CA1128917A - Support matrices for immobilized enzymes - Google Patents
Support matrices for immobilized enzymesInfo
- Publication number
- CA1128917A CA1128917A CA337,572A CA337572A CA1128917A CA 1128917 A CA1128917 A CA 1128917A CA 337572 A CA337572 A CA 337572A CA 1128917 A CA1128917 A CA 1128917A
- Authority
- CA
- Canada
- Prior art keywords
- matrix
- set forth
- enzyme
- solid support
- aminopolystyrene
- 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.)
- Expired
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/04—Reduction, e.g. hydrogenation
Abstract
SUPPORT MATRICES FOR IMMOBILIZED ENZYMES
ABSTRACT
Organic-inorganic support matrices for immobilized enzymes comprise a solid, porous, inorganic, water insoluble support com-bined with a copolymeric material resulting from the reaction of aminopolystyrene and a bifunctional monomer. The matrix may be prepared by depositing a salt of aminopolystyrene within the pores of the solid support from an aqueous solution at a pH less than 7 after which the resulting composite is then reacted with an excess of a bifunctional reactive monomer thus forming a copolymeric organic material in situ substantially entrapped in the pores of the support and containing functionalized pendent groups to which an enzyme may be coupled to form an immobilized enzyme conjugate.
ABSTRACT
Organic-inorganic support matrices for immobilized enzymes comprise a solid, porous, inorganic, water insoluble support com-bined with a copolymeric material resulting from the reaction of aminopolystyrene and a bifunctional monomer. The matrix may be prepared by depositing a salt of aminopolystyrene within the pores of the solid support from an aqueous solution at a pH less than 7 after which the resulting composite is then reacted with an excess of a bifunctional reactive monomer thus forming a copolymeric organic material in situ substantially entrapped in the pores of the support and containing functionalized pendent groups to which an enzyme may be coupled to form an immobilized enzyme conjugate.
Description
SUPPORT MATRICES FOR IMMOBILIZED ENZYMES
BACKGROUND OF THE INVENTION
It is known that enzymes, which are proteinaceous in nature and which are commonly water soluble, comprise biological catalysts which serve to regulate many and varied chemical reactions which occur in living organ-isms. The enzymes may also be isolated and used in analytical, medical ; 5 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 in the use of enzymes such as 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 carbohydrases in starch hydrolysis, sucrose inversion, , .
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BACKGROUND OF THE INVENTION
It is known that enzymes, which are proteinaceous in nature and which are commonly water soluble, comprise biological catalysts which serve to regulate many and varied chemical reactions which occur in living organ-isms. The enzymes may also be isolated and used in analytical, medical ; 5 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 in the use of enzymes such as 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 carbohydrases in starch hydrolysis, sucrose inversion, , .
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~Z89~7 glucose isomerization, etc.; the use of nucleases in flavor control; or 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 delin-eated in the literature.
As hereinbefore set forth, inasmuch as enzymes are co~monly water soluble as well as being generally unstable and readily deactivated, they are also difficult either to remove from the solutions in which they are utilized for subsequent reuse or it is difficult to maintain their cata-lytic activity for a relatively extended period of time. The aforementioned difficulties will, of course, lead to an increase cost in the use of en-zymes for commercial purposes due to the necessity for frequent replacement of the enzyme, this replacement being usually necessary with each applica-tion. 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 through various systems hereinafter set forth in greater detail, it is possible to stabilize the enzymes in a relative manner and, therefore, to permit the reuse of the enzyme which may otherwise undergo deactivation or be lost in the reaction medium. Such immobilized or insolubilized enzymes may be employed in various reactor systems such as in packed columns, stirred tank reactors, etc., depending upon the nature of the substrate which is utilized therein. In general, the immobilization of the enzymes provides a more favorable or broader environmental and structural stability, a minimum of effluent problems and materials handling as well as the possibility of upgrading the activity of the enzyme itself.
As hereinbefore set forth, several general methods, as well as many modifications thereof, have been described by which the immobiliza-tion of enzymes may be effected. One general method is to adsorb the
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~Z89~7 glucose isomerization, etc.; the use of nucleases in flavor control; or 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 delin-eated in the literature.
As hereinbefore set forth, inasmuch as enzymes are co~monly water soluble as well as being generally unstable and readily deactivated, they are also difficult either to remove from the solutions in which they are utilized for subsequent reuse or it is difficult to maintain their cata-lytic activity for a relatively extended period of time. The aforementioned difficulties will, of course, lead to an increase cost in the use of en-zymes for commercial purposes due to the necessity for frequent replacement of the enzyme, this replacement being usually necessary with each applica-tion. 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 through various systems hereinafter set forth in greater detail, it is possible to stabilize the enzymes in a relative manner and, therefore, to permit the reuse of the enzyme which may otherwise undergo deactivation or be lost in the reaction medium. Such immobilized or insolubilized enzymes may be employed in various reactor systems such as in packed columns, stirred tank reactors, etc., depending upon the nature of the substrate which is utilized therein. In general, the immobilization of the enzymes provides a more favorable or broader environmental and structural stability, a minimum of effluent problems and materials handling as well as the possibility of upgrading the activity of the enzyme itself.
As hereinbefore set forth, several general methods, as well as many modifications thereof, have been described by which the immobiliza-tion of enzymes may be effected. One general method is to adsorb the
-3-~128917 enzyme at a solid surface as, for example, when an enzyme such as amino acid acylase is adsorbed on a cellulosic derivative such as DEAE-cellulose;
papain or ribonuclease is adsorbed on porous glassi catalase is adsorbed on charcoal; trypsin is adsorbed on quartz glass or cellulose, chymotryp-sin is adsorbed on kaolinite, etc. Another general method is to trap anenzyme in a gel lattice such as glucose oxidase, urease, papain, etc., being entrapped in a polyacrylamide gel; acetyl cholinesterase being entrapped in a starch gel or a silicone polymer; glutamic-pyruvic transaminase being entrapped in a polyamide or cellulose acetate gel, etc. A further general method is a cross-linking by means of bifunctional reagents and may be effected in combination with either of the aforementioned general methods of immobilization. When utilizing this method, bifunctional or polyfunc-tional reagents which may induce intermolecular cross-linking will coval-ently bind the enzymes to each other as well as on a solid support. This method may be exemplified by the use of glutaraldehyde or bisdiazobenzi-dine-~,2'-disulfonic acid to bind an enzyme such as papain on a solid support, etc. A still further method of immobilizing an enzyme comprises ! the method of a covalent binding in which enzymes such as glucoamylase, trypsin, papain, pronase, amylase, glucose oxidase, pepsin, rennin, fungal protease, lactase, etc., are immobilized by covalent attachment to a polymeric material which is attached by various means 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 -A-"
~28917 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, in such cases it is specified that the pore size of the support cannot exceed a diameter of about 1000 Angstroms. In view of this weak bond, the 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 ex-tensively deactivated due to its lack of mobility or due to interaction between the support and the active site of the enzyme. Another process which may be employed is the entrapment of enzymes in gel lattices which can be effected by polymerizing an aqueous solution or emulsion containing the monomeric form of the polymer and the enzyme 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, it also has disadvantages in that the conju-gate 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 sizes which are large enough to permit the loss of enzyme. In addition, some pore sizes may be sufficîently small so 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. The disadvantages which are present when immobilizing an enzyme by intermolecular cross-linkage, as already noted, are due to the lack of mobility with resulting deactivation because .5~
~128917 of inability of the enzyme to assume the natural configuration necessary for maximum activity, particularly when the active site is involved in the binding process.
Covalent binding 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 func-tional groups of the molecule, such as, for example, gamma-aminopropyltri-ethoxysi;lane, 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 non-essential for its catalytic activity such as free amino groups, carboxyl groups, hydroxyl groups, phenolic groups, sulfhydryl groups, etc. These functional groups will also react with a wide variety of other functional groups such as an aldehydo, iso-cyanato, acyl, diazo, azido, anhydro activated ester, etc., to produce co-valent 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 im-mobilizing enzymes which, however, in various ways fail to meet the require-ments of economical industrial use. However, as will hereinafter be discussed 11289~7 in greater detail, none of the prior art compositions comprise the composi-tion of matter of the present invention which constitutes an inorganic porous support containing a copolymer, forn~d in situ from a polyfunctional monomer, a low molecular weight polymer~ a polymer hydrolysate, or a pre-formed polymer, of natural or synthetic origin by reaction with a bifunc-tional monomer, the copolymer formed being substantially entrapped within the pores of said support, and which contains terminally functionalized, pendent groups extending therefrom; the enzyme being covalently bound to the active moieties at the terminal reactive portions of the pendent groups, thus permitting the freedom of movement which will enable the enzyme to exercise maximum activity. A variable portion of the enzyme will also be adsorbed upon the matrix, but this will be recognized as an unavoidable consequence of almost all immobilization procedures involving porous inor-ganic supports and is not to be considered a crucial aspect of this inven-tion. Furthermore, the bond between the inorganic support and the organiccopolymer which has been prepared in situ in the pores of the support is not covalent but rather physico-chemical and mechanical in nature and the inorganic-organic matrix so produced presents high stability and resistance to disruption. As further examples of prior art, 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 binding agent, the enzyme being covalently coupled to a glass carrier by means of an intermediate silane coupling agent, the silicon portion of the coupling agent being attached to the carrier while the organic l~Z8917 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 a water-insoluble polymer such as polyacrolein is deposited - 5 on an inorganic carrier and an enzyme is then covalently linked to the alde-hyde groups of the polymer. However, according to most of the examples set forth in this patent, it is necessary to first hydrolyze the composite - prior to the deposition of the enzyme on the polymer. Additionally the product which is obtained by the method of this patent suffers a number of disadvantages in that it first requires either the deposition, or initially the formation, of the desired polymer in an organic medium followed by its deposition on the inorganic carrier with a subsequent clean-up operation involving distillation to remove the organic medium. In addition to this, in another method set forth in this reference, an additional hydrolytic reaction is required in order to release the aldehyde groups from the initial acetal configuration in which they occurred in the polymer. Inasmuch as these aldehyde moieties are attached directly to the backbone of the polymer, the enzyme is also held adjacent to the surface of the polymer inasmuch as it is separated from the surface of the polymer by only one carbon atom of the reacting aldehyde group and, therefore, the enzyme is obviously sub-~ected to the physico-chemical influences of the polymer as well as being relatively immobilized and inhibited from assuming its optimum configuration.
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 adsorp-tion thereof, the enzyme is further immobilized in part and cannot act freely as in its native state as a catalyst. The cross-linkage of enzymes in effect links them together, thereby preventing a free movement of the enzyme and decreases the mobility of the enzyme which is a necessary pre-requlsite for maximum activity.
U.S. Patent No. 3,654,083 discloses a water-soluble enzyme con-jugate which is prepared from an organic water-soluble support to which the enzyme is cross-linked and whose utility is limited only to cleaning compositions and pharmaceutical ointments. However, this enzyme composition also suffers from the disadvantages of the close proximity and interlocking of the enzyme and support, as well as the poor mechanical strength which is generally exhibited by enzyme conjugates based on organic polymeric supports.
U.S. Patent No. 3,796,634 also discloses an immobilized biologi-cally active enzyme which differs to a considerable degree from the immobi-lized enzyme conjugates of the present invention. The enzyme conjugate of this patent consists of an inorganic support comprising colloidal particles possessing a particle size of from 50 to 20,000 Angstroms with a polyethyl-eneimine, the latter being cross-linked with glutaraldehyde ~o attach the cross-linked polymer so formed as a monolayer on the surface of the colloidal particles, followed by adsorption of the enzyme directly onto this mono-layer. Following this, the enzyme which is adsorbed as a monolayer on the surface of the colloidal particles is then cross-linked with additional glutaraldehyde to other adsorbed enzyme molecules to prevent them from being readily desorbed while in use. There is no indication of any coval-ent binding between enzyme and polymer matrix as is present in the present invention. By the enzyme molecules being cross-linked together on the surface of the support, this conjugate, therefore, is subjected to deac-tivation by both the cross-linking reaction and by the electronic and g_ ~, .
~lZ8917 steric effects of the surface, said enzyme possessing limited mob11ity.
Inasmuch as the product of this patent is colloidal in nature, it also possesses a very limited utility for scale-up to commercial operation, since it cannot be used in a continuous flow system such as a packed column because it would either be carried along and out of the system in the flow-ing liquid stream or, if a restraining membrane should be employed, the particles would soon become packed against the barrier to for~ an imper-vious layer. In addition, such a colloidal product could not readily be utilized in a fluidized bed apparatus, thereby limiting the chief utility to a batch type reactor such as a stirred tank type reactor from which it would have to be separated by centrifugation upon each use cycle. In contrast to this, the immobili~ed enzyme conjugates of the present inven-tion may be employed in a wide variety of batch or continuous type reactors and therefore are much more versatile with regard to their modes of appli-cation.
In addition, another prior art reference United States Patent3,959,080 relates to a carrier matrix for immobili2ing biochemical effec-tive substances. However, the matrix which is produced according to this ; reference constitutes the product derived from the reaction of an organic polymer containing cross-linkable acid hydrazide or acid azide groups with a bifunctional cross-linking agent such as glutaraldehyde. However, this matrix also suffers from the relatively poor mechanical stability and other deficiencies which are characteristic of organic enzyme supports as well as the relatively complex organic reactions employed in preparing such polymeric hydrazides, etc.
As will hereinafter be shown in greater detail, the organic-inorganic matrix of the present invention will provide a support to which llZ8917 an enzyme may be covalently bound to provide a catalytic composite which will maintain its activity and stability over a relatively long period of use.
SPECIFICATION
This invention relates to compositions of matter comprising sup-port matrices for immobilized enzymes. More specifically. the invention is concerned with support matrices consisting of an organic-~norganic com-posite in which the inorganic support material is combined with an organic copolymer prepared in situ and substantially entrapped within the pores of the inorganic support. The copolymer is formed by the reaction of - aminopolystyrene with a sufficient excess of a bifunctional monomer con-taining suitable reactive moieties to provide a copolymeric product con-taining-terminally functionalized pendent groups capable of co~alently binding enzymes at the terminal reactive portions thereof. In~addition, the 1nvention is also concerned with a process for preparing these matrices.
As hereinbefore set forth, the use of enzymes in analytical, medical or industrial applications may be greatly enhanced if said enzymes are in an immobilized condition, that is, said enzymes, by being in combin-ation with other solids 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 co~mercially usable ~n an aqueous insoluble state.
It is therefore an object of this invention to provide novel compositions of matter in which enzymes may be covalently bound in an immobilized state.
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~28917 A further object of this invention is to provide a process for preparing combined inorganic-organic support matrices which are utilized for covalently binding an enzyme to the functionalized pendent groups at the reactive terminal portions thereof.
In one aspect an embodiment of this invention resides in an organic-inorganic matrix comprising a porous, inorganic, water-insoluble solid support in combination with a copolymeric material resulting from the reaction of aminopolystyrene and a bifunctional monomer~
- A further embodiment of this invention is found in a method for preparing an organic-inorganic matrix by depositing a salt of aminopoly-styrene on a sol~d support from an aqueous solution at a pH less than 7 and thereafter reacting the resultant aminopolystyrene-solid support composite with a bifunctional monomer to form the desired organic-inorganic matrix.
A specific embodiment of this invention is found in an organic-inorganic matrix which comprises gamma-alumina having combined therewith a copolymer resulting from the reaction of aminopolystyrene and an excess of glutaraldehyde.
Another specific embodiment of this invention is found in a process for preparing an organic-inorganic matrix which comprises depositing ; 20 the hydrochloric acid salt of aminopolystyrene on gamma-alumina from an aqueous solution at a p~ in the range of from about 1 to about 4, there-after reacting the resultant aminopolystyrene-gamma-alumina composite with an excess of glutaraldehyde, and recovering the resultant organic-inorganic matrix.
' Another specific embodiment of this invention resides in a pro-cess for the preparation of an organic-inorganic matrix which comprises treat-ing a solid porous, inorganic, water-insoluble support with a styrene monomer, polymerizing the styrene-solid supp~rt composite at polymerizing conditions, nitrating the resultant polystyrene-solid support composite, reducing the nitrated polystyrene-solid support composite, and thereafter reacting the resultant aminopolystyrene-solid support composite with a bifunctional monomer to form the desired organic-inorganic matrix.
Other objects and embodiments will be found in the following futher detailed description of the present invention.
As hereinbefore set forth the present invention is concerned with support matrices which are used to immobilize enzymes, said matrices com-prising an organic-inorganic composite consisting of an inorganic support - material of the type hereinafter set forthjn greater detail combined with - 15 a copolymeric organic material which, in the case of a porous support, is substantially entrapped in the pores of said porous support. The copoly-meric composite will contain pendent groups extending therefrom, said pendent groups containing terminally positioned functional moieties which will enable an enzyme to be covalently bound to said group at the reactive terminal portions thereof. Furthermore, the invention is also concerned with a process for preparing these support matrices using relatively inex-pensive reactants as well as util~zing more simple steps in theprocedure for preparing said compositions. In addition, the mechanical strength and stabllity of enzyme conjugates which result from the covalent binding of enzymes to these support matrices will be greater than that which is possessed by the immobilized enzymes of the prior art. Therefore, it will be readily apparent that the compositions of matter of the present inven-tion possesses economical advantages which are useful for inàustrial applications.
llZ8917 Examples of inorganic supports which may be utilized as one component of the support matrices of the present invention will consist of a wide variety of materials including porous supports such as alum;na which possess pore diameters ranging from 100 Angstroms up to 55,000 Angstroms and which also possess an Apparent 8ulk Density (ABD~ in the range of from 0.1 to 0.6. The surface area of the particular inorganic porous support will also vary over a relatively wide range, said range being from 1 to 500 m2/gm, the preferred range of surface area being from 5 to 400 m2/gm. The configuration of the inorganic porous support material will vary, depending upon the particular type of support which is utilized.
For example, the support material may be in spherical form, particulate form ranging from fine particles to macrospheres, as a ceramic monolith which may or may not be coated with a porous inorganic oxide, a me~brane, ceramic fibers, alone or woven into a cloth, silica, mixtures of metallic oxides, sand particles, zeolites or mica. The particle size may also vary over a wide range, again depending upon the particular type of support which is em-ployed and also upon the substrate and the type of installation in which the enzyme conjugate is to be used. For example, if the support is in spherical form, the spheres may range in size from 0.25 to 6.35 mm in diameter, the preferred size ranging from 0.79 to 3.17 mm in diameter.
When the support is in particulate form, the particle size may also range between the same limits. In terms of U.S. standard mesh sizes, such part~cles may range from 2.5 to 100 mesh, with 10-40 mesh sizes preferred.
Likewise, if the support is in the shape of ceramic fibers, the fibers may range from 0.5 to 20 microns in diameter or, if in the form of a membrane, the membrane may comprise a ceramic material which is cast into a thin sheet.
It is to be understood that the aforementioned types of support configuration and size of the various supports are given merely for purposes of illustration, and it is not intended that the present invention be necessarily limited thereto.
l~Z8917 It is also contemplated that the porous support materials may be coated with various oxides of the type hereinbefore set forth, or consists of mixtures thereof, or may have incorporated therein various other inor-ganic materials such as boron phosphate, these inorganic materials impart-ing special properties to the support material. A particularly useful form of support will constitute a ceramic body which may have the type of porostiy herein described for materials of the present invention or it may be honeycombed with connecting macro size channels throughout, such materials being commonly known as monoliths, and which may be coated with various types of porous alumina, zirconia or titanium oxide. The use of such a type of support has the particular advantage of permitting the free flow of highly viscous substrates which are often encountered in commercial enzyme catalyzed reactions. One component of the organic por-tion of the support matrix comprises an aminopolystryene whil~ the other component of the organic portion comprises a bifunctional monomer. ~he bifunctional monomer reactant is present in sufficient excess as needed to produce pendent terminally functionalized groups, said bifunctional - monomer being present in a range of from 2 to 50 moles relative to the reactive moieties of the support composite, the preferred range being from 4 to 25 moles of excess.
The functional groups which are present on the bifunctional mono-mer will comprise well-known reactive moieties capable of bonding readily with amino groups such as carbonyl, acyl or isocyanato, moieties. 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 of the bifunctional compounds are therefore capable of covalently bonding with both the aminopolystyrene component of the support matrix and subsequently, after washing out unreacted llZ8917 materials, also with the amino groups of the enzyme which is to be added in a subsequent step, said enzyme being then covalently bound to the re~
active functional group at the terminal portion of the pendent chain. After addition of the enzyme to this composition, a relativley stable enzyme con-jugate will be produced which possesses high activity and high stability.
The unreacted enzyme can also be recovered for reuse. Due to the large excess of intermediate, or spacer bifunctional monomeric molecules which are used, the matrix will contain pendent groups comprising the spacer molecules, said molecules extending from the matrix and having reactive moieties available at the terminal portions thereof which are capable of reacting with and binding the enzyme to the aforesaid spacer molecules via covalent bonds. Therefore, it is readily apparent that a suitable organic-inorganic matrix which is app1icable in binding enzymes will be formed, provided that a large enough excess of the bifunctional molecule is used to provide reactive pendent - 15 groups which are capable of subsequently reacting with the enzyme to be immob11ized. By utilizing these functional pendent gro~ps as a binding s1te 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 with ad~acent enzyme molecules by means of bifunctional reagents, etc. Not all formulations, however, will produce equivalent results in tenms of stability or activity.
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Examples of enzymes which may be immobilized by a covalent bond-ing reaction and which contain an amino group capable of reacting with an aldehydric, isocyanato, acyl or ester moiety of the pendent group which is attached to a polymeric material substantially entrapped in the pores of li28917 a porous support material will include trypsin, papain, hexokinase, beta-galactosidase (lactase), ficin, bromelain, lactate dehydrogenase, gluco-amylase, chymotrypsin, pronase, glucose, isomerase, acylase, invertase, amylase, glucose oxidase, pepsin, rennin, protease, xylanace and cellulase.
In general any enzyme whose active site is not involved in the coval-ent bonding can be used although not necessarily with equivalent results.
While the aforementioned discussion was centered about pendent groups which contain as a functional moiety thereon an aldehydic or isocyanato group, it is also contemplated within the scope of this invention that the pendent group can contain other functional moieties capable of reaction with car-boxyl, sulfhydryl or other moieties usually present in enzymes. However, the covalent bonding of enzymes containing these other moieties with other pendent 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 inven-tion is not necessarily limited thereto.
The preparation of the compositions of matter of the present invention is preferably effected in a batch type operation. In a pre-ferred method of preparation, the inorganic support material will be treated with a solution, preferably aqueous in nature, of a salt of aminopolysty~ene, the aqueous solution being maintained at a pH less than 7 and preferably in a range of 1 to 4. Examples of salts of aminopolystyrene which may be employed will include the hydrochloric acid salt, the sulfuric acid salt, the nitric acid salt and the phosphoric acid salt of aminopoly-styrene. The pH of the aqueous solution is maintained at the desired level by the addition of an acid such as those hereinbefore set forth.
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~28917 - Upon completion of the addltion of the acid salt of the aminopolystyrene which in a preferred embodiment of the invention is effected at ambient temperature and atmospheric pressure, the mixture is preferably placed under vacuum for a period of time which may range from 0.5 up to 4 hours or more in duration.. Upon completion of the reaction time, the unadsorbed solution is removed and the treatedsupport allowed to air dry. Thereafter the - organic-inorganic compositeiscontacted with a sufficiently ~arge excess of a bifunctional monomer of from 2 to 50 moles proportion relative to the amine content of the initial aminopolystyrene to provide pendent groups extending from the resultant copolymer, said pendent groups containing un-reacted terminal functional moieties. The bifunctional monomer is also preferably added in an~aqueous solution which, after reaction with the aminopolystyrene, is removed and the resultant matrix washed to separate any bifunctional monomer which may still be present. The procedure may be conducted in a temperature range of from 4 to 60C., preferably from 20 to 25C.
In another preferred method of preparation, an emulsion system is prepared consisting of styrene a1Ong with additives such as sodium hy-drogen phosphate or sodium lauryl sulfate in an aqueous solution. The ; 20 solid support which may be in a form hereinbefore set forth in greater '~ detail such as pellets, beads or monoliths, is placed in the solution and adsorption of the emulsion solution is allowed to proceed for a period of time which may range from 0.5 up to 2 hours in duration, said adsorption being preferably effected at subatmospheric pressures, usually in a vaccum.
Upon completion of the adsorption period the mixture is then heated to a temperature which may range from 50 to 100C. for a period of time utiliz-ing a polymerization catalyst such as potassium persulfate. After allowing the polymerization of the styrene to proceed for a period of time which 1~289~7 :`
may range from 2 to 4 hours in duration, the heating is discontinued and the solid, composite support containing the polymerized styrene is recovered from the aqueous solution, washed with distilled water and a solvent such as acetone or benzene to remove any unreacted styrene. Alternatively, the styrene may be polymerized without resorting to an emulsion type of polym-; erization. When this method of preparation is effected the support is added to only the liquid styrene monomer and the styrene allowed ., to adsorb preferably under vacuum for a predetermined period of time.
; At the end of this adsorption period, water and a catalyst such as potassium persulfate is added, the mixture is then heated to the aforesaid temperature range and the polymerization is allowed to proceed for a predetermined period of time. At the end of the polym-erization period, the polystryene-solid support composite is recov-ered in a manner similar to that hereinbefore set forth.
Nitration of the polystyrene-solid support composite is then effected by adding the composite to a nitrating agent such as nitric acid, and preferably 90X fuming nitric acid. The addition is effected at a relatively slow rate while maintaining the nitric acid preferably at subambient temperatures ranging from 0 to 10C. by means of ex-2a ternal cooling means such as an ice bath or cooling coils. However such low temperatures are not critical to the success of the nitration reaction and higher temperatures may be employed, if so desired. The nitration of the polystryene-solid support composite is allowed to proceed for a period of time which may range from 1 to 4 hours or more fol-lowing which the nitrated polystyrene-solid support composite is re-covered and again wahsed with water and a solvent.
Thereafter the nitropolystyrene-solid support composite is then added to an aqueous solution containing a reducing agent such as ~Z89~7 sodium dithionite following which the solution is heated to a tempera-ture in the range of 50 to 1~0C. and maintained thereat for a period - of time which may range from 0.5 to 2 hours. At the end of this period of time, the aminopolystyrene-support matrix is recovered, washed, dried under vacuum and preferably maintained under a nitrogen blanket. To prepare the desired support matrix which will contain pendent groups to which an enzyme may be immobilized, the aminopolystyrene-solid sup-port matrix is then contacted with a sufficiently large excess of bi-functional monomer of from 2 to 50 mole proportions relative to the : 10 amine groups of the aminopolystyrene which will react therewith to provide pendent groups extending from the resulting copolymer which contains unreacted terminal functional moieties. This bifunctional monomer is added preferably in an aqueous solution whereby the co-polymer which is formed will be substantially entrapped in the pores of the inorganic support. The support matrix is then washed with dis-tilled water to remove any unreacted bifunctional monomer.
As hereinbefore set forth, the use of an excess of the bifunc-tional monomer inthe preferred methods of preparation will result in pendent groups extending from the matrix which contain unreacted terminal functional moieties. The unreacted functional moieties are then avail-able for covalent binding to the enzyme, which is added to the matrix, again usually in an aqueous solution. After removal of the unreacted materials by conventional means such as washing, the enzyme which is covalently bound to the pendent functionalized groups rema~ns attached at the terminal portions thereof. It is therefore readily apparent - that the entire immobilized procedure can be conducted in a simple and ~nexpensive manner, for example, in a column packed with the inorganic supports, utilizing an aqueous or inexpensive solvent media. The pro-~Z8917 cedure is effected by utilizing a minimal of operating steps and, in addi-tion, permitting a ready recovery of the excess reactants, unbound enzyme and finished composition of matter, the excess reactants and unbound enzymes being available for reuse thereof.
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. One method which may be employed is ~- effected by placing a quantity of the solid support in an appropriate apparatus usually consitituting a column. As in the case of the batch type operation, the solid support material may be in any form desired such as powder, pellets or monoliths, and is placed in the column after which a preferably aqueous solution of a salt of aminopolystyrene is also charged and contacts the support until the latter is saturated with the solution.
The aqueous solution is maintained at a pH less than 7 and preferably at a pH in a range of from 1 to 4 by the addition of an appropriate acid.
After saturation of the support has been accomplished an excess is then drained and an intermediary spacer compound such as a reactive bifunctional monomer molecule, preferably in aqueous solution, is charged to the column, said bifunctional molecule being present in an excess in a range of from 2 to 50 moles relative to the amine content of the aminopolystyrene.
While the formation of the matrix is effected during a period of time which may range from 1 to 22 hours in duration! the formation is usually accom-plished during a relatively short period of time. Following the com-pletion of the desired resldence time the excess spacer reactant such as glutaraldehyde is removed by draining followed by a thorough water washing to remove any unreacted materials.
To form an immobilized enzyme conjugate, an aqueous solution of an enzyme of the type hereinbefore set forth in greater detail is then , ' ' ~28917 passed through the column containing the thus formed support matrix thereby effecting a covalent bonding of the enzyme to the terminal reactive groups of the functionalized pendent moieties which extend from the matrix. This occurs until there is no further covalent binding of the enzyme to the S pendent molecules. The excess enzyme is recovered in the effluent which is continuously withdrawn after draining and may be recycled to the column for further use. After washing the column, the column is then ready for use in chemical reactions in which the catalytic effect of the enzyme is to take place. These procedures are conducted within the time, temperature and concentration parameters hereinbefore set forth described in the batch type procedure and will result in comparable immobilized enzyme complexes.
It is also contemplated with~n the scope of this invention that with suit-able modifictions of reaction parameters it will be obvious to those skilled in the~art that the process may be applied to a wide variety of supports, bifunctional monomers and enzymes.
~ The following examples are given for purposes of illustrating the novel compositions of matter of the present invention and to the methods for preparing the same. However, it is to be understood that these examples llZ8917 are given merely for purposes of illustration and that the present inven-tion is not necessarily limited thereto.
EXAMPLE I
In this example 1 gram of a porous alumina base having a particle size of from 25 to 40 mesh, an Apparent Bulk Density (ABD) of 0.34 and pore size ranging from 200 Angstroms to 10,000 Angstroms was admixed with i 10 ml of a 5X weight by volume of aminopolystyrene having a molecular weight of 22,000 dissolved in aqueous 0.1 M hydrochloric acid. The two components were mixed at room temperature and allowed to stand for a period of 1 hour.
At the end of this time the solution was degassed, filtered and the solid support containing the aminopolystyrene adsorbed thereon was dried. Follow-ing this, the composite was mixed with 10 ml of a 1.5X aqueous solution of glutaraldehyde whlch had a pH of 1.4 and maintained for a period of 1 hour at room temperature. At the end of this 1 hour period the excess glutar-aldehyde was decanted and the organic-inorganic matrix was thoroughly washed with water several t~mes. The final immobilized conjugate was then pre-pared by treating the matrix with 6482 units of a commercial glucoamylase ~ Jradehl~rJc t~ sold under the Tradcnamc "Ambazyme". The immobilization of the enzyme was effected during a period of 16 hours while maintaining the temperature of the composite at 4C. by means of an ice bath. At the end of the 16 hour period the residual and unbound enzyme was washed out with water and a sodium chloride solution.
The immob~lized enzyme conjugate was packed in a column and a 30% weight by volume solution of starch sold under the commcrcial Trade-1ta~e~ "Maltrin-150" was passed over the beads while maintaining the tempera-ture at 60C., said starch feed being passed over the beads for a period of 2 hours at a flow rate of 2 ml/min. At the end of the 2 hour period, .
1~28917 the amount of glucose formed was assayed. It was found that the activity of the enzyme conjugate was 3240 units/gram at the flow rate of 2 ml/min.i the unit being defined as the micromoles of glucose formed per minute per gram of immobilized enzyme conjugate.
S EXAMPLE II
The above experiment was repeated with the exception that the enzyme, namely, glucoamylase was purified by means of an isopropanol pre-cipitation procedure well known in the art, resulting in a 1.3 fold increase of purity of the enzyme~ When this purified enzyme was immobilized in a manner similar to that set forth above and utilized to convert starch to glucose, it was found that the immobilized enzyme coniugate had an activity of 4070 units/gram at a flow rate of 2 ml/min.
EXAMPLE III
In a manner similar to that set forth in Example I above, 1 gram of an alumina base having a particle size of from 25-35 mesh and an ABD of 0.3 was added to 10 ml of a solution comprising 5X by volume of aminopoly-styrene dissolved in aqueous 0.1 M hydrochloric acid. The mixture was maintained at room temperature for a period of 1 hour after which it was degassed, filtered and the aminopolystyrene-alumina matrix was dried. The dried beads were then added to 10 ml of a 1.5% aqueous solution of glutar-aldehyde which had a pH of 1.4. The mixture was held for a period of 1 hour at room temperature and thereafter the excess glutaraldehyde was decanted.
The organic-inorganic matrix was washed several times with water and thereafter was treated with 1300 units of glucose isomerase which had a specific activity of 8.5 units/mg of protein. The coupling was effected at a temperature of 4C. for a period of 22 hours. The resulting immobilized glucose isomerase conjugate in which the enzyme was covalently bound to the free aldehyde functions of the copolymeric material which arose from the use of excess glutaraldehyde, was washed thoroughly with water and a 2 M
aqueous sodium chloride solution to remove any unreacted enzyme.
The immobilized enzyme conjugate was packed in a suitable column and a 45X solution of fructose which possessed a pH of 8 and contained 5 x 10 3 molar magnesium chloride was passed through the conjugate bed at a temperature of 60C. for a period of 2 hours. The glucose and flow rate were assayed and it was found that this immobilized enzyme conjugate had an activity of 700 units/gram at a flow rate of 2 ml/min., with a coupling efficiency of 60~. It is further determined that the enzyme con-jugate possessed a half-life of 22 days at 60- in a continuous column operation using the flow rate of 2 ml/min. of feed over the conjugate.
EXAMPLE IV
- In this example an alumina base having a particle size of from 60-80 mesh and an ABD of 0.3 was treated with aminopolystyrene and an excess of glutaraldehyde in a manner similar to that set forth above to form an organic-inorganic support matrix. This support matrix was treated with glucose isomerase which possessed a specific activity of 15 units/mg. The coupling was effected by offering 2800 units of enzyme to the support matrix at a temperature of 4~. for a period of 22 hours. After thorough washing of the immobilized enzyme conjugate, lt was packed in a column and a fructose feed similar to that described in Example III above was passed through the conjugate bed for a period of 2 hours at a temperature of 60-C.
The product was assayed and it was found that the immobilized enzyme conju-gate had an activity of 1200 units/gram with a coupling efficiency of 54g.
' 11289~7 EXAMPLE ~
In this example 1 gram of porous alumina pellets, 10 mesh, having an apparent bulk density (ABD) of 0.344 and pore si~es rang-; ing from 200 Angstroms to 10,000 Angstroms were placed in an emul-sion prepared from 0.5 grams of sodium hydrogen phosphate, 0.5 grams of sodium lauryl sulfate, 2 ml of styrene and 20 ml of distilled wa-ter. The peltets were allowed to adsorb in this emulsion under vacuum for a period of 1 hour following which the mixture was then - heated for a period of 3 hours at a temperature of 100C. in the presence of a polymerization catalyst comprising 0.5 grams of potas-sium persulfate. At the end of the 3 hour period, the beads were filtered to remove liquid, washed with a distilled water and ! ml of acetone. Thereafter 1 gram of the polystyrene-alumina support was~slowly added to 50 ml of 90X fuming nitric acid. The addition of the~support to the acid was effected at a temperature of about 0C. to 10C., the temperature being attained by placing the appara-tus in an ice bath. After a period of 2 hours the nitrated support was removed, washed with water and acetone. To effect the reduction of the nitrated polystyrene-alumina support, 1 gram of the composite along with 1 gram of sodium dithionite was added to 100 ml of dis-tilled water. The mixture was then heated to boiling and maintained at this temperature for a period of 1 hour. Following this the i~
aminopolystyrene-alumina composite support was washed with distilled water, dried under vacuum and maintained under a nitrogen atmosphere.
The aminopolystyrene-alumina support prepared according to the above paragraph was then contacted with 10 ml of a 50% aqueous solution of glutaraldehyde which had a pH of 3.5 for a period of 1 hour at room temperature. At the end of the 1 hour period the excess `` ~
1~28917 glutaraldehyde was decanted and the organic-inorganic matrlx was thoroughly washed with water several times in order to effectively remove unreacted reagents. The final immobilized enzyme conjugate was then prepared by treating the matrix with 8300 units of a com-~ r~a de ~
5 ~ mercial glucose amylase sold under the tr~dena~ Novo PPAG-12, said immobilization of the enzyme being effected during a period of 16 hours while maintaining the temperature of the mixture at about 4C.
by means of an ice bath. At the end of the 16 hour period the re-sidual and unbound enzyme was washed out with water and a sodium chloride solution.
The immobilized enzyme conjugate was then packed in a col-~ umn ànd a 30% weight by volume solution of starch, 6omncrQial Trade-s~ r~
~u*e~ Maltrin-150, was passed over the beads at a flow rate of 2 ml/
min. while maintaining the temperature at 60C. for a period of 2 hours. At the end of the 2 hour period the amount of glucose formed was assayed. It was found that the activity of the enzyme conjugate was 695 units/gram at the flow rate of 2 ml/minute; the term "unit"
being defined as the micromoles of glucose formed per minute per gram of immobllized enzyme coniugate.
- EXAMPLEVI
Experiment V was repeated with the exception that the enzyme was purified by means of an isopropanol precipitation pro-cedure well known to those skilled in the art. When the purified en-zyme was immobilized in a manner simiiar to that set forth above and utilized to convert starch to glucose, it was found that the immobil-ized enzyme coniugate had an activity of 852 units/gram at a flow rate of 2 ml/min.
,,
papain or ribonuclease is adsorbed on porous glassi catalase is adsorbed on charcoal; trypsin is adsorbed on quartz glass or cellulose, chymotryp-sin is adsorbed on kaolinite, etc. Another general method is to trap anenzyme in a gel lattice such as glucose oxidase, urease, papain, etc., being entrapped in a polyacrylamide gel; acetyl cholinesterase being entrapped in a starch gel or a silicone polymer; glutamic-pyruvic transaminase being entrapped in a polyamide or cellulose acetate gel, etc. A further general method is a cross-linking by means of bifunctional reagents and may be effected in combination with either of the aforementioned general methods of immobilization. When utilizing this method, bifunctional or polyfunc-tional reagents which may induce intermolecular cross-linking will coval-ently bind the enzymes to each other as well as on a solid support. This method may be exemplified by the use of glutaraldehyde or bisdiazobenzi-dine-~,2'-disulfonic acid to bind an enzyme such as papain on a solid support, etc. A still further method of immobilizing an enzyme comprises ! the method of a covalent binding in which enzymes such as glucoamylase, trypsin, papain, pronase, amylase, glucose oxidase, pepsin, rennin, fungal protease, lactase, etc., are immobilized by covalent attachment to a polymeric material which is attached by various means 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 -A-"
~28917 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, in such cases it is specified that the pore size of the support cannot exceed a diameter of about 1000 Angstroms. In view of this weak bond, the 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 ex-tensively deactivated due to its lack of mobility or due to interaction between the support and the active site of the enzyme. Another process which may be employed is the entrapment of enzymes in gel lattices which can be effected by polymerizing an aqueous solution or emulsion containing the monomeric form of the polymer and the enzyme 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, it also has disadvantages in that the conju-gate 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 sizes which are large enough to permit the loss of enzyme. In addition, some pore sizes may be sufficîently small so 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. The disadvantages which are present when immobilizing an enzyme by intermolecular cross-linkage, as already noted, are due to the lack of mobility with resulting deactivation because .5~
~128917 of inability of the enzyme to assume the natural configuration necessary for maximum activity, particularly when the active site is involved in the binding process.
Covalent binding 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 func-tional groups of the molecule, such as, for example, gamma-aminopropyltri-ethoxysi;lane, 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 non-essential for its catalytic activity such as free amino groups, carboxyl groups, hydroxyl groups, phenolic groups, sulfhydryl groups, etc. These functional groups will also react with a wide variety of other functional groups such as an aldehydo, iso-cyanato, acyl, diazo, azido, anhydro activated ester, etc., to produce co-valent 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 im-mobilizing enzymes which, however, in various ways fail to meet the require-ments of economical industrial use. However, as will hereinafter be discussed 11289~7 in greater detail, none of the prior art compositions comprise the composi-tion of matter of the present invention which constitutes an inorganic porous support containing a copolymer, forn~d in situ from a polyfunctional monomer, a low molecular weight polymer~ a polymer hydrolysate, or a pre-formed polymer, of natural or synthetic origin by reaction with a bifunc-tional monomer, the copolymer formed being substantially entrapped within the pores of said support, and which contains terminally functionalized, pendent groups extending therefrom; the enzyme being covalently bound to the active moieties at the terminal reactive portions of the pendent groups, thus permitting the freedom of movement which will enable the enzyme to exercise maximum activity. A variable portion of the enzyme will also be adsorbed upon the matrix, but this will be recognized as an unavoidable consequence of almost all immobilization procedures involving porous inor-ganic supports and is not to be considered a crucial aspect of this inven-tion. Furthermore, the bond between the inorganic support and the organiccopolymer which has been prepared in situ in the pores of the support is not covalent but rather physico-chemical and mechanical in nature and the inorganic-organic matrix so produced presents high stability and resistance to disruption. As further examples of prior art, 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 binding agent, the enzyme being covalently coupled to a glass carrier by means of an intermediate silane coupling agent, the silicon portion of the coupling agent being attached to the carrier while the organic l~Z8917 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 a water-insoluble polymer such as polyacrolein is deposited - 5 on an inorganic carrier and an enzyme is then covalently linked to the alde-hyde groups of the polymer. However, according to most of the examples set forth in this patent, it is necessary to first hydrolyze the composite - prior to the deposition of the enzyme on the polymer. Additionally the product which is obtained by the method of this patent suffers a number of disadvantages in that it first requires either the deposition, or initially the formation, of the desired polymer in an organic medium followed by its deposition on the inorganic carrier with a subsequent clean-up operation involving distillation to remove the organic medium. In addition to this, in another method set forth in this reference, an additional hydrolytic reaction is required in order to release the aldehyde groups from the initial acetal configuration in which they occurred in the polymer. Inasmuch as these aldehyde moieties are attached directly to the backbone of the polymer, the enzyme is also held adjacent to the surface of the polymer inasmuch as it is separated from the surface of the polymer by only one carbon atom of the reacting aldehyde group and, therefore, the enzyme is obviously sub-~ected to the physico-chemical influences of the polymer as well as being relatively immobilized and inhibited from assuming its optimum configuration.
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 adsorp-tion thereof, the enzyme is further immobilized in part and cannot act freely as in its native state as a catalyst. The cross-linkage of enzymes in effect links them together, thereby preventing a free movement of the enzyme and decreases the mobility of the enzyme which is a necessary pre-requlsite for maximum activity.
U.S. Patent No. 3,654,083 discloses a water-soluble enzyme con-jugate which is prepared from an organic water-soluble support to which the enzyme is cross-linked and whose utility is limited only to cleaning compositions and pharmaceutical ointments. However, this enzyme composition also suffers from the disadvantages of the close proximity and interlocking of the enzyme and support, as well as the poor mechanical strength which is generally exhibited by enzyme conjugates based on organic polymeric supports.
U.S. Patent No. 3,796,634 also discloses an immobilized biologi-cally active enzyme which differs to a considerable degree from the immobi-lized enzyme conjugates of the present invention. The enzyme conjugate of this patent consists of an inorganic support comprising colloidal particles possessing a particle size of from 50 to 20,000 Angstroms with a polyethyl-eneimine, the latter being cross-linked with glutaraldehyde ~o attach the cross-linked polymer so formed as a monolayer on the surface of the colloidal particles, followed by adsorption of the enzyme directly onto this mono-layer. Following this, the enzyme which is adsorbed as a monolayer on the surface of the colloidal particles is then cross-linked with additional glutaraldehyde to other adsorbed enzyme molecules to prevent them from being readily desorbed while in use. There is no indication of any coval-ent binding between enzyme and polymer matrix as is present in the present invention. By the enzyme molecules being cross-linked together on the surface of the support, this conjugate, therefore, is subjected to deac-tivation by both the cross-linking reaction and by the electronic and g_ ~, .
~lZ8917 steric effects of the surface, said enzyme possessing limited mob11ity.
Inasmuch as the product of this patent is colloidal in nature, it also possesses a very limited utility for scale-up to commercial operation, since it cannot be used in a continuous flow system such as a packed column because it would either be carried along and out of the system in the flow-ing liquid stream or, if a restraining membrane should be employed, the particles would soon become packed against the barrier to for~ an imper-vious layer. In addition, such a colloidal product could not readily be utilized in a fluidized bed apparatus, thereby limiting the chief utility to a batch type reactor such as a stirred tank type reactor from which it would have to be separated by centrifugation upon each use cycle. In contrast to this, the immobili~ed enzyme conjugates of the present inven-tion may be employed in a wide variety of batch or continuous type reactors and therefore are much more versatile with regard to their modes of appli-cation.
In addition, another prior art reference United States Patent3,959,080 relates to a carrier matrix for immobili2ing biochemical effec-tive substances. However, the matrix which is produced according to this ; reference constitutes the product derived from the reaction of an organic polymer containing cross-linkable acid hydrazide or acid azide groups with a bifunctional cross-linking agent such as glutaraldehyde. However, this matrix also suffers from the relatively poor mechanical stability and other deficiencies which are characteristic of organic enzyme supports as well as the relatively complex organic reactions employed in preparing such polymeric hydrazides, etc.
As will hereinafter be shown in greater detail, the organic-inorganic matrix of the present invention will provide a support to which llZ8917 an enzyme may be covalently bound to provide a catalytic composite which will maintain its activity and stability over a relatively long period of use.
SPECIFICATION
This invention relates to compositions of matter comprising sup-port matrices for immobilized enzymes. More specifically. the invention is concerned with support matrices consisting of an organic-~norganic com-posite in which the inorganic support material is combined with an organic copolymer prepared in situ and substantially entrapped within the pores of the inorganic support. The copolymer is formed by the reaction of - aminopolystyrene with a sufficient excess of a bifunctional monomer con-taining suitable reactive moieties to provide a copolymeric product con-taining-terminally functionalized pendent groups capable of co~alently binding enzymes at the terminal reactive portions thereof. In~addition, the 1nvention is also concerned with a process for preparing these matrices.
As hereinbefore set forth, the use of enzymes in analytical, medical or industrial applications may be greatly enhanced if said enzymes are in an immobilized condition, that is, said enzymes, by being in combin-ation with other solids 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 co~mercially usable ~n an aqueous insoluble state.
It is therefore an object of this invention to provide novel compositions of matter in which enzymes may be covalently bound in an immobilized state.
'~' }, ,. . .
,~ :
~28917 A further object of this invention is to provide a process for preparing combined inorganic-organic support matrices which are utilized for covalently binding an enzyme to the functionalized pendent groups at the reactive terminal portions thereof.
In one aspect an embodiment of this invention resides in an organic-inorganic matrix comprising a porous, inorganic, water-insoluble solid support in combination with a copolymeric material resulting from the reaction of aminopolystyrene and a bifunctional monomer~
- A further embodiment of this invention is found in a method for preparing an organic-inorganic matrix by depositing a salt of aminopoly-styrene on a sol~d support from an aqueous solution at a pH less than 7 and thereafter reacting the resultant aminopolystyrene-solid support composite with a bifunctional monomer to form the desired organic-inorganic matrix.
A specific embodiment of this invention is found in an organic-inorganic matrix which comprises gamma-alumina having combined therewith a copolymer resulting from the reaction of aminopolystyrene and an excess of glutaraldehyde.
Another specific embodiment of this invention is found in a process for preparing an organic-inorganic matrix which comprises depositing ; 20 the hydrochloric acid salt of aminopolystyrene on gamma-alumina from an aqueous solution at a p~ in the range of from about 1 to about 4, there-after reacting the resultant aminopolystyrene-gamma-alumina composite with an excess of glutaraldehyde, and recovering the resultant organic-inorganic matrix.
' Another specific embodiment of this invention resides in a pro-cess for the preparation of an organic-inorganic matrix which comprises treat-ing a solid porous, inorganic, water-insoluble support with a styrene monomer, polymerizing the styrene-solid supp~rt composite at polymerizing conditions, nitrating the resultant polystyrene-solid support composite, reducing the nitrated polystyrene-solid support composite, and thereafter reacting the resultant aminopolystyrene-solid support composite with a bifunctional monomer to form the desired organic-inorganic matrix.
Other objects and embodiments will be found in the following futher detailed description of the present invention.
As hereinbefore set forth the present invention is concerned with support matrices which are used to immobilize enzymes, said matrices com-prising an organic-inorganic composite consisting of an inorganic support - material of the type hereinafter set forthjn greater detail combined with - 15 a copolymeric organic material which, in the case of a porous support, is substantially entrapped in the pores of said porous support. The copoly-meric composite will contain pendent groups extending therefrom, said pendent groups containing terminally positioned functional moieties which will enable an enzyme to be covalently bound to said group at the reactive terminal portions thereof. Furthermore, the invention is also concerned with a process for preparing these support matrices using relatively inex-pensive reactants as well as util~zing more simple steps in theprocedure for preparing said compositions. In addition, the mechanical strength and stabllity of enzyme conjugates which result from the covalent binding of enzymes to these support matrices will be greater than that which is possessed by the immobilized enzymes of the prior art. Therefore, it will be readily apparent that the compositions of matter of the present inven-tion possesses economical advantages which are useful for inàustrial applications.
llZ8917 Examples of inorganic supports which may be utilized as one component of the support matrices of the present invention will consist of a wide variety of materials including porous supports such as alum;na which possess pore diameters ranging from 100 Angstroms up to 55,000 Angstroms and which also possess an Apparent 8ulk Density (ABD~ in the range of from 0.1 to 0.6. The surface area of the particular inorganic porous support will also vary over a relatively wide range, said range being from 1 to 500 m2/gm, the preferred range of surface area being from 5 to 400 m2/gm. The configuration of the inorganic porous support material will vary, depending upon the particular type of support which is utilized.
For example, the support material may be in spherical form, particulate form ranging from fine particles to macrospheres, as a ceramic monolith which may or may not be coated with a porous inorganic oxide, a me~brane, ceramic fibers, alone or woven into a cloth, silica, mixtures of metallic oxides, sand particles, zeolites or mica. The particle size may also vary over a wide range, again depending upon the particular type of support which is em-ployed and also upon the substrate and the type of installation in which the enzyme conjugate is to be used. For example, if the support is in spherical form, the spheres may range in size from 0.25 to 6.35 mm in diameter, the preferred size ranging from 0.79 to 3.17 mm in diameter.
When the support is in particulate form, the particle size may also range between the same limits. In terms of U.S. standard mesh sizes, such part~cles may range from 2.5 to 100 mesh, with 10-40 mesh sizes preferred.
Likewise, if the support is in the shape of ceramic fibers, the fibers may range from 0.5 to 20 microns in diameter or, if in the form of a membrane, the membrane may comprise a ceramic material which is cast into a thin sheet.
It is to be understood that the aforementioned types of support configuration and size of the various supports are given merely for purposes of illustration, and it is not intended that the present invention be necessarily limited thereto.
l~Z8917 It is also contemplated that the porous support materials may be coated with various oxides of the type hereinbefore set forth, or consists of mixtures thereof, or may have incorporated therein various other inor-ganic materials such as boron phosphate, these inorganic materials impart-ing special properties to the support material. A particularly useful form of support will constitute a ceramic body which may have the type of porostiy herein described for materials of the present invention or it may be honeycombed with connecting macro size channels throughout, such materials being commonly known as monoliths, and which may be coated with various types of porous alumina, zirconia or titanium oxide. The use of such a type of support has the particular advantage of permitting the free flow of highly viscous substrates which are often encountered in commercial enzyme catalyzed reactions. One component of the organic por-tion of the support matrix comprises an aminopolystryene whil~ the other component of the organic portion comprises a bifunctional monomer. ~he bifunctional monomer reactant is present in sufficient excess as needed to produce pendent terminally functionalized groups, said bifunctional - monomer being present in a range of from 2 to 50 moles relative to the reactive moieties of the support composite, the preferred range being from 4 to 25 moles of excess.
The functional groups which are present on the bifunctional mono-mer will comprise well-known reactive moieties capable of bonding readily with amino groups such as carbonyl, acyl or isocyanato, moieties. 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 of the bifunctional compounds are therefore capable of covalently bonding with both the aminopolystyrene component of the support matrix and subsequently, after washing out unreacted llZ8917 materials, also with the amino groups of the enzyme which is to be added in a subsequent step, said enzyme being then covalently bound to the re~
active functional group at the terminal portion of the pendent chain. After addition of the enzyme to this composition, a relativley stable enzyme con-jugate will be produced which possesses high activity and high stability.
The unreacted enzyme can also be recovered for reuse. Due to the large excess of intermediate, or spacer bifunctional monomeric molecules which are used, the matrix will contain pendent groups comprising the spacer molecules, said molecules extending from the matrix and having reactive moieties available at the terminal portions thereof which are capable of reacting with and binding the enzyme to the aforesaid spacer molecules via covalent bonds. Therefore, it is readily apparent that a suitable organic-inorganic matrix which is app1icable in binding enzymes will be formed, provided that a large enough excess of the bifunctional molecule is used to provide reactive pendent - 15 groups which are capable of subsequently reacting with the enzyme to be immob11ized. By utilizing these functional pendent gro~ps as a binding s1te 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 with ad~acent enzyme molecules by means of bifunctional reagents, etc. Not all formulations, however, will produce equivalent results in tenms of stability or activity.
, ~
Examples of enzymes which may be immobilized by a covalent bond-ing reaction and which contain an amino group capable of reacting with an aldehydric, isocyanato, acyl or ester moiety of the pendent group which is attached to a polymeric material substantially entrapped in the pores of li28917 a porous support material will include trypsin, papain, hexokinase, beta-galactosidase (lactase), ficin, bromelain, lactate dehydrogenase, gluco-amylase, chymotrypsin, pronase, glucose, isomerase, acylase, invertase, amylase, glucose oxidase, pepsin, rennin, protease, xylanace and cellulase.
In general any enzyme whose active site is not involved in the coval-ent bonding can be used although not necessarily with equivalent results.
While the aforementioned discussion was centered about pendent groups which contain as a functional moiety thereon an aldehydic or isocyanato group, it is also contemplated within the scope of this invention that the pendent group can contain other functional moieties capable of reaction with car-boxyl, sulfhydryl or other moieties usually present in enzymes. However, the covalent bonding of enzymes containing these other moieties with other pendent 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 inven-tion is not necessarily limited thereto.
The preparation of the compositions of matter of the present invention is preferably effected in a batch type operation. In a pre-ferred method of preparation, the inorganic support material will be treated with a solution, preferably aqueous in nature, of a salt of aminopolysty~ene, the aqueous solution being maintained at a pH less than 7 and preferably in a range of 1 to 4. Examples of salts of aminopolystyrene which may be employed will include the hydrochloric acid salt, the sulfuric acid salt, the nitric acid salt and the phosphoric acid salt of aminopoly-styrene. The pH of the aqueous solution is maintained at the desired level by the addition of an acid such as those hereinbefore set forth.
.
~28917 - Upon completion of the addltion of the acid salt of the aminopolystyrene which in a preferred embodiment of the invention is effected at ambient temperature and atmospheric pressure, the mixture is preferably placed under vacuum for a period of time which may range from 0.5 up to 4 hours or more in duration.. Upon completion of the reaction time, the unadsorbed solution is removed and the treatedsupport allowed to air dry. Thereafter the - organic-inorganic compositeiscontacted with a sufficiently ~arge excess of a bifunctional monomer of from 2 to 50 moles proportion relative to the amine content of the initial aminopolystyrene to provide pendent groups extending from the resultant copolymer, said pendent groups containing un-reacted terminal functional moieties. The bifunctional monomer is also preferably added in an~aqueous solution which, after reaction with the aminopolystyrene, is removed and the resultant matrix washed to separate any bifunctional monomer which may still be present. The procedure may be conducted in a temperature range of from 4 to 60C., preferably from 20 to 25C.
In another preferred method of preparation, an emulsion system is prepared consisting of styrene a1Ong with additives such as sodium hy-drogen phosphate or sodium lauryl sulfate in an aqueous solution. The ; 20 solid support which may be in a form hereinbefore set forth in greater '~ detail such as pellets, beads or monoliths, is placed in the solution and adsorption of the emulsion solution is allowed to proceed for a period of time which may range from 0.5 up to 2 hours in duration, said adsorption being preferably effected at subatmospheric pressures, usually in a vaccum.
Upon completion of the adsorption period the mixture is then heated to a temperature which may range from 50 to 100C. for a period of time utiliz-ing a polymerization catalyst such as potassium persulfate. After allowing the polymerization of the styrene to proceed for a period of time which 1~289~7 :`
may range from 2 to 4 hours in duration, the heating is discontinued and the solid, composite support containing the polymerized styrene is recovered from the aqueous solution, washed with distilled water and a solvent such as acetone or benzene to remove any unreacted styrene. Alternatively, the styrene may be polymerized without resorting to an emulsion type of polym-; erization. When this method of preparation is effected the support is added to only the liquid styrene monomer and the styrene allowed ., to adsorb preferably under vacuum for a predetermined period of time.
; At the end of this adsorption period, water and a catalyst such as potassium persulfate is added, the mixture is then heated to the aforesaid temperature range and the polymerization is allowed to proceed for a predetermined period of time. At the end of the polym-erization period, the polystryene-solid support composite is recov-ered in a manner similar to that hereinbefore set forth.
Nitration of the polystyrene-solid support composite is then effected by adding the composite to a nitrating agent such as nitric acid, and preferably 90X fuming nitric acid. The addition is effected at a relatively slow rate while maintaining the nitric acid preferably at subambient temperatures ranging from 0 to 10C. by means of ex-2a ternal cooling means such as an ice bath or cooling coils. However such low temperatures are not critical to the success of the nitration reaction and higher temperatures may be employed, if so desired. The nitration of the polystryene-solid support composite is allowed to proceed for a period of time which may range from 1 to 4 hours or more fol-lowing which the nitrated polystyrene-solid support composite is re-covered and again wahsed with water and a solvent.
Thereafter the nitropolystyrene-solid support composite is then added to an aqueous solution containing a reducing agent such as ~Z89~7 sodium dithionite following which the solution is heated to a tempera-ture in the range of 50 to 1~0C. and maintained thereat for a period - of time which may range from 0.5 to 2 hours. At the end of this period of time, the aminopolystyrene-support matrix is recovered, washed, dried under vacuum and preferably maintained under a nitrogen blanket. To prepare the desired support matrix which will contain pendent groups to which an enzyme may be immobilized, the aminopolystyrene-solid sup-port matrix is then contacted with a sufficiently large excess of bi-functional monomer of from 2 to 50 mole proportions relative to the : 10 amine groups of the aminopolystyrene which will react therewith to provide pendent groups extending from the resulting copolymer which contains unreacted terminal functional moieties. This bifunctional monomer is added preferably in an aqueous solution whereby the co-polymer which is formed will be substantially entrapped in the pores of the inorganic support. The support matrix is then washed with dis-tilled water to remove any unreacted bifunctional monomer.
As hereinbefore set forth, the use of an excess of the bifunc-tional monomer inthe preferred methods of preparation will result in pendent groups extending from the matrix which contain unreacted terminal functional moieties. The unreacted functional moieties are then avail-able for covalent binding to the enzyme, which is added to the matrix, again usually in an aqueous solution. After removal of the unreacted materials by conventional means such as washing, the enzyme which is covalently bound to the pendent functionalized groups rema~ns attached at the terminal portions thereof. It is therefore readily apparent - that the entire immobilized procedure can be conducted in a simple and ~nexpensive manner, for example, in a column packed with the inorganic supports, utilizing an aqueous or inexpensive solvent media. The pro-~Z8917 cedure is effected by utilizing a minimal of operating steps and, in addi-tion, permitting a ready recovery of the excess reactants, unbound enzyme and finished composition of matter, the excess reactants and unbound enzymes being available for reuse thereof.
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. One method which may be employed is ~- effected by placing a quantity of the solid support in an appropriate apparatus usually consitituting a column. As in the case of the batch type operation, the solid support material may be in any form desired such as powder, pellets or monoliths, and is placed in the column after which a preferably aqueous solution of a salt of aminopolystyrene is also charged and contacts the support until the latter is saturated with the solution.
The aqueous solution is maintained at a pH less than 7 and preferably at a pH in a range of from 1 to 4 by the addition of an appropriate acid.
After saturation of the support has been accomplished an excess is then drained and an intermediary spacer compound such as a reactive bifunctional monomer molecule, preferably in aqueous solution, is charged to the column, said bifunctional molecule being present in an excess in a range of from 2 to 50 moles relative to the amine content of the aminopolystyrene.
While the formation of the matrix is effected during a period of time which may range from 1 to 22 hours in duration! the formation is usually accom-plished during a relatively short period of time. Following the com-pletion of the desired resldence time the excess spacer reactant such as glutaraldehyde is removed by draining followed by a thorough water washing to remove any unreacted materials.
To form an immobilized enzyme conjugate, an aqueous solution of an enzyme of the type hereinbefore set forth in greater detail is then , ' ' ~28917 passed through the column containing the thus formed support matrix thereby effecting a covalent bonding of the enzyme to the terminal reactive groups of the functionalized pendent moieties which extend from the matrix. This occurs until there is no further covalent binding of the enzyme to the S pendent molecules. The excess enzyme is recovered in the effluent which is continuously withdrawn after draining and may be recycled to the column for further use. After washing the column, the column is then ready for use in chemical reactions in which the catalytic effect of the enzyme is to take place. These procedures are conducted within the time, temperature and concentration parameters hereinbefore set forth described in the batch type procedure and will result in comparable immobilized enzyme complexes.
It is also contemplated with~n the scope of this invention that with suit-able modifictions of reaction parameters it will be obvious to those skilled in the~art that the process may be applied to a wide variety of supports, bifunctional monomers and enzymes.
~ The following examples are given for purposes of illustrating the novel compositions of matter of the present invention and to the methods for preparing the same. However, it is to be understood that these examples llZ8917 are given merely for purposes of illustration and that the present inven-tion is not necessarily limited thereto.
EXAMPLE I
In this example 1 gram of a porous alumina base having a particle size of from 25 to 40 mesh, an Apparent Bulk Density (ABD) of 0.34 and pore size ranging from 200 Angstroms to 10,000 Angstroms was admixed with i 10 ml of a 5X weight by volume of aminopolystyrene having a molecular weight of 22,000 dissolved in aqueous 0.1 M hydrochloric acid. The two components were mixed at room temperature and allowed to stand for a period of 1 hour.
At the end of this time the solution was degassed, filtered and the solid support containing the aminopolystyrene adsorbed thereon was dried. Follow-ing this, the composite was mixed with 10 ml of a 1.5X aqueous solution of glutaraldehyde whlch had a pH of 1.4 and maintained for a period of 1 hour at room temperature. At the end of this 1 hour period the excess glutar-aldehyde was decanted and the organic-inorganic matrix was thoroughly washed with water several t~mes. The final immobilized conjugate was then pre-pared by treating the matrix with 6482 units of a commercial glucoamylase ~ Jradehl~rJc t~ sold under the Tradcnamc "Ambazyme". The immobilization of the enzyme was effected during a period of 16 hours while maintaining the temperature of the composite at 4C. by means of an ice bath. At the end of the 16 hour period the residual and unbound enzyme was washed out with water and a sodium chloride solution.
The immob~lized enzyme conjugate was packed in a column and a 30% weight by volume solution of starch sold under the commcrcial Trade-1ta~e~ "Maltrin-150" was passed over the beads while maintaining the tempera-ture at 60C., said starch feed being passed over the beads for a period of 2 hours at a flow rate of 2 ml/min. At the end of the 2 hour period, .
1~28917 the amount of glucose formed was assayed. It was found that the activity of the enzyme conjugate was 3240 units/gram at the flow rate of 2 ml/min.i the unit being defined as the micromoles of glucose formed per minute per gram of immobilized enzyme conjugate.
S EXAMPLE II
The above experiment was repeated with the exception that the enzyme, namely, glucoamylase was purified by means of an isopropanol pre-cipitation procedure well known in the art, resulting in a 1.3 fold increase of purity of the enzyme~ When this purified enzyme was immobilized in a manner similar to that set forth above and utilized to convert starch to glucose, it was found that the immobilized enzyme coniugate had an activity of 4070 units/gram at a flow rate of 2 ml/min.
EXAMPLE III
In a manner similar to that set forth in Example I above, 1 gram of an alumina base having a particle size of from 25-35 mesh and an ABD of 0.3 was added to 10 ml of a solution comprising 5X by volume of aminopoly-styrene dissolved in aqueous 0.1 M hydrochloric acid. The mixture was maintained at room temperature for a period of 1 hour after which it was degassed, filtered and the aminopolystyrene-alumina matrix was dried. The dried beads were then added to 10 ml of a 1.5% aqueous solution of glutar-aldehyde which had a pH of 1.4. The mixture was held for a period of 1 hour at room temperature and thereafter the excess glutaraldehyde was decanted.
The organic-inorganic matrix was washed several times with water and thereafter was treated with 1300 units of glucose isomerase which had a specific activity of 8.5 units/mg of protein. The coupling was effected at a temperature of 4C. for a period of 22 hours. The resulting immobilized glucose isomerase conjugate in which the enzyme was covalently bound to the free aldehyde functions of the copolymeric material which arose from the use of excess glutaraldehyde, was washed thoroughly with water and a 2 M
aqueous sodium chloride solution to remove any unreacted enzyme.
The immobilized enzyme conjugate was packed in a suitable column and a 45X solution of fructose which possessed a pH of 8 and contained 5 x 10 3 molar magnesium chloride was passed through the conjugate bed at a temperature of 60C. for a period of 2 hours. The glucose and flow rate were assayed and it was found that this immobilized enzyme conjugate had an activity of 700 units/gram at a flow rate of 2 ml/min., with a coupling efficiency of 60~. It is further determined that the enzyme con-jugate possessed a half-life of 22 days at 60- in a continuous column operation using the flow rate of 2 ml/min. of feed over the conjugate.
EXAMPLE IV
- In this example an alumina base having a particle size of from 60-80 mesh and an ABD of 0.3 was treated with aminopolystyrene and an excess of glutaraldehyde in a manner similar to that set forth above to form an organic-inorganic support matrix. This support matrix was treated with glucose isomerase which possessed a specific activity of 15 units/mg. The coupling was effected by offering 2800 units of enzyme to the support matrix at a temperature of 4~. for a period of 22 hours. After thorough washing of the immobilized enzyme conjugate, lt was packed in a column and a fructose feed similar to that described in Example III above was passed through the conjugate bed for a period of 2 hours at a temperature of 60-C.
The product was assayed and it was found that the immobilized enzyme conju-gate had an activity of 1200 units/gram with a coupling efficiency of 54g.
' 11289~7 EXAMPLE ~
In this example 1 gram of porous alumina pellets, 10 mesh, having an apparent bulk density (ABD) of 0.344 and pore si~es rang-; ing from 200 Angstroms to 10,000 Angstroms were placed in an emul-sion prepared from 0.5 grams of sodium hydrogen phosphate, 0.5 grams of sodium lauryl sulfate, 2 ml of styrene and 20 ml of distilled wa-ter. The peltets were allowed to adsorb in this emulsion under vacuum for a period of 1 hour following which the mixture was then - heated for a period of 3 hours at a temperature of 100C. in the presence of a polymerization catalyst comprising 0.5 grams of potas-sium persulfate. At the end of the 3 hour period, the beads were filtered to remove liquid, washed with a distilled water and ! ml of acetone. Thereafter 1 gram of the polystyrene-alumina support was~slowly added to 50 ml of 90X fuming nitric acid. The addition of the~support to the acid was effected at a temperature of about 0C. to 10C., the temperature being attained by placing the appara-tus in an ice bath. After a period of 2 hours the nitrated support was removed, washed with water and acetone. To effect the reduction of the nitrated polystyrene-alumina support, 1 gram of the composite along with 1 gram of sodium dithionite was added to 100 ml of dis-tilled water. The mixture was then heated to boiling and maintained at this temperature for a period of 1 hour. Following this the i~
aminopolystyrene-alumina composite support was washed with distilled water, dried under vacuum and maintained under a nitrogen atmosphere.
The aminopolystyrene-alumina support prepared according to the above paragraph was then contacted with 10 ml of a 50% aqueous solution of glutaraldehyde which had a pH of 3.5 for a period of 1 hour at room temperature. At the end of the 1 hour period the excess `` ~
1~28917 glutaraldehyde was decanted and the organic-inorganic matrlx was thoroughly washed with water several times in order to effectively remove unreacted reagents. The final immobilized enzyme conjugate was then prepared by treating the matrix with 8300 units of a com-~ r~a de ~
5 ~ mercial glucose amylase sold under the tr~dena~ Novo PPAG-12, said immobilization of the enzyme being effected during a period of 16 hours while maintaining the temperature of the mixture at about 4C.
by means of an ice bath. At the end of the 16 hour period the re-sidual and unbound enzyme was washed out with water and a sodium chloride solution.
The immobilized enzyme conjugate was then packed in a col-~ umn ànd a 30% weight by volume solution of starch, 6omncrQial Trade-s~ r~
~u*e~ Maltrin-150, was passed over the beads at a flow rate of 2 ml/
min. while maintaining the temperature at 60C. for a period of 2 hours. At the end of the 2 hour period the amount of glucose formed was assayed. It was found that the activity of the enzyme conjugate was 695 units/gram at the flow rate of 2 ml/minute; the term "unit"
being defined as the micromoles of glucose formed per minute per gram of immobllized enzyme coniugate.
- EXAMPLEVI
Experiment V was repeated with the exception that the enzyme was purified by means of an isopropanol precipitation pro-cedure well known to those skilled in the art. When the purified en-zyme was immobilized in a manner simiiar to that set forth above and utilized to convert starch to glucose, it was found that the immobil-ized enzyme coniugate had an activity of 852 units/gram at a flow rate of 2 ml/min.
,,
Claims (20)
1. An organic-inorganic matrix comprising a porous inorganic, water insoluble, solid support in combination with a copolymeric material resulting from the reaction of aminopolystyrene and a bifunctional monomer.
2. The organic-inorganic matrix as set forth in Claim 1 which is prepared by depositing a salt of aminopolystyrene on a solid support from an aqueous solution at a pH less than 7 and thereafter reacting the resultant aminopolystyrene-solid support composite with a bifunctional monomer to form the desired organic-inorganic matrix.
3. The matrix as set forth in Claim 2 in which the pH is in a range of from 1 to 4.
4. The matrix as set forth in Claim 2 or 3 in which the salt of aminopolystyrene is the hydrochloric acid salt.
5. The matrix as set forth in Claim 2 or 3 in which the salt of aminopolystyrene is sulfuric acid salt.
6. The matrix as set forth in Claim 2 or 3 in which the bifunctional monomer is in excess over the aminopolystyrene.
7. The organic-inorganic matrix as set forth in Claim 1 which is prepared by treating a solid support with a styrene monomer, polymeriz-ing the styrene-solid support composite at polymerizing conditions, nitrating the resultant polystyrene-solid support composite, reducing the nitrated polystyrene-solid support composite and thereafter reacting the resultant aminopolystyrene-solid support composite with a bifunctional monomer to form the desired organic-inorganic matrix.
8. The matrix as set forth in Claim 7 in which said polymerization conditions include a temperature in the range of from 50° to 100°C.
9. The matrix as set forth in Claim 7 in which said polymeriza-tion is effected in the presence of a polymerization catalyst.
10. The matrix as set forth in Claim 9 in which said polymerization catalyst is potassium persulfate.
11. The matrix as set forth in Claim 7 in which said nitration is effected by treating said polystyrene-solid support composite with nitric acid at subambient temperatures.
12. The matrix as set forth in Claim 11 in which said temperatures are in a range of from 0° to 10°C.
13. The matrix as set forth in Claim 7 in which said nitrated polystyrene-solid support composite is reduced by treat-ment with sodium dithionite at an elevated temperature.
14. The matrix as set forth in Claim 13 in which said elevated temperature is 50° to 100 C.
15. The matrix as set forth in Claim 1 in which said solid support comprises a metallic oxide.
16. The matrix as set forth in Claim 15 in which said metallic oxide is an alumina.
17. The matrix as set forth in Claim 16 in which said alumina is gamma-alumina.
18. The matrix as set forth in any of Claims 1, 2 or 7 in which said solid support is a ceramic monolith coated with a porous metallic oxide.
19. The matrix as set forth in any of Claims 1, 2 or 7 in which said solid support is a porous silica.
20. The matrix as set forth in any of Claims 1, 2 or 7 in which the bifunctional monomer is glutaraldehyde.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US951,948 | 1978-10-16 | ||
| US05/951,948 US4193910A (en) | 1978-10-16 | 1978-10-16 | Preparation of support matrices for immobilized enzymes |
| US05/951,949 US4206259A (en) | 1978-10-16 | 1978-10-16 | Support matrices for immobilized enzymes |
| US951,949 | 1978-10-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1128917A true CA1128917A (en) | 1982-08-03 |
Family
ID=27130323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA337,572A Expired CA1128917A (en) | 1978-10-16 | 1979-10-15 | Support matrices for immobilized enzymes |
Country Status (7)
| Country | Link |
|---|---|
| CA (1) | CA1128917A (en) |
| DE (1) | DE2941881C2 (en) |
| DK (1) | DK436379A (en) |
| ES (1) | ES485047A1 (en) |
| FR (1) | FR2453898A1 (en) |
| GB (1) | GB2033399B (en) |
| IT (1) | IT1125483B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0062968B2 (en) * | 1981-03-18 | 1991-07-10 | FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. | Support material for use in serological testing and process for the production thereof |
| DE3130924A1 (en) * | 1981-08-05 | 1983-02-17 | Röhm GmbH, 6100 Darmstadt | SURFACE-BASED SYSTEMS FOR FIXING SUBSTRATES CONTAINING NUCLEOPHILE GROUPS |
| CN113061600A (en) * | 2021-03-05 | 2021-07-02 | 宁波泓森生物科技有限公司 | Preparation method of immobilized threonine aldolase, immobilized threonine aldolase and application |
| CN114540332B (en) * | 2022-03-01 | 2023-07-21 | 江苏恰瑞生物科技有限公司 | Preparation method and application of uricase immobilized resin |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3860486A (en) * | 1973-05-24 | 1975-01-14 | Owens Illinois Inc | Insolubilizing enzymes with polystyrene derivatives |
| GB1485122A (en) * | 1973-08-06 | 1977-09-08 | Nat Res Dev | Biologically active matrices |
| US4001085A (en) * | 1973-09-10 | 1977-01-04 | Owens-Illinois, Inc. | Immobilization of enzymes on an inorganic matrix |
| GB1485123A (en) * | 1974-07-25 | 1977-09-08 | Nat Res Dev | Support for biologically active material |
| PT64790B (en) * | 1975-02-18 | 1977-07-07 | Uop Inc | Immobilized enzyme conjugates |
| US4141857A (en) * | 1976-04-30 | 1979-02-27 | Uop Inc. | Support matrices for immobilized enzymes |
-
1979
- 1979-10-15 CA CA337,572A patent/CA1128917A/en not_active Expired
- 1979-10-16 DE DE2941881A patent/DE2941881C2/en not_active Expired
- 1979-10-16 DK DK436379A patent/DK436379A/en not_active Application Discontinuation
- 1979-10-16 FR FR7925672A patent/FR2453898A1/en active Granted
- 1979-10-16 ES ES485047A patent/ES485047A1/en not_active Expired
- 1979-10-16 IT IT26535/79A patent/IT1125483B/en active
- 1979-10-16 GB GB7935953A patent/GB2033399B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| FR2453898B1 (en) | 1983-11-18 |
| IT1125483B (en) | 1986-05-14 |
| DE2941881C2 (en) | 1983-11-10 |
| FR2453898A1 (en) | 1980-11-07 |
| IT7926535A0 (en) | 1979-10-16 |
| DE2941881A1 (en) | 1980-04-17 |
| GB2033399A (en) | 1980-05-21 |
| DK436379A (en) | 1980-04-17 |
| GB2033399B (en) | 1982-11-24 |
| ES485047A1 (en) | 1980-05-16 |
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| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |