U.S. patent application number 10/847567 was filed with the patent office on 2004-10-21 for enzyme-containing polyurethanes.
This patent application is currently assigned to Agentase, LLC. Invention is credited to LeJeune, Keith E., Russell, Alan J..
Application Number | 20040209339 10/847567 |
Document ID | / |
Family ID | 32595365 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040209339 |
Kind Code |
A1 |
LeJeune, Keith E. ; et
al. |
October 21, 2004 |
Enzyme-containing polyurethanes
Abstract
A method of increasing loading of active enzyme immobilized in a
polyurethane polymer including the steps of: synthesizing the
polyurethane polymer in a reaction mixture containing water and
enzyme; and including a sufficient amount of a surfactant in the
reaction mixture to increase enzyme activity at an enzyme
loading.
Inventors: |
LeJeune, Keith E.;
(Pittsburgh, PA) ; Russell, Alan J.; (Gibsonia,
PA) |
Correspondence
Address: |
Craig G. Cochenour, Esq.
Buchanan Ingersoll PC
301 Grant Street
One Oxford Centre, 20th Floor
Pittsburgh
PA
15219
US
|
Assignee: |
Agentase, LLC
Pittsburgh
PA
|
Family ID: |
32595365 |
Appl. No.: |
10/847567 |
Filed: |
May 17, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10847567 |
May 17, 2004 |
|
|
|
09441592 |
Nov 17, 1999 |
|
|
|
6759220 |
|
|
|
|
Current U.S.
Class: |
435/180 |
Current CPC
Class: |
C12N 11/098 20200101;
C12N 11/093 20200101; C12N 11/18 20130101 |
Class at
Publication: |
435/180 |
International
Class: |
C12N 011/08 |
Claims
1-10. cancelled.
11. A polyurethane polymer containing an enzyme loading of more
than approximately 0.1 weight percent enzyme, the polyurethane
polymer having been synthesized in the presence of a sufficient
amount of a surfactant to increase enzyme activity at the enzyme
loading.
12. The polyurethane polymer of claim 11 wherein the surfactant is
nonionic.
13. The polyurethane polymer of claim 12 wherein the enzyme loading
is greater that approximately 0.5 percent weight of the
polyurethane polymer.
14. The polyurethane polymer of claim 12 wherein the enzyme loading
is greater that approximately 1 percent by weight of the
polyurethane polymer.
15. The polyurethane polymer of claim 12 wherein enzyme immobilized
in the polyurethane polymer includes at least one of an
oxidoreductase, a transferase, a proteolytic enzyme, a lyase, an
isomerase or a ligase.
16. The polyurethane polymer of claim 12 wherein enzyme immobilized
in the polyurethane polymer includes at least one of a protease, a
lipase, a peroxidase, a tyrosinase, a glycosidase, a nuclease, a
aldolase, a phosphatase, s sulfatase, a hydrolase, or a
dehydrogenase.
17. The polyurethane polymer of claim 12 wherein at least two
species of enzyme are co-immobilized within the polyurethane
polymer.
18. The polyurethane polymer of claim 17 wherein the two species of
enzyme are within the same class of enzyme.
19. The polyurethane polymer of claim 12 wherein the surfactant
comprises between 0.5 to 5.0 weight percent of the aqueous
component of a reaction mixture.
20. The polyurethane polymer of claim 12 wherein the enzyme is a
hydrolase and the surfactant is nonionic.
21. A method of improving enzymatic activity in a polyurethane
polymer synthesized with an enzyme loading of more than
approximately 0.1 weight percent enzyme, the method comprising the
step of: adding a sufficient amount of a surfactant during
synthesis of the polyurethane polymer to increase enzyme activity
at the enzyme loading.
22. The method of claim 21 wherein the surfactant is nonionic.
23. The method of claim 22 wherein the enzyme loading is greater
that approximately 0.5 percent by weight of the polyurethane
polymer.
24. The method of claim 22 wherein the enzyme loading is greater
that approximately 1 percent by weight of the polyurethane
polymer.
25. The method of claim 22 wherein enzyme immobilized in the
polyurethane polymer includes at least one of an oxidoreductase, a
transferase, a proteolytic enzyme, a lyase, an isomerase or a
ligase.
26. The method of claim 22 wherein enzyme immobilized in the
polyurethane polymer includes at least one of a protease, a lipase,
a peroxidase, a tyrosinase, a glycosidase, a nuclease, a aldolase,
a phosphatase, a sulfatase, a hydrolase, or a dehydrogenase.
27. The method of claim 22 wherein at least two species of enzyme
are co-immobilized within the polyurethane polymer.
28. The method of claim 27 wherein the two species of enzyme are
within the same class of enzyme.
29. The method of claim 22 wherein the surfactant comprises between
0.5 to 5.0 weight percent of the aqueous component of the
mixture.
30. The method of claim 22 wherein the enzyme is a hydrolase and
surfactant is nonionic.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to enzyme-containing
polyurethanes, and, especially, to enzyme-containing polyurethanes
of relatively high enzyme loading and relatively high catalytic
(enzyme) activity.
BACKGROUND OF THE INVENTION
[0002] It has been known for some time that one can incorporate
proteins within polyurethane polymers during polymer synthesis. For
example, U.S. Pat. Nos. 3,928,138, 3,929,574, 4,098,465, 4,195,127,
and 4,250,267 describe enzymes bound within a hydrophilic
polyurethane polymer. Although enzyme activity was evident in those
polymers, no attempt was made to quantify the degree of enzyme
activity within the polymers.
[0003] Academics have more recently begun to revisit the synthesis
of enzymatic polyurethane. For example, Dias et al. assessed the
performance of lipase incorporated within polyurethane foams. Dias,
S. F., Vilas-Boas, L., Cabral, J. M. S., and Fonseca, M. M. R.,
Biocatalysis, 5, 21 (1991). That study described the synthesis of
enzymatic polymers without the use of additives, enzyme
stabilizers, or enzyme pre-modification. Enzyme concentration
within the polymers was varied over a broad range in the course of
this study. Those studies indicated an apparent reduction in enzyme
activity retention at high enzyme loading (for example, greater
than 0.1 weight percent).
[0004] Storey et al described the immobilization of
amyloglucosidase enzyme within several types of crosslinked
polyurethane matrices Storey, K. B., Duncan, J. A., Chakrabarti, J.
A., Appl. Biochem. Biotechnol. 23, 221 (1990). The enzyme
concentrations employed in that study were relatively dilute and
the use of additives or other non-essential components was not
explored.
[0005] Recent studies of general polyurethane synthesis
(irrespective of incorporation of enzyme therein) have shown that
incorporation of a surfactant in the reaction mixture can lead to
desirable physical properties of the polyurethane polymer product.
It is believed that surfactants stabilize the carbon dioxide
bubbles that are formed during synthesis and are responsible for
foaming. For example, certain surfactants have been found to
promote the creation of small carbon dioxide bubble, resulting in
formation of a polymer product having a morphology similar to a
fabric. Other surfactant have been found to promote relatively
large carbon dioxide bubbles, resulting in a polymer product having
a morphology similar to a sponge. Given the control that
surfactants enable over the physical/morphological characteristics
of polyurethanes, suppliers of polyurethane prepolymer typically
recommend that surfactant be added to a polyurethane reaction
mixture.
[0006] Thus, recent studies of the synthesis of enzyme-containing
polyurethanes have employed surfactants to alter/control the
physical properties of the resultant polymers. For example, a
number of studies describe the immobilization of organophosphorus
hydrolase using a polyurethane polymer synthesis strategy in which
a variety of non-ionic surfactants were used as additives to alter
the physical properties polymers. Havens, P. L., Rase, H. F., Ind.
Eng. Chem. Res., 32, 2254 (1993); LeJeune, K. E., Swers, J. S.,
Hetro, A. D., et al. Biotechnol. Bioeng., 64, 2, 250 (1999);
LeJeune, K. E., et al. Biotechnol. Bioeng., 54, 105, (1997);
LeJeune, K. E. and Russell, A. J. Biotechnol. Bioeng., 51, 450
(1996). In general, these surfactants were used in an attempt to
optimize the performance of the polyurethane sponge product in a
particular application. For example, the studies of Havens and Rase
were focused upon using the resultant polymers as column packing
material and as adsorbent sponges to decontaminate pesticide
spills. The studies reported varying surfactant hydrophobicity
could produce polymers that were better suited for a particular
application. The enzyme concentration/loading employed in the
studies of Havens and Rase and the other studies was quite low (in
general, well below 0.1 weight percent of the polymer).
[0007] It is desirable to develop enzyme containing polymers and
methods of synthesis of such polymers in which enzyme loading and
enzyme activity are improved.
SUMMARY OF THE INVENTION
[0008] The present inventors have discovered that certain
surfactants not only enable control of polyurethane physical
properties/morphology, but enhance the activity of immobilized
enzymes at relatively high enzyme loading. As used herein, the term
"enzyme" refers to a protein that catalyzes at least one
biochemical reaction. A compound for which a particular enzyme
catalyzes a reaction is typically referred to as a "substrate" of
the enzyme. Enzymes typically have molecular weights in excess of
5000.
[0009] In general, six classes or types of enzymes (as classified
by the type of reaction that is catalyzed) are recognized. Enzymes
catalyzing reduction/oxidation or redox reactions are referred to
generally as EC 1 (Enzyme Class 1) Oxidoreductases. Enzymes
catalyzing the transfer of specific radicals or groups are referred
to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis
are referred to generally as EC 3 hydrolases. Enzymes catalyzing
removal from or addition to a substrate of specific chemical groups
are referred to generally as EC 4 Lyases. Enzymes catalyzing
isomeration are referred to generally as EC 5 Isomerases. Enzymes
catalyzing combination or binding together of substrate units are
referred to generally as EC 6 Ligases.
[0010] In one aspect, the present invention provides a method of
increasing loading of active enzyme immobilized in a polyurethane
polymer including the steps of:
[0011] synthesizing the polyurethane polymer in a reaction mixture
containing water and enzyme; and
[0012] including a sufficient amount of a surfactant in the
reaction mixture to increase enzyme activity at an enzyme loading
(as compared to a polymer of the same enzyme loading synthesized
without surfactant).
[0013] As used herein, the term "surfactant: refers generally to a
surface active agent that is reduces the surface tension of a
liquid (water, for example) in which it is dissolved.
[0014] Preferably, the surfactant is nonionic and comprises between
0.5 to 5.0 weight percent of the aqueous component of the mixture.
In the synthesis of the polyurethanes of the present invention,
urethane prepolymers were mixed with water. The aqueous component
of the reaction mixture included water, enzyme, surfactant and
buffer salts. The weight percent surfactant in the aqueous
component is thus calculated by dividing the weight of the
surfactant by the weight of the entire aqueous component and
multiplying the result by 100%. The enzyme loading in the present
invention can be greater than approximately 0.1 percent by weight
of the polyurethane polymer (weight of enzyme/[weight of
enzyme-containing polymer product]*100%) while retaining
substantial enzyme activity. Relatively high activity is maintained
even when the enzyme loading is greater that approximately 0.5
percent by weight of the polyurethane polymer. Indeed, relatively
high activity is maintained even when the enzyme loading is greater
that approximately 1 percent by weight of the polyurethane
polymer.
[0015] The polyurethane polymers of the present invention
preferably include at least one of an oxidoreductase, a
transferase, a hydrolase, a lyase, an isomerase or a ligase.
Examples of enzymes suitable for use in the present invention
include, but are not limited to, a lipase, a peroxidase, a
tyrosinase, a glycosidase, a nuclease, a aldolase, a phosphatase, a
sulfatase, or a dehydrogenase.
[0016] More than one type of enzyme are easily co-immobilized
within the polyurethane polymer. The enzymes can be within the same
class (for example, two hydrolases) or a within different classes
of enzyme.
[0017] In another aspect, the present invention provides a
polyurethane polymer containing an enzyme loading of more than
approximately 0.1 weight percent. The polyurethane polymer is
synthesized in the presence of a sufficient amount of a surfactant
(preferably, nonionic) to increase enzyme activity at the enzyme
loading of the polymer (as compared to the case when no surfactant
is used).
[0018] In still another aspect, the present invention provides a
method of improving enzymatic activity of a polyurethane polymer
synthesized with an enzyme loading of more than approximately 0.1
weight percent. The method includes the step of:
[0019] adding a sufficient amount of a surfactant (preferably,
nonionic) to during synthesis of the polyurethane polymer to
increase enzyme activity at the enzyme loading.
[0020] The polymers and methods of the present invention provide
enhanced enzyme activity retention as the enzyme loading or enzyme
content of such polymers is increased (for example, to above
approximately 0.1 weight percent of the polymer). Relatively large
quantities of enzymes are immobilized within the polymers of the
present invention while retaining a significant portion of the
native enzyme specific activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 illustrates an embodiment of a synthetic scheme
synthesis of enzyme-containing polymers.
[0022] FIG. 2 illustrates the effect of using surfactant in the
synthesis of subtilisin-containing polyurethane polymers.
[0023] FIG. 3 illustrates the effect of using surfactant in the
synthesis of urease-containing polyurethane polymers.
[0024] FIG. 4 illustrates the effect of surfactant concentration
upon the catalytic activity of polymer containing subtilisin
carlsberg.
[0025] FIG. 5 illustrates a study of the utility of non-ionic
surfactants in polymer synthesis as compared to surfactants that
are cationic or anionic in nature.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The enzyme-containing polyurethane polymers of the present
invention can, for example, be synthesized by reaction of
relatively hydrophilic polyurethane prepolymer with aqueous
solution to produce a urethane foam. The polyurethane prepolymers
used in the present studies were urethanes that were capped (that
is, functionalized at chain ends) with multiple isocyanate
functionalities. Prepolymers containing multiple isocyanate
functionalities have the ability to form chemical crosslinks upon
reaction with a diol or water. Water reacts with isocyanates,
initiating a foaming reaction in which a carbamic acid intermediate
is formed. The carbamic acid quickly degrades to an amine and
evolves CO.sub.2. The carbon dioxide bubbles through the highly
viscous reacting polymer solution, creating a porous foam
structure. Because amines readily react with isocyanates, a
multi-functional prepolymer in aqueous solution results in a
crosslinked polyurethane matrix.
[0027] Because the vast majority of enzymes are most active in
aqueous solution, water not only serves to initiate the
prepolymeric reaction, but also provides a route to deliver an
enzyme to the reaction. Proteins such as enzymes have many amine
groups present via lysine residues and can readily react with
isocyanate functionalities to form a crosslinked polymer-protein
network through multi-point attachments of the enzyme and polymer.
A schematic of the reactions occurring in this process is
illustrated in FIG. 1. In FIG. 1, R.sub.1 represents a prepolymer
molecule (for example, having a molecular weight of approximately
300 to approximately 10,000) having multiple isocyanate
functionalities/groups. R.sub.2 and R represent other prepolymer
molecules with isocyanate functionalities. E represents an enzyme
with a reactive amine functionality present via lysine residues and
at the N-terminus of the protein.
[0028] It is believed that the surfactants used in synthesis of
enzyme-containing polyurethane polymers of the present invention
enhance the activity of biocatalytic polymers when the enzymatic
content of the composite materials is sufficiently high to
overwhelm the capacity of the polymer to provide the enzyme
incorporated therein with sufficient access to bind substrate or to
release product at a rate equivalent to the maximum achievable
catalytic rate. In that regard, several studies of the present
invention have demonstrated that polymers with excessive enzyme
content are diffusionally limited in their ability to catalyze
reactions. It is believed, that the use of certain surfactants over
a range of concentrations eliminates the diffusional limitations
imposed by a polymeric superstructure within which relatively large
amounts of enzyme have been incorporated.
EXPERIMENTAL PROCEDURES
[0029] 1. Enzyme Polymer Synthesis
[0030] As known in the art, variation of reaction conditions
affects both the physical properties polyurethane foams and the
degree of enzyme-foam interaction. Described below is a typical
procedure for biopolymer synthesis used in the present studies.
Initially, 4 ml of pH 7.8 Tris buffer (10 mM) containing a specific
surfactant at a particular concentration (approximately 0 to 8
weight percent in the studies of the present invention) were placed
into a narrow cylindrical mixing vessel. Subsequently, an enzyme
solution (for example approximately 1 ml of 1.5 mg/ml urease in the
same buffer, for example) was added. Finally, approximately 4 ml of
Hypol prepolymer, available from Hampshire Chemical Corp., a
subsidiary of Dow Chemical Company, (preheated to 30.degree. C. to
limit handling problems resulting from high viscosity) were added
to the mixture. The solutions was then intimately mixed. During the
initial "cream" period, the solution was injected into a
cylindrical mold where it rose and then set within 2 to 5 minutes.
Polymer synthesis was complete in less than 10 minutes. The
CO.sub.2 evolved during the reaction of water and isocyanate lifted
the foam to a final volume of approximately 50 to 60 ml.
[0031] After the initial 10 minute "set-up" time, foam samples were
treated in several ways. Some foam samples were immediately sealed
in vials, while others were pre-rinsed. Bulk foam samples were
typically placed in a fume hood or lyophilizer to facilitate the
removal of residual water and CO.sub.2 still present from the
reaction. Foams were stored under a wide range of conditions until
being assayed for enzyme activity.
[0032] The mixing system used in the present studies required 30 to
40 seconds of mixing at 2500 rpm to create a high quality foam with
Hypol 3000, a toluene di-isocyanate based prepolymer. The mixing
system included an oar-shaped metal loop having a height of 3.2 cm
and a diameter of 1.3 cm. Hypol 5000 (methylene bis(p-phenyl
isocyanate) based), a more hydrophobic prepolymer, required
additional mixing. Insufficient mixing can result in un-reacted
residual prepolymer dispersed within a dense hard mass of
polyurethane. Overmixing does not allow the evolving CO.sub.2 to
act in lifting the foam. Properly mixed foam will typically
increase approximately six-fold in volume throughout the course of
the reaction.
[0033] In one embodiment of the present invention, an aqueous
solution of enzymes and surfactant was contacted with an
isocyanate-based prepolymer under sufficient agitation to initiate
reaction. The enzyme can, for example, be added as a freeze-dried
powder or aqueous solution that is either pure or impure. The term
"impure" a used herein refers generally to enzymes containing, for
example, other proteins/enzymes and biological molecules. Virtually
any enzyme or combination of enzymes can be co-immobilized within
the same polymer in the present invention.
[0034] In model studies of the present invention, enzyme-containing
polymers were synthesized both with and without a series of
surfactants. Enzymes incorporated into the polymers of the present
invention included, for example, organophosphorus hydrolase (OPH),
organophosphorus acid anhydrolase (OPAA), butyrylcholinesterase
(BChE), urease, and subtilisin carlsberg. The benefit of using
certain surfactants in the synthesis of the enzyme-containing
polymers of the present invention was demonstrated with in series
of kinetic experiments discussed below.
[0035] 2. Increasing Enzyme Activity in Highly Loaded Polymers
through the use of Surfactants
[0036] Using the procedures described above for polymer synthesis,
enzyme-containing polymers were synthesized both with and without
the use of surfactants. For example, subtilisin carlsberg and
urease enzymes were individually incorporated within polyurethane
polymers over a range of enzyme concentrations from approximately
10 .mu.g to approximately 20 mg enzyme per gram polymer (that is,
approximately 0.001 to approximately 2% by weight. Multiple
polymers were synthesized at each enzyme concentration, some with
the use of 1 weight percent Pluronic F-68 non-ionic surfactant
present and some without surfactant. The polymers were placed in a
fume hood for 12 hours after synthesis to facilitate the removal of
residual water and CO.sub.2 before their catalytic activity was
assessed.
[0037] Subtilisin-containing polymers were assayed for their
hydrolytic activity on N-succinyl-ALA-ALA-PRO-PHE p-nitroanilide in
10% MeOH/50 mM Tris buffer (pH 8.0) solutions. Substrate hydrolysis
was monitored with the use of a spectrophotometer. Reaction rates
were determined by placing polymer samples (100 mg) within 10 ml
substrate solutions and taking and subsequently replacing aliquots
from the reacting system at regular intervals. FIG. 2 illustrates
the benefit achieved by including surfactant within the polymer
formulation. Without the addition of surfactants during the polymer
synthesis, very little if any benefit is incurred by increasing the
enzyme content within the polymers, whereas those polymers
synthesized in the presence of surfactant exhibited activity levels
which were closely related to enzyme content.
[0038] The activity of urease polymers (150 mg samples) was assayed
in 300 mM urea within 10 mM Phosphate buffer at pH 7.25 (15 ml).
Urea hydrolysis was assessed by monitoring solution pH, since urea
hydrolysis causes a corresponding increase in pH. FIG. 3 shows that
there are no significant diffusional limitations present at low
enzyme concentration. The rates of reaction with or without
surfactant are essentially identical when the urease content of the
polymer is low (see Table 1 for rate data). The rate of catalysis
is proportional to enzyme concentration in the presence of
surfactant. However, the absence of surfactant is believed to
result in diffusional limitations within the system. Apparent
catalytic activity was found to have very little dependence upon
enzyme loading when surfactants are not utilized in polymer
synthesis.
1TABLE 1 Rate data for urease-polymer assays. Polymers Polymers
synthesized synthesized with without Enzyme loading in surfactant
surfactant polymer (.mu.g urease/ Reaction Rate Reaction Rate g
polymer) (.DELTA.pH/min) * 10.sup.3 (.DELTA.pH/min) * 10.sup.3 170
8.2 6.4 430 15.0 8.7 1700 63.0 9.8
[0039] Diffusional limitations for other enzymes (including
organophosphorus hydrolase, organophosphorus acid anhydrolase, and
butyrylcholinesterase) have also been measured.
[0040] 3. Surfactant Concentration
[0041] The amount of surfactant present during polymer synthesis
was found to affect the retention of enzyme activity in the
enzyme-containing polymers of the present invention. In the limit
as surfactant concentration approaches zero, the resulting material
exhibits the same polymer properties and subsequent diffusional
limitations present when no surfactant is employed. The effect of
the amount of surfactant used was studied by synthesizing
biocatalytic polymers with sufficient enzyme loading to cause
diffusional limitation in the absence of surfactant. Nearly
identical synthetic procedures were also carried out for polymers
in which Pluronic F-68 surfactant content was gradually increased
to near its solubility limit in water (7.5%).
[0042] Subtilisin-containing polymers were synthesized with an
enzyme loading of approximately 250 .mu.g enzyme per gram polymer.
This degree of loading is believed to be sufficient to incur
diffusional limitations within polymers not formulated for high
enzyme loading (see FIG. 2) through the use of sufficient
surfactant during synthesis. FIG. 4 illustrates a study of the
effect of surfactant concentration upon the rate of catalysis
observed for subtilisin-polymers (100 mg) having an enzyme loading
of 250 .mu.g enzyme per gram polymer that were assayed against
N-succinyl-ALA-ALA-PRO-PHE p-nitroanilide in 10% MeOH/50 mM Tris
buffer (pH 8.0) solutions (10 ml). Surfactant concentrations of
less than 0.5 weight percent of the aqueous synthesis component
were found to be insufficient to overcome the diffusional
limitations imposed at this level of enzyme loading. The data
indicate that an "optimum" surfactant concentration exists between
approximately 0.5 and approximately 5.0 weight percent. The data of
Table 2 indicates that increasing surfactant concentration beyond
the optimum concentration did not further improve activity. It is
possible that diffusional limitations were overcome at the enzyme
loading studied at the lower surfactant concentration.
2TABLE 2 Rate data for urease-polymer assays. Surfactant
concentration used in polymer synthesis Observed Rate (wt % aqueous
phase) (.DELTA.Abs.sub.400 nm/min) * 10.sup.2 0.01 1.5 0.1 2.7 0.5
7.7 2.5 9.1 5.0 8.8 7.5 7.9
[0043] 4. The Nature of the Surfactant
[0044] The above studies demonstrated that retention of enzyme
activity is improved through the use of surfactants in polymer
synthesis. There are, however, many types of surfactants which one
might envision using to synthesize polymers. The broadest
classification of surfactants is based upon the charge of the head
group. The available surfactant pool includes of anionic, cationic,
and non-ionic surfactants. In several studies, two representative
surfactants were selected from each group and employed in polymer
synthesis at a loading of 1 weight percent of the aqueous component
of the synthesis mixture. Polymers without surfactant and without
enzyme were synthesized as controls for the activity assays.
[0045] Subtilisin was used as a model enzyme in these studies. The
procedures described above were employed to synthesize the enzyme
polymers (200 .mu.g subtilisin/gram polymer). Anionic (lauryl
sulfate, octyl sulfate), cationic (cetylpyridinium chloride,
dodecyltrimethylammonium bromide) and non-ionic (Pluronic.TM. F-68
available from BASF Corp., Mount Olive, N.J. and Tween.TM. 20
available from ICI Americas, Wilmington Del.) surfactants were each
used in synthesizing individual enzyme polymer samples. The
polymers were exposed to open air in a fume hood for several hours
before assay to facilitate the removal of residual water and
CO.sub.2.
[0046] The resulting polymers were assayed for their hydrolytic
activity on N-succinyl-ALA-ALA-PRO-PHE p-nitroanilide in 10%
MeOH/50 mM Tris buffer (pH 8.0) solutions. Substrate hydrolysis was
monitored with the use of a spectrophotometer at 400 nm. Reaction
rates were determined by placing polymer samples (100 mg) within 10
ml substrate solutions. The data of FIG. 5 indicate that, while
polymers synthesized without surfactant or with cationic or anionic
surfactants have appreciable catalytic activity (compared to the
corresponding polymer without enzyme), one preferably uses
non-ionic surfactant(s) in polymer synthesis to maximize the
retained activity of the enzyme immobilized therein. Table 3
further illustrates this phenomenon. The relative reaction rates
are ratios based upon the catalytic rates achieved when no
surfactant is employed.
3TABLE 3 Relative catalytic rates when employing different
surfactants in subtilisin polymer synthesis. Surfactant used in
polymer Relative Surfactant synthesis Reaction Classification (1 wt
% of aqueous phase) Rates None 1.0 Anionic Lauryl sulfate 1.0 Octyl
sulfate 1.1 Cationic Cetylpyridinium chloride 1.5
Dodecyltrimethylammonium 1.3 bromide Non-ionic Pluronic F-68 5.8
Tween 20 6.2
[0047] Although the present invention has been described in detail
in connection with the above examples, it is to be understood that
such detail is solely for that purpose and that variations can be
made by those skilled in the art without departing from the spirit
of the invention except as it may be limited by the following
claims.
* * * * *