Note: Descriptions are shown in the official language in which they were submitted.
1128917
SUPPORT MATRICES FOR IMMOBILIZED ENZYMES
BACKGROUND OF THE INVENTION
It is known that enzymes, which are proteinaceous in nature and
which are commonly water soluble, comprise biological catalysts which serve
to regulate many and varied chemical reactions which occur in living organ-
isms. The enzymes may also be isolated and used in analytical, medical
; 5 and industrial applications. For example, they find use in industrial
; applications in the preparation of food products such as cheese or bread
, as well as being used in the preparation of alcoholic beverages. Some
,:,
specific uses in industry may be found in the use of enzymes such as in the
resolution of amino acids; in the modification of penicillin to form various
substrates thereof; the use of various proteases in cheese making, meat
- tenderizing, detergent formulations, leather manufacture and as digestive
aids; the use of carbohydrases in starch hydrolysis, sucrose inversion,
, .
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-2-
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~Z89~7
glucose isomerization, etc.; the use of nucleases in flavor control; or
the use of oxidases in oxidation prevention and in the color control of
food products. These uses as well as many others have been well delin-
eated in the literature.
As hereinbefore set forth, inasmuch as enzymes are co~monly water
soluble as well as being generally unstable and readily deactivated, they
are also difficult either to remove from the solutions in which they are
utilized for subsequent reuse or it is difficult to maintain their cata-
lytic activity for a relatively extended period of time. The aforementioned
difficulties will, of course, lead to an increase cost in the use of en-
zymes for commercial purposes due to the necessity for frequent replacement
of the enzyme, this replacement being usually necessary with each applica-
tion. To counteract the high cost of replacement, it has been suggested
to immobilize or insolubilize the enzymes prior to the use thereof. By
immobilizing the enzymes through various systems hereinafter set forth in
greater detail, it is possible to stabilize the enzymes in a relative
manner and, therefore, to permit the reuse of the enzyme which may otherwise
undergo deactivation or be lost in the reaction medium. Such immobilized
or insolubilized enzymes may be employed in various reactor systems such
as in packed columns, stirred tank reactors, etc., depending upon the nature
of the substrate which is utilized therein. In general, the immobilization
of the enzymes provides a more favorable or broader environmental and
structural stability, a minimum of effluent problems and materials handling
as well as the possibility of upgrading the activity of the enzyme itself.
As hereinbefore set forth, several general methods, as well as
many modifications thereof, have been described by which the immobiliza-
tion of enzymes may be effected. One general method is to adsorb the
-3-
~128917
enzyme at a solid surface as, for example, when an enzyme such as amino
acid acylase is adsorbed on a cellulosic derivative such as DEAE-cellulose;
papain or ribonuclease is adsorbed on porous glassi catalase is adsorbed
on charcoal; trypsin is adsorbed on quartz glass or cellulose, chymotryp-
sin is adsorbed on kaolinite, etc. Another general method is to trap anenzyme in a gel lattice such as glucose oxidase, urease, papain, etc., being
entrapped in a polyacrylamide gel; acetyl cholinesterase being entrapped
in a starch gel or a silicone polymer; glutamic-pyruvic transaminase being
entrapped in a polyamide or cellulose acetate gel, etc. A further general
method is a cross-linking by means of bifunctional reagents and may be
effected in combination with either of the aforementioned general methods
of immobilization. When utilizing this method, bifunctional or polyfunc-
tional reagents which may induce intermolecular cross-linking will coval-
ently bind the enzymes to each other as well as on a solid support. This
method may be exemplified by the use of glutaraldehyde or bisdiazobenzi-
dine-~,2'-disulfonic acid to bind an enzyme such as papain on a solid
support, etc. A still further method of immobilizing an enzyme comprises
! the method of a covalent binding in which enzymes such as glucoamylase,
trypsin, papain, pronase, amylase, glucose oxidase, pepsin, rennin,
fungal protease, lactase, etc., are immobilized by covalent attachment
to a polymeric material which is attached by various means to an organic
or inorganic solid porous support. This method may also be combined with
the aforesaid immobilization procedures.
The above enumerated methods of immobilizing enzymes all possess
some drawbacks which detract from their use in industrial processes. For
example, when an enzyme is directly adsorbed on the surface of a support,
the binding forces which result between the enzyme and the carrier support
-A-
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~28917
are often quite weak, although some prior art has indicated that relatively
stable conjugates of this type have been obtained when the pore size of the
support and the spin diameter of the enzyme are correlated. However, in
such cases it is specified that the pore size of the support cannot exceed
a diameter of about 1000 Angstroms. In view of this weak bond, the enzyme
is often readily desorbed in the presence of solutions of the substrate
being processed. In addition to this, the enzyme may be partially or ex-
tensively deactivated due to its lack of mobility or due to interaction
between the support and the active site of the enzyme. Another process
which may be employed is the entrapment of enzymes in gel lattices which
can be effected by polymerizing an aqueous solution or emulsion containing
the monomeric form of the polymer and the enzyme or by incorporating the
enzyme into the preformed polymer by various techniques, often in the
presence of a cross-linking agent. While this method of immobilizing
enzymes has an advantage in that the reaction conditions utilized to effect
the entrapment are usually mild so that often there is little alteration
or deactivation of the enzyme, it also has disadvantages in that the conju-
gate has poor mechanical strength, which results in compacting when used
,~ in columns in continuous flow systems, with a concomitant plugging of the
column. Such systems also have rather wide variations in pore size thus
leading to some pore sizes which are large enough to permit the loss of
enzyme. In addition, some pore sizes may be sufficîently small so that
large diffusional barriers to the transport of the substrate and product
will lead to reaction retardation, this being especially true when using
a high molecular weight substrate. The disadvantages which are present
when immobilizing an enzyme by intermolecular cross-linkage, as already
noted, are due to the lack of mobility with resulting deactivation because
.5~
~128917
of inability of the enzyme to assume the natural configuration necessary
for maximum activity, particularly when the active site is involved in
the binding process.
Covalent binding methods have found wide applications and may be
used either as the sole immobilization technique or as an integral part of
many of the methods already described in which cross-linking reactions are
employed. This method is often used to bind the enzyme as well as the
support through a bifunctional intermediary molecule in which the func-
tional groups of the molecule, such as, for example, gamma-aminopropyltri-
ethoxysi;lane, are capable of reacting with functional moieties present in
both the enzyme and either an organic or inorganic porous support. A wide
variety of reagents and supports has been employed in this manner and the
method has the advantage of providing strong covalent bonds throughout the
conjugate product as well as great activity in many cases. The covalent
linkage of the enzyme to the carrier must be accomplished through functional
groups on the enzyme which are non-essential for its catalytic activity
such as free amino groups, carboxyl groups, hydroxyl groups, phenolic
groups, sulfhydryl groups, etc. These functional groups will also react
with a wide variety of other functional groups such as an aldehydo, iso-
cyanato, acyl, diazo, azido, anhydro activated ester, etc., to produce co-
valent bonds. Nevertheless, this method also often has many disadvantages
involving costly reactants and solvents, as well as specialized and costly
porous supports and cumbersome multi-step procedures, which render the
method of preparation uneconomical for commercial application.
The prior art is therefore replete with various methods for im-
mobilizing enzymes which, however, in various ways fail to meet the require-
ments of economical industrial use. However, as will hereinafter be discussed
11289~7
in greater detail, none of the prior art compositions comprise the composi-
tion of matter of the present invention which constitutes an inorganic
porous support containing a copolymer, forn~d in situ from a polyfunctional
monomer, a low molecular weight polymer~ a polymer hydrolysate, or a pre-
formed polymer, of natural or synthetic origin by reaction with a bifunc-
tional monomer, the copolymer formed being substantially entrapped within
the pores of said support, and which contains terminally functionalized,
pendent groups extending therefrom; the enzyme being covalently bound to
the active moieties at the terminal reactive portions of the pendent groups,
thus permitting the freedom of movement which will enable the enzyme to
exercise maximum activity. A variable portion of the enzyme will also be
adsorbed upon the matrix, but this will be recognized as an unavoidable
consequence of almost all immobilization procedures involving porous inor-
ganic supports and is not to be considered a crucial aspect of this inven-
tion. Furthermore, the bond between the inorganic support and the organiccopolymer which has been prepared in situ in the pores of the support is
not covalent but rather physico-chemical and mechanical in nature and the
inorganic-organic matrix so produced presents high stability and resistance
to disruption. As further examples of prior art, U.S. Patent No. 3,556,945
relates to enzyme composites in which the enzyme is adsorbed directly to an
inorganic carrier such as glass. U.S. Patent No. 3,519,538 is concerned
with enzyme composites in which the enzymes are chemically coupled by means
of an intermediary silane coupling agent to an inorganic carrier. In
similar fashion, U.S. Patent No. 3,783,101 also utilizes an organosilane
composite as a binding agent, the enzyme being covalently coupled to a
glass carrier by means of an intermediate silane coupling agent, the silicon
portion of the coupling agent being attached to the carrier while the organic
--7--
l~Z8917
portion of the coupling agent is coupled to the enzyme, the composition
containing a metal oxide on the surface of the carrier disposed between
the carrier and the silicon portion of the coupling agent. In U.S. Patent
No. 3,821,083 a water-insoluble polymer such as polyacrolein is deposited
- 5 on an inorganic carrier and an enzyme is then covalently linked to the alde-
hyde groups of the polymer. However, according to most of the examples
set forth in this patent, it is necessary to first hydrolyze the composite
- prior to the deposition of the enzyme on the polymer. Additionally the
product which is obtained by the method of this patent suffers a number of
disadvantages in that it first requires either the deposition, or initially
the formation, of the desired polymer in an organic medium followed by its
deposition on the inorganic carrier with a subsequent clean-up operation
involving distillation to remove the organic medium. In addition to this,
in another method set forth in this reference, an additional hydrolytic
reaction is required in order to release the aldehyde groups from the initial
acetal configuration in which they occurred in the polymer. Inasmuch as
these aldehyde moieties are attached directly to the backbone of the polymer,
the enzyme is also held adjacent to the surface of the polymer inasmuch as
it is separated from the surface of the polymer by only one carbon atom of
the reacting aldehyde group and, therefore, the enzyme is obviously sub-
~ected to the physico-chemical influences of the polymer as well as being
relatively immobilized and inhibited from assuming its optimum configuration.
Another prior art patent, namely, U.S. Patent No. 3,705,084 discloses a
macroporous enzyme reactor in which an enzyme is adsorbed on the polymeric
surface of a macroporous reactor core and thereafter is cross-linked in
place. By cross-linking the enzymes on the polymeric surface after adsorp-
tion thereof, the enzyme is further immobilized in part and cannot act
--8-
1128917
freely as in its native state as a catalyst. The cross-linkage of enzymes
in effect links them together, thereby preventing a free movement of the
enzyme and decreases the mobility of the enzyme which is a necessary pre-
requlsite for maximum activity.
U.S. Patent No. 3,654,083 discloses a water-soluble enzyme con-
jugate which is prepared from an organic water-soluble support to which
the enzyme is cross-linked and whose utility is limited only to cleaning
compositions and pharmaceutical ointments. However, this enzyme composition
also suffers from the disadvantages of the close proximity and interlocking
of the enzyme and support, as well as the poor mechanical strength which
is generally exhibited by enzyme conjugates based on organic polymeric
supports.
U.S. Patent No. 3,796,634 also discloses an immobilized biologi-
cally active enzyme which differs to a considerable degree from the immobi-
lized enzyme conjugates of the present invention. The enzyme conjugate of
this patent consists of an inorganic support comprising colloidal particles
possessing a particle size of from 50 to 20,000 Angstroms with a polyethyl-
eneimine, the latter being cross-linked with glutaraldehyde ~o attach the
cross-linked polymer so formed as a monolayer on the surface of the colloidal
particles, followed by adsorption of the enzyme directly onto this mono-
layer. Following this, the enzyme which is adsorbed as a monolayer on the
surface of the colloidal particles is then cross-linked with additional
glutaraldehyde to other adsorbed enzyme molecules to prevent them from
being readily desorbed while in use. There is no indication of any coval-
ent binding between enzyme and polymer matrix as is present in the present
invention. By the enzyme molecules being cross-linked together on the
surface of the support, this conjugate, therefore, is subjected to deac-
tivation by both the cross-linking reaction and by the electronic and
g_
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~lZ8917
steric effects of the surface, said enzyme possessing limited mob11ity.
Inasmuch as the product of this patent is colloidal in nature, it also
possesses a very limited utility for scale-up to commercial operation,
since it cannot be used in a continuous flow system such as a packed column
because it would either be carried along and out of the system in the flow-
ing liquid stream or, if a restraining membrane should be employed, the
particles would soon become packed against the barrier to for~ an imper-
vious layer. In addition, such a colloidal product could not readily be
utilized in a fluidized bed apparatus, thereby limiting the chief utility
to a batch type reactor such as a stirred tank type reactor from which it
would have to be separated by centrifugation upon each use cycle. In
contrast to this, the immobili~ed enzyme conjugates of the present inven-
tion may be employed in a wide variety of batch or continuous type reactors
and therefore are much more versatile with regard to their modes of appli-
cation.
In addition, another prior art reference United States Patent3,959,080 relates to a carrier matrix for immobili2ing biochemical effec-
tive substances. However, the matrix which is produced according to this
; reference constitutes the product derived from the reaction of an organic
polymer containing cross-linkable acid hydrazide or acid azide groups with
a bifunctional cross-linking agent such as glutaraldehyde. However, this
matrix also suffers from the relatively poor mechanical stability and
other deficiencies which are characteristic of organic enzyme supports as
well as the relatively complex organic reactions employed in preparing
such polymeric hydrazides, etc.
As will hereinafter be shown in greater detail, the organic-
inorganic matrix of the present invention will provide a support to which
-10-
llZ8917
an enzyme may be covalently bound to provide a catalytic composite which
will maintain its activity and stability over a relatively long period of
use.
SPECIFICATION
This invention relates to compositions of matter comprising sup-
port matrices for immobilized enzymes. More specifically. the invention
is concerned with support matrices consisting of an organic-~norganic com-
posite in which the inorganic support material is combined with an organic
copolymer prepared in situ and substantially entrapped within the pores
of the inorganic support. The copolymer is formed by the reaction of
- aminopolystyrene with a sufficient excess of a bifunctional monomer con-
taining suitable reactive moieties to provide a copolymeric product con-
taining-terminally functionalized pendent groups capable of co~alently
binding enzymes at the terminal reactive portions thereof. In~addition,
the 1nvention is also concerned with a process for preparing these matrices.
As hereinbefore set forth, the use of enzymes in analytical,
medical or industrial applications may be greatly enhanced if said enzymes
are in an immobilized condition, that is, said enzymes, by being in combin-
ation with other solids materials, are themselves in such a condition
whereby they are not water soluble and therefore they may be subjected
to repeated use while maintaining the catalytic activity of said enzyme.
In order to be present in an immobilized state, the enzymes must be bound
in some manner to a water insoluble carrier, thereby being co~mercially
usable ~n an aqueous insoluble state.
It is therefore an object of this invention to provide novel
compositions of matter in which enzymes may be covalently bound in an
immobilized state.
'~'
-11-
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~28917
A further object of this invention is to provide a process for
preparing combined inorganic-organic support matrices which are utilized
for covalently binding an enzyme to the functionalized pendent groups at
the reactive terminal portions thereof.
In one aspect an embodiment of this invention resides in an
organic-inorganic matrix comprising a porous, inorganic, water-insoluble
solid support in combination with a copolymeric material resulting from
the reaction of aminopolystyrene and a bifunctional monomer~
- A further embodiment of this invention is found in a method for
preparing an organic-inorganic matrix by depositing a salt of aminopoly-
styrene on a sol~d support from an aqueous solution at a pH less than 7 and
thereafter reacting the resultant aminopolystyrene-solid support composite
with a bifunctional monomer to form the desired organic-inorganic matrix.
A specific embodiment of this invention is found in an organic-
inorganic matrix which comprises gamma-alumina having combined therewith a
copolymer resulting from the reaction of aminopolystyrene and an excess of
glutaraldehyde.
Another specific embodiment of this invention is found in a
process for preparing an organic-inorganic matrix which comprises depositing
; 20 the hydrochloric acid salt of aminopolystyrene on gamma-alumina from an
aqueous solution at a p~ in the range of from about 1 to about 4, there-
after reacting the resultant aminopolystyrene-gamma-alumina composite with
an excess of glutaraldehyde, and recovering the resultant organic-inorganic
matrix.
'
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1128917
Another specific embodiment of this invention resides in a pro-
cess for the preparation of an organic-inorganic matrix which comprises treat-
ing a solid porous, inorganic, water-insoluble support with a styrene monomer,
polymerizing the styrene-solid supp~rt composite at polymerizing conditions,
nitrating the resultant polystyrene-solid support composite, reducing the
nitrated polystyrene-solid support composite, and thereafter reacting the
resultant aminopolystyrene-solid support composite with a bifunctional monomer
to form the desired organic-inorganic matrix.
Other objects and embodiments will be found in the following
futher detailed description of the present invention.
As hereinbefore set forth the present invention is concerned with
support matrices which are used to immobilize enzymes, said matrices com-
prising an organic-inorganic composite consisting of an inorganic support
- material of the type hereinafter set forthjn greater detail combined with
- 15 a copolymeric organic material which, in the case of a porous support, is
substantially entrapped in the pores of said porous support. The copoly-
meric composite will contain pendent groups extending therefrom, said
pendent groups containing terminally positioned functional moieties which
will enable an enzyme to be covalently bound to said group at the reactive
terminal portions thereof. Furthermore, the invention is also concerned
with a process for preparing these support matrices using relatively inex-
pensive reactants as well as util~zing more simple steps in theprocedure
for preparing said compositions. In addition, the mechanical strength
and stabllity of enzyme conjugates which result from the covalent binding
of enzymes to these support matrices will be greater than that which is
possessed by the immobilized enzymes of the prior art. Therefore, it will
be readily apparent that the compositions of matter of the present inven-
tion possesses economical advantages which are useful for inàustrial
applications.
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llZ8917
Examples of inorganic supports which may be utilized as one
component of the support matrices of the present invention will consist
of a wide variety of materials including porous supports such as alum;na
which possess pore diameters ranging from 100 Angstroms up to 55,000
Angstroms and which also possess an Apparent 8ulk Density (ABD~ in the
range of from 0.1 to 0.6. The surface area of the particular inorganic
porous support will also vary over a relatively wide range, said range being
from 1 to 500 m2/gm, the preferred range of surface area being from 5 to
400 m2/gm. The configuration of the inorganic porous support material will
vary, depending upon the particular type of support which is utilized.
For example, the support material may be in spherical form, particulate
form ranging from fine particles to macrospheres, as a ceramic monolith which
may or may not be coated with a porous inorganic oxide, a me~brane, ceramic
fibers, alone or woven into a cloth, silica, mixtures of metallic oxides,
sand particles, zeolites or mica. The particle size may also vary over a
wide range, again depending upon the particular type of support which is em-
ployed and also upon the substrate and the type of installation in which
the enzyme conjugate is to be used. For example, if the support is in
spherical form, the spheres may range in size from 0.25 to 6.35 mm in
diameter, the preferred size ranging from 0.79 to 3.17 mm in diameter.
When the support is in particulate form, the particle size may also range
between the same limits. In terms of U.S. standard mesh sizes, such
part~cles may range from 2.5 to 100 mesh, with 10-40 mesh sizes preferred.
Likewise, if the support is in the shape of ceramic fibers, the fibers may
range from 0.5 to 20 microns in diameter or, if in the form of a membrane,
the membrane may comprise a ceramic material which is cast into a thin sheet.
It is to be understood that the aforementioned types of support configuration
and size of the various supports are given merely for purposes of illustration,
and it is not intended that the present invention be necessarily limited
thereto.
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l~Z8917
It is also contemplated that the porous support materials may be
coated with various oxides of the type hereinbefore set forth, or consists
of mixtures thereof, or may have incorporated therein various other inor-
ganic materials such as boron phosphate, these inorganic materials impart-
ing special properties to the support material. A particularly useful
form of support will constitute a ceramic body which may have the type
of porostiy herein described for materials of the present invention or it
may be honeycombed with connecting macro size channels throughout, such
materials being commonly known as monoliths, and which may be coated with
various types of porous alumina, zirconia or titanium oxide. The use
of such a type of support has the particular advantage of permitting the
free flow of highly viscous substrates which are often encountered in
commercial enzyme catalyzed reactions. One component of the organic por-
tion of the support matrix comprises an aminopolystryene whil~ the other
component of the organic portion comprises a bifunctional monomer. ~he
bifunctional monomer reactant is present in sufficient excess as needed
to produce pendent terminally functionalized groups, said bifunctional
- monomer being present in a range of from 2 to 50 moles relative to
the reactive moieties of the support composite, the preferred range
being from 4 to 25 moles of excess.
The functional groups which are present on the bifunctional mono-
mer will comprise well-known reactive moieties capable of bonding readily
with amino groups such as carbonyl, acyl or isocyanato, moieties. The
reactive groups of the bifunctional compounds are preferably, but not
necessarily, separated by chains containing from 4 to 10 carbon atoms.
The reactive moieties of the bifunctional compounds are therefore
capable of covalently bonding with both the aminopolystyrene component
of the support matrix and subsequently, after washing out unreacted
-15-
llZ8917
materials, also with the amino groups of the enzyme which is to be added
in a subsequent step, said enzyme being then covalently bound to the re~
active functional group at the terminal portion of the pendent chain. After
addition of the enzyme to this composition, a relativley stable enzyme con-
jugate will be produced which possesses high activity and high stability.
The unreacted enzyme can also be recovered for reuse. Due to the large
excess of intermediate, or spacer bifunctional monomeric molecules which are
used, the matrix will contain pendent groups comprising the spacer molecules,
said molecules extending from the matrix and having reactive moieties available at
the terminal portions thereof which are capable of reacting with and binding
the enzyme to the aforesaid spacer molecules via covalent bonds. Therefore,
it is readily apparent that a suitable organic-inorganic matrix which is
app1icable in binding enzymes will be formed, provided that a large enough
excess of the bifunctional molecule is used to provide reactive pendent
- 15 groups which are capable of subsequently reacting with the enzyme to be
immob11ized. By utilizing these functional pendent gro~ps as a binding
s1te for the enzymes, it will permit the enzymes to have a greater mobility
and thus permit the catalytic activity of the enzyme to remain at a high
level for a relatively longer period of time than will be attained when the
enzyme has been immobilized by any of the other methods such as entrapment
in a gel lattice, adsorption on a solid surface or cross-linkage of the
enzyme with ad~acent enzyme molecules by means of bifunctional reagents,
etc. Not all formulations, however, will produce equivalent results in
tenms of stability or activity.
, ~
Examples of enzymes which may be immobilized by a covalent bond-
ing reaction and which contain an amino group capable of reacting with an
aldehydric, isocyanato, acyl or ester moiety of the pendent group which
is attached to a polymeric material substantially entrapped in the pores of
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li28917
a porous support material will include trypsin, papain, hexokinase, beta-
galactosidase (lactase), ficin, bromelain, lactate dehydrogenase, gluco-
amylase, chymotrypsin, pronase, glucose, isomerase, acylase, invertase,
amylase, glucose oxidase, pepsin, rennin, protease, xylanace and cellulase.
In general any enzyme whose active site is not involved in the coval-
ent bonding can be used although not necessarily with equivalent results.
While the aforementioned discussion was centered about pendent groups which
contain as a functional moiety thereon an aldehydic or isocyanato group,
it is also contemplated within the scope of this invention that the pendent
group can contain other functional moieties capable of reaction with car-
boxyl, sulfhydryl or other moieties usually present in enzymes. However,
the covalent bonding of enzymes containing these other moieties with other
pendent groups may not necessarily be effected with equivalent results and
may also involve appreciably greater coSts in preparing intermediates. It
is to be understood that the aforementioned listing of porous solid supports,
monomers, hydrolysates, polymers and enzymes are only representative of the
various classes of compounds which may be used, and that the present inven-
tion is not necessarily limited thereto.
The preparation of the compositions of matter of the present
invention is preferably effected in a batch type operation. In a pre-
ferred method of preparation, the inorganic support material will be treated
with a solution, preferably aqueous in nature, of a salt of aminopolysty~ene,
the aqueous solution being maintained at a pH less than 7 and preferably
in a range of 1 to 4. Examples of salts of aminopolystyrene which may
be employed will include the hydrochloric acid salt, the sulfuric acid
salt, the nitric acid salt and the phosphoric acid salt of aminopoly-
styrene. The pH of the aqueous solution is maintained at the desired
level by the addition of an acid such as those hereinbefore set forth.
-17-
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~28917
- Upon completion of the addltion of the acid salt of the aminopolystyrene which
in a preferred embodiment of the invention is effected at ambient temperature
and atmospheric pressure, the mixture is preferably placed under vacuum
for a period of time which may range from 0.5 up to 4 hours or more in
duration.. Upon completion of the reaction time, the unadsorbed solution
is removed and the treatedsupport allowed to air dry. Thereafter the
- organic-inorganic compositeiscontacted with a sufficiently ~arge excess
of a bifunctional monomer of from 2 to 50 moles proportion relative to
the amine content of the initial aminopolystyrene to provide pendent groups
extending from the resultant copolymer, said pendent groups containing un-
reacted terminal functional moieties. The bifunctional monomer is also
preferably added in an~aqueous solution which, after reaction with the
aminopolystyrene, is removed and the resultant matrix washed to separate
any bifunctional monomer which may still be present. The procedure may
be conducted in a temperature range of from 4 to 60C., preferably from
20 to 25C.
In another preferred method of preparation, an emulsion system
is prepared consisting of styrene a1Ong with additives such as sodium hy-
drogen phosphate or sodium lauryl sulfate in an aqueous solution. The
; 20 solid support which may be in a form hereinbefore set forth in greater
'~ detail such as pellets, beads or monoliths, is placed in the solution
and adsorption of the emulsion solution is allowed to proceed for a period
of time which may range from 0.5 up to 2 hours in duration, said adsorption
being preferably effected at subatmospheric pressures, usually in a vaccum.
Upon completion of the adsorption period the mixture is then heated to a
temperature which may range from 50 to 100C. for a period of time utiliz-
ing a polymerization catalyst such as potassium persulfate. After allowing
the polymerization of the styrene to proceed for a period of time which
-18-
1~289~7
:`
may range from 2 to 4 hours in duration, the heating is discontinued and
the solid, composite support containing the polymerized styrene is recovered
from the aqueous solution, washed with distilled water and a solvent such
as acetone or benzene to remove any unreacted styrene. Alternatively, the
styrene may be polymerized without resorting to an emulsion type of polym-
; erization. When this method of preparation is effected the support
is added to only the liquid styrene monomer and the styrene allowed
.,
to adsorb preferably under vacuum for a predetermined period of time.
; At the end of this adsorption period, water and a catalyst such as
potassium persulfate is added, the mixture is then heated to the
aforesaid temperature range and the polymerization is allowed to
proceed for a predetermined period of time. At the end of the polym-
erization period, the polystryene-solid support composite is recov-
ered in a manner similar to that hereinbefore set forth.
Nitration of the polystyrene-solid support composite is then
effected by adding the composite to a nitrating agent such as nitric
acid, and preferably 90X fuming nitric acid. The addition is effected
at a relatively slow rate while maintaining the nitric acid preferably
at subambient temperatures ranging from 0 to 10C. by means of ex-
2a ternal cooling means such as an ice bath or cooling coils. However
such low temperatures are not critical to the success of the nitration
reaction and higher temperatures may be employed, if so desired. The
nitration of the polystryene-solid support composite is allowed to
proceed for a period of time which may range from 1 to 4 hours or more fol-
lowing which the nitrated polystyrene-solid support composite is re-
covered and again wahsed with water and a solvent.
Thereafter the nitropolystyrene-solid support composite is
then added to an aqueous solution containing a reducing agent such as
-1 9-
~Z89~7
sodium dithionite following which the solution is heated to a tempera-
ture in the range of 50 to 1~0C. and maintained thereat for a period
- of time which may range from 0.5 to 2 hours. At the end of this period
of time, the aminopolystyrene-support matrix is recovered, washed, dried
under vacuum and preferably maintained under a nitrogen blanket. To
prepare the desired support matrix which will contain pendent groups
to which an enzyme may be immobilized, the aminopolystyrene-solid sup-
port matrix is then contacted with a sufficiently large excess of bi-
functional monomer of from 2 to 50 mole proportions relative to the
: 10 amine groups of the aminopolystyrene which will react therewith to
provide pendent groups extending from the resulting copolymer which
contains unreacted terminal functional moieties. This bifunctional
monomer is added preferably in an aqueous solution whereby the co-
polymer which is formed will be substantially entrapped in the pores
of the inorganic support. The support matrix is then washed with dis-
tilled water to remove any unreacted bifunctional monomer.
As hereinbefore set forth, the use of an excess of the bifunc-
tional monomer inthe preferred methods of preparation will result in
pendent groups extending from the matrix which contain unreacted terminal
functional moieties. The unreacted functional moieties are then avail-
able for covalent binding to the enzyme, which is added to the matrix,
again usually in an aqueous solution. After removal of the unreacted
materials by conventional means such as washing, the enzyme which is
covalently bound to the pendent functionalized groups rema~ns attached
at the terminal portions thereof. It is therefore readily apparent
- that the entire immobilized procedure can be conducted in a simple and
~nexpensive manner, for example, in a column packed with the inorganic
supports, utilizing an aqueous or inexpensive solvent media. The pro-
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~Z8917
cedure is effected by utilizing a minimal of operating steps and, in addi-
tion, permitting a ready recovery of the excess reactants, unbound enzyme
and finished composition of matter, the excess reactants and unbound enzymes
being available for reuse thereof.
It is also contemplated within the scope of this invention that
the formation of the finished composition of matter may also be effected
in a continuous manner of operation. One method which may be employed is
~- effected by placing a quantity of the solid support in an appropriate
apparatus usually consitituting a column. As in the case of the batch
type operation, the solid support material may be in any form desired such
as powder, pellets or monoliths, and is placed in the column after which a
preferably aqueous solution of a salt of aminopolystyrene is also charged
and contacts the support until the latter is saturated with the solution.
The aqueous solution is maintained at a pH less than 7 and preferably
at a pH in a range of from 1 to 4 by the addition of an appropriate acid.
After saturation of the support has been accomplished an excess is then
drained and an intermediary spacer compound such as a reactive bifunctional
monomer molecule, preferably in aqueous solution, is charged to the column,
said bifunctional molecule being present in an excess in a range of from
2 to 50 moles relative to the amine content of the aminopolystyrene.
While the formation of the matrix is effected during a period of time which
may range from 1 to 22 hours in duration! the formation is usually accom-
plished during a relatively short period of time. Following the com-
pletion of the desired resldence time the excess spacer reactant such as
glutaraldehyde is removed by draining followed by a thorough water washing
to remove any unreacted materials.
To form an immobilized enzyme conjugate, an aqueous solution of
an enzyme of the type hereinbefore set forth in greater detail is then
, '
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'
~28917
passed through the column containing the thus formed support matrix thereby
effecting a covalent bonding of the enzyme to the terminal reactive groups
of the functionalized pendent moieties which extend from the matrix. This
occurs until there is no further covalent binding of the enzyme to the
S pendent molecules. The excess enzyme is recovered in the effluent which
is continuously withdrawn after draining and may be recycled to the column
for further use. After washing the column, the column is then ready for
use in chemical reactions in which the catalytic effect of the enzyme is
to take place. These procedures are conducted within the time, temperature
and concentration parameters hereinbefore set forth described in the batch
type procedure and will result in comparable immobilized enzyme complexes.
It is also contemplated with~n the scope of this invention that with suit-
able modifictions of reaction parameters it will be obvious to those
skilled in the~art that the process may be applied to a wide variety of
supports, bifunctional monomers and enzymes.
~ The following examples are given for purposes of illustrating the
novel compositions of matter of the present invention and to the methods
for preparing the same. However, it is to be understood that these examples
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llZ8917
are given merely for purposes of illustration and that the present inven-
tion is not necessarily limited thereto.
EXAMPLE I
In this example 1 gram of a porous alumina base having a particle
size of from 25 to 40 mesh, an Apparent Bulk Density (ABD) of 0.34 and
pore size ranging from 200 Angstroms to 10,000 Angstroms was admixed with
i 10 ml of a 5X weight by volume of aminopolystyrene having a molecular weight
of 22,000 dissolved in aqueous 0.1 M hydrochloric acid. The two components
were mixed at room temperature and allowed to stand for a period of 1 hour.
At the end of this time the solution was degassed, filtered and the solid
support containing the aminopolystyrene adsorbed thereon was dried. Follow-
ing this, the composite was mixed with 10 ml of a 1.5X aqueous solution of
glutaraldehyde whlch had a pH of 1.4 and maintained for a period of 1 hour
at room temperature. At the end of this 1 hour period the excess glutar-
aldehyde was decanted and the organic-inorganic matrix was thoroughly washed
with water several t~mes. The final immobilized conjugate was then pre-
pared by treating the matrix with 6482 units of a commercial glucoamylase
~ Jradehl~rJc
t~ sold under the Tradcnamc "Ambazyme". The immobilization of the enzyme was
effected during a period of 16 hours while maintaining the temperature of
the composite at 4C. by means of an ice bath. At the end of the 16 hour
period the residual and unbound enzyme was washed out with water and a
sodium chloride solution.
The immob~lized enzyme conjugate was packed in a column and a
30% weight by volume solution of starch sold under the commcrcial Trade-
1ta~e~ "Maltrin-150" was passed over the beads while maintaining the tempera-
ture at 60C., said starch feed being passed over the beads for a period
of 2 hours at a flow rate of 2 ml/min. At the end of the 2 hour period,
.
1~28917
the amount of glucose formed was assayed. It was found that the activity
of the enzyme conjugate was 3240 units/gram at the flow rate of 2 ml/min.i
the unit being defined as the micromoles of glucose formed per minute per
gram of immobilized enzyme conjugate.
S EXAMPLE II
The above experiment was repeated with the exception that the
enzyme, namely, glucoamylase was purified by means of an isopropanol pre-
cipitation procedure well known in the art, resulting in a 1.3 fold increase
of purity of the enzyme~ When this purified enzyme was immobilized in a
manner similar to that set forth above and utilized to convert starch to
glucose, it was found that the immobilized enzyme coniugate had an activity
of 4070 units/gram at a flow rate of 2 ml/min.
EXAMPLE III
In a manner similar to that set forth in Example I above, 1 gram
of an alumina base having a particle size of from 25-35 mesh and an ABD of
0.3 was added to 10 ml of a solution comprising 5X by volume of aminopoly-
styrene dissolved in aqueous 0.1 M hydrochloric acid. The mixture was
maintained at room temperature for a period of 1 hour after which it was
degassed, filtered and the aminopolystyrene-alumina matrix was dried. The
dried beads were then added to 10 ml of a 1.5% aqueous solution of glutar-
aldehyde which had a pH of 1.4. The mixture was held for a period of 1 hour
at room temperature and thereafter the excess glutaraldehyde was decanted.
The organic-inorganic matrix was washed several times with water and
thereafter was treated with 1300 units of glucose isomerase which had a
specific activity of 8.5 units/mg of protein. The coupling was effected
at a temperature of 4C. for a period of 22 hours. The resulting immobilized
glucose isomerase conjugate in which the enzyme was covalently bound to the
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1128917
free aldehyde functions of the copolymeric material which arose from the
use of excess glutaraldehyde, was washed thoroughly with water and a 2 M
aqueous sodium chloride solution to remove any unreacted enzyme.
The immobilized enzyme conjugate was packed in a suitable column
and a 45X solution of fructose which possessed a pH of 8 and contained
5 x 10 3 molar magnesium chloride was passed through the conjugate bed at
a temperature of 60C. for a period of 2 hours. The glucose and flow
rate were assayed and it was found that this immobilized enzyme conjugate
had an activity of 700 units/gram at a flow rate of 2 ml/min., with a
coupling efficiency of 60~. It is further determined that the enzyme con-
jugate possessed a half-life of 22 days at 60- in a continuous column
operation using the flow rate of 2 ml/min. of feed over the conjugate.
EXAMPLE IV
- In this example an alumina base having a particle size of from
60-80 mesh and an ABD of 0.3 was treated with aminopolystyrene and an excess
of glutaraldehyde in a manner similar to that set forth above to form an
organic-inorganic support matrix. This support matrix was treated with
glucose isomerase which possessed a specific activity of 15 units/mg. The
coupling was effected by offering 2800 units of enzyme to the support
matrix at a temperature of 4~. for a period of 22 hours. After thorough
washing of the immobilized enzyme conjugate, lt was packed in a column and
a fructose feed similar to that described in Example III above was passed
through the conjugate bed for a period of 2 hours at a temperature of 60-C.
The product was assayed and it was found that the immobilized enzyme conju-
gate had an activity of 1200 units/gram with a coupling efficiency of 54g.
'
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11289~7
EXAMPLE ~
In this example 1 gram of porous alumina pellets, 10 mesh,
having an apparent bulk density (ABD) of 0.344 and pore si~es rang-
; ing from 200 Angstroms to 10,000 Angstroms were placed in an emul-
sion prepared from 0.5 grams of sodium hydrogen phosphate, 0.5 grams
of sodium lauryl sulfate, 2 ml of styrene and 20 ml of distilled wa-
ter. The peltets were allowed to adsorb in this emulsion under
vacuum for a period of 1 hour following which the mixture was then
- heated for a period of 3 hours at a temperature of 100C. in the
presence of a polymerization catalyst comprising 0.5 grams of potas-
sium persulfate. At the end of the 3 hour period, the beads were
filtered to remove liquid, washed with a distilled water and ! ml
of acetone. Thereafter 1 gram of the polystyrene-alumina support
was~slowly added to 50 ml of 90X fuming nitric acid. The addition
of the~support to the acid was effected at a temperature of about
0C. to 10C., the temperature being attained by placing the appara-
tus in an ice bath. After a period of 2 hours the nitrated support
was removed, washed with water and acetone. To effect the reduction
of the nitrated polystyrene-alumina support, 1 gram of the composite
along with 1 gram of sodium dithionite was added to 100 ml of dis-
tilled water. The mixture was then heated to boiling and maintained
at this temperature for a period of 1 hour. Following this the
i~
aminopolystyrene-alumina composite support was washed with distilled
water, dried under vacuum and maintained under a nitrogen atmosphere.
The aminopolystyrene-alumina support prepared according to
the above paragraph was then contacted with 10 ml of a 50% aqueous
solution of glutaraldehyde which had a pH of 3.5 for a period of 1
hour at room temperature. At the end of the 1 hour period the excess
`` ~
1~28917
glutaraldehyde was decanted and the organic-inorganic matrlx was
thoroughly washed with water several times in order to effectively
remove unreacted reagents. The final immobilized enzyme conjugate
was then prepared by treating the matrix with 8300 units of a com-
~ r~a de ~
5 ~ mercial glucose amylase sold under the tr~dena~ Novo PPAG-12, said
immobilization of the enzyme being effected during a period of 16
hours while maintaining the temperature of the mixture at about 4C.
by means of an ice bath. At the end of the 16 hour period the re-
sidual and unbound enzyme was washed out with water and a sodium
chloride solution.
The immobilized enzyme conjugate was then packed in a col-
~ umn ànd a 30% weight by volume solution of starch, 6omncrQial Trade-
s~ r~
~u*e~ Maltrin-150, was passed over the beads at a flow rate of 2 ml/
min. while maintaining the temperature at 60C. for a period of 2
hours. At the end of the 2 hour period the amount of glucose formed
was assayed. It was found that the activity of the enzyme conjugate
was 695 units/gram at the flow rate of 2 ml/minute; the term "unit"
being defined as the micromoles of glucose formed per minute per
gram of immobllized enzyme coniugate.
- EXAMPLEVI
Experiment V was repeated with the exception that
the enzyme was purified by means of an isopropanol precipitation pro-
cedure well known to those skilled in the art. When the purified en-
zyme was immobilized in a manner simiiar to that set forth above and
utilized to convert starch to glucose, it was found that the immobil-
ized enzyme coniugate had an activity of 852 units/gram at a flow
rate of 2 ml/min.
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