JPH0560357B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] 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 using dried microbial cells. Phospholipids can be used not only as emulsifiers but also as base materials for liposomes, drug carriers, artificial blood,
Applications to artificial cells or immunodiagnosis have attracted attention in recent years, and as they themselves have physiological and pharmacological activities, various applications are being considered in the medical, 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. [Prior art and problems to be solved by the invention] Conventionally, methods for producing such phospholipids include reactions using various purified phospholipase D enzymes derived from cabbage or microorganisms directly or by immobilization. A method has been proposed (Japanese Unexamined Patent Publications No. 63-36790, No. 63-36792, No. 63-91087). 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. In addition, the entrapment immobilization method using a gelling agent or the like has many disadvantages such as complicated immobilization operations and a reduction in reaction rate due to diffusion control, and improvements are desired. An object of the present invention is to provide a base exchange reaction method for phospholipids that can improve these drawbacks and obtain the desired product in high yield by directly subjecting dried bacterial cells containing phospholipase D to the reaction. . [Means for Solving the Problems] That is, in producing a phospholipid whose base structure has been changed, the present invention combines a raw material phospholipid and a receptor having a hydroxyl group with dried bacterial cells of actinomycetes having phospholipase D. The subject matter is a method for producing phospholipids, which is characterized in that the reaction is carried out in contact with 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,
Examples include defatted soybean lecithin, egg yolk lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and mixtures 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 availability, and reactivity with enzymes, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine are particularly preferred as they are industrially highly effective. Examples of receptors in the present invention include nitrogen-containing alcohols such as choline, ethanolamine, serine, N-methylethanolamine, N,N-dimethylethanolamine, polyols such as glycerol, glucose, and fructose, and monosaccharides. I can do it. 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, petroleum ether, diethyl ether, methyl ethyl ether, diisopropyl ether, chloroform, dichloromethane,
Solvents such as carbon tetrachloride, dichloroethane, n-hexane, cyclohexane, n-octane, isooctane, ethyl acetate, dioxane, benzene, or a mixed solvent system thereof, or acetone,
A mixed solvent system containing a polar solvent such as acetonitrile may be used. 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 actinomycetes used in the present invention, all can be used as long as they produce phospholipase D, but in particular, Streptomyces genus, Streptoverticillium genus, Micromonospora genus Examples include microorganisms belonging to the genus Nocardia, Nocardiopsis, Actinomadura, and the like. More specifically, St. chromofuscus
IFO 12851), St. mediocidicus IFO 13202, St. lavendulae
subsp.lavendulae IFO 12789), St.prunicolor IFO
13075), St. antibioticus (St.antibioticus IFO 12652), St. verticillium griseocalneum (Stv.
griseocarneum IFO 12776), Stv.cinnamoneum (Stv.cinnamoneum)
subsp.cinnamomeum IFO 12852), Streptoverticillium hachijiyoense (Stv.
hachijoense IFO 12782), Micromonospora
M.chalcea ATCC 12452, Nocardia mediterranei
mediterranei IFO 13142), Nocardiopsis dassonvillei
IFO 13908), Actinomadura libanotica IFO 14095, and other known microorganisms may be used. In order for the above-mentioned microorganisms to work efficiently as reaction catalysts, it is important to use a culture method that allows the microorganisms to contain a large amount of phospholipase D, and to use drying conditions that allow phospholipase D within the microorganisms to easily come into contact with receptors, while also not reducing the activity as much as possible. Become. As the medium used for culture, those commonly used for culturing microorganisms can be widely used. Carbon sources include assimilable carbon compounds such as glucose, sucrose, 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 set as appropriate within the range that allows the bacteria to grow and produce phospholipase D, but it is usually 15 to 40°C.
is cultivated in Cultivation time varies depending on conditions.
The culture may be terminated when the intracellular phospholipase D activity reaches its maximum, which is usually 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 and can be applied to an effective reaction system such as continuous production or 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, examples of porous polymer materials include polyethylene,
Polyolefin systems such as polypropylene; diene systems such as butadiene or isoprene; vinyl polymers such as polyvinyl chloride, polyvinyl alcohol, acrylamide or polystyrene; condensation systems such as polyether, polyester, polycarbonate or polyamide; polyurethane, silicone and fluorine Materials such as resins, ceramics, glass, activated carbon, porous metals, metal processing materials, etc. can be used as inorganic materials. In order to immobilize microorganisms well on the carrier in any material, porous materials with a porosity of 60 to 90% and a pore diameter of 2 to 2000 μm, or a porous material with a porosity of 60 to 99% are used. % is preferable. These 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 approximately 1 to 100 mm, and if it is block shaped, it is approximately 1 to 100 mm on a side. things are used. Further, immobilization of microorganisms on the above-mentioned carrier can be easily achieved using commonly known batch, half-batch, continuous culture, etc. To remove water from the cells obtained as above, in principle, drying at a temperature (40 to 60°C) that does not deactivate the enzyme is sufficient; however, simply evaporating water does not affect the cell composition. Shrinkage occurs and the tissue becomes extremely stiff, cutting off contact between phospholipase D within the tissue and the outside world, making it difficult to express activity.
Therefore, a method that does not involve shrinkage of cell tissue must be used to dry the bacterial cells. For this reason, contraction of the cell tissue can be suppressed 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. In this case, drying methods include vacuum drying, freeze drying,
Known drying methods such as low temperature drying can be used. Furthermore,
By immersing the bacterial cells in a 5% by weight or less glutaraldehyde aqueous solution to fix the cell tissue before immersing the bacterial cells in the solvent, shrinkage of the cell tissue can be suppressed more effectively. The water content of the bacterial cells obtained by such a method is usually adjusted to a water content of 1 to 20% by weight. The dry bacterial cells prepared in this manner are suspended in a mixture of reactants, ie, raw material phospholipids, and receptors to react.The ratio of water to organic solvent is 1:0.5 by weight. Although it can be used in the range of ~0.1:10, it is preferable to control the water content to 40% by weight or less in order to prevent the formation of phosphatidic acid caused by side reactions as much as possible. The reaction temperature may be any temperature appropriate for the intracellular enzyme used, and is usually in the range of 20 to 60°C. However, this does not apply when the solvent used has a low boiling point. The reaction time is approximately 0.5 to 0.5 in batches.
40 hours, even in semi-batch and continuous methods, by setting the reaction time to match the reaction conditions.
The desired reaction can be carried out. In addition, as for the reaction mode, it is sufficient to disperse or suspend the microbial cells in a solvent in a container and carry out a contact reaction, such as rotational stirring or ultrasonic stirring, filling in a column, etc., bubble column reactor, etc. Any type of reaction mode can be applied, such as fluidization by. [Action/Effect] By using the method and conditions for preparing dried bacterial cells as described above, dried bacterial cells can be used for the base exchange reaction of phospholipids for the first time, and phospholipids can be converted by enzymes in the bacterial cells. The present invention is the first to successfully conduct a lipid base exchange reaction. 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 dry bacterial cells immobilized on a porous microbial carrier are used, phospholipase D in the bacterial cells can be reduced.
Production is significantly improved since the enzyme is more effectively stabilized and continuous or repeated batch reaction methods can be applied. [Examples] The present invention will be specifically described below based on Examples, but the present invention is not limited thereto. In Examples 1 to 6, the composition analysis and purity test of phospholipids were performed using thin layer chromatography (TLC). The developing solvent was chloroform-acetone-methanol-acetic acid-water (50:
20:10:15:5), color was developed using a Deuttomer 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 Iatroskian. That is,
A phospholipid solution extracted with chloroform-methanol (2:1) was spotted on Chromatorod SII (silica gel rod manufactured by Yatron), and chloroform-acetone-methanol-acetic acid-water (6.5:1) was added.
2:1:1:0.3) as a developing solvent for about 10 cm, and subjected to Iatroscan (Iatroscan TH-10 manufactured by Yatron) to determine the weight ratio from the peak area ratio. Example 1 Streptomyces chromophuascus
IFO12851, glucose 1% (weight%, same below), meat extract 0.75%, peptone 0.75%, NaCl 0.3
%:MgSO0.1% containing medium (PH7.2), temperature 30
C. for about 3 days with aeration. The obtained bacterial cells were washed twice with pure water, and then added to a 50% acetone solution for 10 minutes.
The sample was soaked in 100% acetone for 5 minutes, filtered, and then dried under vacuum at 30°C. The moisture content of the dried bacterial cells thus obtained was approximately 5%. Add 2.0 g of phosphatidylcholine (Asahi Kasei Industries, Ltd.), 500 ml of diethyl ether, 100 ml of 1M ethanolamine/hydrochloric acid buffer (PH6.0), and 25 g of dried bacterial cells to the stirred reactor shown in Figure 1 at 30°C.
The mixture was reacted for 2 hours. After the reaction was completed, phospholipids were extracted with chloroform and analyzed. As a result of analysis, phosphatidylethanolamine
95%, 4% phosphatidic acid, and 1% phosphatidylcholine. Example 2 Glycerol instead of ethanolamine
The reaction and operating conditions were exactly the same as in Example 1, except that 100 ml of 1M acetic acid buffer (PH6.0) was used as a buffer. As a result of analysis, phosphatidylglycerol 90
%, phosphatidic acid 5%, and phosphatidylcholine 5%. Example 3 Glucose instead of ethanolamine
In addition to the addition of mM, the other reaction conditions and operating conditions were exactly the same as in Example 2. As a result of analysis, phosphatidyl glucose 75%,
Phosphatidic acid 12%, Phosphatidylcholine 13
It was %. Example 4 100mM serine instead of ethanolamine
In addition, the other reaction conditions and operating conditions were exactly the same as in Example 2. As a result of analysis, the content was 85% phosphatidylserine, 10% phosphatidic acid, and 5% phosphatidylcholine. Example 5 A microorganism carrier (manufactured by Bridgestone Corporation, Everlight HR-40) consisting of a block-shaped polyurethane foam of 4 mm on each side was added to the same medium as in Example 1.
100 cells/100 ml of medium were added and cultured to immobilize the microbial cells 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 bacterial cells in the microorganism carrier is approximately 5
mg/piece. 1000 microorganism carriers with dried microbial cells immobilized thereon 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 is complete, remove all the reaction solution and add new reaction solution again.
A similar 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】

[Table] From Table 1, it can be seen that the enzyme activity of the dried microbial cells immobilized on the microbial carrier is stable and can be used repeatedly. Example 6 A reaction was carried out under exactly the same conditions as in Example 1, except that Streptoverticillium cinnamoneum IFO12852 was used instead of the microorganism used in Example 1. As a result of analysis, phosphatidylethanolamine
86%, phosphatidic acid 12%, and phosphazylcholine 2%. Example 7 Streptoverticillium hachijiyouense
IFO 12782, glucose 1%, meat extract 0.75
%, peptone 0.75%, NaCl 0.3%, MgSO4 _0.1 %
The cells were cultured in a medium containing (PH7.2) at a temperature of 30° C. for about 3 days with aeration. The obtained bacterial cells were washed twice with pure water, and then immersed in a 50% acetone aqueous solution for 10 minutes.
After further soaking in 100% acetone for 5 minutes, it was filtered and then vacuum dried at 30°C. The moisture content of the dried bacterial cells thus obtained was approximately 5%. 4 g of glycerol in a 100 ml separating funnel,
Phosphatidylcholine (PC) 200mg, diethyl ether 10ml, 0.2M acetic acid buffer 6ml (PH
5.6 (containing 0.04M Ca ions) was added to 0.2 g of dried bacterial cells and reacted at 30°C for 7 hours in a shaker. After the reaction, chloroform-methanol-(2:1)
Phospholipids were extracted and analyzed. As a result of the analysis, phosphatidylglycerol (PG) was 81%, phosphatidic acid (PA) was 10%, and phosphatidylcholine (PC) was 9%. Example 8 Streptoberticillium hanjiyouense
Streptomyces chromophuascus IFO 12851, Micromonospora charcea ATCC 12452, Nocaldeia mediterranei IFO 13142, Nocaldeiopsis IFO 12782 instead of IFO 12782
The same operation as in Example 7 was carried out except that Dason Belay IFO 13908 and Actinomadyura libanotica IFO 14095 were used. The analysis results are shown in Table 2.

[Table] Example 9 (A) Ethanolamine 60 instead of glycerol
mg, (B) glucose 500mg, and (C) L-serine 200mg
mg was used. The reaction conditions and operating conditions were exactly the same as in Example 7, except that 1M ethanolamine/hydrochloric acid buffer was used as the buffer in (A), and phosphatidylethanolamine (PE),
Phosphatidylglucose (PGL) and phosphatidylserine (PS) were produced. The analysis results are shown in Table 3.

[Table] Example 10 Using a 500 ml shaking flask, 100 microorganism carriers (manufactured by Bridgestone, Everlite HR-40) made of block-shaped polyurethane foam with a side of 4 mm were added to 100 ml of the same medium as in Example 1. The cells were cultured and immobilized on a microorganism carrier. The immobilized bacterial cells were dried in the same manner as in Example 7 to prepare dried bacterial cells. The amount of dry bacterial cells in the microbial carrier was approximately 5 mg/cell. Next, 50 of the immobilized microorganism carriers were placed in the reactor shown in FIG. 2, and reacted under the same reaction conditions as in Example 7. After the reaction was completed, all the reaction solution was taken out, new reaction solution was added again, and the same reaction was repeated. Repeat this reaction 3 times.
I went around. The reaction solutions obtained in each reaction were as shown in Table 4.

[Table] From the results in Table 4, it can be seen that the enzyme activity of the dried bacterial cells immobilized on the microbial carrier 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... Dry bacterial cells, 3...
Jacket hot water, 4...Reactor, 5...Water bath, 6...Magnetic stirrer, 7...
Stirrer, 8...Microorganism support.

Claims (1)

[Scope of Claims] 1. In producing a phospholipid with a converted base structure, a raw material phospholipid and a receptor having a hydroxyl group are brought into contact with dried bacterial cells of an actinomycete having phospholipase D to carry out a reaction. A method for producing phospholipids characterized by: 2. The production 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 actinomycete is at least one species selected from the genus Streptomyces, Streptoverticillium, Micromonospora, Nocardia, Nocardiopsis, and Actinomadyula. 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.