JPS6391081A - Modified preformed chemical mediator - Google Patents

Abstract

PURPOSE:To disperse a complex of a preformed chemical mediator and a magnetic material into an organic solvent stably and to make it possible to carry out organic synthetic reactions incapable of being done by a conventional bioreactor using an aqueous solution, by modifying the preformed chemical mediator with a lipophilic molecule and bonding the magnetic material to the preformed chemical mediator through the lipophilic molecule to prepare the preformed chemical mediator. CONSTITUTION:A preformed chemical mediator modified with a lipophilic molecule and a complex of a magnetic material and the preformed chemical mediator wherein the magnetic material is bonded through the lipophilic molecule to the preformed chemical mediator. 4-20C alkane, polyethylene terephthalate, etc., are used as the lipophilic molecule. A transition metal, ion or oxide thereof or a compound of the transition metal, etc., and another element may be cited as the magnetic material. When formation reaction of the magnetic material is simultaneously carried out, reaction of ferrous ion and ferric ion can be used. The complex of the magnetic material and the preformed chemical mediator can be rapidly and simply recovered from an organic solvent by magnetic separation and can be reused.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a biologically active substance modified with a lipophilic (hydrophobic) molecule and a complex formed by binding a magnetic substance and a physiologically active substance via the lipophilic molecule. It exhibits physiological activity when stably dispersed or dissolved as a colloid in a hydrophobic organic solvent.
Furthermore, the present invention relates to a bioreactor that makes it possible to recover physiologically active substances in liquid form using magnetism.

[Conventional technology]

For example, enzymes are generally treated as bioreactors in water (fdH), but if the seven physiological activities are expressed in an organic solvent and used for organic synthesis, it is possible to apply biotechnology. You can greatly expand your sexuality. If the 416 enzyme is dissolved in an organic solvent and exhibits high catalytic efficiency, it can be used for many reactions, such as the reverse reaction of hydrolysis and the enzymatic treatment of hydrophobic substances that are difficult to dissolve in water. becomes. In addition, it is also possible to solubilize proteins other than enzymes in organic solvents and to express the functions of proteins and proteins in organic solvents.

However, in order to further expand the use of physiologically active substances, it is necessary to recover and reuse them.

Conventional methods for separating and recovering enzymes and proteins include
In place of methods in which proteins are bound to polymeric or inorganic carriers to be insolubilized and recovered by sedimentation or centrifugation, magnetic separation methods have recently been attracting attention because they are quick, simple, and enable rapid separation. This method involves directly adsorbing proteins onto the surface of magnetic particles, or adsorbing or coating organic polymers (dextran, starch, bovine serum albumin, cellulose) on the surface of the particles, and then adsorbing protein and protein onto the surface of the particles. It is a method of combining magnetic materials, and while a general electromagnet can separate magnetic materials with a particle diameter of up to about 30 nm, a superconducting electromagnet can separate even smaller particles. However, these complexes are stably dispersed only in water bath liquids, and aggregate in organic solvents to form large clumps, making them unusable. Therefore, as a result of intensive research into technology for dispersing or dissolving complexes of B-forms and physiologically active substances such as proteins in solvents as colloids, we have completed this research.

[Structure of the invention]

The present invention relates to a physiologically active substance modified with a lipophilic molecule that dissolves in an @d medium, and a magnetic physiologically active substance complex in which a magnetic substance and a physiologically active substance are bonded via the lipophilic molecule.

The residual oil molecule has 4 to 20 carbon atoms, preferably 8 to 20 carbon atoms.
16 alkane, carbon number 6-25, preferably 10-1
8 alkenes or alkynes are mentioned. In addition, polystyrene, polyethylene, polyzolopyrene, or polyacrylamide soluble in an organic medium such as toluene may be used as chloroform or trichlene.

Lipophilic molecules with larger carbon numbers can also be used, but the yield of modified bioactive substances is reduced.

These lipophilic molecules have two or more calgexyl groups, amine groups, or hydroxyl groups in one molecule in order to bind to physiologically active substances and magnetic substances. The following compounds are used as such active alkane visiting conductors.

Anovic acid, pimelic acid, speric acid, sepacic acid, 1
, 9-nonanedicardane e, 1.10-7'candicardic acid, 1.11-undecanenocarmenic acid, 1,12
-dodecanedicarmenic acid, 1.13-)lidecannocarboxylic acid, 1.14-tetradecanedicarboxylic acid, 1.
15-pentadecane dicarboxylic acid, 1,16-hexazocannotol/acid, 1,17-hedetadecane dicarboxylic acid, 1,18-octadecane dicarboxylic acid! Nocarmenic acids such as 1.19-nonadekatsunocarboxylic acid, 1.20-eicosandicarboxylic acid; ω-aminovaleric acid, ω-aminocaproic acid, ω-aminocaprylic acid, ω- Amino lauri/aminocarboxylic acid like brew; ω
- and hydroxyl acids such as droxyeicosatetraenoic acid, ω-hydroxyvaleric acid, ω-hydroxystearic acid, ω-hydroxymyristic acid, ω-hydroxynonadecane powder, ω-hydroxylacric acid, and ω-hydroxypalmitic acid. Cardic acid: 1,4-diaminobutane, 1.
5-noaminobentane, 1,6-noaminohexane, 1
．． 7-Diaminohebutane, 1,8-noaminooctane,
More diamines such as 1.9-diaminononane, 1.10-noaminodecane; 1,4-butanediol, 1.5-bentanediol, 1,6-hexatsunool, 1.7-
hepta/diol, 1,8-octatsunool, 1,9
Diols such as -nonatuol, 1,10-deca7ol, pinacol can be used.

As this externally oily molecule, polyethylene terephthalate, nylon, polyurethane, polycarbonate, polyoxymethylene, polysulfide, polyurea, polyvinyl acetal, polyphenyleneoxy P, etc. can be used.

These polymers bind to physiologically active substances and magnetic substances through terminal hydroxyl groups, carboxyl groups, amino groups, or sulfhydryl groups.

The reaction for preparing the complex of the present invention can be carried out in the following two reaction modes. The magnetic material is M1, the bioactive substance is E1, the lipophilic molecule is P1, the modified bioactive substance of the present invention is P-E, and the magnetic bioactive substance complex is M-P'-B.

Reaction mode (1): First, a lipophilic molecule and a physiologically active substance are chemically bonded to synthesize P-E, and this P-E is magnetized to form M
-Synthesize P-E.

i) P+E-+P-E-■71)
M+P-E→M-P-E -■Reaction pattern (2
): First, a lipophilic molecule is magnetized to synthesize a complex M-P, and a physiologically active substance E is chemically bonded to this M-P to form M-P.
-Synthesize E.

1) M+P-+M-P-■It)
M-P + E -+ M-P-E -■P
The bond (■) between and E and the bond (■) between M-P and E are preferably covalent bonds, and the hydroxyl group, carboxyl group,
The amino group covalently bonds with a hydroxyl group, amino group, carboxyl group, sulfhydryl group, etc. of a physiologically active substance, such as an acid amide bond, an ester bond, or a nosulfuryl-P bond.

The reaction (2) in which M and P-E combine and the reaction (2) in which M and P combine are magnetization reactions. In the magnetization reaction, magnetic particles prepared in advance may be used, and magnetic particles may be generated. The reactions may be carried out simultaneously.

The bond between M and P may be in any form such as a covalent bond, a chemical bond such as an ionic bond, or a physical chemical adsorption, as long as it is irreversible. For example, if a lipophilic molecule has a carboxyl group, hydroxyl group, amine group, phenol group, etc., it may have a covalent bond or coordination bond with the hydroxyl group of the magnetic substance, or a coordination bond with the metal ion of the magnetic substance, or between these groups. 7A/Der Waals force coupling can be used.

For magnetization, transition metals, their ions, their oxides, or compounds of these with other elements, such as metals such as iron, cobalt, and nickel, and their oxides, magnetite, ferrite, garnet-type oxides, and corundum. type oxide, perovskite type oxide, rutile type oxide,
Erbium, lutetium, ferritin, high-spin type deoxyhemoglobin, heme, or a conductor thereof can be used.

In order to carry out the reaction to generate magnetic substances simultaneously, the reaction of ferrous ions and ferric ions in neutral-alkali, the reaction of oxidation of ferrous ions, the reaction of lepidocrocite or ferric ions with ferrous ions, etc. Reaction with iron ions, oxidation or spontaneous reaction of green rust, reaction of amorphous ferric oxide with iron ions, etc. can be used.

In order to stabilize the magnetic physiologically active substance complex in an all-organic solvent, the particle diameter of the magnetic substance may be 1 μm or less, preferably 50 nm or less.

The size of the magnetic physiologically active substance composite particles will be the size of the magnetic particles when magnetization prepared in advance is used, and the size of the magnetic body particles will be the size of the magnetic body particles when magnetization prepared in advance is used, and the size of the magnetic body biologically active substance composite particles will be the size of the magnetic body particles when magnetization prepared in advance is used, and the size of the magnetic body biologically active substance composite particles will be the size of the magnetic body particles when pre-prepared magnetization is used, and when the magnetic body production reaction is performed, the size of the magnetic body biologically active substance composite particles will be the size of the magnetic body particles.
)−! or P (reaction mode (2)) and the amount of reagent used to generate the magnetic material. For example, a particle size of 50 nm
When ferrite is used, the result is a magnetic bioactive substance composite with a particle size of 50 nm. 64η chloride to polyethylene terephthalate lipase CP-F,
When iron and 1511Ni ferric chloride are reacted, a complex with a particle size of 30 nm is formed.If the amount of these iron ions is reduced, for example, when 6.4 ~ ferrous chloride and 15 ■ ferric chloride are reacted. In this case, it becomes a composite with a particle size of 10 nm.

The physiological activity of a magnetic physiologically active substance complex, such as enzyme activity, depends on its particle size, and the smaller the particle size, the greater the activity. This is due to the fact that the surface area of the particles increases as the particle size decreases. For example, the ester synthesis activity in benzene of the magnetic lipase complex synthesized using C1ocargenic acid (C1ocargenic acid -1J)? -ze (P-E in the above reaction scheme (1)
), the mother body I with a particle size of 702 nm and 30 nm
J No! -The activity of the enzyme complex is (C1o-nocarboxylic acid)-
10 and 60% of the activity of I7pase, respectively.

The modified bioactive substance and the magnetic bioactive substance complex are stably dispersed in an organic solvent. For example, modification 1) ノ? −
and the magnetic linose complex in benzene at 200%
No precipitation occurs when centrifuged at 0.times.g for 5 minutes, and it is dispersed in an organic static medium such as benzene for 2 to 3 days. Organic solvents other than benzene include chlorinated hydrocarbons such as toluene and chloroform, hydrocarbons such as hexane, dioxa/
It exhibits activity when dispersed and dissolved in organic solvents such as acetone, trimethyl sulfoxide, dimethylformamide, and alcohols such as methanol and ethanol.

The magnetic substance that makes up the entire magnetic bioactive substance complex is a ferromagnetic substance with a particle size of approximately 30 nm or more, which is considered a permanent magnet and! Separation can be done with a magnet, but with other magnetic materials, ultra-low 4 particles can be separated at the exit surface regardless of the particle size. For example, the particle size is about 70
100 nanometer ferromagnetic material (ferrite)-linose coatings are recovered in 5 minutes in a magnetic field of 300 oersted (Oe) at intervals of 1.7 am using an electromagnet. In addition, particles with a particle size of 30 nm can be prepared using an electromagnet.
, 100 chips are collected in 7 minutes in a magnetic field of 5,000 (Oe) at 7N intervals. In addition, a paramagnetic material (erbium)-physiologically active substance complex is produced using ultra-low conductivity stone.
) = 20,000 (Oe) in a magnetic field, 100% is recovered in 5 minutes.

The present invention is generally applicable to enzymes, proteins, antibodies, antigens, polysaccharides, lipids, amino acids, prosthetic groups (heme, sulfur flavin, etc.), hormones, vitamins, etc., and their constituent components. For example, enzymes include lipases, esterases, hydrolytic enzymes such as chymotrypsin, trypsin, and subtilisin, and oxidoreductases such as peroxidase and catalase, which are used when the substrate or product is water-insoluble, and are used in organic solvents. It also includes cases where the reaction can proceed reversibly by performing the reaction. When hemin is used, it can be used in various reactions using hemin as a catalyst. Also, a specific antibody, concanavalin A1
Substances such as polysaccharides, amino acids, vitamins, lipids, hormones, etc. can be used to separate, f#, and recover substances that have an affinity for them in organic solvents.

〔Effect of the invention〕

The modified physiologically active substance of the present invention has l) lipophilicity and is therefore dissolved or dispersed as a colloid in an organic solvent. 2) It enables the expression of organic synthesis reactions or biological activities that are not possible with conventional bioreactors using aqueous solution. Furthermore, a complex in which a magnetic substance and a physiologically active substance are bound via a lipophilic molecule can be rapidly and easily recovered from an organic solvent by magnetic separation. 4) The magnetically separated material can be redissolved or dispersed in the used organic solvent and reused.

Examples of the present invention are shown below.

Example I L) 4.6 f (20 mmol) of 1,10-
Decanedicargenic acid and 2.3 t (20 mmol)
N-hydroxysuccinimide was dissolved in 801M dioxane, and 4.5f dicyclohexylcarbodiimide was added to activate the carboxyl group of the zocallic acid. Add 250 µ of the above activated nocargenic acid to 20 ml of a phosphate buffered aqueous solution (pi(7,5)) containing linosase obtained from 200 q of Shutomonas fluorescens cells to convert - to 6.5 - 2 while keeping it at 7.5.
The reaction was carried out at 5° C. for 1 hour to obtain modified linose.

b) Add 93 Misaki's modified lipase to 3 parts of water, adjust the - to 9.0 with aqueous ammonia, and add 0.1N water bath solution containing 13 parts of ferrous chloride and 30 rq of ferric chloride to this solution. ″f:
Added dropwise. During the dropwise addition, adjust the pH from 9.0 to 9 with ammonia water.
．． 5 and stirred thoroughly at room temperature. After sufficiently precipitating in water, the magnetic substance-lipase complex was obtained by freeze-drying.

The magnetic content of this basic substance-lipase complex was 30%, and the protein content was 55%. The synthetic activity of lauryl laurate in benzene, one of the bath media, was 15μm01AIFF/η protein. It was completely recovered in 5 minutes in a magnetic field of 2,000 Oe. It was dissolved into the reaction solution again as a colloid and showed similar activity.

Magnetic material dissolved as a colloid in benzene/9-
The enzyme complex did not sediment when centrifuged at 11,000X, but at 600X.
The turbidity measurement at nm did not change for 1-3 hours, indicating that it was stably dispersed in the organic solvent. Similar dispersion stability was observed in aqueous solutions. According to electron microscope observation, the particles were ultrafine particles with a particle size of 20-40 nm.

Example 2 a) Add 5j' of polyethylene terephthalate to 50 mB of chloroform, stir for 2 days, take out the supernatant, evaporate the chloroform, and add dioxane to pH 9.
After 7,0 phosphoric acid solution, 10 mj Ic solution was dissolved. Furthermore, di-9-ase 100 obtained from Shutomonas fluorescens cells was added to the polyethylene terephthalate ag.
Add the cape and 200 μl of water-bathable calmediimide and heat at 25°C.
The reaction was allowed to proceed with stirring for 0 minutes. After thorough dialysis against water containing 10% dioxane, freeze-drying and polyethylene tele7
A talate-lipase complex was obtained.

b) Add 100 q of this polyethylene tele-7 tallateli/4-ase complex to 3 ml of distilled water containing 10 thidioxane, adjust the pH to 9.5 with ammonia, and add 1
A 0.1-water bath solution containing 311 iF ferrous chloride and 30 iv ferric chloride was added dropwise.

During the dropwise addition, the pH was maintained at 9.0-9.5 with aqueous ammonia, and the mixture was sufficiently stirred at room temperature. After thorough dialysis against water, a magnetic material-polyethylene terephthalate-lipase complex was obtained by freeze-drying.

The magnetic material content of this magnetic material-polyethylene tele7 talatori/4-ase composite is 50% and the protein content is 3%.
The synthetic activity of lauryl laurate in benzene, one of the typical hydrophobic organic solvents, was 25 μm.
Between ol/III/■tan/buttock quality. The magnetic material-polyethylene terephthalate-lipase complex was completely recovered from the reaction solution in 5 minutes in a magnetic field of 6,000 Oe. This was again dissolved in the reaction solution as a colloid and showed similar activity.

The magnetic substance-polyethylene terephthalate-lipase complex dissolved as a colloid in benzene is 1100Ox
It does not sediment when centrifuged at
It was found that there was no change for -3 hours and that it was stably dispersed in the organic solvent. According to microscopic observation, the particle size is 20-40
The particles were ultrafine particles.

Example 3 a) 92! 1.10-noaminodecane (IV) and molecular sieve (0.5f) were added and stirred for 3 hours to dehydrate the mixture. Then, 118cm of dicyclohexylcarminoide was added and reacted at 25°C for 2 days. 200% chloroform was added to the reaction solution, filtered to remove impurities, and the solvent was evaporated. After adding 100% water to the residue to hydrolyze unreacted calmetzimide, chloroform (200%
m) Extraction and drying under reduced pressure yielded ci(C,.diamine)-hemin.

b) 100 capes of (C,.diamine)-hemin to 31 of 1
01 Add to water containing dioxane and add - to 9 with ammonia water.
．． 5, and a 0.11 aqueous solution containing 13 wq of ferrous chloride and 30 μ of ferric chloride was added dropwise to this solution.During the dropwise addition, - was maintained at 9.0-9.5 with aqueous ammonia. The mixture was thoroughly stirred at room temperature.

After thorough dialysis against water containing 10% dioxane, a magnetic material-(C1o diamine)-hemin complex was obtained by freeze-drying.

Using this magnetic material -(C1゜diamine)-hemin as a catalyst, the amount of peroxide can be determined at 0.1 μM in chloroform using reduced crystal violet as a hydrogen donor.
I was able to do this with higher sensitivity. The magnetic material content of this composite was 60 inches, and the hemin content was 30 inches. The S-isomer-(010 noamine)-hemin complex was completely recovered from the reaction solution in a magnetic field of 5,000 Oe in 5 minutes. This was again dissolved in the reaction solution as a colloid and showed similar activity. The magnetic material-(C,.diamine)-hemin complex dissolved as a colloid in benzene did not sediment when centrifuged at 1000 x g, but at 600 nm.
It was found that the turbidity did not change for 1-3 hours and was stably dispersed in the organic solvent.

Example 4 a) 500 μl of Elch oxide in chloroform 511117 containing 52 1.10-decafdicar diphosphoric acid:) A
(particle size: 200 nm), pulverized with l-lumil for 7 days, completely removed chloroform, and bonded N-hydroxysuccinylimide to the carboxyl group of the magnetic material - decanedicarboxylic acid in dioxane according to a conventional method. , an activated magnetic substance-decanedicarboxylic acid was synthesized.

b) 4M phosphate buffer i (p)1 containing sono9-ase obtained from 5w9 Noseudomonas fluorescens cells
7.0), 75Xv activated magnetic material-decanocarboxylic acid was dispersed in 0.4μ dioxane and heated at 37°C for 90°C.
After reacting for a minute, a magnetic substance-lipase complex having similar properties was obtained.

The magnetic substance content of this magnetic substance-lipase complex was 45%, and the protein content was 30%.Synthetic activity of lauryl laurate in benzene, one of the 6 representative hydrophobic organic hydrogen media. was 20 μmol/hour/■ tan・9 lithium. The lipase-lipase complex was added to the reaction solution (5 m
9/rul) was completely recovered in 5 minutes in a hot water at 20,000 Oe = 2 Tesla tT+ in the presence of a wire. This was dissolved again in a 11C reaction bath as Colloid P, and showed similar activity.

The magnetic material + IJ pase complex dissolved as Colloid P in benzene did not sediment when centrifuged at 2000 x g, but at 60
Turbidity measurement at 0 nm did not change for 24-40 hours, indicating that it was stably dispersed in the organic solvent. According to electron microscopic observation, the particles were ultrafine particles with a particle size of 50 nm.

Example 5 a) 7.8 r of 1,18-octadecanedicarboxylic acid and 2.3 t of N-hydroxyl <l [-] are dissolved in 80 ml of dioxane and 4.5 t of dicyclohexyl carmetzimide are added to the solution. The carboxyl group of dicarboxylic acid was activated. 20 μg of phosphate buffered water solution containing 200 Hq of anti-p-nitrophenol antibody [(pH 7,5)
Add 50η of the above activated dicarboxylic acid to pHi 6.
A modified anti-p-nitrophenol antibody was obtained by reacting at 25° C. for 1 hour while maintaining the temperature at 5-7.5.

b) Add 93rIIg of modified anti-p-nitrophenol antibody to water containing 3μ of 10 thidioxane, bring the - to 9.0 with aqueous ammonia, and add 13μ of ferrous chloride and 3μ of ferrous chloride to this solution.
0.11 aqueous solution containing 0wI ferric chloride? ? One drop was added. During dripping, use ammonia water -'i 9.0-9.5
and stirred thoroughly at room temperature. After thorough dialysis against water containing 10 thidioxane, a complex of cymagnetic material-octadecanocarboxylic acid-anti-p-nitrophenol antibody was obtained by rapid drying.

The maternal shoulder content of this magnetic substance-octadecanedicarboxylic acid-anti-p-nitrophenol antibody was 40%, and the protein content was 60%. p-Nitrophenol, which is present in benzene, one of the typical hydrophobic organic solvents, was recovered by this complex. The magnetic substance-octadecanocarinic acid-anti-p-nitrophenol antibody complex was completely recovered from the reaction solution in 5 minutes in a magnetic field of 6,000 Oe.

This again dissolved fiK in the reaction bath as a colloid and showed similar activity.

The magnetic substance-octadecanesocargenic acid-p-nitrophenol antibody complex dissolved as a colloid in benzene did not precipitate by centrifugation at 1100Ox, did not change in turbidity measurement at 600nm for 1-3 hours, and did not change in organic solvent. It was found that it was stably dispersed in the medium. According to electron microscope observation, the particles were found to be ultrafine particles with a particle size of 220-40 nm.

Claims (1)

[Claims] 1. A physiologically active substance modified with a lipophilic molecule. 2. The substance according to claim 1, wherein the lipophilic molecule is an alkane having 4 to 20 carbon atoms. 3. The substance according to claim 1, wherein the lipophilic molecule is polyethylene terephthalate. 4. The substance according to claim 1, wherein the physiologically active substance is an enzyme. 5. The substance according to claim 4, wherein the enzyme is lipase. 6. A magnetic physiologically active substance complex in which a magnetic substance and a physiologically active substance are bonded via lipophilic molecules. 7. The complex according to claim 6, wherein the lipophilic molecule is an alkane having 4 to 20 carbon atoms. 8. The composite according to claim 6, wherein the lipophilic molecule is polyethylene terephthalate. 9. The composite according to claim 6, wherein the magnetic substance is a product of a reaction between ferrous ions and ferric ions. 10. The complex according to claim 6, wherein the physiologically active substance is an enzyme.