JPH01503299A - Method for producing phosphatidylinositol from biological objects - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Alternative method for producing phosphatidylinositol from biological objects field of invention The present invention relates to a technology for producing lipids, and more particularly, the present invention relates to a technology for producing lipids, and more specifically, the production of phosphatides from biological objects. The present invention relates to a method for producing luinositol.

Phosphatidylinositol belongs to the phospholipid class and plays a role in biological processes. Creation of a membrane system that simulates; functions to transfer foreign genetic information into living cells Formation of liposomes: transport of foreign proteins, vaccines and enzymes through membrane barriers biochemical reagents for delivery; release of pharmaceutical formulations and other agents to target tissues; It is useful.

Conventional technology from various biological objects such as animal hearts and brains, and from bacteria and seeds. Several methods are known in the art for producing sphatidylinositol. Ru. Methods for producing phosphatidylinositol from yeast are also known in the art. [Lipid Production Chemistry 1 Edited by L. D. Bergelsson on) and E.V. Dyatlovitska ya), 1981, Moscow, °Nauka' Publishing House (-Nauka' Pu blishers), page 178].

The method for producing phosphatidylinositol from yeast is to ferment it with toluene for 5 hours. Let the mother bacteria self-decompose and incubate with a chloroform:methanol (1:1) mixture for 7 hours. to extract the phospholipids; filter the extract and wash the filter cake with the above organic solvent mixture. The filtrate was stirred with distilled water for 20 hours; the filtrate was added to the chloroform phase and the water phase. Separate; evaporate the chloroform phase; Consisting of treating with Ropatool and then filtering it through a glass filter . The phospholipids are then first chloroformed from the resulting filtrate in a silica gel column. 5-7 liters of form-methanol-ammonia (80:20:2) mixture The solution is separated by successive elution with 1-2 liters of ethanol. However, in this method, the ammonium salt of phosphatidylinositol is recovered and subsequently converted to the sodium salt.

This method requires expensive organic solvents with very high consumption rates. therefore, To produce 1 g of phosphatidylinositol, 5 g or more of chloroform, It is necessary to use methanol of 39 or higher, ethanol of about 1Ω and other solvents. become. Moreover, the method requires many time-consuming and labor-intensive operations. child All of these significantly impact the cost of the final product [Calbiochem According to the catalog of iochem), 1 g of phosphatidylinositol is 6.9 50 US dollars]. Operations using large amounts of organic solvents are not normal for workers. creating harmful working conditions that interfere with environmental conditions.

Disclosure of invention Reduce labor intensity and consumption time, improve working conditions, and significantly reduce product costs phosphatide, which can substantially reduce the rate of organic solvent consumption. It is an object of the present invention to provide a method for producing luinositol.

The purpose is to homogenize and extract phosphorus from biological objects. achieved by a method for the production of atidylinositol, i.e. according to the invention The method uses salts with a molar concentration of 0.005 to IM at a knee pH of 5.0 to 11.0. Homogenization of biological objects in aqueous solution; Centrifugation at 3,000 to 30,000 g or pore size of 3 to 50 μm Determination of protein-phosphatidylinositol complexes by filtration with a filter. into a cell-free saline phase containing particles and a precipitate containing unbroken cells and other phospholipid particles. , separation of the homogenate obtained; - protein from the cell-free saline phase - phosphatide Collection of dylinositol complex particles; Phosphate from recovered protein-phosphatidylinositol complex particles Extraction of atidylinositol;

Recovery of protein-phosphatidylinositol complex particles from cell-free saline phase is a gel chromatography method using an adsorbent that can separate substances with a molecular mass of 1 million Daltons or more. by ultrafiltration using a filter with a pore size of 0.2 μm or less. It can be done by Additionally, protein-phosphatidylino from the saline phase Recovery of the sitol complex particles is achieved by its treatment with alkaline or neutral proteases. As a result, these particles agglomerate and become easy to separate and stick together. . In order to recover protein-phosphatidylinositol complex particles, This method is preferred because it is easy to implement, and the reagents mentioned above, especially the This is because tease is easily available and relatively inexpensive.

Methanol is extracted from the recovered protein-phosphatidylinositol complex particles. by extraction with chloroform or a mixture thereof with chloroform or distilled water The product is isolated. Processed seeds of grasses are used as biological objects. The reason for this is that these raw materials are relatively easily available and inexpensive. This is because the desired product can be obtained in high yield.

According to the method of the present invention, the consumption rate of organic solvent is several hundred times higher than that of the prior art method. This reduces the operating time by 2 to 4 times, significantly reduces manufacturing costs, and It can make it possible to improve working conditions.

The operation can be easily carried out on a commercial scale.

The advantages of the present invention are as follows: It will become clearer from the explanation.

Biological objects include plant seeds, their processed products, roots, leaves, stems, and animals. tissue and microorganisms can be used; however, grass seeds and It is preferable to use processed products such as siftings for sifting. Delicious. This is because these raw materials are readily available, inexpensive, and can be phosphorized in high yields. This is due to the fact that fatidylinositol can be obtained.

Biological objects can be removed using a mortar or blade homogenizer or any other suitable Homogenization by grinding is carried out in a suitable apparatus.

Homogenization of biological objects by the method of the invention can be carried out at molar concentrations between 0.005 and I It is carried out in an aqueous salt solution of M. This salt concentration range is less than 0.005 ki It is determined based on the fact that the salt concentration in the water is undesirable, i.e. This causes dissolution of the protein-phosphatidylinositol complex, which This is because it interferes with product manufacturing. The upper limit of the salt molarity is the salt used at room temperature. The value can be greater than 1 h1 until maximum dissolution is reached.

However, it is desirable to set the upper limit of the salt concentration to INi. Beyond IM Increasing the salt molarity to such an extent does not increase the yield of the desired product; leading to excessively high consumption rates of reagents. The optimum molar concentration of salt is 0.05-0. INl range It is within.

Although any organic or inorganic salt can be used for the preparation of the aqueous salt solution, However, it was not possible to degrade the protein-phosphatidylinositol complex. Salts with chaotropic properties are excluded. sodium chloride um, sodium or potassium salts of phosphoric acid, and trishydroxyamino Neutral or weak alkali such as methane chloride, phosphate or sulfate It is most preferred to use salts that are soluble.

According to the invention, the homogenization of biological objects can be carried out at a pH within the range of 5 to 11. It is carried out in a saline solution. This limitation of pH in salt aqueous solutions is less than 5 and 11 Protein-phosphatidylinositol complex particles break down when the pH exceeds The decision is based on the fact that it has been destroyed and this cannot be tolerated.

Homogenization of this object in the saline solution described above results in cells of the biological object. is decomposed to leave protein-phosphatidylinositol complex particles in the saline phase. release to. Protein-phosphatase from non-destructive cells and other phospholipid-containing particles To separate the tidylinositol complex particles, the homogenate according to the invention is Subjected to centrifugation or filtration. Centrifugation of the homogenate This is done with gravitational acceleration values within the range of 000 g. This limitation applies to items less than 3,000g. In the case of centrifugation, other phospholipid-containing particles remain in the brine phase without settling and their Is it the fact that phosphatidylinositol is contaminated with other phospholipids? has been determined.

For centrifugation above 30,000 g, protein-phosphatidylino The citol complex particles precipitate, substantially reducing the product yield. gravity The optimum range of acceleration values is s,ooo to 10,000g.

Separation of the homogenate should be performed by filtration through a filter with a pore size of 3 to 50 μm. Can be done. Filtration of homogenates through filters with pore sizes less than 3 μm is undesirable. However, the reason is that there is a protein-phosphatidylinositol complex on the filter. This may result in residual particles remaining, resulting in a lower yield of the desired product. be. Homogenate filtration with a filter with a pore size exceeding 50 um is also undesirable. However, this is because other phospholipid-containing particles migrate into the brine phase and remove the desired product. This is because the quality will substantially deteriorate.

As a result of homogenate separation by the above technique, protein-phosphatidyl A cell-free saline phase containing nositol complex particles is obtained, while other remaining The phospholipid-containing precipitate is discarded.

Protein-phosphatidylinositol complex particles have a molecular mass of 1 million Dal Gel chromatography on adsorbents capable of separating over 1,000 tons of substances from the brine phase It can be recovered from. Separates substances with a molecular weight of less than 11 million Daltons. Vine adsorbents can separate protein-phosphatidylinositol complex particles. It cannot be tolerated because there is no such thing. The best adsorbent is “Pharmacia” ( PharlIacia) (Sweden Company) Commercially available (7)”-t’770-s° (Sepharose), “Sephacryl” (Sephacryl), “Ur Ultrogel (LKB, Sweden), Bio Gel A" (Biogel A) ["Bjorad" ) Inc., USA), “Toyapearl” C’Toya Soda ° (Toyasoda) Co., Ltd. 1 Japan].

Separation of protein-phosphatidylinositol complex particles from cell-free saline phase is a pore diameter of 0. Can be performed by ultrafiltration with a filter of 2 μm or less . It is undesirable to use a filter with a pore size exceeding 0.2 μm, and the reason is Protein-phosphatidylinositol complex particles move (penetrate) into the filtrate. This is because the yield of the desired product decreases.

Recovery of protein-phosphatidylinositol complex particles from cell-free saline phase is that at a gravitational acceleration value of 30,000 to 100,000 g in an ultracentrifuge. This can also be done by sedimentation. This limitation is for values less than 30,000g. If the value exceeds 100,000 g, it is not possible to limit the value. External filtration excessively increases the power consumption rate without increasing the yield of the desired product. This decision was made based on the fact that there is the inconvenience of

The above embodiments regarding separation of protein-phosph-7tidylinositol complex particles Additionally, in the present invention, by treatment of the cell-free saline phase with alkaline or neutral proteases. Those minutes M! We also provide three types. In the presence of such proteases, most proteins Protein is removed from these particles, which cause them to clump together and become paper or They are easily separated by simple filtration through glass filters, or they are sedimentation by centrifugation at low gravitational acceleration values of 00 to 10,000 g . In this embodiment, the protease is used in an amount of 5 to 100 μg per gram of saline phase. be done. This limitation is due to the protein-phosphorus 7 is insufficient for complete recovery of the tidyl complex particles, whereas 100 of them If the amount exceeds μg/I), the reagent usage rate will be excessively increased without producing any additional effect. The decision was made based on the fact that there is the inconvenience of

of protein-phosphatidylinositol complex particles by the above protease. The separations are preferred compared to other aspects of their separation. This is an alkali or protein-phosphatidylinositol complex in the presence of a neutral protease. The body particles stick to each other and can be easily and easily separated by paper or glass filters. Due to the fact that they can be separated. Proteases are relatively inexpensive and readily available. It is an easy-to-handle product.

The separated protein-phosph-7tidylinositol complex particles are then deposited there. diethyl ether, acetone or any other to remove lipid impurities from with similar organic solvents. Afterwards, the product is purified with or with methanol. Extracted with loloform or a mixture with distilled water. phosphatidylinosyto The yield of L is greater than 90% of its content in the starting biological object.

As is clear from the detailed description of the present invention, according to the method of the present invention, its implementation operation is Easy to use, does not require high organic solvent consumption rates, improves working conditions and reduces operating time 2 to 4 times faster, significantly lowering product costs.

BEST MODE FOR CARRYING OUT THE INVENTION As a biological object, grain sieves are used as products, e.g. wheat sieves are used as products. Ru. They are p! until homoanate is obtained. (s, 3-8.5 trishydride By grinding in a 0.05-0.1M aqueous solution of roxyaminomethane chloride. homogenized. In this way, the cells of the product are destroyed and the sieve content is The protein-phosphatidylinositol complex particles are transferred into the saline phase.

The resulting homoanate is centrifuged at 10.000 g for 10 minutes. to this Thus, protein-phosphatidyl can be extracted from non-disrupted cells and other phospholipid-containing particles. The cell-free saline phase containing the inositol complex particles is separated. obtained in this way Protein-phosphatidylinositol complex particles are extracted from the cell-free saline phase. ．． alkaline or neutral protease in an amount of 0.05 mg (i.e. 50 gg)/ml of phase. This phase is separated by treatment.

Through this treatment, protein-phosphatidylinositol complex particles adhere to each other. and they are separated from the above phase by centrifugation at 10,000 g. Obtained The protein-phosphatidylinositol complex particle residues are mixed into the product. Washed with diethyl ether to remove lipid impurities. protein - From the purification residue of phosphatidylinositol complex particles, the product is methanol. is extracted. The yield of the product is approximately 93% of its initial content in the product. %.

For a better understanding of the present invention, we will explain the method for producing phosphatidylinositol. Some specific examples are provided below.

example] 10 kg of corn flour was added to 0.0 kg of disodium phosphate at pH 9.0 in a porcelain mortar. Grind until homogeneous in 05M aqueous solution. To recover the cell-free saline phase , the homoanate is filtered through a 5 μm pore size filter. Cephalometric cell-free saline phase Protein-phosphatide was subjected to gel chromatography using a base 4B column. A fraction containing dylinositol complex particles is collected. This fraction is transferred to a vacuum evaporator. Concentrate to dryness. Treat the dried residue with 50 ml of acetone to remove lipid impurities. . The desired product is extracted with distilled water. The desired product yield is 0.09 g ( 8.9%).

Example 2 Grind 10 g of Ento particles in a mill, and then add the ground mass to N a with a pH of 11.0. Homogenize with a blade homogenizer in 2C030, 1M solution. homoa Centrifuge the nates at 10.000 g for 10 minutes. The resulting cell-free saline phase was ,000g ultracentrifugation. The residue is washed with 50 ml of diethyl ether.

The product is extracted with 5 ml of methanol. The yield of product is 0. 02g (65%) It is.

Example 3 Grind 10g of acorns in a mill. The obtained powder was adjusted to a pH of 5 and a concentration of 0. IM acetic acid Grind in aqueous sodium solution. The homogenate thus obtained was ．． Centrifuge at 000g for 10 minutes. Toyapearl HW adsorbent for cell-free saline phase 65 and subjected to gel chromatography. Protein - Phosphatidylinosyto The fraction containing the complex particles was evaporated using a vacuum evaporator, and 50ml of diethyl ether was added. 1 to remove lipid impurities. The product is extracted with 5 ml of methanol. Living The yield of the product is 0. Olg (20%).

Example 4 For the sifted barley grains, 10g of the product was added to a 0.005M aqueous solution of potassium chloride at pH 7. Grind. The resulting homogenate was centrifuged at 10,000g for 10 minutes. vinegar.

The cell-free saline phase containing protein-phosphatidylinositol complex particles was Treat with papain in an amount of gg/ml of the above phase for 4 hours. Protein-phosphatide The aggregated particles of the dylinositol complex were centrifuged at 10,000g for 10 minutes. Let it settle. Wash the precipitate with 50 ml of diethyl ether to remove lipid impurities. and extract the product with 5 ml of methanol-chloroform (2:1) mixture. Living The yield of product is 0.05 g (50%).

Example 5 10g of corn grains were crushed and homogenized in a 0.05M aqueous solution of sodium chloride at pH 7. Genize. The resulting homogenate was centrifuged at 3,000g for 10 minutes. vinegar.

The cell-free saline phase is separated and treated with trypsin in an amount of 50 gg/ml of above phase.

The aggregated particles of the protein-phosphatidylinositol complex were filtered through filter paper. I collect it. Wash the filter cake with 50 ml of petroleum ether to remove lipid impurities. Ru. The product is extracted with 5 ml of methanol. Product yield was 0.7 g (90% ).

Example 6 For the corn sieve, place 10g of the product in a porcelain mortar and add Trishydro at a concentration of 0.05M. Homogenize in aqueous oxyaminomethane phosphate solution. The obtained homogene Centrifuge at 30,000g for 10 minutes. 50 gg/cell-free saline phase Treat with m+ second chymotrypsin for 8 hours. Protein - Phosphatidylino The aggregated particles of the sitol complex were sedimented by centrifugation at 10,000g for 10 minutes. let The residue is treated with 50 ml of diethyl ether to remove lipid impurities. Generate Extract the material with 5 ml of methanol. Product yield is 0.02g (18%) wheat For the sieve, 10g of the product was mixed with trishydroxyaminomethane at a pH of 8.5 and a concentration of 0.1M. Homogenize for 10 minutes in an aqueous sulfate solution. The obtained homogenate Filter through a filter with a pore size of 50 μm. 50 gg/top of separated cell-free saline phase Treat with papain in an amount of 1 ml for 4 hours. Protein - Phosphatidylino The adhered particles of the sitol complex were sedimented by centrifugation at 10,000g for 10 minutes. let

The resulting residue is washed with 20 ml of diethyl ether to remove lipid impurities. Living The product was extracted with 2 ml of methanol. Product yield was 0.07g (85%) be.

■Dan For the wheat sifter, 10g of the product was mixed with trishydroxyamide at a pH of 8.5 and a concentration of 0.05M. Grind in an aqueous nomethane chloride solution. 10,000 of the obtained homogenate Centrifuge for 10 minutes at g. The cell-free saline phase was treated with papain in an amount of 50 gg/ml. Process for 8 hours. Aggregated particles of protein-phosphatidylinositol complex is sedimented by centrifugation at 10.000 g for 10 minutes.

The precipitate is washed with 50 ml of diethyl ether to remove lipid impurities. the product Extract the precipitate with 5 ml of methanol. The yield of product is 0. l1g (95% ).

凰2 For the wheat sifter, place 10g of the product in a homogenizer at pH 7 to a concentration of 0.05M. Homogenize in aqueous sodium chloride solution. The obtained homogenate was diluted with 10.0 Centrifuge at 00g for 10 minutes. The separated cell-free saline phase was separated into a pore size of 0.2 μm. The sample is subjected to filtration using an ultrafiltration membrane. Remove the cake from the filter and soak it in diethyl ether. Wash with 50 ml of water to remove lipid impurities. Convert the product from the precipitate into methanol Extract with 2 ml of water. Product yield is 0.06 g (63%).

Industrial applicability The invention can be implemented in the chemical industry, biotechnology and medicine. Ru.

International investigation report

Claims (7)

[Claims] 1. homogenization of this object and extraction of the product from the biological object. A method for producing sphatidylinositol, comprising: - Salt aqueous solution with a molar concentration of 0.005 to 1M within the range of pH 5.0 to 11.0 homogenization of biological objects in - Centrifugation at gravitational acceleration values of 3,000-30,000 g or pore size of 3-50 μm Protein-phosphatidylinositol complex by filtration through a filter of into a cell-free saline phase containing particles of , separation of the homogenate; - protein from the cell-free saline phase - phosphatidylino Separation of sitol complex particles; - Separated protein-phosphatide from phosphatidylinositol complex particles Extracting atidylinositol. 2. Homogenization of biological objects in salt water with a molar concentration range of 0.05-0.1M 2. The method according to claim 1, which is carried out in liquid. 3. of protein-phosphatidylinositol complex particles from the cell-free saline phase. Separation is achieved by gelation with an adsorbent that allows the separation of substances with a molecular mass of 100 Daltons or more. 2. The method according to claim 1, which is carried out by chromatography. 4. of protein-phosphatidylinositol complex particles from the cell-free saline phase. Claim in which the separation is carried out by ultrafiltration through a filter with a pore size of 0.2 μm or less The method described in 1. 5. of protein-phosphatidylinositol complex particles from the cell-free saline phase. The method according to claim 1, wherein the separation is carried out with an alkaline or neutral protease. . 6. Phosphate from separated protein-phosphatidylinositol complex particles Extraction of fatidylinositol can be carried out using methanol or chloroform or 2. The method according to claim 1, carried out in a mixture with distilled water. 7. Claims 1 to 5, wherein a processed product of seeds of a grass family plant is used as the plant object. The method described in any one of the above.