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Description  |
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This application relates to immobilized biologically active proteins and to
a process for preparing the same.
In enzymatic reactions, immobilized enzymes are superior to natural ones in
their convenient recovery from the reaction mixtures. While many
immobilization methods have been reported, most have shortcomings, such as
low enzymatic activity of the immobilized products, weak bond strength
between enzyme and carrier, or low mechanical strength of the products.
Covalent bonding is superior to other immobilization methods in bond
strength, but the enzymatic activity or mechanical strength of the known
products is unsatisfactory.
It is an object of the present invention to provide immobilized
biologically active proteins which retain a significant amount of their
original biological activity.
In accordance with the present invention, there is provided a biologically
active composition by contacting a polypeptide having repeating acidic
amino acid units of which at least one carboxyl group in a side chain is
converted to a carbonyl azide group and optional repeating units of a
neutral and/or basic amino acid (polypeptide azide) with a biologically
active protein having an amino acid unit including an amino group in
position .alpha. or .omega., the biologically active protein being
covalently bonded to the polypeptide azide through reactive groups.
The term "polypeptide azide" as used herein refers to a polypeptide having
repeating units of the formulas (I) or (II), jointly represented by
formula (Ia),
##STR1##
WHEREIN N IS 1, 2, 3 OR 4.
The polypeptide azide usually has unreacted repeating units of the original
polypeptide, of which the formulas are
##STR2##
wherein n is as above, and R is hydrogen, lower alkyl having one to four
carbon atoms, or metal.
When the polypeptide azide is prepared from polypeptide molecules having
repeating units of formulas (III) or (IV) by the hydrazine method
described below, some repeating units are cross linked with an adjacent
polypeptide molecule through hydrazine, as shown by formulas (V), (VI) or
(VII), and the cross-linking so produced remains in the polypeptide azide.
##STR3##
The advantages of this invention are also available in copolymers in which
the above units are connected by peptide bonds to repeating units of one
or more netural and/or basic amino acids in the backbone of the molecule.
Repeating units of a neutral amino acid make the polypeptide
water-insoluble.
The neutral amino acids which may link the repeating acidic amino acid
units of Formulas (III) and (IV) in a peptide chain forming the backbone
of the polymeric molecule include leucine, alanine, phenylalanine, serine,
threonine, cysteine, methionine, and their derivatives, such as
O-substituted hydroxy-amino acids and S-substituted sulfur-containing
amino acids. Suitable basic amino acids are lysine, ornithine, arginine,
histidine and their lower acyl derivatives.
The amino acids mentioned above may be in the optically active form, in the
racemic form, or be mixtures of the two forms.
There are many methods for converting a carboxyl group to an acyl azide
group, and most can be applied to the present invention.
In one of the most suitable methods, a polypeptide containing repeating
units of a lower alkyl ester of an acidic amino acid is reacted with
hydrazine, and the hydrazide produced is reacted with nitrous acid until
the hydrazide groups in a portion of the units are converted to azide
groups.
The homopolymers or copolymers which have repeating units of a lower alkyl
ester of an acidic amino acid are prepared in any manner known, and the
carboalkoxy groups in a portion of the units are converted to primary
hydrazide moieties as described, for example, in Nature 189, p. 576
(1961), where it was reported that trypsin and chymotrypsin are
immobilized on carboxymethyl cellulose whose carboxymethyl groups have
been converted to azide groups. After the reaction with hydrazine, a
portion of the units is usually cross-linked by the hydrazine, while other
carboalkoxy groups remain unchanged.
The reaction product is separated from the reactant solution by filtration
or centrifuging, washed with water until the washings are neutral, and, if
desired, the product is further washed with a chilled, weakly acidic,
aqueous solution and/or chilled aqueous solution of an organic solvent to
remove most of the soluble constituents.
The washed product is immersed in 0.02 - 0.5 N mineral acid at a
temperature below 20.degree. C, preferably below 5.degree. C, thereafter
nitrous acid or an aqueous solution of a nitrite, for example, a 1-20%
solution of sodium nitrite, is added to the mineral acid solution in one
batch or within a few minutes, and the mixture is moderately stirred for 5
- 60 minutes at low temperature.
The polypeptide azide so produced is separated from the reactant solution,
washed several times with chilled water and/or chilled buffer solution of
a pH not to impair the activity of the biologically active immobilized
protein. Thereafter, if desired, the polypeptide azide is further washed
with a chilled aqueous solution of an organic solvent.
The weakly acidic aqueous solution which can be employed in the first
washing process described above may be a dilute solution of a mineral
acid, such as 0.01 N to 0.1 N hydrochloric acid or phosphoric acid. The
mineral acid solution may contain an organic acid, such as acetic acid, or
organic solvent which is water-miscible, such as methanol, ethanol, benzyl
alcohol or dimethylformamide.
Suitable aqueous washing solutions of organic solvents include 10 to 70%
(vol.) aqueous solutions of methanol, ethanol, ether, acetone, and
dioxane.
A polypeptide azide may also be produced by reaction of a polyamino acid
chloride with sodium azide.
The biologically active proteins to which the present invention is
applicable include enzymes, such as urease, uricase, urokinase, amino acid
acylase, aspartase, amylase, lipase, glucose oxydase and protease, natural
proteins of animal and plant origin, and natural peptides, such as
antigens, antibodies and peptide hormones.
These biologically active proteins may easily be immobilized by immersing a
polypeptide azide in solutions of the proteins buffered to a pH at which
the activity of the biologically active protein to be immobilized is not
adversely affected, and whose temperature is kept below 40.degree. C,
preferably below 10.degree. C, and by moderately stirring the mixture for
6 to 24 hours.
The polypeptide on which a biologically active protein is immobilized may
have any shape and be a membrane, tube, fiber, porous shaped body, bead or
viscous liquid.
In order to enhance the mechanical strength of the composition, the
polypeptide may be deposited on various carriers. When the coated carrier
is immersed in a solution, the coating may peel off unless fastened to the
surface of the carrier by an adhesive. The adhesive may also fix
water-soluble constituents of the polypeptide. Since the original
polypeptide is usually a linear polymer and is appreciably water-soluble,
some constituents which are only sparingly cross-linked and whose carboxyl
groups are not fully converted to carboalkoxy groups may be dissolved even
when combined with biologically active proteins.
Suitable adhesives include those capable of cross-linking, such as
polyurethane resin, epoxy resin and polyester resin, and those incapable
of cross-linking, such as polyvinyl chloride, polyvinyl acetate,
polyvinylidene chloride, polyacrylate, and polyamide. Examples of such
adhesives are reaction products of three parts by weight of
polyurethanediisocyanate and one part by weight of epichlorohydrin, also
reaction products of 3, 9-bis(3-aminopropyl)- 2,4,8,10
tetraoxospiro-[5,5]-undecane of the formula
##STR4##
and epichlorohydrin.
The carriers capable of being coated with the polypeptide include beads of
glass, synthetic resins, such as acrylic resin and vinyl chloride resin,
or stainless steel, and the inner walls of glass and stainless steel
tubes.
The adhesive is first applied to the surface of the carrier, a solution or
a thin layer of the polypeptide is deposited on the coated surface, and
the coating is dried. Alternatively, a solution of the polypeptide is
first mixed with the adhesive, and the mixture is deposited on the surface
of the carrier and dried. When a carrier is not used, the adhesive is
still effective for fixing the water-soluble constituents of the
composition and for increasing its mechanical strength.
The superiority of the present invention over known covalent bonding
methods is demonstrated by the following two experiments.
EXPERIMENT 1
Small pieces of a copolymer of L-methionine and L-glutamic acid
.gamma.-methyl ester (mole ratio 1:1) weighing one gram were suspended in
10 ml methanol, 2 ml 80% hydrazine hydrate were added to the suspension,
and the mixture was kept at 50.degree. C for 2 hours. The hydrazide
produced thereby was filtered off and washed four times with 50% methanol.
Thereafter, the washed product was immersed in 30 ml of a mixture of equal
volumes of methanol and 0.5 N hydrochloric acid, allowed to stand for a
few minutes, and filtered off. The immersing procedure was repeated once
more, the hydrazide product was washed with methanol and dried. The
hydrazide weighed 0.8 gram and contained 1.1 meq/g hydrazino groups.
100 mg Hydrazide was suspended in 20 ml methanol cooled in an ice bath and
10 ml chilled 0.5 N hydrochloric acid, and then 2.0 ml chilled aqueous 3%
sodium nitrite solution were added to the suspension. The mixture was
stirred for 20 minutes at 0.degree. - 4.degree. C. The azide produced
thereby was recovered by centrifuging at 3,000 r.p.m. for 2 minutes and
washed three times with chilled 50% methanol and subsequently twice with
chilled water.
The recovered azide was immersed in 3.0 ml chilled 0.1 M phosphate buffer
solution of pH 8.7 containing 2.55 mg/ml urease and 1 mM EDTA, and the
mixture was stirred at 4.degree. C for 48 hours. The urease employed was
produced by SIGMA Chemical Co. (Type III, 2,2 U/ml). The resulting
composition containing immobilized urease was centrifuged off, and washed
successively once with 0.05 M phosphate buffer solution of pH 7.0, twice
with the same buffer solution additionally containing 1 M sodium chloride,
and twice with the same buffer solution without sodium chloride. The total
volume of the filtrate was about 45 ml. The urease activities of the
immobilized enzyme and of the filtrate were 8.6 U (86 U/g carrier) and 8.0
U, respectively.
One unit of urease activity produces 1 mg nitrogen as ammonia from 0.25 M
urea solution of pH 7.0 at 25.degree. C in 5 minutes when the ammonia is
colorimetrically determined by the indophenol method.
In a comparison test, the same urease was immobilized on a carboxymethyl
cellulose in the manner described in Nature, 189, p. 576 (1961).
Carboxymethyl cellulose (CMC) was washed successively with water, sodium
hydroxide, hydrochloric acid, methanol and ether, and dried. The hydroxyl
groups of the washed CMC were converted to methyl ester groups in the
conventional method, one gram of the methylated CMC was suspended in 10 ml
methanol, 2 ml 80% hydrazine hydrate was added to the suspension, and the
suspension was refluxed for one hour. The reaction mixture was allowed to
stand for 2 hours at room temperature and centrifuged. The precipitated
hydrazide was washed five times with methanol and dried.
100 mg Hydrazide was suspended in 10 ml 0.5 N hydrochloric acid cooled in
an ice bath, and 2.0 ml chilled aqueous 3% sodium nitrite solution was
added to the suspension. The mixture was stirred for 20 minutes, and then
centrifuged at 3,000 r.p.m. for 2 minutes. The precipitate was washed
three times with chilled 50% methanol and subsequently twice with chilled
water.
On the carboxymethyl cellulose azide so produced, the urease was
immobilized in the same manner as described above with reference to the
copolymer of L-methionine and L-glutamic acid .gamma.-methyl ester. An
immobilized enzyme having 3.3 U (33 U/g carrier) of urease activity and a
filtrate having 12.0 U of urease activity were obtained.
When the same urease was immobilized on polymethacrylic acid in the same
manner as described with reference to CMC, the urease activities of the
immobilized enzyme and of the washing solution were 1.4 U (14 U/g carrier)
and 12.3 U, respectively.
EXPERIMENT 2
Azides of L-methionine-L-glutamic acid .gamma.-methyl ester copolymer, of
carboxymethyl cellulose, and of polymethacrylic acid were prepared in the
same manner as in Experiment 1.
100 mg Batches of the three azide compounds were immersed each in 3.0 ml of
chilled 0.1 M borate buffer solution of pH 8.5 containing 2.0 mg/ml of
uricase, and stirred at 4.degree. C for 48 hours. The uricase employed was
yeast uricase having 2.86 U/mg of uricase activity. The resulting
compositions containing immobilized uricase were centrifuged off, and
washed successively once with 0.1 M borate buffer solution of pH 8.5,
twice with the same buffer solution containing 1 M sodium chloride, and
twice with the same buffer solution without sodium chloride.
The uricase activities of the immobilized enzymes and of the filtrates are
shown in Table 1.
Table 1
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Immobilized Enzyme
Filtrate
Copolymer 14.4 U(144 U/g carrier
2.2 U
CMC 1.4 (14) 12.4
Polymethacrylic acid
0.67 (6.7) 13.7
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To determine uricase activity, 0.1 - 0.2 ml of a sample containing about 5
- 30mU uricase was mixed with 5.0 ml of a 0.1 mM solution of uric acid in
0.1 M borate buffer solution of pH 8.5, and the mixture was stirred at
25.degree. C for 5 minutes. In order to stop the enzyme reaction, 0.2 ml
20% trichloroacetic acid was then added, and the residual uric acid was
determined colorimetrically at 292.5 m.mu.. One unit of uricase activity
corresponds to 1 .mu.mol of uric acid digested per minute.
EXPERIMENT 3
Several batches of filaments of poly-D-glutamic acid hydrazide were washed
neutral with water on a filter having a bottom of sintered glass. Some
batches were washed subsequently three times with 0.08 N hydrochloric
acid, and thereafter again with water until the washings were neutral.
Hydrazide groups of the washed poly-D-glutamic acid hydrazide were
converted to azide groups in the conventional manner at 4.degree.-
5.degree. C, and the reaction mixture was filtered through a glass filter.
The azide on the sintered glass disk was washed three or four times with
cold water, and in several instances subsequently three or four times with
cold, 50% aqueous, organic solvent solution.
The poly-D-glutamic acid azide was immersed in 0.05 M phosphate buffer
solution of pH 8.5 containing 1 - 3 mg/ml of acylase and in some instances
20% ethanol. The immobilization mixture was stirred by a magnetic stirrer
or by shaking for 2 - 5 days at 4.degree. C. The acylase-bearing solid was
filtered off through a glass filter and washed with 0.05 M phosphate
buffer solution of pH 7.0 - 7.2 until no acylase activity could be
detected in the filtrate. The acylase activities of the immobilized enzyme
and of the filtrate were determined in the following manner.
1.0 Ml of an acylase solution or of a suspension of a solid bearing
immobilized acylase was added to 10.0 ml of a solution of 22 mM
N-acetyl-DL-phenylalanine, 0.1 mM cobalt chloride, and 50 mM Veronal
buffer solution of pH 8.0 at 37.degree. C. The mixture was centrifuged for
1.0 minute, and a first sample of 0.2 - 0.3 ml supernatant was collected
in a micropipette. The remaining mixture was stirred at 37.degree. C for
30 minutes, and another sample of the same volume of supernatant was
collected. Each sample was held for 5 minutes in water above 95.degree. C
to inactivate the acylase, and the phenylalanine present was determined by
the colorimetric method of Yamm, Cocking, et al.
One unit of acylase activity produces one .mu.mol of phenylalanine.
Table 2 lists, for each batch, the amount of poly-D-glutamic acid hydrazide
filaments in terms of the polymethyl-D-glutamate filaments from which the
hydrazide was prepared, the use (+) or non-use (-) of 0.08 N HCl in
washing the hydrazide filaments, the solvent used for washing the azide,
the solvent used in the immobilization mixture, the initial amount of
acylase, and the distribution of acylase between the filtrate and the
immobilized enzyme.
Table 2
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PMG Hydrazide
Azide Solvent in
Initial
Acylase Re-
Immob'd
Filaments
Washed with
Washing Immobilize'n
Acylase
covered From
Enzyme U/g
mg 0.08 NHCl
Solvent Mixture U/ml Filtrate
Activity
Carrier
__________________________________________________________________________
150 - Water Water 53U/1.5 ml
87% 7.5 U (14%)
52
150 + Water Water " 75% 14 U (25%)
93
150 + Dioxane 50%
Water 130U/2 ml
80% 34 U (26%)
231
150 + Ethanol 50%
Ethanol 20%
" 77% 44 U (34%)
292
500 - Water Water 450U/10 ml
93% 13 U (3%)
26
500 + Ethanol 50%
Ethanol 20%
" 83% 109 U (24%)
218
__________________________________________________________________________
EXPERIMENT 4
A film of poly-.gamma.-methyl-D-glutamate having an intrinsic viscosity of
1.2 was cut into pieces 50 mm square and 32 .mu. thick, and the pieces
were immersed in 100 ml of a hydrazine solution in an aqueous organic
solvent (benzyl alcohol, N,N-dimethylformamide, or methanol) at 21.degree.
C. The hydrazide produced was filtered off, washed with water until free
from hydrazine and solvent, and air-dried.
The hydrazide was analyzed for nitrogen content and methoxy group content
(F. Viebock and C. Brecher), and the methoxy group content, hydrazide
group content, and cross-linking group content were calculated and are
shown in Table 3.
The Table shows the variation in the composition of the side chains due to
the hydrazine concentration, to the nature of the solvent employed with
the hydrazine, and the reaction time. All percentages are by weight.
Table 3
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Reaction Side Chains, Mole %
Hydrazine Solution
Time Hydra- Cross-
Methyl
%N.sub.2 H.sub.4
Solvent Hours zide linked
ester
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60 63% Benz.alc.
1 49 14 37
60 63% Benz.alc.
3 93 5 2
67 50% Benz.alc.
1 35 21 44
67 50% Benz.alc.
2 82 11 7
67 33% Benz.alc.
2 42 32 26
67 33% Benz.alc.
3 80 12 8
40 83% DMF 6 28 49 23
60 63% DMF 6 54 29 27
40 83% Methanol 6 36 27 37
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The following Examples further illustrate the invention. All percentage
values and parts referred to in these Examples are by weight unless stated
otherwise.
EXAMPLE 1
150 mg Poly-.gamma.-methyl-D-glutamate film was immersed in 50 ml of a
mixture of 10 volumes 80% hydrazine hydrate with one volume pyridine for 1
hour at 50.degree. C, and filtered off.
The polyglutamate hydrazide so produced was washed with water and three
times with 50 ml 0.05 N hydrochloric acid. The washed hydrazide was
immersed in 10 ml ice-cold 0.1 N hydrochloric acid for 15 seconds, 2 ml
aqueous 3% sodium nitrite solution was added, and the mixture was stirred
for 20 minutes. It was then filtered, and the solid material was washed
several times with ice-cold 0.05 M phosphate buffer solution of pH 7.5,
and several times with ice-cold 50% ethanol.
The azide so produced was shaken 48 hours at 4.degree. C with 4 ml 0.05 M
phosphate buffer solution of pH 8.5 containing 2 mg urease isolated from
jack beans and having 5.6 U urease activity and 20% ethanol by volume. The
reaction mixture was filtered, and the recovered solids were washed
several times with ice-cold 0.05 M phosphate buffer solution of pH 7.5
containing 1 M sodium chloride until no urease activity could be detected
in the filtrate.
The activity of the immobilized urease was 4.3 U and that of the filtrate
was 1.2 U when determined as in Experiment 1.
when the procedure described above was repeated, but without washing the
hydrazide with dilute hydrochloric acid, without washing the azide with
aqueous ethanol, and without adding ethanol to the urease immobilizing
mixture, the urease activity of the immobilized enzyme was 2.0 U.
EXAMPLE 2
150 mg Poly-.gamma.-methyl-D-glutamate filaments were treated with
hydrazine and washed with water in the manner of Example 1, and then
washed three times with 50 ml of 0.02 N hydrochloric acid.
The hydrazide was treated with nitrite and washed with 50% ethanol also as
described in Example 1. The azide so produced was stirred 48 hours at
4.degree. C with 4 ml 0.05 M phosphate buffer solution of pH 8.5
containing 4 mg jack bean urease having a urease activity of 11.2 U and
10% by volume acetone. The urease-bearing substance was washed as in
Example 1, and the activities of the washed immobilized enzyme and of the
filtrate were 9.8 U and 1.4 U respectively.
The urease activity of the immobilized enzyme was 2.7 times the activity of
the enzyme immobilized in the otherwise identical procedure without
washing of the hydrazide with dilute hydrochloric acid, without washing of
the azide with aqueous ethanol, and without acetone in the urease
immobilizing mixture.
EXAMPLE 3
Six parts poly-.gamma.-methyl-D-glutamate, three parts polyurethane
diisocyanate, and one part epichlorohydrin were dissolved in 300 parts
ethylene dichloride. 10 g Porous glass beads of 80 - 120 mesh were stirred
with 30 g of this solution, and the solvent was removed. The glass beads
coated with the polyglutamate were dried at 100.degree. C for 5 hours, and
1.0 g dried beads were immersed in a hydrazine solution to convert the
poly-.gamma.-methyl-D-glutamate coatings of the beads to the hydrazide as
described in Example 1.
The beads then were washed with 0.08 N hydrochloric acid, treated with
nitrite, and washed with cold 50% (vol.) acetone.
The azide coated beads were shaken 48 hours at 4.degree. C with 1.5 ml of
0.05 M phosphate buffer solution of pH 8.5 containing 3.0 mg acylase (110
U) produced by Aspergillus, 20% by volume ethanol and 0.05 M sodium
acetate and filtered off. They were washed thereafter with 0.05 M
phosphate buffer solution of pH 7.5 containing 0.05 M sodium phosphate
until no acylase activity could be detected in the filtrate.
The activity of the immobilized acylase was found to be 56 U (56 U/g), and
that of the filtrate was 55 U.
The enzyme activity of the immobilized enzyme was 3.3 times that of the
enzyme immobilized in the same procedure except for the washing processes.
When the immobilized acylase was held at 70.degree. C for 30 minutes, it
retained 73% of its initial activity.
EXAMPLE 4
150 mg Beads (30 - 80 mesh) of a copolymer of DL-methionine and L-glutamic
acid .gamma.-methyl ester (mole ratio 1:1) were treated with hydrazine and
washed with dilute hydrochloric acid in the manner of Example 1. The
hydrazide was treated with nitrite and washed with cold 50% (vol.)
dioxane.
The azide bearing beads so produced were stirred 24 hours at 4.degree. C
with 2 ml 0.05 M phosphate buffer solution of pH 8.5 containing 3 mg jack
bean urease having an activity of 8.4 U and 20% (vol.) ethanol. The beads
carrying the immobilized urease were washed as in Example 1, and the
activity of the washed immobilized urease was found to be 8.2 U (55 U/g).
This was about 2.2 times the activity of the immobilized enzyme produced
in the same procedure except for the washing processes.
When stored at 25.degree. C for 6 months, the immobilized urease retained
91% of its initial activity.
EXAMPLE 5
A solution of 6 parts poly-.gamma.-methyl-D-glutamate having an intrinsic
viscosity of 1.2, 3 parts polyurethane diisocyanate, 1 part
epichlorohydrin, and 300 parts of a solvent consisting of 9 volumes
dichloroethane and one volume toluene, was stirred in an amount of 50 g
with 10 g porous glass beads (80 - 120 mesh). The organic solvents were
removed on a rotary evaporator, and the coated glass beads were dried at
100.degree. C for 5 hours.
They were then immersed in 50 ml of a mixture of 10 volumes 80% hydrazine
hydrate with one volume pyridine at 50.degree. C for 3 hours.
The hydrazide bearing beads were filtered off, washed with water until free
from hydrazine and solvent, and suspended in 100 ml chilled 0.1 N
hydrochloric acid for 15 seconds. 15 ml Chilled, aqueous, 3% sodium
nitrite solution was added to the suspension which was then stirred at
4.degree. C for 20 minutes. The azide coated glass beads were filtered
off, washed with ice-cold water and subsequently with 0.05 M phosphate
buffer solution of pH 8.5, immediately immersed in 10 ml chilled 0.05 M
phosphate buffer solution of pH 8.5 containing 30 mg jack bean urease
having an activity of 63 U, and moderately stirred at 4.degree. C for 24
hours. The glass beads were filtered off, and washed several times with
0.05 M phosphate buffer solution of pH 7.5 until no urease activity could
be detected in the filtrate and subsequently once or twice with the same
buffer solution containing 1 M NaCl
The urease activity of the immobilized enzyme was 5.0 U/g and that of the
filtrate was 12 U.
When the beads carrying the immobilized enzyme were packed in a column, and
enzyme reactions were repeated at 25.degree. C for 6 months using the
column, the urease activity of the immobilized enzyme was scarcely
lowered. The coating did not separate from the beads and was not eluted.
EXAMPLE 6
10 g Glass beads coated with azide modified polyglutamate were prepared as
in Example 5, immersed in 5 ml of a solution of 10 mg Aspergillus acylase
(650 U) in 5 ml 0.05 M phosphate buffer solution of pH 8.5, and further
treated as described in Example 5. The acylase activity of the immobilized
enzyme was 45 U/g and that of the filtrate was 190 U.
When the beads carrying the immobilized enzyme were packed in a column, and
enzyme reactions were repeated at 37.degree. C for 2 months using the
column, the acylase activity of the immobilized enzyme was scarcely
lowered and the flow rate did not change.
EXAMPLE 7
In the manner of Example 5, 60 g polyglutamate solution was deposited on 15
g alumina beads (100 - 200 mesh) and treated with hydrazine and nitrite.
The azide bearing beads were immersed in a solution of 30 mg jack bean
urease (70 U) in 20 ml 0.05 M phosphate buffer solution of pH 8.5, and
further treated as in Example 5. The urease activities of the immobilized
enzyme and of the filtrate were 5.3 U/g and 2 U, respectively.
When the beads carrying the immobilized enzyme wee stirred in a 200 ml
beaker with a Teflon agitator of 3 cm diameter while an enzyme reaction
was carried out for six hours, the urease activity of the immobilized
enzyme was scarcely lowered, and neither separation nor elution of the
coatings from the beads occurred.
EXAMPLE 8
30 g Glass beads of about 2 mm diameter were stirred with a glass rod in a
mixture of 5 g 3% epichlorohydrin and 2 g 3% 3,9-bis
(3-aminopropyl)-2,4,8,10 tetraoxospiro-[5,5]-undecane until the solvent
was evaporated and the surfaces of the beads were almost dry.
The beads were stirred in 5 g of a mixture of dichloroethane and
tetrachloro ethane containing 5% polymethyl-D-glutamate having an
intrinsic viscosity of 1.8. When enough solvent was evaporated to make the
mixture viscous, it was added little by little to a large amount of
ethanol with stirring. After the addition was completed, the ethanol was
removed, and the glass beads were dried at 100.degree. C for 5 hours.
The glass beads coated with the polyglutamate were treated with hydrazine
and nitrite, and reacted with acylase in the same manner as in Example 6.
The activity of the immobilized acylase was 0.6 U/g. When the beads
carrying the immobilized enzyme were packed in a column and employed for
enzyme reactions, the acylase activity of the column was scarcely lowered
and the flow rate of 10 cm/min did not change.
EXAMPLE 9
6 g 10% Epichlorohydrin was mixed with 2 g 10% 3,9-bis
(3-aminopropyl)-2,4,8,10 tetraoxospiro-[5,5]-undecane. To the mixture, 2 g
of a mixture of dichloroethane and tetrachloroethane containing 10% of
polymethyl-D-glutamate having an intrinsic viscosity of 1.8 was added with
stirring. The liquid then was sealed and allowed to stand at room
temperature for 2 weeks. It became white, turbid, and very viscous.
It was stirred gradually into a large amount of methanol and then
comminuted in a Waring blender (homogenizer) for 1 - 2 minutes. Methanol
was evaporated, and the residue was dried at 100.degree. C for 3 hours.
3 g White porous beads (30 - 100 mesh) produced in this manner were treated
with hydrazine, and subsequently with nitrite, and reacted with acylase in
the same manner as in Example 6. The acylase activity of the immobilized
enzyme was 170 U/g. When the enzyme composition was packed in a column and
employed for enzyme reactions at 37.degree. C for one month, 92% of the
initial acylase activity was retained and no difficulties occurred due to
changes in the flow rate.
EXAMPLE 10
5 g 5% Polyvinyl chloride solution was mixed with 10 g 5%
polymethyl-D-glutamate solution (intrinsic viscosity 1.8). A glass tube, 5
mm in inside diameter and 45 cm long, was coated internally with the
solution. The solvent was evaporated while the tube was rotated, and then
the tube was dried by heating at 80.degree.- 100.degree. C for 5 hours.
Using a small circulating pump, the polymethyl glutamate coating on the
inner wall of the tube was treated with hydrazine and subsequently with
nitrite. 5 mg (10.5 U) Uricase extracted from yeast was dissolved in 3 ml
0.1 M borate buffer solution of pH 8.5, the solution was injected into the
glass tube, and the tube was rotated and shaken slowly at 4.degree. C for
48 hours. The glass tube was washed in a conventional manner. The uricase
activity of the immobilized enzyme was 9.3 U. When the inmobilized enzyme
was employed for reaction with a passing substrate solution at a flow rate
of 1 ml/min for 24 hours, its uricase activity was scarcely lowered and no
elution of the coating occurred.
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