KR101446475B1 - Method for Synthesis of reconstituted phospholipid - Google Patents

Abstract

The present invention relates to a method for synthesizing reconstituted phospholipid using an enzymatic method, and more specifically, to a very effective method which can substitute fatty acids combined to phospholipid, to a desired fatty acid, through an exchange esterification reaction between phospholipid and fatty acids. The present invention selects a type of enzyme suitable for synthesizing the reconstituted phospholipid and uses an immobilization method to raise efficiency of the reaction. Through the exchange esterification reaction using enzyme and resynthesis using a vacuum system, the content of the desired fatty acid can be increased in phospholipid.

Description

Method for Synthesis of Reconstituted Phospholipid

The present invention relates to a method for synthesizing a reconstituted phospholipid using an enzymatic method.

Phospholipids are complex lipids containing phosphorus as natural emulsifiers present in nature. It is possible to use it as surfactant and emulsifier because it has amphiphilic property by having phosphate group and fatty acid. There are four types of phospholipids: phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylserine (PS) It is known that it has functionalities such as cholesterol control, blood circulation improvement and brain nutrition supply though it shows a slight difference.

The most common phospholipids that can be obtained from food and are widely used are lecithin, a substance mainly composed of phosphatidylcholine. This was obtained by purifying the obtained gum as a raw material as a result of degumming. Previously, lecithin was a valuable product because it was extracted from yolk of eggs, but nowadays it can be mass-produced in soybeans, so it is widely used at low cost. Soybean lecithin is not only an excellent emulsifier for food, but also nutritional value. Lecithin, which is currently produced in large quantities, is obtained from soybeans, and other scum such as corn also contains phospholipids.

Phospholipids with such functionalities can be combined with useful fatty acids to synthesize a reconstituted phospholipid to improve the physiological function of the phospholipid and also to synthesize phospholipids having various emulsifying properties. Among them, synthesis using enzymes is a safe and environmentally friendly method, and the reaction using enzymes can be performed at a lower temperature than other chemical methods, and due to the specificity of the enzyme, There are advantages. However, when it is used as an enzyme itself, it is not efficient in the process such as economical aspect or problem of enzyme filtration, and thus, an immobilization process of the enzyme is essential. Thus, there are covalent bonds, ionic bonds, physical adsorption, encapsulation, encapsulation, and the like. However, the methods mainly used for immobilizing lipases include ion-binding methods using polar fixed bodies and non- (Porous polymer) is used. In the immobilization of the enzyme, the activity of the enzyme is greatly changed by the fixing method as well as the fixing agent.

Accordingly, the present inventors have found that phospholipase A1 is immobilized and the phospholipase A1 is subjected to an exchange esterification reaction with a phospholipid and a fatty acid to synthesize a reconstituted phospholipid, and a reconstituted phospholipid having a desired fatty acid content by using a vacuum system during the esterification reaction The yield can be improved, and the present invention has been completed.

Korean Patent Laid-Open Publication No. 10-2011-0034116 discloses a technique for improving the selectivity and productivity of fatty acid substitution of phospholipids by the immobilized phospholipase A2. However, a difference in the type of the enzyme used in the present invention have.

Korean Patent Publication No. 10-2011-0034116

The present invention is based on the finding that phospholipase A1 derived from Thermomyceslanguinosus or Fusarium oxysporum is immobilized with an enzyme by using a fixative and phosphatidylcholine and fatty acid It is an object of the present invention to provide a method for synthesizing a reconstituted phospholipid having a desired fatty acid content through an exchange esterification reaction using docosahexaenoic acid (DHA) as a substrate.

In order to achieve the above object,

The present invention provides a process for the production of a phospholipid, comprising: a) selecting an enzyme for the exchange esterification reaction of a phospholipid;

b) immobilizing the enzyme selected in step a) using a fixative;

c) evaluating the degree of immobilization and activity of the enzyme immobilized in step b);

d) mixing phosphatidylcholine as a phospholipid and docosahexaenoic acid (DHA) as a fatty acid;

e) adjusting the moisture content of the mixture of phospholipids and fatty acids mixed in step d) to 0.2 to 1.4%;

f) reacting the mixture of step e) with the enzyme immobilized in step b) at a reaction temperature of 35 to 65 ° C; And

g) adding the Candida antarctica-derived immobilized enzyme to the reactant while maintaining the vacuum system at 0 to 10 torr, and reacting the reactant with the immobilized enzyme.

According to the method for synthesizing the phospholipid of the present invention, the conditions of the enzyme suitable for the exchange esterification reaction using the phospholipid and the fatty acid can be selected, and the immobilization conditions for increasing the reactivity of the enzyme can be set. Therefore, there is an advantage that a reconstituted phospholipid having an increased content of a desired fatty acid can be effectively synthesized without containing a solvent and cofactors.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram briefly illustrating the overall steps for synthesizing a reconstituted phospholipid. FIG.
2 is a schematic diagram briefly showing a method for immobilizing an enzyme to be used for reconstituting phospholipid synthesis.
FIG. 3 is a graph showing experimental results for selecting an enzyme to be used for synthesis of a reconstituted phospholipid.
4 is a graph showing experimental results for selecting a solid body to be used for enzyme immobilization for synthesizing a reconstituted phospholipid.
5 is a graph showing experimental results for selecting a pH range of a buffer solution to be used for enzyme immobilization for synthesizing a reconstituted phospholipid.
6 is a graph showing experimental results for selecting the concentration of an enzyme dilution solution to be used for enzyme immobilization for synthesizing a reconstituted phospholipid.
FIG. 7 is a graph showing experimental results according to the initial moisture content in the reconstituted phospholipid synthesis method. FIG.
FIG. 8 is a graph showing experimental results according to the reaction temperature in the reconstituted phospholipid synthesis method. FIG.
9 is a graph showing the results of experiments according to the amount of immobilized enzyme in the reconstituted phospholipid synthesis method.
FIG. 10 is a graph showing experimental results when a vacuum system is used in the reconstituted phospholipid synthesis method. FIG.
11 is a graph showing the results of reconstituted phospholipid synthesis according to the amount of immobilized enzyme when Lewatit VP OC 1600 is used as a fixative.
FIG. 12 is a graph showing the effect of reconstituting phospholipid re-synthesis by time of vacuum introduction time.

Hereinafter, the present invention will be described in detail.

According to the present invention,

a) selecting an enzyme for the exchange esterification reaction of a phospholipid;

b) immobilizing the enzyme selected in step a) using a fixative;

c) evaluating the degree of immobilization and activity of the enzyme immobilized in step b);

d) mixing phosphatidylcholine as a phospholipid and docosahexaenoic acid (DHA) as a fatty acid;

e) adjusting the moisture content of the mixture of phospholipids and fatty acids mixed in step d) to 0.2 to 1.4%;

f) reacting the mixture of step e) with the enzyme immobilized in step b) at a reaction temperature of 35 to 65 ° C; And

g) adding the Candida antarctica-derived immobilized enzyme to the reactant while maintaining the vacuum system at 0 to 10 torr, and reacting the reactant with the immobilized enzyme.

Preferably, the fatty acid is selected from the group consisting of alpha-linolenic acid (ALA, 18: 3 (n3)), docosahexaenoic acid (DHA, 22: 6 (Eicosa Pentaenoic Acid, EPA, 20: 5 (n-3)), SDA (stearidonic acid) and ETA (eicosatetraenoic acid), and docosahexaenoic acid Acid: DHA).

The a) with the enzyme used in step Thermo My process called leakage No suspension (Lipozyme TL IM, Li jomu cor Mie Hay (Lipozyme RM IM, Candida hits arcs urticae (Candida Antarctica) derived from Rhizomucormiehei) derived from Thermomyceslanuginosus) derived from a lipase (lipase), the phospholipase a ₂ derived from porcine pancreas (PhospholipaseA ₂₎ and Thermo My process called leakage no suspension (Thermomyceslanuginosus) or Fusarium oxy's Forum (Fusariumoxysporum) containing the origin Novozym 435 from A phospholipase A ₁ , or a phospholipase A ₁ derived from Thermomyceslanuginosus or Fusarium oxysporum . The phospholipase A ₁ may be one selected from the group consisting of phospholipase A _{1, 1} ).

The enzyme is preferably contained in an amount of 2.5 to 20% by weight based on the weight of the phospholipid and the fatty acid. When included in the above range, the content of DHA bound to phosphatidylcholine increases as the amount of enzyme increases.

As a non-polar fixative, Lewatit VP OC 1600 (Novo Nordis Bioindustry Ltd. Korea), Accrurel MP 1000 (Membrana GmbH Accurel system, Germany), Amberlite XAD4 (Sigma Aldrich, Germany) as fixators used for immobilizing the enzyme in step b) (Sigma Aldrich, Korea) or Duolite A568 (Rohm and Haas France SAS, France), Amberlite XAD 7HP (Sigma Aldrich, Korea) as a polar fixing material, , Celite 545 (Sigma Aldrich, Korea), and Dowex 50w x8 (Sigma Aldrich, Korea).

The pH used to immobilize the enzyme in the step b) is in the range of 5.5 to 8.5, more preferably 6.5 to 8.5. When the pH is in the above range, the amount of the immobilized protein is increased and the most amount of the enzyme is immobilized.

The dilution ratio of the enzyme solution is preferably 30 to 90% based on the weight of the solid body. In the concentration range of the enzyme solution, the transesterification reaction of DHA to phosphatidylcholine was possible, and as the concentration of the solution increased, the amount of enzyme immobilized on the solidified body increased.

Phosphatidylcholine as a phospholipid and Docosa hexaenoic acid (DHA) as a fatty acid react with an enzyme as a substrate, and the molar ratio is preferably 1: 2 to 1: 8. When the molar ratio is 1: 2 to 1: 8, the synthesis yield and economical efficiency of the reconstituted phospholipid having an increased content of the desired fatty acid are excellent.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental Example

Experimental Example  1: Immobilization of enzyme

Immobilization by nonpolar solid body

Lewatit VP OC 1600 (Novo Nordis Bioindustry Ltd. Korea), Accrurel MP 1000 (Membrana GmbH Accurel system, Germany), Amberlite XAD4 (Sigma Aldrich, Korea) and Octyl silica (Sigma Aldrich, Korea) were used as non-polar fixatives.

In the immobilization method using a nonpolar solid body, the nonpolar solid body was immersed in 95% ethanol for 3 hours to swell, then the ethanol was removed with a phosphate buffer solution (pH 7.5), and the swollen solid body was filled with the buffer solution. And diluted with phosphate buffer solution so that the concentration of the phospholipase A ₁ solution becomes 70%. 10 mL of the phospholipase A ₁ solution diluted to 1 g of the fixative weight was mixed at 30 DEG C and 250 rpm for 16 hours. Thereafter, the flask was washed with 40 mL of phosphate buffer solution on the basis of 1 g of fixative weight. The immobilized phospholipase A ₁ was dried at room temperature for 24 hours and then dried in a vacuum dryer for 24 hours. Until the reaction, they were refrigerated and stored in a closed container with a water activity of 0.64 for the reaction. FIG. 2 briefly outlines a method for immobilizing an enzyme to be used for the reconstitution phospholipid synthesis.

Immobilization by Polar Solids

(Sigma Aldrich, Korea), Celite 545 (Sigma Aldrich, Korea) and Dowex 50w x8 (Sigma Aldrich, Korea) were used as the polarizer.

The immobilization method using the polarity fixture was carried out in the same manner as the nonpolar fixation method except for the swelling process of the fixture.

Experimental Example  2: Measurement method of enzyme immobilization degree

To measure the degree of immobilization of the enzyme, the protein of the initial phospholipase A ₁ solution is quantified. After the phospholipase A ₁ is immobilized, the amount of protein remaining in the buffer solution and the amount of the protein that has exited into the buffer solution after washing are quantified. The reagent A used in the Lowry method was 2% Na ₂ CO ₃ And 0.4 g of 0.1 N NaOH in 100 mL of distilled water. Reagent B1 is prepared by mixing 0.5 g of 1% CuSO ₄ · 5H ₂ O in 50 mL of distilled water. Reagent B2 is prepared by adding 2% NaK tartrate · 4H ₂ O to 50 mL of distilled water. Immediately before storage and use in the form of reagents A, B1 and B2, mix reagent C in volume proportions of 100: 1: 1, respectively. 200 μL of the sample was mixed with 1 mL of reagent C, vortexed, and left for 10 minutes. Then, 50 μL of the Folin reagent was added, vortexed, and left for 30 minutes to develop color (blue). The absorbance of the developed sample was measured at a wavelength of 750 nm.

<Formula 1>

Amount of immobilized enzyme (mg / g) =

W1: protein amount of phospholipase A ₁ stock solution (mg)

W2: Amount of protein remaining in buffer solution after immobilization (mg)

W3: Fixture Weight (g)

Experimental Example  3: Exchange Esterification  reaction

Phosphatidylcholine (PC) and 73% DHA - containing fatty acids were dissolved at 50 ℃ and mixed at a constant molar ratio, and water content was measured by Karl fisher. 3 g of the substrate mixture was added to the reactor, and a predetermined amount of the enzyme (the amount calculated on the basis of the amount of the substrate mixture) was added thereto. The mixture was stirred at 250 rpm, reacted for a predetermined time, The reaction temperature was set by controlling the temperature of the water circulator connected to the water jacket of the reactor. After the first reaction, the sample was taken after a certain reaction time while continuing the reaction with agitation at a vacuum of 5 torr after the esterification reaction in order to increase the functional PC yield. The yield of PC measured in the present invention was calculated to include the phosphatide in which the fatty acid was esterified at both the 1st and 2nd carbon of glycerin.

Experimental Example  4: Analysis method

a) PC extraction using TLC

50 mg of the reaction product was dissolved in 150 μL of chloroform and loaded on a TLC plate (silica gel 60 F ₂₅₄ , Merck, Germany) and developed with a solution of chloroform / methanol / acetic acid / water (75: 40: 8: 3, . After separating PC and LPC from the plate, 1 mg of internal standard (C17: 0) was added and methylation was performed by using BF ₃ (14% Boron fluoride in methanol).

b) GC-FID analysis conditions

A column of Superlcowax ^™ 10 (30 m × 0.25 mm, 0.25 μm, Supelco, USA) was attached to a GC (gas chromatograph, Buruker cp-3800, USA). The column oven temperature was maintained at 180 ° C. for 1 minute, / min and allowed to stand for 5 minutes. The temperature of the injection part was set at 240 ° C and the temperature of the flame ionization detector was set at 250 ° C. The mobile phase gas was analyzed at a split ratio of 50: 1 with helium flowing at 1.5 mL / min.

<Formula 2>

<Expression 3>

Comparative Example  1. Selection of Enzymes for Synthesis of Recombinant Phospholipids

The present invention is intended to synthesize a reconstituted phospholipid by an exchange esterification reaction using phosphatidylcholine and a functional fatty acid. In this comparative experiment, an experiment for selecting an enzyme having the highest activity for efficiently binding a target fatty acid to a phospholipid The results are shown in Fig. The enzymes used were Lipase OF derived from Candida rugosa , Lipase AK derived from Pseudomonasfluorescens , Lipase PS derived from Burkholderiacepacia , Penicillium camembertia And Lipase G from Penicillium camemberti. A total of 3 g of phosphatidylcholine and 73% DHA-containing fish oil fatty acid were mixed in a molar ratio of 1: 8, and the mixture was reacted for 24 hours at 50 rpm with shaker at 250 rpm. Enzymes were used in an amount of 3% based on the weight of the substrate. Among the enzymes, Lipase OF, Lipase AK, Lipase PS, and Lipase G were powdery enzymes. As shown in FIG. 3, the conjugated linoleic acid (CLA), which is a desired fatty acid, was not synthesized when these enzymes were used.

Comparative Example  2: Enzyme immobilization for synthesis of reconstituted phospholipids Middle fixture  selection

In this Comparative Example 2, in order to confirm the effect of immobilization of phospholipase A ₁ on immobilization and activity, Dowex 50w x8 and Celite 545 were used as the fixed bodies, and the results are shown in FIG. 4 . In the case of two solid bodies, it was confirmed that phospholipase A ₁ could be immobilized but no reconstituted phospholipid was synthesized at all.

Example  1. Selection of Enzymes for Synthesis of Recombinant Phospholipids

In this Example 1, experiments were conducted to select enzymes for the reconstituted phospholipid synthesis through exchange esterification of phospholipids and desired fatty acids. A total of 3 g of phosphatidylcholine and 73% DHA-containing fish oil fatty acid were mixed in a molar ratio of 1: 8, and the mixture was reacted for 24 hours at 50 rpm using a shaker at 250 rpm. The enzymes were used in an amount of 3% based on the weight of the substrate and the results are shown in FIG. The enzymes used are derived from a porcine pancreas phospholipase _{A 2 (Lecitase10L, PhospholipaseA 2)} , Thermo My process called leakage No suspension (Thermomyceslanuginosus) or Fusarium oxy scan the phospholipase A ₁ (LecitaseUltra derived from Forum (Fusariumoxysporum) , Lipozyme TL IM from Thermomyces luginosus , Lipozyme RM IM from Rhizomucormiehei , and Novozym 435 from Candida Antarctica. . Of the enzymes, Novozym 435, Lipozyme RM IM and Lipozyme TL IM were immobilized enzymes, while phospholipase A ₁ and phospholipase A ₂ were liquid enzyme. As shown in FIG. 3, all of the five enzymes used in Example 1 were able to carry out the exchange esterification reaction with DHA. In particular, it was confirmed that the immobilized phospholipase A ₁ binds DHA to phosphatidylcholine in a large amount.

Therefore, phospholipase A ₁ was used as an enzyme for the present invention, and experiments on the efficient immobilization conditions of phospholipase A ₁ were performed prior to this experiment.

Example  2. Enzyme immobilization for synthesis of reconstituted phospholipids Middle fixture  selection

In order to confirm the difference in activity appears to power and reaction for immobilizing a Phospholipase A ₁ according to the fixed body, the polarity fixing body was used as the Amberlite XAD 7HP, Duolite A568, a non-polar stationary body is Lewatit VP OC 1600, Octyl silica , Accrurel MP 1000 and Amberlite XAD4 were used.

The results are shown in FIG. 4, and the phospholipase A ₁ was immobilized on all the fixators used, but particularly good fixation was obtained when Lewatit VP OC 1600, Amberlite XAD 7HP, and Duolite A 568 fixatives were used. In addition, the effect of DHA exchange esterification on phosphatidylcholine was significantly different. Especially excellent results were obtained when Lewatit VP OC 1600, Amberlite XAD 7HP, and Duolite A568 were used.

From the results of Example 2, the immobilization conditions and the synthesis conditions of the reconstituted phospholipids were tested using Duolite A568 as a typical immobilizing material of the present invention.

Example  3. Enzyme immobilization to synthesize reconstituted phospholipids pH  selection

FIG. 5 shows the results of the immobilization of the enzyme on the degree of immobilization on the Duolite A568 fixture according to the pH and the activity to bind DHA to the phosphatidylcholine. The amount of the protein (enzyme) immobilized on the fixative increased with increasing pH, and the amount of the protein immobilized at pH 6.5 to 8.5 was increased, so that the enzyme was immobilized at pH 8.5. However, the amount of DHA bound to phosphatidylcholine was found to be the lowest at pH 8.5.

Therefore, it was shown that phospholipase A ₁ was most efficiently immobilized at pH 7.5.

Example  4. Selection of concentration of enzyme solution during enzyme immobilization for synthesis of reconstituted phospholipids

Fig. 6 shows the result of investigation of the concentration of the phospholipase A ₁ solution capable of binding the maximum to the Duolite A568 fixative. The enzyme dilution was prepared by diluting a phospholipase A ₁ solution with a phosphate buffer solution at a pH of 7.5 to a dilution ratio of 10, 30, 50, 70 and 90%, respectively.

As shown in the results of FIG. 6, it was confirmed that as the concentration of the phospholipase A ₁ solution increases, the amount of the protein (enzyme) immobilized on the solid body increases. When the concentration of phospholipase A ₁ solution increased from 10 to 50%, the amount of immobilized protein increased from 21 to 125 mg / g, but increased from 125 to 147 mg / g in the range of 50 to 90% When the amount of the lipase A ₁ solution was 50% or more, the increase rate of the amount of the immobilized protein was decreased as compared with the increase of the enzyme.

In terms of the reactivity to bind DHA to phosphatidylcholine, it was confirmed that the DHA exchange esterification reaction did not occur when the concentration of the phospholipase A ₁ solution was 10%.

Therefore, the concentration of the phospholipase A ₁ solution capable of the exchange esterification reaction of DHA with phosphatidylcholine is 30% or more, more preferably 50 to 90%. However, when the enzyme solution was diluted to 70% and 90%, the activity did not change during storage, but it was confirmed that the enzymes diluted to 50% and immobilized had a longer storage time and decreased enzyme activity.

Therefore, phospholipase A ₁ solution was most efficiently diluted to 70% with pH 7.5 buffer solution.

Example  5. Reconstitution phospholipid synthesis by initial moisture content

FIG. 7 shows the results of experiments at various moisture contents in order to synthesize DHA-conjugated phosphatidylcholine. In Example 5, 10% of phospholipase A ₁ immobilized on a substrate mixed with phosphatidylcholine and 73% DHA-containing fatty acid at a molar ratio of 1: 8 was added together and reacted at 55 ° C with stirring at 250 rpm to obtain reaction The yield of phosphatidylcholine and the content of DHA bound to phosphatidylcholine were measured over time.

The yield of phosphatidylcholine decreased rapidly during hydrolysis during the initial 3 hour reaction and steadily decreased thereafter. After 3 hours, the yield of phosphatidylcholine decreased with increasing water content, and the yield of phosphatidylcholine was highest at 36% and 36% at 0.2% moisture content and 30% and 0.8% at 0.6% moisture content, respectively. %, The yield of phosphatidylcholine was 25 mol%.

The content of DHA bound to phosphatidylcholine increased with increasing reaction time. In the early stage of the reaction, the binding rate of DHA increased rapidly as the moisture content increased, and the DHA content of phosphatidylcholine continued to increase with increasing reaction time at a water content of 0.8% or more after 9 hours of reaction, The contents of DHA bound to phosphatidylcholine did not change with the contents. As a result, the content of DHA bound to phosphatidylcholine was 28 mol% at the moisture content of 0.8% or more after 24 hours.

Therefore, the change in the yield of phosphatidylcholine after 9 hours and the change in the content of DHA bound to phosphatidylcholine were observed, and it was determined that the moisture content of 0.8 to 1.0% was the most suitable range.

Example  6. Reconstitution of phospholipid according to reaction temperature

Experimental results on the reaction temperature for synthesizing DHA-conjugated phosphatidylcholine are shown in FIG. In Example 6, phosphatidylcholine mixed with 1: 8 molar ratio and 73% DHA-containing fatty acid were mixed with immobilized phospholipase A _{1 at} 35, 45, 55 and 65 ° C. The amount of immobilized enzyme was 10% based on the total amount of substrate, and the reaction was carried out with stirring at 250 rpm under atmospheric pressure at a moisture content of 0.8%.

As the temperature increased, the yield of phosphatidylcholine tended to decrease more and the yield of phosphatidylcholine at 55 and 65 ℃ showed similar tendency. The content of DHA bound to phosphatidylcholine tended to increase with increasing reaction temperature as opposed to the yield of phosphatidylcholine. The DHA content of phosphatidylcholine at 55 and 65 ℃ was similar to that of phosphatidylcholine gave. Reactivity at 45 ° C The results of the reaction up to 12 hours showed that the content of DHA bound to phosphatidylcholine was smaller than that at 55 ° C and 65 ° C. However, in the reaction at 24 hours, the content of DHA bound to phosphatidylcholine at 45, The contents of DHA showed similar values.

Example  7. Immobilization In the amount of enzyme  Recombinant phospholipid synthesis

Experimental results on the amount of enzyme required to synthesize DHA-conjugated phosphatidylcholine are shown in FIG. In Example 7, phosphatidylcholine and DHA mixed at a molar ratio of 1: 8 were mixed with immobilized phospholipase A ₁ and reacted at a temperature of 55 ° C. The amount of immobilized enzyme was set to 2.5, 5, 10, 20% based on the total substrate amount, and the reaction was carried out with stirring at 250 rpm under atmospheric pressure at a moisture content of 0.8%.

As a result, the yield of phosphatidylcholine and the content of DHA bound to phosphatidylcholine were greatly different according to the amount of enzyme. As the amount of enzyme increased, the yield of phosphatidylcholine decreased, and the content of DHA bound to phosphatidylcholine increased. Especially, the content of DHA bound to phosphatidylcholine significantly varied between 5 and 10%. When the enzyme content was increased up to 20%, the amount of DHA bound to phosphatidylcholine was continuously increased. However, the yield of phosphatidylcholine was greatly decreased, and hydrolysis was observed to occur.

Therefore, immobilized phospholipase A ₁ was found to be most effective to use 10% of the amount of substrate.

Example  8. Reconstitution of phospholipids by vacuum

In order to synthesize DHA-conjugated phosphatidylcholine, phospholipase A ₁ , in which phosphatidylcholine and 73% DHA-containing fatty acids were mixed in a molar ratio of 1: 8 and immobilized with Duolite A568, was used in an amount of 10% based on the total mass of the substrate, After adding Novozym 435 (10% based on the total base weight) to the reaction mixture at a reaction temperature of 55 ° C and a moisture content of 0.8% for 6 hours, the product was reacted with a 5 torr vacuum system. 10.

The yield of phosphatidylcholine was 33 mol% after 6 hours of reaction and PC synthesis rate was increased after using vacuum system. It was concluded that the esterification reaction was promoted by the reduction of water produced by the vacuum system. In addition, it can be confirmed that the ratio of DHA synthesis is increased, and Novozym 435 is added to the immobilized phospholipase A ₁ as an enzyme, and it is found that using a vacuum system is an effective method for synthesizing phosphatidylcholine having an increased DHA content I could.

Example  9. Other Fixing  Synthesis of Recombinant Phospholipids Using Immobilized Enzyme

In the same manner as the immobilization method using Duolite A568, the phospholipase A ₁ was immobilized using a fixed Lewatit VP OC 1600, and then a test for the synthesis of the reconstituted phospholipid was carried out.

Reconstitution Phospholipid synthesis To investigate the effects of the reconstituted phospholipid synthesis according to the amount of immobilized enzyme in the experimental conditions, phosphatidylcholine and DHA mixed at a molar ratio of 1: 8 were mixed with immobilized phospholipase A ₁ and reacted at a temperature of 55 ° C. The results are shown in Fig. Other reaction conditions were optimized with immobilized enzyme using Duolite A568, ie, the amount of immobilized enzyme was set at 2.5, 5, 10, 20, 30% based on the total substrate amount, 0.8%, and reacted under stirring at 250 rpm under atmospheric pressure.

As a result, it was confirmed that the content of DHA bound to phosphatidylcholine increased sharply as the amount of enzyme increased from 2.5% to 20%. However, as the enzyme content increased from 20% to 30%, it was confirmed that the content of DHA bound to phosphatidylcholine was not significantly changed. It was confirmed in Example 8 that the yield of phosphatidylcholine could be improved by increasing the amount of enzyme. It was confirmed in Example 8 that the yield of phosphatidylcholine could be improved by using a vacuum system. Therefore, in Example 10 in which the amount of enzyme was 20% Was carried out as follows.

Example  10. Re-synthesis of reconstituted phospholipids by time of vacuum introduction

Phospholipase A ₁ , in which phosphatidylcholine and 73% DHA-containing fatty acids were mixed at a molar ratio of 1: 8 and immobilized with Lewatit VP OC 1600, was used at a rate of 20% based on the total mass of the substrate, a reaction temperature of 55 ° C, a moisture content of 0.8 % Under vacuum condition using 5 torr vacuum system. In order to investigate the effects of reconstitution of phospholipid according to the timing of introduction of vacuum, the vacuum introduction time was selected as 0 hour of reaction initiation, 3 hours of reaction initiation, and 9 hours after reaction initiation. As a criterion for setting the vacuum introduction time, based on the results of the experiment in Example 9, it was determined that DHA bound to phosphatidylcholine sharply increased for 3 hours, the content was almost equilibrated for 9 hours, Experiments were conducted. The results are shown in Fig.

As a result, it was confirmed that the results were different even though the same vacuum was introduced at the time of vacuum introduction. As the reaction time increased after the introduction of the vacuum, the content of DHA bound to phosphatidylcholine was slightly decreased at 3 hours and 9 hours except for the control group, and the yield of phosphatidylcholine rapidly increased. The amount of DHA bound to phosphatidylcholine was not significantly different between 3 hours and 9 hours except for the control group as the reaction time was 96 hours after the introduction of the vacuum. However, the yield of the phosphatidylcholine was lowered to the vacuum Was significantly higher than when the vacuum was applied at 9 hours. Therefore, it was confirmed that introducing a vacuum at 3 hours of reaction time is an effective method.

Claims (5)

a) selecting an enzyme for the exchange esterification reaction of a phospholipid;
b) immobilizing the enzyme selected in step a) using a fixative;
c) evaluating the degree of immobilization and activity of the enzyme immobilized in step b);
d) mixing phosphatidylcholine as a phospholipid and docosahexaenoic acid (DHA) as a fatty acid;
e) adjusting the moisture content of the mixture of phospholipids and fatty acids mixed in step d) to 0.2 to 1.4%;
f) reacting the mixture of step e) with the enzyme immobilized in step b) at a reaction temperature of 35 to 65 ° C; And
g) adding the Candida antarctica-derived immobilized enzyme to the reactant while maintaining the vacuum system at 0 to 10 torr, and then reacting the reactant with the immobilized enzyme. The method according to claim 1, wherein the enzyme is Thermo My process called leakage No suspension (Thermomyceslanuginosus) or Fusarium oxy's forum a Phospholipase A ₁ derived from (Fusariumoxysporum) (phospholipaseA _1), and 2.5 to weight ratio of the phospholipids and fatty acids 20% by weight, based on the total weight of the reconstituted phospholipids. [3] The method according to claim 1, wherein the fixative used for immobilizing the enzyme in step b) is at least one selected from the group consisting of Duolite A568, Amberlite XAD 7HP, Lewatit VP OC 1600, Accrurel MP 1000, Amberlite XAD4, Lt; RTI ID = 0.0 &gt; phospholipid &lt; / RTI &gt; The method according to claim 1, wherein the pH used to immobilize the enzyme in step b) is in the range of 5.5 to 8.5, and the concentration of the enzyme solution is in the range of 30 to 90% Methods for the synthesis of reconstituted phospholipids. The method according to claim 1, wherein the molar ratio of phosphatidyl choline to docosahexa and docosahexaenoic acid (DHA) as a fatty acid is from 1: 2 to 1: 8. .

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