Abstrict An insolubilized mineral-supported enzyme composite having outstanding
catalytic activity and mechanical stability comprises an enzyme
covalently attached to silanized porous, attrition-resistant granules
of heat-activated attapulgite clay.
Claims I claim:
1. An insolubilized enzymatically active composite in the form
of mechanically stable particles comprising sized granules essentially
all of which are larger than 50 microns and smaller than 1000 microns
of heat-activated attapulgite clay having a volatile matter content
below 18% by weight, a surface area in the range of about 100 to
150 m.sup.2 /g. as determined by the nitrogen absorption method
of Brunauer, Emmett and Teller and a porosity in the range of about
0.5 to 0.6 ml./g. wherein the pore distribution is such that the
major contribution to the total pore volume is derived from pores
with radii larger than 500 Angstrom units, an organosilane coated
on the surface of said granules, a crosslinking agent covalently
bound to said organosilane and an enzyme covalently bound to said
crosslinking agent.
2. The composite of claim 1 wherein said organosilane is an omega-aminoalkyltrialkoxysilane
and the crosslinking agent is an aldehyde.
3. The composite of claim 2 wherein the aldehyde is glutaraldehyde.
4. The composite of claim 1 wherein the enzyme is glucose oxidase.
5. The composite of claim 1 wherein the enzyme is catalase.
6. The composite of claim 2 wherein the enzyme is glucose oxidase.
7. The composite of claim 2 wherein the enzyme is catalase.
8. An insolubilized enzymatically active composite in the form
of mechanically stabilized particles comprising sized granules essentially
all of which are in the range of 50 to 1000 microns of heat-activated
attapulgite clay having a volatile matter content below 18% by weight,
a surface area in the range of about 100 to 150 m.sup.2 /g. as determined
by the nitrogen absorption method of Brunauer, Emmett and Teller
and a porosity in the range of about 0.5 to 0.6 ml./g. wherein the
pore distribution is such that the major contribution to the total
pore volume is derived from pores with radii larger than 500 Angstrom
units, gamma-aminopropyltrioxysilane coated on the surface of said
granules, glutaraldehyde covalently bound to said silane and a redox
enzyme bound to said crosslinking agent.
9. The composite of claim 8 wherein said redox enzyme is glucose
oxidase.
10. The composite of claim 8 wherein said redox enzyme is catalase.
11. The composite of claim 1 wherein said heat-activated attapulgite
clay has a volatile matter content in the range of about 6% to 10%
by weight and contains no free moisture and the granules have a
particle size of about 125 microns to about 250 microns.
12. The composite of claim 1 wherein said heat activated attapulgite
clay has a volatile matter content in the range of about 12% to
16% by weight and contains about 3% to 7% free moisture and the
granules have a particle size of about 125 microns to about 250
microns.
Description BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to particulate insolubilized enzyme composites
comprising a solid support which can be used and reused as a catalyst
to initiate or promote enzyme-catalyzed chemical reaction. More
particularly, the invention concerns relatively inexpensive solid-supported
insolubilized biochemical catalyst compositions capable of functioning
for long periods and useable in continuous processes.
Enzymes are known to be highly active and selective catalysts for
many applications involving aqueous solutions of substrate materials.
The enzymes are water-soluble protein-like substances. Consequently,
when employed in water-soluble form, the enzyme is lost, used up
or destroyed to prevent contamination of the ultimate product.
Recently attempts have been made to prolong the activity of enzymes
by rendering them insoluble and thus amenable to reuse and recovery
by fixing the enzyme upon a water-insoluble support. In the technique
of primary interest there is a direct chemical attachment by covalent
chemical bonds of the enzyme to an organic polymeric matrix or a
porous inorganic solid. Particulate insoluble enzymatically-active
enzymes have been produced using solid siliceous support material
such as porous glass by reacting the particulate solid siliceous
support material with certain organosilanes, e.g., gamma-aminopropyltrimethoxy
silane, and attaching an enzyme to the reacted organosilane with
a crosslinking agent such as a dialdehyde exemplified by glutaraldehyde.
Porous glass particles and nickel-coated screen or other nickel-coated
solids are currently the most popular supports, the porous glass
being especially preferred because of the high catalytic activity
per unit weight of support. Porous glass is, however, very expensive
and the cost of enzyme catalysts based upon such support material
is prohibitive for many possible industrial applications. Furthermore,
porous glass is friable. During catalyst preparation, use and reuse,
especially under conditions of strong agitation or high flow rates,
the supported catalyst particles are subjected to mechanical forces.
When, as in the case of supports based upon porous glass, the particles
lack mechanical strength, they tend to break down into fines which
are too small to be useful in catalytic reactors because of excessive
pressure drops. Breakdown has been reported to result in leaching
of the enzyme when it occurs after fixation of enzyme to porous
glass.
The widespread industrial usage of insolubilized particulate enzyme
catalysts awaits the availability of reasonably low cost products
having high catalytic activity and in the form of small granular
particles which resist breakdown under dry and wet conditions.
2. Prior Art
The following patents suggest the use of various clays as possible
supports for enzyme catalysts wherein the catalyst compositions
are obtained by covalently binding enzymes to a silanized support
through various crosslinking agents.
U.S. Pat. No. 3519538 - R. A. Messing et al - July 7 1970
U.S. Pat. No. 3669841 - R. E. Miller - June 13 1972
SUMMARY OF THE INVENTION
In accordance with the present invention, insoluble enzymatically
active composites in the form of mechanically stable particles comprise
sized aggregates (granules) of heat-activated attapulgite clay having
a surface area (B.E.T.) in the range of about 100 to 150 m.sup.2
/g. and a porosity in the range of about 0.5 to 0.6 ml./g., wherein
the pore distribution is such that the major contribution to the
total pore volume is derived from pores with radii larger than 500
Angstrom units, and an enzyme coupled to the aggregates by an intermediate
silane coupling agent.
The insoluble particulate enzyme catalysts of the invention are
characterized by unusually high enzyme activity per unit weight,
the activity for any given size particles being of an order obtained
with porous glass of similar size. The cost of the enzyme catalyst
is a fraction of the cost of a catalyst based upon a porous silica
support. In addition to this benefit, the catalysts of the invention
are unusually resistant to breakdown in dry form and when slurried
in water. Thus the catalysts of the invention maintain their physical
form under conditions which may result in breakdown of other granular
supported enzyme catalysts. It has been found that simple adsorption
will not immobilize common enzymes on granules of heat-activated
attapulgite clay. Although the intitial activity of such a supported
enzyme catalyst may be high, the catalytic activity may decline
rapidly. Presumably this occurs because the enzyme is washed from
the substrate.
It will be noted that the support material useful in the practice
of the invention has a higher surface area than materials commonly
used as supports for enzyme catalysts. Generally materials having
surface areas of the order of 40 to 50 m.sup.2 /g. are selected
as supports for enzyme catalysts in order to avoid any interaction
between active sites on the supports and reactants. Porous activated
attapulgite clay has been demonstrated to be an outstanding support
in spite of the fact that it has a high surface area.
DETAILED DESCRIPTION
An essential feature of the invention resides in the selection
of attapulgite clay as the clay support and the use of such clay
in the heat-activated (calcined) form. Uncalcined attapulgite clay
is not suitable in the practice of my invention for the reason,
among others, that granules of uncalcined disintegrate in water
and the requisite granular form is not preserved.
Bentonites and kaolins, which constitute the most common clays,
are also unsuitable and are therefore outside the scope of the invention.
Bentonite can be produced in the form of granules which resist breakdown
but such clay granules lack the sorptivity of granules of heat-activated
attapulgite clay and do not result in a viable support. Kaolin granules
tend to be lacking in mechanical strength and are inadequately sorptive.
The clay particles used in carrying out the invention are obtained
by calcining naturally-occurring attapulgite clay (Georgia-Florida
fullers earth) at a temperature in the range of 600.degree.F. to
1300.degree.F. for a time sufficient to reduce the volatile matter
to a value below 18 percent by weight. The term "volatile matter"
(V.M.) is well known in the art. See, for example, U.S. Pat. No.
3174826 to Allegrini et al. The naturally occurring clay is commonly
called "Attapulgus clay" or "attapulgite" after
the predominating mineral constituent. A mined typical sample of
such clay containing 70 percent to 80 percent attapulgite, 10 percent
to 15 percent other clays, 4 percent to 8 percent quartz and 1 percent
to 5 percent calcite or dolomite.
The mineral attapulgite is a hydrated magnesium aluminum silicate,
typically about 1 micron long and 0.01 micron wide, with a unique
chain structure that imparts to the clay unusual sorptive and colloidal
properties. Heat activation results inter alia in loss of all or
a substantial proportion of chemically held water (water of crystallization).
This heat treatment profoundly affects the physical and chemical
properties of the clay. For example, activation results in the collapse
of the open channels of the colloidal crystalline attapulgite needles
and a rigid porous structure is developed. Heat activation decreases
surface area, e.g., typical non-heat treated attapulgite has a B.E.T.
surface area of about 210 m.sup.2 /g. Heat activated attapulgite
has a B.E.T. surface area in the range of about 100 to 150 m.sup.2
/g., typically 125 m.sup.2 /g. The designation "B.E.T."
refers to the nitrogen absorption method described by Brunauer,
S., Emmett, P. H. and Teller, E. J., J. AM. CHEM. SOC., 60 309
(1938) using molecular size data of Livingston, H. K., J. AM. CHEM.
SOC. 66 569 (1944). The effects of heat activation on surface area
and porosity of attapulgite clay is discussed by McCarter, W. S.
W. et al, IND. ENG. CHEM. 42 529 (1950).
To prepare the heat-activated clay, the raw clay is crushed and
nonclay impurities may be removed. If desired, the raw clay may
be extruded in known manner after being mixed with a small amount
of water. The raw clay, extruded or not extruded as the case may
be, is calcined in suitable equipment, usually a rotary calciner.
The calcined crushed clay must be placed in the form of fairly uniform
sized granules. For example, the discharge from the calciner may
be processed in a corrugated roll mill and the milled clay classified
to segregate particles of desired size. Alternatively the clay can
be sized before heat activation. Generally, the finer the particles
of the calcined attapulgite clay support, the higher the activity
of any given enzyme attached thereto. However, as the particles
become finer the excessive pressure drop becomes a problem. Consequently,
ultrafine particles, e.g., particles finer than 44 microns or 325
mesh, are undesirable. Typical supports are: 500/1000 microns, corresponding
to 18/35 mesh, U.S. Standard Sieve; 250/500 microns and 50/100 microns.
The designation indicates that essentially all of the particles
are larger than the particle size preceding the slash mark and at
least as fine as the particle size following the slash and can be
within a range of 50 microns to 1000 microns, all sizes being determined
by conventional dry screening.
To attach the enzyme covalently to the granules of heat-activated
attapulgite clay through an intermediate coupling agent, the clay
is silated in known manner to introduce functional groups which
may then be linked to the enzyme by crosslinking agents, also known
in the art. The disclosure of U.S. Pat. No. 3669841 (supra) relative
to the silation step, classes and species of substituted organosilanes
and crosslinking agents are hereby incorporated by reference. Of
the substituted organosilanes, hydrolyzable aminosilanes of the
formula given in U.S. Pat. No. 3669841 at column 1 lines 72 to
75 are preferred. Especially preferred are omega-aminoalkyl and
aminoaryltrialkoxysilanes exemplified by gammaaminopropyltrimethoxysilane,
aminopropyltriethoxysilane and aminophenyltriethoxysilane. Examples
of preferred crosslinking agents which form a covalent bond with
a reactive group on the enzyme that is not essential to the enzyme
activity and which also form a covalent bond with a funtional group
of the preferred aminosilanes are aldehydes including, for example,
formaldehyde, glyoxal, glutaraldehyde and acrolein. Also suitable
are bispropiolates and disulfoxyhalides. See U.S. Pat. No. 3669841.
Another known method for attaching enzymes to inorganic carriers,
which is described in U.S. Pat. No. 3519538 (supra), may be used
and involves covalent attachment of the enzyme through an intermediate
organofunctional group. In carrying out this method the clay is
silated as in U.S. Pat. No. 3669841 using preferably the same
type of aminoalkylsilanes to bond the silane to the clay, and then
bonding the enzyme to the silanized clay by reacting the amino group
of the silane with p-nitrobenzoic acid, reducing the nitro group
to the amide and then diazotyzing with nitrous acid. Alternatively,
after the hydrolyzable aminoalkylsilane is reacted with the clay
granules, the amino group is reacted with thiophosgene to prepare
the isothiocyanoalkylsilane derivative. The enzyme is than reacted
with the organofunctional portion of the silane coupling agent.
Active enzymes useful in practice of the invention are set forth
in U.S. Pat. No. 3669841 column 2 line 33 to column 4 line
37 which disclosure is incorporated herein by reference. The enzymes
include: the redox enzymes that catalyze oxidation or reduction
reactions, e.g., glucose oxidase and catalase; hydrolytic enzymes
which hydrolyze proteins and transferase enzymes.
Any inert medium may be used to bond the enzyme, the crosslinking
agent and the organosilane which is reacted with sites on the activated
clay. Usually an aqueous medium is used. The pH and temperature
employed are selected to avoid or minimize deactivation of the enzyme.
With most enzymes, the temperature is at room temperature or below.
Generally the enzyme is dissolved in a buffer solution at appropriate
pH and temperature and the solution is usually assayed. The silanized
clay is added and the glutaraldehyde or other immobilizing agent
is added to the slurry. The resulting slurry is maintained at a
desired temperature for a suitable time, generally 1 to 72 hours,
while maintaining pH at desired level. The bonded enzyme may then
be assayed. The bonded enzyme may be stored in water or in a buffered
solution at room temperature or below. In some cases the bonded
enzyme may be dried mildly prior to use.
Insolubilized enzyme composites of the present invention are useful
in analytic and chemical processes using enzymes and are of special
value in industrial enzymatic processes carried out in continuously
operated columns or stirred tank reactors. In conducting such processes
the physical form and mechanical stability of the particulate catalysts
of the invention is of utmost value. Industrial processes capable
of utilizing the insolubilized enzyme catalysts include, by way
of example, starch conversion and sugar processing, dairy processing
and brewing.
The following examples illustrate the preparation and utility of
enzymatic catalysts of the invention by coupling redox enzymes (glucose
oxidase and catalase) to granules of heat-activated attapulgite
by means of an intermediate silane coupling agent and using glutaraldehyde
as the insolubilizing agent. These examples are given for illustrative
purposes and the invention is not to be construed as being limited
to the specific embodiments that are illustrated therein.
The clays used in the test were commercial granular grades of heat-activated
Attapulgus clay. One was an "RVM" grade and the other
an "LVM" grade, these designations referring respectively
to regular and low volatile matter contents. Samples of these clays
were ground in a hammer mill and sieved to different particle size
ranges. Typical properties of these clays are as follows:
Considering the total pore volume (0.519 ml./g.), most of the pore
volume in the calcined clay is contributed by bores with radii >
500 A (i.e., 0.370 ml./g.)
In illustrative examples, glucose oxidase was bonded to samples
of the sieved activated attapulgite clay via a gamma-aminopropyl-trioxysilane
coupling agent with glutaraldehyde employed as the immobilizing
agent. The procedure used for silanizing the clay granules and incorporating
the enzyme and couping agent are described in a publication by Herring,
Laurence and Kittrell, "IMMOBILIZATION OF GLUCOSE OSIDASE ON
NICKEL-SILICA ALUMINA," Biotechnology and Bioengineering, Vol.
XIV, pages 975-984 (1972). This procedure was modified by using
glutaraldehyde as the immobilizing agent in conventional manner,
as described for example in U.S. Pat. No. 3669841 Example 3.
Assays of enzyme activity were by the polargraphic technique referred
to at page 978 in the publication of Herring et al (supra) using
the initial rate of oxygen uptake as the catalyst activity. Glucose
was used as the substrate. In making the tests with the immobilized
glucose oxidase, 30 mg. of immobilized catalyst was immersed in
1.2 ml. of 5.5 pH citrate phosphate buffer. The solution was saturated
with oxygen. A 2M glucose solution (0.12 ml.) was injected into
the cell at 25.degree.C. and the suspension was stirred vigorously.
After one day the stored samples were assayed. The activity (representing
the initial rate of change of oxygen concentration with time) was
reported as moles O.sub.2 /mg. catalyst-sec.
The activities for catalysts prepared with the two different samples
of activated attapulgite were essentially the same at all mesh sizes.
Consequently, the activity values summarized below in table form
represent average values for the two different grades of activated
attapulgite clay.
The activity values for the glucose oxidase coupled with glutaraldehyde
to activated attapulgite clay were compared to values for similarly
sized catalysts obtained by immobilizing glucose oxidase on other
known supports with glutaraldehyde. In all cases, the catalyst prepared
with the heat-activated attapulgite supports were significantly
more active than catalyst prepared with supports other than porous
glass. For example, the 250/500 micron fraction of a catalyst produced
with a support composed of 18 percent nickel oxide of kieselguhr
(known to be a support providing high activity for glucose oxidase)
was 155 .times. 10.sup..sup.-12 moles O.sub.2 /gm.-sec. This was
only about 56 percent of the activity of the catalysts obtained
with 250/500 micron particles of heat-activated attapulgite clay.
The activity of the 500/1000 micron fraction of the catalyst supported
on the nickel-treated kieselguhr was 89 .times. 10.sup..sup.-12
moles O.sub.2 /sec.-mg. as compared to 111 .times. 10.sup..sup.-12
for the activated attapulgite clay sample of similar size.
Immobilized catalase samples were prepared using the same procedures
employed with the glucose oxidase catalysts. These samples were
maintained on a solution buffered at pH 7 (26.degree.C.) and used
with a glucose substrate.
Assays of tests made with catalase coupled to various supports
were made two days after immobilization. The activity for the catalysts
prepared with 250/500 micron attapulgite supports averaged 752 .times.
10.sup..sup.-12 moles O.sub.2 removed per gram per second. The catalyst
supported on nickel-coated kieselguhr (250/500 micron fraction)
was 381 .times. 10.sup..sup.-12 half that of the attapulgite clay-supported
catalyst. At all given mesh sizes, except for the fine powdered
samples (0/125 micron) the attapulgite clay-supported catalase catalysts
had a higher activity. |