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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to a method for the immobilization of an enzyme by
use of light.
At present, enzymes are extensively utilized for the manufacture of
pharmaceutical products and food products, for the decomposition,
synthesis and determination of various substances and for other purposes.
In enzymatic reactions carried out for these purposes, enzymes immobilized
as by being entrapped within microorganic cells, deposited on carriers,
combined with each other or similarly treated exhibit enhanced activities
for longer lengths of time as compared with enzymes which are used in
unimmobilized forms and are, furthermore easier to handle. Thus, there
have heretofore been proposed numerous methods for immobilizing enzymes by
use of specific reagents or cross-linking agents. Examples of U.S. patents
which disclose these methods are U.S. Pat. No. 3,843,442, U.S. Pat. No.
3,950,222, U.S. Pat. No. 3,985,617, U.S. Pat. No. 3,985,618 and U.S. Pat.
No. 3,933,587.
Besides, a method whereby immobilization of an enzyme is effected by use of
light has been disclosed in "Chemical Abstracts," Vol. 85, page 244,
106024f. This method immobilizes a given enzyme by using an oligomer as a
carrier and subjecting the enzyme to a cross-linking thermal reaction and
a cross-linking photoreaction in the presence of an initiator for thereby
causing the enzyme to be entrapped in the lattice of the resultant gelled
polymer.
An object of the present invention is to provide a method for easily
immobilizing a given enzyme with substantially no inactivation of the
enzyme by use of light.
Another object of this invention is to provide a method for producing an
immobilized enzyme preparation possessing desired mechanical properties.
SUMMARY OF THE INVENTION
To accomplish the objects described above in accordance with the present
invention, there is provided a method for the immobilization of an enzyme
which comprises mixing a given enzyme with an azido compound containing at
least one aromatic group and a water-soluble high molecular compound,
forming the mixture in a desired shape and exposing the formed mixture to
light for thereby causing the enzyme to be bound to the azido group in the
azido compound.
Since the method of this invention causes the enzyme to be bound to the
azido group, the enzyme will not be dissolved out of the enzyme
preparation obtained while the enzyme preparation is in use. Since none of
the treatments involved in the immobilization by the method of the present
invention is performed under any harsh condition, the enzyme undergoes
absolutely no inactivation throughout the entire course of immobilization.
Further, the mechanical properties of the immobilized enzyme preparation
obtained by this method can freely be changed by suitably selecting the
amount and kind of the high molecular compound to be incorporated during
the preparation of the mixture. Thus, an enzyme preparation possessed of
flexibility or an enzyme preparation excelling in permeability to liquids,
for example, can be obtained by making a suitable selection.
DESCRIPTION OF PREFERRED EMBODIMENT
It has heretofore been known in the art to immobilize a given enzyme by
combining the enzyme with a carrier or with another enzyme in some way or
other through the medium of a cross-linking reagent or by entrapping the
enzyme with a carrier.
The conventional method which accomplishes the desired immobilization of an
enzyme by use of a cross-linking reagent, however, has not proved
completely satisfactory because of inactivation of the enzyme during the
treatment for immobilization, insufficient strength of immobilization and
high production cost.
The inventors continued devoted studies with a view to overcoming the
various disadvantages suffered by the conventional immobilized enzymes.
They have consequently made a discovery that a specific azido compound is
caused through the agency of light to react as with the
##STR1##
group, the >CH.sub.2 group, etc. of the carrier or the amino acid which is
one component of the enzyme proteins to give rise to a cross-linkage and
that this formation of the cross-linkage is effective in the
immobilization of an enzyme. The present invention has been accomplished
on the basis of this discovery.
To be specific, the method according to the present invention effects the
manufacture of an immobilized enzyme preparation by mixing and dissolving
the given enzyme with an azido compound and a high molecular compound as a
carrier, then through evaporation forming the resultant mixed solution
into a film or pellets and thereafter exposing the formed mixture to light
for thereby causing the enzyme to be bound to the azido group of the azido
compound.
The azido compound to be used in the present invention is an azido compound
which has at least one aromatic group of the generic formula:
##STR2##
wherein, X, Y and Z each represent a member selected from the group
consisting of halogen atoms, alkyl, alkoxyl and hydroxyl groups and salts
thereof, sulfonic acid group and salts thereof, carboxyl group and salts
thereof, l, m and n each represent an integer having a value of from 0 to
4 and r represents an integer having a value of from 1 to 5.
In the case of azido compounds containing two such aromatic groups, there
are those in which the two aromatic groups are directly connected to each
other and those in which they are connected by means such as of S, O, CO,
NH, CH.dbd.CH--, --CH.sub.2, etc.
The azido compounds satisfying the foregoing requirement are broadly
divided into two types, i.e. aromatic azido compounds and water soluble
high molecular compounds containing an azido group. When a compound of the
former type is used, the reaction proceeds so that the enzyme units (E)
are immobilized to the high molecular compound units (carrier) (C) via the
compound units (B) in a configuration wherein either end of each compound
unit (B) is connected to an enzyme unit (E) or a carrier unit (C) such as
diagrammatically illustrated below, for example.
--(C)--(B)--(E)--(B)--(E)--(B)--(C)--(B)--(E)--
when there is used a compound of the latter type containing an azido group,
since the compound contains a high molecular compound from the beginning,
it is not always required to add a high molecular compound as a carrier
during the preparation of a mixed solution. In this case, the reaction
proceeds so that the enzyme units (E) are bound to the azido groups of the
compound units (A). One example of the reaction is as illustrated below.
##STR3##
In the former reaction, there is a possibility that the azido compound
units will bind the high molecular compound (carrier) units mutually in
the manner of cross-linkage to give rise to a reticular configuration. In
the latter reaction, it is not unlikely that high molecular compound units
each containing an azido group will mutually react with one another and
will consequently be bound to one another, thus giving rise to a reticular
configuration. It is, therefore, quite possible that the enzyme units will
be immovably enclosed in such a reticular configuration even if they are
not allowed to react directly with azido group units.
Of the azido compounds usable for this invention, the aromatic diazido
compounds or bis-azido compounds are represented by the following three
generic formulas:
##STR4##
wherein, l, m and n each represent an integer having the value of from 0
to 4, X, Y and Z each represent one member selected from the class
consisting of halogen atoms, alkyl, alkoxyl and hydroxyl groups and salts
thereof, sulfonic acid group and salts thereof and carboxyl group and
salts thereof and R represents --CH.sub.2 --.sub.p, --CH.dbd.CH--.sub.q
(where, p and q each represent an integer having the value of from 1 to
3), an oxygen atom, a sulfur atom, SO.sub.2 group or NH group.
In the case of 4,4'-diazido-stilbene-2,2'-disulfonic acid, as an example of
the aromatic diazido compound, the formation of the cross-linkage by the
photoreaction proceeds as shown below.
##STR5##
The soluble high molecular compounds containing an azido group are
represented by the following two generic formulas:
##STR6##
wherein, P represents a water soluble high molecular compound moiety
selected from the group consisting of polyvinyl alcohols, cellulose,
novolak resins, agar and scleroproteins (gelatin, collagen, fibroin and
keratin), l represents an integer having the value of from 0 to 4 and r
represents an integer having the value of from 1 to 5.
The water soluble high molecular compounds containing an azido group can
easily be synthesized by the reaction of the water soluble high molecular
compounds and azido compounds which reaction involves the esterification
between the hydroxyl group constituting part of the structures of the high
molecular compounds and the carboxyl group present in the azido compounds.
In the case of polyvinyl alcohol-p-azido-benzoic acid ester as one example
of the high molecular compounds containing an azido group, the formation
of the cross-linkage by the photoreaction is estimated to proceed as shown
below.
##STR7##
wherein x and y are positive integers having respective values such as to
satisfy 100<x+y<2,000
##EQU1##
The enzymes which can be immobilized by the azido compounds described above
include those of the classes of oxidoreductases, transferases, hydrolases,
lyases, isomerases, ligases, etc. To be more specific, examples of
oxidoreductases include glutamate dehydrogenase, lactate dehydrogenase,
glyceraldehyde-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase,
malate dehydrogenase, isocitrate dehydrogenase, alcohol dehydrogenase,
glucose oxidase, D-amino-acid oxidase, L-amino-acid oxidase, xanthine
oxidase, protocatechuate 3,4-oxygenase, lipoxygenase, tyrosinase and
pyridinenucleotide transhydrogenase.
Examples of transferases include pyruvate kinases, creatine kinase and
t-RNA nucleotidyltransferase.
Examples of hydrolases include .alpha.-glucosidase, .beta.-glucosidase,
.alpha.-galactosidase, .beta.-galactosidase, invertase, glucoamylase,
lysozyme, hyaluronidase, urease, acid phosphatase, alkaline phosphatase,
asparaginase, ficin, apyrase, prolidase, trypsin, .alpha.-chymotrypsin,
papain, pepsin, pronase, subtilisin, carboxypeptidase A, carboxypeptidase
B, lipase, ATPase, RNase, acetylcholinesterase and penicillinase.
Examples of lyases include aldolase, citrate synthase, ATP deaminase and
AMP deaminase.
Examples of isomerases include glucose isomerase, glucose-phosphate
isomerase and alanine racemase.
An example of ligase is succinyl-CoA synthetase.
As the carrier, any high molecular compound can be used insofar as it
possesses a
##STR8##
group or a >CH.sub.2 group. Examples of high molecular compounds usable
for this purpose include water-soluble natural high molecular compounds
such as cellulose, agar, scleroproteins (gelatin, collagen, fibroin and
keratin) and water-soluble synthetic high molecular compounds such as
polyvinyl alcohols, polyvinyl pyrrolidone and polyacrylamides.
To obtain the immobilized enzyme by the method of this invention, the
proportions in which the aromatic diazido compound or bis-azido compound,
the enzyme and the carrier are admixed are such that, for the fixed
proportion of about 5% by weight of the compound, the proportion of the
enzyme falls in the range of from about 1 to about 20% by weight and that
of the carrier falls in the range of from about 94 to about 75% by weight.
When a high molecular compound containing an azido group is used as the
azido compound in the preparation of the mixture, since the high molecular
compound functions not merely as a cross-linking agent but also as a
carrier, there is no particular need for using a high molecular compound
specifically designed as a carrier. In this case, the immobilized enzyme
can be advantageously obtained by using about 99 to about 80% by weight of
the high molecular compound containing an azido group and about 1 to about
20% by weight of a given enzyme. The mechanical properties of the
immobilized enzyme preparation to be obtained by the method of this
invention rely upon the properties of the high molecular compound in any
event. It is, therefore, possible to modify the mechanical properties of
the immobilized enzyme preparation to be obtained finally by suitably
decreasing the proportion of the high molecular compound to be added in
the preparation of the mixture and adding the high molecular compound
functioning solely as a carrier in a proportion to make up for the
decrease. In this case, the proportion of the high molecular compound to
be added as the carrier has an upper limit of about 70% by weight.
Depending on the purpose for which the immobilized enzyme is used, the
mixing proportions of the azido compound, the enzyme and the high
molecular compound can be freely changed within their respective ranges
mentioned above. Particularly by changing the kind and the proportion of
addition of the high molecular compound, there can easily be obtained
immobilized enzyme preparations whose mechanical properties such as
permeability to liquids and plasticity are varied as desired.
When collagen is selected as the high molecular compound and is added in a
proportion of about 50%, for example, there is obtained a supple
immobilized enzyme membrane having high plasticity. When keratin is added
as the high molecular compound in a proportion of about 50%, there is
obtained a rigid immobilized enzyme membrane having low plasticity. By
pulverization, this membrane is converted into an immobilized enzyme in
the form of pellets.
When glycerin is added in a proportion of 20 to 30%, for example, besides
the high molecular compound, there is obtained a highly plastic and porous
immobilized enzyme membrane excelling in permeability.
The immobilized enzyme in the form of a film is obtained by dissolving the
azido compound, the enzyme and the high molecular compound in water,
casting the resultant mixed solution on a horizontal plate and evaporating
water from the solution. The immobilized enzyme in the form of pellets is
obtained by spray drying the mixed solution in a hot-air fluidizing zone
maintained at a temperature not so high as to inactivate the enzyme.
The formed immobilized enzyme obtained as described above is then exposed
to light. As the source of this light, there can be used a high-pressure
mercury lamp, a carbon arc, a luminescent lamp, sunlight, or the like.
The duration of the exposure to the light depends upon various factors such
as the kind and concentration of the azido compound, the shape of the
formed immobilized enzyme, the intensity of the light used for the
exposure, etc. The photoreaction caused in the formed mixture by the
agency of light can be considered to be complete when the cumulative
decrease in the azido group content of the formed mixture totals slightly
over 50%. And the cumulative decrease can be easily traced by testing the
formed mixture for infrared absorption at 2100 to 2300 cm.sup.-1 at proper
intervals.
For example, when the mixture formed in the shape of a film 0.05 to 0.2 mm
in thickness is exposed to the light from a 500-watt high-pressure mercury
lamp, a duration of from 10 to 60 minutes suffices for required exposure,
the variation depending somewhat on the type and concentration of the
azido compound in use. A duration of from 10 to 24 hours is required,
however, where the exposure is performed by use of a 40-watt luminescent
lamp.
As for the temperature of the formed mixture during its exposure to light,
the only requirement is that this temperature should not be so high as to
inactivate the enzyme contained in the mixture. Where the enzyme happens
to be of a type readily inactivated by heat, the photoreaction can easily
be caused at amply low temperatures such as +10.degree. to -50.degree. C.,
for example.
The immobilized enzyme preparation thus obtained as a consequence of the
photoreaction can be used similarly to any ordinary enzyme. In the case of
an immobilized enzyme preparation in the shape of a film, effective use
thereof can be obtained by immersing the preparation in a given reaction
solution and agitating the solution. Effective use of an immobilized
enzyme preparation in the shape of pellets is obtained by packing a column
with the pellets and passing a given reaction solution through the column.
Since, in the case of the present invention, the immobilized enzyme
preparation can be produced in the form of a film of any desired shape, an
immobilized enzyme membrane having permeability may be formed in a shape
conforming to the cross section of a reaction cell and placed in a
reaction vessel to partition the interior of the reaction vessel into two
halves. The reaction vessel thus partitioned is usable in an operation
wherein a high molecular substrate is placed in one of the two halves of
the vessel interior and a low molecular substrate produced by the
enzymatic reaction involving the immobilized enzyme present in the
partitioning membrane is obtained in the remaining half of the vessel
interior. Of course, the immobilized enzyme preparation in the form of a
film can be cut up into pellets and used in the form of pellets.
As is evident from the foregoing description, the immobilization of a given
enzyme according to this invention relies on the binding of the azido
group of the azido compound to the enzyme. Thus, the enzyme is retained so
strongly by the azido group that it will not be dissolved out while the
immobilized enzyme preparation is in use in the reaction. Further since
this immobilization is wholly carried out effectively at temperatures
below the inactivating temperature of the enzyme, substantially all the
enzyme that is used in the preparation of the mixture can be immobilized.
By changing the type and the amount of addition of the high molecular
compound also incorporated in the preparation, the resultant immobilized
enzyme preparation can be given desired mechanical properties. Moreover,
the immobilized enzyme preparation can easily be formed into any desired
shape.
Now, the present invention will be described specifically with reference to
examples, which are solely illustrative of and not limitative of the
present invention.
EXAMPLE 1
1 g of commercially available gelatin (having a molecular weight of
100,000) was, as a carrier, placed in 10 ml of water and heated at about
60.degree. C. to dissolve in the water. After the temperature of the
resultant solution was lowered to 30.degree. C., the solution was mixed
with 5 ml of an aqueous 20 mg/ml .beta.-glucosidase solution and 5 ml of
an aqueous 10 mg/ml 4,4'-diazidostilbene-2,2'-disulfonate solution and
agitated to uniformity, with the temperature kept at 30.degree. C. The
resultant solution was cast to a uniform thickness on a titanium plate
having a smooth surface and left to stand overnight at room temperature
(about 20.degree. C.) so as to be dried. Consequently, there was obtained
a thin membrane about 145 mm in length, about 120 mm in width and about
0.06 mm in thickness and about 1.1 g in weight.
Then, this thin membrane was exposed to the light from a 40-W luminescent
lamp at 20.degree. C. for 12 hours, washed with water and again left to
stand on the plate to dry in the air.
The immobilized enzyme preparation thus obtained in the form of a film was
assayed for protein content and tested for enzyme activity. The results
indicate that in the course of the manufacture of the immobilized enzyme
membrane, losses of the carrier and the enzyme were negligibly small. The
weight ratio of the enzyme to the entire membrane was found to be about
9%.
An enzyme reaction was carried out as described below by using the
immobilized enzyme membrane as the catalyst, to determine the yield of the
activity of the membrane. In this reaction,
p-nitrophenyl-.beta.-D-glucopyranoside was used as a substrate and the
activity was determined by measuring the amount of p-nitrophenol liberated
in the absorption at 410 nm.
In 10 ml of water containing 180 mg of
p-nitrophenyl-.beta.-D-glucopyranoside, 20 mg of the immobilized enzyme
membrane (containing 1.8 mg of enzyme) was gently shaken for 60 minutes
under the conditions of 25.degree. C. of temperature and pH 5.7 to induce
a reaction. As the result of this reaction, a yield of the activity of 52%
was obtained. The reason why the yield of the activity became one half of
that of the native enzyme is not that the immobilization brings about the
inactivation of the enzyme, but presumably that the rate at which the
substrate is diffused in the membrane is slow and that the concentration
of the substrate is lower in the solid state phase membrane than in the
aqueous phase.
Further, the membrane was washed with water, dried in air and put to use
again. When the membrane was tested for enzyme activity, there was
obtained a yield of the activity of 48%. These results indicate that the
immobilized enzyme preparation manufactured by the present invention
retains a high stable activity.
For the purpose of determining the amount of enzyme dissolved out during
the reaction, the membrane was removed from the solution and the remaining
solution was similarly tested for enzyme activity. The solution showed no
discernible enzyme activity in either of the two tests. The results
indicate that in the immobilized enzyme membrane manufactured by the
present invention, the enzyme is strongly immobilized.
The "yield of the activity" as used herein means the relative activity per
unit weight of the enzyme immobilized, with the activity per unit weight
of the initially incorporated enzyme taken as 100.
EXAMPLE 2
A thin membrane prepared by following the procedure of Example 1 was
exposed to the light from a 500-W high-pressure mercury lamp at 20.degree.
C. for 10 minutes, then washed with water and dried. The resultant
immobilized enzyme membrane was tested for yield of the activity. The
yield was found to be about 54%. The yield of the activity was about 49%
when the duration of the exposure to the light was increased to 60
minutes. Absolutely no dissolution of the enzyme was observed in either of
the tests.
EXAMPLE 3
The procedure of Example 1 was repeated, except .alpha.-glucosidase and
.beta.-galactosidase were used as enzymes, to afford immobilized enzyme
preparations each in the form of a film.
The film incorporating immobilized .alpha.-glucosidase and the film
incorporating immobilized .beta.-galactosidase were subjected to enzyme
reaction, using p-nitrophenyl-.alpha.-D-glucopyranoside and
p-nitrophenyl-.beta.-D-galatopyranoside respectively as substrates, for 60
minutes under the conditions of 25.degree. C. of temperature and pH 5.7,
to determine their enzyme activities. The yield of the activity was found
to be 47% for the former film and 49% for the latter film. Absolutely no
dissolution of enzyme was recognized in either of the films.
EXAMPLE 4
In 5 ml of water heated to about 60.degree. C., 0.5 g of gelatin was
dissolved. The resultant solution was cooled to 30.degree. C. This aqueous
solution was mixed with 5 ml of an aqueous solution containing 0.5 g of
polyvinyl alcohol (having a molecular weight of 60000). From this point
onward, the procedure of Example 1 was repeated: The mixed aqueous
solution was mixed with the same aqueous solution of .beta.-glucosidase
and 4,4'-diazidostilbene-2,2'-disulfonate, followed by thorough agitation
to uniformity. The resultant mixture was then formed into a membrane and
exposed to the light to afford an immobilized enzyme membrane. By
repeating the procedure but using .beta.-galactosidase instead of
.beta.-glucosidase, another immobilized enzyme membrane was similarly
obtained.
The membrane incorporating immobilized .beta.-glucosidase and the membrane
incorporating immobilized .beta.-galactosidase were subjected to enzymatic
reaction, using p-nitrophenyl-.beta.-D-glucopyranoside and
p-nitrophenyl-.beta.-D-galactopyranoside respectively as substrates, for
60 minutes under the conditions of 25.degree. C. of temperature and pH
5.7, to determine their enzyme activities. The yield of the activity was
found to be 48% for the former membrane and 45% for the latter membrane.
Absolutely no dissolution of enzyme was recognized in either of the tests.
EXAMPLE 5
The immobilized enzyme membranes prepared in Example 4 were retained at
room temperature (about 20.degree. C.) for 50 days. Thereafter, they were
tested for enzyme activities similarly to Example 4. The yield of activity
was found to be 45% for the membrane incorporating immobilized
.beta.-glucosidase and 41% for the membrane incorporating immobilized
.beta.-galactosidase. Absolutely no dissolution of enzyme was recognized
in either of the tests.
EXAMPLE 6
A polyvinyl alcohol (having a molecular weight of 60000) had its hydroxyl
group partially esterified with p-azido-benzoic acid to an extent of being
not completely deprived of water solubility. Two 10-ml portions of an
aqueous solution each containing 1 g of the partial esterification product
were mixed with 5 ml of an aqueous solution containing 50 mg of
.beta.-glucosidase and 5 ml of an aqueous solution containing 50 mg of
.beta.-galactosidase respectively, followed by thorough agitation to
uniformity. The resultant mixtures were formed into membranes as in
Example 1. These membranes were exposed to light as in Example 1, washed
with water and then tested for enzyme activity. The yield of the activity
was found to be 43% for the former membrane and 40% for the latter
membrane. Absolutely no dissolution of enzyme was recognized in either of
the tests.
EXAMPLE 7
To 8 ml of an aqueous solution containing 1 g of the same polyvinyl
alcohol-p-azido-benzoic acid ester as prepared in Example 6, there was
added 4 ml of an aqueous solution which contained 0.5 g of gelatin
dissolved at 60.degree. C. and which was thereafter cooled to 30.degree.
C. The resultant mixed solution was mixed with 3 ml of an aqueous solution
having dissolved therein 50 mg of .alpha.-glucosidase, followed by
thorough agitation to uniformity. This procedure was repeated, except
there was used 3 ml of an aqueous solution having dissolved therein 50 mg
of .beta.-galactosidase. These mixtures were formed each in the form of a
membrane, exposed to a light and washed with water similarly to Example 1.
The resultant immobilized enzyme membranes were tested for enzyme activity
similarly to Example 2.
The yield of the activity was found to be 39% for the former membrane and
38% for the latter membrane. Absolutely no dissolution of enzyme was
recognized in either of the tests.
EXAMPLE 8
A polyvinyl alcohol (having a molecular weight of 60000) had its hydroxyl
group partially esterified with p-azido-cinnamic acid to an extent of
being not completely deprived of water solubility. 10 ml of an aqueous
solution containing 1 g of the partial esterification product was mixed
with 5 ml of an aqueous solution containing 50 mg of .beta.-galactosidase,
followed by thorough agitation to uniformity. The resultant mixture was
formed into a membrane as in Example 1. The membrane was exposed to the
light from a 500-W high-pressure mercury lamp at 20.degree. C. for 20
minutes, washed with water and then tested for enzyme activity. The yield
of activity was found to be 39%. Absolutely no dissolution of enzyme was
recognized in this test.
* * * * *
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Description |
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