NO148600B - IMMOBILIZED ORGANIC-INORGANIC ENZYM CONJUGATE AND PROCEDURE FOR THE PREPARATION OF THIS - Google Patents

Abstract

Immobilized organic-inorganic enzyme conjugate, and process for its preparation.

Description

It is known that enzymes, which are of a protein nature and are most often water-soluble, contain biological catalysts that regulate many and varied chemical reactions that take place in living organisms. The enzymes can also be isolated and used for analytical, medical or industrial purposes. E.g. they are used for industrial purposes for the production of foodstuffs such as cheese or brbd and for the production of alcoholic beverages. Some special applications are found in industry, such as e.g. separation of amino acids, modification of penicillin for the production of different substrates, use of different proteases for cheese production, meat curing, detergent compositions, leather production and as digestive aids, use of carbohydrases in starch hydrolysis, sucrose inversion, glucose isomerisation etc., use of nucleases for regulation of taste and smell or the use of oxidases to prevent oxidation and for color regulation of foodstuffs. These applications and many others are well described in the literature.

As the enzymes are usually water-soluble and generally unstable and easily deactivated, they are also difficult either to remove from the solutions in which they are used, so as to be able to use them again, or it is difficult to maintain their catalytic effect over a longer period of time. These difficulties will naturally result in increased costs when using enzymes on a technical scale due to the necessity for frequent replacement or replacement of the enzyme, usually at each use. To counteract these high costs, it has been proposed to make the enzymes insoluble or to fix the enzymes for use. By deactivating the enzymes in different ways, which will be described in more detail later, it is possible to relatively stabilize the enzymes and therefore to reuse enzymes that would otherwise be deactivated or lost in the reaction medium. Such undissolved enzymes can be used in different reactor systems such as e.g. packed columns, reactors with rbring etc, depending on the type of mixture or substrate used. Generally, insoluble production of enzymes will provide better or wider environmental stability, minimal breeding and processing problems and the possibility of activation of the enzyme.

As mentioned, several general methods and modifications of such methods have been described for insolubilizing or fixing enzymes. A general method is to adsorb the enzyme on a solid surface such as when an enzyme such as amino acid acylase is adsorbed on cellulose derivatives such as DEAE cellulose, papain or ribonuclease is adsorbed on porous glass, catalase is adsorbed on charcoal, trypsin is adsorbed on quartz glass or cellulose, chymotrypsin is adsorbed on kaolinite etc. Another general method is to capture enzymes in a gel network such as e.g. glucose oxidase, urease, papain etc. which are captured or embedded in a polyacrylamide gel, acetylcholinesterase which is embedded in a starch gel or a silicone polymer, glutamic acid-pyruvic acid transaminase which is embedded in a polyamide or cellulose acetate gel etc. Another general method is cross-linking with bifunctional reagents which can be carried out in combination with one of the other general methods mentioned. When using the latter method, the bifunctional or multifunctional reagents that induce intermolecular cross-linking will bind the enzymes covalently to each other and to the solid substrate. Examples of the method are the use of glutardialdehyde or bisdiazobenzidine-2,2'-disulfonic acid for attaching an enzyme such as papain to a solid substrate or support material. Yet another method for attaching an enzyme consists in the covalent binding of enzymes such as glucoamylase, trypsin, papain, pronase, amylase, glucoseoxidase, pepsin, rennin, fungal protease, lactase etc. covalently to a polymeric compound which in turn is bound or attached to an organic or inorganic solid porous substrate. This method can also be combined with the aforementioned indissolving methods.

The aforementioned methods for insolubilizing enzymes are all affected by some disadvantages which make the method correspondingly less applicable industrially. When e.g. an enzyme is adsorbed directly on the surface of a support material, the binding forces that arise between the enzyme and the support material will often be weak, even though it has been stated in previous works that relatively stable preparations of this type can be obtained when the pore size of the support material or substrate and the spin diameter of the enzyme is voted off. However, the carrier material's pore size cannot exceed a diameter of approx. o,l^um. Because of these weak bonds, enzymes are often desorbed in the presence of the mixture being treated. In addition, the enzyme will be completely or substantially deactivated due to lack of mobility or due to reaction between the substrate and the enzyme's active groups. Another process that can be used is the embedding of enzymes in gel networks which can be done by polymerizing an aqueous solution or emulsion containing the polymer in monomer form as well as the enzyme, or by incorporating the enzyme into a pre-prepared polymer in different ways, often in the presence of cross-linking agent. Although this method for insolubilizing the enzyme has an advantage in that the reaction conditions used during the embedding process are usually mild so that major changes or deactivation of the enzyme rarely occur, it also has disadvantages in that the preparation has poor mechanical strength, which lead to compression when used in columns or in systems with continuous flow, with consequent resealing of the column. Such systems also have fairly large variations in pore size and possibly individual pores that are large enough for the enzyme to be lost. In addition, some pore spaces can be sufficiently small that diffusion barriers are formed for the transport of substrate and product, which in turn causes a delay in the reaction, and this applies in particular when using high molecular weight mixtures. The disadvantages that exist when one insolubilizes an enzyme by intermolecular cross-linking are due, as mentioned, to the lack of mobility or motility which in turn causes deactivation due to the enzyme's inability to adopt the natural configuration that is necessary to achieve maximum activity, especially when the enzyme's active area or group is included in the binding process.

Covalent bonding processes have been used either as the only insoluble release technique or as part of the many above-mentioned methods where cross-linking reactions are used. Covalent binding processes are often used to attach the enzyme and carrier material via a bifunctional intermediate molecule where the molecule's functional groups, such as e.g. gamma-aminopropyltriéthoxysilane, is capable of reacting with functional groups both in the enzyme and in an organic or inorganic porous support material. Many different reagents and carrier materials■have■been used in this way and the method has the advantage that you get strong covalent bonds in the preparation and at the same time great activity in the enzyme. The covalent bond between the enzyme and the support material must take place via functional groups on the enzyme that are not essential for the enzyme's catalytic activity, e.g. free amino groups, carboxyl groups, hydroxyl groups and phenol groups, thiol groups etc. These functional groups will also react with a large number of other functional groups such as e.g. aldehyde, isocyanate, acyl, diazo, azido, anhydro, activated ester etc. while forming covalent bonds. Nevertheless, this method also often has disadvantages consisting of expensive reactants and solvents, as well as special and expensive porous carrier materials and complicated multi-step processes which make the method unsuitable for technical commercial use.

The known technique in the area is therefore full of different methods for insolubilizing or fixing enzymes which are, however, in various ways unable to fulfill all industrial requirements. As will be discussed in more detail later, however, none of the previously known preparations contain the composition of compounds provided by the present invention and which consist of an inorganic porous carrier material containing a polymeric compound formed in situ from a monomer or a pre-produced polymer of natural or synthetic origin which is embedded and also partially adsorbed in the pores of the support material and which contains functionalized outer groups, and where the enzyme is partially adsorbed on the lattice and at the same time covalently bound to the active groups on or near the end parts of said outer groups as that the enzyme is given the opportunity for free movement, which ensures maximum activity. For example US patent 3.556.9'+5 deals with enzyme preparations where the enzyme is adsorbed directly on an inorganic support medium such as glass. US patent 3,519,538 relates to enzyme preparations where the enzyme is chemically coupled via an intermediate silane coupling agent to an inorganic carrier medium. In a similar way, US patent 3,783,101 also uses an organosilane medium as a binding agent, in that the enzyme is covalently linked to the glass carrier material via an intermediate silane coupling agent, where the silicon group in the coupling agent is bound to the carrier medium while the organic part of the coupling medium is coupled to the enzyme, whereby the preparation contains a metal oxide on the surface of the carrier material, arranged between the carrier material and the silicon group of the coupling agent. In US patent 3,821,083, the inert carrier is coated with a pre-made polymer such as polyacrolein which is bound to an enzyme. According to most of the examples mentioned in the patent, however, it is first necessary to acid hydrolyze the preparation for the enzyme to be deposited on the polymer. Another patent in the area, namely US patent 3-705.08U- indicates a macroporous enzyme reactor where the enzyme is adsorbed on the polymeric surface of a macroporous reactor core and is then cross-linked in place. When cross-linking the enzymes on the polymer surface after adsorption of the polymer, the enzyme will be further blocked and cannot work freely as in the original catalyst state. Cross-linking of the enzymes will bind or hang them together so that the free movement of the enzyme is prevented with the following reduced mobility, which, as mentioned, is necessary for maximum activity.

The present invention relates to immobilized organic-inorganic enzyme conjugate comprising an inorganic,

porous carrier and an organic polymeric material, which polymeric material is selected from water-soluble polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexamethylenediamine and polyethyleneamine, water-insoluble polyamines such as methylenedicyclohexylamine and methylenedianiline, and natural and synthetic partially hydrolyzed polymers and formed polymers such as nylon, collagen, polyacrolein, polymaleic anhydride, alginic acid, casein hydrolyzate and gelatin, which polymeric material is adsorbed on the inorganic support, and which enzyme is adsorbed on the porous support and the organic polymeric material, and also covalently

bound to the organic polymeric material, which conjugate

is characterized by the fact that the polymeric material is embedded in the pores of the inorganic, porous support, forming a combined organic-inorganic matrix, and that the polymeric material contains associated groups, which associated groups are

formed by reacting the polymeric material with a bifunctional monomer containing functional groups such as amino, hydroxyl, carboxyl, thiol and carbonyl, which functional groups are preferably separated by chains containing from 4 to 10 carbon atoms, which enzyme is covalently bound to the functional parts of the associated groups at or in the immediate vicinity of their end positions.

As previously mentioned, the use of enzymes for analytical and medical or industrial use can be extended considerably if the enzymes are in an insoluble or immobile state, i.e. that the enzymes, by being in combination with other solid substances, "in themselves are in such a state that they are not water soluble and therefore can undergo multiple uses while retaining the enzyme's catalytic activity.To fulfill this solid or insoluble

condition, the enzymes must be bound in some way to a water-insoluble carrier whereby the enzymes can be used commercially in a condition where they do not dissolve in water.

A special embodiment of the invention consists of an insoluble enzyme preparation in the form of an organic-inorganic mass consisting of a porous support material of silicon oxide-aluminium oxide with a low bulk density and relatively high surface area, which can also contain inorganic additives, as well as a polymer produced in situ from tetraethylenepentamine-glutaraldehyde etc. which is adsorbed and embedded in the pores of the silicon oxide-aluminium oxide, as well as an enzyme consisting of glucoamylase which is covalently bound to the glutaraldehyde groups on the polymer in or near the end parts of the groups as well as partially absorbed on said organic-inorganic component.

As previously mentioned, the invention relates to insoluble enzyme preparations which consist of a combined organic-inorganic mass in the form of an inorganic porous carrier material containing an organic polymer adsorbed and embedded in the pores of the inorganic carrier material. In addition, the polymeric compound must contain external groups which, by covalent bonding, retain an enzyme on or near the end parts of the groups and, in addition, the enzyme is also partially adsorbed in said organic-inorganic mass or component. In contrast to the mixtures containing insolubilized enzymes known from the past, the immobilized enzyme conjugate according to the present invention can be produced using relatively cheap reactants and using simpler process steps. In addition, the mechanical strength and stability of the enzyme conjugates according to the invention will be greater than for the previously known preparations with insolubilized enzymes. It will therefore be clear that the present conjugates have economic advantages that can be exploited industrially.

The conjugates according to the invention can be made in a relatively simple way. In a preferred method, the inorganic porous support material is treated with a solution, preferably aqueous, of a bi- or multi-functional monomer, a polymer hydrolyzate or a previously prepared polymer, after which the unabsorbed solution is removed in a known manner such as e.g. runoff etc. It is also conceivable that other cheap organic solvents such as acetone, tetrahydrofuran etc. can be used as carriers for said monomer or polymer. After removal of unabsorbed solution, the wet porous support material is brought into contact with a relatively large excess of 5 to 20 mole percent of a second bifunctional monomer in which the reactive groups are preferably separated from each other by a chain containing from h to 10 carbon atoms, this second bifunctional monomers are also added in aqueous solution during which a polymer mass is formed which is both adsorbed and embedded in the pores of the carrier material and from which protruding or outer groups of the other monomer will protrude. These protruding groups will contain unreacted functional residues due to the fact that an excess of said second bifunctional monomer was used to treat the support material. The unreacted functional groups are then available for covalent binding to the enzyme which is added to the formed organic-inorganic molecular lattice, again usually in aqueous solution. After removal of unreacted compounds e.g. in the case of treatment of the type of washing etc., the enzyme next to being covalently bound to the projecting functional groups on or near their end parts will also be partially adsorbed on the base material. It is therefore clear that the entire undissolving process can. happen in a simple and cheap way with aqueous and cheap solvents whereby the method is carried out within a temperature range that can. be lower than room temperature (approx. 5°C and up to higher temperatures of approx. 60°C, but preferably at room temperature) (20-25°C)) using a minimal number of steps which additionally enables easy recovery of excess reactant and processing of end product.

Many inorganic support media discussed in previous publications specify "regulated pore compounds" such as glass, aluminum oxide, etc. having a pore diameter of 0.05 to 0.07 µm

, . for approx. 96% of the compound and a maximum pore diameter of o o.l^um, surface area hO - 70 m 2 /g and particle size ^0-80 mesh. In addition, these carrier materials can be coated with metal oxides such as zirconium oxide and titanium oxide to achieve greater stability. In contrast to these carrier materials, it is within the scope of the invention that the inorganic porous carrier materials used have pore diameters of between 0.01 and 5.5 µm

in that up to 25 to 60% of the porous carrier material has pores with a diameter above 2.0 µm and a surface area of 150 to 200 m 2 /g. The particle size can also vary over a large range of from 10 to 20 mesh and down to a fine powder, as the particle size depends on the particular system in which the material is to be used. It is also possible that the porous support material can be coated with different oxides of the type that is indicated or may have incorporated various other inorganic compounds such as boron phosphate etc. which give special properties to the carrier material. A particularly suitable form of carrier material consists of a ceramic substance which may have the porosity described above or may be provided with interconnected macro-channels like a beehive, such compounds are usually referred to as monoliths, and which may be coated with different types of porous aluminum oxide, zirconium oxide etc-. The use of such a carrier material has the particular advantage that it enables the free flow of even highly viscous substances that often appear in technical enzymes

catalyzed reactions.

The inorganic porous carrier materials used as one component in the combined organic-inorganic carrier mass will contain certain metal oxides such as aluminum oxide and especially gamma aluminum oxide, silicon oxide, zirconium oxide, or mixtures of metal oxides such as silicon oxide-aluminium oxide, silicon oxide-zirconium oxide, silicon oxide-magnesium oxide, silicon oxide-zirconium oxide-aluminium oxide etc., or gamma-aluminium oxide containing other inorganic compounds such as boron phosphate etc., ceramic materials etc. as well as combinations of these where one can serve as a coating on the other which then forms the substrate material.

The polymeric compounds which are formed in situ in such a way that they are partly adsorbed and partly embedded in the pores of the inorganic support material as described, can be made on it. general way previously mentioned, i.e. by first adsorbing a solution containing from 2 to 25% bi- or multi-functional monomer, polymerized hydrolyzate or a pre-prepared polymer where the monomer or polymer is synthetic or natural and which is preferably soluble in water or other solvents. which are inert to the reactants used. As mentioned, it is within the scope of the invention to add another bifunctional monomer in a similar way - for the production of an organic-inorganic mass by reaction - with the original addition adsorbed on the inorganic support material. The functional groups found on the bifunctional monomer consist of well-known reactive groups such as amino, hydroxyl, carboxyl, thiol, carbonyl etc. As mentioned, the reactive groups on the bifunctional compounds are preferably, but not necessarily, separated by molecular chains containing from h to 10 carbon atoms. The reactive groups can be covalently bound to both the original additions and then, after washing out unreacted compounds, to an enzyme that is added at a later stage and then covalently bound to the functional group on or near the functional the end part of the chain as well as being adsorbed on the mass. After adding enzymes to this composition, a relatively stable enzyme preparation is formed which has high activity and high stability. In addition, this preparation also has extensive diversity in use in addition to the other mentioned advantages in that one can refrain from using inorganic carrier material which is soluble in either acidic or alkaline mixtures. As described in more detail later, and depending on the reaction participants used, the preparations will retain their stability in such media when they are prepared in combination with an inorganic carrier medium as previously known in the field. By having these properties, it becomes possible to expand the area of application for the preparations.

Special examples of the bi- or multi-functional monomers, polymer hydrolysates or pre-made polymers that can be initially adsorbed on the inorganic carrier medium include water-soluble polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexamethylenediamine, pplyethyleneimine, etc.; water-insoluble polyamines such as methylenedicyclohexylamine, methylenedianiline, etc.; natural and synthetic partially hydrolyzed polymers and pre-made polymers such as nylon, collagen, polyacrolein, polymaleic anhydride, alginic acid, casein hydrolyzate, gelatin etc. Some special examples of bifunctional intermediates that can be added to the above products to form an organic-inorganic mass with the necessary properties includes compounds such as glutardialdehyde, adipoyl chloride, sebacoyl chloride, toluene diisocyanate, hexamethylene diisocyanate, etc. It will be seen that when a polyethyleneamine of the type described is reacted with glutardialdehyde in the absence of an inorganic porous carrier medium, an aqueous acid-soluble substance is obtained, while when a polyethyleneamine is reacted with diisocyanate or acyl halide, a water-insoluble product is obtained. Conversely, with a reaction complex without an inorganic carrier medium and containing free carboxyl groups, an al.potassium-soluble complex can be obtained. Due to the large excess of interspersed or spacer bifunctional molecules used, the polymer lattice formed will contain protruding or outer groups comprising the spacer molecules, the latter protruding from the matrix and having reactive groups available at or near their end parts which are capable of reacting with and binding the enzyme to the distance-ordering molecules via covalent bonds. In addition, when the enzyme is added after unreacted reagents have been removed from the organic-inorganic mass by washing out, it will also be partially adsorbed on the mass at the same time. Binding of the enzyme only to the organic mass will not usually affect the solubility's dependence on pH, but when the inorganic carrier material is added as described, the overall preparation has high stability over a relatively large pH range from 3 to 9, as the stability naturally is also a function of the optimum pH properties of the particular enzyme and the properties of the inorganic support medium used. It will therefore be clear that a suitable organic-inorganic base mass can be formed which can be used in many situations by adsorbing on the carrier material substances which are mentioned above and which are known in the field and which are then treated with a bifunctional molecule which is also known in the field and advantageously functionalized for reaction with the original addition, provided that a large enough excess of the bifunctional molecule is used to form protruding groups capable of subsequent reaction with the enzyme to be insolubilized. By using these functional protruding groups as binding points for the enzymes, the enzymes will gain greater mobility and the enzyme will thereby gain high catalytic activity over a relatively longer period of time than when the enzyme is made insoluble by other methods such as e.g. adsorption on a solid surface or cross-linking of the enzyme using bifunctional reagents etc. However, not all preparations will give equally good results with regard to stability or activity.

Examples of enzymes that can be insolubilized by covalent binding reaction and that contain an amino group that can react with an aldehyde or isocyanate residue on the protruding groups that are attached to the embedded polymeric material adsorbed in the pores of the porous support material include trypsin, papain, hexokinase, betagalactosidase, ficin, bromelain, lactic dehydrogenase, glucoamylase, chymotrypsin, pronase, acylase, invertase, amylase, glucoseoxydase, pepsin, rennin, fungal protease, etc. In general, you can use all enzymes whose active region or molecular part is not included in the covalent bond. While the above discussion concerned protruding groups that contained an aldehyde or isocyanate group as a functional residue, it is also within the scope of the invention that the protruding group may contain other functional residues that can react with carboxy, sulfhydryl or other residues that are normally found in enzymes. However, the covalent binding of enzymes containing these other groups to other protruding groups does not necessarily give equally good results and will possibly mean considerably higher costs for the production of intermediate products. It will be understood that the aforementioned porous solid support material, monomers, hydrolysates, polymers and enzymes are only representatives of the different compound classes that can be used and that the invention is not necessarily limited to these.

Production of conjugates according to the invention preferably takes place in batches as already described in detail, although it is also within the framework of the invention to produce the finished product continuously. When continuous reaction is used, a quantity of the porous solid support material is applied. in suitable apparatus, usually a column. The porous solid support material can have any desired shape such as; powder, pellets, monoliths etc. and the column is filled after which, a preferably aqueous solution of e.g. a multi-functional amine is brought into contact with the porous support material until the latter is saturated with amine solution, after which excess-. it drains. A spacer or interstitial bifunctional molecule such as glutardialdehyde is brought into contact with the saturated support material. The formation of the polymeric mass thus takes place in an aqueous system and the reaction is carried out over the course of 1 to 10 hours, but usually over a shorter time. After removing excess glutardialdehyde by draining and washing out water-soluble and unreacted material, which in the case of polyamine is preferably done with a buffer solution with pH approx. ^, an aqueous enzyme solution is recirculated through the column and this causes covalent bonding between the enzyme and the end-placed aldehyde groups in the functionalized protruding molecules from said mass. This takes place until there is no longer any physical adsorption and/or covalent binding of enzymes to the organic-inorganic lattice mass and the protruding molecules. The excess enzyme is recovered in the waste water after draining and rinsing the column. The column is thus ready for use in chemical reactions where the enzyme's catalytic effect is to be used. These processes are mostly carried out within the times, temperatures and concentration parameters described for the batch process and will give similar undissolved enzyme complexes. It is also covered by the invention that with suitable modifications of pH and temperature which will be obvious to those skilled in the art, the method can be applied to a large number of different inorganic porous support materials, polymer-forming reactants and enzymes.

The following examples shall illustrate the new preparations according to the invention and the associated methods.

Example I

In this example, 2 grams of porous silicon oxide-aluminium oxide with added boron phosphate and with a particle size of 0-80 mesh, pore diameter 0.01 to 5.5 µm and surface area 150-200 m 2 /g are used as inorganic carrier material for the new preparations according to the invention. The carrier material was burned at approx. 26CTC to remove any adsorbed moisture. The carrier material was then treated with 25 ml of high-% aqueous tetraethylenepentamine solution at room temperature for 1 hour in a vacuum to facilitate the penetration of the solution into the pores of the carrier material. The excess of unadsorbed solution was decanted from, approx. 25$ of the tetraethylenepentamine was adsorbed in the pores of the carrier material. The wet support material was then treated with 25 ml of a 5% aqueous solution of glutardialdehyde at room temperature and a practically immediate reaction was obtained with the formation of an insoluble reaction product both on the surface and inside the pores of the support material. The excess of glutardialdehyde solution was decanted from and the organic-inorganic complex washed to remove unreacted and unadsorbed reagents, washing first with water and then with 0.02 molar acetate buffer. pH ^.2, at <l>h5°C. An enzyme solution was then added containing 200 mg of glucoamylase per 25 ml of water and this reacted with the above complex at room temperature over the course of 1 hour. After approx. 1 hour, the excess of glucoamylase solution was decanted from and the enzyme preparation washed with water to remove unbound and/or non-adsorbed enzyme. The composition was leached over 2 hours with acetate buffer solution with the same composition as above. The amount of adsorbed and/or covalently bound enzyme was measured by micro-Dumas gas chromatography both before and after addition of enzyme. The enzyme preparation's activity was measured by the amount of glucose produced with ^, 0% diluted starch solution as substrate with pH ^.2 and 60°C with Worthington's glucostat method for the analysis of glucose, the latter being considered safer for measuring the applicability of the preparation. An activity of 28 units per gram carrier material with an enzyme level of 29 mg per grams of carrier material was reached in this way (1 unit denotes the production of 1 gram of glucose per hour at 60°C according to forsbks specifications). It will be seen that despite the known solubility at pH 2.2 for the enzyme preparation when it is prepared in the absence of inorganic carrier material, an insignificant loss of enzyme was obtained from the combined inorganic-organic complex when leaching with buffer solution pH '+, 2. This was demonstrated by examining the outcome of this treatment.

Example II

In this example, the procedure from example I was repeated with except that the inorganic porous carrier had a particle size of 10 to 30 mesh. The silicon oxide-aluminum oxide mass containing boron phosphate was treated with tetraethylenepentamine, glutardialdehyde and glucoamylase as above. An active undissolved enzyme complex with somewhat less activity was obtained, probably due to diffusion problems caused by the greater particle size of the preparation.

Example III

In a similar manner as described in Example I above,

2 grams of lithium oxide-aluminum oxide with the same physical properties with regard to particle size, pore diameter and surface area as indicated -in example- I treated with an acetone solution of tetraethylenepentamine followed by a toluene diisocyanate solution, also in acetone, instead of aqueous glutardialdehyde. After decanting off excess diisocyanate solution and washing with water, the organic-inorganic complex was further treated with aqueous glucoamylase solution. As in Example I, the finished product contained an active and completely insoluble enzyme complex.

Example IV

To illustrate the fact that different concentrations of solutions can be used for the production of the desired product, the procedure from Example I above was repeated with the difference that higher concentrated solutions of the various reagents were used. E.g. 2 grams of 10-30 mesh silicon oxide-aluminium oxide were treated with 25 ml of a 20% tetraethylenepentamine solution and after decantation, 50 ml of a 25% glutardialdehyde solution was added. After washing, the complex was treated with aqueous glucoamylase to produce an insoluble enzyme preparation which showed an activity of approx.

12 units per gram based on the glucostat test.

Example V

To a silicon oxide-aluminium oxide which consisted of 2 grams of 10-30 mesh particles, 25 ml of a 5% aqueous, partially hydrolyzed collagen solution was added instead of tetraethylenepentamine. After decantation and treatment with glutardialdehyde, the organic-inorganic mass was washed and treated with glucoamylase solution. The finished substance mixture was treated in a similar way as described in example I by decanting, washing and leaching with buffer solution (pH M-.2) to produce an insoluble enzyme preparation with an activity of approx. 10 units per gram.

Example VI

In this example, silicon oxide-alumina with a particle size of 10-30 mesh, pore diameter from 0.01 to approx.

5.5/ Um and surface area approx. 150-200 m <p>/g processed

by adding tetraethylenepentamine in a 1% partially hydrogenated aqueous collagen solution where the collagen was used as an external

more binding agent. After draining and reaction with glutardialdehyde, the organic-inorganic mass was treated with glucoamylase solution as described in example I for the production of an active enzyme preparation.

Example VII

To show that different enzymes can be used for the preparation of the desired preparation, a silicon oxide-aluminum oxide containing boron phosphate was treated with tetraethylenepentamine solution, decanted, washed, after which glutardialdehyde solution was added and the resulting mixture was treated with aqueous lactase solution. This gave an active enzyme preparation. Similar methods can be used to bind enzymes such as proteases, glucose isomerase and glucose oxidase to form active preparations.

Example VIII

In this example, a column with an internal diameter of 20 mm was used containing 1^.2 grams of active enzyme preparation made from glucoamylase, bound to 10-30 mesh silicon oxide-aluminium oxide as a porous support material containing boron phosphate embedded, the preparation was prepared as described in example I. The column was used continuously over 30 days at <1>+5°C for the hydrolysis of aqueous 30% dilute starch solution buffered to pH .2. The effluent was measured with respect to glucose production by the Worthingtori glucostat method. No significant loss of enzyme activity was found during this period of time and that the percentage conversion of starch to glucose at this temperature and flow rate was approx. 150 ml per hour was 62%,.

Example IX

To show that different substrates or carrier materials can be used for the production of the desired preparations, an aluminum oxide-coated monolith consisting of ceramic material with a honeycomb structure in the form of macro-channels was treated^ in the same way as in example I above, i.e. that the monolith was treated with solutions of tetraethylenepentamine, glutardialdehyde and glucoamylase enzyme in a sequence comprising decanting, washing and leaching as described. The original ceramic monolith had a dry weight of 256 g, of which 13% consisted of aluminum oxide coating. The finished undissolved enzyme preparation was filled onto a column in a glass tube with an internal diameter of 70 mm where it could be operated continuously by means of suitable pump equipment within a temperature-regulated container which was kept at +5°C. Over a period of 10 days where the column was used continuously for the hydrolysis of a 3°$-ig buffered dilute starch solution, it was found that only approx. 3% of the original activity of the enzyme preparation was lost while maintaining a strain rate of approx. 85 ml per hour. Furthermore, it was found that in the last part of this ^O day period, an approx. 80$ conversion of the starch to glucose. In order to study the system's properties further, different variations in the flow rate were made and it was then discovered that at a flow rate of approx. 38 ml per hour it was possible to achieve a conversion rate of 92-93$ starch to glucose. The relatively long period during which the enzyme was used for the conversion of starch to glucose without significant loss of enzyme activity by either desorption or deactivation shows the catalyst's long half-life. -

Example X

In this example, a monolith preparation filled column was prepared in a similar manner as in Example IX above with the difference that the enzyme used for the preparation of the complex consisted of lactase instead of glucoamylase. The preparation was examined with regard to stability under continuous straining at a temperature of 37°C over 29 days. It was again found that there was no significant loss of activity in the insoluble enzyme preparation. This insolubilized enzyme was used for the treatment of 5% lactose solution which had been buffered to pH <!>+.2 and which was filled into a column in an amount of ^+ ml per hour. During the last 29 days, a 35% conversion of lactose to glucose and galactose was found.

Claims (15)

1. Immobilized organic-inorganic enzyme conjugate comprising an inorganic, porous carrier and an organic polymeric material, which polymeric material is selected from water-soluble polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexamethylenediamine and polyethyleneamine, water-insoluble polyamines such as methylenedicyclohexylamine and methylenedianiline, and natural and synthetic partially hydrolyzed polymers and formed polymers such as nylon, collagen, polyacrolein, polymaleic anhydride, alginic acid, casein hydrolyzate and gelatin, which polymeric material is adsorbed on the inorganic support, and which enzyme is adsorbed on the porous support and the organic polymer ~material, and also covalently bound to the organic polymeric material, characterized in that the polymeric material is embedded in the pores of the inorganic, porous support while forming a combined organic-inorganic matrix, and that the polymeric material contains lder attached groups, which attached groups are formed by reacting the polymeric material with a bifunctional monomer containing functional groups such as amino, hydroxyl, carboxyl, thiol and. carbonyl, which functional groups are preferably separated by chains containing from 4 to 10 carbon atoms, which enzyme is covalently bound to the functional parts of the associated groups at or in the immediate vicinity of their terminal positions. 2. Enzyme conjugate according to claim 1, characterized in that the inorganic, porous support material is aluminum oxide. 3. Enzyme conjugate according to claim 1, characterized in that the inorganic, porous support material is silicon oxide-aluminium oxide. 4. Enzyme conjugate according to claim 1, characterized in that the inorganic, porous carrier material is silicon oxide-aluminium oxide containing boron phosphate. 5. Enzyme conjugate according to claim characterized in that the inorganic, porous carrier material is a ceramic material coated with porous aluminum oxide provided with beehive-like macrochannels. 6. Enzyme conjugate according to claims 1 - 5, characterized in that the organic polymer material is the reaction product between polyethyleneimine and glutardialdehyde. 7. Enzyme conjugate according to claim 6, characterized in that the organic polymer material is the reaction product between tetraethylenepentamine and glutardialdehyde. 8. Enzyme conjugate according to claim 6, characterized in that the organic polymer material is the reaction product between tetraethylenepentamine and toluene diisocyanate. 9. Enzyme conjugate according to claim 6, characterized in that the organic polymer material is the reaction product between a mixture of tetraethylenepentamine and partially hydrolysed collagen, and glutardialdehyde. 10. Enzyme conjugate according to claim 6, characterized in that the organic polymer material is the reaction product between pentaethylenehexamine and glutardialdehyde. 11. Enzyme conjugate according to one or more of claims 1 - 10, characterized in that the inorganic porous support material has a pore diameter of 0.01 - 5.5 µm where up to 25 - 60% of the material has a diameter over 2.0 /Um, as well as a surface area of 150 - 200 m 2/g. 12. Method for producing immobilized enzyme conjugate according to claims 1-11, characterized in that the inorganic porous support material is treated with a first solution of a bi- or multi-functional monomer, a polymer hydrolyzate or a pre-prepared polymer, non-adsorbed solution is removed, the inorganic, porous carrier material is brought into contact with a relatively large excess of a second bifunctional monomer solution while forming the organic-inorganic matrix, an enzyme solution is added to the formed organic-inorganic matrix and unreacted compounds are removed. 13. Method as stated in claim 12, characterized in that a first solution, a second solution and an enzyme solution which is aqueous are used. 14. Method according to claim 12, characterized in that cheap organic solvents such as acetone and tetrahydrofuran are used as carrier media for said bi- or multi-functional monomers, polymer hydrolysates or pre-produced polymers. 15. Method according to one or more of claims 12 - 14, characterized in that a second bifunctional monomer with reactive groups which are separated from each other by a molecular chain containing 4-10 carbon atoms is used.