Note: Descriptions are shown in the official language in which they were submitted.
11 1076047
BACKGROUND OF THE INVENTION
Porous cellulose beads provide a relatively low-
cost, stable material possessing versatile chemical prop-
erties such that they can be useful as a carrier for immo-
bilized enzymes and other active biological agents.
While ordinary cellulose particles and regenerated
cellulose powders meet most of the desired requirements of
good carriers to which enzymes can be immobilized, they
suffer from configural disadvantages which cause column
reactors to become tightly packed resulting in reduction of
flow and sometimes channeling, and thus insufficient contact
between the immobilized enzyme and reaction fluid. The
immobilization of enzymes on an insoluble carrier is a
widely-accepted technique for a practical application of
enzymes, avoiding the necessity of employing fresh enzymes
for each desired use. Through immobilization of the enzyme,
stabilization is achieved which provides for efficient enzyme
use and provides for the design and operation of enzyme reactors
in a continuous mode.
To a large degree, the success of an immobilized
enzyme for use in practical application depends upon the
properties of the carriers employed for immobilization.
Accordingly, a good carrier should meet the requirements of
being inexpensive ar~d should be of such a physical shape
that it is easy to be employed in reactors. In this regard,
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1~76047
the shape of a spherical bead is particularly desirable,
since it is useful in a packed bed, fluidized bed, expanded
bed, stirred tank, or other common types of chemical reactor
designs. Such a carrier should also have the proper physical
and mechanical strength such that it will not be crushed or
deformed when packed in a tall column. Crushing and defor-
mation results in the column becoming tightly packed, thereby
blocking the flow of liquid reagents through the column,
thus decreasing the efficiency of the chemical reactor.
Suitable carriers should also possess versatile chemical
properties such that the immobilization of enzymes and other
biological agents onto the carrier through ionic or chemical
covalent bonding, as well as surface absorption, can be
readily achieved. In this regard, the carrier should have a
high capacity for forming a large number of bonds such that
each unit of the carrier can immobilize large amounts of the
enzyme desired. Thus, a carrier having a high deg~ee of
porosity and uniformly distributed internal void spaces
is particularly desirable. Such porosity provides for
good diffusion of chemical reagents or reaction products
into and out of the internal void spaces of the cellulose
beads. Carriers should be chemically stable, physically
strong, and made of inert material which resists micro-
biological attack causing carrier deterioration in order to
provide an immobilized enzyme system having a prolonged
active life.
~ ~ 1076047
Currently, porous glass and porous ceramic particles
are commonly employed for the immobilization of enzymes and whil
such particles meet most of the above requirements for an
acceptable particle, they are relatively expensive. Furthermore,
S the number of chemical reactions which may be used for immobili-
zation of enzymes to glass and ceramic carriers is limited.
In U. S. Patent Nos. 3,947,325; 3,905,954; 3,573,277;
3,505,299; 3,501t419; 3,397,198; 3,296,000; 3,251,824;
3,236,669; 2,843,583; 2,773,027; 2,543,928 and 2,465,343,
there is described the preparation of a variety of cellulose
materials in a variety of forms, some of which are described
as suitable for use in fixing biologically-active materials
such as enzymes or ion-exchange groups thereto. However, these
processes seem to suffer also from the disadvantage of being
lS expensive and the products obtained generally are of an
undesirable physical shape for use in such chemical reactors as
packed beds and fluidized beds. In particular, the prior art
fails to provide a means for producing spherical shaped cellulose
beads having a uniform distribution of pores throughout the
surface and a large uniformly porous internal void space.
Furthermore, the cellulose particles and powders of the prior
art generally are of such a small particle size that they are
not suited for use in chemical reactors. In addition, the
cellulose powders and particles of the prior art often have a
hard surface skin which causes severe diffusional hinderance and
inefficient use in chemical reactors.
, ~, ..
107~047
In our application, we describe the process
of making highly porous cellulose beads of uniform porosity
which were found highly suitable for immobilizing enzymes.
We have found that these beads may also be useful in the
purification and separation of enzymes, proteins, nucleic
acids and the like. Furthermore, the beads may be useful
to separate metallic ions from dilute solutions containing
same.
Accordingly, the primary object of the present
invention is to provide a means for preparing inexpensive,
highly-porous, stable particles having versatile chemical
properties whereby they may be useful as a carrier to which
enzymes or other biologically-active materials can be
immobilized,
A further object of the present invention is to
provide a method for the transformation of cellulose deri-
vatives into highly-porous particles having good mechanical
stability such that it will provide for adequate passage
of liquid therethrough when operated in packed bed reactors.
Still yet another object of the present invention
is to provide a porous cellulose bead having sufficiently
large surface area to provide high immobilization capacity
of enzymes.
Still a further object of the present invention is
to provide a porous cellulose bead having improved physical
and mechanical strength so that it will not be crushed and
deformed when used in chemical reactors.
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. .
1076047
Yet a further object of the invention is to pro-
vide an improved means for the purification and/or separa-
tion of enzymes, proteins, nucleic acids and the like.
Yet another object of our invention is to provide
a means for the separation of metallic ions from dilute
solutions containing same.
These and other objects of the present invention
will be more fully apparent from the discussion set forth
hereinbelow.
" ` ~ 1076017
DESCRIPTION OF THE INVENTION
According to the present invention, a process is
provided for the preparation of porous cellulose beads
which are suitable for use as a carrier of enzymes and
other biological agents. The invention also provides a
means for the modification of the chemical and physical
property of porous beads made from cellulose derivatives, as
well as techniques for immobilizing enzymes and other biolo-
gical active agents onto the porous beads so formed. While
orginary microcrystalline cellulose and other particles
made from cellulose satisfy many of the general require-
ments for a suitable carrier of enzymes, such particles
suffer from the tendency to pack together tightly under
pressure and also fail to provide sufficient porosity to
attach a sufficiently-large amount of enzymes thereto.
Cellulose derivatives are generally inexpensive and when
treated according to our invention provide a highly-versatile
material for chemical reactions being generally biologi-
cally inert. Thus, the cellulose derivative beads herein
provide many desirable properties for use as a carrier of
immobilized enzymes.
Our process for the modification of the physical
properties of cellulose derivatives, in order to produce
porous cellulose beads, involves the steps of:
a) dissolving a cellulose derivative in an
inert organic, water-miscible solvent to form a solution
having a density greater than that of the precipitation
solution as defined hereinbelow;
1076047
b) distributing said solution in the form of
droplets into a precipitation solution whereby said
cellulose derivative is precipitated in the form of
uniformly porous beads;
c) separating the precipitated beads from
said solution;
d) washing the separated porous beads with
water;
e) hydrolyzing the washed beads to convert
the beads to cellulose and to increase the active sites
for attachment of enzymes and other blological agents;
f) washing the hydrolyzed beads to obtain
porous cellulose beads.
According to the present invention, by dissolving a
cellulose derivative in a selected solvent and distributing
same into a selected precipitation solution, we are able to
produce cellulose beads of high uniform porosity and superior
chemical and physical properties. The beads produced in
accordance with the present invention are highly porous.
The pores are generally uniformly distributed over the
surface and throughout the interior of the bead. By
proper selection of solvents and precipitation solutions,
the pore size of the beads may be controlled. It is of
particular advantage that in accordance with the process we
are able to control both the pore size and pore distribution.
With reference to Figures 2, 4 (A) and (B), it will be seen
that the pore openings are uniformly distributed over the
surface of the bead and were estimated to be about 1,000 A
iO76047
which is a proper size for movement of enzyme and reagent
molecules in the pores.
The inert organic water miscible solvent may be
a single liquid or a combination of liquids. It is important
S that one employ a correct combination of inert organic solvent
and precipitation solution in order to obtain the porous
cellulose beads of desired shape and porosity. The inert
organic water-miscible solvent may be a combination of liquids
which together with the cellulose derivative provide a solution
which when mixed with the precipitating solution results in a
phase inversion whereby the cellulose derivative is coagulated
in the form of a porous bead. The inert organic solvent thus
contains a component (a) which is characterized as a liquid
which is capable of dissolving the cellulose derivative, such
as cellulose acetate, and is soluble in the precipitation
solution.
A second component (b) of the solvent system is a
liquid which is soluble in component (a) and also in the
precipitation solution and which is present in the solvent
solution in an amount sufficient that the density of the final
solvent solution (together with the cellulose derivative) is
sufficiently higher than the density of the precipitation
solution so that upon distributing the solvent solution in the
form of droplets into the precipitation solution the cellulose
will coagulate and precipitate out as a porous bead of desired
size and porosity. Component (b) of the solvent is used to
control the surface activity of the solvent solution such that
the droplets of solvent solution will maintain their shape
1076047
upon contact with the precipitation solution. Component ~b)
also sexves to control the pore size and porosity of the
precipitated beads. In some instances, component (a) and
component (b) may be the same. In other instances, it may
be appropriate to employ one or more liquids in preparing
component (a) and/or componen~ (b).
As used herein, the term "precipitation solution"
is defined as a liquid solution which is a non-solvent for
the cellulose derivative and is miscible with the above inert
organic, water-mis¢ible solvent. ~y means of illustration,
the precipitation solution may be w~ter or an aqueous
solution. The precipitation solution thus is miscible
with both solvent components (a) and (b). Thus, it will
be appreciated that when one dissolves the cellulose
derivative in tbe organic solvent, and subsequently adds a
drop of the resulting solvent solution to the precipitation
solution, the cellulose derivative will coagulate and
precipitate out due to the phase inversion which the cellulose
derivative undergoes thereby forming the desired porous
cellulose bead.
As will be apparent from the discussion herein, a
number of variations are possible in the above-described process
in preparing the desired porous cellulose beads. In
addition to cellulose acetate, other cellulose derivatives
may be employed as a starting material for the preparation
of the porous beads, for example, cellulose nitrate and methyl
cellulose. The terms "cellulose derivative" and "hydrolyzable
cellulose derivative" as used herein are intended to include
materials from which cellulose may be regenerated such
as by means of, for example, hydrolysis or hydrogenation.
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1076047
The organic solvent components (a) and (b) for
the cellulose derivative can vary, but should be chemically
inert to the cellulose derivative and wholly or substantially
miscible with the precipitation solution. It is of prime
importance that the density of the solvent solution formed
by adding the cellulose derivative to the inert solvent be
greater than that of the precipitation solution into which
it is distributed such that when droplets of the solvent
solution are distributed into the precipitation solution,
the droplets will sink when the aqueous solution is not
agitated. Suitable single solvents, when using an aqueous
precipitation solution, include among others, for example,
dimethylsulfoxide and methyl acetate. It should be understood
that commercially available materials may be employed as
solvent components (a) and/or (b), and that these materials
may contain moisture, which in some in tances has been found
to be advantageous.
When employing an aqueous precipitation solution,
one may suitable use as solvent component (a) a member
from the group consisting of acetone, formamide a mixture
of acetone and methanol or ethanol, methyl acetate, a mixture
of methylene dichloride and methanol, methyl ethyl ketone
and dimethyl sulfoxide. The solvent component (b) may thus
be suitably chosen from a member selected from the group
consisting of dimethyl sulfoxide, formamide, methyl acetate,
cyclohexanone, methylene dichloride, ethylene dichloride, a
mixture of methylene dichloride and methanol, and a mixture
o ethylene dichloride and methanol.
-11- 1
- 107604'7
A preferred solvent component ~a) is acetone, but other
solvents can be suitably employed, and when using an aqueous
precipitation solution one may select a component (a) from the
following materials (the ratio of mixtures being the minimum ratio
desirable on a volume basis):
Component (a) Minimum Ratio
(Volume)
Acetone ----
Acetone + Methanol 60:40
Acetone + Ethanol 60:40
Methyl acetate ----
Methylene dichloride + Methanol 80:20
Dimethyl Sulfoxide ----
Methyl Ethyl Ketohe ----
Formamide ----
As noted above, the primary function of component (a) is to
disslove the cellulose derivative. The addition of component (b)
is necessary in order to provide a solvent solution having the
requisite density such that the cellulose derivative will
; 20 precipitate out in the precipitation solution. Component (b) also
provides for the control of pore size and uniform porosity of the
beads.
The solvent component (b) therefore provides for the
desired specific gravity of the solvent solution and when employing
an aqueous precipitation solution it is preferred to use dimethyl
sulfoxide as component (b). As will be appreciated, in some
instances component (a) and component (b) may be the same, i.e.
dimethyl sulfoxide, formamide or methyl acetate when used with
aqueous precipitation solutions. Various materials which may
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'.` 1076(~47
be used suitably as component (b) when employing an aqueous
precipitation solution are outlined below.
Component_(b) Minimum Ratio
(Volume)
Dimethyl sulfoxide
Ethylene dichloride ~ methanol 60:40
Methylene dichloride + methanol 60:40
Ethylene dichloride
Methylene dichloride ----
Formamide ----
Cyclohexanone ----
-¦ The solution of cellulose derivative and inert
solvent should have a controlled cellulose derivative-to-
I solvent ratio since such will have an effect on the eventual
15 ¦ porosity of the beads prepared. Generally, a small ratio
(larger content of solvent) results in beads having a larger
porosity. A cellulose-to-solvent (including components (a)
and (b) ratio of from 1:20 and 1:3 (weight/volume) has been
found suitable for preparing cellulose beads having various
specific applications. Preferably, a cellulose derivative-
to-solvent ratio of 1:10 to 1:6 (weight/volume)is employed
to provide an easy-to-handle solution which results in porous
cellulose beads of desirable properties having a void space
of at least 50~ by volume, preferably 75 to 95% and most
suitably about 75 to 80%. Beads having a higher porosity
will generally have a larger proportion of uniformly
distributed internal void spaces providing less diffusion
hindrance, but will be somewhat weaker in physical strength
than beads of lower porosity.
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10~6047
The preferred precipitation solution into which
the solution of cellulose derivative is to be distributed
generally consists of water, but may be an aqueous solution
which contains suitable amounts of non-ionic or ionic sur-
factants to reduce the surface tension thereof and facilitate
formation of the porous beads. The precipitation solution
can also suitably contain a mixture of water and methanol
or ethanol (volume ratio 50:50). It is also envisioned that
the precipitation may be non-aqueous so long as the cellulose
derivative is insoluble therein and the necessary density
requirement is met. Thus, hydrocarbon solutions may be used
such as cyclohexane, hexane, decane, benzene and the like so
long as they are liquid in form, possess a density less than
that of the inert organic solvent and are miscible therewith.
When the cellulose derivative solution is distributed by
spraying via a suitable means such as a spray nozzle, the
pressure drop and miscibility of the inert solvent in the
aqueous solution results in a dispersion and ultimate precipi-
tation of porous beads of the cellulose derivative.
As will be appraciated by those skilled in the art,
in precipitating the cellulose beads, a sufficient amount of
solvent component; (b) must be present in order that the solvent
containing cellulose derivative possess the requisite higher
density than that of the precipitation solution. Table 1
ZS sets for a numbe~ of inert organic solvents for tbe precipi-
` 1076047
tating of a cellulose derivative in an aqueous solution.
The ratios set forth are the minimum needed in order to pro-
vide a solvent solution having a density greater than that of
water. As can be seen, the greater the specific gravity of
component (b), the less of that component is needed in order
to achieve the minimum density.
TABLE 1
Solvent Minimum
Volume Ratio
Component (a) Component (b) a:b
Acetone Dirnethyl sulfoxide 70:30
Acetone Ethylene dichloride 80:20
Acetone Methylene dichloride 80:20
Acetone Formamide 75:25
I5 Acetone Cyclohexanone 45:55
Acetone Methyl acetate 35:65
After precipitation of the porous beads, cellulose
is regenerated from the derivative by hydrolysis in order to
create more active sites for enzyme attachment. In regenerating
cellulose from its derivative after formation of the beads,
one can remove the substituting groups (such as acetate from
cellulose acetate) in order to regenerate all the hydroxyl
groups normally present in the cellulose material. The
higher the degree of regeneration, the more stability is to be
found in the resulting beads. In some cases, wherein enzymes
are to be immobilized on the cellulose bead carriers, it is
desirable to convert the hydroxy or substituting groups into
functional chemical groups, such as amino groups, which facilitate
enzyme attachment.
~076047
BRIEF DESCRIPTION OF THE DR~WINGS
FIGURE 1 is a illustration of the particle size
distribution of the porous beads.
FIGURE 2 is a scanning el~ctromic~rograph of a porous
cellulose bead.
FIGURE 3 is a plot of the pressure-drop-characteristics
of the porous cellulose beads as illustrated on the same page
as Figure 1,
FIGURE 4 (A) is a scanning electron michrograph of the
surface of a porous cellulose bead (20,000x).
FIGURE 4 (B) is a scanning electron micrograph of the
interior of a porous cellulose bead (20,000x).
Reference is made to Figure 1 which illustrates the size
distribution of the final porous beads obtained by distributing
(by spraying) a solution of cellulose derivative through a spray
nozzle, according to the detailed procedure outlined hereinbelow.
Beads which are either too large or too small, depending upon the
intended end use, may be collected and re-dissolved in the
appropriate solvent, if desired. Generally speaking, if employed
in a column type chemical reactor, beads of a uniform size are
preferred. The desired particle size may vary depending on the
projected use of the beads, e.g. the type of enzyme to be
immobilized.
The porous cellulose beads prepared by the process
described above generally have a very high porosity and a
controlled pore size ranging from 0.05 to 30 microns. When a
cellulose-to-solvent ratio of 1:10 (weight/volume) is used in ~.
preparing the cellulose/solvent solution, the final beads formed
- 16 -
:: :
-`` 1076047
have a high porosity of about 90~ void. A scanning electro- -
micrograph of a porous cellulose bead prepared by the process
shown in Figures 2, 4(A) and 4(B). From these views, one can
observe several important features of the beads produced. Firstly,
it can be seen that the beads are generally spherical in shape and
porous openings are uniformly distributed over thè surface of the
beads. For most applications, this is desirable because it can
provide an immobilized en~yme catalyst of uniform activity. The
void phase of the cellulose beads is continuous. This is a desir-
able feature because a discontinuous, discrete "bubble" would
result in useless and nonaccessible dead space in an immobilized
enzyme system. Thirdly, there is no hard "skin" at the bead
surface. A hard skin will cause serious diffusional hindrance.
Finally, the pore sizes are quite uniform. As a result, all of
the interior surface area of the internal void spaces of the beads
will be accessible for enzymé immobilization and for enzyme
catalyzed reactions. Both the high porosity and other noted
features have made the porous cellulose beads of this invention
uniquely suited for use in immobilization of enzymes and other
biologically-active agents.
An important property of an enzyme carrier is the
pressure drop it causes at various liquid flow rates through an
enzyme reactor containing the carrier. For example, DEAE-cellulose
is currently used in industry and an enzyme carrier for the
conversion of glucose into fructose. For DEAE-cellulose , the
pressure drop is very high and consequently only shallow beds can
be used to obtain a reasonable
- 17 -
; ' , . ~ ~ : ' '
~` ' ;` `'
.: . ' ::.:'
107~>047
rate of fluid flow. The pressure drop characteristics of
the porous cellulose beads of this invention in a packed
column operation is shown by Curve A in Figure 3. The
nominal linear flow velocity is calculated by dividing the
volumetric flow rate of the feed liquid to the column by the
column cross-sectional area. In practical operations, the
nominal linear flow velocity in industrial column reactors
will be less than 0.5 cm/sec. For example, with a reactor
column of two feet (60.96 cm) inside diameter, a linear
velocity of 0.5 cm/sec is equivalent to a volumetric flow
rate of 1389 gal/hr (5254 liters/hr). In a typical indus-
trial operation for producing fructose from glucose, the
sugar concentration in the feed is about 5 lb. sugar/gallon.
The above flow rate will yield more than 60 million pounds
of the product per 2 feet column per year. Because of the
residence time requirement of the enzymatic reaction, the
linear flow rate is usually less than 0.5 cm/sec. Therefore,
it can be seen that the porous cellulose beads of this
invention do not pose any serious engineering problems with
regard to ~ressure drop, when used in column type chemical
reactors as a carrier to which enzymes and other biologically-
active agents can be immobilized. When the porous cellulose
beads, after proper derivatization, are used for other poten-
tial applications (e.g. removal of tannin from fruit juice,
wine or beer as well as metallic ions from dilute solutions)
the liquid flow rate through a reactor column could be much
larger than that of 0.5 ~m/sec cited here.
1076047
The flow characteristics and other physical and
mechanical properties of the porous cellulose beads can be
improved by cross-linking with bi- and/or mult~-functional
compounds. Curve B in Figure 3 shows the pressure drop
requirement of the porous cellulose beads after the treat-
ment with tolylene-2, 4-diisocyanate and enzyme immobilization.
Above a nominal linear velocity of 2 cm/sec, the untreated
cellulose beads (Curve A) become compressed and deformed
considerably, resulting in a drastic increase of the pressure
drop. Curve B concaves upward only slightly indicating
little deformation, if any, of the treated beads.
Treatment of the porous cellulose beads with a
cross-linking agent, either before or after hydrolysis of
the beads, results in an increase of their physical strength.
Attachment of enzymes onto the beads will also increase
their physical strength. After treatment with, for example,
a diisocyanate (e.g., tolylene-2, 4-diisocyanate or hexa-
methylene diisocyanate), the beads in fact become quite
rigid and strong. Cross-linking with epichlorohydrin also
improves the physical properties of the porous cellulose
beads. The chemistry of cross-linking of polysaccharides,
including cellulose and starch, is a well-developed branch
of physical science. Other suitable cross-linking agents
among othersinclude formaldehyde in hydrochloric acid
solution or glutaraldehyde. Many other carbohydrate cross-
linXing agents are well known, as shown, for example, by
Jones et al, U. S. Patent No. 3,905,954.
1076047
In general, the porous beads of the present
invention are prepared according to the following steps:
a) a hydrolyzable form of cellulose is dissolved
in an inert organic water-miscible solvent in a controlled
ratio of cellulose derivative-to-solvent which is generally
in the range of 1:20 to 1:3 (weight:volume) to produce a
solvent solution. The solvent should be wholly or substan-
tially miscible with the precipitation solution and the
density of the -solvent solution should be sufficient that
upon contact with the precipitation solution, the solvent
becomes readily miscible with the precipitation solution and
the cellulose derivative precipitates therein.
b) a solvent solution is distributed (e.g., by
spraying) in the form of droplets into a precipitation
solution. Upon contact with the precipitation solution,
which may contain a surfactant, the solvent is dispersed
within the solution media and porous beads of the cellulose
material fonm as they coagulate and precipitate to the
bottom of the tank holding the precipitation solution. The
cellulose derivative solution may suitably be sprayed under
pressure through an atomizing nozzle into a precipitation
solution bath. If desired, the bath may be agitated to
enhance the formation of the beads.
c) the precipitated beads, after being washed,
are then hydrolyzed in order to regenerate cellulose,
1076047
thereby providing a porous cellulose bead having active
sites for enzyme attachment. If desired, in order to
increase the stability of the porous beads or provide
suitable reaction sites, one can chemically modify the beads
in a number of ways. For example, the beads may be cross-
linked in order to provide greater stability and increased
physical strength. Also, one can chemically substitute
either positively-charged or negatively-charged groups to
alter the surface-absorption properties of the cellulose
bead. The cellulose itself is generally hydrophilic and,
thus, by altering the reaction sites thereof, one can alter
its hydrophilic properties.
The present invention further provides for a
method by which enzymes and other biological active agents
may be immobilized by attachment onto the porous cellulose
beads described hereinbefore. For example, one may convert
porous cellulose beads, as described above, to diethylamino-
ethyl ~DEAE) cellulose by reacting said beads with N,N-
diethyl 2-chloroethylamine hydrochloride in a conventional
manner. Beads so obtained contain DEAE-cellulose and were
successfully used to attach glucose isomerase, derived from
a streptomyces culture. We have also employed a procedure
involving cyanogen bromide to immobilize the glucose isomerase.
Another procedure for enzyme immobilization on the
porous cellulose beads involves the use of tolylene-2,4-
diisocyanate. Diisocyanate was employed to cross-link
cellulose to improve the physical strength of the porous
1076047
beads. However, we have found that the porous cellulose
beads of the present invention when treated with diisocyanate,
can immobilize enzymes on the surface thereof by simply
mixing the diisocyanate-treated beads together with an
enzyme solution. For example, when glucoamylase was used,
the diisocyanate beads attached more than 1000 international
units of the enzyme per gram of dry beads. While not wishing
to be limited in any way by the following theory, it appears
that when dry porous cellulose beads are in dry acetone with
tolylene-2,4-diisocyanate in the presence of a catalyst (for
example, triethylamine), a considerable degree of cross-
linking occurs between cellulose molecules in light of the :
improved physical strength of the beads. After a sufficient
length of time for reaction, the beads were washed with dry
acetone to remove free diisocyanate residues. The cellulose
beads appear to possess a large number of attached isocyanate
groups. Upon mixing the treated beads with an aqueous
enzyme solution, enzyme molecules appear to be covalently
bonded to the cellulose beads through the isocyanate groups.
It has also been found that washing the treated beads with
water results in converting isocyanate groups to amino
groups. In such a manner, we were successful in immobi-
lizing an enzyme, glucoamylase, to the amino cellulose beads
with glutaraldehyde, an agent well known for its capability
of reacting and cross-lin~ing amino groups (on the beads and
the enzyme).
10 76047
The porous cellulose beads produced ~n accordance
with the present invention also find use in the separation
and purification of enzymes, proteins, nucleic acids and
the like. The porous cellulose beads produced in accordance
with the process of the present invention may be derivatized
to produce DEAE-porous cellulose beads which possess
excellent flow properties and yet are able to efffectively
separate enæymes, proteins, nucleic acids and the like as
effectively as current commercial products according to
the technique known as column chromatography.
Also, one may derivatize the porous cellulose
beads of the p.esent invention (in situ) with groups other
than DEAE. Thus, the porous cellulose beads of the present
invention are applicable for a wide variety of specific
applications. For example, one can attach a specific
functional group to the porous cellulose beads and the then
derivatized beads may be used to, for example, remove
tannin from fruit juice by passing the juice through a
bed of the derivatized porous cellulose beads with protein.
In a similar fashion, one may remove metallic ions
from dilute solutions containing same. Such a method would
provide for the recovery of valuable metallic ions ~i.e.,
copper ions and gold ions) from dilute mining solutions,
and would find particular applicability to current solution
mining techniques whereby metals are extracted from ores by
acid solutions.
1076047
The following examples are offered to more fully
describe the invention, but are not to be construed as
limiting the scope thereof:
EXAMPLE I
Fifty (50) grams of cellulose acetate ~Visc 3 from
Eastman Xodak Chemicals) were dissolved in 400 ml of solvent
A (composed of acetone and dimethyl sulfoxide in a volume
ratio of 6-to-4) to form a 12.5% (weight/ volume) solution.
With a spray gun (paint sprayer from Sears Roebuck & Co.),
the cellulose solution was then sprayed at an air pressure
of 20 psi as fine droplets into a water tank containing 40
gallons of water and four drops of common household detergent.
Upon contacting the surface of the water, the cellulose
acetate droplets coagulate into porous beads and sink to the
bottom. The porous beads were then collected and washed.
The washed beads were then deacetylated with about a; 0.15 N
of sodium hydroxide overnight at room temperature. The
deacetylated beads were then washed and suction-dried,
yielding a porous cellulose bead having a void space greater
than 50~ by volume ready for use in enzyme immobilization.
Figure 1 illustrates the size distribution of the porous
beads obtained. Electron micrographs revealed that the
beads were generally spherical, with the interior and surface
thereof having the same structure. The pore sizes were quite
uniform and the pores were distributed uniformly throughout
the entire bead as illustrated in Figures 2, 4(A) and 4(B).
-24-
`` 1076047
The pore size of the beads was determined from scanning
electron micrographs. The scanning micrographing requires
dry samples and since the drying of the beads in air results
in a size shrinkage, the beads were dried by the critical
point technique with liquid carbon dioxide. ~he pore size
was determined to be about 1000 A .
EXAMPLE II
Using a 10~ (weight/volume) cellulose acetate
solution in solvent A, according to the process of Example
I, porous beads were also formed and were suitable for use
in enzyme immobilization.
EXAMPLE III
A 10~ (weight/volume) cellulose acetate (Visc 3
from Eastman Kodak Chemicals) solution was prepared in
solvent B (acetone and formamide in a volume ration of
7-to-3). The cellulose acetate soluti~n was then sprayed
and hydrolyzed according to the procedure in Example I
above. ~ighly porous cellulose beads were obtained having a
void space greater than 50~ by volume.
EXAMPLE IV
The procedures outlined in Example II, above, were
repeated using a solution prepared with cellulose acetate of
Visc 45 type (available from Eastman Kodak Chemicals).
Porous beads were also obtained having excellent properties
for enzyme immobilization.
1076047
EXAMPLE V
The procedures outlined in Example II, above, were
carried out using a 10% weight /volume solutio~ of cellulose
triacetate (available from Eastman Kodak Chemicals) in -?
solvent A. The beads resulting therefrom exhibited excellent
porosity for enzyme immobilization. As we have noted,
cellulose can be used as a supporting material for the
immobilization of enzymes and other biologically active
agents, Many workers have chosen cellulose as a support
because cellulose is inexpensive, chemically stable, and it
is resistant to microbiological contamination. Also, cellulose
has three hydroxyl groups on each anhydro-glucose unit which
provides high versatility as well as large capacity for the
immobilization of a desired substance.
The major disadvantage of using cellulose as a
supporting material is that cellulose has a fibrous shape
and lacks the necessary mechanical strength. ~eactors packed
with cellulose have poor flow properties, develop severely
high pressure drop, and sometimes channelling. To overcome
these problems, we prepared cellulose into a bead form
according to the present invention which exhibited a better
mechanical strength and provided enhanced flow properties than
prior materials. However, since the structure of our cellulose
beads differs from that of regular cellulose, the loading
of enzymes and stability of the immobilized enzymes may differ
from that with regula; cellulose. The chemistry involved
in the preparation of immobilized enzymes not only affects
the loading and stability of the enzyme on the cellulose
beads, but also affects the mechanical strength of the
~076047
cellulose beads. Any chemical procedures for immobilization
of enzymes, which increase mechanical strength of cellulose
beads, would improve the flow properties in a reactor, as will
be apparent from the examples.
EXAMPLE VI
One (1) gram of porous cellulose beads, produced
according to Example I, was dispersed in 15 ml water which
was adjusted to pH 11.5 with sodium hydroxide and kept at
a constant temperature of 20C. One (1) gram of cyanogen
bromide was added to this dispersion. The pH was maintained
at 11.5 with 1 N NaOH. After lS minutes, the beads were
washed with a phosphate buffer (0.1 M) at pH = 7.0 and 0C.
Fifteen (15) ml of glucoamylase solution (30 mg/ml) were
then added to the beads. The mixture was left overnight.
The beads so prepared contained 1830 units of enzyme activity
per gram dry weight of cellulose bead at 60C. using 5~
maltose as substrate. One unit of enzyme activity is defined
to be that which produces one micromole of product per
minute.
EXAMPLE VII
Porous cellulose beads (0.2 gm), obtained as in
Example I, were dispersed in 5 ml acetone. 0.2 ml tri-
ethylamine was added to the dispersion as was 0.2 ml of
tolylene-2,4-diisocyanate. After 30 minutes, the beads were
washed with acetone and then an acetate ~buffer at pH 4.75.
Five (5) ml of glucoamylase solution (25 mg/ml) were added.
107604~7
The enzyme was thereby immobilized on the beads with an
activity of 2,000 units/gm cellulose beads.
EXAMPLE VIII
Two hundred (200) mg glucose isomerase in maleic
acid buffer solution was immobilized onto 2 gm of cellulose
beads by the same procedure as described in Example VII.
The cellulose beads contained 90 units of enzyme activity
per gm of cellulose beads at 60 C. using 9~ fructose as
. the substrate.
EXAMPLE IX
Three hundred (300) mg of invertase in 5 ml of
acetate buffer were immobilized onto 0.5 gm of porous
cellulose beads using the procedure described in Example
VII. The cellulose beads contained 3000 units activity per
lS gm of cellulose used.
EXAMPLE X
Fifty (50) mg of lactase in phosphate buffer
(pH = 7.0) were immobilized onto 0.5 gm cellulose beads
using the procedure, described in Example VI. The resulting
cellulose beads contained about 80 units enzyme activity per
. gm of cellulose beads at 30C. using 1% lactose as substrate.
10760 17
EXAMæLE XI
Five hundred (500) mg of glucose isomerase were
dissolved in 150 ml maleic acid buffer (0.01 M, pH = 5.5).
The enzyme solution was pumped through 5 gm porous cross-
linked cellulose beads prepared as described in Example XVI.
The DEAE cellulose beads thus contained 100 units of enzyme
activity per gm of beads.
EXAMPLE XII
One-quarter ~0.25) gm of porous cellulose beads,
produced in accordance with Example I, was soaked in 3% of
glutaraldehyde and 0.1 M MgC12. After drying, using vacuum
suction on a Buchner funnel, the samples were heated at 80
C. for 30 minutes. Five (5) ml of glucoamylase (25 mg/ml)
were added to the beads. After standing overnight, the
beads thus prepared contained about 200 units of enzyme
activity per gm of dry cellulose beads.
EXAMæLE XIII
One (1) gm of porous cellulose beads was cyano-
ethylated with 10 ml acrylonitrile (C = CC~N) at 50C. The
so-treated cellulose beads were then treated with hydorxylamine
at a pH 6.5 - 6.7 at 50 - 100C. for 4 hours. The
resulting modified porous bead product contained
NH2
- C = NOH groups and is suitable for absorbing heavy ions
such as ferric, ferrous, and cupric.
. '
-29-
107604~
EXAMPLE XIV
A suspension of 2.5 gm porous cellulose beads was
treated with 2.5 ml hexamethylene diisocyanate and triethyl-
amine, followed by hydrolysis in water. The product was then
S treated with 50 ml of 0.5 M 0- methyl iso-urea at pH 5. The
product obtained has the following funtional group:
~L NH2~3
N - C - NH2
which is useful as an anionic ion exchanger.
EXAMPLE XV
Five (5) grams of porous cellulose beads, obtained
according to Example I, were added to 100 ml of 36% formal-
dehyde and 200 ml of 37~ hydrochloric acid. After standing
for 1-1/2 hours at room temperature, the beads were filtered
and subsequently washed with water and 0.2% sodium carbonate
solution. The beads were then dried at 75 to 80C. The
resulting cross-linked porous cellulose beads exhibited
strong physical strength.
- EXAMPLE XVI
Three ~3) grams of porous cellulose beads were
cross-linked by formaldehyde according to the process in
Example XV. The beads were then treated with 3 grams of
2-chlorotriethylamine. After heating the mixture for a period
of 35 minutes at a temperature of 80 to 85C., the beads
were then washed sequentially with sodium chloride, sodium
hydroxide, hydrochloric acid, water and ethanol. The
cross-linked porous DEAE cellulose beads so obtained exhibited
1076047
excellent porosity having a void space greater than 50% by
volume.
- EXAMPLE XVII
A dispersion was formed of 0.5 grams porous cellulose
beads in 5 ml of 0.2 N sodium hydroxide and 5 ml epichlorohydrin.
The dispersion was then heated for several minutes to a
temperature of 80C. Subsequently, the beads were washed
and the cross-linked porous beads so treated exhibited greater
strength than the porous cellulose beads prior to cross-
linking. Wet cellulose beads, obtained according to the
procedure of Example I, were washed in acetone. The washed
beads were then suspended in dry acetone containing 0.6 ml of
triethylamine for each gram of cellulose. Tolylene-2,4-
diisocyanate, in an amount of 1.6 ml per gram of cellulose
beads was added to the suspension at 0C. After a period of
30 minutes, the beads were washed with dry acetone and sub-
sequently filtered. The resulting porous cellulose beads
contain isocyanate-reactive groups which could then be
hydrolyzed to an amino group by the addition of water.
EXAMPLE XVIII
Two tenths g of the cellulose beads produced as in
Example I were suspended in 10 ml of distilled water, the pH
was adjusted to 11.5 by the addition of 1 N NaOH at 20C.
Two tenths g of CN~r was added to the cellulose beads suspension,
a small portion at a time, and the pH was maintained by an
auto-titratometer with 1 N NaOH. After 20 minutes the beads
were washed with ice cold distilled water and an appropriate
-
1076047
buffer solution. Enzymes dissolved in a proper buffer solution
were added to the washed cellulose beads. Cellulose (Solka
floc) used in this method was mercerized with 18~ (w/v) NaOH
for four hours then washed with distilled water.
EXAMPLE XIX
Two g of suction dried cellulose beads of Example I
were washed with acetone to remove moisture and were suspended
in 10 ml of acetone. One tenth ml of triethylamine or
dibutyltin diacetate were added as catalyst. One tenth ml
of tolylene-2,4-diisocyanate or hexamethylene diisocyanate
were added to the cellulose bead suspension. After 45 minutes
of reaction at ambient temperature, the cellulose beads
were washed with acetone to remove excess diisocyanate and
-i water was then used to wash the cellulose beads to remove
acetone. Enzymes dissolved in an appropriate buffer solution
were added to the cellulose beads. The cellulose beads were
stored at 4C overnight.
EXAMPLE XX
Aryl diisocyanate was attached to cellulose beads
as described in Example XIX. Before the enzyme solution was
added, the cellulose beads were suspended in distilled water.
One tenth ml of triethylamine was added to catalyæe the
2S reaction between isocyanate and water to form aryl amine
cellulose beads. The arylamine derivative was then diazotized
with NaN02 in HCl. Enzymes suspended in a proper buffer
solution were then attached to the cellulose beads.
~ ~V7604
ExAMæLE XXI
Diisocyanate attached on the cellulose according to
Example XIX reac~s with water to form amino group with or
without a tertiary amine as catalyst. Glutaraldehyde is
used to couple the enzyme onto the cellulose beads by cross-
linking amino groups on enzymes and on cellulose beads.
EXAMPLE XXII
Two g of suction-dried beads produced in accordance
with Example I were suspended in 10 ml of 3% glutaraldehyde
which was 0.1 M in Mg C12. The suspension was heated at
100C for 30 minutes. The cellulose beads were then washed
with distilled water. Enzymes dissolved in an appropriate
buffer solution were added to the cellulose beads. The reaction
was allowed to continue overnight at 4C.
EXAMPLE XXIII
One g of cellulose beads produced according to the
procedure of Example I was refluxed with 10ml of 10% 3-
aminopropyltriethoxysilane in toluene for 4 hours. The
cellulose beads were then filtered and washed with acetone.
2.5% (w/v) glutaraldehyde solution in 0.1 M phosphate buffer
pH = 7.0) was added to the cellulose beads at ambient temper-
ature for one hour with occasional stirring. The cellulose
beads were then washed thoroughly with water and an appropriate
buffer solution. Enzymes dissolved in an appropriate buffer
solution were added to the cellulose beads. The reaction was
allowed to continue overnight at 4C.
1076047
EXAMPLE XXIV
Porous cellulose beads produced in accordance with
the procedure of Example I were first cross-linked by 36%
formaldehyde and 37% HCl with a volume ratio of 5 to 1. The
crosslinked cellulose beads (S g dry weight) were suspended
in 50 ml of cold 1.5 N NaOH solution. Six g of 2-Chloro-
triethylamine hydrochloride were added to the cellulose beads.
The mixture was then heated at 80 - 85C for 35 minutes. The
mixture was cooled in an ice bath and filtered. The celiulose
beads were washed with 500 ml of 2M NaCl and then were washed
with 200 ml of lN NaOH and 200 ml of lN NaOH, alternately
for three times. After washing with another 200 ml of lN NaOH,
the cellulose beads were washed with distilled water until
T5 the pH of washing water became neutral. The reaction with
2-chlorotriethylamine hydrochloride was repeated again for a
higher degree of substitution. Enzymes dissolved in an
appropriate buffer were added to the cellulose beads for
overnight at 4C.
EXAMPLE XXV
Hexamethylene diisocyanate was attached to cellulose
beads as described in Example XIV and then the isocyanate
groups were hydrolized to form amino group as described in
Example XX. O-methyl isourea was added to the cellulose beads
to incorporate the guanidino function into the derivatized
beads.
76047
Examples XVIII through XXIII provide for immobiliza-
tion of enzymes by covalent bonding, whereas Examples XXIV
and XXV utilize ionic adsorption. Glucoamylase, glucose
isomerase and invertase were loaded onto various of the beads
of Examples XVIII to XXV and the amount of enzyme loading
measured.
The enzymes immobilized by covalent bonding were
washed with 2M NaCl solution to remove the absorbed enzymes.
Some properties of the immobilized enzymes are shown in Table
2. It indicates that the same chemistry used for regular
cellulose can also be used for cellulose beads. The fact that
cellulose beads have higher enzyme loading capacity than
regular cellulose may indicate a larger surface area in the
porous cellulose beads.
EXAMæLE XXVI
Protein and enzymes may be separated and purified
according to the following procedure. 2 gm of glucose
isomerase (Strep. albus obtained from Miles Laboratory) was
suspended in 20 ml of O.OlM phosphate buffer (pH = 7.0) and
the suspension centrifuged. The supernatant was added to a
column of DEAE-porous cellulose beads produced according to
Example XVI. The bed volume was 30 ml and the column diameter
1.5 cm. The column was washed with O.OlM phosphate buffer
(pH = 7.0). The column was eluded with NaCl gradient solution
in 0.01 M phosphate buffer. Glucose isomerase began to
elude out of the column in the NaCl fractions with concentra-
tions ranging from 0.25 to 0.45 M.
Il 107~047
Table 2 Enzyme Loadin~ on Porous Cellulose Beads
Enzyme loading on
cellulose,
IU*/g (~alculated
Method of from initial
Enzymes Immobili- reaction rate) Assay conditions
zation Porous
Example regular Fellulose
cellulose beads
XVIII 8201,800 10% Maltose, 60C
xrx 550 ll
Glucoamylase XX 275 10/~ Maltose, 40C
(A. Oryzae) XXI 530 5% Maltose, 60C
. XXII 80190 10% Maltose, 60.. C
XXIII 200
Glucoamylase XXIV3,000 9,000
(A. Niger)XXV 1,000
XIX 90 0.5M Fructose,60C
Glucose
IsomeraseXXIV 300 ,.
albusj XXV 160
InvertaseXIX 1,140 0.125M Sucrose,45C
(Cand daXXIV 2,000 . ,.
XXV 1,840 ~.
*IU - international units
1076~47
EXAMPLE XXVII
The porous cellulose beads of Example XIII are added
to a 0.05 M sodium acetate solution (pH = 5.2) which contains
1,600 ppm of cupric ion. After one hour, the cellulose beads
picked up 6.3% cupric ion by weight of the beads.
We have also found that when the porous cellulose
beads of the present invention are dried and/or heated, e.g.
at 100C prior to use, the resulting beads exhibit an increased
physical strength.
The invention, in its broadest aspects, is not
limited to the specific details shown and described, but
departures may be made from such details within the scope
of the accompanying claims without departing from the principles
of the invention. Furthermore, the invention may comprise,
consist, or consist essentially, of the hereinbefore-recited
materials and steps.
