JP4012117B2 - Method for producing immobilized enzyme - Google Patents

Description

【００３１】
【発明の効果】
本発明方法により、酵素の失活を招く乾燥を行うことなく水分量を調整して、高活性なエステル化反応用固定化酵素を製造することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing an immobilized enzyme having high activity, which is used as a catalyst in an esterification reaction of a fatty acid and an alcohol or an ester exchange reaction of an oil (mono, di or triglyceride).
[0002]
[Prior art]
In order to produce a new glyceride by exchanging the esterified product of a fatty acid and an alcohol and the acyl group of a fat (mono, di or triglyceride), there are an increasing number of cases where an enzyme for decomposing oil or fat is used as a catalyst. In particular, when producing oils and fats having functionality, lipases having position specificity are often used. One method for recovering and reusing this enzyme is to use an immobilized enzyme.
[0003]
Immobilized enzymes that are currently available are all provided in dry form, such as Lipozyme RM IM, Lipozyme TL IM, Novozym 435, etc. sold by Novozymes. The reason why it is in the form of a dry product is that it takes into account the suppression of enzyme deactivation during storage and good handling properties. However, in the process of drying the immobilized enzyme under reduced pressure, vacuum, or heating, the adsorbed enzyme is likely to be deactivated, and the maximum activity at the time of adsorption is not often exhibited when the actual activity is exhibited.
[0004]
In view of this, a method has been proposed in which an enzyme is adsorbed and immobilized on an immobilization carrier and then directly brought into contact with a reaction substrate without drying to carry out an esterification reaction (see Patent Document 1). According to this method, although the reaction is delayed by the influence of a large amount of water contained in the immobilized enzyme in the initial reaction, high activity can be achieved in the second and subsequent reactions. However, it needs to be brought into contact with the reaction substrate promptly and is not suitable for storage in the form of an immobilized enzyme. In addition, a method has been proposed in which the activity expression is enhanced by drying the immobilized enzyme in the contact with a fatty acid derivative (see Patent Document 2). However, in this method, it is necessary to dry slowly, which is not efficient and complicated in setting conditions, and requires expensive equipment, which is not practical.
[0005]
[Patent Document 1]
JP 2000-166589 A [Patent Document 2]
Japanese Patent Laid-Open No. 62-134090 [0006]
[Problems to be solved by the invention]
The enzyme deactivation occurs when the fat and oil-degrading enzyme is changed to a dry immobilized enzyme. Forcibly removing moisture by drying causes damage to the enzyme (for example, destruction of higher-order structures). It is thought that it will end. Therefore, the present invention provides a method for producing an immobilized enzyme for highly active esterification reaction (including transesterification reaction) by adjusting the amount of water without performing drying that causes inactivation of the enzyme. Objective.
[0007]
[Means for Solving the Problems]
The inventor suppresses inactivation of the enzyme and expresses high activity by adjusting the residual water content of the immobilized enzyme using a specific method when producing the immobilized enzyme for esterification reaction. I found that I can do it.
[0008]
That is, in the present invention, the enzyme for fat and oil decomposition is adsorbed and immobilized on an immobilizing carrier, and then is contacted with a fatty acid triglyceride or a fatty acid partial glyceride without drying. The present invention provides a method for producing an immobilized enzyme for an esterification reaction in which the enzyme moisture is adjusted so that the amount of moisture is adjusted.
[0009]
In the present invention, the enzyme for decomposing oil and fat is adsorbed and immobilized on an immobilizing carrier, and then directly contacted with 20 to 3000% by weight of fatty acid, fatty acid triglyceride or fatty acid partial glyceride without drying. An object of the present invention is to provide a method for producing an immobilized enzyme for esterification reaction in which the enzyme moisture is adjusted so that the residual moisture content is 1 to 50% by weight based on the weight of the carrier by dehydration.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The carrier for immobilization used in the present invention is an inorganic carrier such as celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics, ceramic powder, polyvinyl alcohol, polypropylene, chitosan, ions An organic polymer such as an exchange resin, a hydrophobic adsorption resin, a chelate resin, or a synthetic adsorption resin may be mentioned, and an ion exchange resin is particularly desirable.
[0011]
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.
[0012]
A lipase is preferable as the oil-degrading enzyme used in the present invention. As the lipase, not only those derived from animals and plants but also commercially available lipases derived from microorganisms can be used. The microbial origin lipase, Rhizopus (Rizopus) genus Aspergillus (Aspergillus) genus, Mucor (Mucor) genus Pseudomonas (Pseudomonas) genus, Jiotorikemu (Geotrichum) genus Penicillium (Penicillium) genus Candida (Candida) of the genus, etc. The thing of origin is mentioned. Especially for the purpose of producing functional fats and oils, the Aspergillus genus Rizopus, which is a position-specific lipase that can selectively form a bond at the target position of glycerin, is a position-specific lipase. (Aspergillus) genus Mucor (Mucor) genus Pseudomonas (Pseudomonas) genus Jiotorikemu (Geotrichum) genus, it is desirable to utilize a Penicillium (Penicillium) genus.
[0013]
When immobilizing these enzymes, the carrier and the enzyme may be adsorbed directly, but in order to achieve an adsorption state that expresses high activity, the carrier is treated with a fat-soluble fatty acid or a derivative thereof in advance before the enzyme adsorption. May be used. Examples of the fat-soluble fatty acid to be used include saturated or unsaturated, linear or branched fatty acids having 8 to 18 carbon atoms, which may be substituted with a hydroxyl group. Specifically, capric acid, lauric acid, myristylic acid, oleic acid, linoleic acid, α-linolenic acid, ricinoleic acid, isostearic acid and the like can be mentioned. Examples of the derivatives include esters of these fatty acids with mono- or polyhydric alcohols, phospholipids, and derivatives obtained by adding ethylene oxide to these esters. Specific examples include methyl esters, ethyl esters, monoglycerides, diglycerides, ethylene oxide adducts thereof, polyglycerin esters, sorbitan esters, and sucrose esters of the above fatty acids. Two or more of these fat-soluble fatty acids or derivatives thereof may be used in combination.
[0014]
As a method for contacting these fat-soluble fatty acids or derivatives thereof and the carrier, these may be added directly to water or an organic solvent, but in order to improve dispersibility, the fat-soluble fatty acids or derivatives thereof are once dispersed in the organic solvent. After dissolution, it may be added to a carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, ethanol, and the like. The amount of the fat-soluble fatty acid or derivative thereof used is preferably 1 to 500%, particularly preferably 10 to 200%, based on the weight of the carrier. The contact temperature is preferably 0 to 100 ° C., particularly preferably 20 to 60 ° C., and the contact time is preferably about 5 minutes to 5 hours. The carrier after this treatment is collected by filtration, but may be dried. The drying temperature is preferably room temperature to 100 ° C, and drying under reduced pressure may be performed.
[0015]
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.
[0016]
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. Moreover, the enzyme solution can also use what was refine | purified by the supernatant obtained by removing the insoluble part by centrifugation, ultrafiltration, etc. as needed. The amount of enzyme used is preferably 5 to 1000%, particularly 10 to 500%, based on the weight of the carrier.
[0017]
In the present invention, after the fat and oil-degrading enzyme is adsorbed and immobilized on the immobilizing carrier, it is not dried, and is contacted with (A) fatty acid triglyceride or fatty acid partial glyceride, or (B) fatty acid, fatty acid triglyceride or The residual water content is adjusted by dehydrating while contacting the fatty acid partial glyceride. In the present invention, “without drying” means “without subjecting to drying by reduced pressure, vacuum or heating”.
[0018]
The residual water content is adjusted to 5 to 50% by weight in the case of treatment (A), but preferably 15 to 50% by weight. In the case of treatment (B), the content is adjusted to 1 to 50% by weight, preferably 1 to 30% by weight.
[0019]
Fatty acid triglyceride and fatty acid partial glyceride to be contacted with the immobilized enzyme in the moisture adjustment treatment (A) include vegetable liquid oils such as rapeseed oil, soybean oil, sunflower oil, fish oil such as sardine oil, tuna oil, bonito oil, Sea animal oil such as whale oil, monoglycerides and diglycerides derived therefrom, and mixtures thereof, and transesterified fats and oils obtained from these fats and oils can also be used. Two or more of these may be used in combination. In the water adjustment treatment (B), not only the fatty acid triglyceride and the fatty acid partial glyceride, but also fatty acids generated from these compounds can be used. The fatty acid used in the treatment (B) is preferably a vegetable liquid oil such as rapeseed oil, soybean oil or sunflower oil, or a fatty acid produced from fish oil such as sardine oil, tuna oil or bonito oil. The fatty acid, fatty acid triglyceride or fatty acid partial glyceride used in these treatments (A) or (B) is an oil in an actual esterification reaction or transesterification reaction using the immobilized enzyme prepared by the method of the present invention. It is preferable to select what is used as a phase substrate.
[0020]
The amount of fatty acid glyceride used in the treatment (A) is preferably 500 to 5000% based on the weight of the carrier from the viewpoint of sufficient contact with the immobilized enzyme and avoiding waste due to use of an excessive amount. Further, 800 to 4000%, particularly 1000 to 3000% is preferable. Further, when the residual water content may be 15 to 50% by weight relative to the carrier weight, the amount of fatty acid glyceride used is preferably 500 to 3000%, particularly preferably 800 to 2500%, based on the carrier weight. When the water content is reduced to 5 to 15% by weight based on the carrier weight, the amount of fatty acid glyceride used is preferably 2000 to 5000%, particularly 2500 to 4000%, based on the carrier weight. Further, the amount of the fatty acid or fatty acid glyceride used in the treatment (B) is 20 to 3000% based on the weight of the carrier from the same viewpoint as described above and from the viewpoint of improving fluidity and improving dehydration efficiency. 100 to 1000% is preferable.
[0021]
The contacting method of the immobilized enzyme and fatty acid glyceride in the treatment (A) may be any method such as immersion, stirring, or passing through a column filled with the immobilized enzyme with a pump or the like. The contact temperature may be any temperature as long as the oil phase does not solidify during the contact, and can be appropriately determined according to the characteristics of the fatty acid glyceride used and the characteristics of the enzyme. preferable. The contact time is suitably 0.1 to 72 hours, but the contact time may be longer than this time, or the contact time may be stored while being in contact with the fatty acid glyceride. In addition, when dehydrating while contacting with fatty acid or fatty acid glyceride in the treatment (B), the temperature at the time of dehydration is the same as in the case of the treatment (A), and the dehydration time is suitably 0.5 to 24 hours. In the case of the treatment (B), rapid dehydration is possible, and the dehydration process can be completed in a short time by setting the rate of water decrease per hour to 50% or more, preferably 60% or more, particularly 70% or more. be able to. As the dehydration method, a known method such as a method using a dehydrating agent such as molecular sieves or a method of treating in a reduced pressure system is adopted, but if a dehydrating agent is used, an operation such as removal of the dehydrating agent is necessary after the treatment. In view of this, it is desirable to perform the treatment in a reduced pressure system.
[0022]
When the enzyme is adsorbed and immobilized on a carrier, the water content of the immobilized enzyme is usually in the range of 120 to 200% of the weight of the carrier. Residual moisture can be reduced. Further, when dehydrating while contacting with (B) fatty acid or fatty acid glyceride, the residual moisture can be reduced from 1 to 50% to the weight of the carrier. After the contact treatment or dehydration treatment is completed or before the immobilized enzyme is used, the immobilized enzyme is recovered by filtration and used in the actual reaction.
[0023]
Thus, according to the present invention, by adjusting the amount of water using a fatty acid or fatty acid glyceride, damage to the enzyme that occurs during forced removal of water as in normal drying is reduced as much as possible. Can do. For this reason, it is possible to prepare an immobilized enzyme having high activity expression. It is also considered that excess water is removed by contacting the immobilized enzyme with fatty acid glycerides and the like, and the vicinity of the enzyme forms a reaction field suitable for the reaction. Furthermore, the immobilized enzyme whose water content has been adjusted by the method of the present invention can be stored for a long period of time either in a state separated from the contacted fatty acid glyceride or the like or in a state of being kept in contact.
[0024]
【Example】
Example 1
100 g of Duolite A-568 (Rohm and Hass) was stirred in 1 L of N / 10 NaOH solution for 1 hour. After filtration, it was washed with 1 L of distilled water, and the pH was equilibrated with 1 L of 500 mM acetate buffer (pH 5). Thereafter, pH equilibration was performed twice for 2 hours with 1 L of 50 mM acetate buffer (pH 5). The carrier was recovered by filtration, and then replaced with 500 mL of ethanol for 30 minutes. After filtration, 500 mL of an ethanol solution containing 100 g of ricinoleic acid was brought into contact with the carrier for 30 minutes. After filtration, buffer replacement was performed 4 times for 0.5 hours with 500 mL of 50 mM acetate buffer (pH 5). After filtration, the enzyme was adsorbed by contacting with 1000 mL of a 10% strength lipase solution (manufactured by Nagase Sangyo Co., Ltd.) at room temperature for 4 hours. After adsorption, it was filtered and washed with 500 mL of 50 mM phosphate buffer (pH 5) for 0.5 hour. The immobilized enzyme was recovered by filtration after washing. The residual water content of the immobilized enzyme at this time was 168% with respect to the weight of the adsorption carrier.
To this immobilized enzyme, 1000 g of rapeseed oil was added, stirred at 40 ° C. for 24 hours, and then filtered to recover the immobilized enzyme. The residual moisture content of the immobilized enzyme was 29% based on the weight of the adsorption carrier.
8 g of the immobilized enzyme thus obtained was weighed as a dry weight and charged into a 200 mL four-necked flask. Thereto was added 80 g of a mixture of oleic acid and glycerin (oleic acid / glycerin = 2.0 in molar ratio), and an esterification reaction was performed at 40 ° C. under reduced pressure of 400 Pa. After the reaction, the reaction solution and the immobilized enzyme were separated by filtration, and oleic acid and glycerin were charged again so that the above charging was performed, and the reaction was performed. After this reaction, the reaction was performed once again in the same manner. That is, a total of three esterification reactions were performed using the same immobilized enzyme.
The glyceride composition of each reaction solution was analyzed by gas chromatography after trimethylsilylation of the reaction solution. Table 1 shows the time when the total of diglyceride (DG) + triglyceride (TG) was 70%. The first and second and subsequent reaction times were 1.67 hr and 1.53 hr, respectively, which were sufficiently fast and highly active.
[0025]
Example 2
The enzyme was adsorbed by the same method as in Example 1, washed with a buffer solution, and the immobilized enzyme was recovered by filtration. 3000 g of rapeseed oil was added to this immobilized enzyme, stirred for 24 hours at 40 ° C., and then filtered to recover the immobilized enzyme. The residual moisture content of the immobilized enzyme was 11% with respect to the weight of the adsorption carrier.
Using the immobilized enzyme thus obtained, the reaction was carried out in the same manner as in Example 1. As a result, the first and second and subsequent reaction times were 1.69 hr and 1.52 hr, respectively, which were sufficiently fast and highly active.
[0026]
Example 3
The enzyme was adsorbed by the same method as in Example 1, washed with a buffer solution, and the immobilized enzyme was recovered by filtration. 400 g of oleic acid was added to the immobilized enzyme, and the mixture was stirred for 0.5 hours under reduced pressure at 40 ° C. and 400 Pa, and then filtered to recover the immobilized enzyme. The residual moisture content of the immobilized enzyme was 31% with respect to the weight of the adsorption carrier.
Using the immobilized enzyme thus obtained, the reaction was carried out in the same manner as in Example 1. As a result, the first and second and subsequent reaction times were sufficiently fast, 1.63 hr and 1.58 hr, respectively, and were highly active.
[0027]
Example 4
The enzyme was adsorbed by the same method as in Example 1, washed with a buffer solution, and the immobilized enzyme was recovered by filtration. 400 g of oleic acid was added to the immobilized enzyme, and the mixture was stirred for 18 hours under reduced pressure at 40 ° C. and 400 Pa, and then filtered to recover the immobilized enzyme. The residual moisture content of the immobilized enzyme was 2.4% with respect to the weight of the adsorption carrier.
Using the immobilized enzyme thus obtained, the reaction was carried out in the same manner as in Example 1. As a result, the first and second reaction times were 1.45 hr and 1.43 hr, respectively, sufficiently early from the first reaction and highly active. there were.
[0028]
Comparative Example 1
The enzyme was adsorbed by the same method as in Example 1, washed with a buffer solution, and the immobilized enzyme was recovered by filtration. The residual water content of the immobilized enzyme at this time was 178% with respect to the weight of the carrier. The immobilized enzyme was dried as it was at 40 ° C. under a reduced pressure of 100 Pa for 24 hours. The residual moisture content of the immobilized enzyme after drying was 3% with respect to the weight of the adsorption carrier.
Using the immobilized enzyme thus obtained, the reaction was carried out in the same manner as in Example 1. As a result, the reaction times for the first time and the second time and later were 2.27 hr and 2.23 hr, respectively. Thus, the esterification activity was inferior.
[0029]
Comparative Example 2
The enzyme was adsorbed by the same method as in Example 1, washed with a buffer solution, and the immobilized enzyme was recovered by filtration. The residual water content of the immobilized enzyme at this time was 178% with respect to the weight of the carrier.
Using the immobilized enzyme thus obtained, the reaction was carried out in the same manner as in Example 1. As a result, the first and second reaction times were 3.52 hr, 1.88 hr and the second and subsequent times, respectively, which were sufficiently fast and highly active. However, the first reaction was slower than in Examples 1 to 4.
[0030]
[Table 1]

[0031]
【The invention's effect】
According to the method of the present invention, a highly active immobilized enzyme for esterification reaction can be produced by adjusting the amount of water without drying that causes inactivation of the enzyme.

Claims (4)

After the lipase is adsorbed and immobilized on the carrier for immobilization, it is not dried, and only the fatty acid triglyceride or the fatty acid partial glyceride is brought into contact with the enzyme so that the residual water content is 5 to 50% by weight based on the weight of the carrier. A method for producing an immobilized lipase for esterification reaction that adjusts moisture.   The method for producing an immobilized lipase according to claim 1, wherein the amount of the fatty acid triglyceride or the fatty acid partial glyceride is 500 to 5000% based on the weight of the carrier. After the lipase is adsorbed and immobilized on the carrier for immobilization, the carrier weight is obtained by dehydrating while contacting only 20 to 3000% by weight of fatty acid, fatty acid triglyceride or fatty acid partial glyceride with respect to the weight of the carrier without directly drying. A method for producing an immobilized lipase for an esterification reaction, wherein the moisture content of the enzyme is adjusted so as to have a residual moisture content of 1 to 50% by weight.   The method for producing an immobilized lipase according to any one of claims 1 to 3, wherein the fatty acid, fatty acid triglyceride or fatty acid partial glyceride to be contacted with the immobilized lipase after adsorbing the lipase is an oil phase substrate of lipase.