KR100817775B1 - A method for pretreating lipase before immobilizing the same on carrier - Google Patents

Abstract

A method for pretreating lipase before its immobilization is provided to improve activity of lipase when it is immobilized on the carrier without use of complicated procedures such as control of carrier pore size or addition of other additives during immobilization, so that the expensive lipase is reused repeatedly without loss of activity. A pretreatment of lipase is carried out by reacting lipase with a fatty acid as a substrate of lipase or oil before its immobilization to combine the fatty acid to the active site of lipase, so that the active site of lipase is protected, wherein the fatty acid is a fatty acid having the carbon number of 16-18 such as linoleic acid, oleic acid, stearic acid or palmitic acid, and the oil is vegetable oil such as olive oil, soybean oil or palm oil.

Description

A method for pretreating lipase before immobilizing the same on carrier

1 is Candida Lugosa rugosa ) is a graph showing the effect of pretreatment time on the activity of immobilized lipase in the case of immobilization of lipase.

FIG. 2 shows a lyzofoss uraise ( Rhizopus) oryzae ) is a graph showing the effect of pretreatment on the activity of lipase in the case of immobilization of lipase.

Figure 3 is a graph showing the change of lipase activity when reused immobilized Candida Lugosa lipase after a pretreatment process.

Figure 4 is a graph showing the change in the activity of lipases when reusing the immobilized lyphos lipase lipase after pretreatment.

5 is a simplified schematic of pretreatment of lipase prior to immobilization. Here, A shows that when the untreated lipase is immobilized on the activated silica gel, many amino groups are covalently bound near the active site, resulting in a significant decrease in the activity of the lipase, while B is a lipase pretreated with soybean oil. It is shown that steric hindrance by fatty acids promotes covalent bond production at lipase sites that are not related to lipase function and consequently remain active while lipase activity is immobilized.

6 is a graph showing the activity of immobilized lipase during immobilization. Where A is for untreated lipases and B is for lipases pretreated with soybean oil.

7 is a graph showing the effect of pretreatment material on the activity of immobilized lipase. Here, NO represents a non-pretreated lipase, SO represents a soybean oil pretreated lipase, LA represents a linoleic acid pretreated lipase, and GL represents a glycerol pretreated lipase.

8 is a graph showing the reusability of immobilized lipase. Where? Represents an untreated lipase, and ○ represents a case of soybean oil pretreated lipase.

9 is a graph showing the effect of pretreatment temperature on immobilized lipase activity. Where ● is soybean oil, ○ is olive oil, ▼ is linoleic acid, and ▽ is oleic acid.

10 is a graph showing the effect of stirring speed on the immobilized lipase activity. Where ● is soybean oil, ○ is olive oil, ▼ is linoleic acid, and ▽ is oleic acid.

11 is a graph showing the effect of pretreatment concentration on immobilized lipase activity. Where ● is soybean oil, ○ is olive oil, ▼ is linoleic acid, and ▽ is oleic acid.

12 is a graph showing the effect of pretreatment time on immobilized lipase activity. Where ● is soybean oil, ○ is olive oil, ▼ is linoleic acid, and ▽ is oleic acid.

13 is a graph showing the reusability of immobilized lipase. Where ● represents soybean oil, and ○ represents linoleic acid.

The present invention relates to a method of pretreatment of lipase before immobilization on the carrier, and more particularly, the lipase activity is increased even after immobilization on the carrier by a simple pretreatment process in which the lipase is reacted with fatty acids or oils before immobilization on the carrier. The present invention relates to a pretreatment method for immobilization of a lipase that can be maintained as well as maintain high activity upon reuse.

The bioprocess using enzymes is energy-saving compared to chemical processes due to the reaction proceeding at room temperature and pressurized conditions, and the process is simple, which has the advantage of low process cost. However, the price of the enzyme used is expensive and the stability of the enzyme is a disadvantage to be overcome. To solve this problem, immobilization of enzymes is used. By immobilizing the enzyme, the enzyme used in the reaction can be reused in the next reaction, thereby solving the problem of the price of the enzyme and increasing the stability than the enzyme without immobilization, so that it can be used more efficiently in the process.

The method of immobilization of the enzyme involves adsorbing a polyamine to a metal oxide such as alumina treated with an excess of a bifunctional agent such as glutaraldehyde to crosslink the amine, enclosing the resulting polymer in the pores of the metal oxide, binding aldehyde groups and enzymes. It is described in US Pat. No. 4,141,857 by Levi and Push as contacting an enzyme and a lump to form a covalent bond between amino groups. The useful life of an immobilized enzyme system is limited to the continuous decrease in enzyme activity. Many of the mechanisms that lead to inactivation of enzymes in this system include enzyme bonders and enzymes that cause enzymes to be read as impurities in the raw material, cause other chemical variations of the enzymes during use, denature the enzymes, or cause degradation of the enzymes. This may be due to the rupture of the bond or the breakdown of the bond between the bonder and the intermediate bond layer, or the loss of the bond layer due to chemical cleavage or physical cleavage that binds the enzyme to the support.

Lipase is an enzyme that can cause various reactions such as hydrolysis of copper and vegetable oil, esterification which is the reverse reaction, and esterification by transesterification of alcohol and oil. . In addition, its industrial value is increasing day by day, used to separate and produce optically active materials. The preparation of immobilized lipases is essential for such lipases to be used in real industry.

Various studies have been made to prepare immobilized lipases, and adsorption and covalent bonding methods are typically used. Lipase immobilization by adsorption method has the advantage of high activity of lipase, but the loss of lipase easily occurs, which is not suitable for the primary reuse of immobilization. However, lipase immobilization by covalent bonding is an immobilization method suitable for multiple reuses because lipase loss hardly occurs.

By covalent bonding, a method of covalently bonding an aldehyde group attached to a carrier with an amino group present in a lipase is used. However, if the amino group near the active site of the lipase is covalently bonded to the aldehyde group of the carrier, the active site of the lipase may not function properly or it may be difficult to contact the substrate, thereby significantly reducing its activity.

Therefore, in order to prevent the degradation of the lipase activity during immobilization, the conventional method relies on complex methods such as controlling the pore size of the carrier or immobilizing other proteins without enzyme activity (Cleide M. F Soares et al., Biotechnol . Prog) . ., 19, pp803-807, 2003) .

Accordingly, the inventors of the present invention can maintain the activity of the enzyme itself by performing a pretreatment process before immobilizing the lipase to the carrier in a simple manner without using the pore size or other additives of the carrier when the lipase is immobilized. While studying, the active site of lipase can be protected by masking that glutaraldehyde and the enzyme's functional region on the surface of the carrier can be prevented from forming covalent bonds. The present invention was completed by providing a pretreatment method capable of preventing covalent bonds from being formed near the active site upon immobilization and confirming that the lipase immobilized by this pretreatment can be repeatedly reused several times.

An object of the present invention is to perform a pretreatment process by adding a fatty acid or oil to the lipase before the lipase is immobilized on the carrier to maintain the activity of the immobilized lipase as well as to maintain high activity even after several reuses. It is to provide a method for pretreatment of cylipase.

In order to achieve the above object, the present invention is characterized by undergoing a simple pretreatment process of reacting lipase with fatty acid or oil to bind the lipase substrate to the active site of lipase before immobilizing the lipase on the carrier. The pretreated lipase as described above can maintain high activity when immobilized on the carrier.

Hereinafter, the present invention will be described in detail.

The present invention provides a pretreatment method for immobilizing a lipase using a carrier, wherein the lipase is reacted with a fatty acid or an oil to bind the fatty acid, which is a substrate of the lipase, to the active site of the lipase before the lipase is immobilized on the carrier.

The present invention provides a lipase immobilization method comprising immobilizing a lipase on a carrier after pretreatment of binding a lipase substrate to an active site of the lipase before immobilizing the lipase on the carrier.

When oil is reacted with lipase to induce the hydrolysis reaction of oil, the oil is broken down into glycerol and fatty acid by lipase. At this time, since the produced fatty acid is lipophilic, it is difficult to diffuse into the aqueous solution, so that some remain in the active site of the lipase, which protects the active site of the lipase during immobilization, thereby allowing the immobilized lipase to have high activity.

In addition, the lipase has a structural lead (Lid) has a feature of surface activation (Interfacial activation) that increases the activity when the substrate is combined with the lead. Through the pretreatment process of the present invention, the lipase is made to have a state of surface activity, and the lipase is immobilized in such a state to be immobilized at a later time, so that the lipase can have high activity when immobilized.

Fatty acids having at least 16 carbons are preferred as fatty acids that may be used for the pretreatment of lipase in the present invention, but more preferably, linoleic acid (18 carbon atoms), oleic acid (18 carbon atoms), stearic acid (16 to 18 carbon atoms) 16 carbon atoms) and palmitic acid (16 carbon atoms) are preferred, and vegetable oils are preferred as oils, but olive oil, soybean oil, and palm oil are more preferable.

The lipase immobilization method of the present invention may be any method known in the art as a method of immobilizing lipase to a carrier by a covalent bond method, and any carrier may be used if it is known in the art. Specific examples include silica gel.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. The following Examples and Experimental Examples are merely illustrative of the present invention and do not limit the present invention.

Example  1: Preparation of Lipase

Kendida Lugosa ( Candida rugosa Lipase

Lipase OF ^™ , a nonspecific lipase made from Candida rugosa , was purchased from Meito Sangyo, Japan. 1 g of the lipase was suspended in 100 mL of 1 mM phosphate buffer (pH 7) and then centrifuged at 4000 g for 15 minutes at 4 ° C. The supernatant obtained thereafter was used for immobilization while stored at 4 ° C.

Riposos Orize ( Rhizopus oryzae Lipase

Rhizopus oryzae ) KCCM 11970 was incubated for 2 days at 50 ° C. in PDB medium at 28 ° C. and 200 rpm. The cultures were grown in 50 ml of distilled water in 4% olive oil, 8% bactopeptone, and 0.1% KH ₂ PO _4. , 0.1% NaNO ₃ , 0.05% MgSO ₄ · 7H ₂ O was added to the production medium to adjust the pH to 6.0 as a medium medium spawn inoculation at a rate of 10% v / v 28 ℃. Lipases were produced by incubation at 250 rpm for 5 days.

Cells were removed by centrifugation of 50 ml of lyphos oriase culture at 10,000 rpm for 15 minutes at 4 ° C. The lipase was precipitated by adding 60% (g / v) ammonium sulfate to the supernatant containing lipase at 4 ° C. for 6 hours. The supernatant was removed by centrifugation at 10,000 rpm for 15 minutes at 4 ° C. The precipitated lipase was again dissolved in 10 ml of 0.05 M phosphate buffer (pH 7.0) to prepare an aqueous lipase solution. Ultrafiltration was used to remove substances other than proteins, including salt, from the solution to obtain a lipase partially purified solution.

Example  2: for lipase immobilization Carrier  Produce

Activated silica gel was prepared as a carrier.

First, 1 g of dried silica gel was mixed with 20 mL of an acetone solution of 10% (v / v) 3-aminopropyltriethoxysilane and reacted at 50 ° C. for 2 hours with constant stirring. The silica gel reacted as above was washed with water and dried at 60 ° C. for 2 hours. The dried silica gel was suspended in 20 mL of 1 mM phosphate buffer (pH 7). 2 mL of a 25% (w / v) glutaraldehyde solution was added to the silica gel suspension and reacted at 20 ° C. for 2 hours to activate the silica gel. The activated silica gel was washed with water and then dried at 60 ° C. for 2 hours.

Example  3: pretreatment of lipase with olive oil

200U Candida Lugosa liposa 10 ml of lipase (Lipase OF ^™ , Meito Co., Japan) was reacted with 10 ml of olive oil for 5, 10, 30 50 minutes to induce hydrolysis of olive oil to pretreat lipase. .

100 U of lyphos Orisase obtained in Example 1 ( Rhizopus oryzae ) 10 ml of lipase aqueous solution was reacted with 10 ml of olive oil for 30 minutes to induce hydrolysis of olive oil to pretreat lipase.

Example  4: using olive oil Preprocessed  Immobilization of Lipase

500 mg of the activated silica gel prepared in Example 2 and 10 mL of each lipase solution pretreated in Example 3, that is, Candida Lugosa lipase solution or lyphos lipase lipase solution, were mixed and then reacted at 20 ° C. for immobilization. I was. Each immobilized lipase was recovered by filtration, washing with water and drying at room temperature overnight.

Experimental Example  1: Effect of Pretreatment of Lipase with Olive Oil on the Activity of Immobilized Lipase

Candida Lugosa  Lipase

In Example 4, the activity of the immobilized Candida Lugosa lipase was observed in the following manner, except that Candida Lugosa lipase, which was not subjected to the same pretreatment as Example 3, was used. The activity of Candida Lugosa lipase immobilized in the same manner as was observed.

10 ml of isooctane dissolved in 10 ml of distilled water and 10% (g / v) olive oil was added to a 100 ml Erlenmeyer flask, followed by addition of 1 ml of lipase immobilized in Example 4, 37 ° C. for 30 minutes. The hydrolysis reaction was performed at 150 rpm. Fatty acids from the breakdown of olive oil by lipase are dissolved in the isooctane layer. After 30 minutes of reaction, the reaction was terminated with 1 ml of 5N HCl, and 2 ml of isooctane dissolved in the dissolved fatty acid was taken and reacted with 0.5 ml of Cu-acetate-pyridine solution. Fatty acid is produced by the saponification reaction with Cu ^{2 +} , the absorbance was measured at 715nm. Knowing the absorbance, it is possible to know the amount of fatty acids produced by decomposition through the reference curve. The activity of the lipase was calculated based on the number of μmol of the fatty acid produced for 1 minute at 1 U.

As a result, as shown in FIG. 1, when the 200 U Candida Lugosa lipase was immobilized on the carrier, the activity of the immobilized lipase which was not pretreated was 1.3 U / g, and most of the activity was observed. However, the activity of the immobilized lipase after pretreatment was higher than 81 U / g, indicating that the activity was maintained very high. Among them, the activity of immobilized lipase after pretreatment for 30 minutes was the most excellent at 90 U / g. I could confirm it.

Riposos Orize ( Rhizopus oryzae Lipase

In Example 4, the activity of the immobilized lyphos lipase lipase was observed in the following manner, except that the control was used as a lyphos lipase lipase that did not perform the same pretreatment as in Example 3 above. The activity of the lyphos lipase lipase immobilized in the same manner as in Example 4 was observed.

As a result, as shown in FIG. 2, it was confirmed that the lipase activity was significantly increased when the lipase was pretreated than when not pretreated. Therefore, it was confirmed that this pretreatment method can be used for immobilization of all lipases according to the present experimental example.

Experimental Example  2: Investigation of the activity change upon reuse of lipase immobilized after pretreatment with olive oil

Candida Lugosa  Lipase

Since the purpose of immobilizing the lipase was to reuse the lipase after the reaction was completed, the change in activity upon reuse was investigated. After the pretreatment in Example 3 for 30 minutes, the activity change was examined in the same manner as in Experiment 1 by reusing 5 times the immobilized Candida lugosa lipase as in Example 4. Olive oil was hydrolyzed using the immobilized lipase for 30 minutes, and after the reaction, the immobilized lipase was collected and subjected to hydrolysis again to observe the change in activity.

As a result, as shown in Figure 3 it was confirmed that the activity of the immobilized lipase is maintained about 100% even after repeated use five times, thereby confirming that the pre-immobilization pretreatment method of the present invention is very effective.

Riposos Orize ( Rhizopus oryzae Lipase

After the pretreatment in Example 3, the activity change upon reuse of the lysophoric lipase lipase immobilized in Example 4 was observed by the same method as the method for investigating the activity change upon reuse of the Candida Lugo.

As a result, as shown in Figure 4 it was confirmed that the lipase activity is maintained 100% even after 5 times reuse.

Example  5: Soybean oil , Linoleic acid  Or pretreatment of lipase with glycerol

Pretreatment materials, that is, 200 mg of soybean oil, linoleic acid or glycerol, respectively, were added to 20 mL of the lipase solution prepared in Example 1 and mixed. The mixture was reacted at 37 ° C. for 30 minutes while stirring at a constant rate. The resulting 10 mL of lipase solution was used for immobilization.

Example  6: Soybean oil , Linoleic acid  Or using glycerol Preprocessed  Immobilization of Lipase

500 mg of the activated silica gel prepared in Example 2 and 10 mL of the lipase solution pretreated in Example 5 were mixed and then reacted at 20 ° C. for immobilization. The reaction solution was filtered, washed with water and dried at room temperature overnight to recover the immobilized lipase.

Experimental Example  3: Soybean oil , Linoleic acid  Effect of Pretreatment of Lipase with Glycerol on the Activity of Immobilized Lipase

In Example 6, the activity of the immobilized Candida lugosa lipase was observed in the following manner, except that Candida Lugosa lipase, which was not subjected to the same pretreatment as Example 5, was used. The activity of Candida Lugosa lipase immobilized in the same manner as was observed.

10 mL of isooctane, dissolved in 10% (w / v) soybean oil, was added to 10 mL of 50 mM phosphate buffer (pH 7) mixed with 200 mg of immobilized lipase. The reaction mixture was reacted for 30 minutes with stirring at 150 rpm in a 37 ° C vacuum bath. 2 mL of the supernatant was taken into a test tube and reacted with 0.5 ml of a copper-acetate-pyridine solution. Free fatty acids were quantified by measuring absorbance at 715 nm. The activity of the lipase was calculated based on the number of μmol of the fatty acid produced for 1 minute at 1 U.

Lipase immobilization was performed in hydrophilic buffer. Since fatty acids are lipophilic, once fatty acids surround the active site, it is difficult to escape the active site and remain in the active site to protect (mask) the active site. In addition, it is expected that the amino group present in the enzyme site far from the active site will react with the glutaraldehyde present in the silica gel upon immobilization.

Figure 5 shows a schematic diagram of the lipase pretreatment process before immobilization. Soybean oil was added to the lipase solution and reacted for 30 minutes in a stirred reactor to protect the active site of the lipase. In the reaction mixture, lipase hydrolyzed soybean oil to fatty acids and glycerol. Glycerol is hydrophilic, so it is easily separated into an aqueous layer, but fatty acids remain on lipase active sites. Lipase remaining in the mixture was then immobilized on the activated silica gel. The reaction was named pretreatment step.

To test the pretreatment effect on the activity of the immobilized lipase, activated silica gel was added to the untreated and pretreated lipases. It took 2 hours to immobilize the unprepared lipase and the activity of the immobilized lipase was 32 U / g-matrix. However, the pretreated lipase had an activity of 532 U / g-matrix after immobilization for 12 hours or more (FIG. 6). Therefore, it was found that pretreatment of lipase before immobilization improved the activity of immobilized lipase by 16 times or more. Furthermore, it took significantly more time for immobilization for pretreated lipases than for untreated lipases, due to the steric effect of fatty acids present in the active site, and for pretreated lipases to react with glutaraldehyde on the surface. It was judged that this was because the number of amino groups that could be decreased.

The effects of glycerol and fatty acids in the pretreatment step were examined by treating lipases with glycerol and linoleic acid, the main constituents of soybean oil, under the same conditions, respectively. Figure 7 shows that glycerol is not effective for the pretreatment of lipases. This is because lipase pretreated with glycerol is similar to the activity of unpretreated lipase. On the other hand, when lipase was pretreated with linoleic acid, its immobilization activity was found to be 400 U / g-matrix, which was found to be effective for masking the active site of lipase in the case of linoleic acid.

Experimental Example  4: Soybean oil , Linoleic acid  Effect of Pretreatment of Lipase with Glycerol on Recycling of Immobilized Lipase

The Candida Lugosa lipase immobilized in the same manner as in Example 6 was used in a series of hydrolysis reactions, except that Candida Lugosa lipase immobilized in Example 6 and Candida Lugosa lipase without pretreatment were used. It was. After a single batch reaction, the immobilized lipase was filtered off, washed with water and then reused in the next batch reaction. 8 shows that the immobilized lipase activity retains at least 90% of the initial activity even after 10 reuses. It was confirmed that the lipase immobilized after being pretreated with soybean oil or linoleic acid does not lose its activity despite several reuses.

Experimental Example  5: Investigation of the activity of immobilized lipase according to the kind of pretreatment

With each of various oils or fatty acids as a pretreatment material, 200 mg of each pretreatment material was added to 20 mL of the lipase solution prepared in Example 1 and mixed. The mixture was reacted at 37 ° C. for 30 minutes while stirring at a constant rate. The resulting 10 mL of lipase solution was used for immobilization.

500 mg of the activated silica gel prepared in Example 2 and 10 mL of the lipase solution pretreated in Example 7 were mixed and then reacted at 20 ° C. for immobilization. The reaction solution was filtered, washed with water and dried at room temperature overnight to recover the immobilized lipase.

As a result of examining the activity of the lipase immobilized by different pretreatment materials as described above, it was as Table 1 below.

Effect of Pretreatment Types on Immobilized Lipase Activity matter Active (U / g matrix) matter Active (U / g matrix) Linoleic acid 397.7 ± 7.3 Acetic acid 23.3 ± 3.7 Oleic acid 367.7 ± 4.3 Soybean oil 530.7 ± 6.3 Stearic acid 308.0 ± 5.0 Olive oil 513.3 ± 5.7 Palmitic acid 343.3 ± 2.7 Palm oil 350.0 ± 10.0 Dodecanoic acid 163.3 ± 1.7 Tributyrin 32.7 ± 2.3 Octanoic acid 89.3 ± 5.7 Triacetin 33.0 ± 3.0 Butyric acid 27.7 ± 2.3 Glycerol 31.7 ± 1.3 Propionic acid 42.0 ± 2.0 No pretreatment 32.3 ± 1.7

As described in the above experimental examples, pretreatment of lipase before immobilization causes fatty acids to remain in the active site and exerts a three-dimensional effect. As a result, glutaraldehyde on the surface is covalently bonded to amino groups located far from the active site. Therefore, in view of the above, it can be assumed that the longer fatty acid is more effective in protecting the lipase active site upon immobilization. As can be seen from Table 1, it can be seen that fatty acids having 16 or more carbons, such as linoleic acid, oleic acid, stearic acid, and palmitic acid, are very effective in pretreatment of lipases. After pretreatment with these substances, the activity of the immobilized lipase increased more than 10 times compared to the lipase immobilized without pretreatment. In addition, the lipase immobilized after linoleic acid or oleic acid has higher activity than the immobilized lipase pretreated with stearic acid, indicating that the unsaturated fatty acid is more effective as a pretreatment material than the saturated fatty acid. This should be investigated further, but it is speculated that the curved structure of unsaturated fatty acid can protect the active site of lipase better than the linear structure of saturated fatty acid. Fatty acids with fewer carbons were not effective for lipase pretreatment. Dodecanoic acid and octanoic acid only increased the activity of immobilized lipase by 5 and 3 times, respectively. Furthermore, acetic acid, propionic acid and butyric acid having 2, 3 and 4 carbon atoms, respectively, were ineffective in lipase pretreatment, whereas acetic acid and butyric acid reduced the activity of immobilized lipase. Through the above results, it was confirmed that the active site can be protected during lipase pretreatment due to the steric effect of fatty acids.

In addition, vegetable oils (vegetable oils) are also effective for lipase pretreatment and can be found to increase the activity of the immobilized lipase more than 10 times compared to the lipase immobilized without pretreatment. In particular, the soybean oil was very effective, and the activity of the immobilized lipase after pretreatment was 17 times higher than that without pretreatment with 537 U / g-matrix. However, triacetin and tributyrin were not effective for pretreatment with lipases, and the immobilized lipase after pretreatment with these substances was less than 40 U / g-matrix similar to immobilized lipase without pretreatment. In contrast to vegetable oils consisting primarily of fatty acids of 16 or more carbon atoms, triacetin and tributyrin are each composed of acetic acid and butyric acid. During the pretreatment step, the vegetable oils are hydrolyzed with glycerol and acetic acid or butyric acid, respectively. Since these fatty acids have 2 or 3 carbon atoms, it was determined that triacetin and tributyrin were not effective for lipase pretreatment.

Experimental Example  6: investigation of activity of immobilized lipase with temperature

20 mL of lipase solution at various temperature ranges from 25 to 45 ° C. was added to 200 g of soybean oil, olive oil, linoleic acid, and oleic acid, which were found to be more effective than lipase pretreatment, followed by pretreatment for 30 minutes with stirring at 150 rpm. After immobilization on the activated silica gel prepared in the present invention was investigated the activity of the immobilized lipase with temperature.

The results are shown in FIG. 9 it can be seen that the optimum temperature of the lipase pretreatment is 40 ℃. Soybean and olive oils showed similar activity at 35 ° C to 40 ° C. However, at 25 ° C., the activity of lipase was drastically reduced. Pretreatment with fatty acids showed similar activity at 25 ° C to 45 ° C. Therefore, it was found that the pretreatment of lipase with oil is more dependent on temperature than the pretreatment with fatty acid. In the case of oil, the pre-treatment temperature during the pretreatment using fatty acid is increased because fatty acid and glycerol are formed through the hydrolysis reaction during pretreatment and the fatty acids thus formed protect the active sites of lipase. It was judged not to be an important variable.

Experimental Example  7: At the stirring speed  Activity of immobilized lipase

20 mL of lipase solution in various stirring speed ranges was added to 200 g of soybean oil, olive oil, linoleic acid and oleic acid, which were found to be more effective in lipase pretreatment than other materials, and then pretreated for 30 minutes at 40 ° C. After immobilization on silica gel, the activity of immobilized lipase was investigated according to the stirring speed.

The results are shown in FIG. 10 shows that the optimum stirring speed during the lipase pretreatment was 200 rpm, and the activity of the immobilized lipase was slightly decreased at 150 rpm and 250 rpm. In particular, during oil pretreatment, the activity of immobilized lipase rapidly decreased at 100 rpm. On the other hand, the pretreatment with fatty acid showed similar activity at 150 rpm to 250 rpm and did not decrease rapidly at 100 rpm. Agitation promotes oil hydrolysis. Therefore, in the case of pretreatment using oil, it was found that the stirring speed would be more important than that of fatty acids.

Experimental Example  8: Investigation of the activity of immobilized lipase according to the pretreatment concentration

Various amounts of the pretreatment material were added to the lipase solution, pretreated for 30 minutes with stirring at 200 rpm at 40 ° C., and then immobilized on activated silica gel, and the activity of the immobilized lipase according to the pretreatment concentration was investigated.

The results are shown in FIG. In case of pretreatment with lipase using 0.05% soybean oil, the activity of immobilized lipase was reduced by 30% compared to immobilized lipase after pretreatment with 0.1% soybean oil. The activity of the immobilized lipase was similar when pretreated with soybean oil of 0.1% to 2%, but slightly decreased with 3% to 4% soybean oil. On the other hand, when using 0.1% to 3% olive oil showed a similar activity, when the concentration was reduced to 0.05% activity of the immobilized lipase was reduced by 25% compared to the activity when 0.1% treatment. When linoleic acid and oleic acid were treated at 0.1% to 4%, the activity did not change in this concentration range. However, pretreatment with 0.05% linoleic acid and oleic acid reduced the activity by 10% compared to the activity at 0.1%. Through these results, the optimal pretreatment concentration was determined to be 0.1%.

Experimental Example  9: Investigation of the activity of immobilized lipase with pretreatment time

20 mg of the pretreatment material was added to 20 mL of the lipase solution, and the pretreatment time was varied with stirring at 200 rpm at 40 ° C., and the immobilized lipase activity was investigated after immobilization on the activated silica gel.

The results are shown in FIG. The optimal pretreatment time for lipase pretreatment using soybean oil and olive oil was 45 minutes. The activity of the immobilized lipase after pretreatment for 45 minutes with soybean oil was 630 U / g-matrix, but the activity of immobilized lipase decreased after pretreatment for more than 45 minutes. In particular, when lipase was pretreated with soybean oil for 120 minutes, the activity of immobilized lipase was reduced by 20% compared to 45 minutes pretreatment. However, when lipase was pretreated with linoleic acid and oleic acid, the pretreatment time did not affect activity.

Experimental Example  10: Investigation of the Reusability of Immobilized Lipase by Pretreatment with Oil or Fatty Acid

Immobilized lipase was prepared by adding 20 mg of soybean oil and linoleic acid to 20 mL of lipase solution, pretreating for 45 minutes with stirring at 200 rpm at 40 ° C., and immobilizing on activated silica gel.

The immobilized lipase was used to hydrolyze soybean oil in a repeated batch reaction. Hydrolysis of soybean oil was carried out for 30 minutes while maintaining at 37 ℃ in a stirred water bath. Immobilized lipase was filtered and washed before reuse. The 100% relative activity of soybean oil pretreated immobilized lipase was 630 U / g-matrix and that of linoleic acid pretreated immobilized lipase was 405 U / g-matrix. Afterwards, changes in relative activity according to reuse were investigated.

The results are shown in FIG. In both cases, more than 90% of the initial activity was maintained after 10 repeated uses. Therefore, it was found that both oils and fatty acids enable reuse of immobilized lipase when pretreated with them.

The present invention relates to a method for pretreatment before lipase immobilization on a carrier. The present invention relates to a method of pretreatment of lipase by immobilizing fatty acids or oils to lipase before immobilization of the lipase. Not only can the lipase be immobilized on the carrier while maintaining the lipase activity without using complicated methods such as controlling or using other additives, but the lipase activity is also used when the pretreated and immobilized lipase is reused several times. This is a very useful invention for the chemical industry because it has a very good effect that can be perfectly maintained and repeatedly reused economically expensive enzymes.

Claims (3)

A method of immobilizing a lipase, characterized in that the lipase is immobilized on the carrier after a pretreatment process in which the lipase is reacted with a fatty acid or an oil before the lipase is immobilized on the carrier by covalent bonding. The method of claim 1, wherein the fatty acid is a fatty acid having 16 to 18 carbons and the oil is a vegetable oil. The method of claim 2, wherein the fatty acid having 16 to 18 carbons is linoleic acid, oleic acid, stearic acid or palmitic acid, and the vegetable oil is olive oil, soybean oil or palm oil.

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