JP2709721B2 - Method for modifying phospholipid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to the transesterification of phospholipids by reacting phospholipids with a fatty acid or a lower alcohol ester of a fatty acid with an enzyme having a fatty acid transesterification effect of the phospholipids. And a method for modifying a phospholipid comprising:

(Prior Art) Phospholipids are formed by bonding a hydrophilic phosphorus-containing compound such as phosphorylcholine, phosphorylethanolamine, or phosphorylinositol with glycerol and a fatty acid. Functional properties such as activity are largely affected by the type of bound fatty acid.

As one of means for adjusting the physical properties and the like by changing the type of fatty acid bound to the glycerol residue, there is a method of transesterifying a fatty acid by utilizing the action of an enzyme having transesterification ability. Many studies have been made on the modification of fats and oils (triglycerides) by this method, and there are many patent applications related to this (for example, Japanese Patent Application Laid-Open No. 61-149097, The main patent applications and their evaluations are described).

On the other hand, modification of phospholipids by enzymes causes enzymes such as snake venom phospholipase and pancreatin, which have the ability to cleave acyl bonds of phospholipids, to decompose a part of them and convert them into hydrophilic lyso-type phospholipids. Methods for improving the emulsifying properties are known (for example, see JP-A-53-7151).
7).

However, regarding the reaction of fatty acids of phospholipids by enzymes, Z. Naturforsch
Vol. 25b, pp. 581-586 (1970) only reports that HPFranck et al. Have found that phospholipase A has the ability to bind fatty acids to lysophospholipids. is there. However, later reports revealed that this was also a false conclusion due to experimental deficiencies [J. Goerke et al .: Biochimica et Biophysica Acta.
et Biophysica Acta) 248: 245-253 (197
1)].

Recently, a method for transesterification of phosphatidylcholine with a lipase modified with a polyalkylene glycol has been proposed (JP-A-63-105686), but it is said that unmodified lipase has no such transesterification function.

Accordingly, the present inventors have examined the fatty acid transesterification ability of phospholipids for a large number of enzymes, and found that certain enzymes have the same action.Based on this, a method for modifying phospholipids by transesterification was found. Proposed as Japanese Patent Application No. 62-15181.

This method itself is an epoch-making process that is unprecedented, but for example, even when the reaction is performed under the same reaction conditions using the same enzyme, the transesterification rate and the yield may vary. Problems were scattered.

(Problems to be Solved by the Invention) The present inventors have conducted detailed studies on the above-mentioned causes and found that the transesterification reaction largely involves the water content of the system.

Regarding the water content of the reaction system of transesterification by enzymes,
Conventionally, studies have been made on a system using fats and oils (triglyceride) as a substrate, but it is desirable to reduce the water content of the reaction system in order to suppress the ester hydrolysis reaction that occurs simultaneously with the transesterification. 52-1 Kaisho
Tanaka et al. [Fermentation and Industry, Vol. 41, No. 5
No. 375-381 (1983)] recommends an extremely low range such as 0.25% of the organic solvent such as n-hexane used for dissolving the substrate.

However, transesterification using a phospholipid as a substrate proved to be completely contrary to the above-mentioned findings on triglycerides.

In other words, in order to obtain a phospholipid having a high transesterification rate in a stable and high yield, a knowledge with a conventional triglyceride of a system in which the volume ratio of the organic solvent phase and the aqueous phase is 1: 9 to 9: 1. Could not have expected. It has been found that it is better to work in a high moisture system. The present invention has been completed based on such findings.

(Constitution of the Invention) The present invention uses a phospholipid and a fatty acid or a fatty acid lower alcohol ester as substrates, and the volume ratio of the organic solvent phase to the aqueous phase is
In a system in which the ratio is 1: 9 to 9: 1, an enzyme having a phospholipid fatty acid transesterification effect is actuated to thereby provide a phospholipid modification method characterized by performing a phospholipid transesterification. .

Phospholipids to be subjected to transesterification by the enzyme of the present invention include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and other simple phospholipids, for example, soybean lecithin, yolk lecithin and other commercial lecithins of animal and plant origin, and these phospholipids There is a so-called lyso-type phospholipid from which a part of the bound fatty acid is removed, a so-called extracted soap slag containing a phospholipid component, which is obtained in a degumming step which is one of the steps of refining vegetable oil.

As the fatty acid, linear saturated fatty acids, unsaturated fatty acids and polyunsaturated fatty acids having about 8 to 24 carbon atoms can be used, and examples thereof include palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid. Can be. The lower alcohol ester of a fatty acid refers to an ester compound of the above fatty acid and a linear saturated monohydric alcohol having 1 to 6 carbon atoms, and examples thereof include methyl palmitate, methyl stearate, methyl linoleate, methyl arachidonic acid, and palmitic acid. Ethyl, ethyl stearate, ethyl linoleate, ethyl arachidate and the like.

Enzymes that act on these phospholipids and fatty acids or fatty acid lower alcohol esters to change the type and position of the fatty acids bound to the phospholipids, that is, enzymes having a transesterification effect include, for example, Aspergillus,
Rhizopus, Mucor, Pseudomonas, Arisobacter, Candida, Chromobacterium,
Examples include enzymes produced by microorganisms belonging to the genus Penicillium and Streptomyces, and lipolytic enzymes derived from the pancreas.

Examples of microorganisms belonging to the above genera include Aspergillus niger in Aspergillus.
r) or Aspergillus wen
ti), Rhizopus dremar (Rhizopus d
elemar), Rhizopus javanic
us), Rhizopus niveus, Mucor miehei for the genus Mucor, Mucor javanicus for the genus Mucor, Pseudomonas fluorescens for the genus Pseudomonas
monus fluorescence), Pseudomonas frage (Ps
eudomonus fragi), and Arisrobacter ureafaciens (Arthrobacter ureafac)
iens), Candida cylindracea for Candida, and Chromobacterium viscosum for Chromobacterium
m viscosum), Penicillium cyclopium (Penicillium cyclopium), Streptomyces genus Streptomyces violaceoruber (streptomyces violaceoruber) and the like, and the enzymes produced by these have lipase or phospholipase activity In addition, since it has a transesterification activity of phospholipids, it can be suitably used in the present invention.

The reaction is conducted by adding a phospholipid and a fatty acid or a fatty acid lower alcohol ester to a nonpolar solvent such as hexane in an amount of 1 to 50% by weight.
Water containing an amount of enzyme equivalent to 10 to 50,000 units of lipase activity measured by the method of Yamada et al. {Journal of the Japanese Society of Agricultural Chemistry 36 , 860 (1962)} per gram of phospholipid. Or add a buffer solution.

In the present invention, the volume ratio of the organic solvent phase and the aqueous phase of the system is 1: 9 to 9: 1, more preferably 1: 1, based on the entire reaction system.
It is essential to add water or buffer solution to make the system 8-8: 1.

The reasons for limiting the water content of the reaction system to the above are as follows. That is, when the volume ratio of the aqueous phase is 9: 1 (aqueous phase) or less, ester hydrolysis is suppressed, but the transesterification does not proceed sufficiently, and conversely, the volume ratio of the aqueous phase is 1: 9 (water phase). If the amount exceeds (phase), the hydrolysis reaction is accelerated, and in any case, the transesterification rate is significantly reduced.

The reaction solution is shaken at 20 to 60 ° C, more preferably 25 to 60 ° C.
React at 40 ° C for 1 to 48 hours. The ratio of phospholipid and fatty acid or fatty acid lower alcohol ester of the reaction system is 1: 0.
It is desirable to be in the range of 2 to 1: 5.

When a substance having a high degree of unsaturation, such as arachidonic acid or an ester thereof, is used as the substrate, it is desirable that tocopherol and other substances having antioxidative properties coexist in an organic solvent.

As a means for increasing the activity of the enzyme, a celite-based support and an activating agent such as ethanol and propanol can be appropriately added to the reaction system. In addition, bioreactors variously devised for the enzymatic reaction can also be used.

The reaction solution after the lapse of a predetermined time, the lipase is inactivated by heating or the like, if necessary, concentrated by an appropriate concentrator as needed, and further subjected to appropriate drying means such as spray drying and freeze drying. Concentrate or dry. Further, the reaction product is extracted and separated from the reaction system by using an appropriate solvent such as benzene or chloroform at any of the above-mentioned stages, and after performing solvent separation, purification such as column treatment as necessary, the solvent is distilled off. If this is the case, a higher-purity modified phospholipid can be obtained.

(Example) Example 1 An enzyme derived from Rhizopus delemar (manufactured by Tanabe Seiyaku Co., Ltd.
100 U) were dissolved in TES buffer (pH 6.5). To this was added 10 mg of dipalmitoyl phosphatidylcholine and 20 mg of oleic acid in n-hexane. The total amount of the buffer and n-hexane was 1 ml in total, and the mixing ratio of both was changed from 5:95 to 9: 1. The reaction vessel is sealed and 3
The reaction was carried out at 0 ° C. for 40 hours with reciprocal shaking (180 rpm, shaking 2 cm).

After drying the reaction mixture, the oil content is extracted with chloroform, the remaining amount of phosphatidylcholine is quantified by Iatroscan, and the phosphatidylcholine fraction is collected by silica gel thin-layer chromatography (developing solvent: chloroform: methanol: water 60: 30: 3). The fatty acid composition was examined by gas chromatography.

As shown in FIG. 1, the oleic acid content of the fatty acid in the phosphatidylcholine fraction, which indicates the degree of transesterification, increased as the water content of the system increased, and the organic solvent phase and the aqueous phase in the system increased. Volume ratio of 8: 2
The equilibrium was almost reached in the range of 22: 8, and after the ratio exceeded 1: 9, it began to decrease. On the other hand, it was revealed that the residual amount of phosphatidylcholine continued to decrease as the water content increased, and that the water content promoted the hydrolysis of the ester bond.

Example 2 An enzyme derived from Rhizopus delemar (Seikagaku Corporation)
(Purified enzyme), 200 U was dissolved in TES buffer (pH 6.5).

To this was added 10 mg of dipalmitoyl phosphatidylcholine and 30 mg of oleic acid in n-hexane. The total volume of the buffer and n-hexane was 1 ml in total, and the volume ratio of both was changed from 1: 9 to 9: 1.

The reaction vessel was sealed and reacted at 35 ° C. for 42 hours with reciprocal shaking (180 rpm, 2 cm shaking). The reaction solution after the elapse of time was analyzed in the same manner as in Example 1. The results are as shown in FIG. 2, and the transition of the residual ratio of phosphatidylcholine was the same as in Example 1. However, in the phosphatidylcholine fraction, the highest value of the oleic acid content in the fatty acid was 1% by volume ratio between the organic solvent phase and the aqueous phase. : 1 to 1: 4.

Example 3 100 U of an enzyme derived from Rhizopus delemar (Talipase manufactured by Tanabe Seiyaku Co., Ltd.) was dissolved in 500 μl of TES buffer (pH 6.5). To this was added 500 μl of n-hexane containing 10 mg of phospholipids and 20 mg of fatty acids. Phospholipids include dipalmitoyl phosphatidylcholine (DPPC), dioleoyl
Phosphatidylcholine (DOPC) and dipalmitoyl phosphatidylethanolamine (DPPE) were used. As fatty acids, capric acid, myristic acid, palmitic acid, oleic acid, γ-linolenic acid and methyl oleate were used.

The reaction vessel was sealed and reacted at 35 ° C. for 20 hours with reciprocal shaking (180 rpm, shaking 2 cm). The reaction solution after the elapse of time was analyzed in the same manner as in Example 1. Table 1 shows the results.
As shown in the figure, it was shown that transesterification occurs with any combination of substrates.

Example 4 Four types of commercially available lipases (from Aspergillus niger; from Amano Pharmaceutical Lipase A, Pseudomonas fluorescens); from Amano Pharmaceutical Lipase P, from Mucor Javanicas; from Amano Pharmaceutical Lipase M, from Candida Cylindracea; ) 100 U of each was dissolved in 500 μl of TES buffer (pH 6.5). To this was added 500 μl of n-hexane containing 10 mg of dipalmitoyl phosphatidylcholine and 30 mg of oleic acid.

The reaction vessel was sealed and reacted at 35 ° C. for 20 hours with reciprocal shaking (180 rpm, shaking 2 cm). The reaction solution after the elapse of time was analyzed in the same manner as in Example 1. Table 2 shows the results.
The results show that transesterification occurs in any of the enzymes.

(Effect of the Invention) The present invention has developed an efficient transesterification technique for phospholipids using an enzyme. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to arbitrarily adjust physical properties such as a melting point and fluidity of a phospholipid, and it becomes possible to prepare a phospholipid most suitable for the use in the food industry and other industries. Phospholipids are also a material for preparing liposomes, which are attracting attention as carriers for various drugs and enzymes. By using a phospholipid whose fatty acid composition has been changed by the method of the present invention, its physiological action can be significantly enhanced.

Since the enzyme of the present invention is used, there is no degradation of the highly unsaturated fatty acid as compared with the chemical synthesis method, and the desired fatty acid is introduced into a specific site of the phospholipid utilizing the specificity of the enzyme. Yes, there are advantages.

[Brief description of the drawings]

1 and 2 show the analysis of the reaction solution in Examples 1 and 2, respectively. In each figure, ● indicates the content of oleic acid in the fatty acid in the phosphatidylcholine fraction, は indicates the residual ratio of phosphatidylcholine, and the abscissa indicates the volume of the buffer when the total volume of n-hexane and the buffer was assumed to be 10. Show.

Continued on the front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical indication (C12P 9/00 C12R 1: 785) (C12P 9/00 C12R 1:38) (C12P 9/00 C12R 1: 06) (C12P 9/00 C12R 1:72) (C12P 9/00 C12R 1:01) (C12P 9/00 C12R 1:80) (C12P 9/00 C12R 1: 465)

Claims (3)

(57) [Claims] The present invention relates to a method comprising the steps of: (1) using a phospholipid and a fatty acid or a fatty acid lower alcohol ester as substrates;
A method for modifying a phospholipid, wherein a phospholipid is subjected to fatty acid transesterification by causing an enzyme having a phospholipid fatty acid transesterification action to act in a system of 1: 9 to 9: 1. 2. The enzyme having a fatty acid transesterification effect of phospholipids is selected from the group consisting of Aspergillus, Rhizopus, Mucor,
2. The method for modifying a phospholipid according to claim 1, wherein the enzyme is an enzyme produced by one of microorganisms belonging to the genera Pseudomonas, Arisobacter, Candida, Chromobacterium, Penicillium, and Streptomyces. 3. The method for modifying a phospholipid according to claim 1, wherein the enzyme having a fatty acid transesterification activity of the phospholipid is a pancreatic lipolytic enzyme.