tissue 1 to 1 in 100 parts by weight of a resin that swells but does not dissolve in fats and oils, fatty acid esters of glycerin, or fatty acids.
The present invention provides an immobilized lipase comprising 1 part of 1,000 iftl, and 100 parts by weight of a resin that swells but does not dissolve in fats and oils, fatty acid esters of glycerin, or fatty acids, and lipase or biological IJl fabrics containing lipase. 1,000 parts by weight of the enzyme are dissolved and dispersed in a volatile organic solvent to prepare an enzyme suspension resin solution, and then the volatile organic solvent is removed. It is something.

The oils and fats, fatty acid esters of glycerin, or resins that swell in but do not dissolve in fatty acids that can be used to produce the immobilized lipase of the present invention are determined by the following resin oil absorption evaluation method based on the weight of the resin: It is capable of absorbing fats and oils in a range of from 10% to 500% by weight, preferably from 10% to 300% by weight.

In addition, the oil absorbency evaluation of the resin is performed as follows.

That is, a predetermined amount of resin is dissolved in a volatile organic solvent, cast, and cast to a thickness of 0.05 m to 1 m to prevent air bubbles from forming.
．． Create an On film. After confirming that the obtained film has a constant weight, it is immersed in excess soybean white squeezed oil at 30° C. for 24 hours in a stationary state to absorb oil. After treatment, the fats and oils adhering to the resin surface are washed away with a volatile organic solvent (a good solvent for soybean oil, but a poor solvent for the resin). After confirming that the organic solvent for cleaning has been sufficiently removed and that the amount of oil-absorbed resin is constant, the oil absorption is calculated using the following formula.

(oil absorption ht% vs. resin amount) - Resin weight after oil absorption - Resin weight before oil absorption Resin weight before oil absorption Such resins that can be used in the present invention include, for example, natural rubbers, butadiene polymers, butadiene polymers, etc. Diene-based synthetic rubbers such as styrene copolymers, butadiene-acrylonitrile copolymers, isoprene polymers, chloroprene polymers, isobutylene-diene copolymers, ethylene-propylene copolymers (EPM), as a third component in EPM EPrlM with a small amount of unsaturation added, olefin synthetic rubbers such as chlorosulfonated polyethylene, polysulfide synthetic rubbers such as alkylene sulfide polymers, silicone rubbers, fluorine rubbers, polyester rubbers, etc.
Urethane-based synthetic rubbers such as isocyanate daily materials, polyether/isocyanate condensates, propylene oxide rubber, epichlorohydrin rubber, nitroso rubber, ethylene acetate rubber, recycled rubber, and other rubbers;
Acrylic ester (co)polymer, alkyl styrene (
Examples include co)polymers, ABS resins, etc., but any resin may be used as long as it meets the conditions for oil absorption evaluation described above, but rubber-based resins are preferred, and diene-based rubbers are particularly preferred. used.

These materials may be used alone or in combination of two or more.

In the present invention, post-crosslinked versions of the above resins can also be used. As a method for post-crosslinking, for example, UV
, self-crosslinking by irradiation such as EB, crosslinking by vulcanization, addition of a different type of monomer, etc.

The volatile organic solvent used in producing the immobilized lipase of the present invention may be one that dissolves the above-mentioned oil-absorbing resin and vaporizes at a temperature of 70'C or less under normal pressure or reduced pressure. Either one is fine. Examples of volatile organic solvents that can be used in the present invention include, but are not limited to, tetrahydrofuran, chloroform, toluene, methyl ethyl ketone, ethyl acetate, benzene, and hexane.

The lipase or dried body tissue containing lipase used in the present invention may be produced by microorganisms, or may be obtained from animal organs, plant seeds, etc.

In the present invention, if these are pulverized in advance using a pulverizer or the like, good results can be obtained since they can be well dispersed when preparing the enzyme suspension resin solution.

The enzyme suspension resin solution in the present invention is prepared using (Al oil, fatty acid ester of glycerin, or a resin that swells with but does not dissolve in fatty acids, FBL lipase or a dried body tissue containing lipase, and (C) a volatile organic solvent. However, (a) is added to 1 part by weight (bl is 0.01
It is preferable to use it in a range of 10 parts by weight. The amount of the volatile organic solvent for the resin is preferably 5 to 50 parts by weight, more preferably 7 to 30 parts by weight, per 1 part by weight of +al. Particularly preferred.

The enzyme suspension resin solution can be prepared by simply mixing the oil-absorbing resin, lipase powder, and volatile organic solvent, but preferably, the lipase powder is first stirred and dispersed in the volatile organic solvent, and then the oil-absorbing resin The preparation method of dissolving the resin gives good results as a well-dispersed system of lipase powder is obtained.

The immobilized lipase of the present invention can be prepared by removing the volatile organic solvent from the enzyme suspension resin solution.

The immobilized lipase in the present invention can be supported on any type of carrier, or can be used as is.

Examples of materials for supporting the immobilized lipase of the present invention include metals such as aluminum, activated alumina, and iron;
Inorganic materials such as silica, glass, ceramics, bisque ceramics, and rocks; organic materials such as cross-linked polystyrene beads, carbon, vulcanized rubber, polyethylene beads, and polypropylene beads; The material is not limited in any way as long as it is not soluble in the solvent.

The form of the immobilized lipase of the present invention is not particularly limited and can take any form. For example, film-like, spherical,
The shape may be fibrous, prismatic, lumpy, ring-like, etc., but any other shape may be used.

The size of the immobilized lipase of the present invention is arbitrary, but in consideration of the diffusion movement of the substrate and product within the resin, it is preferably spherical with a diameter of 1 cflI or less, particularly preferably 511 or less. From the point of view of diffusion and movement of substrates and products within the resin, the smaller the diameter of spherical particles, the thinner the film thickness of membrane-shaped particles, the better.

In the present invention, lipase was immobilized in a lipophilic resin, but the method of the present invention can also be applied to enzymes other than lipase. Applicable enzymes are not particularly limited, but enzymes that use lipophilic substances as substrates are preferred. Specific examples include alcohol dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, cholesterol oxidase, aldehyde dehydrogenase, acyl-CoA dehydrogenase, 3-oxosteroid Δ1-dehydrogenase, lipoxygenase, alkane 1-monooxygenase. , steroid 21-monooxygenase, Δ24-sterol methyltransferase, acetyl-CoA acyltransferase, phospholipase A2, lysophospholipase, acetyl esterase, cholesterol esterase,
Phospholipase AI, riboprotein lipase, steroid lactonase, phospholipase C1 phospholipase D1 α-glucosidase, β-glucosidase, phosphatidylserine decarboxylase, steroid lactonase
Δ-isomerase, cholesterol/Δ-isomerase, butyryl-CoA/synthetase, acyl-CoA/
Examples include synthetase, acetyl-CoA carboxylase, and the like.

〔Example〕

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Candida cylinderase (Candida cyl)
0.1 g of lipase produced by i-ndracea (trade name: Lipase Kano, manufactured by Meito Sangyo, 360,000 yen/g) powder
into a 501 beaker, and then add tetrahydrofuran l.
og and stirred with a stirrer at room temperature to thoroughly pulverize the lipase powder.

1,2-polybutadiene (
Add 1.0 g of RB-810 (manufactured by Nippon Synthetic Rubber Co., Ltd.) and dissolve by stirring at room temperature. After the resin is dissolved, 10 g of activated alumina granules (4 to 6 rich and short diameter, manufactured by Gyudon Chemical Co., Ltd.) are added, and the surface of the activated alumina granules is coated with the enzyme suspension rubber solution using the dipping method. . The obtained coated product is dried at room temperature and under normal pressure and then at room temperature and under reduced pressure (48 hours), washed with ion-exchanged water, and filtered to obtain immobilized lipase.

By the above method, per 10g of activated alumina granules,
Immobilized lipase on which 20 μg of lipase leaf powder was supported was obtained.

Application example 1: 100 ml of 20 g of white soybean oil and 20 g of ion exchange water
Add the entire amount of the immobilized lipase prepared in Example 1 to an Erlenmeyer flask with a stopper, and incubate at 30°C for 150 strokes.
The mixture was shaken for 24 hours and the reaction was carried out for 24 hours. After the reaction is complete,
After filtering, the reaction solution was subjected to heat treatment and centrifugation, and the acid value (AV) and saponification value (SV) of the oil layer were measured to determine the hydrolysis rate (AV/SV). On the other hand, the recovered immobilized lipase was washed with water and directly added to the above reaction system to perform the same reaction. This operation was repeated 40 times, but the activity of the immobilized lipase did not decrease, and it was observed that the reuse stability was good. The results are shown in Figure 1.

Application example 2 The immobilized lipase obtained in Example 1 was left as is for one month without any modification at room temperature and atmospheric pressure, and reused using soybean white squeezed oil in the same manner as in Application example 1. Stability was evaluated. There was no decrease in the activity of the immobilized enzyme due to standing, and good storage stability was observed. The results are shown in Table 1.

Table 1 Example 2 Lipase OF powder 0.1g, tetrahydrofuran 10g
1 Using 1 g of high styrene rubber (manufactured by Japan Synthetic Rubber ■) and log of activated alumina granules, immobilized lipase was prepared in the same manner as in Example 1.
An immobilized lipase carrying 30 μg of gluepase OF powder was obtained. When this product was evaluated in the same manner as Application Example 1, it was found that there was no decrease in the activity of the immobilized enzyme, and that it had good reuse stability. The results are shown in Table 2.

Table 2 Example 3 Lipase OF powder 0.1g, tetrahydrofuran 10g
, chloroprene rubber (DCR-50 manufactured by Denki Kagaku Kogyo ■)
An immobilized lipase was prepared and evaluated by the method of Example 1 using 10 g of Ig and zeolite (manufactured by Wako Pure Chemical Industries, Ltd.). An immobilized lipase was obtained in which 30 μ of lipase OF powder was supported on 10 g of boiling stone. There was no decrease in the activity of the immobilized enzyme, and good reuse stability was observed. The results are shown in Table 3.

Table 3 Example 4 Lipase OF powder 0.1g, tetrahydrofuran 10g
, epichlorohydrin rubber (CHR21 manufactured by Nippon Zeon)
01) 1 g, activated alumina granules (4-6 diameter)
Immobilized lipase was prepared by the method of Example 1 using 10 g and evaluated. 27.5 per 10g of activated alumina
Immobilized lipase carrying lipase OF powder was obtained. There was no decrease in the activity of the immobilized enzyme, and good reuse stability was observed. The results are shown in Table 4.

Table 4 Example 5 Lipase leaf powder 0.1g, tetrahydrofuran 10g
, ABS resin (manufactured by East side, TOYOLAC 900) 1
g, immobilized lipase was prepared by the method of Example 1 using 10 g of activated alumina granules (4 to 611 diameter),
evaluated. An immobilized lipase was obtained in which 32.1 μg of gluepase OF powder was supported on activated alumina]Og. There was no decrease in the activity of the immobilized enzyme, and good reuse stability was observed. The results are shown in Table 5.

Table 5 Comparative Example 1 A comparative study was conducted using calcium alginate gel that does not have oil absorption performance. Sodium alginate (Wako Pure Chemical Industries)
0.5g of the product (manufactured by Kawasaki Co., Ltd.) was placed in a 1001 beaker, 25g of ion-exchanged water was added, and the mixture was stirred with a stirrer at room temperature to dissolve. To the resulting aqueous solution, 0.0% lipase OF powder was added.
2g was added and dissolved. On the other hand, as a gelling agent, 2%
50 g of calcium chloride aqueous solution was prepared. Lipase OF
10g of activated alumina granules (manufactured by Gyudon Chemical Co., Ltd.) are immersed in the solution in which the activated alumina is dissolved, filtered, and then immersed in a gelling agent to coat the surface of the activated alumina with enzyme-immobilized calcium alginate gel. An immobilized lipase was obtained. An immobilized lipase was obtained in which 86 μg of gluepase OF powder was supported on 10 g of activated alumina granules. The evaluation was carried out in the same manner as in Application Example 1 using 50 μm of lipase OF powder. It was observed that the activity of the immobilized enzyme decreased with repeated reuse. The results are shown in Table 6.

Table 6 Application Example 3 Using the apparatus shown in FIG. 2, oils and fats were continuously hydrolyzed in a circulation system. Immobilized lipase is lipase leaf powder 0.8
g, 150 g of tetrahydrofuran.

Chloroprene rubber (rlcR-50 manufactured by Denki Kagaku Kogyo ■)
Example 1 using 9 g and 110 g of activated alumina granules.
It was prepared by the method described in . Activated alumina granules 1
An immobilized lipase in which 500 μg of lipase OF powder was supported on 10 g was obtained.

The evaluation was performed using the apparatus shown in FIG. The device is a 500ml tank with a mechanical stirring device for mixing oil and water.
Separable flask 1, jacketed packed bed reactor (diameter 4.5 cm) 2. Constant temperature water bath 3 to keep 1 and 2 at constant temperature, pump 4 to supply constant temperature water to the jacket, flask 1 It consists of a pump 5 for supplying the contents to the packed bed reactor 2, and a silicone tube 6 for connecting each part. The reaction is soybean white squeezed oil 200
Put 200 g of ion-exchange water and 150 g of ion-exchanged water into flask 1.
The entire amount of immobilized lipase described above (equivalent to 500 μl of lipase OF powder) was charged into reactor 2, and the mixture in flask 1 was poured onto reactor 2 at a flow rate of 0.5 n/hr. The process was carried out using a circulation system in which the liquid was pumped downward from the flask 5 and returned to the flask 1. The constant temperature water bath was maintained at 30°C. Such a reaction was continued for 24 hours to form the first fat/oil hydrolysis. After the reaction was completed, the reaction solution was replaced with 200 g of freshly squeezed soybean oil and 200 g of ion-exchanged water, and a second fat/oil hydrolysis was performed under the same conditions as the first. Evaluations were repeated in the same manner. There was no decrease in the activity of the immobilized enzyme, and good reuse stability was observed. The results are shown in Table 7.

Table 7 [Effects of the Invention] As specifically shown in the Examples, the immobilized lipase of the present invention allows the lipase to be reused over a long period of time and is highly stabilized. It is an enzyme preparation product.

It also has excellent storage stability at room temperature and under normal pressure. Furthermore, since the entrapment method is used as the immobilization method, the activity of the enzyme itself is reduced during immobilization.
is hardly recognized. Furthermore, the difficulty in resin diffusion of substrates and products, which is thought to be a drawback of the entrapment immobilization method, could be solved by selecting an oil-absorbing resin that is easily compatible with oil. In addition, the comprehensive immobilization method is simple and highly practical.

Furthermore, the method for producing immobilized lipase of the present invention is an extremely simple process and advantageous compared to conventional methods.

As described above, the present invention can greatly save the use of expensive lipase, and the present invention makes a significant contribution to industry.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the evaluation results of the reuse stability of immobilized lipase in Application Example 1 of the present invention. FIG. 2 is a schematic cross-sectional view of the apparatus used for the reaction in Application Example 3. 1: Flask 2: Packed bed reactor 3: Constant temperature water tank 4: Constant temperature water supply pump 5: Reaction liquid supply pump 6: Silicone tube

Claims (1)

[Scope of Claims] 1. Immobilization in which 1 to 1000 parts by weight of lipase or a lipase-containing biological tissue is encased in 100 parts by weight of fats and oils, fatty acid esters of glycerin, or resins that swell in but do not dissolve in fatty acids. Lipase. 2. The immobilized lipase according to claim 1, wherein the resin that swells but does not dissolve in fats and oils, fatty acid esters of glycerin, or fatty acids is polybutadiene, high styrene rubber, or chloroprene rubber. 3. 100 parts by weight of fats and oils, fatty acid esters of glycerin, or resins that swell but do not dissolve in fatty acids, and 1 to 1000 parts by weight of lipase or a lipase-containing biological tissue are dissolved and dispersed in a volatile organic solvent, respectively. A method for producing immobilized lipase, which comprises removing the volatile organic solvent after preparing an enzyme-suspended resin solution. 4. The method for producing immobilized lipase according to claim 3, wherein the resin that swells but does not dissolve in fats and oils, fatty acid esters of glycerin, or fatty acids is polybutadiene, high styrene rubber, or chloroprene rubber. 5. The method for producing immobilized lipase according to claim 3 or 4, wherein the volatile organic solvent is vaporized at a temperature of 70° C. or lower under normal pressure or reduced pressure. 6. The method for producing immobilized lipase according to claim 3 or 4, wherein the volatile organic solvent is tetrahydrofuran, chloroform, or toluene.