IE42482B1 - Immobilized enzyme conjugates - Google Patents

Abstract

The enzyme fixed to a support is composed of a combined organic-inorganic base material, which consists of an inorganic porous support having an organic polymer, which contains functional groups, partially adsorbed and enclosed in its pores, and of an enzyme which is partially adsorbed to the base material and bound covalently to the functional groups of the organic polymer in the terminal regions of this polymer. Such immobilised enzymes are of use in the biochemical and fermentation industries. They are obtained, in particular, by drenching a porous support with a solution of a polyfunctional organic compound and treating the impregnated support with an excess of a bifunctional monomer. Subsequently, the enzyme is added, part of which enzyme then binds covalently to reactive groups of the polymer which has been formed and which is present in and on the support. During this procedure, the biochemically active residues of the enzyme must not be blocked or altered.

Description

The invention relates to an immobilized enzyme conjugate and to a method of preparing such immobilized enzyme conjugate.

It is known that enzymes, which are proteinaceous in nature and which are commonly water-soluble, act as biological catalysts which serve to regulate many and varied chemical reactions which occur in living organisms. The enzymes may also be isolated and used in analytical, medical and industrial applications. For example, they find use in industrial applications in the preparation of food products such as cheese or bread as well as being used in the preparation of alcoholic beverages. Some specific uses in industry may be found, for example, in the use of enzymes in the resolution of amino acids: in the modification of penicillin to form various substrates thereof; the use of various proteases in cheese-making, meat tenderizing, detergent formulations, leather manufacture and as digestive aids, the use of carbonhydrases, for exanple, in starch hydrolysis, sucrose inversion and glucose isomerization; the use of nucleases in flavour control;. . and., the use Of oxidases in oxidation prevention and in the' color control of food products. These uses, as· well as many, others, have been well delineated in' the literature.

• -As hereinbefore 'set forth, inasmuch as enzymes are commonly water-soluble as well as being generally unstable and readily deactivated, they are also difficult either - 3 to remove for reuse from solutions in which they are utilized or it is difficult to maintain their catalytic activity for a relatively extended period of time. These difficulties lead to an increased cost in the use of enzymes for commercial purposes due to the necessity in practice for frequent replacement of the enzyme, this replacement being usually necessary with each application. To counteract the high cost of replacement, it has been suggested to immobilize or insolubilize the enzymes prior to the use thereof. By immobilizing the enzymes by various known means hereinafter set forth in greater detail, it is possible to stabilize the enzymes and, therefore, to permit the reuse of the enzyme, which may otherwise undergo deactivation or be lost in the reaction medium in connection with which it is used. Such immobilized or insolubilized enzymes may be employed in various reactor systems, for example in packed columns and stirred tank reactors, depending upon the nature of the substrate which is utilized therein. In general, the immobilization of the enzymes provides a more favourable or broader environmental stability, a minimum of effluent problems and materials handling as well as the possibility of upgrading the activity of the enzyme itself.

As already indicated several general methods, as well as many modifications thereof, have been described by which the immobilization of enzymes may be effected. One general method is to adsorb the enzyme at a solid surface as, for example, to adsorb an enzyme such as amino acid acylase on a cellulose derivative such as DAE-cellulose, to adsorb papain or ribonuclease on porous glass, to adsorb catalase on charcoal, to adsorb trypsin on quartz glass or cellulose or to adsorb chymotrypsin on kaollinite.

Another general method is to trap an enzyme in a gel - 4 43483 lattice, for example to entrap glucose oxidase, urease or papain in a polyacrylamide gel, acetyl cholinesterase in a starch gel or a silicone polymer, or glutamic-pyruvic transaminase in a polyamide or cellulose acetate gel. A further general method is a cross-linking by means of bifunctional reagents, which may be effected in combination with either of the aforementioned general methods of immobilization. When utilizing this method, bifunctional or polyfunctional reagents which may induce intermolecular cross-linking will covalently bind the enzymes to each other as well as to a solid support. This method may be exemplified by the use of glutardialdehyde or bisdiazobenzidine - 2.21 - disulfonic acid to bind an enzyme such as papain to a solid support. A still further method of immobilizing an enzyme comprises a covalent binding in which an enzyme such as glucoamylase, trypsin, papain, pronase, amylase, glucose oxidase, pepsin, rennin, fungal protease or lactose is immobilized by covalent attachment to a polymeric material which is attached to an organic or inorganic solid porous support. This method may also be combined with the aforesaid immobilization procedures.

The above-enumerated methods of immobilizing enzymes all possess some drawbacks Which detract from their use in industrial processes. For example, when an enzyme is directly adsorbed on the surface of a support, the binding forces which result between the enzyme and the carrier support are often quite weak, although some prior art has indicated that relatively stable conjugates of this type have been obtained when the pore size of the support and the spin diameter of the enzyme are correlated. However, the pore size of the support then cannot exceed a diameter of about 100 Angstroms. In view of this weak bond, the - 5 enzyme is often readily desorbed in the presence of solutions of the substrate being processed. In addition to this, the enzyme may be partially or extensively deactivated due to its lack of mobility or due to interaction between the support and the active site of the enzyme. As already mentioned, enzyme immobilization may alternatively be effected, for example by entrapment of enzyme in gel lattices and this can conveniently be effected by polymerizing an aqueous solution or emulsion containing the enzyme and a monomer polymerizable to form a lattice-structured polymer gel, or by incorporating the enzyme into the preformed polymer by various techniques, often in the presence of a cross-linking agent. while this method of immobilizing enzymes has an advantage in that the reaction conditions utilized to effect the entrapment are usually mild so that often there is little alteration or deactivation of the enzyme (unless the cross-linking agent mentioned above is present during the entrapment procedure), it also has disadvantages in that the conjugate has poor mechanical strength, which results in compacting when used in columns in continuous flow systems, with a concomitant plugging of the column. Such systems also have rather wide variations in pore size thus leading to some pore sixes whieh are large enough to permit the loss of enzyme. In addition, some pore sizes may be sufficiently small that large diffusional barriers to the transport of the substrate and product will lead to reaction retardation, this being especially true when using a high molecular weight substrate. When the mentioned cross-linking agent is present, there are other disadvantages, these being due, as already noted, to the lack of mobility with resulting deactivation because of inability of the enzyme to assume 42483 - 6 the natural configuration necessary for maximum activity, particularly when the active site is involved in the binding process.

Covalent birding methods have found wide applications and may be used either as the sole immobilization technique or as an integral part of many of the methods already described in which cross-linking reactions are employed. This method is often used to bind the enzyme as well as the support through a bifunctional intermediary molecule in which the functional groups of the molecule, for example gamma-amino-propyltriethoxysilane, are capable of reacting with functional moieties present in both the enzyme and either an organic or inorganic porous support. A wide variety of reagents and supports has been employed in this manner and the method has the advantage of providing strong covalent bonds throughout the conjugate product as well as great activity in many cases. The covalent linkage of the enzyme to the carrier must be accomplished through functional groups on the enzyme which are non20 essential for its catalytic activity such as free amino groups, carboxyl groups, hydroxyl groups, phenolic groups or sulfhydryl groups. These functional groups will also react with a wide variety of other functional groups such as aldehydo, isocyanato, acyl, diazo, azido, anhydro and activated ester, to produce covalent bonds. Nevertheless, this method also often has many disadvantages involving costly reactants and solvents, as well as specialized and costly porous supports and cumbersome multi-step procedures, which render the method of preparation uneconomical for commercial application.

The prior art is therefore replete with various methods for immobilizing enzymes which, however, in various ways fail to meet the requirements of industrial use. 42483 - 7 For example, U.S. Patent No. 3,556,945 relates to enzyme composites in which the enzyme is adsorbed directly to an inorganic carrier such as glass. U.S. Patent No. 3,519,538 is concerned with enzyme composites in which the enzymes are chemically coupled by means of an intermediary silane coupling agent to an inorganic carrier. In similar fashion, U.S. Patent No. 3,783,101 also utilizes an organosilane composite as a hinding agent, the enzyme being covalently coupled to a glass carrier by means of an intermediate 3ilane coupling agent, the silicon portion of the coupling agent being attached to the carrier while the organic portion of the coupling agent is coupled to the enzyme, the composition containing a metal oxide on the surface of the carrier disposed between the carrier and the silicon portion of the coupling agent. In U.S. Patent No. 3,821,083, an inert carrier is coated with a preformed polymer such as polyacrolein which has bonded thereto an enzyme. However, according to most of the examples set forth in the patent, it is necessary first to acid hydrolize the composite prior to the deposition of the enzyme on the polymer. Another prior art patent, namely U.S. Patent No. 3,705,084 discloses a macroporous enzyme reactor in which an enzyme is adsorbed on the polymeric surface of a macroporous reactor core and thereafter is cross-linked in place. By cross-linking the enzymes on the polymeric surface after adsorption thereof, the enzyme is further immobilized in part and cannot act freely, as in its native state, as a catalyst. The cross-linking of enzymes in effect links them together, thereby preventing a free movement of the enzyme and decreases the mobility of the enzyme.

As hereinbefore set forth, the use of enzymes in analytical medical or industrial applications may be - 8 424S2 greatly enhanced if said enzymes are in an immobilized condition, that is, said enzymes, by being in combination with other solid materials, are themselves in such a condition whereby they are not water-soluble and therefore they may be subjected to repeated use while maintaining the catalytic activity of said enzyme. In order to be present in an immobilized state, the enzymes must be bound in some manner to a water-insoluble carrier, thereby being commercially usable in a water-insoluble state.

As will be appreciated from the Examples which follow, in contradistinction to the compositions of matter containing immobilized enzymes as set forth in the prior art, the invention enables immobilized enzyme conjugates to be prepared by utilizing relatively inexpensive reactants as well as utilizing more simple steps in the procedure for preparing the immobilized enzyme conjugates. In addition, the mechanical strength and stability of the enzyme conjugates of the present invention Will, as will also be appreciated from the Examples, be greater than that which is possessed by the immobilized enzymes of the prior art.

According to the invention there is provided an immobilized enzyme conjugate comprising an organicinorganic matrix which comprises an inorganic porous support and an organic polymeric material contained in the support both by entrapment in the pores thereof and by adsorption of so-entrapped organic polymeric material onto the support material, and an enzyme contained in said matrix both by adsorption onto the material of said matrix and by covalent bonding of enzyme so-adsorbed to pendant functional moieties of said organic polymeric material. - 9 The term pendant functional moieties of the organic polymeric material refers to moieties of the adsorbed organic polymeric material which are reactive with the enzyme to form covalent linkage therewith and which extend from the surface of the matrix so that covalently bound enzyme molecules will be disposed in positions removed from the matrix surface. In practice, the enzyme will preferably be bound to a portion of the pendant functional moiety disposed the greatest distance from the point of attachment of such moiety to the polymer body.

The immobilized enzyme conjugates of the invention may be prepared in a relatively 'simple manner. In a preferred method of preparation, the inorganic porous support is contacted with a solution, preferably aqueous in nature, of a polyfunctional monomer (i.e. a bifunctional monomer), a polymer hydrolyzate or a preformed polymer to effect adsorption of the polyfunctional monomer, polymer hydrolyzate or preformed polymer onto the support material; following this procedure, unadsorbed solution is removed.

Such removal may be accomplished by any means known in the art, for example by allowing draining of the unadsorbed solution. It is also contemplated that one or more organic solvents, such as acetone and tetrahydrofuran, may alternatively be used as the carrier for the aforementioned monomers or polymers. Following the removal of the unadsorbed solution, the porous support, usually whilst still wet, is contacted with an excess, for example a 5 to 20 mole percent excess, of a second bifunctional monomer, preferably one in whioh the reactive groups are separated by a chain containing from 4 to 10 carbon atoms, in the form of a solution, preferably an aqueous solution, whereby an organic polymeric material which is both adsorbed and - 10 entrapped in the pores of the support will be formed and from which unreaeted pendant functional moieties extend due to the fact that an excess amount of the second bifunctional monomer whose solution was contacted with the porous support in the third above-mentioned stage was employed.

The unreaeted functional moieties are then available for covalent binding to the enzyme, which is added to the resulting organic-inorganic matrix by contact of the matrix with the enzyme, as a solution, preferably an aqueous solution. After removal of the unreaeted materials, for example by washing, the enzyme, besides being covalently bound to the pendant functional moieties, will also be adsorbed onto the matrix. It is therefore readily apparent that the entire immobilization procedure can be conducted in a simple and inexpensive manner utilizing an aqueous or organic solvent medium, the procedure being conducted by utilizing a minimum of operating steps and, in addition, permitting a ready recovery of the excess reactants and finished immobilized enzyme conjugate. The procedures described above may conveniently be conducted at subambient temperature (e.g. 5°C) or elevated temperature (e.g. 60°C). However, ambient temperatures will preferably be used, for example a temperature of from 20°C to 25°C.

Many of the inorganic supports reported in the prior art specify controlled pore materials such as glass or alumina having a pore diameter of from 500 to 700 Angstroms for about 96% of the material and a maximum pore diameter 2 of 1000 Angstroms, a surface area of 40-70 m /gm and 40-80 mesh size (United States Standard Series) particles. In addition, these supports may be coated with a metallic oxide such as zirconium oxide or titanium oxide for greater stability. In contradistinction to these supports, it is - 11 contemplated within the scope of this invention that the inorganic porous supports which are utilized herein, will be materials in general possessing pore diameters ranging from 100 Angstroms to 55,000 Angstroms, with up to 60% (e.g. from 25% to 60%) of the porous support material possessing pores having diameters above 20,000 Angstroms, and which have surface areas ranging from 150 to 200 m /gm. The particle size may also vary over a wide range, in general in the range from 10 mesh (United States Standard Series) to a fine powder (e.g. from 20 mesh (United States Standard Series) to a fine powder), the precise particle size characteristics depending upon the particular system in which the particles are to be used.

The inorganic porous support material may comprise a metal oxide such as alumina (and particularly gammaalumina) silica, zirconia, a mixture of metal oxides (such as silica-alumina, silica-zirconia, silica-magnesia and silica-zirconia-alumina) and silica-alumina containing one or more other inorganic compounds such as boron phosphate.

Combinations of materials, such as the aforementioned materials, one material serving as a coating for another material comprising the support, may alternatively be used as the inorganic support. In particular, as a preference, a porous support material coated with an oxide of the type hereinbefore mentioned may be used. Apart from the addition to silica-alumina of, for example, boron phosphate, as mentioned above, it will be appreciated that an inorganic support material of other than silica-alumina may have an addition of an inorganic material such as boron phosphate to impart special properties to the support material. A particularly useful form of support will constitute a ceramic body which may have the type of porosity just 43483 - 12 described or it may be honeycombed with connecting macro size channels throughout, such materials being commonly known as monoliths, and coated with a porous material, for example porous alumina or porous zirconia (preferably, a porous material which comprises alumina). The use Of such a type of support has the particular advantage of permitting the free flow through the macro sized channels of even highly viscous substrates which will often be encountered when using the immobilized enzyme conjugate of the invention in commercial enzyme catalyzed reactions.

A specific embodiment of this invention is found in an immobilized enzyme conjugate comprising an organicinorganic matrix consisting of a low bulk density, porous silica-alumina support of relatively high surface area which may also contain inorganic additives (e.g. as mentioned earlier) and an in situ-prepared tetraethylenepentamineglutaraldehyde polymeric material which is adsorbed as well as entrapped in the pores of said silica-alumina? and an enzyme comprising glucoamylase being covalently bound to the glutaraldehyde pendant groups of the polymeric material and also being adsorbed onto the matrix.

The polymer material may conveniently be produced according to any of the prior art general methods hereinbefore described, that is, for example, by first adsorbing a solution containing from 2 to 25% of a polyfunctional (e.g. bifunctional) monomer, polymeric hydrolysate, or a preformed polymer, the monomer or polymer being synthetic or natural in origin and preferably being used as a solution in water.

The pendant functional moieties, derived from the bifunctional monomer added using the second solution referred to earlier conveniently will comprise suoh wellknown reactive moieties as amino, hydroxyl, carboxy. - 13 thiol or carbonyl moieties. As was hereinbefore set forth, the reactive groups of the bifunctional compounds are preferably, but not necessarily, separated by chains containing from 4 to 10 carbon atoms. The reactive moieties are capable of covalently bonding with both the initial additives (adsorbed onto the inorganic support using the first solution referred to) and subsequently, after removing (e.g. by washing out) unreacted materials, with the enzyme which is to be added in a subsequent step, said enzyme being then covalently bound to the functional group as well as concomitantly adsorbed on the matrix.

After addition of the enzyme to this composition, it will be appreciated from the Examples which follow that a relatively stable enzyme conjugate can be obtained which possesses high activity and high stability.

Specific examples of bi- or polyfunctional monomers, polymer hydrolysates or preformed polymers which may be initially adsorbed on the inorganic support will include water-soluble polyamines such as ethylenediamine, a poly20 ethyleneamine, for example, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine or hexamethylenediamine; water-insoluble polyamines such as rnethylenedicyclohexylamine and methylenedianiline; natural and synthetic polymers such as nylon, collagen, polyacrolein, polymaleic anhydride, alginic acid, casein hydrolysate and gelatin; and partially hydrolyzed polymers such as partially hydrolyzed collagen. Some specific examples of intermediate bifunctional materials which may be added to the above-enumerated products to produce an organic-inorganic matrix and which possess the necessary characteristics hereinbefore set forth include compounds such as glutardialdehyde, adipoyl chloride, sebacoyl - 14 chloride, toluenediisocyanate and hexamethylenediisocyanate. It will be noted that when a polyethyleneamine of the type hereinbefore set forth is reacted with glutardialdehyde in the absence of an inorganic porous support, an aqueous acid-soluble material is obtained, whereas when a polyethyleneamine is reacted with a diisocyanate or acyl halide, a water-insoluble product is obtained. Conversely, if a reaction complex without the inorganic support contains free carboxyl groups, an alkaline-soluble complex can be obtained. Due to the excess of intermediary, or spacer, bifunctional molecules which are used, the polymeric matrix which ie formed will contain pendant groups comprising the spacer molecules, said molecules extending from the matrix and having reactive moieties available, for example at or adjacent to terminal portions thereof, which are capable of reacting with and binding the enzyme to the spacer molecules via covalent bonds. In addition, the enzyme, when applied after the unreaeted reagents have been removed from the organic-inorganic matrix for example by washing, will also concomitantly undergo adsorption in part With said matrix. Binding the enzyme solely to the organic matrix will not usually affect the dependency of the solubility of the aggregate on the pH of the solution but when the inorganic support is included as heretofore described, the total conjugate exhibits high stability over a relatively wide pH range from 3 to 9, the stability of course also being a function of the optimal pH characteristics of the particular enzyme employed as well as the inorganic support used. Therefore, it is readily apparent that a suitable organic-inorganic matrix which is applicable in many situations will be formed with the support material by adsorbing any of the type of materials hereinbefore - 15 described which are known to the art and then treated with any bifunctional molecule which is also known to the art and is suitably functionalized to react with the original additive, provided that a large enough excess of the bifunctional molecule is used to provide pendant groups which are capable of subsequently reacting with the enzyme which is desired to be immobilized. By utilising these functional pendant groups as a binding site for the enzymes, it will permit the enzymes to have a greater mobility and thus permit the catalytic activity of the enzyme to remain at a high level for a relatively longer period of time than will be attained when the enzyme has been immobilized by any of the other methods such as entrapment in a gel lattice, adsorption on a solid surface or cross-linkage of the enzyme by means of bifunctional reagents. Not all formulations, however, will produce equivalent results in terms of stability or activity.

Examples of enzymes which may be immobilized by a covalent bonding reaction and which contain an amino group capable of reacting with an aldehyde or isocyanato moiety of the pendant group which is attached to a polymeric material entrapped and adsorbed in the pores of a porous support material will include trypsin, papain, hexokinase, betagalactosidase, ficin, bromelain, lactic dehydrogenase, lactase, glucose isomerase, glucoamylase, chymotrypsin, pronase, acylase, invertase, amylase, glucose oxidase, pepsin, rennin and fungal protease. In general, any enzyme whose active site is not involved in the covalent bonding can be used. While the aforementioned discussion was centred about pendant groups which contain as a functional moiety thereon an aldehydic or isocyanato group, it is also contemplated within the scope of this invention - 16 SH<»o β that ths pendant group can contain other functional moieties capable of reaction with carboxy, sulfhydryl or other moieties usually present in enzymes. However, the covalent bonding of enzymes containing these other moieties with other pendant groups may not necessarily be effected with equivalent results and may also involve appreciably greater costs in preparing intermediates. It is to be understood that the aforementioned listing of porous solid supports, monomers, hydrolysates, polymers and enzymes are only representative of the various classes of compounds which may be used, and that the present invention is not necessarily limited thereto.

The preparation of the compositions of matter of the present invention is preferably effected in a batch type operation as hereinbefore already described in detail, although it is also contemplated within the scope of this invention that the formation of the finished composition of matter may also be effected in a continuous manner of operation. When a continuous type operation is used, a quantity of the porous solid support material is placed in an appropriate apparatus, usually constituting a column.

The porous solid support material may be in any form desired such as powder, pellets or monoliths, and is charged to the column, after which a preferably aqueous solution of, for example, a polyfunctional amine is contacted with the porous support until the latter is saturated with the amine solution and the excess is then drained. A spacer or intermediary bifunctional molecule such as glutardialdehyde is then contacted with the satur30 ated support. The formation of the polymeric matrix is thus effected in an aqueous system, said reaction being effected during a period of time which may range from 1 to - 17 43482 hours in duration, but is usually of short duration. After removing the excess glutardialdehyde by draining and washing out any water-soluble and unreacted materials, which in the case of a polyamine is preferably done with a buffer solution possessing a pH of about 4, an aqueous solution of the enzyme is contacted or recycled through the column, this step effecting a covalent bonding of said enzyme to the terminal aldehydic groups of the functionalized pendant molecules which extend from the matrix. This occurs until there is no further physical adsorption and covalent bonding of the enzyme to the organic-inorganic matrix and pendant molecules. The excess enzyme is recovered in the effluent after draining and washing the column. The column is thus ready for use in chemical reactions in which the catalytic effect of the enzyme is to take place. The procedures are, for the most part, conducted within the time, temperature and concentration parameters hereinbefore described in the batch type procedure and will result in comparable immobilized enzyme complexes. It is also contemplated within the scope of this invention that with suitable modifications of pH and temperature parameters which will be obvious to those skilled in the art, the process may be applied to a wide variety of inorganic porous supports, polymer forming reactants and enzymes.

The following Examples are intended to illustrate the invention. In the Examples all percentages are on a weight basis and the mesh sizes mentioned United States Standard Series mesh sizes.

EXAMPLE 1 In this example 2 grams of a porous silica-alumina composite which contained boron phosphate incorporated - 18 therein having a particle size of 40-80 mesh, a pore diameter ranging from 100 to 55,000 Angstroms and a surface area of 150-200 m /gm was utilized as the inorganic support for the immobilized enzyme conjugate of the present invention.

This support was calcined at a temperature of about 500°F. to remove any adsorbed moisture contained therein. Thereafter the support was treated with 25 ml of a 4% aqueous solution of tetraethylenepentamine at ambient temperature for a period of 1 hour in vacuo to facilitate the penetrat10 ion of the solution into the pores of the support. The excess unadsorbed solution was then decanted, about 25% of the tetraethylenepentamine having been adsorbed into the pores of the support. Following this, the wet support was then treated with 25 ml of a 5% aqueous solution of glutar15 dialdehyde at ambient temperature and an almost immediate reaction took place with the formation of an insoluble reaction product both on the surface and within the pores of the support. The excess glutardialdehyde solution was then decanted and the organic-inorganic complex was washed to remove unreaeted and unadsorbed reagents, said washing being accomplished first with water followed by washing with a 0.02 molar acetate buffer solution which possessed a pH of 4.2, the washing operation being effected at a temperature of 45°C. Thereafter an enzyme solution containing about 200 mg of glucoamylase per 25 ml of water was added and allowed to react with the composite at ambient temperature for a period of 1 hour. At the end of this 1-hour period, the excess glucoamylase solution was decanted and the enzyme conjugate was washed with water to remove any unbound and/or unadsorbed enzyme. The composition was then leached for a period of 24 hours with an acetate buffer solution similar to that hereinbefore - 19 described. The amount of adsorbed and/or covalently bonded enzyme was determined by micro Dumas gas chromatography analyses both before and after the addition of the enzyme. The activity of the enzyme conjugate was then determined by the amount of glucose produced using 30% thinned starch solution as substrate at a pH of 4.2 and 60°C., and employing Worthington's glucostat procedure for analysing glucose, the latter being considered the more reliable procedure for determining the utility of the conjugate. An activity of 28 units per gram of support with an enzyme loading of 29 mg/gm of support was obtained by this procedure (one unit representing the production of 1 gram glucose per hour at 60°c. according to the assay specifications). It will be noted that despite the known solubility at pH of 4.2 of the enzyme conjugate when prepared in the absence of inorganic support, negligible loss of enzyme from the combined inorganic-organic complex occurred during leaching with the 4.2 pH buffer solution. This was demonstrated by assaying the effluent from this treatment.

EXAMPLE II In this example the procedure of Example I was followed with the exception that the inorganic porous support had a particle size of 10-30 mesh. This silicaalumina composite containing boron phosphate incorporated therein was treated with tetraethylenepentamine, glutardialdehyde and glucoamylase in a manner similar to that set forth above. An active immobilized enzyme complex was obtained although of decreased activity probably because a diffusion problem is produced by the larger particle size of the composite. - 20 EXAMPLE III In a manner similar to that set forth in Example I above, 2 grams of a silica-alumina composite possessing the same physical characteristics of particle size, pore diameter and surface area as that set forth in Example I was treated with an acetone solution of tetraethylenepentamine and followed by a toluenediisocyanate solution also in acetone instead of aqueous glutardialdehyde. After decanting the excess diisocyanate solution and washing with water, the organic-inorganic complex was further treated with an aqueous glucoamylase solution. As in Example I, the finished product comprised an active completely insoluble enzyme complex.

EXAMPLE IV To illustrate the point that various concentrations of solutions can be used to prepare the desired product, the procedure set forth in Example I above was repeated with the exception that more highly concentrated solutions of the various reagents were used. For example, 2 grams of a 10-30 mesh silica-alumina composite was treated with 25 ml of a 20% tetraethylenepentamine solution and after decanting 50 ml of a 25% glutardialdehyde solution was added thereto. This complex, after washing, was then treated with aqueous glucoamylase to prepare an immobilized enzyme conjugate which showed an activity of about 12 units per gram assayed by the glucostat test.

EXAMPLE V To a silica-alumina composite comprising 2 grams of 10-30 mesh particles was added 25 ml of a 5% aqueous, partially hydrolyzed collagen solution which was in place of the tetraethylenepentamine. After decanting and treating with glutardialdehyde, the organic-inorganic - 21 matrix was washed and then treated with a glucoamylase solution. The finished composition of matter was treated in a manner similar to that set forth in Example I above by decanting, washing and leaching with a buffered (pH Of 4.2) solution to give an immobilized enzyme conjugate whieh had an activity of about 10 units per gram.

EXAMPLE VI In this example a silica-alumina composite having a particle size of 10-30 mesh, a pore diameter ranging from 100 to 55,000 Angstroms and a surface area of from 150 to 200 m /gm was treated by adding tetraethylenepentamme in a 1% partially hydrolyzed aqueous collagen solution, the collagen being utilized as an additional bonding agent. After draining and reacting with glutardialdehyde, the organic-inorganic matrix was then treated with a glucoamylase solution according to the general procedure of Example I to prepare an active enzyme conjugate.

EXAMPLE VII To illustrate that various enzymes can be used in preparing the desired compositions of matter, a silicaalumina composite containing boron phosphate incorporated therein was treated with a tetraethylenepentamine solution, decanted, washed, followed by addition of a glutardialdehyde solution and the resulting composite was then treated with an aqueous lactase solution. This produced an active enzyme conjugate. Similar procedures can be used to bind enzymes such as proteases, glucose isomerase, and glucose oxidase to produce active conjugates.

EXAMPLE VIII In this example a column possessing an inside diameter of 20 mm contained 14.2 grams of an active enzyme - 22 Q /> ‘Λ conjugate prepared from glucoamylase which was bonded to a 10-30 mesh silica-alumina porous support containing boron phosphate incorporated therein, the conjugate having been prepared in a manner similar to that set forth in Example I above. The column was used continuously for a period of 30 days at a temperature of 45°C. to hydrolyze an aqueous 30% thinned starch solution which had been buffered to a pH of 4.2. The effluent was monitored for the glucose production using the Worthington glueostat procedure. It was found that there was no apparent loss of enzyme activity during this period of time and that the percentage of conversion of starch to glucose at this temperature and at a flow rate of about 150 ml per hour was 62%.

EXAMPLE IX To illustrate the fact that various substrates or supports may be utilized to prepare the desired conpositions of matter, an alumina coated monolith which consisted of a ceramic body honeycombed with connecting macro si2e channels was treated in a manner similar to that set forth in Example I above, that is, the monolith was treated with solutions of tetraethylenepentamine, glutardialdehyde and a glucoamylase enzyme, the treatment being carried out in a sequential operation which included decanting, washing, and leaching procedures hereinbefore described. The original ceramic monolith possessed a dry weight of 256 grams, of which 13% consisted of an alumina coating. The finished immobilized enzyme conjugate was elaborated into a column within a glass tube having an inside diameter of 70 mm in order that it could be operated continuously by means of a suitable pumping apparatus within a temperature controlled container, said container being maintained at a temperature of 45°C. Over a 40-day period of continuous - 23 usage for the hydrolysis of a 30% buffered thinned starch solution, it was found that only about 3% of the original activity of the enzyme conjugate was lost while maintaining a flow rate of about 85 ml per hour. In addition, it was found that during the 40-day period there was an approximate 80% conversion of the starch to glucose. In order to further study the properties of the system, subsequent variations in flow rate were made during which it was found that at a flow rate of about 38 ml per hour it was possible to obtain a conversion in the range of from 92-93% of starch to glucose. The relatively long period of time during which this enzyme was used to convert starch to glucose without a significant loss of enzyme activity either by desorption or deactivation indicated a long half life of the catalyst.

EXAMPLE X In this example a monolith type of conjugate and column similar to that described in Example IX above was prepared, the exception being that the enzyme which was used to prepare the complex comprised lactase in place of glucoamylase. The conjugate was tested for stability under a continuous flow while maintaining the temperature at 37°C. for a period of 29 days. It was again found that there was no apparent loss of activity of the immobilized enzyme conjugate. This immobilized enzyme was used in the treatment of a 5% lactose solution which had been buffered to a pH of 4.2, said lactose solution being charged to the column at a rate of 54 ml per hour. It was found during the 29-day period that there was about 35% conversion of lactose to glucose and galactose.

Claims (21)

1. CLAIMS:1. An immobilized enzyme conjugate comprising an organic-inorganic matrix which comprises an inorganic porous support and an organic polymeric material contained 5 in the support both by entrapment in the pores thereof and by adsorption of so-entrapped organic polymeric material onto the support material, and an enzyme contained in said matrix both by adsorption onto the material of said matrix and by covalent bonding of enzyme so-adsorbed to pendant 10 functional moieties of said organic polymeric material.

2. An immobilized enzyme conjugate as claimed in claim 1 in which said organic polymeric material is one which has been formed in situ from a monomer, a hydrolyzed polymer or a polymer of natural or synthetic origin by 15 reaction with a bifunctional reactive monomer which latter provides said pendant functional moieties.

3. An immobilized enzyme conjugate as claimed in claim 1 or claim 2 in which said inorganic porous support material comprises alumina, silica or zirconia. 20

4. An immobilized enzyme conjugate as claimed in claim 3 in which said inorganic porous support material is gamma-alumina or silica-alumina.

5. An immobilized enzyme conjugate as claimed in claim 3 in which said inorganic porous support material is 25 silica-alumina containing boron phosphate.

6. An immobilized enzyme conjugate as claimed in claim 1 or claim 2 in which said inorganic porous support material is a ceramic body which is coated with a porous material comprising alumina and which is honeycombed with 30 connected channels through which, in use of the enzyme conjugate, to effect a flow of material for contact with the enzyme. - 25 43482

7. An immobilized enzyme conjugate as claimed in any preceding claim in which said organic polymeric material is the product of reaction of a polyethyleneamine with glutardialdehyde or toluenediisocyanate.

8. An immobilized enzyme conjugate as claimed in claim 7 in which said polyethyleneamine is tetraethylenepentamine .

9. An immobilized enzyme conjugate as claimed in any one of claims 1 to 6, in which said polymeric material is the reaction product of a mixture of tetraethylenepentamine and partly hydrolyzed collagen with glutardialdehyde.

10. An immobilized enzyme conjugate as claimed in claim 7 in which said polyethyleneamine is pentaethylenehexamine and said organic polymeric material is the reaction product therewith of glutardialdehyde.

11. An immobilized enzyme conjugate as claimed in any preceding claim in which said enzyme is lactic dehydrogenase, glucoamylase, lactase, fungal protease, glucose isomerase or glucose oxidase.

12. An immobilized enzyme conjugate as claimed in any preceding claim in which said inorganic porous support is a material which has pore diameters ranging from 100 to 55,000 Angstroms with from 25% to 60% of the porous support material having diameters above 20,000 Angstroms, and which has a surface area ranging from 150 to 200 m /gm.

13. An immobilized enzyme conjugate as claimed in claim 1 and substantially as hereinbefore described.

14. A method for preparing an immobilized enzyme conjugate as claimed in claim 1 which method comprises treating said inorganic support with a first solution, of a polyfunctional monomer, a polymer hydrolysate or a preformed polymer, to effect adsorption of the polyfunctional monomer, polymer hydrolysate or preformed polymer - 26 onto the support material; removing unadsorbed solution; contacting said inorganic support with an excess of a second solution, of a bifunctional monomer, to form said organic-inorganic matrix; contacting an enzyme solution 5 with the organic-inorganic matrix to provide said enzyme, contained in the matrix in the manner specified in claim 1; and removing the unreacted materials.

15. A method as claimed in claim 14 in which said first solution, said second solution, and said enzyme 10 solution are aqueous solutions.

16. A method as claimed in claim 14 in which the first solution is a solution in an organic solvent.

17. A method as claimed in claim 16 wherein the organic solvent is acetone or tetrahydrofuran. 15

18. A method as claimed in any one of claims 14 to 17 in which said bifunctional monomer of said second solution is one having reactive groups which are separated by a chain containing from 4 to 10 carbon atoms.

19. A method as claimed in claim 14 and substantially 20. As hereinbefore described.

20. An immobilized enzyme conjugate whenever obtained by a method as claimed in any one of claims 14 to 19.

21. An immobilized enzyme conjugate as claimed in any one of claims 1 to 13 wherein the combined organic25 inorganic matrix is one substantially as hereinbefore described in any one of the foregoing specific Examples.