JP3836824B2 - Regeneration method of immobilized enzyme - Google Patents

Description

  The present invention relates to a method for regenerating an immobilized enzyme for fat hydrolysis by effectively utilizing the residual activity of a used immobilized enzyme in an oil hydrolysis reaction.

  In order to use an enzyme efficiently when hydrolyzing fats and oils using an oil-degrading enzyme, the immobilized enzyme which fixed the fat-and-oil degrading enzyme to the inorganic or organic support | carrier is used. As this immobilized enzyme is used for a hydrolysis reaction over a long period of time, its activity decreases. Therefore, it is necessary to collect the immobilized enzyme when the activity has decreased to some extent and replace it with a new immobilized enzyme.

  As a means for effectively using the recovered used immobilized enzyme, a method of removing all the oil and protein adhering thereto and reusing it as a carrier can be easily considered. However, this method generates a large amount of washing waste liquid several hundred times as large as that of the enzyme carrier, and has an environmental problem, and also has a problem that the activity remaining in the used immobilized enzyme cannot be completely used.

In addition, a method of eliminating an inhibitory factor of an enzyme reaction by solvent washing from an immobilized lipase having a decreased activity used for transesterification or transesterification (Patent Document 1), water is desorbed by a reaction under low moisture. There is a method (Patent Documents 2 and 3) for reactivating the remaining lipase by controlling the moisture that contributes to the reaction by wet treatment with a solvent or a solvent and a phospholipid for the enzyme that has been trapped. However, these methods do not regenerate the immobilized enzyme whose activity has decreased due to the elimination of a part of the lipase, but are a method of reactivating the remaining lipase.
JP-A-5-137574 JP-A-9-56379 JP 11-75834 A Japanese Unexamined Patent Publication No. 1-153090

  Therefore, the present invention effectively utilizes the residual activity of the used immobilized enzyme in the fat hydrolysis reaction, and is an oil and fat hydrolysis immobilized enzyme having the same performance as before use at low cost and in a small amount of waste liquid. The object is to provide a method of reproduction.

  By the way, when immobilizing fat and oil hydrolase, it is known that a highly active immobilized enzyme can be obtained by previously adsorbing a fatty acid on an enzyme carrier (see Patent Document 4). The present inventor, without completely removing a large amount of fatty acid adhering to the immobilized enzyme used in the fat hydrolysis reaction, after treating with a solvent so that an amount effective for the activation remains, It has been found that by adsorbing an enzyme having an activity reduced due to the desorption of the enzyme, the used immobilized enzyme can be regenerated to an activity equivalent to that of the new immobilized enzyme in an environmentally and economically advantageous manner. .

  That is, the present invention is to wash the fat and oil immobilized enzyme used for the fat hydrolysis reaction by adding a solvent, adjust the fatty acid equilibrium concentration in the washing liquid, remove the washing liquid, The present invention provides a method for regenerating an immobilized enzyme for hydrolyzing fats and oils to be adsorbed.

  According to the present invention, the remaining activity of the used immobilized enzyme in the fat hydrolysis reaction is effectively used, and the immobilized enzyme for fat hydrolysis has the same performance as before use at low cost and in a small amount of waste liquid. Can be played.

  Examples of the carrier for immobilization in the immobilized enzyme that is the target of the regeneration method of the present invention include celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic carriers, ceramic powder Organic polymers such as polyvinyl alcohol, polypropylene, chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, and synthetic adsorption resin are exemplified, and ion exchange resin is particularly desirable.

  As the ion exchange resin, a porous anion exchange resin is preferable. Since such a porous carrier has a large surface area, a larger amount of enzyme adsorbed can be obtained. The particle diameter of the resin is preferably 100 to 1000 μm, and the pore diameter is preferably 10 to 150 nm. Examples of the material include phenol formaldehyde, polystyrene, acrylamide, divinylbenzene and the like, and phenol formaldehyde resin (for example, Duolite A-568 manufactured by Rohm and Hass) is particularly desirable.

  As the oil-degrading enzyme used in the present invention, lipase is preferable. As the lipase, not only those derived from animals and plants but also commercially available lipases derived from microorganisms can be used. Examples of microorganism-derived lipases include the genus Rizopus, Aspergillus, Mucor, Pseudomonas, Geotrichum, Penicillium, and Candida. The thing of origin is mentioned.

  The used immobilized enzyme that is the target of the regeneration method of the present invention is one that is used for the hydrolysis reaction of fats and oils and has a reduced activity. Examples of the oils and fats include vegetable oils such as soybean oil, olive oil, palm oil, and rapeseed oil, and animal oils such as beef tallow, pork fat, and fish oil.

  Examples of the solvent used for washing the used immobilized enzyme include ethanol, n-hexane, acetone and the like, and ethanol and n-hexane are particularly preferable. In addition, n-hexane is more preferable in that there is no desorption / inactivation of the remaining enzyme and the amount of enzyme used can be reduced.

  When the used immobilized enzyme is washed with these solvents for a certain period of time (about 30 minutes), the fatty acid concentration in the washing solution and the remaining (adsorbed) fatty acid amount of the immobilized enzyme reach an equilibrium. The fatty acid concentration in the cleaning liquid is expressed, for example, by the dissolved fatty acid weight per unit weight of the solution. The amount of fatty acid remaining in the immobilized enzyme is expressed, for example, by the weight of fatty acid per unit weight of the immobilized enzyme. In the present invention, it is necessary to adjust the fatty acid equilibrium concentration in the washing solution in order to obtain a highly active regenerated immobilized enzyme. The fatty acid equilibrium concentration may be appropriately adjusted depending on the form of the enzyme to be adsorbed after removing the washing solution, preferably 4 to 28% by weight, more preferably 5 to 25% by weight, particularly 5 to 22% by weight, and particularly 5 to 20%. % By weight is preferred. The amount of the solvent used may be an amount in which the fatty acid equilibrium concentration of the washing solution falls within the above range in consideration of the amount of fatty acid contained in the immobilized enzyme before washing. About 3 to 20 times by weight is preferable, 3 to 15 times by weight is more preferable, 3 to 10 times by weight, particularly 5 to 10 times by weight, particularly 5 to 8 times by weight is more preferable. The washing temperature of the immobilized enzyme with a solvent is preferably 0 to 60 ° C., particularly 5 to 40 ° C. at which the remaining enzyme does not deactivate.

  After washing with the solvent, the washing solution is removed by filtration and the immobilized enzyme is washed with a buffer solution. The remaining solvent is completely removed by washing with this buffer solution. At this time, the solvent may be removed by distillation. In particular, when the solvent is n-hexane, distillation is preferably performed. This distillation may be atmospheric distillation or vacuum distillation. As the buffer, it is preferable to use a buffer that is used for the subsequent immobilization of the enzyme.

  The temperature at which the enzyme is immobilized can be determined depending on the properties of the enzyme, but is preferably 0 to 60 ° C., particularly 5 to 40 ° C. at which the enzyme is not deactivated. Moreover, the pH of the enzyme solution used at the time of immobilization may be in a range where no denaturation of the enzyme occurs, and can be determined by the characteristics of the enzyme as well as the temperature, but is preferably pH 3-9. In order to maintain this pH, a buffer solution is used. Examples of the buffer solution include an acetate buffer solution, a phosphate buffer solution, and a Tris-HCl buffer solution.

  It is desirable that the enzyme concentration in the enzyme solution is not more than the saturation solubility of the enzyme and sufficient concentration from the viewpoint of immobilization efficiency. The amount of enzyme to be adsorbed may be adjusted according to the residual activity of the used immobilized enzyme. As the enzyme solution, a supernatant obtained by removing an insoluble part by centrifugation, if necessary, or a solution purified by ultrafiltration can be used.

  After immobilization of the enzyme, the immobilized enzyme in a wet state can be recovered by filtration and used as it is in the subsequent oil hydrolysis reaction, and if necessary, the oil described in JP-A-12-166552 can be used. The moisture may be removed by a method of treatment and drying.

  The immobilized enzyme activity (1 U) is 1 μmol per minute when hydrolyzed at 40 ° C. for 30 minutes with stirring and mixing a mixture of oil: water = 100 parts by weight: 25 parts by weight. The resolution of enzymes that produce fatty acids is shown.

Preparation of used immobilized enzyme
10 parts by weight of Duolite A-568 (Rohm and Hass) was stirred for 1 hour in 100 parts by weight of an aqueous N / 10 sodium hydroxide solution. After filtration, it was washed with 100 parts by weight of ion-exchanged water, and the pH was equilibrated with 100 parts by weight of 500 mM phosphate buffer (pH 7). Thereafter, the pH was equilibrated twice with 100 parts by weight of 50 mM phosphate buffer (pH 7) for 2 hours. Thereafter, filtration was performed and the carrier was recovered, followed by ethanol replacement with 40 parts by weight of ethanol. After filtration, a mixed solution of 10 parts by weight of ricinoleic acid and 31.6 parts by weight of ethanol was added, and ricinoleic acid was adsorbed on the carrier for 30 minutes. Thereafter, the carrier was recovered, washed with 50 parts by weight of 50 mM phosphate buffer (pH 7) four times for 30 minutes to remove ethanol, and filtered to recover the carrier. Thereafter, 3.88 parts by weight of lipase (Lipase AY Amano 30, Amano Pharmaceutical Co., Ltd.) was contacted with an enzyme solution dissolved in 180 parts by weight of 50 mM phosphate buffer (pH 7) for 2 hours for immobilization. At this time, when the immobilization rate was determined from the difference between the residual activity of the enzyme solution after immobilization and the activity of the enzyme solution before immobilization, it was 70%. After immobilization, the immobilized enzyme was recovered by filtration, and then washed with 50 parts by weight of 50 mM phosphate buffer (pH 7) for 30 minutes to remove unimmobilized enzyme and protein. The wet immobilized enzyme was recovered by filtration after washing. Next, the immobilized enzyme recovered and 40 parts by weight of rapeseed oil were brought into contact with each other for 2 hours, and the immobilized enzyme subjected to oil treatment by filtration was recovered. All the above operations were performed at 20 ° C.
The immobilized enzyme prepared by the above operation had an initial expression activity of 2,800 U / g. This immobilized enzyme was packed in a column, and the hydrolysis operation performed by circulating through the column while mixing the reaction substrate (rapeseed oil: water = 100 parts by weight: 60 parts by weight) was continuously repeated. The expression activity of the enzyme decreased with repeated reaction operations, and used immobilized enzymes (activity-reducing enzymes) having various remaining activities were collected.

Example 1
72 parts by weight of ethanol was added to 10 parts by weight (based on dryness) of the used immobilized enzyme having a residual activity of 770 U / g, and the mixture was stirred for 30 minutes. At this time, the fatty acid concentration in the cleaning liquid after the stirring operation was 12.9% by weight. After filtration, the mixture was washed with 50 parts by weight of 50 mM phosphate buffer (pH 7) four times for 30 minutes, ethanol was removed, and the carrier was recovered by filtration. Thereafter, 3.88 parts by weight of lipase (Lipase AY Amano 30, Amano Pharmaceutical Co., Ltd.) was contacted with an enzyme solution dissolved in 180 parts by weight of 50 mM phosphate buffer (pH 7) for 2 hours for immobilization. At this time, when the immobilization rate was determined from the difference between the residual activity of the enzyme solution after immobilization and the activity of the enzyme solution before immobilization, it was 67%. After immobilization, the immobilized enzyme was recovered by filtration, and then washed with 50 parts by weight of 50 mM phosphate buffer (pH 7) for 30 minutes to remove unimmobilized enzyme and protein. The wet immobilized enzyme was recovered by filtration after washing. All the above operations were performed at 20 ° C.
The immobilized enzyme regenerated by the above operation had an initial expression activity of 2,600 U / g.

Example 2
The same procedure as in Example 1 was performed except that the ethanol used was 40 parts by weight. The fatty acid concentration in the washing solution was 22.3% by weight, the enzyme immobilization rate was 73.1%, and the expression activity was 2,401 U / g.

Example 3
The same procedure as in Example 1 was performed except that the residual activity of the used immobilized enzyme was 800 U / g and the ethanol used was 40 parts by weight. The fatty acid concentration in the washing solution was 19.7% by weight, the enzyme immobilization rate was 70.9%, and the expression activity was 2,910 U / g.

Example 4
The same procedure as in Example 1 was conducted except that the used immobilized enzyme had a residual activity of 1,440 U / g and the ethanol used was 72 parts by weight. The fatty acid concentration in the washing solution was 13.1% by weight, the enzyme immobilization rate was 69.6%, and the expression activity was 3,014 U / g.

Example 5
The used immobilized enzyme was dispersed in the same manner as in Example 1 using n-hexane instead of ethanol, and the stirring operation was performed for 30 minutes. At this time, the fatty acid concentration in the solvent after the stirring operation was 12.8% by weight. After filtration, 50 parts by weight of 50 mM phosphate buffer (pH 7) was added, and the mixture was stirred at 20 ° C. under a reduced pressure of 30 Torr for 30 minutes to remove the solvent. After filtration, 50 mM phosphate buffer solution (pH 7) was washed with 50 parts by weight three times for 30 minutes, and filtered to recover the carrier. Subsequent lipase adsorption was carried out in the same manner as in Example 1. The enzyme immobilization rate was 70%, and the expression activity after regeneration was 2,672 U / g.

Example 6
The same operation as in Example 5 was carried out except that the n-hexane used was 110 parts by weight. The fatty acid concentration in the washing solution was 8.9% by weight, the enzyme immobilization rate was 81.4%, and the expression activity was 2,489 U / g.

Example 7
The same procedure as in Example 5 was performed except that the used immobilized enzyme had a residual activity of 1,440 U / g and the n-hexane used was 68 parts by weight. The fatty acid concentration in the washing solution was 13.7% by weight, the enzyme immobilization rate was 67.5%, and the expression activity was 2,768 U / g.

Example 8
The residual activity of the used immobilized enzyme is 605 U / g, 182 parts by weight of n-hexane used, 36.8 parts by weight of lipase (Lipase AY Amano 25L, Amano Pharmaceutical Co., Ltd.), 148 parts by weight of 50 mM phosphate buffer (pH 7) The same procedure as in Example 5 was carried out except that the enzyme solution dissolved in the part was contacted for 2 hours to perform immobilization. The fatty acid concentration in the washing solution was 6.0% by weight, the enzyme immobilization rate was 81.8%, and the expression activity was 2,814 U / g.

Example 9
The same procedure as in Example 8 was performed except that the used immobilized enzyme had a residual activity of 604 U / g and the amount of n-hexane used was 89 parts by weight. The fatty acid concentration in the washing solution was 11.7% by weight, the enzyme immobilization rate was 76.3%, and the expression activity was 3,016 U / g.

Comparative Example 1
The same procedure as in Example 1 was performed except that the residual activity of the used immobilized enzyme was 700 U / g and the ethanol used was 530 parts by weight. The fatty acid concentration in the washing solution was 2% by weight, the enzyme immobilization rate was 81.8%, and the expression activity was 1,744 U / g.

Comparative Example 2
The same operation as in Example 5 was carried out except that the n-hexane used was 24 parts by weight. The fatty acid concentration in the washing solution was 30.4% by weight, the enzyme immobilization rate was 59.8%, and the expression activity was 2,167 U / g.

Comparative Example 3
The same operation as in Example 5 was performed except that n-hexane used was 337 parts by weight. The fatty acid concentration in the washing solution was 3.1% by weight, the enzyme immobilization rate was 83.8%, and the expression activity was 2,200 U / g.

Comparative Example 4
The same procedure as in Example 8 was conducted except that the used immobilized enzyme had a residual activity of 607 U / g and the n-hexane used was 552 parts by weight. The fatty acid concentration in the washing solution was 2.0% by weight, the enzyme immobilization rate was 79.0%, and the expression activity was 2,334 U / g.

  FIG. 1 shows the relationship between the fatty acid concentration in the used immobilized enzyme washing solution and the expression activity of the obtained regenerated immobilized enzyme in each Example and Comparative Example.

It is a figure which shows the relationship between the fatty acid density | concentration in the washing | cleaning liquid of the used fixed enzyme in each Example and a comparative example, and the expression activity of the obtained reproduction | regeneration fixed enzyme.

Claims (2)

The fatty acid equilibrium concentration in the cleaning solution is adjusted to 4 to 28% by washing by adding a solvent to the fat and oil-immobilized enzyme used in the fat hydrolysis reaction, and then the cleaning solution is removed. A method for regenerating an immobilized enzyme for hydrolyzing fats and oils that adsorbs degrading enzymes. Solvent, method of reproduction according to claim 1, wherein ethanol or n- hexane.

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