WO2003091263A1

WO2003091263A1 - Method for producing phosphatidylethanolamine and lysophosphatidylethanolamine using non-organic solvent system

Info

Publication number: WO2003091263A1
Application number: PCT/KR2003/000834
Authority: WO
Inventors: Guk-Hoon Chung; Jung-Jun Han; Young-Lae Yang; Hun-Min Jung; Ui-Chan Koh
Original assignee: Doosan Corp
Current assignee: Doosan Corp
Priority date: 2002-04-25
Filing date: 2003-04-24
Publication date: 2003-11-06
Anticipated expiration: 2004-10-25
Also published as: KR20030084200A; AU2003224466A1

Abstract

This method for producing phosphatidylethanolamine or lysophosphatidylethanolamine is characterized by the reaction of lecithin comprising at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol in non-organic solvent system comprising calcium chloride and buffer. Therefore, the present invention eliminates the danger or difficulty in purification, which may exist in the case where toxic organic solvent is used and the present invention enables low inactivity of enzyme and high reaction efficiency. In particular, isolation and purification process after the completion of the reaction can be simplified and organic acid used in the isolation and purification process can be easily recovered since the present does not use organic solvent during the enzymatic reaction. Furthermore, even the lecithin having high purity can be dispersed smoothly in water.

Description

Method for Producing Phosphatidylethanolamine and Lysophosphatidylethanolamine Using Non-Organic Solvent System

TECHNICAL FIELD The present invention relates to a method for producing phosphatidylethanolamine and lysophosphatidylethanolamine using a non-organic solvent system and to a water-soluble composition comprising the lysophosphatidylethanolamine produced by the method of the present invention. In particular, the present invention relates to a method for producing phosphatidylethanolamine and lysophosphatidylethanolamine by treating natural or synthetic lecithin (phospholipid), which comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol, etc., with phospholipase D alone or in combination with phospholipase A (Al or A2). More particularly, the present invention relates to a method for producing phosphatidylethanolamine and lysophosphatidylethanolamine by adding 1 to 50% of natural or synthetic lecithin (phospholipid) comprising at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol, etc., to the buffer comprising calcium chloride and then treating it with phospholipase D alone or in combination with phospholipase A (Al or A2).

BACKGROUND ART

Lysophosphatidylethanolamine has been known for its very important roles in the repining and senescence of fruits. Also, it was known that the treatment of lysophosphatidylethanolamine can inhibit senescence of leaves and fruits of tomatoes, thereby enabling extension of storage term of fruits if tomatoes are treated with lysophosphatidylethanolamine after the harvest (See US Patent No. 5,110,34, US Patent No. 5,126,155). In addition, it has been reported that the treatment of apples with lysophosphatidylethanolamine promotes formation of anthocyanin in their skins and inhibits loss of firmness during the storage term of harvested apples. These functions are known to be induced by lowering respiration rates of fruits such as cranberry or tomato, etc. and promoting or repressing production of ethylene gas (Farag, K.M. and J.P. Palta, "Stimulation of Ethylene Production by Erea, Thidiazoron, and Lysophosphatidylethanolamine and Possible sites of this stimulation", Annual meeting of the American Society of Plant Physiologists, April 1989).

Lysophosphatidylethanolamine solution of a proper concentration may be used as a means for extension of shelf life of cut flower (HortScience 32(5): 888-890, 1997). Silver thiosulfate solution containing sugar can inhibit aging of flowers through the treatment of harvested flowers for at least about 20 hours and thus has been used for such purposes. However, ordinary skilled persons in the art are reluctant to use such solutions recently in the United States, since ions contained in the solution cause environmental pollution. In addition to silver thiosulfate solution, it is also known that natural lysophosphatidylethanolamine can improve the storage stabilities of cut flowers in a flower vase. Therefore, the uses of lysophosphatidylethanolamine purified from nature for such purposes are increasing recently.

Practically, for the purpose of shelf life extension of harvested crops, the harvested crops may be immersed in lysophosphatidylethanolamine solution or treated with the solution in the spray form. In order to apply lysophosphatidylethanolamine in these practical uses, water-soluble formulations are preferable. Water-soluble formulations can provide homogenized forms. Therefore, lysophosphatidylethanolamine must be a water-soluble form to maximize the effect of shelf life expansion. No method for producing high-purity lysophosphatidylethanolamine has yet been developed. Merely, the isolation and purification of the high-purity lysophosphatidylethanolamine is being conducted in a small amount on experimental scales by using silica gel column chromatography. These lysophosphatidylethanolamines have been sold by Avanti Polar Lipids, Inc. or Sigma in a small amount for the uses as reagents at very high prices. It is very difficult to isolate lysophosphatidylethanolamine using column chromatography because the movement of lysophosphatidylethanolamine in a column is very similar with other components. Further, if column chromatography is carried out using the lowly toxic organic solvents such as general hexane or ethanol only, lysophosphatidylethanolamine will be very difficult to be purified due to the very low solubility thereof in organic solvents. If a very highly toxic solvent group including chloroform, benzene and methanol is used in column chromatography for purification, good results may be obtained. However, the yield will be low and a great amount of expenses will be incurred for purification from toxic solvents. Generally, lecithin from soybean or yolk is a starting material for the production of lysophospholipids, but the starting material is not limited to the lecithin. Crude soybean lecithin produced as a by-product during production processes of soybean oil consists of polar lipid (phospholipid / glycolipid) 60-70%, soybean oil 27-39%, water 1-3% and other materials 0,5-3%. Polar lipid, among the above components, can be purified by removing soybean oil, which is a neutral lipid contained in crude soybean lecithin. The polar lipid purified by removing soybean oil from the crude soybean lecithin consists of 22-30% of phosphatidylcholine (hereinafter, referred as "PC"), 2-5% of lysophosphatidylcholine (hereinafter, referred as "LPC"), 16-22% of phosphatidylethanolamine (hereinafter, referred as "PE"), 0.5-2% of lysophosphatidylethanolamine (hereinafter, referred as "LPE"), 0.5-8% of phosphatidic acid (hereinafter, referred as "PA"), 0.1-3% of phosphatidylserine), and 6-15% of phosphatidylinositol and the rest. The polar lipid from egg yolk lecithin also consists of 73-83% of phosphatidylcholine (PC), 2-5% of lysophosphatidylcholine(LPC), 13-17% of phosphatidylethanolamine(PE), 1-3% of lysophosphatidylethanolamine(LPE) and the rest. As the above composition of polar lipid in lecithin shows, lecithin contains only a very small amount of lysophosphatidylethanolamine. Therefore, it is nearly impossible to utilize the lysophosphatidylethanolamines isolated directly from this lecithin on a commercial scale. Therefore, it has been necessary to develop methods for producing lysophosphatidylethanolamine by reacting lecithin and ethanolamine under the existence of phospholipase D and phospholipase A, which results in high yield and is convenient for purifing lysophosphatidylethanolamine.

The two-phase system, which uses an organic solvent such as ethyl acetate, diethyl ether, etc. and a buffer, is generally used in phospholipid synthesis with phospholipase D, because phospholipid is not water-soluble. However, lecithin, 20-40% of which is phosphatidylcholine and 20-30% of which is phosphatidylethanolamine (for example, SOYA LECITHIN POWDER 95 from Shindongbang company or FP 40, D, RP40 from CENTRAL SOYA company) has difficulty in being dissolved in ethyl acetate. In particular, the two-phase system consisting of ethyl acetate and water is difficult to stir and thus may cause aggregation of lecithin. Also, the two-phase system shows low reactivity and has a problem of high enzyme inactivation. It is difficult to produce phospholipid on a large scale using diethyl ether as a solvent because of its high volatility and low boiling point. Toluene and benzene, etc. are not desirable either because they cause serious enzyme inactivation and it is difficult to isolate and purify phospholipid from such solvents due to their high toxicity. Hexane is a very efficient solvent to dissolve lecithin, but shows very low reactivity in synthesis transfer reaction.

DISCLOSURE OF THE INVENTION

It is, therefore, an object of the present invention to provide a method for producing phosphatidylethanolamine and lysophosphatidylethanolamine, which enables efficient synthesis, isolation and purification. In particular, it is an object of the present invention to provide a method which can solve the problem of high enzyme inactivation and promotes the safety and high reactivity even without using a toxic organic solvent.

Another object of the present invention is to provide a method, which does not use any organic solvent during enzyme reaction so that isolation and purification process after the completion of the enzyme reaction may be simplified and through which organic solvent used in the isolation and purification process may be recovered easily. Further, another object of the present invention is to provide a method for producing phosphatidylethanolamine and lysophosphatidylethanolamine using a non- organic solvent system, which has a very high reactivity and a very safe property from lecithin with high purity as well as from lecithin with medium or low purity.

Another object of the present invention is to provide a method which can remove the ethanolamines and enzymes used in the reactions, cholines generated after the reactions, and the remained extra ethanolamines after reactions by washing, with water, the results of the reactions, which formed salts after the synthesis of phosphatidylethanolamine with water.

To achieve the above objects, the present invention provides a method for producing phosphatidylethanolamine comprising the step of reacting lecithin and ethanolamine under the existence of phospholipase D, wherein the lecithin comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol and the reaction is carried out in a non-organic solvent system containing calcium chloride and buffer.

Further, the present invention provides a method for producing lysophosphatidylethanolamine comprising the step of reacting lecithin and ethanolamine under the existence of phospholipase D and phospholipase A, wherein the lecithin comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol and the reaction is carried out in a non-organic solvent system containing calcium chloride and buffer. The method for producing lysophosphatidylethanolamine as described above comprises the steps of: conducting the first reaction of lecithin and ethanolamine under the existence of phospholipase D; and conducting the second reaction of the first products generated by the first reaction with phospholipase A added to the first products. In a preferred embodiment of the present invention, the non-organic solvent system consists only of calcium chloride and buffer.

Preferably, the lecithin is added by the ratio of 1 to 50% of the amount of the buffer for the reactions. Preferably, the buffer is pH 4 to 8.

Preferably, the temperature of the reactions is 20 to 60 ^°C .

Preferably, the lecithin is one selected from the group consisting of soybean- derived lecithin, rape seed-derived lecithin, fishes-derived lecithin, mollusk-derived lecithin and yolk-derived lecithin, and the fatty acid chain of the lecithin is saturated or mono- or poly-unsaturated fatty acid chain having 6 to 30 carbons, or one selected from the group consisting of sodium salt, potassium salt, magnesium salt, ammonium salt, phosphate, hydrochloride and sulfate of saturated or mono- or poly-unsaturated fatty acid chain.

Preferably, the phospholipase D is derived from microorganisms or plants. The method for producing phosphatidylethanolamine further comprises the step of washing the reaction solution containing calcium salt generated by the reaction with water or buffer to remove the enzyme, by-products such as choline or inositol, and ethanolamine of unreacted extra substrate in the reaction solution, after the step of conducting the reaction with phospholipase D. The method for producing lysophosphatidylethanolamine further comprises the step of extracting, isolating and purifying the lysophosphatidylethanolamine using ethanol, after the step of conducting the reaction with phospholipase A.

The method for producing lysophosphatidylethanolamine further comprises the step of extracting, isolating and purifying the lysophosphatidylethanolamine using hexane, ethanol and water, after the step of conducting the reaction with phospholipase A.

The method for producing lysophosphatidylethanolamine further comprises the step of treating the purified lysophosphatidylethanolamine with acetone to pulverize it, after the step of extracting, isolating and purifying the lysophosphatidylethanolamine.

The method for producing lysophosphatidylethanolamine or phosphatidylethanolamine further comprises the step of changing the composition of the fatty acid by adding hydrogen to the lecithin, or the phosphatidylethanolamine or lysophosphatidylethanolamine produced by the above-described method. Further, the composition according to the present invention comprises the phosphatidylethanolamine or lysophosphatidylethanolamine produced by the above- described method.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, more detailed description for the present invention is provided. The present invention uses buffer, in which calcium chloride is dissolved, as a reaction medium system. The method according to the present invention produces phosphatidylethanolamine by substituting the polar portion of the commercially available lecithin with ethanolamine and washing the product produced in the form of calcium salt with water to remove the by-products such as choline, enzyme used in the transfer reaction and unreacted extra ethanolamine, etc. so that any impurity cannot be remained in the product and that therefore isolation and purification of phosphatidylethanolamine may be simplified. Lysophosphatidylethanolamines are produced by adding buffer containing phospholipase A and calcium chloride to the lecithin containing the washed phosphatidylethanolamine and then by reacting them.

The buffer used in the method as described above is acetic acid buffer or phosphoric acid buffer having pH 4 to 8, more preferably pH 5 to 7.

The lecithin in the method as described above has identical or different, saturated or mono- or poly- unsaturated fatty acid chains having 6 to 30 carbons, or has a fatty acid composition changed by adding hydrogen to the lecithin, or is a salt form. Any chemically recognized salts may be used as the salt form in the present invention. In particular, sodium salt, potassium salt, magnesium salt, ammonium salt, phosphate, hydrochloride and sulfate. Most preferable salts are sodium salt and potassium salt may be the salt form in the present invention.

The preferable pH range of the buffer is 4 to 8, and the preferable reaction temperature range is 20 ^°C to 60 ^°C . Preferably, the phospholipase D is microorganism-derived enzyme or plant- derived enzyme.

Hereinafter, the present invention's method is described in detail. Ethanolamine is added to a reactor equipped with a stirring device and a temperature-regulating device and then acetic acid buffer or phosphoric acid buffer (preferably, pH is 4 to 8, more preferably 5 to 7, about 0.2M) is added to the reactor. The amount of the buffer is about 3 to 6 times of the amount of lecithin, which is a substrate. The reaction can be carried out at the temperature of 20 to 50 ^°C , depending on the need. Then, a proper amount of an enzyme having the active property of phospholipase D is added to the buffer in which ethanolamine is dissolved. Then, a proper amount of lecithin is added to the reactor and the reactor is stirred for 5 to 24 hours. After the completion of the enzymatic reaction, the reacted solution is filtered under reduced pressure and then clean water is added to the reacted solution. Thereafter, the solution is stirred and then is filtered again. This process is repeated 2 to 3 times. The lecithin containing phosphatidylethanolamine produced by the above treatments is further dried under reduced pressure or optionally treated with acetone, etc. and pulverized, and then is dried under reduced pressure in order to provide phospholipid containing high concentration of phosphatidylethanolamine.

Buffer containing calcium chloride is added, in the amount of 2 to 4 times of the phospholipids, to the phospholipids obtained by reacting and washing as above, and then phospholipase A of a needed amount is added to the buffer containing the phospholipid and the solution is stirred for 5 to 12 hours, to produce lysophosphatidylethanolamine. Then, fatty acids are removed from the solution to recover phospholipid containing high concentration of lysophosphatidylethanolamine. For this procedure, various isolation and purification methods of lysophosphatidylethanolamine can be used as follows.

First, lysophosphatidylethanolamine can be extracted by using ethanol. After the completion of an enzymatic reaction, ethanol is added to the reacted solution so that the density of the ethanol may become 70 to 90%. Then, the reacted solution is stirred under the temperature of 60 to 75 ^°C, and then the extract is filtered or stationed, followed by the steps of drawing the supernatant and then evaporating the supernatant to concentrate. Then, fatty acids are removed by treating the concentrate with acetone and then the concentrate is pulverized to recover high concentration of lysophosphatidylethanolamine. Fatty acids can be removed by supercritical fluid extraction or high-pressure propane gas extraction instead of acetone extraction.

Another method for extracting lysophosphatidylethanolamine uses hexane and ethanol. After the completion of an enzymatic reaction, hexane, ethanol and water are added to the reacted solution and the solution is stirred. Then, the solution is stationed to isolate the phases and the supernatant is concentrated. Then, fatty acids are removed from the concentrate by using acetone, followed by the steps of pulverizing the concentrate to obtain high concentration of lysophosphatidylethanolamine.

Further, the phosphatidylethanolamine or lysophosphatidylethanolamine produced according to the above methods can be modified in terms of the composition of fatty acids by hydrogenation reaction. Hydrogenation is carried out by dissolving raw lecithin or the phosphatidylethanolamine or lysophosphatidylethanolamine produced by transfer reaction in ethanol so that the density may become 10 to 20% and adding 4% palladium carbon to the solution so that it would constitute 3.3 to 5% of the entire solution, followed by the steps of blowing at least 40 psi of hydrogen pressure into the solution three times and finally reacting the solution under the condition of 40 to 60 psi hydrogen pressure(according to the extent of hydrogenation to be needed) at 50 ^°C and 300 to 900 rpm for 6-15 hours, to recover hydrogenated raw lecithin, phosphatidylethanolamine or lysophosphatidylethanolamine.

Hereinafter, the present invention will be described in more detail with references to the following preferred embodiments.

^•determination conditions of the PE or LPE content>

HPLC analysis method is used to determine the content of phosphatidylethanolamine and lysophosphatidylethanolamine. The conditions are follows. LiChrosphere 100 diol (Merck, 4m x 125mm) for HPLC analysis is used. Two solvent system having a concentration gradient, which consists of A phase of hexane/isopropanol/acetic acid/triethylamine = 82 : 17 : 1 : 0.08 and B phase of isopropanol/water/acetic acid/triethylamine •= 85 : 14 : 1 : 0.08 is used. The time schedule is as follows. [ Table 1 ]

The flow rate is 1.5ml/min., ELSD detector is used, the temperature of the detector is 65 ^°C , and the gas pressure is 1.9L/min. <Preferred embodiment 1> 60g of ethanolamine and 8.8g of calcium chloride, and then 2000 unit of phospholipase D derived from Actinomycetes strains are added to 1L of sodium acetate buffer(pH 5.6). Then, the solution mixed as above was stirred at 40 ^°C for the complete dissolution of ethanolamine, and then 200g of lecithin(FP 40, CENTRAL SOYA company, 40% content of phosphatidylcholine) is added to the solution, followed by the step of conducting the reaction of them while the solution is stirred at

200 rpm for 12 hours. After the completion of the reaction, the solution is stirred and filtered with the addition of clean water of 3L. The procedure of the water addition, stirring and filtering as above is repeated 2 times and then the solution is dried under reduced pressure. As a result, the yield obtained is 90%, and the content of phosphatidylethanolamine is 70%. <Preferred embodiment 2>

Lysophosphatidylethanolamine is produced by further treating the solution with phospholipase A after the transfer reaction of the above preferred embodiment 1.

60g of ethanolamine and 8.8g of calcium chloride, and then 2000 unit of phospholipase D derived from Actinomycetes strains are added to 1L of sodium acetate buffer (pH 5.6). Then, the solution mixed as above is stirred at 40 ^°C for the complete dissolution of ethanolamine, and then 200g of lecithin(FP 40, CENTRAL SOYA company, 40% content of phosphatidylcholine) is added to the solution, followed by the reaction conducted while the solution is stirred at 200 rpm for 12 hours. After the completion of the reaction, the solution is stirred and filtered with the addition of clean water of 3L. The procedure of the water addition, stirring and filtering as above is repeated 2 times, followed by further adding 400ml of sodium acetate buffer (pH 5.6) and then 3.5g of calcium chloride and 6ml of phospholipase A2 (8,000 IU/ml, as the brand name of LECITASE 10L, NOVO NORDISK Co.), which is derived from a pig's pancreas, to the solution and then by the reaction at 37 °C for 6 hours. Following the completion of the reaction, 1000 ml of hexane, 600 ml of ethanol and 400ml of water are added to the solution and then the solution is stirred for 30 minutes, followed by standing. After phase isolation takes place, the supernatant drawn from the solution is concentrated by evaporation under the reduced pressure and then is treated by 1L of acetone. The resulting yield is 35%, and the content of lysophosphatidylethanolamine is 70%.

<Preferred embodiment 3> Lysophosphatidylethanolamine is isolated and purified by using ethanol and acetone after the transfer reaction of the above preferred embodiment 2.

60g of ethanolamine and 8.8g of calcium chloride, and then 2000 unit of phospholipase D derived from Actinomycetes strains are added to 1L of sodium acetate buffer (pH 5.6). Then, the solution mixed as above is stirred at 40 ^°C for the complete dissolution of ethanolamine, and then 200g of lecithin(FP 40, CENTRAL SOYA company, 40% content of phosphatidylcholine) is added to the solution, followed by the reaction conducted while the solution is stirred at 200 rpm for 12 hours. After the completion of the reaction, the solution is stirred and filtered with the addition of clean water of 3L. The procedure of the water addition, stirring and filtering as above is repeated 2 times, followed by further adding 400ml of sodium acetate buffer (pH 5.6) and then 3.5g of calcium chloride and 6ml of phospholipase A2 (8,000 IU/ml, as the brand name of LECITASE 10L, NOVO NORDISK Co.), which is derived from a pig's pancreas, to the solution and then by the reaction at 37°C for 6 hours. Following the completion of the reaction, 1.6L of ethanol is added to the solution and then the solution is stirred at 70°C to extract lysophosphatidylethanolamine, followed by extracting and filtering. The filtrate is concentrated by evaporation and stirred using

1L of acetone 2 times to remove fatty acids. Then, lysophosphatidylethanolamine is pulverized. The resulting yield is 40%, and the content of lysophosphatidylethanolamine is 64%.

Lysophosphatidylethanolamine is isolated and purified by dissolving it in ethanol without the use of acetone and then cooling after the completion of the reaction of the above preferred embodiment 2. 60g of ethanolamine and 8.8g of calcium chloride, and then 2000 unit of phospholipase D derived from Actinomycetes strains are added to 1L of sodium acetate buffer (pH 5.6). Then, the solution mixed as above is stirred at 40 ^°C for the complete dissolution of ethanolamine, and then 200g of lecithin(FP 40, CENTRAL SOYA company, 40% content of phosphatidylcholine) is added to the solution, followed by the reaction conducted while the solution is stirred at 200 rpm for 12 hours. After the completion of the reaction, the solution is stirred and filtered with the addition of clean water of 3L. The procedure of the water addition, stirring and filtering as above is repeated 2 times, followed by further adding 400ml of sodium acetate buffer (pH 5.6) and then 3.5g of calcium chloride and 6ml of phospholipase A2 (8,000 IU/ml, as the brand name of LECITASE 10L, NOVO NORDISK Co.), which is derived from a pig's pancreas of a pig, to the solution and then by reaction at 37°C for 6 hours. Following the completion of the reaction, 1.6L of ethanol is added to the solution and then the solution is stirred at 70°C to extract lysophosphatidylethanolamine, followed by cooling to -10°C to precipitate and then followed by filtrating them. The resulting yield is 30%, and the content of lysophosphatidylethanolamine is 90.2%.

In preferred embodiment 5, the reaction of preferred embodiment 1 is conducted as described above, provided, however, that lecithin derived from yolk is used as a raw material of phosphatidylethanolamine. lOOg of ethanolamine and 8.8g of calcium chloride, and then 6000 unit of phospholipase D derived from Actinomycetes strains are added to 1L of sodium acetate buffer (pH 5.6). Then, the solution mixed as above is stirred at 40 °C for the complete dissolution of ethanolamine, and then 200g of lecithin(PL95, Doosan Corporation Biotech BU, 75% content of phosphatidylcholine) is added to the solution, followed by the reaction conducted while the solution is stirred at 200 rpm for 24 hours. After the completion of the reaction, the solution is stirred and filtered with the addition of clean water of 3L. The procedure of the water addition, stirring and filtering as above is repeated 2 times, followed by drying under reduced pressure. The resulting yield is 90%, and the content of lysophosphatidylethanolamine is 90%. <Preferred embodiment 6>

In preferred embodiment 6, the reaction of preferred embodiment 3 is conducted as described above provided, however, that lecithin derived from yolk is used as a raw material of lysophosphatidylethanolamine. lOOg of ethanolamine and 8.8g of calcium chloride, and then 6000 unit of phospholipase D derived from Actinomycetes strains are added to 1L of sodium acetate buffer (pH 5.6). Then, the solution mixed as above is stirred at 40 ^°C for the complete dissolution of ethanolamine, and then 200g of lecithin(PL95, Doosan Corporation Biotech BU, 75% content of phosphatidylcholine) is added to the solution, followed by the reaction conducted while the solution is stirred at 200 rpm for 24 hours. After the completion of the reaction, the solution is stirred and filtered with the addition of clean water of 3L. The procedure of the water addition, stirring and filtering as above is repeated 2 times, followed by further adding 400ml of sodium acetate buffer (pH 5.6) and then 3.5g of calcium chloride and 10ml of phospholipase A2 (8,000 IU/ml, as the brand name of LECITASE 10L, NONO NORDISK Co.), which is derived from a pig's pancreas, to the solution and then by the reaction at 37°C for 6 hours. Following the completion of the reaction, 1.6L of ethanol is added to the solution and then the solution is stirred at 80°C to extract lysophosphatidylethanolamine, followed by extracting and filtering. After the filtrate is concentrated by evaporation and then is stirred using IL of acetone 2 times to remove fatty acids, it is pulverized. The resulting yield is 60%, and the content of lysophosphatidylethanolamine is 94%. <Preferred embodiment 7> Phospholipase D derived from plants can be used as an enzyme as well as that derived from Actinomycetes strains. For example, phospholipase D derived from cabbage or soybean can be used. Preferred embodiment 7 illustrates an example wherein phosphatidylethanolamine is synthesized using cabbage extract showing phospholipase D activity. 60g of ethanolamine and 8.8g of calcium chloride, and then 2000 unit of cabbage extract having phospholipase D activity are added to IL of sodium acetate buffer(pH 5.6). Then, 200g of lecithin(CENTROLEX D, CENTRAL SOYA company) is added to the solution, followed by the reaction conducted while the solution is stirred at 200 rpm for 12 hours. After the completion of the reaction, the solution is stirred and filtered with the addition of clean water of 3L. The procedure of the water addition, stirring and filtering as above is repeated 2 times. Then, 1000 ml of hexane, 600 ml of ethanol and 400 ml of water are added to the solution and then the solution is stirred for 30 minutes, followed by standing. After phase isolation takes place, the supernatant drawn from the solution is concentrated by evaporation under the reduced pressure and then is pulverized using IL of acetone. The resulting yield is 83%, and the content of phosphatidylethanolamine is 40%.

1 kg of the lysophosphatidylethanolamine produced by the above preferred embodiments 2 and 3 is dissolved in ethanol in the concentration of 10%, and then 5% palladium carbon is added so that its weight occupies 2.5% of the entire weight of the solution. After that, the solution is reacted with 14.7psi of hydrogen pressure at 50 ^°C for 15 hours to obtain 952g of lysophosphatidylethanolamine in which almost all of the fatty acids are saturated through hydrogenation. <Preferred embodiment 9> lOg of lysophosphatidylethanolamine produced by the above preferred embodiments 2 and 3 is added to 100ml of water and then stirred to obtain 10% of clear lysophosphatidylethanolamine solution.

Because the invention does not use organic solvent, the present invention eliminates the danger or difficulty in purification, which may exist in the case where toxic organic solvent is used and the present invention enables low inactivity of enzyme and high reaction efficiency. In particular, isolation and purification process after the completion of the reaction can be simplified and organic acid used in the isolation and purification process can be easily recovered since the present does not use organic solvent during the enzymatic reaction. Furthermore, lecithin having high purity as well as lecithin having medium or low purity(less than 50% of the content of lecithin) can be dispersed smoothly. Also, enzyme, ethanolamine and choline produced after the reaction can be removed by washing the product containing calcium salt with water after the synthesis of phosphatidylethanolamine so that impurities such as enzyme, ethanolamine or choline, etc. may not be mixed into the final product.

Claims

What Is Claimed Is:

1. A method for producing phosphatidylethanolamine comprising the step of conducting the reaction of lecithin and ethanolamine under the existence of phospholipase D, wherein: the lecithin comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol; and the reaction is carried out in non-organic solvent system containing calcium chloride and buffer.

2. A method for producing lysophosphatidylethanolamine comprising the step of conducting the reaction of lecithin and ethanolamine under the existence of phospholipase D and phospholipase A, wherein: the lecithin comprises at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and phosphatidylinositol; and the reaction is carried out in non-organic solvent system containing calcium chloride and buffer.

3. The method for producing lysophosphatidylethanolamine according to Claim 2, comprising the steps of: conducting the first reaction of lecithin and ethanolamine under the existence of phospholipase D; and conducting the second reaction of the first products generated by the first reacting upon adding phospholipase A to the first products.

4. The method according to any one of Claims 1 to 3, wherein the non-organic solvent system consists only of calcium chloride and buffer.

5. The method according to any one of Claims 1 to 3, wherein the lecithin is added to the buffer for the reactions in such amount that the lecithin occupies 1 to 50% of the buffer.

6. The method according to any one of Claims 1 to 3, wherein the buffer is pH 4 to 8.

7. The method according to any one of Claims 1 to 3, wherein the temperature of the reactions is 20 to 60 °C .

8. The method according to any one of Claims 1 to 3, wherein the lecithin is one selected from the group consisting of soybean-derived lecithin, rape seed-derived lecithin, fishes-derived lecithin, mollusk-derived lecithin and yolk-derived lecithin, and the fatty acid chain of the lecithin is saturated or mono- or poly-unsaturated fatty acid chain having 6 to 30 carbons, or one selected from the group consisting of sodium salt, potassium salt, magnesium salt, ammonium salt, phosphate, hydrochloride and sulfate thereof.

9. The method according to any one of Claims 1 to 3, wherein the phospholipase D is derived from microorganism or plant.

10. The method according to any one of Claims 1 to 3, comprising the step of washing the reaction solution containing calcium salt generated by the reaction with water or buffer to remove the enzyme, by-products such as choline or inositol, and ethanolamine of unreacted extra substrate in the reaction solution after the step of conducting the reaction under the existence of phospholipase D.

11. The method according to Claim 3, further comprising the step of extracting, isolating and purifying the lysophosphatidylethanolamine using ethanol after the step of conducting the reaction under the existence of phospholipase A.

12. The method according to Claim 3, further comprising the step of extracting, isolating and purifying the lysophosphatidylethanolamine using hexane, ethanol and water after the step of conducting the reaction under the existence of phospholipase A.

13. The method according to Claims 11 or 12, further comprising the step of treating the purified lysophosphatidylethanolamine with acetone to pulverize it after the step of extracting, isolating and purifying is conducted.

14. The method according to any one of Claims 1 to 3, further comprising the step of changing the composition of the fatty acid by adding hydrogen to the lecithin, or the phosphatidylethanolamine or lysophosphatidylethanolamine produced by the method according to Claim 1.

15. A water-soluble composition, comprising the phosphatidylethanolamine produced by the method according to any one of Claims 1 to 14.