Note: Descriptions are shown in the official language in which they were submitted.
11S6580
"REGENERATION OF
AN IMMOBILIZED ENZYME SYSTEM"
BACKGR UND OF THE INVENTION
Enzyme-catalyzed reactions often have the advantages of pro-
ceeding with great chemical specificity under relatively mild conditions,
and often accomplish what man finds difficult, if not impossible, to dupli-
cate in the laboratory. For such reasons there is increasing emphasis onthe use of enzymatic processès on a commercial scale. One example, of
many which could be cited, is the conversion of glucose to fructose using
ylucose isomerase.
Enzymes are water soluble, and if they are merely used in aqueous
solutions recovery of enzyme for reuse is difficult and expensive. Using
the enzyme only once affords a process which is relatively expensive. ~on-
sequently, many techniques have been developed for immobilizing the enzyme
in such a way that substantial enzymatic activity is displayed while the
enzyme itself remains rigidly attached to some water-insoluble support,
thereby permitting reuse of the enzyme over substantial periods of time
and for substantial amounts of feedstock. One illustration of a method
for immobilizing an enzyme is found in Levy and Fusee, ll.S. Patent No.
4,141,857, where a polyamine is adsorbed on a metal oxide such as alumina,
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treated with an excess of a bifunctional reagent, such as glutaraldehyde,
so as to cross-lin~ the amine, thereby entrapping the resultiny polymer
in the pores of the metal oxide~ and then contacting the mass with enzyme
to form covalent bonds between the pendant aldehyde groups and an amino
group on the enzyme.
The useful life oF an immobilized enzyme system is limited Dy
a continual decrease in enzymatic activity. Among the many mechanisms
which lead to enzyme deactivation in such systems are: poisoning of the
enzymes by impurities in the feedstock; other chemical modification of
the enzyme; denaturation of the enzyme; rupture of the bond between the
pendant group and the enzyme leading to dissolution of the enzyme; cleavage
of the bond between the pendant group and the intermediate binding layeri
loss of the bindin~ layer as, for example, by physical ablation or cleavage
of the chemical bond which hold it to the support.
Whatever the mechanism of the enzyme deactivation, reactivation
of a deactivated immobilized enzyme system would prove to be a substantial
advance in the art as well as being economically highly desirable. At
least conceptually, two distinct approaches to reactivation are possible.
One mode would be to rejuvenate the enzyme itself, i.e., assuming no
physical loss of enzyme the transformations which rendered it inactive
would be reversed and the enzyme would revert to its initial active state.
The alternative is to restore the immobilized enzyme system to that state
initially present immediately prior to attachement of enzyme, so that it --
~ould be capable o-F binding fresh, active enzyme once again. This invention
relates to the latter approach.
SUMMARY OF THE INVENTION
An object Or this invention is to regenerate an immobilized enzyme
system which has become substantially deactivated. An embodiment of this in-
ll~6ssn
vention resides in a process for regeneratin~ an immobilized enzyme
system comprising treating the system with a base as an enzyme stripping
agent, removing the base, treating the system with a bifunctional organic
molecule which serves as a pendant group, and removing the excess of bi-
functional organic molecule, so as to put the system in a state where
fresh, active enzyme can be immobilized by suitable means. A more specific
embodiment of this invention resides in the application of this process to
a system wherein the binding layer is an organic polymeric material and
the pendant functional moiety can bond covalently with an enzyme without
greatly destroying its activity. Another more specific embodiment of this
invention is the application of this process whereinthe enzyme is glucose
isomerase and the stripping agent is an alkali metal hydroxide, such as
sodium hydroxide and potassium hydroxide. Other objects and embodimenis
will be apparent from the description provided herein.
It is to be emphasized again that enzymes are merely representative
of one class of reactive chemical entities which may be immobilized to act
in some chemical process. Therefore, this invention encompasses regenera-
tion of any immobilized reactive chemical entity which has become substantially
deactivated.
1 20 DESCRIPTION OF THE FIGURE
! Many immobilized enzyme systems, such as that described above,
have a common conceptual basis which is depicted pictorially in the Figure.
It is to be understood that enzymes are merely one class of reactive chemical
entities which may be immobilized and subsequently utilized in a chernical
process.
There is a central core support, A, whose primary purpose is to
provide rnechanical and thermal stability to the system and which is
chemically inert in the enzymatic reaction. The intermediate primary
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layer, B, provides an interface between the core and the pendant groups,
C. This layer may be held to the core either by physical entrapment, as
within the pores of A, by strong short-range physical and/or chemical
forces, as by surface adsorption or absorption, or by chemiral binding
to the surface of the core support. The pendant groups, C, may be
part oF the molecular structure of the binding 1ayer, or may be chemically
bonded to a suitable site on the binding layer. Such pendant groups
are characterized by the presence of a chemically reactive functionality,
usually terminally situa~ed, which can covalently bond to some part oF
the enzyme, or other reactive chemical entity, sufficiently removed
from its "active site" so as not to interfere substantially with its
catalytic activity.
DESCRIPTION OF THE INVENTION
Although several kinds of immobilized enzyme systems are avail-
able, those wherein the enzyme is covalently bonded to a support seem to
offer the best compromise between enzyme availability to feedstoc~ and
long-term immobility on a supporting structure. Accordingly, emphasis
is placed on stripping deactivated enzyme and regenerating an active im-
mobilized enzyme in such a system, This invention relates to the structure
depicted in the Figure. The central core support, A in the figure, may be
a metal oxide, preferably alumina and silica, glass, a ceramic or a metal
It needs to provide structural integrity, especially mechanical strength,
have good characteristics in a system where there is a liquid flow, and
provide a surface, wholly or in part, to which a layer of organic material
can be attached either by physical or chemical means, or by a combination
of the latter.
The binding layer, B, may be an organic polymer or a resin.
1 156~8P
Examples of such binding layers include functionalized polyethylenes,
polyamines cross-linked with agents such as dialdehydes and diisocyanates,
and others known to those skilled in the art. In a preferred embodiment,
the binding layer is a polyamine such as polyethyleneimine, tetraethylene-
pentamine, ethylenediamine, diethylenetriamine, triethylenetetramine,pentaethylen2hexamine, hexamethylenediamine, phenylenediamine, and the
like, cross-linked via a reagent selected from the group consisting of
dialdehydes and the diisocyanates, as for example glutaraldehyde, succin-
dialdehyde, toluenediisocyanate, and the like. In another preferred embodi-
ment the binding layer is a functionalized polystyrene, such as aminopoly-
styrene, cross-linked by one of the aforementioned reagents.
The pendant group, C, may be an independently functionalized
group of the polymer, as for example an aldehydic moiety attached via medi-
ating carbon atoms to a polyethylene chain, an independently functionalized
group of a resin, or an unreacted terminus of the cross-linking agent wherein
the other terminus is covalently bonded to the binding layer. In a preferred
embodiment, the pendant group arises from a cross-linking agent selected
from the group consisting of dialdehydes and diisocyanates.
In some instances the demarcation between core support, A, binding
iayer B, and pendant group C may seem indistinct. For example, the binding
layer may appear to be part of the core, and might even contain a functional
group which can covalently bond to an enzyme, thereby providing an immobilized
enzyme system. A representative of this class is a chemically modified
glass whose surface bears an organic residue having a functional group
capable of covalently bonding to an enzyme. This invention relates to such
a system, and to all systems which are functionally equivalent to, or can
be functionally described by, the representation in the figure, however
that may be attained in any particular immobilized enzyme system. The combina-
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tion of structures A, B and C forms a support system; addition oF enzymeforms an immobilized enzyme system.
The method of stripping and regeneration taught herein may be
applied to any reactive molecule which can react with the pendant functional
group without substantial loss of chemical activity; enzymes form an
important class of such reactive molecules. Examples oF such enzymes in-
clude glucose isomerase, glucose amylase, lactase, cellulase, glucose
oxidase, trypsin, papain, hexokinase, chymotrypsin, acylase, invertase,
protease, pepsin, rennin, xylanase, etc. It is to be understood that these
enzymes are cited solely for illustrative purposes and it is not to be
construed as a limitation of this invention. Other enzymes may be utilized,
but not necessarily with equivalent results.
The physical form of the immobilized enzyme system generally is
determined by factors extraneous to the stripping-regeneration process.
Thus, the system may be in the form of pellets of, for example, 1/16 inch
size, or it may be in the form of smaller spheres of, for example, 60-80
mesh. Although the form in which the immobilized enzyme system is used may
necessitate different optimum parameters in the stripping-regeneration process,
the basic method remains unchanged.
Immobilized enzyme systems in which the enzyme has become totally
inact;ve, or nearly so, may be unpacked from the columns where they had been
used and placed in containers. To this may be added sufficient enzyme
stripping reagent such that the pellets or spheres are completely covered
with liquid. Among the stripping reagents which are suitable for use are
alkaline materials. Examples of such reagents include the alkali metal
hydroxides and carbonates, such as those of lithium, sodium, potassium,
cesium and rubidium, ammonia, ammonium carbonate, quaternary ammonium
hydr~ides such as tetramethylammonium hydroriue, benzyltrimethylammonium
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hydroxide, cetylpyridinium hydroxide, etc. The concentration of the re-
agent and amount used are not critical, provided that there is sufficient
reagent to remove all inactive enzyme, and that the volume is sufficient
to provide adequate contact with the pellets or spheres. Concentrations
of base employed may range from about 0.01 to about 5 molar. The tempera-
ture at which stripping is conducted may be from about 20C. to about 75~.,
preferably from about 50C. to about 70C. Contact time may be from about
1 to about 30 minutes, preferably from about 1 to about 10 minutes, and may
- be accompanied by agitation. In one embodiment, the reagent is sodium
hydroxide.
After the material has been treated with the base for an appropriate
time, excess reagent may be removed by decantation. The pellets or spheres
are then washed thoroughly with water to remove any base adhering to the
surface. When no more base is present the system is ready for contacting
with a solution which furnishes the pendant group. For example, the
solution may be one of glutaraldehyde in water, where the concentration of
glutaraldehyde is not material so long as there is present sufficient
material to replace any pendant groups lost in its prior history. Where
the pendant group is reactive toward water, the enzyme support system may
have to be dried, by means which will be obvious to those skilled in the art,
erior to treatment with the reagent. Although the system generally will
be treated with a solution which furnishes the pendant group originally pre-
sent, it may be treated with a solution which furnishes a different pendant
group. Representatives of materials furnishing a pendant group enumerated
solely for the purpose of illustration, include glutaraldehyde, succindialde-
hyde, terepthaladehyde, and toluenediisocyanate.
When the enzyme support system has been contacted with a solution
furnishing the pendant groups for a time sufficient to replace all those pre-
115658n
viously lost, which time may vary from about 30 minutes to about 5 hours,depending on the nature of the support system, its history, the stripping
reagent used and the nature of the pendant group, it is washed thoroughly
to remove unreacted but adhering molecules which furnish the pendant group.
At this stage the support system is reiuvenated, which is to say that it
approximates its condition prior to initial enzyme immobiliza-tion. The
support system is now ready to accept fresh, active enzyme to regenerate
an immobilized enzyme system whose activity approximates that obtained with
a new support. In the case, for example, of a polyethyleneimine binding
layer cross-linked with excess glutaraldehyde, glucose isomerase may be
immobilized by contacting the support system, with agitation, with an aqueous
solution of the enzyme for 2 to 24 hours at a temperature from about ~ to
about 50C. preferably from about 0 to about 10C. However, it is not an
object of this invention to teach how enzymes are best immobilized given a
! 15 particular support, thus it suffices to say that the support regenerated
by the method of this invention is treated with enzyme in whatever way is
appropriate for immobilization of that particular enzyme on a particular
support system.
- The description above is for a stripping-regeneration process run
in a batchwise method. However, the process of this invention may be done
in a continuous manner where such a mode is advantageous. Thus, as an
example where the stripping agent is potassium carbonate and the reagent fur-
nishing the pendant group is glutaraldehyde, the deactivated immobilized
enzyme system in a column may be treated with a potassium carbonate solution
recirculated or passed through the column for a time sufficient to remove
all enzyme. Thereafter the column may be washed with water until all traces
of alkaline material are removed, followed by treatment with recirculated
glutaraldehyde solution until there is no further uptake of the latter reagent.
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Unreacted but adhering glutaraldehyde may be removed by
treatment with fresh water, after which active enzyme may be
immobilized by suitable means.
Whether the stripping portion of the process of this
invention consists of selective removal of spent enzyme from
the pendant group, or whether it consists of removal of the
pendant group from the binding layer, or some combination
thereof, is not known. This invention is meant to encompass
removal of spent enzyme from an enzyme support system of the
type described herein whatever the mechanism of removal.
The following examples serve merely to illustrate
the process of this invention, and it is to be understood that
this invention is not limited thereto.
EXAMPLE 1
An immobilized enzyme system based on polyethyleneimine
(PEI) on alumina cross-linked with glutaraldehyde and bearing
glucose isomerase had an initial activity of 1400 units per
gram. The bed, composed of 60-80 mesh particles, was treated
with 2 M NaOH in an amount of 20 ml solution of base per gram
of bed material. This mixture was heated with stirring at
60F. for five minutes, after which the solution was removed
by decantation. The solid was then stirred with water suf-
ficient to cover all material present and liquid was decanted.
This washing procedure was repeated until the wash liquid was
neutral (pH 7). At this stage the material displayed no
enzymatic activity. Regeneration was accomplished by adding
a 2.5~ aqueous solution of glutaraldehyde in an amount equal
to 18 ml. per gram of bed for about one hour. Excess glutara-
ldehyde was removed by thorough washing with water. A prepara-
tion of fresh, active glucose isomerase was contacted with the
regenerated support system for 18 hours at 4C. with continual
shaking. The immobilized enzyme system was thoroughly washed
with water to remove adhering but mobile enzyme. The resulting
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immobilized enzyme system had a glucose isomerase activity
of 820 units per gram, or 59% of the activity originally
present.
EXAMPLE 2
~ The immobilized enzyme system was like that of
Example 1 but in 1/16" pellets. Its initial activity was 141
units per gram of bed. Deactivated bed material was treated
with sodium hydroxide using the procedure given in Example 1.
After reimmobilization of glucose isomerase the sytem showed
a glucose isomerase activity of 121 units per gram, or 86%
that originally present.
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