Note: Descriptions are shown in the official language in which they were submitted.
~.$^~3~
HIGH PERFORMANCE IMMOBILIZED ENZYME COMPOSITIONS
l BY MULTI-LAYERING IMMOBILIZATION OFFERING A MIGH AMOUNT
i OF ACTIVITY PER UNIT VOLUME
¦IIntroduction
This invention relates to an immobilized composition
of a biologically active material that can be prepared to
have an unusually high amount of activity per unit volume.
IIIMore particularly, the invention relates to an immobilized
~lenzyme composition in which the enzyme is immobilized in
novel fashion.
~¦ Background
Enzymes are proteinaceous catalytic materials that have
great industrial potential. Enzymes also are often very
expensive materials. They are generally soluble in their
respective substrates and except where the conversion product
is of great value, recovery of the enzyme for reuse may be
difficult or impossible. In some cases, the processing
conditions may destroy the enzyme. Where the enzyme is not
destroyed, it may be necessary to destroy it, as in some
1~ food products, where continued activity would have an unwanted
effect.
To avoid these problems, fixed or immobilized enzyme
systems have been developed in recent years. Procedures such
as adsorption, encapsulation, and covalent bonding are
; routinely used with many enzymes. The immobilization procedure
selected, from the many available, produces a composition
that can be used in either batch or continuous processes,
but that is most advantageously used in a continuous process
~or economy.
~ ''~
~ ~ ~ 33~ ~
While the term "insolubilized enzyme" has been used in
the past on occasion, as in United States patent 3,519,538,
to refer to an enzyme coupled by covalent chemical bonds to
lan insoluble inorganic carrier, and thus rendered not soluble
5 l~l in water, the term "immobilized" is used herein to refer to
such an enzyme, or other biologically active material, fixed
to any kind of carrier, i.e., organic or inorganic.
The term "stabilized" is used herein to refer to a
Illbiologically active material, such as an enzyme, that has
l¦been stabilized against the loss of activity that would
otherwise occur because of aging or exposure to an elevated
lltemperature, or use in a reaction as a catalyst.
I In the process of immobilizing an enzyme, there are many
l important practical considerations. There should be as little
15 ¦ loss of enzyme activity as possible. The cost of immobilization
should be low. The carrier material should be one that does not
have a deleterious effect on the action of the enzyme during
the process in which it is to be used. The immobilized enzyme
should not leak enzyme or any other material into the reaction
mixture~ especially in food processing applications. The
activity of the enzyme should remain high over a long period
of operating (reaction) time, generally measured, in industrial
processes, as the half~ e. In addition, the immobilized
enzyme should offer good hydraulic characteristics, to permit
reasonable throughput rates. Equally importantly, the
immobilized enzyme should be able to withstand reasonable
operating temperatures, to permit practical operating rates,
I with the least feasible loss of activity.
For economy, it is also desirable that recharging of the
carrier be possible, to reactivate spent immobilized enzyme,
preferably by as simple an operation as possible.
~33`~8
Work in the field has progressed from concern simply
with trying to immobilize an enzyme on a water-insoluble
carrier to more sophisticated wor~c in which the objective was
Ito produce an immobilized enzyme that would deal successfully
llwith all of the practical considerations mentioned above.
Several United States patents describe advances in the
~art that are representative of what has been done.
In United States patent 3,519,538J Messing and Weetall
~Idescribe an immobilized enzyme composition in which the
llenzyme is covalently coupled to an inorganic carrier through
an intermediate silane coupling agent, the silicon portion of
~the coupling agent being attached to the carrier and the
¦organic portion of the coupling agent being attached to the
enzyme. While glass of controlled porosity was the preferred
carrier material, a wide variety of inorganic carrier materials,
often siliceous, are disclosed as being useful.
In United States patent 3,556,945, Messing disclosed an
immobilized enzyme composition which was said to be characterized
by no loss of activity because of the immobilization. The
enzyme was one having available amine groups, and it was
coupled to a porous glass carrier through reactive silanol
groups, by means of amine-silicate bonds and by hydrogen bonding.
In United States patent 3,669,841, Miller ~escribes
immobilized enzyme compositions in which the enzyme is attached
to siliceous materials by a process involving first, the
silation of the siliceous carrier, to introduce functional
groups, and the linking of the functional groups to an
enzyme by means o~ cross-linking agents. The cross-linking
agents identified by Miller include formaldehyde, other
¦monoaldehydes polyaldehydes, bispropiolates, and dis~lfonyl
I -3-
q~
halides. In Example 1 of the patent, gamma-aminopropyltri-
methoxysilane is reacted with particulate silica, then an
enzyme is added wi~h stirring, and then an aqueous formaldehyde
~;olution is added.
Tomb and Weetall in U.S. patent 3,783,101, describe the
covalent coupling of enzyme to a silanated carrier by the
use of glutaraldehyde.
, In United States patent 3,796,634, Haynes and Walsh
lldescribe an immobilized enzyme composition in which the enzyme
1 is said to be adsorbed as a monolayer, enveloping colloidal
¦silica particles. The monolayer is produced by cross-linking
the enzyme with a cross-linking agent.
In United States patent 3,836,433, a polyaldehyde is used
'Ito fix an enzyme to a gel of an organic material such as, for
example, a polyacrylamide or a polysaccharide.
The literature also reports a great rnany immobilized enzyme
compositions and ways in which they may be used. For example,
Olsen and Stanley, in the Journal of Agricultural and Food
Chemistry, vol. 21, No. 3 (1973), pages 440-445, and in U.S.
patent 3,767,531, describe immobilized enzyme compositions
in which lactase and other enzymes are bound to a phenol-
formaldehyde resin with glutaraldehyde.
Other biologically active materials can also be
~ 1 ~.5~ f~
k~ ~ immobilized for useful purposes. For example, in~3,839,153,
1 conjugates of biologically active materials, such as human
choriongonadotrophine, insulin, and cortisol, are conjugated
with different enzymes respectively by a reaction with
glutaraldehyde. The conjugates are useful in immunoassays.
Brief Summary of the Present Invention
In one aspect, the present invention is in a process for
preparing novel immobilized compositions of biologically
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33~8
active materials, partlcularly proteinaceous materials such
as, for example, immunoreactants, but especially, of enzymes.
A broad process aspect of this invention resides in an
improved process for immobilizing a second biologically
active material having available amine groups, comprising
covalently coupling the material to another, first biologically
active material having available amine groups that is
immobilized on a carrier.
In this process, a suitable carrier material is treated
to activate its surface to have residual hydroxyl groups
thereon. A hydrolyzable silane having an amine substituent,
is then coupled to the hydroxyl groups on the activated
surface of the carrier. A poly~functional coupling agent is
then reacted with the amine group of the silane. Preferably,
the polyfunctional material is a polyaldehyde or a bis-imidate,
but other such materials may be used. Next, after removal
of unreacted material, the biologically active material, such
as an enzyme, having available amine groups, is reacted with
thè free (unreacted) aldehyde groups. This step covalently
bonds the enzyme or other material to the carrierl without
substantial loss of activity.
At this stage, there is a single amount or "layer" of
material, such as enzyme, immobilized by covalent bonding to
the carrier. The immobilized material, preferably enzyme,
has available amine groups, and these are reacted in turn
with an additional amount of a polyaldehyde, preferably
glyoxal. After washing, additional material, such as enzyme,
is added, which covalently bonds through its available amine
groups with the unreacted aldehyde groups, to form a covalent
bond between the added material and the initially immobilized
material.
-5-
~1
cb/ n~
~1~33~8
When the immobilized material is an enzyme, in
effect, there are two immobilized enzyme "layers",
one covalently bonded to the carrier, and the other
covalently bonded to the initially immobilized "layer"
of enzyme. The word "layer" is not aptly descriptive;
the term is used for convenience and because those
skilled in the art will understand it.
The process may be repeated as often as desired,
to form a multilayered immobllized composition. This
composition is a second aspect of the invention. It
can be prepared so as to retain high activity, has high
activity per unit volume, and can be prepared to have
unusually good thermal stability, good half-life, and
practical mechanical strength.
A more specific aspect of this invention resides
in an enzymatically reactive composite comprising a
solid, insoluble,carrier having plural, sequentially
applied amounts of enzyme immobilized thereto, the
carrier providing high surface area per unit volume and
having chemically reactive groups at its surface, there
being a first amount of a first enzyme immobilized to
the carrier by covalent bonding through a covalent
chemical coupling means comprising a polyfunctional re- `
agent, the coupling means being chemically coupled to
the carrier through the reactive groups and also
chemically reacted with the first enzyme, and a second
` amount of the same or a different enzyme immobilized
by covalent bonding through a second amount of the
polyfunctional reactant to the immobilized first enzyme,
the enzymes substantially retaining their respective
activities, the total enzyme activity of the enzymatic-
ally reactive composite being greater than that of the
,-,1 mab/ ~
.
:1~ 433V8
first amount of immobilized enzyme.
In a preferred embodiment, the carrier material
is a finely divided, free-flowing particulate material,
most preferably a silica gel, and the immobilized
material is the enzyme, lactase (EC 3.2.1.23). One
feature of the invention is the use of this immobilized
lactase in the treatment of whey, to convert the whey
into more useful products.
Brief Description of the Drawings
In the drawings:
Fig. 1 is a graph plotting the units of activity
per ml. of single layer immQbi~ zed enzyme composition
prepared in accordance with certain embodiments of the
invention, where lactase is immobilized on silica gel
through the use of each of four different dialdehyde
cross-linking reagents, employed at different concen-
trations for comparative purposes, showing the concen-
1.. . .
tration dependence of the dialdehydes;
Fig. 2 is a graph plotting protein (enzyme) con-
centration against reaction time in minutes, showing
the progress of immobilization and the decrease in
enzyme concentration for first layer immobilization
of lactase with glutaraldehyde, Curve ~, and for first
layer and second layer immobilization with glyoxal,
Curves B and C respectively;
Fig. 3 is a plot of the activity of immobilized
lactase on ortho-nitrophenyl galactopyranoside (ONPG)
as a substrate, at different temperatures, and
Fig. 4 is a plot of the activity of immobolized
lactase against time of incubation with ONPG as a
substrate, at
- 6a -
mab/('~>
3~ ~
~different ~emperatures, the arrows on the several curves
indicating the applicable time scale, i.e., whether the time
'Iwas measured in minutes or days.
5 1l Detailed Description of the Invention
Il To make an immobilized enzyme composition in accordance
ilwith the present invention, the carrier material is preferably
~lin particulate form, most preferably finely divided and free-
~ flowing, but in addition, may be in the form of fibers, tubes,
jsheets, beads, or porous glass. In any form, it should provide
lla very high surface area per unit volume.
¦ The carrier may be any chemically inert natural or
synthetic material, such as, for example, a polymer that is
capable of forming a gel in aqueous media. Generally,
siliceous materials are preferred. These materials include
granulated, fibrous and finely particulate silica and silicates.
The carrier material may also be, for example, porous glass,
asbestos, diatomaceous earth, wollastonite, fosterite,
feldspar, mullite, several different kinds of clay, and in
general, any material that has or can be formed to have a
shape that makes processing practical in the desired
end use, that offers a high surface area per unit volume, and
that either has or can be treated to have active hydroxyl
groups at its surface that can react with hydrolyzable groups
of an organosilane or cyanogen bromide, that acts as a
coupling agent.
¦ In a preferred embodiment, a silica gel is treated with
a strong acid or a strong base, in order to activate it by
Igenerating hydroxyl groups at th~ surface of the gel particles.
¦The activated carrier is then reacted with a coupling agent,
preferably a silane that couples to the carrier at one portion
of its molecule, and that provides at a remo~e part of its
~rnolecule a reactive amine group.
The preferred kind of silane coupling agent has the
formula:
H2N - R - Si(ORl)3
where R is an alkylene group, and Rl, of which there are
l~three per molecule, is preferably alkyl, most preferably
l~lower alkyl, and the three Rl substituents may be the same
l! or different on a given molecule.
10 1 In the next step, a suitable amount of a polyfunctional
reactant, preferably a polyaldehyde, and most preferably
glyoxal, in a suitable solvent medium, is brought into contact
with the amine-reactive silica gel or other carrier. In
preferred embodiments, this makes the silica gel aldehyde-
reactive or aldehyde-functional.
The aldehyde-functional carrier is then mixed with
enzyme (or other biologically active material) having available
amine groups. The available amine groups of the enzyme
react with the free aldehyde groups of the carrier, to
immobilize the enzyme on the carrier. The composite is
then washed to remove excess unreacted materials.
The immobilized enzyme now consists of a carrier to which
a first amount or layer of enzyme is covalently bonded. The
enzyme is one having available amine groups. In the next step,
this immobilized enzyme composition is reacted with a poly-
functional material, most preferably glyoxal, so that it
becomes aldehyde-functional. It is then reacted with a
second amount of enzyme, which in turn becomes covalently
bonded, this time to the initially immobilized enzyme. This
process can then be repeated to add as much ~ enzyme as
desired to the composition. Generally not more than 10
3~8
llayers are practical, and most preferably a total of four
`l layers are applied when the enzyme is lactase and the carrier
is silica gel. When proper procedures are employed with careful
~Icontrol over the amount of reactants and the removal of excess
1! reactants, there is relatively little observed loss of enzyme
activity.
The reaction of aldehyde and amine groups takes place
readily even at low temperatures, so that the reaction can
l be conducted at 5C. in solution, and from slight acidity to
l¦a moderate pH range. The pK values of the alpha-amino groups
¦in most enzymes and other polypeptides fall in the range from
about 7 to about 8. Thus, most enzymes and other such
polypeptide materials may be immobilized at a pH that is
very close to being neutral, which is a mild condition that
sustains activity.
In practicing the present invention, it is important to
avoid unwanted cross-linking that may occur. The extent of
cross-linking can be limited by careful control over the
amount and concentration of cross-linking agent, such as
glyoxal, that is employed, and by washing to remove unreacted
excess cross-linker as soon as the covalent bonding has had
a reasonable opportunity to go to completion.
With proper limitation of the cross-linker and of its
reaction, when lactase is immobilized according to the
invention in multiple layers on silica gel, using glyoxal
¦ as the polyfunctional agent, the amount of enzyme activity
retained corresponds to the activity described by the ratio,
for a three layer structure, of 100% to 70%-95% to 70%-95%.
This ratio relationship is employed as a descriptor of
cumulative activity, but at this time it is not known in
which layer (if in any single layer) the decrease occurs.
I _g_
~3~8
~`
II.imited cross-linking stabilizes the immobilized enzyme;
`Itoo much reduces the activity. Some cross-linking, with
consequent reduction in activity, seemingly cannot be avoided.
~ Immobilized multilayered lactase on a i~ia~l gel carrier,
prepared in accordance with the invention, is generally
characterized by advantageously high activity per unit volume;
¦high mechanical and thermal stability; and prolonged half-life.
'l¦ The invention will now be further illustrated by
¦several specific demonstrations o the practice of preferred
¦lembodiments thereof. In this application, all parts and
'percentages are by weight, and all temperatures in degrees
¦~Celsius, unless expressly stated to be otherwise.
I Example 1
Preparation of Chemically Active Groups on
Surface of Su ort Matrix - Silica Gel
PP
Step A. Preparation of Propylamine Silica Gel
10 g. of silica gel, (SiO2)n, 35-70 mesh, ASTM, from
E. Merck, Darmstadt, Germany, was activated by suspending
it in 50 ml of 2% NaOH solution. The mixture was heated and
maintained at 40C. for 1 1/2 hours with occasional gentle
stirring. The alkaline solution was then filtered on a
plastic frit-funnel, and the gel was suspended in 50ml of
20% HNO3 solution to neutralize the residual alkali.
The resulting hydrophilic silica gel was then added to
50 ml of 4% gamma-aminopropyl triethoxy silane solution
which had been adjusted to pH 5.0 with acetic acid. The
~ gel-silane reagent mixture was heated and maintained at 65-
; 30 75C. for 1 1/2 hours with stirring. The silane solution
was then decanted.
--10--
~1~33~8
The propylamine silica gel product was neutralized with
14% KOH solution to about pH 7.5, then washed exhaustively with
clistilled water on a plastic frit-funnel, and then vacuum
llclried for storage. It could be used as is, without drying.
5 1I This propylamine silica gel product can be used as a carrier
l~for immobilization thereto by covalent bonding, as with a
¦¦dialdehyde such as glyoxal, of any biologically active
compound that has an available amine group, such as
~ enzymes, hormones, immunoreactants, and the like. The general
technique is particularly useful for the preparation of an
enzyme electrode.
Step B. Prepara~ion of ~ldehyde Silica Gel
25 ml of the propylamine silica gel was mixed with 50 ml
l of 0.1% ethanedial (glyoxal) solution in O.lM potassium phosphate
15 1 buffer, pH 8.0, which contains 1% reagent alcohol in a flask,
immediately evacuated and filled with N2 gas, then heated to
about 40C. for 1 1/2 hours with occasional gentle shaking. The
aldehyde silica gel was then filtered on a plastic frit-funnel,
washed with distilled H20, and immediately vacuum dried for
storage. The container was filled with N2 gas to prevent
oxidation.
Step C. Preparation of Immobilized
Lactase EnzYme on Silica Gel
25 ml of the aldehyde silica gel was added to 50 ml of
diluted Lactozym 750L lactase (NOVO Industri AS, Denmark)
in O.lM potassium phosphate - 5mM MgS04 - pH 7.3, the amount
of enzyme being in excess of the amount required for coupling.
The reaction vessel was immediately evacuated. The reaction
proceeded at room temperature for 1 hour with occasional
gentle shaking.
3~4.~8
The lactase-silica gel product was filtered on a plastic
ifrit-funnel to remove the excess enzyme, and washed with
washing buffer (0.02M potassium phosphate, 5mM MgS04
'~pH 7.0). The lactase-silica gel was then suspended in
enzyme buffer (0.04M potassium phosphate, 5mM MgS04
p~l 7.0) and stored at 4C.
Step D. Multiple Layer Immobilization of Enzyme
25 ml of lactase silica gPl (sedimented gel volume) was
ladded to 50 ml of 0.1% ethanedial solution in enzyme buffer
10 ~I(O.lM potassium phosphate, 5~I MgS04 pH 7.3) which contained
1% reagent alcohol, and the flask was then evacuated. The
¦mixture was reacted at room temperature for 1 1/2 hours with
¦occasional mild shaking.
~! The aldehyde-functional enzyme gel was filtered and washed
1 with washing buffer at pH 7.0, then immediately added to 50 ml
of diluted Lactozym 750L lactase (20 x dilution by volume,
NOVO Industri AS, Denmark) solution, again in enzyme buffer
(O.lM potassium phosphate, 5mM MgS04 pH 7.3). This mixture
was reacted about 1 hour at room temperature.
The silica gel carrier, now having two t'layers" or
applications of lactase immobilized thereon, was filtered and
washed with washing buffer at pH 7.0, suspended in enzyme
buffer at pH 7.0, and stored at 4C.
l Third, fourth and even more "layers" of enzyme have been
¦ immobilized by repeating this same procedure, with relatively
little loss in activity.
Step _E. Regeneration of Enzyme Silica Gel Activity
After use, spent lactase silica gel, which may retain
some relatively low level of lactase activity, can be
regenerated to increase and restore its lactase activity by
following a similar procedure to that described in Step D.
I'he spent enzyme gel still contains covalently linked
proteinaceous material, having available amine groups, which
Ican be reacted with ethanedial, followed by lactase
immobilization as in Step D.
Example 2
Multiple Enzyme Immobilization
lll A procedure similar to that in Step D of Example 1 was
followed, except that, for the layers of enzyme applied
subsequent to the first, lipase (Marshall lipase, Miles
Laboratories, Inc.) was used instead of Lactozym 750L.
~¦ This immobilized enzyme composition exhibits the enzyme
¦activities of both lactase and lipase. It is therefore useful
Ifor the production of lipolyzed cream and butter oil. The
controlled-lipolysis of such products can enhance the
buttery flavor and/or can be used in a variety of products.
Similarly, the procedure of Step D of Example 1 was
followed, except that the enzyme applied, for layers
subsequent to the first, was the protease bromelin (Midwest
! Biochemical Corp. U.S.A.). This immobilized enzyme composition
exhibited the enzyme activities of both lactase and protease.
It was useful for the hydrolysis of the sugars and proteins
l in cheese whey.
¦ In similar fashion, following a procedure similar to
that in Step D of Example 1, the enzyme employed for application
~subsequent to the first layer may be a mixture of lipase and
protease. The resulting immobilized enzyme composition will
exhibit the activity of all three enzymes, that is, of lactase,
lipase, and protease. Such a composition is useful in the
~ 3 3
processing of dairy products for the controlled hydrolysis o~
~lactose, and whey proteins, as well as the controlled
lipolysis of lipids for enhanced flavors.
Alternatively, spent immobilized enzyme may be employed
as the base on which to immobilize enzymes other than the
original enzymes, following generally the procedure of
Step E of Example 1.
Example 3
! Evaluation and Comparison of Different
Dialdehydes in Immob-ilization
The procedure of Example 1 was followed to immobilize
ilactase on silica gel, with the use of glyoxal as the
coupling agent, and for comparative purposes, with the use
~lof other dialdehydes in place of glyoxal.
1, Four structurally preferred, different dialdehydes were
1 tested for suitability for immobilizing. Their chemical
formulae are as follows:
! ethanedial OHC-CHO
(glyoxal)
l n-pentanedial OHC-CH2-CH2-CH2CHO
(glutaraldehyde)
~l o-phthaldialdehyde CHO
~ ~ CHO
; p-phthaldialdehyde OHC ~ CHO
The concentration dependence of the dialdehydes as immobilizing
l reagents is shown in Fig. 1. The ethanedial appears to be
1 the best one, with highest enzyme activity retention (con-
centration of ethanedial from about 0.03% to about 1%). The
glutaraldehyde and O-phthaldialdehyde are less effective
and only comparably effective in a very narrow low
llconcentration range (~ 0.3%). p-phthaldialdehyde appears
Ito exhibit a very low level of usefulness.
l~ -14-
3~8
The amount of enzyme and the immobilization reaction
kinetics for lactase immobilized on aldehyde silica gel
with ethanedial and with glutaraldehyde respectively are
~lisomewhat similar. The immobilization time course and the
Ildi.sappearance of enzyme concentration in the supernatant
~liquid for first layer and second layer immobilization of
~lactase are shown in Figure 2.
The temperature dependence and the temperature stability
¦of the immobilized lactase activity are shown in Figures 3
land 4 respectively. The comparison of thermal stability of
lactase immobilized with ethanedial and with glutaraldehyde
is shown in Tables 1 and 2. The enzyme activity and its
active conformat.ional stability can be effectively increased
by multi-layer immobilization technique, as shown in
Tables 2, 3 and 4.
Again, the relative thermal stability and immobilized
lactase activity by layered-immobilization are both
significantly improved, and appear to be much better with
ethanedial than with glutaraldehyde. Generally speaking,
glutaraldehyde is a good immobilizing reagent, but ethanedial
is preferred.
The immobilized enzyme composition of E~. 1 is useful
in treating whey solutions to improve their sweetness.
When the immobilized enzyme is a combination of lactase and
glucose isomerase, a very sweet product is produced.
Definitions
Enzyme Activity Unit and Analytical Method.
Lactase activity - 1 ONPG unit is defined for free
enzyme as the hydrolysis of 1 umo~ of ONPG per
minute at 30C in buffer ~0.02M potassium phosphate,
10 ~ ~ C12 pU 7.0); and for immobilized enzyme,
in 0.066 mole potassium phosphate at pH 6.75.
ONPG - ortho-nitrophenyl galactopyranoside
Lactose determination - use Shaffer-Somogyi
micro-method, A.O.A.C., 31.052.
Glucose determination - use Worthington
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3~3~8
,
Table 4. Average half-life of immobilized lactase at
1 50C under assay buffer conditions. Lactase
¦ was immobilized on silica gel with ethanedial
as immobilizing reagent.
11-
Leyers of ~ Half-life at 50'C
3 layers 11.5 min.
2 layers 8.5 min.
1 layer 5.5 min. .
NOTE: 1. Immobilized lactase activity was
determined at its optimum pH.
2. ONPG was used as lactase substrate.
-20-
General
To practice the invention, the siliceous material can
be reacted with the organosilane in any convenient manner
by contacting the former with the latter to obtain the
lldesired bonding through hydrolyzable groups of the organosilane.
¦Usually the organosilane is dissolved in an inert solvent
l¦such as toluene, xylene, or the like, and the resulting
¦Isolution is then applied to the siliceous material.
¦~Aqueous solutions of a soluble silane can also be used.
Il The amount of organosilane coupling agent employed is
~Idependent upon the nature and surface area of the siliceous
material. Usually, at least about 0.01 percent by weight of the
I organosilane, based on the weight of the siliceous material, is
desired. Amounts in the range from about 0.25% to about 2
by weight are preferred.
Suitable organosilanes include substituted organosilanes
which can be represented by the formula
Y,b
Xa - si --[Rn -- Z]c
where X is a hydrolyzable group capable of reacting with a
¦ hydroxyl group, Y is hydrogen or monovalent hydrocarbon
group, R is an alkylene group having from 1 to about 20 carbon
atoms, Z is a functional group capable of reacting with a cross-
linking agent, n is an integer having a value of 0 or 1, a
is an integer having a value of 1 to 3, inclusive, b is an
integer having a value 0 to 2, inclusive, c is an integer
having a value of 1 to 3, inclusive, and the sum of a + b + c
equals 4.
Examples of suitable X groups include halo, hydroxy, alkoxy,
cycloalkoxy, aryloxy, alkoxy-substituted alkoxy such as
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33`~8
beta-methoxyethoxy or the like, alkoxycarbonyl, aryloxycarbonyl,
alkyl carboxylate, and aryl carboxylate groups, preferably
having eight or less carbon atoms.
I Examples of suitable Y groups in the above formula are
llhydrogen~ methyl, e~hyl, vinyl, isobutyl, and other hydrocarbyl
groups, preferably having 10 or less carbon atoms.
The R group in the above formula can be any alkylene
group having up to about 20 carbon atoms, and preferably from
~iabout 2 to about 18 carbon atoms. Examples of such groups
I~are ethylene, the propylenes, the butylenes, the decylenes,
~¦the undecylenes, the octadecylenes, and the like.
The Z groups can be any functional group capable of reacting
with the hereinbelow defined crosslinking agent. Examples of
such groups are amino, primary and secondary amido, epoxy,
isocyanato, hydroxy, alkoxycarbonyl, aryloxycarbonyl,
vinyl, allyl, halo such as chloro or bromo, and the like.
Particularly preferred of such functional groups are amino.
Particularly preferred organosilanes for the purposes of
¦this invention are omega-aminoalkyl and aminoaryltrialkoxysilanes
such as gamma-aminopropyltrimethoxysilane, aminophenyl-
triethoxysilane, and the like.
For the purposes of this invention suitable crosslinking
agents are dialdehydes, bis-imidoesters, bispropiolates and
l disulfonyl halides.
¦ Illustrative dialdehydes are glyoxal, glutaraldehyde,
malonic aldehyde, succindialdehyde, and the like, preferably
containing from 2 to 8 carbon atoms, inclusive.
Illustrative bis-imidoesters are dimethyl adipimidate (DMA),
dimethyl suberimidate (DMS), N,N'bis (z-carboximidoester)
tartarimide dimethyl ester (CETD), dimethyl 3,3'-dithio-
bispropionimidate, and the like.
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1~;33~8
, Illustrative bispropiolates are the diol propiolates such
`las ethylene glycol bispropiolate, propylene glycol bispropiolate,
butylene glycol bispropiolate, h~xamethylene glycol bis-
~ropiolate, decamethylene glycol bispropiolate, cyclohexylene
b,s ~r~P
l~lycol bispropiolate, methylolpropane diol di~.u~iuldL~
~nd the like, as well as bisphenol A propiolate, pentaerythritol
bispropiolate, and the like.
~¦ Illustrative disulfonyl halides are benzene-1,3-disulfonyl
¦~hloride, naphthalene-1,5-disulfonylchloride, naphthalene-
¦~,6-disulfonylchloride, naphthalene-2,5-sulfonylchloride, and
the like.
The amount of crosslinking agent to be used is dependent
rincipally on the amount of enzyme or enzymes that is desired
~o be incorporated into the composite. Usually an enzyme cross-
Linking agent molal ratio is about 1:1 or less. A ratio of~bout 0.01-0.0001/1.0 is preferred.
The bonding of the enzyme, the crosslinking agent and the
~rganosilane, which is present together with the siliceous
~aterial, can be carried out in any convenient inert medium,
lsually an aqueous medium at pH conditions and temperature
~hich do not tend to inactivate the enzyme. Temperatures
~bove about 60C should generally be avoided. The present
?rocess is readily carried out at ambient room temperature.
rhe temperature of choice depends, however, mainly on the
?articular enzyme or mixture of enzymes used. Usually the
temperature can range from about -5C. to about 30C. A
temperature in the range from about 0C to about 10C is
preferred.
Generally the same conditions as mentioned above for
immobilization of the initial enzyme layer apply to the
immobilization of subsequent enzyme layers.
1, . . .
.
33q~8
The dialdehydes are preferred polyfunctional agents
llfor use in the present invention. As Fig. 1 and Table 1
-r~ indicate, the dialdehydes ar~ equivalent in performance.
l Generally, those dialdehydes containing two through four
l~carbon atoms are expected to perform equally well.
Dialdehydes having three and four carbon atoms are not readily
commercially available at the present time.
Glutaraldehyde (n-pentanedial) is not currently believed
~¦to be a simple five carbon molecule. Rather, it is believed
¦~to occur, in its commercially available form, as an oligomer,
~actually a trimer. This makes a substantial difference in the
¦performance of this particular dialdehyde when used in the
¦present invention, since the trimer form would be expected to
¦and apparently does lend itself to the production of cross-
linking between enzyme molecules within a given layer. Suchintra-layer cross-linking is generally not regarded as
desirable, since it apparently tends, based on available data,
to reduce activity.
From Fig. 1, the conclusion can readily be drawn that
the minimum amount of dialdehyde should be used that is
sufficient to produce covalent bonding, and that when more
than the minimum is employed, intra-layer cross-linking occurs
that reduces enzyme activity.
In developing the data that is reproduced in Fig. 1
the consistent practice was to employ one volume of sedimented
immobilized enzyme, on silica gel, with two volumes of the
dialdehyde, at whatever concentration of dialdehyde was
being used.
Glyoxal (ethanedial) is a superior cross-linker,
although the reason for its better performance is not clear.
Apparently, from the data plotted in Fig. 1, if the use
.... .;~. ,
3~
of glyoxal in excess of that required for coupling leads to
intra-layer cross-linking, then the reduction in enzyme activity
is much less than is the case with the other dialdehydes.
Il The choice of cross-linking dialdehyde has some effect
ll upon the way in which the enzyme performs. Thus, the optimum
~pH of lactase immobilized on silica gel in accordance with
the invention is pH 6.75 when the cross-linker is glyoxal,
and pH 6.50 when the cross-linker is glutaraldehyde, as
~compared to pH 7.0 for free lactase. These data suggest that
¦the immobilized lactase retains a more active conformation
when coupled with glyoxal than with glutaraldehyde. This
comparative data was developed through performance evaluation
of immobilized lactase on ONPG at 30C.
The substrate selected also has a bearing on the performance
of immobilized enzyme. Thus, when lactase is immobilized on
silica gel, using glyoxal as the coupling agent, the
immobillzed enzyme generally performs better at a lower pH
on lactose than on ONPG.
Immobilized lactase, on silica gel, ordinarily would be
used for processing whey at a temperature of about 20C
(room or ambient temperature) or less, and at the optimum pH
for the particular lactase. Thus, for lactase from
Asper~illus ni~er, a pH of about 4.5 would be best for enzyme
efficiency.
In Table 1, the loss of lactase activity was observed
when the immobilized lactase acted on ONPG as a substrate,
at 50C. At this temperature, which is well above the
temperature at which the immobilized enzyme would ordinarily
be used, the reaction goes forward rapidly, but the loss of
enzyme activity is also rapid. The Table 1 data demonstrate
that the loss in total activity is less for a double layer
~noblllzed
immobilizcr lactase than for a single layer, indicating that
the enzyme has been stabilized by the immobilization procedure.
~ ~ ~ 33~
In Table 2, the units of enzyme activity per unit
volume, at 30C., are compared as between one, two and
three layers, and where the coupling agent is glyoxal and
'glutaraldehyde. The figures for two layers and for three
llayers report the % increase in activity as compared to a
single layer. The activity "density" for the three layer
jimmobilized enzyme is very high, making this material
~very attractive for use in industrial processes.
1~ Enzyme stability for lactase on silica gel at 50C
~on an ONPG substrate is reported in Table 3, and half-life
is reported in Table 4. The three layer material clearly has
been thermally stabilized to a very significant extent, and
~the half-life significantly extended.
From other experiments, it has been determined that
beyond about 4 layers, the expense of multiple layering
tends to offset the gains, possibly because some cross-linking
between layers may occur. Generally, with lactase immobilized
on silica gel, activity levels in the range from about 7.5
units/ml. to about 30 units/ml., on ONPG at 30C.,
sedimented gel volume, are readily obtained. The lactase
enzyme used in practising the invention may be f~om any
desired source; that from Saccharomyces ~ra~ilis is suitable.
When immobilized on silica gel with glyoxal, in two layers,
a stability as to activity is ordinarily observed such that
at least 20% of the initial activity persists after 7.5
minutes at 50C at a pH of about 6.7.
While the dialdehyde cross-linkers, and specifically
glyoxal, represent preferred materials, the di-imidoesters
and bis-imidates are also preferred materials. The imidoester
dimethyl adipimidate approaches glyoxal in its performance
as a coupling agent.
;
l -26-
~3~
I
Il The enzymes suitable for immobilization are those having
available amine groups. This includes most enzymes of
~proteinaceous nature. Lactase and glucose isomerase are
llcommercially valuable enzymes that can be immobilized in
multiple layers successfully. The same techniques described
~in Example 1 are useful for producing immobilized glucose
~isomerase in multiple layers. The multilayer immobilized
glucose isomerase is especially useful for producing high
fructose corn syrup, by reason of its high activity per unit
volume.
Enzymes may be obtained from any suitable source,
either vegetable, animal or microbiological. In addition
to those mentioned above, the enzymes that act on starch
and on sugars are of particular interest. Other enzymes
that may be used in accordance with the invention include,
for example, cellulase, esterase, nuclease, invertase,
amyloglucosidase, and other types of hydrolases; hydrase,
pectinases, pepsin, rennin, chymotrypsin, trypsin, urease,
agrinase, lysozyme, cytochrome, ll-beta-hydroxylase, and
mixtures of these and other enzymes.
In addition, other biologically active materials may
be immobilized in multi-layer fashion. The immobilized
biologically active material thus obtained has a high level
of activity per unit volume that makes the immobilized material
very valuable for use in diagnostic assay applications,
purification operations, and chromatography applications. For
example, many antibodies and antigens have available amine
groups. When an antibody or antigen is immobilized in
accordance with the present invention, it provides a valuable
means for isolating its complementary immunochemical reactant,
offering potential for diagnostic assays.
33~
Similarly, hormones having available amine groups may be
~i~mobilized in multiple layers to provide highly concentrated
,'sources of hormone activity.
Il Among the features and advantages of the present invention
Ijare the very high activity that is obtainable per unit volume,
l¦the high mechanical stability, the high thermal stability, and
!~the high operational stability or half-life of the immobilized
~Ibiologically active materials. In achieving some of these
¦~advantages and features, the selection of the cross-linking
~ agent and the extent of cross-linking are important. Particularly
outstanding is the performance of multiple layer immobilized
enzyme as a catalyst for a variety of reactions for which
enzymes are useful.
When the carrier is silica gel, the immobilized enzyme
can conveniently be transferred from one container to
another by pouring the particulate, free-flowing silica gel
particles, which act very much like a liquid. This facilitates
use of the immobilized enzyme in conventional reactors such
I as, for example, pressure leaf filters and upright columns.
When lactase is immobilized on silica gel in accordance with
the invention, in four layers, a good performance can be
obtained in converting lactose to sweeter forms that are
more readily assimilable, permitting use of the invention for
the processing of milk, whey, and other dairy materials.
While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications, and this application is
intended to cover any variations, uses, or adaptations of the
invention following in general the principles of the invention
and including such departures from the present disclosure as
come within known or customary practice within the art to which
the invention pertains and as may be applied to the essential
features hereinbefore set forth, and within the scope of the
appended claims.
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