CA1143308A - High performance immobilized enzyme compositions by multi-layering immobilization offering a high amount of activity per unit volume - Google Patents
High performance immobilized enzyme compositions by multi-layering immobilization offering a high amount of activity per unit volumeInfo
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- CA1143308A CA1143308A CA000347295A CA347295A CA1143308A CA 1143308 A CA1143308 A CA 1143308A CA 000347295 A CA000347295 A CA 000347295A CA 347295 A CA347295 A CA 347295A CA 1143308 A CA1143308 A CA 1143308A
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- enzyme
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/18—Multi-enzyme systems
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
ABSTRACT
A high activity immobilized enzyme composite is prepared by covalently bonding a second enzyme layer to a first enzyme layer that is immobilized to u carrier. The process is repeated to apply third, fourth, and more successive enzyme layers. The product has high activity per unit volume, superior stability, and good half-life. Applicable to immunoreactants, hormones, and generally to biologically active material having available amine.
A high activity immobilized enzyme composite is prepared by covalently bonding a second enzyme layer to a first enzyme layer that is immobilized to u carrier. The process is repeated to apply third, fourth, and more successive enzyme layers. The product has high activity per unit volume, superior stability, and good half-life. Applicable to immunoreactants, hormones, and generally to biologically active material having available amine.
Description
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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.
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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 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 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.
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~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/('~>
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~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.
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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.
~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 e ~ ~o~o ~Dn n~
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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.
Leyers of ~ Half-life at 50'C
3 layers 11.5 min.
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.
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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 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 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.
~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.
~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 e ~ ~o~o ~Dn n~
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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.
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.
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 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.
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 .... .;~. ,
1 layer 5.5 min. .
NOTE: 1. Immobilized lactase activity was determined at its optimum pH.
2. ONPG was used as lactase substrate.
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 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.
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.
-2~8-
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.
-2~8-
Claims (34)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An enzymatically reactive composite comprising a solid, insoluble carrier having plural, sequentially applied amounts of enzyme immobilized thereto, said 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 im-mobilized to the carrier by covalent bonding through a covalent chemical coupling means comprising a polyfunc-tional reagent, said coupling means being chemically coupled to the carrier through said reactive groups and also chemically reacted with said first enzyme, and a second amount of the same or a different enzyme immobi-lized by covalent bonding through a second amount of said polyfunctional reactant to said immobilized first enzyme, said enzymes substantially retaining their respective activities, the total enzyme activity of the enzymatically reactive composite being greater than that of said first amount of immobilized enzyme.
2. The composite of claim 1 wherein the carrier is particulate, free-flowing solid siliceous material.
3. The composite of claim 1 or 2 wherein the covalent bond from said first enzyme to said second enzyme is the reaction product of a dialdehyde and an available amine group of each of the said enzymes respectively.
4. The composite of claim 1 or 2 wherein the covalent bond to the enzyme is the reaction product of a diimidoester and an available amine group of the enzyme.
5. The composite of claim 1 wherein the carrier is a silica gel.
6. The composite of claim 5 wherein said first-named enzyme and said second-named enzyme are the same enzyme.
7. The composite of claim 6 wherein the enzyme is lactase.
8. The composite of claim 6 wherein the enzyme is glucose isomerase.
9. The composite of claim 1, 5 or 6 wherein the polyfunctional reactant is ethanedial.
10. The composite of claim 6, 7 or 8 where-in an additional, third amount of enzyme is immobilized to said second-named enzyme through a third quantity of said polyfunctional reagent, the total enzyme activity of the enzymatically reactive composite being greater than that of the combined activities of said first and second amounts of immobilized enzyme.
11. An enzymatically active composition of an enzyme having available amine groups that is chemically coupled to a carrier having chemically reactive hydroxyl groups at its surface, said composite comprising a solid, insoluble inorganic, siliceous carrier in the form of free-flowing, finely divided particles, said carrier particles having plural, sequentially applied amounts of enzyme immobilized thereto, said carrier particles providing high surface area per unit volume, there being a first amount of an enzyme that has available amine groups and that is immobilized to the carrier par-ticles by covalent bonding through a covalent chemical coupling means comprising a first quantity of a di-functional reactant, said coupling means being coupled with the carrier through said hydroxyl groups of the carrier and chemically reacted with the enzyme through the available amine groups of the enzyme, and a second amount of enzyme covalently bonded to the said immobi-lized, first-named enzyme, through a second quantity of difunctional reactant that is reacted with the available amine groups of both of said enzymes respectively.
12. The composition of claim 11 wherein the enzymes are lactase and the carrier is silica gel.
13. The composition of claim 12 having an activity of from at least about 7.5 to about 30 units per ml on ONPG at 30°C.
14. The composition of claim 13 wherein the enzymes are derived from Saccharomyces fragilis, and wherein the immobilized enzyme composition is characterized by a stability as to activity in that at least 20% of the initial activity persists after 7.5 minutes at 50°C
at a pH of about 6.7.
at a pH of about 6.7.
15. The composition of claim 12 having an activity of from at least 15 to about 60 units per ml on lactose at 30°C.
16. The composition of claim 11 wherein the enzymes are derived from Aspergillus niger.
17. The composition of claim 11, 12 or 13 wherein said difunctional reactant is selected from the group consisting of dialdehydes, bis-imidoesters, bis-propiolates and disulfonyl halides.
18. The composition of claim 14, 15 or 16 wherein said difunctional reactant is selected from the group consisting of dialdehydes, bis-imidoesters, bis-propiolates and disulfonyl halides,
19. The composition of claim 11 wherein the enzymes are glucose isomerase.
20. The composition of claim 11 wherein the carrier is silica gel and one of said enzymes is lactase derived from microbial sources and the other of said enzymes is microbial glucose isomerase.
21. The composition of claim 11, 12 or 13 wherein said difunctional reactant is selected from the group consisting of ethanedial and glutaraldehyde.
22. The composition of claim 14, 15 or 16 wherein said difunctional reactant is selected from the group consisting of ethanedial and glutaraldehyde.
23. An immobilized lactase composition according to claim 11, in which the lactase is chemically coupled to a silica gel carrier having chemically re-active hydroxyl groups at its surface, said composite comprising a finely divided, free-flowing, silica gel carrier providing high surface area per unit volume, a first amount of lactase that is covalently bonded to the silica gel carrier particles through said covalent chemical coupling means including a difunctional reactant, said coupling means being reacted with the carrier through hydroxyl groups of the carrier and with the enzyme through available amine groups of the lactase, and a second, sequentially applied amount of lactase that is covalently bonded through a second quantity of said difunctional reactant that has been reacted with available amine groups of said second amount of lactase and with available amine groups of the initially immobilized first amount of lactase, both of said amounts of lactase contributing to the lactase activity of the immobilized lactase composi-tion, said difunctional reactant being selected from the group consisting of ethanedial, glutaraldehyde, and O-phthaldialdehyde.
24. An immobilized lactase composition in accordance with claim 23 wherein a third, sequentially applied amount of lactase is covalently bonded to said immobilized second-applied amount of lactase, through a third amount of said difunctional reactant that is reacted with available amine groups of said second-applied amount of lactase and with the available amine groups of said third-applied amount of lactase, the total enzyme activity of the enzymatically reactive composite being greater than that of the combined activities of said first and second amounts of immobilized enzyme.
25. The immobilized lactase composition of claim 24 wherein a fourth, sequentially applied amount of lactase is covalently bonded to said immobilized, third-applied amount of lactase through the reaction of a fourth amount of said difunctional reactant with the amine groups of said immobilized third-applied amount of lactase and with amine groups of said fourth-applied amount of lactase.
26. The immobilized lactase composition of claim 23, 24 or 25 wherein said difunctional reactant is ethanedial.
27. The immobilized lactase composition of claim 23, 24 or 25 wherein said difunctional reactant is ethanedial, wherein the enzyme is derived from Saccharomyces fragilis, and wherein the immobilized enzyme is characterized by an activity of from at least about 7.5 to about 30 units per ml on ONPG at 30°C, and further characterized by a stability as to activity in that at least 20% of the initial activity persists after 7.5 minutes at 50°C at a pH of about 6.7.
28. The immobilized lactase composition of claim 23, 24 or 25 wherein said difunctional reactant is ethanedial and wherein the enzyme is derived from Aspergillus niger.
29. An improved immobilized enzyme process wherein the immobilized enzyme is brought into contact with an aqueous solution of a substrate on which it is active, permitted to act on said substrate, and then the substrate and said immobilized enzyme are separated from each other, wherein the immobilized enzyme is in the form of the composite of claim 1, 11 or 20.
30. An improved immobilized enzyme process wherein the immobilized enzyme is brough into contact with an aqueous solution of a substrate on which it is active, permitted to act on said substrate, and then the substrate and said immobilized enzyme are separated from each other, wherein the immobilized enzyme is in the form of the composite of claim 23.
31. A process for preparing an enzymatically reactive composite in accordance with claim 1 comprising:
treating said first amount of enzyme that is immobilized on said carrier with a first amount of said polyfunctional reactant that reacts with said first, immobilized enzyme without inactivating it and that im-parts functionality to said immobilized enzyme, then, bringing said functional immobilized enzyme into contact with an aqueous solution of said second amount of enzyme that reacts with said functional immobi-lized enzyme, to itself become immobilized to said first, initially immobilized enzyme through covalent bonding.
treating said first amount of enzyme that is immobilized on said carrier with a first amount of said polyfunctional reactant that reacts with said first, immobilized enzyme without inactivating it and that im-parts functionality to said immobilized enzyme, then, bringing said functional immobilized enzyme into contact with an aqueous solution of said second amount of enzyme that reacts with said functional immobi-lized enzyme, to itself become immobilized to said first, initially immobilized enzyme through covalent bonding.
32. A process for preparing an enzymatically active composition according to claim 11, comprising:
treating said first amount of enzyme that is immobilized on said finely divided carrier particles, and that has available amine groups, with said second quantity of said difunctional reactant that reacts with the amine groups of said first, immobilized enzyme with-out inactivating the enzyme and to impart functionality to said immobilized enzyme, then bringing said functional immobilized enzyme into contact with an aqueous solution of said second amount of enzyme that has available amine groups that react with the functional groups of said functional im-mobilized enzyme, so that said second enzyme itself becomes immobilized to said first, initially immobilized enzyme through covalent bonding.
treating said first amount of enzyme that is immobilized on said finely divided carrier particles, and that has available amine groups, with said second quantity of said difunctional reactant that reacts with the amine groups of said first, immobilized enzyme with-out inactivating the enzyme and to impart functionality to said immobilized enzyme, then bringing said functional immobilized enzyme into contact with an aqueous solution of said second amount of enzyme that has available amine groups that react with the functional groups of said functional im-mobilized enzyme, so that said second enzyme itself becomes immobilized to said first, initially immobilized enzyme through covalent bonding.
33. An improved enzyme process for hydro-lizing lactose, comprising passing a solution of lactose in an aqueous medium into contact with an immobilized enzyme that is active to hydrolyze lactose, wherein the immobilized enzyme is that of claim 7, 12 or 23, and thereafter recovering a solution, separated from the immobilized enzyme, of the hydrolysis products of lactose produced by the action of the immobilized enzyme.
34. An improved enzyme process for hydro-lizing lactose, comprising passing a solution of lactose in an aqueous medium into contact with an immobilized enzyme that is active to hydrolyze lactose, wherein the immobilized enzyme is that of claim 13, 24 or 25, and thereafter recovering a solution, separated from the im-mobilized enzyme, of the hydrolysis products of lactose produced by the action of the immobilized enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000347295A CA1143308A (en) | 1980-03-10 | 1980-03-10 | High performance immobilized enzyme compositions by multi-layering immobilization offering a high amount of activity per unit volume |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000347295A CA1143308A (en) | 1980-03-10 | 1980-03-10 | High performance immobilized enzyme compositions by multi-layering immobilization offering a high amount of activity per unit volume |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1143308A true CA1143308A (en) | 1983-03-22 |
Family
ID=4116432
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000347295A Expired CA1143308A (en) | 1980-03-10 | 1980-03-10 | High performance immobilized enzyme compositions by multi-layering immobilization offering a high amount of activity per unit volume |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2568586A1 (en) * | 1984-08-02 | 1986-02-07 | Nabisco Brands Inc | METHOD FOR CONSTANTLY MAINTAINING THE ACTIVITY OF AN ENZYME IMMOBILIZED IN A REACTOR |
| US5177005A (en) * | 1984-08-02 | 1993-01-05 | Stabra Ag | Method for maintaining immobilized glucose isomerase activity during continuous isomerization of glucose to fructose |
-
1980
- 1980-03-10 CA CA000347295A patent/CA1143308A/en not_active Expired
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2568586A1 (en) * | 1984-08-02 | 1986-02-07 | Nabisco Brands Inc | METHOD FOR CONSTANTLY MAINTAINING THE ACTIVITY OF AN ENZYME IMMOBILIZED IN A REACTOR |
| EP0171258A2 (en) * | 1984-08-02 | 1986-02-12 | Stabra AG | On-column loading |
| EP0171258A3 (en) * | 1984-08-02 | 1989-04-12 | Nabisco Brands, Inc. | On-column loading |
| US5177005A (en) * | 1984-08-02 | 1993-01-05 | Stabra Ag | Method for maintaining immobilized glucose isomerase activity during continuous isomerization of glucose to fructose |
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