CA1100066A - Water-insoluble enzyme compositions - Google Patents
Water-insoluble enzyme compositionsInfo
- Publication number
- CA1100066A CA1100066A CA305,057A CA305057A CA1100066A CA 1100066 A CA1100066 A CA 1100066A CA 305057 A CA305057 A CA 305057A CA 1100066 A CA1100066 A CA 1100066A
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- CA
- Canada
- Prior art keywords
- enzyme
- support material
- composition
- activity
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- 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
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
Abstract
ABSTRACT OF THE DISCLOSURE
An improved process for preparing a water-in-soluble enzyme composition having a high enzymatic activi-ty and containing the enzyme covalently bonded to an in-organic support material is disclosed, wherein such an inorganic support material being capable of covalently bonding the enzyme, which has a pore-size distribution wherein the most frequent pore-diameter is such that in the resulting enzyme composition for any enzyme concen-tration contained therein the enzymatic activity per weight unit of the composition is the highest activity per weight unit of the composition which is obtainable at this enzyme concentration, is contacted with an enzyme solution comprising such an amount of enzyme, which is sufficiently low that the enzyme which is bonded in the resulting enzyme composition has a spe-cific activity which substantially is about the same as that of the enzyme in the free state. The support ma-terial suitably comprises a SiO2-gel of appropriate pore-size distribution.
An improved process for preparing a water-in-soluble enzyme composition having a high enzymatic activi-ty and containing the enzyme covalently bonded to an in-organic support material is disclosed, wherein such an inorganic support material being capable of covalently bonding the enzyme, which has a pore-size distribution wherein the most frequent pore-diameter is such that in the resulting enzyme composition for any enzyme concen-tration contained therein the enzymatic activity per weight unit of the composition is the highest activity per weight unit of the composition which is obtainable at this enzyme concentration, is contacted with an enzyme solution comprising such an amount of enzyme, which is sufficiently low that the enzyme which is bonded in the resulting enzyme composition has a spe-cific activity which substantially is about the same as that of the enzyme in the free state. The support ma-terial suitably comprises a SiO2-gel of appropriate pore-size distribution.
Description
1~0~Q66 BACKGROUND OF THE INVENTION
The present invention relates to a process for preparing a water-insoluble immobilized enzyme composition.
Methods for immobilizing enzymes are well known in the art. ~he terms "immobilizing enzymes"
and "immobilized enzymes" as applied in the present specification mean that these enzymes are rendered essentially insoluble, so that they are rendered re-usable and can be utilized in continuous processes.
For this purpose enzymes are attached to insoluble support materials by means of adsorption or covalent bonding. Conventionally the support material is loaded with the enzyme by treating it with an enzyme solution.
Organic carrier materials (such as cellulose, nylon, or polyac~lamide) exhibit serious disadvantages since they do not possess sufficient mechanical strength, can be affected by the action of solvents, and are sen-sitive against changes in the pH-value or the ion con-centration in the surrounding medium. Furthermore, several of these organic materials are susceptible to being attacked by micro~es which may cause a loosening of the bond between the enzyme and the support material.
Therefore, inorganic materials have been .
suggested as carrier materials onto which enzymes may be attached by means of adsorption or covalent bonding. Which type of attachment is preferred is depending on the properties of the substrate for which, and on the conditions under which the enzyme is to be utilized. If the substrate is comprised in a medium which contains a high salt concentration, an attachment by mere adsorption cannot be used, since desorption of the adsorbed enzyme molecules may occur.
Therefore, a covalent bonding between the enzyme and the carrier is preferred. For this,the carrier sur-face must include a sufficient amount of functional groups which are capable of forming a covalent bond with the respective enzyme. Since most inorganic carrier materials do not contain such specific func-tional groups, a pretreatment of the carrier surface is necessary. A conventional method for providing functional organic groups in the surface of an in-organic carrier material comprises loading the in-organic materials with silanes, which attachto the surface of the carrier material and provide the latter with functional organic groups, preferably alkylamino groups, which are capable of forming a covalent bond with organic compounds. Treatment of
The present invention relates to a process for preparing a water-insoluble immobilized enzyme composition.
Methods for immobilizing enzymes are well known in the art. ~he terms "immobilizing enzymes"
and "immobilized enzymes" as applied in the present specification mean that these enzymes are rendered essentially insoluble, so that they are rendered re-usable and can be utilized in continuous processes.
For this purpose enzymes are attached to insoluble support materials by means of adsorption or covalent bonding. Conventionally the support material is loaded with the enzyme by treating it with an enzyme solution.
Organic carrier materials (such as cellulose, nylon, or polyac~lamide) exhibit serious disadvantages since they do not possess sufficient mechanical strength, can be affected by the action of solvents, and are sen-sitive against changes in the pH-value or the ion con-centration in the surrounding medium. Furthermore, several of these organic materials are susceptible to being attacked by micro~es which may cause a loosening of the bond between the enzyme and the support material.
Therefore, inorganic materials have been .
suggested as carrier materials onto which enzymes may be attached by means of adsorption or covalent bonding. Which type of attachment is preferred is depending on the properties of the substrate for which, and on the conditions under which the enzyme is to be utilized. If the substrate is comprised in a medium which contains a high salt concentration, an attachment by mere adsorption cannot be used, since desorption of the adsorbed enzyme molecules may occur.
Therefore, a covalent bonding between the enzyme and the carrier is preferred. For this,the carrier sur-face must include a sufficient amount of functional groups which are capable of forming a covalent bond with the respective enzyme. Since most inorganic carrier materials do not contain such specific func-tional groups, a pretreatment of the carrier surface is necessary. A conventional method for providing functional organic groups in the surface of an in-organic carrier material comprises loading the in-organic materials with silanes, which attachto the surface of the carrier material and provide the latter with functional organic groups, preferably alkylamino groups, which are capable of forming a covalent bond with organic compounds. Treatment of
2~ the inorganic carrier material with glutardialdehyde, sulfuryl chloride, thionyl chloride,or cyanur chloride, has also been tried.
1100~66 It also is possible to provide the surface of the inorganic carrier material with a coating of a water-insoluble organic polymer which comprises free functional groups, for example, a polyacrolein S which comprises between about 10 and about 70% of free aldehyde qroups relative to the number of mono-mer units.
Aluminum oxides, nickel oxide, iron oxide, titane oxide, zirconium oxide, hydroxyapatite, sili-cates, and porous glass have been proposed as porousinorganic carrier materials. The pore-structure with-in these carrier materials has to be such that the enzyme is able to reach the inner surface of the sup-port particles. Yet, with regard to which additional properties of the carrier material are desirable, i.e., which are the optimum pore-size distributions and/or surface areas, the available information differs largely from each other.
Indepently from the type of bonding between enzyme and support material, and using any of the aforementioned support materials, it has not yet been possible to immobilize enzymes in such a manner that the specific activity of the enzyme in the immobilized state reaches the value of the specific activity of the enzyme in the free state. According to D. L.
Latigue (see Immobilized Enzymes for Industrial Re-actors, London, 1975, p. 127), even under the most ~10Q0~;6 favorable immobilizing conditions only 80~ at the most of the enzyme which is attached to the support material is present in active form.
SU~MARY OF THE INVENTION
It is an object of the present invention to provide a process for preparing an immobilized enzyme composition wherein the enzyme is covalently bonded to an inorganic support material, whereby a maximum activity of the immobilized enzyme compo-sition is achieved by using the lowest possible amount of enzyme.
It is a further object of the present in-vention to provide such a process by which an immo-bilized enzyme composition is obtained which exhi-bits the highest enzymatic activity per weight unitof the composition which is possible at the given enzyme concentration within the composition.
It is a further object of the present in-vention to provide such a process by which a high 2~ specific activity of the enzyme in the immobilized state within the composition is achieved, in particu-lar a specific activity of the enzyme in the immo-bilized state, which is in the same order as that of the enzyme in the free state.
~66 It is a further object of the present in-vention to provide such a process by which immobi-lized enzyme compositions for industrial reactors can be prepared at relatively low costs.
It is a further object of the present in-vention to provide a process for determining the pore-structure of a carrier material and the concentration of an enzyme composition which are most favorable for preparing an immobilized enzyme composition wherein 1~ the enzymatic activity per weight unit of the compo-sition and the specific activity of the immobilized enzyme therein are high.
In order to accomplish the foregoing objects according to the present invention, there is provided an improved process for preparing a water-insoluble enzyme composition having a high enzymatic activity and containing the enzyme covalently bonded to an inorganic support material comprising the steps of a) contacting an inorganic support material which is capable of ~valently binding the enzyme with a solution of the enzyme in an aqueous solvent, whereby a portion of the enzyme is taken up by the support material and covalently bonded there-to, to form a water-insoluble enzyme-containing composition, and b) separating the water-insoluble en2yme-containing ! - 5 ~
110~Q66 composition from the remaining solution, where-in the improvement comprises the inorganic sup-port material, having a pore-size distribution wherein the most frequent pore-diameter is such that in the resulting enzyme composition for any enzyme concentration contained therein, the en-zymatic activity per weight unit of the composi-tion is the highest activity per weight unit of the composition which is obtainable at this en-zyme concentration, and the enzyme solution com-prising such an amount of enzyme which is suffi-ciently low that the enzyme which is bonded in the resulting enzyme composition has a specific - activity which substantially is about the same as that of the enzyme in the free state.
Further objects, features, and advantages of the present invention will become apparent from the detailed description of the invention and its preferred embodiments which follows.
DETAILED DESCRIPTION OF THE INVENTION
AND ITS PREFERRED EMBODIMENTS
Conventional inorganic carrier materials can be utilized within the process according to the present invention. Such carrier materials comprise hydroxy-groups-containing oxides of elements selected from the group consisting of silicium, aluminum, nickel, iron, titanium, zirconium, or mixtures there-of.
In order to obtain a support material which is capable of covalently binding the enzyme, it is preferred to provide, in a conventional manner, the inorganic material with a coupling agent which is sufficiently strongly attached to the carrier material preferably by means of a covalent bond and also is capable of forming a covalent bond with the enzyme.
As mentioned above, various different coupling agents are known in the art and any of these conventional coupling agents can be utilized within the present inventlon .
- Until now organic silanes have been most commonly used and methods for silanizing the inor-ganic carrier material are well known in the art.
Preferably, the inorganic carrier material is treated with a solution of the silane in a high-boiling sol-vent, e.g., a high-boiling aromatic or aliphalic hydrocarbon, in particular benzene or toluene.
The amount of coupling agent must be suit-ably high to provide a sufficient number of function-al groups to the carrier surface and will depend largely on the surface area of the carrier material which is used.
Suitable silane coupling agents comprise ~10Q~66 silanes, having the formula R - (CH2)~ - li ~ (Rl)n (R2)3-n wherein Rl and R2 each represent lower alkyl, pre-x represents 1-5, ~referablv 3 ferably methyl, n represents l-~and R represents a functional group which is capable of forming a covalent bond with the enzyme, e.g., amino or car-bonyl.
Other suitable organic coupling agents comprise lower dialdehydes such as glutardialdehyde.
According to the present invention, the most favorable most frequent pore-diameter of an inorganic support material which is to be used as starting material for preparing a specific enzyme/
- support composition and the most favorable amount of the enzyme which is to be included into the starting enzyme sol-ltion are determined as follows:
Different inorganic carrier materials of the same chemical composition yet which are dis-tinguished from each other by a different most fre~uent pore-diameter are pretreated with a coupling agent to obtain the support material which is cap-able of covalently ~inding the enzyme. Then, vari-ous amounts of the enzyme are offered to each of - the pretreated support materials by contacting them with various solutions of the enzyme, each having a different enzyme content and attaching the enzyme to the support material in a conventional manner by forming a covalent bond between the enzyme and the functional group of the coupling agent. The enzyma-tic activity per weight unit of the thus obtained compositions is determined. The results indicate that regardless of the amount of enzyme which is bonded in the composition, the activity of the com-position is always dependent on the most frequentpore-diameter, and passes through a maximum at a certain pore-diameter range. Furthermore, it has been found that the particle-size of the support material is substantially irrelevant with regard to the pore-diameter range at which the maximum occurs and at the most, might influence the actual value of the maximum. Therefore, the particle-size of the immobilized enzyme composition is only of minor significance within the context of the present in-vention and can be chosen mainly depending on theintended use of the composition, e.g., the viscosity of the substrate for which, and the reaction con-ditions under which it is to be used.
The inorganic support material is selected which has the most favorable most frequent pore-diameter which,independently of the enzyme contents llOOQ66 in the enzyme solutions,yields the enzyme compo-sition having the highest enzymatic activity per weight unit among the group of enzyme compositions obtained from the same enzyme solutions.
Again, various amounts of the enzyme are of-fered to this inorganic support material having the most favorable most frequent pore-diameter by contacting it with solutions containing different amounts of the enzyme. The specific activity of the enzyme which is bonded in the resulting enzyme compositions is determined and is compared with that of the enzyme in free state. From the results, it is appàrent that at a certain enzyme concentration, enzyme compositions are obtained wherein the specific activity of the enzyme in the bond state nearly or completely reaches that of the enzyme in the free state, that is the relative activity of the compo-sition reaches 100%.
According to a preferred embodiment of the present invention, the inorganic carrier material comprises a SiO -gel. Preferably, the SiO -gel is obtained by adjusting the alkali-content expressed as ~ by weight of Na2O to from about 0.1 to about 0.5% by weight, drying the gel, and calcining the gel at a temperature of from about 400 to about 850C, preferably from about 570 to about 750C, in ~a~6~
a flow of water-vapor-containing air during a period of from about 5 to about 10 hours.
It is advisable to carry out the drying at a temperature of between about 180 and about 200C un-der water-vapor-saturated air. The calcining is advantageously effected under a flow of air having a relative humidity of between about 40 and about 80%.
The pore-size distribution in the resulting carrier material is such that the most frequent pore-diameter is in the range of between about 175 and about 3,000 A, preferably in the range of between about 250 and about 500 A, most preferably about 34OA. -lS The process for immobilizing enzymes accord~
ing to the present invention can be applied to any enzymes, e.g., any enzymes which are utilized for technical or analytical purposes, for example, hydrolytic enzymes such as amylases,glucosidases, or proteases, redox enzymes such as glucose-oxidase or catalase, isomerases such as glucose-isomerase, or transferase enzymes such as dextran-sucrose-trans-ferase.
According to an especially preferred embodi-ment of the invention, the enzyme is amylogluco-: sidase which in the free state exhibits a specific ~CI~i6 activity of from about lO to aboùt 15 units/mg, and the support material comprises the SiO2-gel support material according to the above de~ined preferred embodiment. Most preferably the most frequent pore-diameter of this support material is between about 250 and about 600 A, in particular between about 300 and about 400 A, especially about 340 A. The most favorable immobilized amyloglucosidase compo-sition is obtained if this support material is contacted with a solution containing from about 25 to about 75, preferably about 50 mg of amylo-glucosidase per l g of support material.
Any conventional coupling agent may be used in this composition, for example a silane of the above mentioned formula or glutardialdehyde.
According to another especially preferred embodiment of the present invention, the enzyme is glucose-isomerase which in the free state exhibits a specific activity of from about 50 to about 70 units/mg, and the support material comprises the SiO2-gel support material according to the above defined preferred embodiment. Most preferably the most frequent pore-diameter of this support material is between about 250 and about 600 A, in particular between a~out 300 and about 400 A, especially about 340 A. The most favorable immobilized glucose-1~0~6 isomerase composition is obtained if this support material is contacted with a solution containing from about 20 to about 50, preferably about 25 mg of glucose-isomerase per 1 g of support material.
Any conventional coupling agent may be used in this composition, for example, a silane of the above mentioned formula or glutardialdehyde.
The invention will now be further illustrated by the following examples which are intended to be illustrative only.
~Q(~66 EX~PLE 1:
1.0 Preparation of support material No. 1Ø
SiO2-gel, having an alkali-content of 0.3% by weight of Na2O, which has been precipitated from a sodium-silicate solution by means of sulfuric acid, is dried fora period of 3 hours at a temperature of 180C in an at-mosphere of water-vapor-saturated air. 1 kg of this ma-terial is calcined at a temperature of 730C for a period of 6 hours in a stream of air, the relative humidity of which is 80~ and the flow-rate of which is 2 l/min. In the SiO2 resulting from this treatment the pore-size distribution is characterized by a most frequent pore-diameter of about 1400 A. The carrier material is divided into various particle-size fractions by passing it through various sieves of different mesh size~ The fraction, having a particle-size of from about 0.25 to about 0.5 mm, is used for preparing the support material.
A mixture of lS0 g of this carrier material frac-tion and 4 1 of a 10% solution of ~-aminopropyltriethoxy silane in benzene is heated to reflux temperature for a period of 8 hours. ~hen the reaction mixture is cooled and the resulting support material is filtered off and washed
1100~66 It also is possible to provide the surface of the inorganic carrier material with a coating of a water-insoluble organic polymer which comprises free functional groups, for example, a polyacrolein S which comprises between about 10 and about 70% of free aldehyde qroups relative to the number of mono-mer units.
Aluminum oxides, nickel oxide, iron oxide, titane oxide, zirconium oxide, hydroxyapatite, sili-cates, and porous glass have been proposed as porousinorganic carrier materials. The pore-structure with-in these carrier materials has to be such that the enzyme is able to reach the inner surface of the sup-port particles. Yet, with regard to which additional properties of the carrier material are desirable, i.e., which are the optimum pore-size distributions and/or surface areas, the available information differs largely from each other.
Indepently from the type of bonding between enzyme and support material, and using any of the aforementioned support materials, it has not yet been possible to immobilize enzymes in such a manner that the specific activity of the enzyme in the immobilized state reaches the value of the specific activity of the enzyme in the free state. According to D. L.
Latigue (see Immobilized Enzymes for Industrial Re-actors, London, 1975, p. 127), even under the most ~10Q0~;6 favorable immobilizing conditions only 80~ at the most of the enzyme which is attached to the support material is present in active form.
SU~MARY OF THE INVENTION
It is an object of the present invention to provide a process for preparing an immobilized enzyme composition wherein the enzyme is covalently bonded to an inorganic support material, whereby a maximum activity of the immobilized enzyme compo-sition is achieved by using the lowest possible amount of enzyme.
It is a further object of the present in-vention to provide such a process by which an immo-bilized enzyme composition is obtained which exhi-bits the highest enzymatic activity per weight unitof the composition which is possible at the given enzyme concentration within the composition.
It is a further object of the present in-vention to provide such a process by which a high 2~ specific activity of the enzyme in the immobilized state within the composition is achieved, in particu-lar a specific activity of the enzyme in the immo-bilized state, which is in the same order as that of the enzyme in the free state.
~66 It is a further object of the present in-vention to provide such a process by which immobi-lized enzyme compositions for industrial reactors can be prepared at relatively low costs.
It is a further object of the present in-vention to provide a process for determining the pore-structure of a carrier material and the concentration of an enzyme composition which are most favorable for preparing an immobilized enzyme composition wherein 1~ the enzymatic activity per weight unit of the compo-sition and the specific activity of the immobilized enzyme therein are high.
In order to accomplish the foregoing objects according to the present invention, there is provided an improved process for preparing a water-insoluble enzyme composition having a high enzymatic activity and containing the enzyme covalently bonded to an inorganic support material comprising the steps of a) contacting an inorganic support material which is capable of ~valently binding the enzyme with a solution of the enzyme in an aqueous solvent, whereby a portion of the enzyme is taken up by the support material and covalently bonded there-to, to form a water-insoluble enzyme-containing composition, and b) separating the water-insoluble en2yme-containing ! - 5 ~
110~Q66 composition from the remaining solution, where-in the improvement comprises the inorganic sup-port material, having a pore-size distribution wherein the most frequent pore-diameter is such that in the resulting enzyme composition for any enzyme concentration contained therein, the en-zymatic activity per weight unit of the composi-tion is the highest activity per weight unit of the composition which is obtainable at this en-zyme concentration, and the enzyme solution com-prising such an amount of enzyme which is suffi-ciently low that the enzyme which is bonded in the resulting enzyme composition has a specific - activity which substantially is about the same as that of the enzyme in the free state.
Further objects, features, and advantages of the present invention will become apparent from the detailed description of the invention and its preferred embodiments which follows.
DETAILED DESCRIPTION OF THE INVENTION
AND ITS PREFERRED EMBODIMENTS
Conventional inorganic carrier materials can be utilized within the process according to the present invention. Such carrier materials comprise hydroxy-groups-containing oxides of elements selected from the group consisting of silicium, aluminum, nickel, iron, titanium, zirconium, or mixtures there-of.
In order to obtain a support material which is capable of covalently binding the enzyme, it is preferred to provide, in a conventional manner, the inorganic material with a coupling agent which is sufficiently strongly attached to the carrier material preferably by means of a covalent bond and also is capable of forming a covalent bond with the enzyme.
As mentioned above, various different coupling agents are known in the art and any of these conventional coupling agents can be utilized within the present inventlon .
- Until now organic silanes have been most commonly used and methods for silanizing the inor-ganic carrier material are well known in the art.
Preferably, the inorganic carrier material is treated with a solution of the silane in a high-boiling sol-vent, e.g., a high-boiling aromatic or aliphalic hydrocarbon, in particular benzene or toluene.
The amount of coupling agent must be suit-ably high to provide a sufficient number of function-al groups to the carrier surface and will depend largely on the surface area of the carrier material which is used.
Suitable silane coupling agents comprise ~10Q~66 silanes, having the formula R - (CH2)~ - li ~ (Rl)n (R2)3-n wherein Rl and R2 each represent lower alkyl, pre-x represents 1-5, ~referablv 3 ferably methyl, n represents l-~and R represents a functional group which is capable of forming a covalent bond with the enzyme, e.g., amino or car-bonyl.
Other suitable organic coupling agents comprise lower dialdehydes such as glutardialdehyde.
According to the present invention, the most favorable most frequent pore-diameter of an inorganic support material which is to be used as starting material for preparing a specific enzyme/
- support composition and the most favorable amount of the enzyme which is to be included into the starting enzyme sol-ltion are determined as follows:
Different inorganic carrier materials of the same chemical composition yet which are dis-tinguished from each other by a different most fre~uent pore-diameter are pretreated with a coupling agent to obtain the support material which is cap-able of covalently ~inding the enzyme. Then, vari-ous amounts of the enzyme are offered to each of - the pretreated support materials by contacting them with various solutions of the enzyme, each having a different enzyme content and attaching the enzyme to the support material in a conventional manner by forming a covalent bond between the enzyme and the functional group of the coupling agent. The enzyma-tic activity per weight unit of the thus obtained compositions is determined. The results indicate that regardless of the amount of enzyme which is bonded in the composition, the activity of the com-position is always dependent on the most frequentpore-diameter, and passes through a maximum at a certain pore-diameter range. Furthermore, it has been found that the particle-size of the support material is substantially irrelevant with regard to the pore-diameter range at which the maximum occurs and at the most, might influence the actual value of the maximum. Therefore, the particle-size of the immobilized enzyme composition is only of minor significance within the context of the present in-vention and can be chosen mainly depending on theintended use of the composition, e.g., the viscosity of the substrate for which, and the reaction con-ditions under which it is to be used.
The inorganic support material is selected which has the most favorable most frequent pore-diameter which,independently of the enzyme contents llOOQ66 in the enzyme solutions,yields the enzyme compo-sition having the highest enzymatic activity per weight unit among the group of enzyme compositions obtained from the same enzyme solutions.
Again, various amounts of the enzyme are of-fered to this inorganic support material having the most favorable most frequent pore-diameter by contacting it with solutions containing different amounts of the enzyme. The specific activity of the enzyme which is bonded in the resulting enzyme compositions is determined and is compared with that of the enzyme in free state. From the results, it is appàrent that at a certain enzyme concentration, enzyme compositions are obtained wherein the specific activity of the enzyme in the bond state nearly or completely reaches that of the enzyme in the free state, that is the relative activity of the compo-sition reaches 100%.
According to a preferred embodiment of the present invention, the inorganic carrier material comprises a SiO -gel. Preferably, the SiO -gel is obtained by adjusting the alkali-content expressed as ~ by weight of Na2O to from about 0.1 to about 0.5% by weight, drying the gel, and calcining the gel at a temperature of from about 400 to about 850C, preferably from about 570 to about 750C, in ~a~6~
a flow of water-vapor-containing air during a period of from about 5 to about 10 hours.
It is advisable to carry out the drying at a temperature of between about 180 and about 200C un-der water-vapor-saturated air. The calcining is advantageously effected under a flow of air having a relative humidity of between about 40 and about 80%.
The pore-size distribution in the resulting carrier material is such that the most frequent pore-diameter is in the range of between about 175 and about 3,000 A, preferably in the range of between about 250 and about 500 A, most preferably about 34OA. -lS The process for immobilizing enzymes accord~
ing to the present invention can be applied to any enzymes, e.g., any enzymes which are utilized for technical or analytical purposes, for example, hydrolytic enzymes such as amylases,glucosidases, or proteases, redox enzymes such as glucose-oxidase or catalase, isomerases such as glucose-isomerase, or transferase enzymes such as dextran-sucrose-trans-ferase.
According to an especially preferred embodi-ment of the invention, the enzyme is amylogluco-: sidase which in the free state exhibits a specific ~CI~i6 activity of from about lO to aboùt 15 units/mg, and the support material comprises the SiO2-gel support material according to the above de~ined preferred embodiment. Most preferably the most frequent pore-diameter of this support material is between about 250 and about 600 A, in particular between about 300 and about 400 A, especially about 340 A. The most favorable immobilized amyloglucosidase compo-sition is obtained if this support material is contacted with a solution containing from about 25 to about 75, preferably about 50 mg of amylo-glucosidase per l g of support material.
Any conventional coupling agent may be used in this composition, for example a silane of the above mentioned formula or glutardialdehyde.
According to another especially preferred embodiment of the present invention, the enzyme is glucose-isomerase which in the free state exhibits a specific activity of from about 50 to about 70 units/mg, and the support material comprises the SiO2-gel support material according to the above defined preferred embodiment. Most preferably the most frequent pore-diameter of this support material is between about 250 and about 600 A, in particular between a~out 300 and about 400 A, especially about 340 A. The most favorable immobilized glucose-1~0~6 isomerase composition is obtained if this support material is contacted with a solution containing from about 20 to about 50, preferably about 25 mg of glucose-isomerase per 1 g of support material.
Any conventional coupling agent may be used in this composition, for example, a silane of the above mentioned formula or glutardialdehyde.
The invention will now be further illustrated by the following examples which are intended to be illustrative only.
~Q(~66 EX~PLE 1:
1.0 Preparation of support material No. 1Ø
SiO2-gel, having an alkali-content of 0.3% by weight of Na2O, which has been precipitated from a sodium-silicate solution by means of sulfuric acid, is dried fora period of 3 hours at a temperature of 180C in an at-mosphere of water-vapor-saturated air. 1 kg of this ma-terial is calcined at a temperature of 730C for a period of 6 hours in a stream of air, the relative humidity of which is 80~ and the flow-rate of which is 2 l/min. In the SiO2 resulting from this treatment the pore-size distribution is characterized by a most frequent pore-diameter of about 1400 A. The carrier material is divided into various particle-size fractions by passing it through various sieves of different mesh size~ The fraction, having a particle-size of from about 0.25 to about 0.5 mm, is used for preparing the support material.
A mixture of lS0 g of this carrier material frac-tion and 4 1 of a 10% solution of ~-aminopropyltriethoxy silane in benzene is heated to reflux temperature for a period of 8 hours. ~hen the reaction mixture is cooled and the resulting support material is filtered off and washed
3 times with 1000 ml of benzene each and 3 times with 1000 ml of acetone each. After evaporating the solvent under vacuum at room temperature, the support material is washed twice with a 0.05 m phosphate buffer solution (pH 7), 1100~66 3 times with bidistilled water, and then is dried over P2O5 under vacuum. From the average C- and N-content which are determined by elemental analysis, the silane-content of the support is calculated. According to this 5 calculation, t-he support No. 1.0 contains 0.13 m equi-valents of silane/g.
1.1 Preparation of im~obilized enzyme com-position No. 1.1.
10 g of this support material No. 1.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase(Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). The degree of activity of the amyloglucosidase is 11.75 unit/mg, whereby 1 activity - unit is equivalent to the formation of 1 ~ mole of glu-cose/min. at a temperature of 25C. This suspension is kept under vacuum for a period of 20 minutes then air is allowed to reenter, and after a period of 2 hours the vacuum is re-applied for a period of 20 minutes. After a period of 4 hours, the support material is separated from the solution by means of filtration, subsequently is washed 3 times with bidistilled water and finally washed 3 times with a 0.01 m phosphate buffer solution (pH 5).
The resulting final composition 1.1 is stored in a phos-phate buffer solution (pH 5~ at a temperature of 4C. The C-~ N-content which is determined by means of elemental analysis, shows a protein content of 16.5 mg/g.
1.2 Preparation of immobilized enzyme com-position No. 1.2.
10 g of the silanized support material No. 1.0 is suspended in 20 ml of a solution of 0.5 g of amylo-glucosidase (Commercial Product Merck 1330) in 0.05 mphosphate buffer solution (pH 7). The suspension is fur-ther treated as described under 1.1. The C- and N-con-tent of the resulting composition 1.2 indicates a protein content of 9.0 mg/g.
EXAMPLE 2:
2.0 Preparation of support material No. 2Ø
A SiO2-gel, having a Na2O-content of 0.3~ by weight, which has been precipitated from a sodium-silicate solution by means of sulfuric acid, is dried as described in Example 1. 1 kg of this material is calcined for a period of 6 hours at a temperature of 680C in a stream of air, having a relati~e humidity of 80% and a flow-rate of 2 l/min. After this treatment the pore-size distribution of the SiO2 is characterized by a most frequent pore-dia-20 meter of 340 A. The carrier material No. 2.0 is separated into different particle-size fractions by passing it through sieves of appropriate mesh size. The fraction, having a particle-size of 0.25 to n. 5 mm, is used for preparing the support material No. 2Ø
150 g of this fraction of the carrier material is treated with 4 1 of a 10% solution of y-aminopropyl-- triethoxy silane in benzene as is described in Example 1, -~66 ~`
for a period of 8 hours.
The silane content of the support material No. 2.0 is 0.19 m equivalents of silane/g, as calculated from th~ ;
average C- and N-content which is determined by elemental analysis.
2.1 Preparation of immobilized enzyme compo-sition No. 2.1.
10 g of the support material No. 2.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is further treated as described in Example l. The C- and N-content of the resulting composition 2.1 shows a protein content of 30.8 mg/g.
152.2 Preparation of immobilized enzyme compo-sition No. 2.2.
lO g of the silanized support material No. 2.0 are suspended in 20 ml of a solution of 0.5 g of amylo-glucosidase ~Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is further treated as described in Example 1 for the pre-paration of the composition No. 1.2. The C- and N-content of the resulting composition No. 2.2 shows a protein con-tent of 17.4 mg/g.
~10QC~66 EX~IPLE 3:
3.0 Preparation of support material No. 3Ø
A SiO2-gel, having a Na2O-content of 0.3% by weight, which has been precipitated from a sodium silicate solution by means of sulfuric acid, is dried as described in Example 1. 1 kg of this material is calcined for a period of 6 hours at a temperature of 640C in a stream of air having a relative humidity of 60% and a flow-rate of 2 l/min. After this treatment, the particle-size distribution of the SiO2 is characterized by a most fre-quent pore-diameter of 180 A. The carrier material No. 3.0 is separated into various particle-size fractions by passing it through sieves of appropriate mesh size. The fraction, having a particle-size of 0.25 to 0.50 mm, is used for preparing the support material No. 3Ø
150 g of this fraction of carrier material No. 3.0 is treated for a period of 8 hours with 4 1 of a 10% solu-tion of ~-aminopropyltriethoxy silane in benzene as des-cribed in Example 1. The silane-content of the resulting support material No. 3.0 is 0.51 m equivalents of silane/g as calculated from the average C- and N-content which is determined by elemental analysis.
3.1 Preparation of immobilized enzyme compo-sition No. 3.1.
10 g of this support material No. 3.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase ~Q~66 (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). The suspension is further treated as described in Example 1. The C- and N-content of the resulting composition 3.1 indicates a protein-content of 26.2 mg/g.
3.2 Preparation of immobilized enzyme compo-sition No. 3.2.
Another 10 g of the silanized support material No. 3.0 are suspended in 20 ml of a solution of 0.5 g of amyloglucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is treated as described in Example 1.
The C- and N-content of the final composition No. 3.2, indicates a protein-content of 12.7 mg/g.
EXAMPLE 4:
1.1 Preparation of im~obilized enzyme com-position No. 1.1.
10 g of this support material No. 1.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase(Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). The degree of activity of the amyloglucosidase is 11.75 unit/mg, whereby 1 activity - unit is equivalent to the formation of 1 ~ mole of glu-cose/min. at a temperature of 25C. This suspension is kept under vacuum for a period of 20 minutes then air is allowed to reenter, and after a period of 2 hours the vacuum is re-applied for a period of 20 minutes. After a period of 4 hours, the support material is separated from the solution by means of filtration, subsequently is washed 3 times with bidistilled water and finally washed 3 times with a 0.01 m phosphate buffer solution (pH 5).
The resulting final composition 1.1 is stored in a phos-phate buffer solution (pH 5~ at a temperature of 4C. The C-~ N-content which is determined by means of elemental analysis, shows a protein content of 16.5 mg/g.
1.2 Preparation of immobilized enzyme com-position No. 1.2.
10 g of the silanized support material No. 1.0 is suspended in 20 ml of a solution of 0.5 g of amylo-glucosidase (Commercial Product Merck 1330) in 0.05 mphosphate buffer solution (pH 7). The suspension is fur-ther treated as described under 1.1. The C- and N-con-tent of the resulting composition 1.2 indicates a protein content of 9.0 mg/g.
EXAMPLE 2:
2.0 Preparation of support material No. 2Ø
A SiO2-gel, having a Na2O-content of 0.3~ by weight, which has been precipitated from a sodium-silicate solution by means of sulfuric acid, is dried as described in Example 1. 1 kg of this material is calcined for a period of 6 hours at a temperature of 680C in a stream of air, having a relati~e humidity of 80% and a flow-rate of 2 l/min. After this treatment the pore-size distribution of the SiO2 is characterized by a most frequent pore-dia-20 meter of 340 A. The carrier material No. 2.0 is separated into different particle-size fractions by passing it through sieves of appropriate mesh size. The fraction, having a particle-size of 0.25 to n. 5 mm, is used for preparing the support material No. 2Ø
150 g of this fraction of the carrier material is treated with 4 1 of a 10% solution of y-aminopropyl-- triethoxy silane in benzene as is described in Example 1, -~66 ~`
for a period of 8 hours.
The silane content of the support material No. 2.0 is 0.19 m equivalents of silane/g, as calculated from th~ ;
average C- and N-content which is determined by elemental analysis.
2.1 Preparation of immobilized enzyme compo-sition No. 2.1.
10 g of the support material No. 2.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is further treated as described in Example l. The C- and N-content of the resulting composition 2.1 shows a protein content of 30.8 mg/g.
152.2 Preparation of immobilized enzyme compo-sition No. 2.2.
lO g of the silanized support material No. 2.0 are suspended in 20 ml of a solution of 0.5 g of amylo-glucosidase ~Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is further treated as described in Example 1 for the pre-paration of the composition No. 1.2. The C- and N-content of the resulting composition No. 2.2 shows a protein con-tent of 17.4 mg/g.
~10QC~66 EX~IPLE 3:
3.0 Preparation of support material No. 3Ø
A SiO2-gel, having a Na2O-content of 0.3% by weight, which has been precipitated from a sodium silicate solution by means of sulfuric acid, is dried as described in Example 1. 1 kg of this material is calcined for a period of 6 hours at a temperature of 640C in a stream of air having a relative humidity of 60% and a flow-rate of 2 l/min. After this treatment, the particle-size distribution of the SiO2 is characterized by a most fre-quent pore-diameter of 180 A. The carrier material No. 3.0 is separated into various particle-size fractions by passing it through sieves of appropriate mesh size. The fraction, having a particle-size of 0.25 to 0.50 mm, is used for preparing the support material No. 3Ø
150 g of this fraction of carrier material No. 3.0 is treated for a period of 8 hours with 4 1 of a 10% solu-tion of ~-aminopropyltriethoxy silane in benzene as des-cribed in Example 1. The silane-content of the resulting support material No. 3.0 is 0.51 m equivalents of silane/g as calculated from the average C- and N-content which is determined by elemental analysis.
3.1 Preparation of immobilized enzyme compo-sition No. 3.1.
10 g of this support material No. 3.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase ~Q~66 (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). The suspension is further treated as described in Example 1. The C- and N-content of the resulting composition 3.1 indicates a protein-content of 26.2 mg/g.
3.2 Preparation of immobilized enzyme compo-sition No. 3.2.
Another 10 g of the silanized support material No. 3.0 are suspended in 20 ml of a solution of 0.5 g of amyloglucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is treated as described in Example 1.
The C- and N-content of the final composition No. 3.2, indicates a protein-content of 12.7 mg/g.
EXAMPLE 4:
4.0 In order to demonstrate, that the activity of the immobilized enzyme composition is not influenced by the type of coupling agent in the support material, support material No. 4.0 is prepared as follows:
By passing the carrier material No. 2.0 (most frequent pore-diameter 340A) through sieves of appropriate mesh size, the carrier material fraction having a particle-siæe of 0.25 to 0.5 mm, is obtained. 50 g of this fraction are suspended in 500 ml of a 12.5~ aqueous solution of glutardialdehyde and this suspension is stirred for a period of 5 minutes at room temperature. Subsequently 1100~66 500 ml of a saturated NH4Cl-solution are added. The mixture is agitated at room temperature for a period of No. 4.O
4 hours, then the support materiallis filtered off and washed with water until it is void of chloride ions, and then is dried under vacuum over P2O5.
4.1 Preparation of immobilized enzyme compo-sition No. 4.1.
10 g of the support material No. 4.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is further treated as described in Example 1. The C- and N-content of the final composition indicates a protein-content of 29.8 mg/g.
4.2 Preparation of immobilized enzyme compo-sition No. 4.2.
Another 10 g of the support material No. 4.0 are suspended in 20 ml of a solution of 0.5 g of amylo-glucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is treated as described for composition 1.2 in Example 1.
The C- and N-content of the resulting composition 4.2 indicates a protein-content of 17.9 mg.
EXAMPLE 5:
By passing the carrier material No. 2.0 (most frequent pore-diameter 340A) through sieves of appropriate mesh size, the carrier material fraction having a particle-siæe of 0.25 to 0.5 mm, is obtained. 50 g of this fraction are suspended in 500 ml of a 12.5~ aqueous solution of glutardialdehyde and this suspension is stirred for a period of 5 minutes at room temperature. Subsequently 1100~66 500 ml of a saturated NH4Cl-solution are added. The mixture is agitated at room temperature for a period of No. 4.O
4 hours, then the support materiallis filtered off and washed with water until it is void of chloride ions, and then is dried under vacuum over P2O5.
4.1 Preparation of immobilized enzyme compo-sition No. 4.1.
10 g of the support material No. 4.0 are sus-pended in 20 ml of a solution of 1 g of amyloglucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is further treated as described in Example 1. The C- and N-content of the final composition indicates a protein-content of 29.8 mg/g.
4.2 Preparation of immobilized enzyme compo-sition No. 4.2.
Another 10 g of the support material No. 4.0 are suspended in 20 ml of a solution of 0.5 g of amylo-glucosidase (Commercial Product Merck 1330) in a 0.05 m phosphate buffer solution (pH 7). This suspension is treated as described for composition 1.2 in Example 1.
The C- and N-content of the resulting composition 4.2 indicates a protein-content of 17.9 mg.
EXAMPLE 5:
5.1 Preparation of immobilized enzyme compo-sition No. 5.1.
10 g of the support material No. 1.0 (most .
frequent pore-diameter 1400 A) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which con-tains 0.5 g of glucose-isomerase (Y. Takasaki, Agr. Biol.
Chem. 33, No. 11, pp. 1527-1534 ~1969)). The mixture is allowed to react at room temperature for a period of 30 minutes. After periods of 10 minutes each, the re-action-flask is evacuated, and after the reaction is com-pleted, the remaining solution is filtered off with suction. Subsequently, the composition is washed 3 times with water and with 0.05 m phosphate buffer solution (pH 7). The C- and N-content of the resulting composition 5.1 indicates a protein-content of 4.8 mg/g.
5.2 Preparation of immobilized enzyme compo-sition No. 5.2.
]5 ` 10 g of the support material ~o. 1.0 are sus-pended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described und~r 5.1.
The C- and N-content of the resulting composition 5.0 indicates a protein-content of 2.0 mg/g.
EXAMPLE 6:
10 g of the support material No. 1.0 (most .
frequent pore-diameter 1400 A) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which con-tains 0.5 g of glucose-isomerase (Y. Takasaki, Agr. Biol.
Chem. 33, No. 11, pp. 1527-1534 ~1969)). The mixture is allowed to react at room temperature for a period of 30 minutes. After periods of 10 minutes each, the re-action-flask is evacuated, and after the reaction is com-pleted, the remaining solution is filtered off with suction. Subsequently, the composition is washed 3 times with water and with 0.05 m phosphate buffer solution (pH 7). The C- and N-content of the resulting composition 5.1 indicates a protein-content of 4.8 mg/g.
5.2 Preparation of immobilized enzyme compo-sition No. 5.2.
]5 ` 10 g of the support material ~o. 1.0 are sus-pended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described und~r 5.1.
The C- and N-content of the resulting composition 5.0 indicates a protein-content of 2.0 mg/g.
EXAMPLE 6:
6.1 Preparation of immobilized enzyme compo-sition No. 6.1.
10 g of the support material ~Jo. 2.0 (most fre-quent pore-diameter 340 A) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains -~10~Q66 0.5 g of glucose-isomerase. This suspension is further treated as described in Example 5. The C- and N-content of the resulting composition No. 6.1 indicates a protein-content of 22.0 mg/g.
6.2 Preparation of immobilized enzyme compo-sition No. 6.2.
10 g of the support material No. 2.0 are sus-pended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described in Example 5.
The C- and N-content of the resulting composition No. 6.2 indicates a protein-content of 10.2 mg/g.
EX~PLE 7:
10 g of the support material ~Jo. 2.0 (most fre-quent pore-diameter 340 A) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains -~10~Q66 0.5 g of glucose-isomerase. This suspension is further treated as described in Example 5. The C- and N-content of the resulting composition No. 6.1 indicates a protein-content of 22.0 mg/g.
6.2 Preparation of immobilized enzyme compo-sition No. 6.2.
10 g of the support material No. 2.0 are sus-pended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described in Example 5.
The C- and N-content of the resulting composition No. 6.2 indicates a protein-content of 10.2 mg/g.
EX~PLE 7:
- 7.1 Preparation of immobilized enzyme compo-sition No. 7.1.
10 g of the support material No. 3.0 (most fre-quent pore-diameter 1~0 A) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.5 ~ of glucose-isomerase. This suspension is further treated as described in Example 5. The C- and N-content of the resulting composition 7.1 ind~cates a protein-content of 11.2 mg/g.
7.2 Preparation of immobilized enzyme compo-sition No. 7.2.
10 g of the support material No. 3.0 are sus-pended in 40 ml of a 0.05 m phosphate buffer solution 1~0~(~66 (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described in Example 5.
The C- and N-content of the resulting composition 7.2 in-dicates a protein-content of 5.1 mg/g.
EXAMPLE 8:
10 g of the support material No. 3.0 (most fre-quent pore-diameter 1~0 A) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.5 ~ of glucose-isomerase. This suspension is further treated as described in Example 5. The C- and N-content of the resulting composition 7.1 ind~cates a protein-content of 11.2 mg/g.
7.2 Preparation of immobilized enzyme compo-sition No. 7.2.
10 g of the support material No. 3.0 are sus-pended in 40 ml of a 0.05 m phosphate buffer solution 1~0~(~66 (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described in Example 5.
The C- and N-content of the resulting composition 7.2 in-dicates a protein-content of 5.1 mg/g.
EXAMPLE 8:
8.1 Preparation of immobilized enzyme compo-sition No. 8.1.
10 g of support material No. 4.0,which is pre-pared as described in Example 4 (most frequent pore-di-ameter 340 A, carrier material treated with an aqueoussolution of glutardialdehyde) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which con-tains 0.5 g of glucose-isomerase. This suspension is further treated as described in Example 5. The C- and N-content of the resulting composition 8.1 indicates a protein-content of 21.3 mg/g.
8.2 Preparation of immobilized enzyme compo-sition No. 8.2.
10 g of the same support material No. 4.0 are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described in Example 5.
The C- and N-content of the resulting composition 8.2 indiates a protein-content of 9.8 mg/g.
EXAMPLE 9:
The activity of the immobilized enzyme compo-sition No. 1.1, 1.2, 2.1, 2.2, 3.1, 3.2, 4.1, and 4.2 and ~1~66 the activity of the enzyme which is used in these compo-sitions (amyloglucosidase, Commercial Product Merck 1330) is determined by reaction with dinitrosalicylic acid ac-cording to the method described by W. Rick and H. P.
Stegbauer, in H. U. Bergmeyer, "Methoden der Enzymatischen Analyse", Verlag Chemie 1970, p. 848. One activity-unit (U) is equivalent to such an amount of enzyme which li-berates 1 ~ equivalentsof reducing groups (calculated as glucose) per minute under the incubation conditions des-cribed below.
Incubation conditions Substrate: A 2% solution of starch (Commercial Product Zulkowsky-Staerke Merck 1257) in an 0.1 m acetate buffer solution, the pH of which is 5.0; incubation period:
30 minutes; incubation temperature 25C.
In a 40 ml reactor, the immobilized enzyme com-positions are suspended under the aforementioned conditions and under agitation at an agitation rate of ~00 turns/min.
The protein content is calculated from the average C- and N-content which is determined by means of elemental analysis. The most frequent pore-diameter is determined from the pore-size distribution which is de-termined in a high-pressure porosimeter. The character~
istics of the compositions, which are descri~ed in E~-amples 1 to 4, are summarized in Table I below. Thefollowin~ definitions are used:
i ~100066 Most frequent pore-diameter D (A) Enzyme content CE (mg enzyme/g of support~
Activity U ~units/g of composition) Spec. activity of the enzyme Us = U/cE (units/mg in the immobilized state of enzyme) 5 Spec. activity of the enzyme UsF (units/mg in the free state enzyme) Relative activity Urel = 100 . Us (%) TABLE I
mmobilized Amylo~lucosidase Composition Sample Most fre- Enzyme- A C T I V I T Y
of quent pore- Content Compo- diameter sition No. D CE U Us Urel UsF
1.1 1400 16.5 98.4 5.96 51 11.75 1.2 . 9.0 100.5 11.16 95 2.1 340 30.8 208.8 6.77 58 2.2 17.4 203.9 11.71 100 3.1 180 26.2 150.0 5.72 49 3.2 12.7 149.5 11~77 100 4.1 340 29.8 199.7 6.70 57 4.2 17.9 209.4 11.70 100 ~,~OOQ6~
EXAMPLE 10:
The activity of the composition No. 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, and 8.2, which are described in Examples 5 to 8, and the activity of the glucose-iso-5 merase which is used in these compositions, is determinedaccording to the method described by Takasaki (see Y.
Takasaki: Agr. Biol. Chem. Col. 30, No. 12, pp. 1247-1253, (1966), and Z. Dische and E. Borenfreund: J. ~3iol. Chem.
192, 583, (1951)). One activity-unit corresponds to such 10 an amount of enzymes, by which 1 mg of fructose is formed under the incubation conditions described be~ow.
Incubation conditions Temperature 65C
Reaction-period 1 h Substrate 0.1 m glucose xH2O (Merc3s 8342) in 0.05 m phosphate buffer, pH 8.0 containing 0.0004 m MgSO4.
The immobilized glucose-isomerase compositions are suspended in a stirring-reactor under the same con-ditions as described in Example 9.
The protein-content of the compositions is cal-20 culated from the average C- and N-content which is de-termined by means of elemental analysis.
In Table II below the properties of the compo-sitions described in Examples 5 to 8, are su~Nnarized.
The same definitions as in Example 9 are 25 used.
~0~066 TABLE II
Immobilized ~lucose-isomerase compositions Sample 'Most fre- Enzyme-of quent pore- Content A C T I V I T Y
Compo- diameter sition No. D CE U Us Urel USF
5.1 1400 4.8 115.4 24.0 41 58.9 5.2 2.0 117.3 58.7 99 6.1 340 22.0 558.5 25.4 43 6.2 10.2 556.1 54.5 93 7.1 180 11.2 291.0 26.0 44 7.2 5.1 292.3 57.3 97 8.1 340 21.3 543.2 25.5 43 8.2 9.8 544.0 55.5 94 l From the foregoing data it is apparent that:
1. The activity U of the examined compositions passes through a maximum which is dependent on the most frequent pore-diameter of the support material.
2. The specific activity Us is dependent on the enzyme content and, at a certain enzyme-concentration, the specific activity Us of the immobilized enzyme reaches about the same value as the specific activity UsF of the enzyme in the free state, that is, Urel reaches about 100.
If this enzyme-concentration is exceeded, the specific activity is reduced, whereby the value of the product ~QQQ66 Us x cE remains constant.
3. The amount of enzyme which is taken up by the support material is a function of the most frequent pore-diameter.
The system enzyme/support exhibits a maximum activity at a lowest possible enzyme-content, if the best suited carrier 2.0 (most frequent pore-diameter 340 A) has taken up 17.4 mg of amyloglucosidase/g or 10.2 mg of glucose-isomerase/g (compositions No. 2.2 and 6.2).
4. The enzyme-uptake and the activity of the examined compositions, are independent from the utilized coupling agent.
The costs for enzymes are very high and in-crease considerably if an increased degree of purity isrequired. Accordingly, these costs are an important fac-tor in determining the technical possibilities for the in-; dustrial use of enzymes.
10 g of support material No. 4.0,which is pre-pared as described in Example 4 (most frequent pore-di-ameter 340 A, carrier material treated with an aqueoussolution of glutardialdehyde) are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which con-tains 0.5 g of glucose-isomerase. This suspension is further treated as described in Example 5. The C- and N-content of the resulting composition 8.1 indicates a protein-content of 21.3 mg/g.
8.2 Preparation of immobilized enzyme compo-sition No. 8.2.
10 g of the same support material No. 4.0 are suspended in 40 ml of a 0.05 m phosphate buffer solution (pH 7), which contains 0.25 g of glucose-isomerase. This suspension is further treated as described in Example 5.
The C- and N-content of the resulting composition 8.2 indiates a protein-content of 9.8 mg/g.
EXAMPLE 9:
The activity of the immobilized enzyme compo-sition No. 1.1, 1.2, 2.1, 2.2, 3.1, 3.2, 4.1, and 4.2 and ~1~66 the activity of the enzyme which is used in these compo-sitions (amyloglucosidase, Commercial Product Merck 1330) is determined by reaction with dinitrosalicylic acid ac-cording to the method described by W. Rick and H. P.
Stegbauer, in H. U. Bergmeyer, "Methoden der Enzymatischen Analyse", Verlag Chemie 1970, p. 848. One activity-unit (U) is equivalent to such an amount of enzyme which li-berates 1 ~ equivalentsof reducing groups (calculated as glucose) per minute under the incubation conditions des-cribed below.
Incubation conditions Substrate: A 2% solution of starch (Commercial Product Zulkowsky-Staerke Merck 1257) in an 0.1 m acetate buffer solution, the pH of which is 5.0; incubation period:
30 minutes; incubation temperature 25C.
In a 40 ml reactor, the immobilized enzyme com-positions are suspended under the aforementioned conditions and under agitation at an agitation rate of ~00 turns/min.
The protein content is calculated from the average C- and N-content which is determined by means of elemental analysis. The most frequent pore-diameter is determined from the pore-size distribution which is de-termined in a high-pressure porosimeter. The character~
istics of the compositions, which are descri~ed in E~-amples 1 to 4, are summarized in Table I below. Thefollowin~ definitions are used:
i ~100066 Most frequent pore-diameter D (A) Enzyme content CE (mg enzyme/g of support~
Activity U ~units/g of composition) Spec. activity of the enzyme Us = U/cE (units/mg in the immobilized state of enzyme) 5 Spec. activity of the enzyme UsF (units/mg in the free state enzyme) Relative activity Urel = 100 . Us (%) TABLE I
mmobilized Amylo~lucosidase Composition Sample Most fre- Enzyme- A C T I V I T Y
of quent pore- Content Compo- diameter sition No. D CE U Us Urel UsF
1.1 1400 16.5 98.4 5.96 51 11.75 1.2 . 9.0 100.5 11.16 95 2.1 340 30.8 208.8 6.77 58 2.2 17.4 203.9 11.71 100 3.1 180 26.2 150.0 5.72 49 3.2 12.7 149.5 11~77 100 4.1 340 29.8 199.7 6.70 57 4.2 17.9 209.4 11.70 100 ~,~OOQ6~
EXAMPLE 10:
The activity of the composition No. 5.1, 5.2, 6.1, 6.2, 7.1, 7.2, 8.1, and 8.2, which are described in Examples 5 to 8, and the activity of the glucose-iso-5 merase which is used in these compositions, is determinedaccording to the method described by Takasaki (see Y.
Takasaki: Agr. Biol. Chem. Col. 30, No. 12, pp. 1247-1253, (1966), and Z. Dische and E. Borenfreund: J. ~3iol. Chem.
192, 583, (1951)). One activity-unit corresponds to such 10 an amount of enzymes, by which 1 mg of fructose is formed under the incubation conditions described be~ow.
Incubation conditions Temperature 65C
Reaction-period 1 h Substrate 0.1 m glucose xH2O (Merc3s 8342) in 0.05 m phosphate buffer, pH 8.0 containing 0.0004 m MgSO4.
The immobilized glucose-isomerase compositions are suspended in a stirring-reactor under the same con-ditions as described in Example 9.
The protein-content of the compositions is cal-20 culated from the average C- and N-content which is de-termined by means of elemental analysis.
In Table II below the properties of the compo-sitions described in Examples 5 to 8, are su~Nnarized.
The same definitions as in Example 9 are 25 used.
~0~066 TABLE II
Immobilized ~lucose-isomerase compositions Sample 'Most fre- Enzyme-of quent pore- Content A C T I V I T Y
Compo- diameter sition No. D CE U Us Urel USF
5.1 1400 4.8 115.4 24.0 41 58.9 5.2 2.0 117.3 58.7 99 6.1 340 22.0 558.5 25.4 43 6.2 10.2 556.1 54.5 93 7.1 180 11.2 291.0 26.0 44 7.2 5.1 292.3 57.3 97 8.1 340 21.3 543.2 25.5 43 8.2 9.8 544.0 55.5 94 l From the foregoing data it is apparent that:
1. The activity U of the examined compositions passes through a maximum which is dependent on the most frequent pore-diameter of the support material.
2. The specific activity Us is dependent on the enzyme content and, at a certain enzyme-concentration, the specific activity Us of the immobilized enzyme reaches about the same value as the specific activity UsF of the enzyme in the free state, that is, Urel reaches about 100.
If this enzyme-concentration is exceeded, the specific activity is reduced, whereby the value of the product ~QQQ66 Us x cE remains constant.
3. The amount of enzyme which is taken up by the support material is a function of the most frequent pore-diameter.
The system enzyme/support exhibits a maximum activity at a lowest possible enzyme-content, if the best suited carrier 2.0 (most frequent pore-diameter 340 A) has taken up 17.4 mg of amyloglucosidase/g or 10.2 mg of glucose-isomerase/g (compositions No. 2.2 and 6.2).
4. The enzyme-uptake and the activity of the examined compositions, are independent from the utilized coupling agent.
The costs for enzymes are very high and in-crease considerably if an increased degree of purity isrequired. Accordingly, these costs are an important fac-tor in determining the technical possibilities for the in-; dustrial use of enzymes.
Claims (25)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. In a process for preparing a water-in-soluble enzyme composition having a high enzymatic acti-vity and containing the enzyme covalently bonded to an inorganic support material comprising the steps of a) contacting an inorganic support material which is capable of covalently bonding the enzyme with a solution of the enzyme in an aqueous solvent whereby a portion of the enzyme is taken up by the support material to form the water-insoluble enzyme compo-sition; and b) separating the water-insoluble enzyme composition from the remaining solution, the improvement which comprises the inorganic support material having a pore-size distri-bution wherein the most frequent pore-diameter is such that in the resulting enzyme composition for any enzyme concentration contained therein the enzymatic activity per weight unit of the composition is the highest acti-vity per weight unit of the composition which is obtain-able at this enzyme concentration; and the enzyme solution, comprising such an amount of enzyme which is sufficiently low that the enzyme which is bonded in the enzyme composition has a specific activity which substantially is about the same as that of the enzyme in the free state.
2. The process as defined in Claim 1 which further comprises the step of bonding a coupling agent to an inorganic carrier material to obtain an inorganic support material which is capable of covalently bonding the enzyme.
3. The process as defined in Claim 2, wherein the inorganic carrier material comprises a hydroxy-groups-containing oxide of at least one element selected from the group consisting of silicium, aluminum, nickel, iron, titanium, zirconium, or mixtures thereof.
4. The process as defined in Claim 2, wherein the inorganic support material which is capable of co-valently bonding the enzyme is a silanized silicium oxide gel.
5. The process as defined in Claim 1 which further comprises the steps of a') contacting at least 2 different embodiments of the inorganic support material which is capable of co-valently bonding the enzyme, each having a different most frequent pore-diameter, each with at least two different solutions of the enzyme, each having a different enzyme content to form a water-insoluble enzyme composition;
b') determining the enzymatic activity per weight unit of the enzyme compositions obtained in step a');
c') comparing enzymatic activities of the enzyme compositions to determine the sample of the inorganic support material having the pore-size distribution with the most frequent pore-diameter which from each of the enzyme solutions yields the enzyme composition having the highest enzymatic activity amongst the enzyme compositions obtained from this enzyme solu-tion;
d') contacting the inorganic support material having the most frequent pore-diameter determined in step c') with solutions containing different amounts of the enzyme to form water-insoluble enzyme compositions;
e') determining the specific activity of the enzyme which is bonded in the enzyme compositions obtained in step d');
f') comparing the specific activities of the enzyme in the bonded state in the enzyme compositions with the specific activity of the enzyme in the free state;
g') determining the amount of enzyme in the enzyme solu-tion which yields the highest absolute activity and the highest specific activity of the enzyme in the bonded state; and h') using inorganic support material having the most frequent pore-diameter as determined in step c') and a solution containing the amount of enzyme as de-termined in step g') as starting materials in the contacting step a).
b') determining the enzymatic activity per weight unit of the enzyme compositions obtained in step a');
c') comparing enzymatic activities of the enzyme compositions to determine the sample of the inorganic support material having the pore-size distribution with the most frequent pore-diameter which from each of the enzyme solutions yields the enzyme composition having the highest enzymatic activity amongst the enzyme compositions obtained from this enzyme solu-tion;
d') contacting the inorganic support material having the most frequent pore-diameter determined in step c') with solutions containing different amounts of the enzyme to form water-insoluble enzyme compositions;
e') determining the specific activity of the enzyme which is bonded in the enzyme compositions obtained in step d');
f') comparing the specific activities of the enzyme in the bonded state in the enzyme compositions with the specific activity of the enzyme in the free state;
g') determining the amount of enzyme in the enzyme solu-tion which yields the highest absolute activity and the highest specific activity of the enzyme in the bonded state; and h') using inorganic support material having the most frequent pore-diameter as determined in step c') and a solution containing the amount of enzyme as de-termined in step g') as starting materials in the contacting step a).
6. The process as defined in Claim 1, wherein the inorganic support material comprises a SiO2-gel.
7. The process as defined in Claim 6 which further comprises the step of preparing a SiO2-gel by adjusting the alkali content expressed as % by weight of Na2O to from about 0.1 to about 0.5% by weight, drying the gel, and calcining the dried gel at a temperature of from about 400 to about 850°C in a flow of water-vapor-containing air during a period of from about 5 to about 10 hours.
8. The process as defined in Claim 7, wherein the calcining temperature is from about 570 to about 750°C
9. The process as defined in Claim 7, wherein the drying is effected at a temperature of between about 180 and about 200°C under water-vapor-saturated air.
10. The process as defined in Claim 7, wherein the calcining is effected at a flow air having a relative humidity of between about 40 and about 80%
11. The process as defined in Claim 1, wherein the enzyme is selected from the group consisting of hydro-lytic enzymes,redox enzymes,isomerases, and transferase enzymes.
12. The process as defined in Claim 11, wherein the hydrolytic enzymes are selected from the group con-sisting of amylases, glycosidases, and proteases.
13. The process as defined in Claim 12, wherein the enzyme is amyloglucosidase.
14. The process as defined in Claim 12, wherein the enzyme solution contains from about 25 to about 75 mg of amyloglucosidase per 1 g of support material.
15. The process as defined in Claim 14, wherein the amount of amyloglucosidase is about 50 mg per 1 g of support material.
16. The process as defined in Claim 14, wherein the amyloglucosidase exhibits a specific activity in the free state of from about 10 to about 15 units/mg.
17. The process as defined in Claim 14, wherein the support material comprises a SiO2-gel having a most frequent pore-diameter of from about 300 to about 400 .ANG..
18. The process as defined in Claim 11, wherein the enzyme is glucose-isomerase.
19. The process as defined in Claim 18, wherein the enzyme solution contains from about 20 to about 50 mg of glucose-isomerase per 1 g of support material.
20. The process as defined in Claim 19, wherein the amount of glucose-isomerase is about 25 mg per 1 g of support material.
21. The process as defined in Claim 19, wherein the glucose-isomerase exhibits a specific activity in the free state of from about 50 to from about 70 units/mg.
22. The process as defined in Claim 19, wherein the support material comprises a SiO2-gel having a most frequent pore-diameter of from about 300 to about 400.ANG..
23. A process for preparing a water-insoluble enzyme composition wherein an enzyme is covalently bonded to an inorganic support material whereby a maximum activity of the insoluble enzyme composition is achieved with the lowest possible amount of enzyme, said process comprising the steps of:
a) selecting an inorganic support material having the most frequent pore-diameter which produces an enzyme composition having the highest absolute activ-ity when a plurality of inorganic support materials having different most frequent pore-diameters are each individually contacted with an enzyme solution contain-ing a given concentration of enzyme;
b) selecting an aqueous enzyme solution con-taining the concentration of enzyme which produces a composition that has the highest absolute activity and a relative activity of substantially 100% when the sup-port material of step a) is contacted with a plurality of enzyme solutions having different concentrations of enzyme;
c) contacting the support material of step a) with the enzyme solution of step b) whereby a portion of the enzyme from the solution is taken up by the support material to produce a water-insoluble enzyme composition; and d) separating the resulting water-insoluble enzyme composition from the remaining solution.
a) selecting an inorganic support material having the most frequent pore-diameter which produces an enzyme composition having the highest absolute activ-ity when a plurality of inorganic support materials having different most frequent pore-diameters are each individually contacted with an enzyme solution contain-ing a given concentration of enzyme;
b) selecting an aqueous enzyme solution con-taining the concentration of enzyme which produces a composition that has the highest absolute activity and a relative activity of substantially 100% when the sup-port material of step a) is contacted with a plurality of enzyme solutions having different concentrations of enzyme;
c) contacting the support material of step a) with the enzyme solution of step b) whereby a portion of the enzyme from the solution is taken up by the support material to produce a water-insoluble enzyme composition; and d) separating the resulting water-insoluble enzyme composition from the remaining solution.
24. A process according to Claim 23, wherein the inorganic support material is selected by:
a1) contacting a plurality of individual in-organic support materials having different most frequent pore-diameters with at least one enzyme solution to pro-duce insolubilized enzyme compositions;
a2) measuring the absolute activity of each of the resulting insolubilized enzyme compositions;
a3) comparing the measured values for the dif-ferent support materials; and a4) selecting the support material having the most frequent pore-diameter which produces the enzyme composition having the highest absolute activity.
a1) contacting a plurality of individual in-organic support materials having different most frequent pore-diameters with at least one enzyme solution to pro-duce insolubilized enzyme compositions;
a2) measuring the absolute activity of each of the resulting insolubilized enzyme compositions;
a3) comparing the measured values for the dif-ferent support materials; and a4) selecting the support material having the most frequent pore-diameter which produces the enzyme composition having the highest absolute activity.
25. A process according to Claim 23, wherein the enzyme solution is selected by:
b1) contacting the support material of step a) with a plurality of enzyme solutions containing differ-ent concentrations of enzyme to produce insolubilized enzyme compositions;
b2) measuring the amount of enzyme taken up by each of the support materials and the absolute activity of each of the insolubilized enzyme compositions;
b3) determining the relative activity of the enzyme in each of the enzyme compositions;
b4) comparing the measured absolute activities and the relative activities for each of the enzyme compositions; and b5) determining the enzyme solution which produces an insolubilized enzyme composition having the highest absolute activity and a relative activity of substantially 100%.
b1) contacting the support material of step a) with a plurality of enzyme solutions containing differ-ent concentrations of enzyme to produce insolubilized enzyme compositions;
b2) measuring the amount of enzyme taken up by each of the support materials and the absolute activity of each of the insolubilized enzyme compositions;
b3) determining the relative activity of the enzyme in each of the enzyme compositions;
b4) comparing the measured absolute activities and the relative activities for each of the enzyme compositions; and b5) determining the enzyme solution which produces an insolubilized enzyme composition having the highest absolute activity and a relative activity of substantially 100%.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP2726188.7 | 1977-06-10 | ||
| DE2726188A DE2726188C2 (en) | 1977-06-10 | 1977-06-10 | Process for the production of a water-insoluble enzyme preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1100066A true CA1100066A (en) | 1981-04-28 |
Family
ID=6011173
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA305,057A Expired CA1100066A (en) | 1977-06-10 | 1978-06-08 | Water-insoluble enzyme compositions |
Country Status (23)
| Country | Link |
|---|---|
| US (1) | US4230803A (en) |
| EP (1) | EP0000028B1 (en) |
| JP (1) | JPS548789A (en) |
| AR (1) | AR222972A1 (en) |
| AU (1) | AU517551B2 (en) |
| BE (1) | BE868020A (en) |
| BG (1) | BG28720A3 (en) |
| CA (1) | CA1100066A (en) |
| CS (1) | CS216234B2 (en) |
| DD (1) | DD135495A5 (en) |
| DE (2) | DE2726188C2 (en) |
| DK (1) | DK149757C (en) |
| ES (1) | ES470069A1 (en) |
| FI (1) | FI62139C (en) |
| FR (1) | FR2393810A1 (en) |
| GB (1) | GB1600339A (en) |
| HU (1) | HU179727B (en) |
| IT (1) | IT1094879B (en) |
| NL (1) | NL7805996A (en) |
| PL (1) | PL126637B1 (en) |
| RO (1) | RO74644A (en) |
| SE (1) | SE7806679L (en) |
| YU (1) | YU137078A (en) |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2726188C2 (en) * | 1977-06-10 | 1979-05-10 | Kali-Chemie Ag, 3000 Hannover | Process for the production of a water-insoluble enzyme preparation |
| DE3148603C1 (en) * | 1981-12-09 | 1983-07-21 | Kali-Chemie Ag, 3000 Hannover | Process and plant for the production of isomerose |
| EP0093027A1 (en) * | 1982-04-27 | 1983-11-02 | ARGILES & MINERAUX AGS-BMP | Carrier for the fixation of microorganisms |
| FR2525629B1 (en) * | 1982-04-27 | 1985-06-14 | Ags Bmp Argiles Mineraux | SUPPORT FOR FIXING MICROORGANISMS |
| US4530963A (en) * | 1982-08-20 | 1985-07-23 | Devoe-Holbein International, N.V. | Insoluble chelating compositions |
| DE3405035C1 (en) * | 1984-02-13 | 1985-04-25 | Kali-Chemie Ag, 3000 Hannover | Process for the production of isoglucose |
| US4683203A (en) * | 1984-04-14 | 1987-07-28 | Redco N.V. | Immobilized enzymes, processes for preparing same, and use thereof |
| US4654322A (en) * | 1985-08-05 | 1987-03-31 | Devoe-Holbein International, N.V. | Insoluble compositions for removing mercury from a liquid medium |
| US4749653A (en) * | 1985-10-21 | 1988-06-07 | Owens-Corning Fiberglas Corporation | Enzyme immobilization on non-porous glass fibers |
| DE3719324C1 (en) * | 1987-06-10 | 1988-12-15 | Kali Chemie Ag | Process for the production of carrier-bound enzymes |
| US5504042A (en) * | 1994-06-23 | 1996-04-02 | Texas Instruments Incorporated | Porous dielectric material with improved pore surface properties for electronics applications |
| US6319852B1 (en) | 1995-11-16 | 2001-11-20 | Texas Instruments Incorporated | Nanoporous dielectric thin film formation using a post-deposition catalyst |
| US6063714A (en) * | 1995-11-16 | 2000-05-16 | Texas Instruments Incorporated | Nanoporous dielectric thin film surface modification |
| US6037277A (en) * | 1995-11-16 | 2000-03-14 | Texas Instruments Incorporated | Limited-volume apparatus and method for forming thin film aerogels on semiconductor substrates |
| US5807607A (en) | 1995-11-16 | 1998-09-15 | Texas Instruments Incorporated | Polyol-based method for forming thin film aerogels on semiconductor substrates |
| US5753305A (en) * | 1995-11-16 | 1998-05-19 | Texas Instruments Incorporated | Rapid aging technique for aerogel thin films |
| US6130152A (en) | 1995-11-16 | 2000-10-10 | Texas Instruments Incorporated | Aerogel thin film formation from multi-solvent systems |
| US6380105B1 (en) | 1996-11-14 | 2002-04-30 | Texas Instruments Incorporated | Low volatility solvent-based method for forming thin film nanoporous aerogels on semiconductor substrates |
| US5736425A (en) * | 1995-11-16 | 1998-04-07 | Texas Instruments Incorporated | Glycol-based method for forming a thin-film nanoporous dielectric |
| CA2353307A1 (en) * | 2001-07-13 | 2003-01-13 | Carmen Parent | Device and procedure for processing gaseous effluents |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3519538A (en) * | 1968-09-05 | 1970-07-07 | Corning Glass Works | Chemically coupled enzymes |
| US3850751A (en) * | 1973-02-16 | 1974-11-26 | Corning Glass Works | Enzymes immobilized on porous inorganic support materials |
| US3892580A (en) * | 1973-03-26 | 1975-07-01 | Corning Glass Works | Method of making porous inorganic bodies |
| US3930951A (en) * | 1974-05-28 | 1976-01-06 | Corning Glass Works | Bonding enzymes to porous inorganic carriers |
| DE2726188C2 (en) * | 1977-06-10 | 1979-05-10 | Kali-Chemie Ag, 3000 Hannover | Process for the production of a water-insoluble enzyme preparation |
-
1977
- 1977-06-10 DE DE2726188A patent/DE2726188C2/en not_active Expired
-
1978
- 1978-05-05 BG BG7839644A patent/BG28720A3/en unknown
- 1978-05-22 ES ES470069A patent/ES470069A1/en not_active Expired
- 1978-05-29 AR AR272349A patent/AR222972A1/en active
- 1978-05-30 GB GB24510/78A patent/GB1600339A/en not_active Expired
- 1978-05-30 IT IT23967/78A patent/IT1094879B/en active
- 1978-05-31 US US05/911,227 patent/US4230803A/en not_active Expired - Lifetime
- 1978-06-01 NL NL7805996A patent/NL7805996A/en not_active Application Discontinuation
- 1978-06-01 EP EP78100045A patent/EP0000028B1/en not_active Expired
- 1978-06-01 DE DE7878100045T patent/DE2860632D1/en not_active Expired
- 1978-06-05 RO RO7894265A patent/RO74644A/en unknown
- 1978-06-05 FR FR787816697A patent/FR2393810A1/en active Pending
- 1978-06-05 HU HU78KA1505A patent/HU179727B/en not_active IP Right Cessation
- 1978-06-07 CS CS783707A patent/CS216234B2/en unknown
- 1978-06-07 FI FI781821A patent/FI62139C/en not_active IP Right Cessation
- 1978-06-08 CA CA305,057A patent/CA1100066A/en not_active Expired
- 1978-06-08 YU YU01370/78A patent/YU137078A/en unknown
- 1978-06-08 SE SE7806679A patent/SE7806679L/en unknown
- 1978-06-08 DD DD78205886A patent/DD135495A5/en not_active IP Right Cessation
- 1978-06-08 AU AU36926/78A patent/AU517551B2/en not_active Expired
- 1978-06-09 JP JP6973078A patent/JPS548789A/en active Granted
- 1978-06-09 BE BE6046498A patent/BE868020A/en unknown
- 1978-06-09 DK DK257678A patent/DK149757C/en not_active IP Right Cessation
- 1978-06-09 PL PL1978207511A patent/PL126637B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DK149757B (en) | 1986-09-22 |
| US4230803A (en) | 1980-10-28 |
| DK257678A (en) | 1978-12-11 |
| AU3692678A (en) | 1979-12-13 |
| DE2726188C2 (en) | 1979-05-10 |
| EP0000028B1 (en) | 1981-04-22 |
| FI781821A (en) | 1978-12-11 |
| IT1094879B (en) | 1985-08-10 |
| YU137078A (en) | 1983-02-28 |
| FR2393810A1 (en) | 1979-01-05 |
| DK149757C (en) | 1987-03-02 |
| RO74644A (en) | 1980-10-30 |
| ES470069A1 (en) | 1979-01-01 |
| AR222972A1 (en) | 1981-07-15 |
| DD135495A5 (en) | 1979-05-09 |
| JPS548789A (en) | 1979-01-23 |
| SE7806679L (en) | 1978-12-11 |
| BE868020A (en) | 1978-12-11 |
| BG28720A3 (en) | 1980-06-16 |
| EP0000028A1 (en) | 1978-12-20 |
| NL7805996A (en) | 1978-12-12 |
| AU517551B2 (en) | 1981-08-06 |
| FI62139B (en) | 1982-07-30 |
| DE2860632D1 (en) | 1981-07-30 |
| JPS6133557B2 (en) | 1986-08-02 |
| CS216234B2 (en) | 1982-10-29 |
| GB1600339A (en) | 1981-10-14 |
| PL207511A1 (en) | 1979-05-07 |
| PL126637B1 (en) | 1983-08-31 |
| FI62139C (en) | 1982-11-10 |
| HU179727B (en) | 1982-11-29 |
| IT7823967A0 (en) | 1978-05-30 |
| DE2726188B1 (en) | 1978-08-31 |
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| MKEX | Expiry |