JPH022381A - Production of phospholipid - Google Patents

Abstract

PURPOSE:To obtain a phospholipid having modified base structure in high yield at a low cost by contacting and reacting a phospholipid used as a raw material and a receptor having hydroxyl group with a dried microbial cell containing phospholipase D. CONSTITUTION:The objective phospholipid can be produced by contacting and reacting a phospholipid used as a raw material (e.g., phosphatidylcholine or phosphatidylserine) and a receptor having hydroxyl group (e.g., choline ethanolamine) with dried microbial cell containing phospholipase D. The above dried microbial cell is preferably produced by immobilizing microbial cells on a porous carrier for microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing phospholipids, and more particularly to a method for producing phospholipids whose base structure has been converted by dried microbial cells.

In recent years, phospholipids have attracted attention not only for their use as emulsifiers, but also for their use as base materials for liposomes, in drug carriers, artificial blood, artificial cells, and immunodiagnosis.
As a substance that itself has physiological and pharmacological activity, it is
Various uses are being considered in the pharmaceutical and engineering fields. In order to meet such diverse demands, it is of great industrial significance to develop a method for efficiently producing phospholipids having structures suitable for various uses.

[Problems to be solved by conventional technology and invention] Conventionally,
As a method for producing such phospholipids, various purified phospholipases D derived from cabbage or microorganisms are used.
Methods of reacting enzymes directly or by immobilization have been proposed (JP-A No. 63-36790, No. 63-3).
6792, 63-91087), L However, the cost of the enzyme manufacturing process for such extracted or purified enzymes is high, and this poses a major economic problem. Furthermore, in the method of immobilizing the enzyme on a carrier such as Celite, it is difficult for the reactant to diffuse to the enzyme on the carrier, and in particular, the enzyme adsorbed within the pores of the carrier does not substantially participate in the reaction and is not effective. There are problems such as a decrease in the number of working enzymes, which is a major obstacle to commercialization. Also, what about the immobilization operation for comprehensive immobilization using gelling agents? ! It is complicated and has many disadvantages such as a reduction in reaction rate due to diffusion control, and improvements are desired.

The purpose of the present invention is to overcome these drawbacks by directly subjecting dried bacterial cells containing phospholipase D to a reaction, and to provide a base exchange reaction method for phospholipids that yields the desired product in high yield. do.

[Means for solving problems]

That is, the present invention is characterized in that, in producing a phospholipid whose base structure has been changed, a reaction is carried out by bringing a raw material phospholipid and a receptor having a hydroxyl group into contact with dried bacterial cells having phospholipasego. The content is a method for producing phospholipids.

The raw material phospholipid used in the present invention may be any phospholipid extracted from nature, purified after extraction, or synthesized, as long as it can serve as a substrate for phospholipase D. Furthermore, commercially available products or products prepared by known methods may be used. For example, defatted soybean lecithin, egg yolk lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, etc.
or a mixture thereof. In order to maximize the effects of the present invention, it is more convenient to use purified phospholipids or those with a simple composition as raw material phospholipids in terms of obtaining pure reaction products. In addition, from the viewpoints of raw material cost, ease of labor, and reactivity to moss, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine are particularly preferred as they are industrially highly effective.

In the present invention, receptors include choline, ethanolamine, serine, N-methylethanolamine, N, N-
Examples include nitrogen-containing alcohols such as dimethylethanolamine, polyols such as glycerol, glucose, and fructose, and monosaccharides.

The reaction is preferably carried out in the presence of an organic solvent that dissolves or suspends the raw material phospholipid, and any solvent system that does not deactivate microbial enzymes can be used. For example, solvents such as petroleum ether, diethyl ether, methyl ethyl ether, diisopropyl ether, chloroform, dichloromethane, carbon tetrachloride, dichloroethane, n-hexane, cyclohexane, n-octane, isooctane, ethyl acetate, dioxane, benzene, etc.; Examples include a mixed solvent system, or a mixed solvent system in which a polar solvent such as acetone or acetonitrile is mixed with these solvents.

However, since alcohols serve as substrates for the desired reaction, it is not very preferable to use them for purposes other than adding them as substrates.

As the microorganism used in the present invention, any microorganism that produces phospholipase D can be used, but in particular, Streptomyces
s) and the genus I. Leptoverticillium (Strepto
verticilliui), Micromonosvora (
Micromonospora) genus, Nocardia (N
ocardi-a) genus, Nocardiopsis (Noca
rdiopsis), Actinomadura (Acti
Examples include microorganisms belonging to the genus Nomadura. More specifically, Streptomyces chromofuscus (SL, chr-omofuscus IF
O12851), Streptomyces mediocidicus (SL, mediocidicus lFo, 13
202), Streptomyces labenjulae (S
L, Iaven-dulae 5ubsp, Iav
endulae IFO12789), Streptoverticillium griseocarnelum (St, gris
eocarneum IFO12776), Streptoverticillium cinnamomelum (St, cinna
momeum 5ubsp.

cinnamomeum IFO12852) %Streptoberticillium)z Chijoense (St,
hachijoense IFOI2782), Micromonosvora charcea (M, cha-Icea)
ATCC12452), Nocardia mediterranei I
FO13142), Nocardiopsis d-assonvil
lei IFo 1390B), actinomadura
Actinoiadura l1bano
tica IFO14095), and other known microorganisms may also be used.

In order to make the above microorganisms work efficiently as reaction catalysts,
What is important is a culture method that allows the microorganism to contain a large amount of phospholipase D, and drying conditions that allow the phospholipase D within the microorganism to easily come into contact with the receptor and that do not reduce its activity in any way possible.

As the culture medium used for culture, a wide variety of media commonly used for culturing microorganisms can be used. Carbon sources include assimilable carbon compounds such as glucose, ci=! Sugar, lactose, maltose, starch, dextrin, molasses, glycerin, etc. and hydrocarbons are used.

The nitrogen source may be any available nitrogen compound, and organic compounds such as corn steep liquor, soybean flour, wheat gluten, peptone, meat extract, yeast extract, malt extract, and casein hydrolyzate are particularly preferred. In addition, salts such as phosphate, magnesium, calcium, potassium, iron, manganese, and zinc are used as necessary.

The culture temperature can be appropriately selected within a range that allows the bacteria to grow and produce phospholipase D, but the culture is usually carried out at 15 to 40°C. The culture time varies depending on the conditions, and it is sufficient to terminate the culture when the intracellular phospholipase D activity reaches its maximum.
Usually it takes about 1 to 6 days.

Furthermore, in the present invention, the microorganisms can be cultured together with a microorganism carrier made of a porous material, the microorganisms can be attached and grown on the carrier, and the obtained microbial cells can be used. In such a method, a biofilm-like bacterial cell is formed near the surface layer of the carrier, which makes phospholipase D more stable.
Furthermore, since it can be applied to effective reaction systems such as continuous production and repeated batch production, productivity is greatly improved and it is extremely convenient. As the microorganism holder, any material that enables adsorption and proliferation of microorganisms due to the adhesive force and adsorption power possessed by microorganisms can be used. For example, the polymeric porous materials include polyolefin-based materials such as polyethylene and polypropylene; diene-based materials such as butadiene or isoprene; vinyl-based polymers such as polyvinyl chloride, polyvinyl alcohol, acrylamide, and polystyrene; polyethers, polyesters, polycarbonates, etc. Condensation systems such as polyamide; materials such as polyurethane, silicon, and fluorine fiber resin; and as inorganic materials, ceramics, glass, activated carbon, porous metals, and metal processing materials can be used. In any material, the porosity is 60 to 9 in order to properly immobilize microorganisms on the holding body.
It is preferable to use a porous material with a porosity of 0% and a pore diameter in the range of 2 to 2000 μm, a metal processing material with a porosity of 60 to 99%, and the like.

Such various carriers can be appropriately selected depending on the type of microorganism, culture conditions, etc., and can be processed into shapes such as spheres, blocks, or sheets. The dimensions are determined appropriately depending on the type of microorganism, culture conditions, type of reactor, etc., but if it is spherical, the diameter is about 1 to 100 mm, and if it is block-shaped, the diameter is about 1 to 100 mm on a side. is used. Also,
Immobilization of microorganisms on the above-mentioned carrier can be easily achieved using commonly known batch, semi-batch, continuous culture, etc.

In principle, the method for removing water from the bacterial cells obtained as described above is at a temperature that does not deactivate the enzyme (40 to 60°C).
However, if the moisture is simply evaporated, the cellular Mi tissue will shrink and become very stiff, cutting off contact between phospholipase D in the tissue and the outside world, making it difficult to express activity. Therefore, in order to dry the bacterial cells, it is necessary to use a method that does not involve shrinkage of the cell tissue. For this reason, shrinkage of cellular tissue is achieved by immersing the bacterial cells in a water-soluble solvent such as acetone or lower alcohols such as methanol, ethanol, and isopropanol, replacing the tissue with the solvent, and then evaporating the solvent. Dried bacterial cells can be obtained by suppressing the amount of bacteria.

In this case, drying methods include vacuum drying, freeze drying,
Known drying methods such as crystal drying can be used. Furthermore, before immersing the bacterial cells in the solvent, by immersing them in an aqueous solution of geltaraldehyde of 5% by weight or less to fix the cell tissue, the cell Mi
The shrinkage of the fabric can be suppressed more effectively. The moisture content of the bacterial cells obtained by this method is usually 1 to 20% by weight.
The moisture content is adjusted to .

The dried bacterial cells prepared in this way are suspended in a mixture of reactants, ie, starting phospholipids, and receptors to react.The ratio of water to organic solvent is t by weight. :O
, 5 to o, and t: 1o, but in order to prevent as much as possible the production of phosphatidic acid produced by side reactions, it is preferable to limit the water content to 40% by weight or less.

The reaction temperature should be an appropriate temperature for the intracellular enzyme used (
The temperature is usually in the range of 20 to 60°C. However, this does not apply if the solvent used has a low boiling point.

The reaction time is approximately 0.5 to 40 hours in the case of a batch method, and even in a semi-batch method or a continuous method, the desired reaction can be carried out by setting a reaction time that matches the reaction conditions. . In addition, as for the reaction mode, it is sufficient to disperse or suspend microbial cells in a solvent in a container and carry out a contact reaction, and methods include stirring methods using rotary stirring or ultrasonic waves, methods of filling a column, etc., and methods such as a bubble column reactor. Any type of reaction mode can be applied, such as a method of fluidizing the reaction mixture by means of a fluidization method.

[Action/Effect]

As mentioned above, the preparation method and conditions for dried bacterial cells made it possible for the first time to use dried bacterial cells for the base exchange reaction of phospholipids, and the base exchange reaction of phospholipids was successfully carried out using such intracellular enzymes. The present invention is the first to do so.

According to the present invention, the extraction and purification steps of cultured microbial cells can be omitted and they can be directly used in the reaction for producing phospholipids, so the cost of enzymes used in the reaction can be significantly reduced and the desired yield can be achieved. of phospholipids can be obtained.

Furthermore, if dried bacterial cells immobilized on a porous microbial carrier are used, the phospholipase D enzyme within the bacterial cells is more effectively stabilized, and continuous or repeated batch reaction methods can be applied, resulting in improved productivity. is significantly improved.

〔Example〕

Hereinafter, the present invention will be specifically explained based on Examples.
The present invention is not limited to these.

In Examples 1 to 6, the compositional analysis and purity test of phospholipids were performed using thin layer chromatography (TLC). Chloroform-acetone-methanol-acetic acid-water (50:20:10:15:5) was used as a developing solvent, color was developed using a deinodomer reagent, and the composition ratio of the product was measured by densitometry.

Moreover, in Examples 7 to 10, the compositional analysis of phospholipids was determined by IATROScan. That is, a phospholipid solution extracted with chloroform-methanol (2:1) was spotted on Chromarod Sl+ (silica gel rond manufactured by Yatron), and a phospholipid solution extracted with chloroform-acetone-methanol-
Approximately 10 mouths of acetic acid-water (6°5:2:l:1:0.3) were developed as a developing solvent, and the mixture was subjected to Iatroscan (Iatroscan TH-10 manufactured by Yatron) to determine the components from the peak area ratio. The weight ratio was determined.

Example 1 Streptomyces chromofuscus IF01285
1, glucose 1% (weight %, same below) meat extract 0
75%, peptone 0.75%, NaCl 10.3%: in a medium (p) 17.2) containing 1% gsoO,
Aerated culture was carried out at a temperature of 30° C. for about 2 days. The obtained bacterial cells were washed twice with pure water, and then diluted with 50% acetone for 10 minutes.
The sample was soaked for 5 minutes, then immersed in 100% acetone for 5 minutes, filtered, and then vacuum dried at 30°C. The moisture content of the dried bacterial cells thus obtained was approximately 5%.

2.0 g of purified dipalmitoyl phosphatidylcholine (manufactured by Sigma) was placed in the stirred reactor shown in FIG.

Ethanolamine 50mM, diethyl ether 50Om
Lo, 5M acetic acid buffer (pH 5)], Oml,
25 g of dried bacterial cells were added and reacted at 37°C for 2 hours.

The water concentration in the reaction solution was detected using a known moisture sensor (manufactured by Panametric, model: System 1), and if necessary, an acetic acid buffer solution was added. It was controlled to about 100-200 ppm. In this case, the water concentration in the bacterial cells was 5 to 20%. After completion of the reaction, phospholipids were extracted with chloroform and analyzed.

As a result of analysis, phosphatidylethanolamine 95%,
The contents were 4% phosphatidic acid and 1% phosphatidylcholine.

Example 2 70mM of glycerol was added instead of ethanolamine, and the other reaction conditions and operating conditions were exactly the same as in Example 16. As a result of analysis, 90% phosphatidylglycerol, 5% phosphatidic acid, and 5% phosphatidylcholine. Ta.

Example 3 150 mM of glucose was added instead of ethanolamine, and the other reaction conditions and operating conditions were exactly the same as in Example 1.

As a result of analysis, phosphatidyl glucose was 75%, phosphatidic acid was 12%, and phosphatidylcholine was 13%.

Example 4 Adding 100mM serine instead of ethanolamine,
The other reaction conditions and operating conditions were exactly the same as in Example 1.

As a result of analysis, it was found that phosphatidylserine was 85%, phosphatidic acid was 10%, and phosphatidylcholine was 5%.

Example 5 Into the same medium as in Example 1, 100/1 (1
The medium was added to a volume of 0 ml and cultured, and the microbial cells were immobilized on the microorganism carrier. The immobilized cells were dried in the same manner as in Example 1 to prepare dried cells. The amount of dry microbial cells in the microorganism carrier was already 5 mg/cell.

1000 microorganism carriers on which dried microbial cells were immobilized were added to the reactor used in Example 1, and the reaction was carried out under the same reaction conditions as in Example 1. After the reaction was completed, all the reaction solution was taken out, new reaction solution was added again, and the same reaction was repeated. Such a repeated reaction was performed three times.

The phospholipids extracted with chloroform from the reaction solution obtained in each reaction were analyzed. The results are shown in Table 1.

Table 1 From Table 1, it can be seen that the enzyme activity of dried bacterial cells immobilized on the microorganism support is stable and can be used repeatedly. The reaction was carried out under exactly the same conditions as in Example 1, except that Cinnamoeus TPO12852 was used.

As a result of analysis, phosphatidylethanolamine 86%,
The contents were 12% phosphatidic acid and 2% phosphatidylcholine.

Example 7 Streptoberticillium hachijoense IF01
2782, glucose 1%, meat extract 0.75%, peptone 0.75%, NaC 10.3%, Mg5Oa
, 1% in a medium (pH 7.2>, temperature 30°C, aerated culture for about 2 days. The obtained bacterial cells were washed twice with pure water, then immersed in 50% acetone water solution TL for 10 minutes, and further After soaking in 100% acetone for 5 minutes, filter
Vacuum drying was performed at 0°C. The moisture content of the dried bacterial cells thus obtained was approximately 5%.

It can be seen that there are 4 g of glycerol and dipalmitoylphosphatidylcholine (DPPC) in a separatory funnel with a volume of 110° and n1.

200mg, diethyl ether 10mj, 0.2M acetic acid buffer y -6ml (pH 5,6,0,04M-Ca
0.2 g of dried bacterial cells was added to the solution (containing ions) and reacted at 30° C. for 7 hours in a shaker. After the reaction was completed, phospholipids were extracted with chloroform-methanol (2:1) and analyzed.

As a result of analysis, dipalmitoylphosphatidylglycerol (DPPG) 81%, phosphatidic acid (PA)
10% dipalmitoylphosphatidylcholine (DPP
C) It was 9%.

Example 8 Streptoberticillium hanjoense TFO1
Example 7 except that Streptomyces chromofuscus IP01285+, Micromonosvora charcea ATCC12452, Nocardia mediterranei IFO13142, Nocardiopsis dasonvillei IFO13908, and Actinomadura libanotica IFO14095 were used instead of 2782. Performed exactly the same operation.

The analysis results are shown in Table 2.

Table Example 9 (A) Ethanolamine 60■ instead of glycerol
The reaction and operating conditions were exactly the same as in Example 7, except that , (B) glucose 500μ, and (C) L-serine 200μ were used. Glucose (DPGL) and dipalmitoylphosphatidylserine (DPPS) were produced.

The analysis results are shown in Table 3.

Table 3 I did this three times.

The reaction solutions obtained in each reaction were as shown in Table 4.

Table 4 (Unit: %) Example 10 Using a 500-inch shake flask, the same medium 1 as in Example 1 was prepared.
00 ml, 100 microorganism-retaining particles (manufactured by Pridiston, Everlite HR-40) made of block-shaped polyurethane foam with an amount of 41 on each side were added and cultured, and the microorganism-retaining particles were immobilized with bacterial cells. The immobilized bacterial cells were dried in the same manner as in Example 7 to prepare dried bacterial cells. The amount of dry microbial cells in the microorganism-retaining particles was approximately 5 μ/cell.

Next, 50 of the immobilized microorganism-retaining particles were placed in the reactor shown in FIG. 2, and reacted under the same reaction conditions as in Example 7. After the reaction is complete, remove all the reaction solution and start a new reaction again. 8 liquids were added and the same reaction was repeated. From the results of such repeated reactions shown in Table 4, it can be seen that the enzyme activity of the dried bacterial cells immobilized on the microorganism-retaining particles is stable and can be used repeatedly.

[Brief explanation of the drawing]

FIG. 1 and FIG. 2 are schematic diagrams showing the apparatus used in Example 1 and Example 10, respectively. 1... Stirring reactor, 2... Moisture sensor 3.
・・Moisture analyzer 4・・Personal computer 5・・Buffer night storage bottle 6・・Buffer liquid feed pump 7・・Jacket warm water 8・・Dried bacterial cells 9・・・
Reactor, lO... water bath 11...
・Mag fuchisoku cooler

Claims (1)

[Claims] 1. In producing a phospholipid whose base structure has been converted, the raw material phospholipid and a receptor having a hydroxyl group are brought into contact with dried bacterial cells containing phospholipase D to carry out the reaction. A method for producing phospholipids. 2. The manufacturing method according to claim 1, wherein dried microbial cells in which microorganisms are immobilized on a porous microorganism support are used. 3. The production method according to claim 2, wherein the microorganism is at least one species selected from the genus Streptomyces, Streptoverticillium, Micromonosvora, Nocardia, Nocardiopsis, and Actinomadura. . 4. The production method according to claim 1 or 2, wherein the dried bacterial cells are obtained by immersing the bacterial cells in a water-soluble solvent, replacing the solvent in the bacterial tissue, and then evaporating the solvent.