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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to a conjugate (M-P-E) formed by binding a magnetic
material (M) and a physioloqically active substance (E) to each other
through a polyethylene glycol derivative (P) which is an amphiphatic
substance for both of water and organic solvents, having physiological
activity stably dispersed or dissolved as colloid in both of aqueous
solutions and organic solvents, and to a bioreactor enabling application
to or rocovery of physiologically active substances the liquid state by
utilizing the magnetic properties.
For example, enzymes have been generally dealt with as bioreactors in
aquieous solutions, and if their physiological activites can be exerted in
organic solvents and utilized for organic syntheses and the like,
application range and possibility of biotechnology can be greatly
broadened. In short, if enzymes can be catalytic efficiency therein, they
can become utilized for more reactions such as reverse reactions of
hydrolysis or enzymatic treatment of hydrophobic substances only slightly
soluble in water. Furtherwise, other proteins than enzymes can be
solubilized in organic solvents and the functions of the proteins can be
exhibited in organic solvents. Accordingly, the present inventor has made
it possible to solubilize enzymes in orqanic solvents by modification with
amphiphatic polymers (Japanese Unexamined Patent Publication No.
156395/1985). However, for utilizing generally physiologically active
substances, recovery and reutilization of these are required. As the
method for recovery of enzyme proteins, in place of the method in which
enzyme proteins are insolubilized by binding with a polymeric or inorganic
material carrier and recovered by sedimentation or centrifugation,
magnetic separation enabling rapid and simple separation is recently
attracting attention. Among these methods, there may be included the
method in which proteins are directly adsorbed on the surfaces of magnetic
material particles, the method in which the magnetic material particle
surfaces are adsorbed or coated with an organic polymer (polyacrylamide,
dextran, starch, bovine serum albumin, cellulose) and proteins are then
bound thereto, and electromagnets in general can separate magnetic
materials with particle sizes to about 30 nm, and superconductive
electromagnets can separate those with further smaller sizes. However,
these conjugates (M-E) can be dispersed only in aqueous solutions, but
agglomerated in organic solvents to form a great mass, which cannot be
used. Accordingly, studies have been made intensiely about the technique
for dispersing or dissolving as colloid the conjugates (M-E) of magnetic
materials and physiologically by active substances such as proteins in
aqueous solutions and organic solvents and consequently the present
invention has been accomplished.
SUMMARY OF THE INVENTION
The present invention provides a magnetic material-physiologically active
substance conjugate (M-P-E) having a magnetic material (M) and a
physiologically active substance (E) bound to each other through a
polyethylene glycol derivative (P) which is amphiphatic for water and an
organic solvent, and also a conjugate (M-P) having M and P bound to each
other to be used as the chemical modifier therefor.
DETAILED DESCRIPTION OF THE INVENTION By selecting the molecular weight of
the polyethylene glycol derivative according to the molecular weight of
the physiologically active substance and the particle size of the magnetic
material, dispersion stability in an organic solvent can be obtained.
Generally speaking, the molecular weight should be desirably greater in
order that the magnetic material-physiologically active substance
conjugate (M-P-E) can be stably dissolved or dispersed in an organic
solvent such as benzene. A polyethylene glycol derivative should
preferably have a molecular weight of 500 to 200,000. Concerning protein
which is one of physiologically active substances, for example, a polymer
with a molecular weight of 1,000 or more is preferable for lysozyme with
molecular weight of about 14,000; a polymer with a molecular weight of
2,000 or more for lipase with molecular weight of about 33,000; a polymer
with a molecular weight of 10,000 or more for catalase with molecular
weight of about 240,000. On the other hand, when a ferrite with particle
size of 30 nm or larger is used as the magnetic material, a polymer with a
molecular weight of 2,000 or more can be used, while a polymer with a
molecular weight of 500 or more can be used for a magnetic material with a
particle size smaller than 30 nm. Also, for D-asparagine with molecular
weight of 132 which is one of physiologically active substances, a polymer
with a molecular weight of 500 or more can be used.
As the magnetic material, there can be used transition metals or ions
thereof, oxides thereof or compounds of these with other elements, for
example, metals such as iron, cobalt, nickel, erbium, lutetium,
dysprosium, terbium, thrium, gadlinium, or the like and oxides thereof,
magnetite, ferrite, garnets, oxides with corrundum structure, oxides with
perovskite structure, magnetoplumbites, oxides with rutile structure,
further ferritin or highspin type deoxyhemoglobin and heme or derivatives
thereof.
The reaction for preparing the magnetic material-physiologically active
substance conjugate (M-P-E) of the present invention can be performed
according to the two reaction schemes as shown below.
Reaction scheme (1): first, a polyethylene glycol derivative and a
physiologically active substance are chemically bound together to
synthesize a conjugate (P-E), and this P-E is conjugated to M synthesize
M-P-E.
(i) P+E.fwdarw.P-E . . . * .circle.1
(ii) M+P-E.fwdarw.M-P-E . . . * .circle.2
Reaction scheme (2): first, a polyethylene glycol derivative is conjugated
to M as described below to synthesize a conjugate (M-P), and this M-P is
chemically bound with a physiologically active substance E to synthesize
M-P-E.
(i) M+P.fwdarw.M-P . . . * .circle.3
(ii) M-P+E.fwdarw.M-P-E . . . * .circle.4
The bond * .circle.1 between P and E and the bond * .circle.4 between
M-P and E may be preferably covalent bonds, with hydroxyl group, carboxyl
group, methoxycarbonyl group, amide group, amino group, methoxy group,
etc. of the polyethylene glycol derivatives being covalently bonded to
hydroxyl group, amino group, carboxyl group, etc. of the physiologically
active substance to form, for example, acid-amide bond, ester bond, etc.
In the following, their examples are shown. Here, P-0H represents a
polyethylene glycol derivative having hydroxyl group and E represents a
physiologically active substance and E-OH, E-NH2, E-COOH, E-SH represent
physiologically active substances having hydroxyl group, amino group,
carboxyl group, sulfhydryl group. (1) P-OH is allowed to react with
cyanuric chloride in an inert solvent in the presence of a base to obtain
an activated derivative having 1 or 2 P-OH chains bonded thereto. The
activated derivative is allowed to react with a physiologically active
substance in a buffered solution bound to amino oroup or hydroxyl group in
the physiologically active substance molecule.
##STR1##
(2) Through the reaction between P-OH and bromoacetyl bromide in
dibromoacetic acid and dioxane, P-bromoacetate is obtained. The acetyl
derivative is allowed to react with a physiologically active substance.
P-dibromosuccinate prepared by use of dibromosuccinic anhydride can be
also allowed to react with a physiologically active substance.
##STR2##
(3) As the acid azide method, P-OH is allowed to react with chloroacetic
anhydride, then with diazomethane to obtain P-acetic methyl ether, which
is treated with hydrazide to obtain a corresponding hydrazide, followed by
treatment with sodium nitrite to obtain an acid azide derivative. This
active derivative is allowed to react with a physiologically active
substance to effect amide bonding with the free amino group in the
physiologically active substance.
##STR3##
(4) As the diazo method, for example, P-OH is allowed to react with isatoic
anhydride to obtain an anthranylic acid ester, then treated with sodium
nitrite under acidic condition to convert it to a diazonium derivative,
which is subjected to diazo coupling with a physiologically active
substance.
##STR4##
(5) The hydroxyl group of P-OH is convertible to amino group. This method
comprises, for example, allowing tosyl chloride with P-OH to form
P-OH-tosylate, then allowing this to react with a phthalimide salt to
obtain a N-P-substituted phthalimide, which is treated with hydrazine to
obtain an amphiphatic polymer, P-NH.sub.2, having amino group. This
P-NH.sub.2 is allowed to react with the carboxyl group in a
physiologically active substance with a carbodiimide reagent or Woodward
reagent K. Alternatively, P-OH-tosylate or P-OH-bromide obtained by the
reaction with a halogenating agent can be converted with sodium azide into
P-OH-azide, followed by hydrogen reduction to form P-NH.sub.2.
##STR5##
(6) The carboxylic acid derivative of P-OH, otherwise as described above,
can be also allowed to react with a bromoacetic acid ester in the presence
of potassium t-butoxide, followed by hydrolysis to obtain P-carboxymethyl
ether. The amphiphatic polymer, P-O-CH.sub.2 COOH, having carboxylic acid
is allowed to react wtth N-hydroxysuccinimide by utilizing a carbodiimide
reagent to obtain a corresponding succinimide ester, which is allowed to
react with amino group or hydroxyl group in a physiologically active
substance.
##STR6##
(7) Bonding of P onto the sulfhydryl group of E can be done by the above
bonding method to amino group, but in order to be specifically bound to
the sulfhydryl group, for example, pH is lowered to near neutral during
the binding reaction by use of cyanuric chloride, or the reagent shown
below exhibiting specific reactivity for the sulfhydryl group may be
introduced into P.
##STR7##
The reaction * .circle.2 in which M is bound to P-E and the reaction *
.circle.3 in which M is bound to P are magnetization reactions, and in
magnetization reaction, either previously prepared magnetic material
particles may be used or the formation reaction of magnetic material may
be effected at the same time.
Bonding between the magnetic material and the polyethylene glycol
derivative may be effected through coordination bonding or hydrogen
bonding between the oxygen atom of ether bond of the polyethylene glycol
skelaton or the hydrogen atom of ethylene group and hydroxyl group or
metal ion on the surface of the magnetic material,.or in the case of
derivatives when the polyethylene glycol has carboxyl group, hydroxyl
group, methoxycarbonyl group, amide group, amino group, methoxy group,
etc., through covalent bonding or coordination bonding of these groups
with the hydroxyl group of the magnetic material, or coordination bonding
with metal ions of the magnetic material, or through Van der Waals force
between these.
Practically, the reaction for forming such bonding can be performed
according to the two methods. Reaction 1: the method in which magnetic
material particles are allowed to react with polyethylene glycol
derivative in water or an organic solvent by means of a dispersing
machine. Reaction 2: the method in which the formation reaction of a
magnetic material is performed simultaneously in the presence of
polyethylene glycol derivative. As the dispersing machine in Reaction 1, a
ball mill, a vibrating mill, etc. can be used. By elongating the reaction
time in the dispersing machine, the amount of plyethylene glycol
derivative bound to magnetic material can be increased. Also, by
increasing the amount of polyethylene glycol derivatie, the amount of
polyethylene glycol derivative bound to magnetic material can be
increased. For the formation reaction of magnetic material in Reaction 2,
the reaction between ferrous ions and ferric ions under neutral-alkali
condition, the reaction by oxidation of ferrous ions, the reaction between
lipidocrocite or akaganecite and ferrous ions, oxidation or spontaneous
transformation reaction of gneen rusts, the reaction between amorphous
ferric oxide and ferrous ions, etc. can be used.
For example, 3 g of magnetite and 10 g of polyethylene glycol may be
dispersed in water and, after the reaction in a ball mill for 2 days, the
product is dialyzed against water to remove unreaced polyethylene glycol
and obtain a magnetite-polyethylene glycol conjugate (M-P). The particle
size of the magnetite-polyethylene glycol conjugate (M-P) is 30 nm, and
the conjugate has 29% (weight ratio) of polyethylene glycol bound thereto.
Also, the conjugate (M-P) obtained by performing the recovery operation
in-a magnetic field of 6000 (Oersted) Oe in place of dialysis against
water exhibits the same composition (29%). The above binding of the
magnetic material with polyethylene glycol is irreversible and, as shown
in Table 1, the composition (29%) will-not be changed b.y repeating
dialysis in a large amount of water for any number of times or repeating
magnetic separation operation, thus indicating that polyethylene glycol is
bound to the magnetic material in the conjugate.
TABLE 1
______________________________________
Weight ratio of polyethylene glycol
Operation times
Dialysis Magnetic separation
______________________________________
5 29% 30%
10 30% 30%
20 28% 26%
40 31% 29%
______________________________________
This bonding is destroyed under the condition where the magnetic material
in the conjugate (M-P) is decomposed (6N HCl acidity), and this was
confirmed by measurement of the IR absorption spectrum of the polyethylene
glycol recovered by separation by thin layer chromatography after
decomposition. This bonding is coordination bonding or hydrogen bonding
between oxygen atom in the ether bond or hydrogen atom in ethylene group
of polyethylene glycol and iron atom or hydroxyl group on the magnetite
surface. This bonding is very stable, and the conjugate (M-P) will not be
decomposed in organic solvents such as benzene, toluene, chlorofom,
trichloroethylene, carbon tetrachloride, pyridine, acetone, dioxane,
methanol, ethanol, dimethylformamide, dimethyl sulfoxide, etc., or in
aqueous solutions of pH 4 or higher. At a pH of 3 or lower, the crystals
of magnetite become unstable, whereby the conjugate (M-P) is decomposed.
The ferritepolyethylene glycol conjugate (M-P) is stable even at pH 2.
Even if pH may be essentially low, provided that the magnetic material
itself is not decomposed, bonding between polyethylene glycol and the
magnetic material is stable. If the amount of polyethylene glycol in the
reaction using a ball mill is increased to 30 g, the composition of
polyethylene glycol in the magnetic material-polyethylene glycol conjugate
(M-P) becomes 43% (weight). Thus, the bound amount can be increased. If
the reaction time in the ball mill is made 10 days, the particle size of
the conjugate becomes 15 nm.
All of the above magnetite-polyethylene glycol conjugates (M-P) can be
dissolved and dispersed stably in water and organic solvents, and no
agglomerated particle is formed even after standing for 5 days as shown in
Table 2.
TABLE 2
______________________________________
Standing time (hrs.)
Formation of agglomerated particles
______________________________________
0 --
0.5 --
1 --
10 --
24 --
48 --
120 --
______________________________________
It can be understood that dispersion-stability of the conjugate (M-P)
according to the present invention is remarkably high, as compared with
the magnetite particles having no polyethylene glycol bound thereto which
is not substantially dispersed.
By use of a polyethylene glycol derivative having functional groups such as
amino group, carboxyl group, etc., other than the oxygen bond in the ether
bond of the polyethylene glycol skelton mentioned above, bonding also
occurs between these functional groups and iron atoms and hydroxyl groups
on the magnetic material surface. However, these bondings do not occur in
all functional group of the polyethylene glycol derivative molecules.
For example, in the conjugate of .alpha.-methoxycarbonyl-.omega.methoxy
polyethylene glycol and magnetite (M-P), carboxyl groups not participating
in bonding with the magnetic material remain at 10 to 30%, and also in the
conjugate of .alpha.-aminopropyl-.omega.-methoxy polyethylene glycol and
magnetite (M-P), amino groups not participating similarly in bonding
remain at 25 to 50%.
The size of the magnetic material-polyethylene glycol conjugate (M-P) can
be controlled by controlling the reaction time as described above in the
case of carrying out the reaction by means of a dispersing machine
(Reaction 1), and by changing the amount of the reagent used for formation
of the magnetic material and pH during the reaction in the case of
effecting simultaneously the formation reaction of the magnetic material
(Reaction 2). In the method of Reaction 2, for example, when 1 g of a
polyethylene glycol (average molecular weight 5,000) is allowed to react
with 120 mg of ferrous chloride and 300 mo of ferric chloride at pH 8.5, a
conjugate (M-P) with a partile size of 45 nm is obtained, while when
reacted with decreased amounts of these iron ions, namely 64 mg of ferrous
chloride and 151 mg of ferric chloride, a conjugate (M-P) with a particle
size of 30 nm is obtained. On the other hand, at pH 11.0, a conjugate
(M-P) of 20 nm is obtained in both cases. Dispersion stability of these
conjugates in water and organic solvents is similarly high as those
obtained by the reaction by means of a dispersing machine. Further, a
conjugate (M-P) reacted with a polyethylene glycol having a molecular
weight of 500 to 200,000 exhibits similar dispersion stability.
By use of any of the synthetic methods of the magnetic
material-physiologically active substance conjugate (M-P-E) of the above
two reaction schemes, by varying the molecular weight of the polyethylene
glycol derivative, magnetic material-physiologically active substance
conjugate (M-P-E) with the same solubility and dispersibility in organic
solvents, and futher the same stability and activity can be obtained. For
example, when the physiologically active substance is lipase, the magnetic
material-lipase conjugate (M-P-E) obtained by use of a polyethylene glycol
derivative with a molecular weight of 2,000 in the reaction scheme (1) and
the magnetic material-lipase conjugate (M-P-E) obtained by use of a
polyethylene glycol derivative with a molecular weight of 10,000 in the
reaction scheme (2) have the same properties.
The size of the magnetic material-physiologically active substance
conjugate (M-P-E) particles, when employing previously prepared magnetic
material, becomes the size of the magnetic material particles, while it is
determined depending on the P-E (the reaction scheme (1)) or P (the
reaction scheme (2)) and the amounts of the reagents for formation of the
magnetic material when the formation reaction of the magnetic material is
performed. For example, in the case of using a ferrite with a particle
size of 50 nm, it becomes a magnetic material-physiologically active
substance conjugate (M-P-E) with a particle size of 50 nm. On the other
hand, when 1 g of a polyethylene glycol-lipase conjugate (P-E) is allowed
to react with 64 mg of ferrous chloride and 151 mg of ferric chloride, a
conjugate (M-P-E) with a particle size of 30 nm is obtained, and when the
amounts of these iron ions are decreased, for example, to 6.4 mg of
ferrous chloride and 15 mg of ferric chloride, a conjugate (M-P-E) with a
particle size of 10 nm is obtained.
Physiological activity of the magnetic material- physiologically active
substance conjugate (M-P-E), for example, enzyme activity depends on its
particle size, and the activity is greater as the particle size is
smaller. This is because the surface area of the particles is increased as
the particle size is smaller. For example, when the ester synthetic
activity in benzene of the magnetic material-lipase conjugate (M-P-E)
synthesized by use of a polyethylene glycol derivative is compared with a
polyethylene glycol-lipase conjugate (P-E in the above reaction scheme
(1)), the activities of the magnetic material-lipase conjugates (M-P-E)
with particle sizes of 70 nm and 10 nm are respectively 10% and 95% of
that of the polyethylene glycol-lipase conjugate (P-E).
The magnetic material-physiologically active conjugate (M-P-E) is stably
dispersed in an aqueous solution and an organic solvent. For example, the
magnetic material-lipase conjugate (M-P-E) will not be sedimented by
centrifugation in benzene at 2,000.times.g for 5 minutes, but can be
dispersed and dissolved in aqueous solutions and organic solvents such as
benzene for about 2 days to 7 days.
The magnetic material-physiologically active substance conjugate (M-P-E) in
which the constituting magnetic material is a ferromagnetic material with
a particle size of about 30 nm can be separated by a permanent magnet and
an electromagnet, and while the other magnetic materials, by
superconductive electromagnets irrespectively of their particle sizes. For
example, a ferromagnetic material (ferrite)-lipase conjugate (M-P-E) with
a particle size of about 70 nm can be recovered in a magnetic field of 300
Oersted (Oe) by use of permanent magnets at a distance of 1.7 cm in 5
minutes at 100% recovery. On the other hand, with particle size of 30 nm,
100% recovery is possible in 7 minutes in a magnetic field of 5,000 Oe by
use of electromagnets at a distance of 1.7 cm. The paramagnetic material
(erbium)-physiologically active conjugate (M-P-E) can be recovered at 100%
in 5 minutes in a magnetic field of 2 Tesla (T)=20,000 Oe by use of a
superconductive electromagnet.
The present invention is applicable for enzyme, protein, antibody, antigen,
polysaccharide, nucleic acid, lipid, amino acid, co-enzyme (NAD.sup.+,
NADP.sup.+, etc.), high energy phosphoric acid compound (ATP, ADP, etc.),
prosthetic group (heme, riboflavin, etc.), hormone, vitamin, receptor,
ligand, antibiotic, antitumor substance, pharmaceutical, or chloroplast,
mitochondrion, virus, cell and constituents thereof, which are
physiologically active substances. For example, as the enzyme,
hydroxylases such as lipase, esterase, chymotrypsin, trypsin, subtilicin,
redoxidases such as peroxidase, catalase, may be preferably used when the
substrate or the product is water-insoluble, including the case when the
reaction can be proceeded reversibly by carrying out the reaction in an
organic solvent. In the case of using a co-enzyme, the enzymatic reaction
requiring a co-enzyme such as alcohol dehydrogenation reaction can be
carried out. When using ATP, ADP, etc., the present invention can be
utilized for the enzymatic reaction in which these are used as the
substrate. Also, by use of such substances as specific antibodies,
concanavalin A, polysaccharides, amino acids, vitamins, lipids, hormones,
viruses, substances having affinity for these substances can be separated,
purified and recovered. Further, when antibiotics, antitumor substances,
pharmaceuticals are used, since magnetic properties can be imparted to
these substances, they can be made amphiphatic pharmaceuticals having
magnetic properties.
The specific feature of the present invention resides in that the magnetic
material and the physiologically active substance are bound to each other
through a polyethylene glycol derivative. Consequently, the following
effects may be contemplated. (1) The magnetic material-physiologically
active substance conjugate (M-P-E) can be recovered rapidly and simply by
magnetic separation through its magnetic properties from in aqueous
solutions and in organic solvents. (2) Due to amphiphatic property
possessed by the polymer, the magnetic material-physiologically active
substance conjugate (M-P-E) can be dissolved or dispersed as colloid in
aqueous solutions and organic solvent. (3) Organic synthetic reaction or
exhibition of biological activity which have been impossible in
bio-reactors using aqueous solutions of the prior art are rendered
possible. (4) The magnetic material-physiologically active substance
conjugate (M-P-E) can be dissolved or dispersed again in the solvent
employed for reuse. (5) When used as a pharmaceutical, irrespectively of
whether the afflicted site is under hydrophobic or hydrophilic
environment, the pharmaceutical can be magnetically led to the afflicted
site.
The present invention is described below by referring to Examples.
EXAMPLE 1
(a) To a solution of 5 g (2.5 mmol) of .alpha.,.omega.-dicarboxy
polyethylene glycol (average molecular weight 2,000) and 288 mg (2.5 mmol)
of N-hydroxysuccinic imide dissolved in 15 ml of dimethylformamide was
added 1 ml of dimethylformamide containing 618 mg of
dicyclohexylcarbodiimide to activate the carboxyl groups of the
polyethylene glycol derivative. Into 20 ml of an aqueous phosphate
buffered solution (pH 7.0) containing 200 mg of lipase obtained from
Pseudomonas fluorescens cells was added 2 g of the above activated
polyethylene glycol derivative, followed by the reaction at 25.degree. C.
for 1 hour to obtain a polyethylene glycol-lipase conjugate (P-E).
A solution of 1 g of the polyethylene glycol-lipase conjugate (P-E)
dissolved in 1.3 ml of water was adjusted to pH 8.0 with ammonia water,
and 0.6 ml of an aqueous solution containing 64 mg of ferrous chloride and
151 mg of ferric chloride was added dropwise to this solution. During the
dropwise addition, pH was maintained at 8.0 to 8.5 with ammonia water, and
the mixture was well stirred at room temperature. After sufficiently
dialyzed against water, a magnetic material-lipase conjugate (M-P-E) was
obtained by lyophilization.
The magnetic material-lipase conjugate (M-P-E) was found to contain 34% of
the magnetic material and 30% of the protein. Also, it had a hydrolysis
activity of olive oil in water of 1500 units/mg protein, and a synthetic
activity of lauryl laurate of 10 .mu.mol/min./mg protein in benzene which
is a representative hydrophobic organic solvent. The magnetic-lipase
conjugate (M-P-E) was completely recovered from the reaction solution in 5
minutes in a magnetic field of 6,000 Oersted (Oe). This was again
dissolved as colloid in the reaction solution to exhibit similar
activities.
It was found that the magnetic material-lipase conjugate (M-P-E) dissolved
as colloid in the aqueous solution and benzene was not sedimented by
centrifugation at 2,000.times.g, and its turbidity did not change for 24
to 60 hours in turbidity measurement at 600 nm. Thus the conjugate stably
dispersed in an organic solvent. Also, in the aqueous solution, similar
dispersion stability was observed. By observation with an electron
microscope, the conjugate was found to consist of ultra-fine particles
with particle sizes of 10 to 40 nm.
As to organic solvents other than benzene, in solvents in which the
amphiphatic polymer used here is soluble such as toluene, chloroform,
chlorinated hydrocarbons, etc., the magnetic material-lipase conjugate
(M-P-E) exhibited similar properties.
(b) Also by use of .alpha.,.omega.-dicarboxy polyethylene glycols with
molecular weights of 5,000 and 20,000 in place of the
.alpha.,.omega.-dicarboxy polyethylene glycol with molecular weight of
2,000, magnetic material-lipase conjugates (M-P-E) having similar
properties were obtained.
(c) Also by use of .alpha.-amino-.omega.-carboxy polyethylene glycol
(molecular weight 2,000), .alpha.-carboxy-.omega.-methoxy polyethylene
glycol (molecular weight 2,000) or 2,4-bis
(methoxypolyoxyethylene)-6-chloro-S-triazine (molecular weight 10,000) in
place of .alpha.,.omega.-dicarboxy polyethylene glycol, a magnetic
material-lipase conjugate having similar properties was obtained.
(d) Also from the polyethylene glycol-lipase conjugate (P-E) obtained by
use of .alpha.,.omega.-diamino polyethylene glycol in place of
.alpha.,.omega.-dicarboxy polyethylene glycol, a similar magnetic
material-lipase conjugate (M-P-E) was obtained. The polyethylene
glycol-lipase conjugate (P-E) was obtained by adding 200 mg of a
water-soluble carbodiimide into 20 ml of an aqueous phosphate buffered
solution (pH 7.0) containing 2 g of .alpha.,.omega.-diamino polyethylene
glycol and 200 mg of lipase and carrying out the reaction at 37.degree. C.
for 30 minutes, followed by dialysis against water.
EXAMPLE 2
According to the method of Example 1a) by use of 150 mg of subtilicin
derived from Bacillus subtilis in place of lipase, a magnetic
material-subtilicin conjugate (M-P-E) was obtained. The magnetic
material-subtilicin conjugate (M-P-E) contained 36% of the magnetic
material and 25% of the protein. Also, it had a hydrolysis activity of
ethyl acetyltyrosinate in aqueous solution of 600 units/mg protein, and a
synthetic activity of N-benzoyltyrosine butylamide of 4.0 .mu.mol/min./mg
protein in benzene which is a representative hydrophobic organic solvent.
The magnetic material-subtilicin conjugate (M-P-E) was completely
recovered from the reaction solution in 5 minutes in a magnetic field of
5,500 Oersted (Oe). This was dissolved again as colloid in the reaction
solution to exhibit similar activities. Dispersion stability, the particle
size and other properties of the magnetic material-subtilicin conjugate
(M-P-E) dissolved as colloid in the aqueous solution and the organic
solvent were found to be the same as the above magnetic material-lipase
conjugate (M-P-E).
EXAMPLE 3
According to the method of Example 1a) by use of 70 mg of catalase derived
from bovine liver in place of lipase, a magnetic material-catalase
conjugate (M-P-E) was obtained. The magnetic material-catalase conjugate
(M-P-E) contained 25% of the magnetic material and 45% of the protein.
Also, it had a decomposition activity of hydrogen peroxide in aqueous
solution of 60,000 units/mg protein, and a decomposition activity of
hydrogen peroxide of 70,000 units/mg protein in benzene which is a
representative hydrophobic organic solvent. The magnetic material-catalase
conjugate (M-P-E) was recovered completely from the reaction solution in 7
minutes in a magnetic field of 6,000 Oersted (Oe). This was again
dissolved as colloid in the reaction solution to exhibit similar
activities. Dispersion stability and the particle size of the magnetic
material-catalase conjugate (M-P-E) dissolved as colloid in the aqueous
solution and the organic solvent were found to be the same as the above
magnetic material-lipase conjugate (M-P-E).
Also, similar results were obtained by use of esterase in place of lipase.
EXAMPLE 4
A solution of 5 g of .alpha.,.omega.-dicarboxy polyethylene glycol
(molecular weight 2,000) dissolved in 5 ml of water was adjusted to pH 8.0
with an aqueous ammonia, and to this solution was added dropwise 2.4 ml of
distilled water containing 250 mg of ferrous chloride, 50 mg of cobalt
chloride and 750 mg of ferric chloride. During the dropwise addition, pH
was maintained at 8.0 to 8.5 with an aqueous ammonia, and the mixture was
stirred sufficiently at 60.degree. C. The reaction mixture was
sufficiently dialyzed against water to obtain a magnetic
material-polyethylene glycol conjugate (M-P).
Into 4 ml of a phosphate buffered solution (pH 7.0) containing 5 mg of
lipase obtained from Pseudomonas fluorescens cells and 75 mg of the
magnetic materialpolyethylene glycol conjugate (M-P) was added 500 mg of a
water-soluble carbodiimide and, after the reaction at 37.degree. C. for 90
minutes, 5 ml of water was added to the reaction mixture. The magnetic
material-lipase conjugate (M-P-E) formed by magnetic separation was
thoroughly washed with water and then lyophilized to obtain a
magnetic-lipase conjugate (M-P-E).
The magnetic material-lipase conjugate (M-P-E) was found to contain 50% of
the magnetic material and 20% of the protein. Also, it had a hydrolysis
activity of olive oil in water of 500 units/mg protein, and a synthetic
activity of lauryl laurate of 1.5 .mu.mol/min./mg protein in benzene which
is a representative hydrophobic organic solvent. The magnetic-lipase
conjugate (M-P-E) was completely recovered from the reaction solution in 5
minutes in a magnetic field of 300 Oersted (Oe). This was again dissolved
as colloid in the reaction solution to exhibit similar activities.
It was found that the magnetic material-lipase conjugate (M-P-E) dissolved
as colloid in the aqueous solution and benzene was not sedimented by
centrifugation at 2,000.times.g, and its turbidity did not change for 24
to 60 hours in measurement at 600 nm. Thus the conjugate stably dispersed
in an organic solvent. Also, in the aqueous solution, similar dispersion
stability was observed. By observation with an electron microscope, the
conjugate was found to consist of ultra-fine particles with particle sizes
of 60 to 100 nm.
As to organic solvents other than benzene, in solvents in which the
amphiphatic polymer used here is soluble such as toluene, chloroform,
chlorinated hydrocarbons, etc., the magnetic material-lipase conjugate
(M-P-E) exhibited similar properties. Also, to the carboxyl groups of the
magneic materialpolyethylene glycol conjugate (M-P) was bound
N-hydroxysuccinimide according to a conventional method in dioxane to
synthesize an activated matnetic material-polyethylene glycol conjugate
(M-P). And, 75 mg of the activated magnetic material-polyethylene glycol
conjugate (M-P) was added to 4 ml of a phosphate bufferd solution (pH 7.0)
containing 5 mg of lipase obtained from Pseudomonas fragi 22-39B cells
and, after the reaction at 37.degree. C. for 90 minutes, the reaction
product was purified to obtain a magnetic material-lipase conjugate having
similar properties.
EXAMPLE 5
A solution of 5 g of .alpha.,.omega.-dicarboxy polyethylene glycol (average
molecular weight 2,000) and 150 mg of ferrous chloride dissolved in 4 ml
of water was adjusted to pH 8.0 with an aqueous ammonia, and oxidized by
passing oxygen at 65.degree. C. for 1 hour. The reaction mixture was
thoroughly dialyzed against water to obtain a magnetic
material-polyethylene glycol conjuqate (M-P). To the carboxyl groups of
the magnetic material-polyethylene glycol (M-P) was bound
N-hydroxysuucinimide according to a conventional method to synthesize an
activated magnetic materil-polyethylene glycol conjugate (M-P). And, 75 mg
of the activated magnetic material-polyethylene glycol conjugate (M-P) was
added to 4 ml of a phosphate buffered solution (pH 7.0) containing 5 mg of
lipase obtained from Pseudomonas fluorescens cells and, after the reaction
at 37.degree. C. for 90 minutes, the magnetic material-lipase conjugate
(M-P-E) formed by magnetic separation was thoroughly washed with water,
followed by lyophilization, to give a magnetic material-lipase conjugate
(M-P-E). Dispersibility or magnetic separation characteristics, etc. of
this conjugate were found to be the same as the magnetic material-lipase
conjugate (M-P-E) in Ex | | |
