CA1058538A - Enzyme bound to polymer which is entrapped in inorganic carrier - Google Patents
Enzyme bound to polymer which is entrapped in inorganic carrierInfo
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- CA1058538A CA1058538A CA245,940A CA245940A CA1058538A CA 1058538 A CA1058538 A CA 1058538A CA 245940 A CA245940 A CA 245940A CA 1058538 A CA1058538 A CA 1058538A
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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
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/091—Phenol resins; Amino resins
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/0234—Nitrogen-, phosphorus-, arsenic- or antimony-containing compounds
- B01J31/0235—Nitrogen containing compounds
- B01J31/0254—Nitrogen containing compounds on mineral substrates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/06—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing polymers
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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
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/093—Polyurethanes
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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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- General Health & Medical Sciences (AREA)
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- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Inorganic Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
ABSTRACT OF THE disclosure This invention relates to novel compositions of matter comprising immobilized enzyme conjugates. More specifically the invention is concerned with novel compositions of matter which comprise an immobilized enzyme conjugate which consists of an organic-inorganic matrix constituting an inorganic porous support material containing an organic polymeric material which has been formed in situ from a monomer, hydrolyzed polymer, or preformed polymer of synthetic or natural origin by reaction with a bifunctional monomer containing suitable reactive moieties.
Said polymer material is both entrapped and adsorbed in the pores of the aforesaid support material, and is further provided with functionalized pendent groups extending therefrom, the funtional moieties being located at or adjacent to the terminal portions thereof, and an enzyme which is both covalently bound to said functionalized pendent groups as well as being adsorbed in part on the organic-inorganic matrix.
Said polymer material is both entrapped and adsorbed in the pores of the aforesaid support material, and is further provided with functionalized pendent groups extending therefrom, the funtional moieties being located at or adjacent to the terminal portions thereof, and an enzyme which is both covalently bound to said functionalized pendent groups as well as being adsorbed in part on the organic-inorganic matrix.
Description
~1~53~
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 organisms.
The enzymes may al50 be isolated and used in analytical, medical and industrial applications. For example, they find use in industrial applications in the preparation of food products such as cheese or bread as well as bein~ 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 ~odification ; of penicillin to form various substrates thereof; the use of various proteases in cheese making, meat tenderizing, detergentformulations, leather manuEacture and as digestive aids; the use of carbonhydrases in starch hydrolysLs, sucrose inversion, gIucose isomerization, etc.; the use of nucleases in flavor control; or the use of oxidases in oxidation pre-vention and in the color control of food products. These uses as well as many others have been well delineated in the literature.
As hereinbefore set forth, inasmuch as enzymes are commonly water soluble as well as being generally unstable and readily deactivated, they are also difficult either to remove from the solutions in which they are utilized for sub-sequent reuse or it is difficult to maintain their catalytic , . . .
activity for a relatively extended period of time. The a-forementioned difficulties will, of course, lead to an in-creased cost in the use of enzymes for commerical purposes due to the necessity for frequent replacement of the enzyme, `:
r ~ --2--~- .
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this replacement being Isu~l~y necessary with eac1- application.
To counteract the high cost of replacement, i~ has been suggested to immobilize or insolubilize the enzymes prior to use thereof. By immobilizing the enzymes through various systems hereinafter set forth in greater detail, it is possible to stablizied the enzymes in a relative manner and, thereofre, to permit the reuse of the enzyme which may otherwise undergo de-activation 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 o~ the enzymes provides a more favorable or broader environmental stability, a minimum of effluent problems and materials handling as well as the possibility of upgrading the activity of the enzyme itself.
As hereinbefore set forth, several genera] methods, as well as many modiEications thereof, have been described by which the immobilization of enzymes may be effected. One general method is to adsorb the enzyme at a solid surface as, for example, when an enzyme such as amino acid acylase is adsorbed on a cellulosic derivative such as DE~E--cellulose; papain (3.4.4.10) or ribonuc-lease (2.7.7.16/2.7.7.17) is adsorbed on porous glass; catalase (1.11.1.6) is adsorbed on charcoal; trypsin t3.4.4.4) is adsorbed on quartz glass or cellulose; chymotrypsin t3.4.4.5) is adsorbed on kaolin:Lte, etc. ~nother general method is to trap an enzyme in a gell lattice such as glucose oxidase (1.1.3.4), urea (3.5.1.5) papain (3.4.4.10), etc., being entrapped in a polyacryl-amide gel; acetyl cholinesterase (3.1.1.7) being entrapped in a starch gel or a silicone polymer; glutamic-pyruvic transamînase (2.6.1.2) being jl/ ~~~ ~ -3-5~S3~
entrapped in a polyamide or cel]ulvse acetate gel, etc. A
~urther general method is a cross-linking by means of bifunc-tional reagents and may be effected in combination with either of the aforementioned general methods of immobilization. When utilizing this method, bifunctional or polyfunctional reagents which may induce intermolecular cross-linking will covalently bind the enzymes to each other as well as to a solid support.
This method may be exemplified by the use of glutardialdehyde or bisdiazobenzidine-2,21-disulfonic acid to bind an enzyme such as papin (3.4.4.10) to a soild support etc. A still further method of immobilizing an enzyme comprises the method of a co-valent binding in which enzymes such as glucoamylase ~3.2.1.3), trypsin (3.4.4.4), papain ~3.4.4.10), pronase ~3.4.21.4/3.4.24.4) amylase (3.2.1.1/3.2.1.2), glucose oxidase (1.1.3.4), pepsin (3.4.4.1) rennin (3.~ .3) fungal protease, lactase (3.2.1.23), etc., are immobilized by covalent attachment to a polymeric material which is attached to an organic or inorganic solid porous support. This method may also be combined with the afore-said 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 be-tween the enzyme and the carrier support are often quite weak, although some prior art has indicated that relatively stable conjugates of this type have been obtained when the pore size of the support and the spin diameter of the enzyme are correlated.
~lowever, the pore size of the support cannot exceed a diameter of about 1000 Angstroms. In view of this weak bond~ the en-jl/ J C ~, 3~358538 zyme is often readily desorbed in the presence of solution~ of the substrate being processed. In addition to this, the enæyme may ~e partially or extensively deactivated due to its lack of mobility or due to interaction between the support and the ac-.~ ~ .
tive site of the enzyme. Another process which may be employedis the entrapment o enzymes in gel lattices which can be ef~ec-ted by polymerizing an aqueous solution or emulsion containing the ~onomeric form of the polymer and the enzyme or by in~orpox-ating the enzyme into the preformed polymer by various techniques, often in the presence of a cross-linking agent. ~hile this meth-od 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 en-zyme, it also has disadvantages in that the conjugate has poor mechanical strength, which results in compacting when used in ~olumns in continuous flow systems, with a concomitant plugging of the column. Such s~stems also have rather ~tide 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 sufficiently small so that large diffusional bar-riers 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 lmmobilizing an enzyme b-y intermolecular cross-linkage, as already noted, are due to the lack of mobility withresulting deactivation because o~ inability of the enzyme to as-- sume the natural configuration necessary for maximum activity, particularly when the active site is involved in the ~inding process.
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~5~3538 Covalent hindin~ methods have ~ound wide a~Plications and may be used either a~ the sole immohilization techni~ue or as an integral part of many of the methods already described in which cros~-lin~ingreactions are em~loved. This method is often used to bind the enzyme as well as the sup~ort throu~h a bifunctional intermediary molecule in ~hich the functional qroups of the molecule, such as, for example, q3mma-aminoproPvl-triethoxysilane, are capable of reactinq with functional moieties present in ooth the en~yme and either an organic or inorqanic porous support. A wide variety of reaqents and su~ports has been employed in this manner and the method has the advantaqe of provlding strong covalent bonds throuqhout the conju~ate product as well as qreat activity in many cases. The covalent linka~e of the enzyme to the carrier must be accomPllshed throughfunctional groups on the enzyme which are non-es~sential for its catalytic activity such as free amino arouPs, carboxvl groups, hvflroxyl ~roups, phenolic ~rouPs, su]fhYdryl qrouDs, etc. These functional qrou~s will also react with a wide variety of other functional groups such as an a~dehyae, i~ocyanato, acyl, diazo, azido, anhydro, activated ester, etc., to produce covalent bonds. Nevertheless, this method also often has many disadvantages involving costly reactants and solvents, as well as specialized and costly ~orous suP~orts and cumhersome multi-step procedures, which render the method of preparation un-economical for commercial a~plication.
The prior art is thexefore replete with variou~ methodsfor immobilizing enzymes which, however, in various ways fail to meet the reqairements of inAustria] use. ~lowever, as will ' _fi _ ~L~5~ii38 hexeinafter be discu~seæ in ~reater detail, none of the Prior art compositions eomprise the composition of matter of the present invention which constitutes an inorqanic porous sbpport containing a polymeric material fon~ea in situ from a monomer or preformed polymer, of natural or svnthetic'oriclin, which is entrapped and also adsorhed in Part within the pores of said sunport ana wHich contains f~nctionalized, pendent grou~s ex- : ~
tending therefrom; the enzyme bein~ ~artially adsorbe'~ to the '' ~atrix and also covalently boun~ to the active moieties at or adjacent to terminal portions of the pendent qrouPs, thus Per- ' .itting the freedom of ~.ovement which ~ill enab}e the enzy~e to e~ercise maximum activity. For examDle, Il .S~ Patent No.
3,556,945 relates to enzyme compo.sites in which the enzvme is ..
adsorbed directly to an inorganic carrier such as qlass. U.~.
Patent ~o. 3,519,538 is concerned with enzyme composites in Yhich .
the enzymes are chemically couPled bY ~eans o.f an interme~iary silane coupling a~ent to an inorqanic carrier. ~n similar ' ~
fashion, U. S. Patent ~o. 3,7~3,101'also utilizes an orqano~
silane composite as a b'inding aqent, the enzYme being covalently counled to a ~lass carrier hy ~ean.s of an intermediate sil~ne couplinq aqent, the silicon portion of the cou~linq aqent bein~
attached to the carrier wh.ile the orqanic oortion of the counlinq ' . ' a~ent is coupleA to the enæyme, the com~osition containing ~ .
metal oxide on the surface of the carrier disposéd bet~een the ' .
carrier and the silicon portion of the couPling ac.~ent. In ll. S.
Patent No. 3,821,083 the inert carrier is coated ~7ith a preformed polymer such as polyacrolein which has bonded thereto an enzy7~e.
~lowever, according to most of ~he examples set ~orth in the patentr it is necessary to first acicl hydrolvze the comPosite prior to ' ~05~3S38 the deposition oE the enzyme on the polymer. Another prior art patent, namely, U.S, Patent No. 3,705,084 discloses a macroporous enzyme reactor in which an enzyme is adsorbed on the polymeric surface of a macroporous reactor core and thereafter is cross-linked in place. By cross-linking the enzymes on the polymeric surface after adsorption thereof, the enzyme is further immobilized in part and cannot act freely as in its native state as a catalyst. The cross-link-age of enzymes in effect links them together, thereby pre~
venting a free movement of the enzyme and decreases the mobility of the enzyme which is a necessary prereguisite for maximum activity.
This invention rela-tes to novel compositions of matter comprising immobilized enzyme conjugates. More spe-cifically the invention is concerned with novel compositions of matter which comprise an immobilized enzyme conjugate which consists of an organic-inorganic matrix constituting an inorganic porous support material containing an organic polymeric material which has been formed in situ from a monomer, hydrolyzed polymer, or preformed polymer of synthetic ox natural origin by reaction with a bifunctional monomer containing suitable reactive moieties. Said polymer material is both entrapped and adsorbed in t~e pores of the aforesaid support material, and is further provided with functionalized pendent groups extending therefrom, the functional mo.ieties being located at or adjacent to the terminal portions thereof, and an enzyme which is both covalently bound to said function-alized pendent groups as well as being adsorbed in part on the organic-inorganic ~atrix.
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_~ ~8 .
~05~538 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 combination with other 5 solid materials, are themselves in such a ~ondition where-by they are not water soluble and therefore they may be sub-jected to repeated use while maintaining the catalytic activity of said enzyme. In order to be present in an im-mobilized state, the enzymes must be bound in some manner to a water insoluble carrier, thereby being commerically - usable in a non-water soluble statq.
It is therefore an object of this invention to pro-vide novel compositions of matter in which enzymes are present in an immobilized state.
A further object of this invention is to provide compositions of matter in which an enzyme is both adsorbed on an organic-inorganic matrix and covalently bound to functional-ized pendent groups, attached to said matrix, which is, in turn, aiso both adsorbed and ~ntrapped in the pores of the - 20 inorganic porous support material.
- In one aspect an embodiment of this invention re~
sides in an immobilized enzyme conjugate comprising an com-bined organic-inorganic matrix consisting of an inorganic porous support containiny an organic polymeric material ad-sorbed and entrapped in the pores of said support, said polymeric material containing functionalized pendent groups, and an enzyme adsorbed to said matrix and covalently bound to the functional moieties of said pendent groups of said organic polymeric material at or adjacent to the terminal portions thereof.
_g_ .,. , ~ - '' '''''I
~OS853B
A specific embodiment of thi9 invention is found in an immobilized enzyme conjugate comprising an organic-inorganic matrix consisting of a low bulk density, porous silica-alumin support of relatively high surface area which may also contain inorganic additives and an in situ~prepared tetraethylenepentamine-glutaralaehyde polymeric material which is adsorbed as well as e~ntrapped in the pores of sai~
h3 silica-alumina, and an enzyme comprising glucoamy~ase~bein~
covalently bound to the glutaraldehyde pendent groups of the polymeric material at or adjacent to the terminal por-tion of said groups as welI as being adsorbed in part on the matrix.
Other objects and embodiments will be found in the following further detailed description of the present invention.
As hereinbefore set forth the present invention is concerned with immobilized enzyme conjugates comprising a combined organic-inorganic matrix consisting of an inor-ganic porous support material containing an organic polymeric material adsorbed and entrapped in the pores of said inor-ganic porous support. In addition, the polymeric material will contain pendent groups, said pendent groups having an enzyme being covalently bound to said groups at or adjacent to the terminal portions thereof and, in addition, said enzyme is also partially adsorbed on said matrix. In con-tradistinction to the compositions of matter containing immobilzied enzymes as set forth in the prior art the com-positions of matter of this invention may be prepared by utilizing relatively inexpensive reactants as well as ~ . ~.
~058538 utilizing more simple steps in the procedure for preparing said compositions. In addition, the mechanical strength and stability of the enzyme conjugates of the present in-vention will be greater than that wnich is possessed by the immobilized enzymes of the prior art. There~ore, it is readily apparent that the compositions of matter of the present invention possess economical advantages which are useful for industrial applications.
The compositions of matter of the present invention may be prepared in a relatively simple manner. In the pre-ferred-method of preparation, the inorganic porous support material will be treated witn a solution, preferably aqueous in nature, of a bi- or polyfunctional monomer, a polymer hydrolysate or a preformed polymer, following which the unadsorbed solution is removed by any means known in the art as draining, etc. It is also contemplated that other in-expensive organic solvents such as acetone, tetrahyarofuran, etc., may also be used as the carrier for the aforementioned monomers or polymers. Following the removal of the unad-sorbed solution, the wet porous support is then contacted with a relatively large excess of from 5 to 20 mole percent of a second bifunctional monomer in which the reactive groups - are preferably separated by a chain containing from ~ to 10 carbon atoms, this second bifunctional monomer also being added in an aqueous solution, whereby a polymeric matrix which is both adsorbed and entrapped in the pores of the support will be formed and from which pendent groups of the second monomer will extend. These pendent groups will con-tain unreacted functional moieties due to the fact that an excess amount of the second bifunctional monomer was employed .
-11- , ' , ~LIt)5~353~ `
in treating the support. The unreacted functional moieties are then available for covalent binding to the enzyme, which is added to the resulting organic-inorganic matrix, again usually in an aqueous solution. After removal of the un-reacted materials such as by treating, wahsing, etc., theenzyme, besides being covalently bound to the pendent fun-ctionalised groups at or adjacent to the terminal por~ions - thereof, will also, in par~ be adsorbed to the matrix. It is therefore readily apparent that the entire immobili-sation procedure can be conducted in a simple and inex-pensive manner utilising an aqueous or inexpensive solvent media, the procedure being conducted over a temperature differential which may range from subambient (about 5C.) up ~o elevated temperatures of about 60C., and preferably at ambient ~about 20-25C.)temperature, said procedure be_ng effect by utilising a minimum of operating steps and, in addition, permitting a ready recovery of the excess reactants and finished composition of matter.
Many of the inorganic supports reported in the prior art specify "controlled pore" materials such as glass, alumina, etc., having a pore diameter of from 500 to 700 Angstroms for abou 96 ~ of the material and a maximum pore diamter of lO00 Angstroms,a surface area of 40-70 m2~gm and 40-80 mesh size particles. In addition, these supports may be coated with metallic oxides such as zirconium oxide and titaniu~ oxide for greater stability. In contradistinction to these sup-ports, it is contemplated within the scope of this inven~
tion that the inorganic porous supports which are utilised hcrein, will constitute materials which possess pore dia-metres ranging from lO0 Angstrom up ~o SS,oO0 Angstroms ~ 58538 with as much as 25-60% of the porous support material possessing pores having diametres above 20,000 Angstroms and surface areas ranging from 150 to 200 m2/gm. The particle size may also vary over a wide range of from 10-20 mesh to a fine powder, said particle size depending up~n the particular system in which the~ are to be used. It is also contemplated that the porous support materials may be coated with various oxides of the type hereinbe- -fore set forth or may have incorporated therein various other inorganic materials such as boron phos~hate, etc.;
- these inorganic materials imparting special properties to the support material. A particularly useful ~orm of sup-port will constitute a ceramic body which may have the type of porosity herein described for materials of the present invention or it may be honeycombed with connecting macro size channels throughout, such materials being com~
monly known as monoliths, and which may be coated with various types of porous alumina, zirconia, etc. The use of such a type of support has the par-ticular advantage of per-mitting the free flow of even highly viscous substrateswhich are o~ten encountered in commercial enzyme catalyzed reactions.
The inorganic porous support materials which are utilised as one component of the combined organic-inorganic matrix will include certain metal oxides such as alumina, and particularly gamma-alumina, silica, ~irconia or mix-tures of the metal oxides such as silica-alumina, silica-zirconia, silica-magnesia, silica-zirconia-alumina, etc., or gamma-alumina containinq other inorqanic compounds such as boron phosphate, etc., ceramic bodies, etc., as well as combinations of the aforementioned material, one of said materials which may serve as a coating for another material comprising the support.
~05~538 The polymeric materials which are formed in situ in such a manner so that the E,olymeric material is both partially adsorbed and partially entrapped in the pores of the inorganic support of the type hereinbefore set forth may be produced according to the general methods herein- -before described, that is, by first adsorbing a solution containing from 2 to 25~ of a bi- or polyfunctional monomer, polymeric hydrolysate, or a preformed polymer, the monomer or polymer being synthetic or naturally occurring in origin, and which are pre~erably soluble in water or other solvents which are inert to the reactions subsequently employed. As hereinbefore set forth, it is contemplated within the scope of this invention that a second bifunctional monomer is then added in similar manner to form an organic-inorganic matrix by reaction with the original additive adsorbed on the in-organic support. The func-tional groups which are present on the bifunctional monomer wil~ comprise well known reactive moieties such as amino, hydro~yl, carboxy, thiolr carbonyl, etc. moi-eties. As was also hereinbefore set forth, the reactive groups of the bifunctional compounds are preferably, but not neces-sarily, separated by chains containing from 4 to 10 carbons atoms. The reactive moieties are capable of covalently bonding with both the initial additives and subsequently, after washing out unreacted materials, with the enzyme which is to be added in a subsequent step, said enzyme being then covalently bound to the functional group at or adjacent to the terminal portion of the functional chain ' ' " . ' ' .
lQS853~
a~ well as concomitantly adsorbed on the matrix. After addition of the enzyme to this composition, a relatively stable enzyme conjugate will be produced which possesses high activity and high stability. In addition, the com-position of matter of the present invention also possessesappreciable versatility in addition to the other advantages hereinbefore enumerated in that it can be applied to pre-pare conjugates in the absence of an inorganic support which are soluble in either acidic or alkaline media. As will hereinafter be set forth in greater detail, depen-ding upon the reactants employed, the conjugates will re-tain their stability in such media when prepared in com-bination with the inorganic support according to the pro-cesses set forth in the prior art. sy possessing these properties, it .is possible to widen the uses to which these conjugates may be applied.
Specific examples of bi- or polyfunctional monomers, polymer hydrolysates or preformed polymers which may be initially adsorbed on the inorganic support will include water soluble polyamines such as ethylenediamine, diethyl-enetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexamethylenediamine, polyethylene-- imine, etc.; water insolu~le polyamines such as methylene-dicyclohexylamine, methylenedianiline, etc.; natural and syn-thetic, partially hydrolyzed polymers and preformed polymers SUC}I as nylon, collagen, polyacrolein, polymaleic anhydride, alginic acid, casein hydrolysate, gelatin, etc. Some specific examples of intermediate bifunctional materials which may be added to tne above enumerated products to pro-l~S8~38 duce an organic-inorganic matrix and which possess the necessary characteristics hereinbefore set forth include compounds such as glutardialdehyde, adipoyl chloride, sebacoyl chloride, toluenediisocyanate,hexamethylenedi-isocyanate, etc. It will be noted that when a polyeth~l-eneamine of the type hereinbefore set forth is reacted with glutardialdehyde in-the absence o~ an inoxganic porous support, an aqueous acid soluble material is obtained, whereas when a polyethyleneamine is reacted with a di-isocyanate or acyl halide, a water insoluble product is ob-tained. Conversely, if a reaction com~lex without the in-organic support coneains free carboxyl groups, an alka-line soluble complex can be obtained. Due to the large excess of intermediary, or spacer, bifunctional molecules which are used, the polymexic matrix which is formed wi}l contain pendent groups comprising the spacer molecules, said molecules extending from the matrix and having reac-tive moieties available at or ad~acent to the terminal pox- ~ -tions thereof which are capable of reacting with and bind-ing the enzyme to the spacer molecules via covalent bonas.
In addition, the enzyme, when applied after the unreacted reagents have been removed ~rom the organic-inorganic matrix by washing, will also concomitantly undergo adsorption in part with said matrix. Binding the enzyme solely to the organic matrix will not usually affect the de~endency of the solubility of the aggregate on the pll of the solution but when the inorganic support is included as heretoeore des-cribed, the total conjugate exhibits high stability over ~
relatively wide pl~ range from 3 to ~, tne stability of course, .
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l~S~53~
also being a function of the optimal pH characteristics of the particular enzyme employed as well as the inorganic support used. Therefore, it is readily apparent that a suitable organic-inorganic matrix which is applicable in many situations will be formed with the support material by adsorbing any of the type of materials hereinbefore described which are known to the art and then treated with any bifunctional molecule which is also known to the art and ; is suitably unctionalised to react with the original ad-ditive, provided that a large enough excess of the bi-functional molecule is used to provide pendent groups which are capable of subsequently reacting with ~he enzyme which is desired to be immobilised. By utilising these functional pendent groups as a binding site for the enzymes, it will permit the enzymes to have a greater mobility and thus per-mit 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 heen immobllised by any of the other methods such as entrapment in a gel lattice, ad-sorption on a solid surface or cross-linkage of the enzyme by means of bifunctional reagents, etc. Not all formulations, however, will produce equivalent results in terms of stabili-ty or activity.
Examples of enzymes which may be immobilised by a covalent bonding reaction and which contain an amino group capable of reacting with an aldehydic or isocyana-to moiety of the pendent group which is attached to a polymeric ma-terial entrapped and adsorbed in t,he pores of a porou,s sup- ~
~ port material will include trypsin~p:paln)~ ~exo~l~nase~J(~ ta- J
:
~058S38 "
` galactosidase (3.2.1.23), ficin (3.~.4.12), bromela:in (3.~.4.2~), lactic dehydrogenase ~1.1.1.27), glucoamylase (3.2.1.3)~ chymotxypsin (3.~ .5), pronase, acylase, invertase (3.2.1.26), amylase (3.2.1.1/3.2.1.2) glucose oxidase (1.1.3.4), pepsin (3.4.4.1) rennin (3.4.4.3) f~mgal protease, etc.
In general any enzyme whose active site is not involved in the covalent bonding can be used. While the aforementioned discuss:io~ was centred about pendent groups which contain as a functional moiety thereon an aldehydic or isoc~anato group, it is also contemplated within the scope of this invention that the pendent group can contain other functional moieties capable of reaction with carboxy, sulfhydryl or other moieties usually present in enzymes. However, the covalent bonding of enzymes containing these other I moieties with equivalent results and may also involve apprrciably greater costs in preparing intermediates. It is to be understood that the afore-mentioned listing of porous solid supports, monomers, hydrolysates, polymers and enzymes are only representative of the various classes of compounds which may be used, and that the present invention is not necessarily limited thereto.
The preparation of the compositions o matter of the present invention is preferably effected in a batch type operation as heretofore already described in detail, al-though it is also contemplated within the scope of this invention that the formation of the finished composition of matter may also be effected in a continuous manner of operation. When a continuous type operation is used, a quantity of the porous solid support material is placed in an appropriate apparatus, usually constituting a column. The porous solid support jl/ ~ ~ -18-; ` ` ~
3Ll)5~538 material may be in any form desired such as powder, pellets, monoliths, etc., and i9 charged to the column, after which a preferably aqueous solution of, for example, a poly-functional amine is contacted ~ith the porous support un-til the latter is saturated with the amine solution and thee~cess is then drained. A spacer or intermediary bifun-ctional molecule such as glutardialdehyde is then contacted with the saturated support. The formation of the polymeric ; matrix is thus effected in an aqueous system, said reaction being effected during a period of time which may range from 1 to 10 hours in duration, but is usually of short duration.
After removing the ~xcess glutard;aldehyde by draining and washing out any water soluble and unreacted materials, which in the case of a polyamine is preferably done with a buffer solution possessing a p~ of about 4, an aqueous solution of the enzyme is contacted or recycled through the column, this step effecting a covalent bonding of said enzyme to the terminal aldehydic groups of the functionalised pendent molecules which extend from the matrix. This occurs until there is no further physical adsorption andfor covalent binding of the enzyme to the organic-inorganic matrix and pendent molecules. The excess enzyme is recovered in the effluent after draining and washinq the column. The column is thus ready for use in chemical reactions in which the catalytic effect of the enzyme is to take place. The pro-cedures are, for the most part, conducted within the time, temperature and concentration parametres hereinbefore des-cribed in the batch type procedure and will result in - comparable immobilised enzyme complexes. It is also con-' .
, .. ~
~5~53J!3 templated within the scope of this invention that with suitable modifications of pH and temperature parametres w~ich will be obvious to those sXilled in the art, the process may be applied to a wide vaxiety of inorganic porous supports, polymer forming reactants and enzymes.
The following examples are given for purposes of illustration of the novel compositions of matter of the present invention and to methods for preparing the same. -~lowever, these examples are given merely for purposes of illustration and it is to be understood that the present invention is not necessarily limited thereto.
EX~MPLE ~
In tbis example 2 grams of a porous silica-alumina composite which contained boron phosp;late incorporatedthere-in having a particle size of ~0-80 mesh, a pore diametre ranging from about 100 to about 55,000 Angstroms and a sur-face area of about 150-200 m2/gm was utilised as the in-organic support for the novel composition of matter of the present invention. This support was calcined at a temper-ature of about 500F. to remove any adsorbed moisture con-tained tnerein. Thereafter the support was treated with 25 - ml of a ~ aqueous solution of tetraethylenepentamine at ambient temperature for a period of 1 hour in vacuo to facilitate the penetration of the solution into the pores of the support. The excess unadsorbed solution was then decanted, about 25~ of the tetraethylenepentamine having been adsorbed into the pores of the support. Following this, the wet sup-port was then treated with 25 ml of a 5% aqueous solution of glutardialdehyde at ambient temperature and an almost ~ -20-~LOS~538 immediate reaction took place with the formation of an in-soluble reaction product both on the surface and within the pores of the support. The excess glutardialdehyde sol-ution was then decanted and the organic-inorganic complex was washed to remove unreacted and unadsorbed reagent~, said washing being accomplished first with water followed by washing with a 0.02 molar acetate buffer solution which possessed a pH of 4.2, the washing operation being effected at a temperature of 45C. Therea~er an enzyme solution containing about 200 mg of glucoamylase~per 25 ~1 of water was added and allowed to react with the composite at am-bient temperature for a period of 1 hour. At the e~d of this l-hour period, the excess glucoamylas~As~o~u~l~o~ was decanted and the enzyme conjugate was washed with water to remove any unbound and/or unadsorbed enzyme. The composi-tior was then leached for a period of 24 hours with an ace-tate buffer solution similar to that hereinbefore desoribed.
The amount of adsorbed and/or covalently bonded enzyme was determined by micro Dumas gas chromatography analyses both before and after addition of the enzyme. The activity of the en2yme conjugate was then determined by the amount of glucose produced using 30~ thinned starch solution as sub-strate at a pH of 4.2 and 60C., and employing Worthington's glucostat procedure for analysing glucose, the latter being considered the more reliable procedure for determining the utility of the conjugate. An activity of 28 units per gram of support with an enzyme loading of 29 mg/gm of support was obtained by this procedure (one unit representing the pro-duction of l gram glucose per hour at 60 C. according to the \
~058S38 assay specifications~. It will be noted that despi~e the known solubility at p~l of 4.2 of the enzyme conjugate when prepared in the abse~ce of an inorganic support, negligible loss of enz~me from the combined inorganic-organic complex oacurred during leaching with the 4.2 pH buffer solution.
This was demonstrated by assaying the effluent from this treatment.
EXAMPLE II
In th'is example the procedure of Example I was followed with the exception that the inorganic porous sup-port had a particle size of 10-30 mesh. This silica-alumina composite containing boron phosphate incorporated therein was treated with tetraethylenepentamine, glutardialdehyae ~ and glucoamylasel ln ~a ma L er similar to that set forth a-bove. An active immobilised enzyme complex was obtained although of decreased activity probably because a diffusion problem is produced by the larger particle size of the com-posite.
EXAMPLE III
In a manner similar to that set forth in Example I above, 2 grams of a silica-alumina composite possessing the same physical characteristics of particle size, pore diameter and surface area as that set forth in Example I
was treated with an acetone solution of tetraethylenepen-tamine and followed by a tolunediisocyanate solutionalso in acetone instead of aqueous glutardialdehyde. After de-canting the excess diisocyanate solution and washing with water, the organic-inorganic complex was further treated with an aqueous glucoamylase solution. As in Example I, the finished product comprised an active completely in~
soluble enzyme complex.
, '1 -EXAMPLE IV
To illustrate the point that various concentra-tions of solutions can be used to prepare the desired pro-duct, the procedure set forth in ExampleI above was re-S peated with the exception that more highly concentratedsolutions of the various reagents were used. For example,
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 organisms.
The enzymes may al50 be isolated and used in analytical, medical and industrial applications. For example, they find use in industrial applications in the preparation of food products such as cheese or bread as well as bein~ 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 ~odification ; of penicillin to form various substrates thereof; the use of various proteases in cheese making, meat tenderizing, detergentformulations, leather manuEacture and as digestive aids; the use of carbonhydrases in starch hydrolysLs, sucrose inversion, gIucose isomerization, etc.; the use of nucleases in flavor control; or the use of oxidases in oxidation pre-vention and in the color control of food products. These uses as well as many others have been well delineated in the literature.
As hereinbefore set forth, inasmuch as enzymes are commonly water soluble as well as being generally unstable and readily deactivated, they are also difficult either to remove from the solutions in which they are utilized for sub-sequent reuse or it is difficult to maintain their catalytic , . . .
activity for a relatively extended period of time. The a-forementioned difficulties will, of course, lead to an in-creased cost in the use of enzymes for commerical purposes due to the necessity for frequent replacement of the enzyme, `:
r ~ --2--~- .
05853~
this replacement being Isu~l~y necessary with eac1- application.
To counteract the high cost of replacement, i~ has been suggested to immobilize or insolubilize the enzymes prior to use thereof. By immobilizing the enzymes through various systems hereinafter set forth in greater detail, it is possible to stablizied the enzymes in a relative manner and, thereofre, to permit the reuse of the enzyme which may otherwise undergo de-activation 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 o~ the enzymes provides a more favorable or broader environmental stability, a minimum of effluent problems and materials handling as well as the possibility of upgrading the activity of the enzyme itself.
As hereinbefore set forth, several genera] methods, as well as many modiEications thereof, have been described by which the immobilization of enzymes may be effected. One general method is to adsorb the enzyme at a solid surface as, for example, when an enzyme such as amino acid acylase is adsorbed on a cellulosic derivative such as DE~E--cellulose; papain (3.4.4.10) or ribonuc-lease (2.7.7.16/2.7.7.17) is adsorbed on porous glass; catalase (1.11.1.6) is adsorbed on charcoal; trypsin t3.4.4.4) is adsorbed on quartz glass or cellulose; chymotrypsin t3.4.4.5) is adsorbed on kaolin:Lte, etc. ~nother general method is to trap an enzyme in a gell lattice such as glucose oxidase (1.1.3.4), urea (3.5.1.5) papain (3.4.4.10), etc., being entrapped in a polyacryl-amide gel; acetyl cholinesterase (3.1.1.7) being entrapped in a starch gel or a silicone polymer; glutamic-pyruvic transamînase (2.6.1.2) being jl/ ~~~ ~ -3-5~S3~
entrapped in a polyamide or cel]ulvse acetate gel, etc. A
~urther general method is a cross-linking by means of bifunc-tional reagents and may be effected in combination with either of the aforementioned general methods of immobilization. When utilizing this method, bifunctional or polyfunctional reagents which may induce intermolecular cross-linking will covalently bind the enzymes to each other as well as to a solid support.
This method may be exemplified by the use of glutardialdehyde or bisdiazobenzidine-2,21-disulfonic acid to bind an enzyme such as papin (3.4.4.10) to a soild support etc. A still further method of immobilizing an enzyme comprises the method of a co-valent binding in which enzymes such as glucoamylase ~3.2.1.3), trypsin (3.4.4.4), papain ~3.4.4.10), pronase ~3.4.21.4/3.4.24.4) amylase (3.2.1.1/3.2.1.2), glucose oxidase (1.1.3.4), pepsin (3.4.4.1) rennin (3.~ .3) fungal protease, lactase (3.2.1.23), etc., are immobilized by covalent attachment to a polymeric material which is attached to an organic or inorganic solid porous support. This method may also be combined with the afore-said 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 be-tween the enzyme and the carrier support are often quite weak, although some prior art has indicated that relatively stable conjugates of this type have been obtained when the pore size of the support and the spin diameter of the enzyme are correlated.
~lowever, the pore size of the support cannot exceed a diameter of about 1000 Angstroms. In view of this weak bond~ the en-jl/ J C ~, 3~358538 zyme is often readily desorbed in the presence of solution~ of the substrate being processed. In addition to this, the enæyme may ~e partially or extensively deactivated due to its lack of mobility or due to interaction between the support and the ac-.~ ~ .
tive site of the enzyme. Another process which may be employedis the entrapment o enzymes in gel lattices which can be ef~ec-ted by polymerizing an aqueous solution or emulsion containing the ~onomeric form of the polymer and the enzyme or by in~orpox-ating the enzyme into the preformed polymer by various techniques, often in the presence of a cross-linking agent. ~hile this meth-od 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 en-zyme, it also has disadvantages in that the conjugate has poor mechanical strength, which results in compacting when used in ~olumns in continuous flow systems, with a concomitant plugging of the column. Such s~stems also have rather ~tide 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 sufficiently small so that large diffusional bar-riers 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 lmmobilizing an enzyme b-y intermolecular cross-linkage, as already noted, are due to the lack of mobility withresulting deactivation because o~ inability of the enzyme to as-- sume the natural configuration necessary for maximum activity, particularly when the active site is involved in the ~inding process.
.
~5~3538 Covalent hindin~ methods have ~ound wide a~Plications and may be used either a~ the sole immohilization techni~ue or as an integral part of many of the methods already described in which cros~-lin~ingreactions are em~loved. This method is often used to bind the enzyme as well as the sup~ort throu~h a bifunctional intermediary molecule in ~hich the functional qroups of the molecule, such as, for example, q3mma-aminoproPvl-triethoxysilane, are capable of reactinq with functional moieties present in ooth the en~yme and either an organic or inorqanic porous support. A wide variety of reaqents and su~ports has been employed in this manner and the method has the advantaqe of provlding strong covalent bonds throuqhout the conju~ate product as well as qreat activity in many cases. The covalent linka~e of the enzyme to the carrier must be accomPllshed throughfunctional groups on the enzyme which are non-es~sential for its catalytic activity such as free amino arouPs, carboxvl groups, hvflroxyl ~roups, phenolic ~rouPs, su]fhYdryl qrouDs, etc. These functional qrou~s will also react with a wide variety of other functional groups such as an a~dehyae, i~ocyanato, acyl, diazo, azido, anhydro, activated ester, etc., to produce covalent bonds. Nevertheless, this method also often has many disadvantages involving costly reactants and solvents, as well as specialized and costly ~orous suP~orts and cumhersome multi-step procedures, which render the method of preparation un-economical for commercial a~plication.
The prior art is thexefore replete with variou~ methodsfor immobilizing enzymes which, however, in various ways fail to meet the reqairements of inAustria] use. ~lowever, as will ' _fi _ ~L~5~ii38 hexeinafter be discu~seæ in ~reater detail, none of the Prior art compositions eomprise the composition of matter of the present invention which constitutes an inorqanic porous sbpport containing a polymeric material fon~ea in situ from a monomer or preformed polymer, of natural or svnthetic'oriclin, which is entrapped and also adsorhed in Part within the pores of said sunport ana wHich contains f~nctionalized, pendent grou~s ex- : ~
tending therefrom; the enzyme bein~ ~artially adsorbe'~ to the '' ~atrix and also covalently boun~ to the active moieties at or adjacent to terminal portions of the pendent qrouPs, thus Per- ' .itting the freedom of ~.ovement which ~ill enab}e the enzy~e to e~ercise maximum activity. For examDle, Il .S~ Patent No.
3,556,945 relates to enzyme compo.sites in which the enzvme is ..
adsorbed directly to an inorganic carrier such as qlass. U.~.
Patent ~o. 3,519,538 is concerned with enzyme composites in Yhich .
the enzymes are chemically couPled bY ~eans o.f an interme~iary silane coupling a~ent to an inorqanic carrier. ~n similar ' ~
fashion, U. S. Patent ~o. 3,7~3,101'also utilizes an orqano~
silane composite as a b'inding aqent, the enzYme being covalently counled to a ~lass carrier hy ~ean.s of an intermediate sil~ne couplinq aqent, the silicon portion of the cou~linq aqent bein~
attached to the carrier wh.ile the orqanic oortion of the counlinq ' . ' a~ent is coupleA to the enæyme, the com~osition containing ~ .
metal oxide on the surface of the carrier disposéd bet~een the ' .
carrier and the silicon portion of the couPling ac.~ent. In ll. S.
Patent No. 3,821,083 the inert carrier is coated ~7ith a preformed polymer such as polyacrolein which has bonded thereto an enzy7~e.
~lowever, according to most of ~he examples set ~orth in the patentr it is necessary to first acicl hydrolvze the comPosite prior to ' ~05~3S38 the deposition oE the enzyme on the polymer. Another prior art patent, namely, U.S, Patent No. 3,705,084 discloses a macroporous enzyme reactor in which an enzyme is adsorbed on the polymeric surface of a macroporous reactor core and thereafter is cross-linked in place. By cross-linking the enzymes on the polymeric surface after adsorption thereof, the enzyme is further immobilized in part and cannot act freely as in its native state as a catalyst. The cross-link-age of enzymes in effect links them together, thereby pre~
venting a free movement of the enzyme and decreases the mobility of the enzyme which is a necessary prereguisite for maximum activity.
This invention rela-tes to novel compositions of matter comprising immobilized enzyme conjugates. More spe-cifically the invention is concerned with novel compositions of matter which comprise an immobilized enzyme conjugate which consists of an organic-inorganic matrix constituting an inorganic porous support material containing an organic polymeric material which has been formed in situ from a monomer, hydrolyzed polymer, or preformed polymer of synthetic ox natural origin by reaction with a bifunctional monomer containing suitable reactive moieties. Said polymer material is both entrapped and adsorbed in t~e pores of the aforesaid support material, and is further provided with functionalized pendent groups extending therefrom, the functional mo.ieties being located at or adjacent to the terminal portions thereof, and an enzyme which is both covalently bound to said function-alized pendent groups as well as being adsorbed in part on the organic-inorganic ~atrix.
.
.
_~ ~8 .
~05~538 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 combination with other 5 solid materials, are themselves in such a ~ondition where-by they are not water soluble and therefore they may be sub-jected to repeated use while maintaining the catalytic activity of said enzyme. In order to be present in an im-mobilized state, the enzymes must be bound in some manner to a water insoluble carrier, thereby being commerically - usable in a non-water soluble statq.
It is therefore an object of this invention to pro-vide novel compositions of matter in which enzymes are present in an immobilized state.
A further object of this invention is to provide compositions of matter in which an enzyme is both adsorbed on an organic-inorganic matrix and covalently bound to functional-ized pendent groups, attached to said matrix, which is, in turn, aiso both adsorbed and ~ntrapped in the pores of the - 20 inorganic porous support material.
- In one aspect an embodiment of this invention re~
sides in an immobilized enzyme conjugate comprising an com-bined organic-inorganic matrix consisting of an inorganic porous support containiny an organic polymeric material ad-sorbed and entrapped in the pores of said support, said polymeric material containing functionalized pendent groups, and an enzyme adsorbed to said matrix and covalently bound to the functional moieties of said pendent groups of said organic polymeric material at or adjacent to the terminal portions thereof.
_g_ .,. , ~ - '' '''''I
~OS853B
A specific embodiment of thi9 invention is found in an immobilized enzyme conjugate comprising an organic-inorganic matrix consisting of a low bulk density, porous silica-alumin support of relatively high surface area which may also contain inorganic additives and an in situ~prepared tetraethylenepentamine-glutaralaehyde polymeric material which is adsorbed as well as e~ntrapped in the pores of sai~
h3 silica-alumina, and an enzyme comprising glucoamy~ase~bein~
covalently bound to the glutaraldehyde pendent groups of the polymeric material at or adjacent to the terminal por-tion of said groups as welI as being adsorbed in part on the matrix.
Other objects and embodiments will be found in the following further detailed description of the present invention.
As hereinbefore set forth the present invention is concerned with immobilized enzyme conjugates comprising a combined organic-inorganic matrix consisting of an inor-ganic porous support material containing an organic polymeric material adsorbed and entrapped in the pores of said inor-ganic porous support. In addition, the polymeric material will contain pendent groups, said pendent groups having an enzyme being covalently bound to said groups at or adjacent to the terminal portions thereof and, in addition, said enzyme is also partially adsorbed on said matrix. In con-tradistinction to the compositions of matter containing immobilzied enzymes as set forth in the prior art the com-positions of matter of this invention may be prepared by utilizing relatively inexpensive reactants as well as ~ . ~.
~058538 utilizing more simple steps in the procedure for preparing said compositions. In addition, the mechanical strength and stability of the enzyme conjugates of the present in-vention will be greater than that wnich is possessed by the immobilized enzymes of the prior art. There~ore, it is readily apparent that the compositions of matter of the present invention possess economical advantages which are useful for industrial applications.
The compositions of matter of the present invention may be prepared in a relatively simple manner. In the pre-ferred-method of preparation, the inorganic porous support material will be treated witn a solution, preferably aqueous in nature, of a bi- or polyfunctional monomer, a polymer hydrolysate or a preformed polymer, following which the unadsorbed solution is removed by any means known in the art as draining, etc. It is also contemplated that other in-expensive organic solvents such as acetone, tetrahyarofuran, etc., may also be used as the carrier for the aforementioned monomers or polymers. Following the removal of the unad-sorbed solution, the wet porous support is then contacted with a relatively large excess of from 5 to 20 mole percent of a second bifunctional monomer in which the reactive groups - are preferably separated by a chain containing from ~ to 10 carbon atoms, this second bifunctional monomer also being added in an aqueous solution, whereby a polymeric matrix which is both adsorbed and entrapped in the pores of the support will be formed and from which pendent groups of the second monomer will extend. These pendent groups will con-tain unreacted functional moieties due to the fact that an excess amount of the second bifunctional monomer was employed .
-11- , ' , ~LIt)5~353~ `
in treating the support. The unreacted functional moieties are then available for covalent binding to the enzyme, which is added to the resulting organic-inorganic matrix, again usually in an aqueous solution. After removal of the un-reacted materials such as by treating, wahsing, etc., theenzyme, besides being covalently bound to the pendent fun-ctionalised groups at or adjacent to the terminal por~ions - thereof, will also, in par~ be adsorbed to the matrix. It is therefore readily apparent that the entire immobili-sation procedure can be conducted in a simple and inex-pensive manner utilising an aqueous or inexpensive solvent media, the procedure being conducted over a temperature differential which may range from subambient (about 5C.) up ~o elevated temperatures of about 60C., and preferably at ambient ~about 20-25C.)temperature, said procedure be_ng effect by utilising a minimum of operating steps and, in addition, permitting a ready recovery of the excess reactants and finished composition of matter.
Many of the inorganic supports reported in the prior art specify "controlled pore" materials such as glass, alumina, etc., having a pore diameter of from 500 to 700 Angstroms for abou 96 ~ of the material and a maximum pore diamter of lO00 Angstroms,a surface area of 40-70 m2~gm and 40-80 mesh size particles. In addition, these supports may be coated with metallic oxides such as zirconium oxide and titaniu~ oxide for greater stability. In contradistinction to these sup-ports, it is contemplated within the scope of this inven~
tion that the inorganic porous supports which are utilised hcrein, will constitute materials which possess pore dia-metres ranging from lO0 Angstrom up ~o SS,oO0 Angstroms ~ 58538 with as much as 25-60% of the porous support material possessing pores having diametres above 20,000 Angstroms and surface areas ranging from 150 to 200 m2/gm. The particle size may also vary over a wide range of from 10-20 mesh to a fine powder, said particle size depending up~n the particular system in which the~ are to be used. It is also contemplated that the porous support materials may be coated with various oxides of the type hereinbe- -fore set forth or may have incorporated therein various other inorganic materials such as boron phos~hate, etc.;
- these inorganic materials imparting special properties to the support material. A particularly useful ~orm of sup-port will constitute a ceramic body which may have the type of porosity herein described for materials of the present invention or it may be honeycombed with connecting macro size channels throughout, such materials being com~
monly known as monoliths, and which may be coated with various types of porous alumina, zirconia, etc. The use of such a type of support has the par-ticular advantage of per-mitting the free flow of even highly viscous substrateswhich are o~ten encountered in commercial enzyme catalyzed reactions.
The inorganic porous support materials which are utilised as one component of the combined organic-inorganic matrix will include certain metal oxides such as alumina, and particularly gamma-alumina, silica, ~irconia or mix-tures of the metal oxides such as silica-alumina, silica-zirconia, silica-magnesia, silica-zirconia-alumina, etc., or gamma-alumina containinq other inorqanic compounds such as boron phosphate, etc., ceramic bodies, etc., as well as combinations of the aforementioned material, one of said materials which may serve as a coating for another material comprising the support.
~05~538 The polymeric materials which are formed in situ in such a manner so that the E,olymeric material is both partially adsorbed and partially entrapped in the pores of the inorganic support of the type hereinbefore set forth may be produced according to the general methods herein- -before described, that is, by first adsorbing a solution containing from 2 to 25~ of a bi- or polyfunctional monomer, polymeric hydrolysate, or a preformed polymer, the monomer or polymer being synthetic or naturally occurring in origin, and which are pre~erably soluble in water or other solvents which are inert to the reactions subsequently employed. As hereinbefore set forth, it is contemplated within the scope of this invention that a second bifunctional monomer is then added in similar manner to form an organic-inorganic matrix by reaction with the original additive adsorbed on the in-organic support. The func-tional groups which are present on the bifunctional monomer wil~ comprise well known reactive moieties such as amino, hydro~yl, carboxy, thiolr carbonyl, etc. moi-eties. As was also hereinbefore set forth, the reactive groups of the bifunctional compounds are preferably, but not neces-sarily, separated by chains containing from 4 to 10 carbons atoms. The reactive moieties are capable of covalently bonding with both the initial additives and subsequently, after washing out unreacted materials, with the enzyme which is to be added in a subsequent step, said enzyme being then covalently bound to the functional group at or adjacent to the terminal portion of the functional chain ' ' " . ' ' .
lQS853~
a~ well as concomitantly adsorbed on the matrix. After addition of the enzyme to this composition, a relatively stable enzyme conjugate will be produced which possesses high activity and high stability. In addition, the com-position of matter of the present invention also possessesappreciable versatility in addition to the other advantages hereinbefore enumerated in that it can be applied to pre-pare conjugates in the absence of an inorganic support which are soluble in either acidic or alkaline media. As will hereinafter be set forth in greater detail, depen-ding upon the reactants employed, the conjugates will re-tain their stability in such media when prepared in com-bination with the inorganic support according to the pro-cesses set forth in the prior art. sy possessing these properties, it .is possible to widen the uses to which these conjugates may be applied.
Specific examples of bi- or polyfunctional monomers, polymer hydrolysates or preformed polymers which may be initially adsorbed on the inorganic support will include water soluble polyamines such as ethylenediamine, diethyl-enetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexamethylenediamine, polyethylene-- imine, etc.; water insolu~le polyamines such as methylene-dicyclohexylamine, methylenedianiline, etc.; natural and syn-thetic, partially hydrolyzed polymers and preformed polymers SUC}I as nylon, collagen, polyacrolein, polymaleic anhydride, alginic acid, casein hydrolysate, gelatin, etc. Some specific examples of intermediate bifunctional materials which may be added to tne above enumerated products to pro-l~S8~38 duce an organic-inorganic matrix and which possess the necessary characteristics hereinbefore set forth include compounds such as glutardialdehyde, adipoyl chloride, sebacoyl chloride, toluenediisocyanate,hexamethylenedi-isocyanate, etc. It will be noted that when a polyeth~l-eneamine of the type hereinbefore set forth is reacted with glutardialdehyde in-the absence o~ an inoxganic porous support, an aqueous acid soluble material is obtained, whereas when a polyethyleneamine is reacted with a di-isocyanate or acyl halide, a water insoluble product is ob-tained. Conversely, if a reaction com~lex without the in-organic support coneains free carboxyl groups, an alka-line soluble complex can be obtained. Due to the large excess of intermediary, or spacer, bifunctional molecules which are used, the polymexic matrix which is formed wi}l contain pendent groups comprising the spacer molecules, said molecules extending from the matrix and having reac-tive moieties available at or ad~acent to the terminal pox- ~ -tions thereof which are capable of reacting with and bind-ing the enzyme to the spacer molecules via covalent bonas.
In addition, the enzyme, when applied after the unreacted reagents have been removed ~rom the organic-inorganic matrix by washing, will also concomitantly undergo adsorption in part with said matrix. Binding the enzyme solely to the organic matrix will not usually affect the de~endency of the solubility of the aggregate on the pll of the solution but when the inorganic support is included as heretoeore des-cribed, the total conjugate exhibits high stability over ~
relatively wide pl~ range from 3 to ~, tne stability of course, .
.
l~S~53~
also being a function of the optimal pH characteristics of the particular enzyme employed as well as the inorganic support used. Therefore, it is readily apparent that a suitable organic-inorganic matrix which is applicable in many situations will be formed with the support material by adsorbing any of the type of materials hereinbefore described which are known to the art and then treated with any bifunctional molecule which is also known to the art and ; is suitably unctionalised to react with the original ad-ditive, provided that a large enough excess of the bi-functional molecule is used to provide pendent groups which are capable of subsequently reacting with ~he enzyme which is desired to be immobilised. By utilising these functional pendent groups as a binding site for the enzymes, it will permit the enzymes to have a greater mobility and thus per-mit 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 heen immobllised by any of the other methods such as entrapment in a gel lattice, ad-sorption on a solid surface or cross-linkage of the enzyme by means of bifunctional reagents, etc. Not all formulations, however, will produce equivalent results in terms of stabili-ty or activity.
Examples of enzymes which may be immobilised by a covalent bonding reaction and which contain an amino group capable of reacting with an aldehydic or isocyana-to moiety of the pendent group which is attached to a polymeric ma-terial entrapped and adsorbed in t,he pores of a porou,s sup- ~
~ port material will include trypsin~p:paln)~ ~exo~l~nase~J(~ ta- J
:
~058S38 "
` galactosidase (3.2.1.23), ficin (3.~.4.12), bromela:in (3.~.4.2~), lactic dehydrogenase ~1.1.1.27), glucoamylase (3.2.1.3)~ chymotxypsin (3.~ .5), pronase, acylase, invertase (3.2.1.26), amylase (3.2.1.1/3.2.1.2) glucose oxidase (1.1.3.4), pepsin (3.4.4.1) rennin (3.4.4.3) f~mgal protease, etc.
In general any enzyme whose active site is not involved in the covalent bonding can be used. While the aforementioned discuss:io~ was centred about pendent groups which contain as a functional moiety thereon an aldehydic or isoc~anato group, it is also contemplated within the scope of this invention that the pendent group can contain other functional moieties capable of reaction with carboxy, sulfhydryl or other moieties usually present in enzymes. However, the covalent bonding of enzymes containing these other I moieties with equivalent results and may also involve apprrciably greater costs in preparing intermediates. It is to be understood that the afore-mentioned listing of porous solid supports, monomers, hydrolysates, polymers and enzymes are only representative of the various classes of compounds which may be used, and that the present invention is not necessarily limited thereto.
The preparation of the compositions o matter of the present invention is preferably effected in a batch type operation as heretofore already described in detail, al-though it is also contemplated within the scope of this invention that the formation of the finished composition of matter may also be effected in a continuous manner of operation. When a continuous type operation is used, a quantity of the porous solid support material is placed in an appropriate apparatus, usually constituting a column. The porous solid support jl/ ~ ~ -18-; ` ` ~
3Ll)5~538 material may be in any form desired such as powder, pellets, monoliths, etc., and i9 charged to the column, after which a preferably aqueous solution of, for example, a poly-functional amine is contacted ~ith the porous support un-til the latter is saturated with the amine solution and thee~cess is then drained. A spacer or intermediary bifun-ctional molecule such as glutardialdehyde is then contacted with the saturated support. The formation of the polymeric ; matrix is thus effected in an aqueous system, said reaction being effected during a period of time which may range from 1 to 10 hours in duration, but is usually of short duration.
After removing the ~xcess glutard;aldehyde by draining and washing out any water soluble and unreacted materials, which in the case of a polyamine is preferably done with a buffer solution possessing a p~ of about 4, an aqueous solution of the enzyme is contacted or recycled through the column, this step effecting a covalent bonding of said enzyme to the terminal aldehydic groups of the functionalised pendent molecules which extend from the matrix. This occurs until there is no further physical adsorption andfor covalent binding of the enzyme to the organic-inorganic matrix and pendent molecules. The excess enzyme is recovered in the effluent after draining and washinq the column. The column is thus ready for use in chemical reactions in which the catalytic effect of the enzyme is to take place. The pro-cedures are, for the most part, conducted within the time, temperature and concentration parametres hereinbefore des-cribed in the batch type procedure and will result in - comparable immobilised enzyme complexes. It is also con-' .
, .. ~
~5~53J!3 templated within the scope of this invention that with suitable modifications of pH and temperature parametres w~ich will be obvious to those sXilled in the art, the process may be applied to a wide vaxiety of inorganic porous supports, polymer forming reactants and enzymes.
The following examples are given for purposes of illustration of the novel compositions of matter of the present invention and to methods for preparing the same. -~lowever, these examples are given merely for purposes of illustration and it is to be understood that the present invention is not necessarily limited thereto.
EX~MPLE ~
In tbis example 2 grams of a porous silica-alumina composite which contained boron phosp;late incorporatedthere-in having a particle size of ~0-80 mesh, a pore diametre ranging from about 100 to about 55,000 Angstroms and a sur-face area of about 150-200 m2/gm was utilised as the in-organic support for the novel composition of matter of the present invention. This support was calcined at a temper-ature of about 500F. to remove any adsorbed moisture con-tained tnerein. Thereafter the support was treated with 25 - ml of a ~ aqueous solution of tetraethylenepentamine at ambient temperature for a period of 1 hour in vacuo to facilitate the penetration of the solution into the pores of the support. The excess unadsorbed solution was then decanted, about 25~ of the tetraethylenepentamine having been adsorbed into the pores of the support. Following this, the wet sup-port was then treated with 25 ml of a 5% aqueous solution of glutardialdehyde at ambient temperature and an almost ~ -20-~LOS~538 immediate reaction took place with the formation of an in-soluble reaction product both on the surface and within the pores of the support. The excess glutardialdehyde sol-ution was then decanted and the organic-inorganic complex was washed to remove unreacted and unadsorbed reagent~, said washing being accomplished first with water followed by washing with a 0.02 molar acetate buffer solution which possessed a pH of 4.2, the washing operation being effected at a temperature of 45C. Therea~er an enzyme solution containing about 200 mg of glucoamylase~per 25 ~1 of water was added and allowed to react with the composite at am-bient temperature for a period of 1 hour. At the e~d of this l-hour period, the excess glucoamylas~As~o~u~l~o~ was decanted and the enzyme conjugate was washed with water to remove any unbound and/or unadsorbed enzyme. The composi-tior was then leached for a period of 24 hours with an ace-tate buffer solution similar to that hereinbefore desoribed.
The amount of adsorbed and/or covalently bonded enzyme was determined by micro Dumas gas chromatography analyses both before and after addition of the enzyme. The activity of the en2yme conjugate was then determined by the amount of glucose produced using 30~ thinned starch solution as sub-strate at a pH of 4.2 and 60C., and employing Worthington's glucostat procedure for analysing glucose, the latter being considered the more reliable procedure for determining the utility of the conjugate. An activity of 28 units per gram of support with an enzyme loading of 29 mg/gm of support was obtained by this procedure (one unit representing the pro-duction of l gram glucose per hour at 60 C. according to the \
~058S38 assay specifications~. It will be noted that despi~e the known solubility at p~l of 4.2 of the enzyme conjugate when prepared in the abse~ce of an inorganic support, negligible loss of enz~me from the combined inorganic-organic complex oacurred during leaching with the 4.2 pH buffer solution.
This was demonstrated by assaying the effluent from this treatment.
EXAMPLE II
In th'is example the procedure of Example I was followed with the exception that the inorganic porous sup-port had a particle size of 10-30 mesh. This silica-alumina composite containing boron phosphate incorporated therein was treated with tetraethylenepentamine, glutardialdehyae ~ and glucoamylasel ln ~a ma L er similar to that set forth a-bove. An active immobilised enzyme complex was obtained although of decreased activity probably because a diffusion problem is produced by the larger particle size of the com-posite.
EXAMPLE III
In a manner similar to that set forth in Example I above, 2 grams of a silica-alumina composite possessing the same physical characteristics of particle size, pore diameter and surface area as that set forth in Example I
was treated with an acetone solution of tetraethylenepen-tamine and followed by a tolunediisocyanate solutionalso in acetone instead of aqueous glutardialdehyde. After de-canting the excess diisocyanate solution and washing with water, the organic-inorganic complex was further treated with an aqueous glucoamylase solution. As in Example I, the finished product comprised an active completely in~
soluble enzyme complex.
, '1 -EXAMPLE IV
To illustrate the point that various concentra-tions of solutions can be used to prepare the desired pro-duct, the procedure set forth in ExampleI above was re-S peated with the exception that more highly concentratedsolutions of the various reagents were used. For example,
2 grams of a 10-30 mesh silica-alumina composite was treated with 25 ml of a 20% tetraethylenepentamine solution and ` after decanting 50 ml of a 25~ glutardialdehyde solution was added thereto. This complex, after washing, was then treated with aqueous glucoamylas~ to prepare an immobilised enzyme conjugate which showed an activity of akout 12 units per gram based on the glucostat test.
EXAMPLE V
- 15 To a silica-alumina composite comprising 2 grams of 10-30 mesh particles was added 25 ml of a 5% aqueous, partially hydrolyzed collagen solution which was in place of the tetraethylenepentamine. After decanting and treating with glutardialdehyde, the organic-inorganlc matr ~ was washed and then treated with a glucoamyla ~solution. The finished composition of ma-tter was treated in a manner similar to that set forth in Example I above by decanting, washing and leaching with a buffered (pH of 4.2) solution to give an immobilised enzyme conjugate which had an activity of about 10 units per gram.
EX~PLE VI
` In this example a silica-alumina composite having a particle size of 10-30 mesh, a pore diametre ranging from about 100 to about 55,000 Angstroms and a surface area of , ~ . ''' '"' ' ' .
~IS1353~
- ~ from about 150-200 m2/gm was treated by adding tetraethylenepentamine in a 1% partially hydrolyzed aqueous collagen solution, the collagen being utilized as an additional bonding agen~. Afte~ draining and reacting with glutartialdehyde, the organic-inorganic matrix was then treated with a glycoamylase (3.2.1.3) solution according to the general procedure of Example I to prepare an active enzyme conjugate.
EX~MPL~ VII
To illustrate that various enzymes can be used in preparing the desired compositions of matter, a silica-alumina composite containing boron phosphate incorporated therein was treated with a tetraethylenepentamine solution, decanted, washed, followed by addition of a glutartialdehyde solution and the resulting composite was then treated with an aqueous lactase (3.2.1.23) solution. This produced an active enzyme conjugate. Similar procedures can be used to bind enzymes such as proteases, glucose isomerase (5.3.1.18), and glucose oxidase ~1.1.3.4) to produce active conjugates.
EXAMPLE VIII
In this example a column possessing an inside diameter of 20 mm contained 1402 grams of an active enzyme conjugate prepared from glucoamylase (3.2.1.3) which was bonded to a 10-30 mesh silica-alumina porous support containing boron phosphate incorporated therein, the conjugate having been prepared in a manner similar to that set forth in Example I above. The column was used continuously for a period of 30 days at a temperature of 45C to hydrolyze an aqueous 30% thinned starch solution which had been buffered to a pH of 4.2. The eEfluent was monitored for the glucose production using the ~ -2~-~L~5853~!3 Worthington glucostat procedure. ~t was found that there was no apparent loss of enzyme activity during this perioa of time and that the percentage of conversion of qtarch to glucose at this temp2rature and at a flow rate of a-5 bout 150 ml per haur was 62%.EXAMPLE IX
To illustrate the fact that various substrates or supports may be utilised to prepare the desired composi-tions of matter, an alumina coated ~onolith which consisted of a Ceramic body honeycombed with connecting macro size channels was treated in a manner similar to that set forth - . in Example I aboYe, that is, the monolith was treated with olutions of tetrae~hylenepentamine, giutar~ialdehyde and a glucoamylase(~en~zyme) the treatment being carried out in a sequential operation which included decanting~ washing, and leaching ~rocedureshereinbefore described. The original ceramic monolith possessed a dry weight of 256 grams, of whlch 13% consisted of an alumina coating. The finished immobilised enzyme conjugate was elaborated into a column within a glass tube having an inside diametre of 70 mm in order that it could be operated continuously by means of a suitable pUmping apparatus within a temperature controlled container, said container being maintained at a temperature of 45C. Over a 40-day period of continuols usage for the hydrolysis of a 10% buffered thinned starch solution, it was found that only about 3~ of the original activity of the enzyme conjugate was lost while maintaining a flow rate of about 85 ml per hour. In addition, it was found that during the 40-day pe~iod th;re was an approximate 80%
L
` ` ` ...
~qD5~31S3~3 co~e~sion of the starch to glucose. In order to further study the properties of the system, subsequent variations in f low rate were made during which it was found that at a flow rate of about 38 ml per hour it was possible to obtain a conversion in the range of from 92~93% of starch to glu-cose. The relatively long period of timeiduring ~hich this enzyme was used to convert starch to glucose without a significant loss of enzyme activity eith~r by desorption or deactivation indicated a long half life of the catalyst.
EXAMPLE X
In this example a monolith type of conjugate and column similar to that described in Example IX above was pre-pared, the exception being that the enzyme which )was used ~ to prepare the~ omplex comprised lactas(e~n p~ace oE gluco-amylase~ ~ ~ é conjugate was tested for stability under acontinuous flow while maintaining the temperature at 3/C.
for a period of 29 days. It was again found that there was no apparent loss of activity of the immobilised enzyme con-jugate. This immobilised enzyme was used in the treatment of a 5% lactose solution which had been buffered to a pH
o 4.2, said lactose solution being charged to the column at a rate of 54 ml per hour. It was found during the 29-day period that there was about 35~ conversion of lactose to glucose and galactose.
EXAMPLE V
- 15 To a silica-alumina composite comprising 2 grams of 10-30 mesh particles was added 25 ml of a 5% aqueous, partially hydrolyzed collagen solution which was in place of the tetraethylenepentamine. After decanting and treating with glutardialdehyde, the organic-inorganlc matr ~ was washed and then treated with a glucoamyla ~solution. The finished composition of ma-tter was treated in a manner similar to that set forth in Example I above by decanting, washing and leaching with a buffered (pH of 4.2) solution to give an immobilised enzyme conjugate which had an activity of about 10 units per gram.
EX~PLE VI
` In this example a silica-alumina composite having a particle size of 10-30 mesh, a pore diametre ranging from about 100 to about 55,000 Angstroms and a surface area of , ~ . ''' '"' ' ' .
~IS1353~
- ~ from about 150-200 m2/gm was treated by adding tetraethylenepentamine in a 1% partially hydrolyzed aqueous collagen solution, the collagen being utilized as an additional bonding agen~. Afte~ draining and reacting with glutartialdehyde, the organic-inorganic matrix was then treated with a glycoamylase (3.2.1.3) solution according to the general procedure of Example I to prepare an active enzyme conjugate.
EX~MPL~ VII
To illustrate that various enzymes can be used in preparing the desired compositions of matter, a silica-alumina composite containing boron phosphate incorporated therein was treated with a tetraethylenepentamine solution, decanted, washed, followed by addition of a glutartialdehyde solution and the resulting composite was then treated with an aqueous lactase (3.2.1.23) solution. This produced an active enzyme conjugate. Similar procedures can be used to bind enzymes such as proteases, glucose isomerase (5.3.1.18), and glucose oxidase ~1.1.3.4) to produce active conjugates.
EXAMPLE VIII
In this example a column possessing an inside diameter of 20 mm contained 1402 grams of an active enzyme conjugate prepared from glucoamylase (3.2.1.3) which was bonded to a 10-30 mesh silica-alumina porous support containing boron phosphate incorporated therein, the conjugate having been prepared in a manner similar to that set forth in Example I above. The column was used continuously for a period of 30 days at a temperature of 45C to hydrolyze an aqueous 30% thinned starch solution which had been buffered to a pH of 4.2. The eEfluent was monitored for the glucose production using the ~ -2~-~L~5853~!3 Worthington glucostat procedure. ~t was found that there was no apparent loss of enzyme activity during this perioa of time and that the percentage of conversion of qtarch to glucose at this temp2rature and at a flow rate of a-5 bout 150 ml per haur was 62%.EXAMPLE IX
To illustrate the fact that various substrates or supports may be utilised to prepare the desired composi-tions of matter, an alumina coated ~onolith which consisted of a Ceramic body honeycombed with connecting macro size channels was treated in a manner similar to that set forth - . in Example I aboYe, that is, the monolith was treated with olutions of tetrae~hylenepentamine, giutar~ialdehyde and a glucoamylase(~en~zyme) the treatment being carried out in a sequential operation which included decanting~ washing, and leaching ~rocedureshereinbefore described. The original ceramic monolith possessed a dry weight of 256 grams, of whlch 13% consisted of an alumina coating. The finished immobilised enzyme conjugate was elaborated into a column within a glass tube having an inside diametre of 70 mm in order that it could be operated continuously by means of a suitable pUmping apparatus within a temperature controlled container, said container being maintained at a temperature of 45C. Over a 40-day period of continuols usage for the hydrolysis of a 10% buffered thinned starch solution, it was found that only about 3~ of the original activity of the enzyme conjugate was lost while maintaining a flow rate of about 85 ml per hour. In addition, it was found that during the 40-day pe~iod th;re was an approximate 80%
L
` ` ` ...
~qD5~31S3~3 co~e~sion of the starch to glucose. In order to further study the properties of the system, subsequent variations in f low rate were made during which it was found that at a flow rate of about 38 ml per hour it was possible to obtain a conversion in the range of from 92~93% of starch to glu-cose. The relatively long period of timeiduring ~hich this enzyme was used to convert starch to glucose without a significant loss of enzyme activity eith~r by desorption or deactivation indicated a long half life of the catalyst.
EXAMPLE X
In this example a monolith type of conjugate and column similar to that described in Example IX above was pre-pared, the exception being that the enzyme which )was used ~ to prepare the~ omplex comprised lactas(e~n p~ace oE gluco-amylase~ ~ ~ é conjugate was tested for stability under acontinuous flow while maintaining the temperature at 3/C.
for a period of 29 days. It was again found that there was no apparent loss of activity of the immobilised enzyme con-jugate. This immobilised enzyme was used in the treatment of a 5% lactose solution which had been buffered to a pH
o 4.2, said lactose solution being charged to the column at a rate of 54 ml per hour. It was found during the 29-day period that there was about 35~ conversion of lactose to glucose and galactose.
Claims (21)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An immobilized enzyme conjugate comprising a combined organic-inorganic matrix consisting of an inorganic porous support containing an organic polymeric material adsorbed and entrapped in the pores thereof, said polymeric material con-taining functionalized pendent groups, and an enzyme adsorbed to said matrix and covalently bound to the functional moieties of said pendent groups of said organic polymeric material at or adjacent to the terminal portions thereof.
2. The immobilized enzyme conjugate as set forth in Claim 1 in which said organic polymeric material is formed in situ from a monomer, a hydrolyzed polymer or a polymer of natural or synthetic origin by reaction with a bifunctional reactive monomer.
3. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said inorganic porous support material is gamma-alumina.
4. The immobilizad enzyme conjugate as set forth in Claim 1 or 2 in which said inorganic porous support material is silic-alumina.
5. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said inorganic porous support material is silica-alumina containing boron phosphate.
6. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said inorganic porous support material is a ceramic body coated with a porous alumina composite and honeycombed with connecting macro sized channels.
7. The immobilized enzyme conjugate as set forth in Claim 1 in which said organic poloymerica material is the re-action product of polyethyleneimine with glutardialdehyde.
8. The immobilized enzyme conjugate as set forth in Claim 7 in which said organic polymeric material is the re-action product of tetraethylenepentamine with glutardialdehyde.
9. The immobilized enzyme conjugate as set forth in Claim 7 in which said organic polymeric material is the re-action product of tetraethylenepentamine with toluenediiso-cyanate.
10. The immobilized enzyme conjugate as set forth in Claim 7 in which said polymeric material is the reaction pro-duct of a mixture of tetraethylenepentamine and partly hydrol-yzed collagen with glutardialdehyde.
11. The immobilized enzyme conjugate as set forth in Claim 7 in which said organic polymeric material is the re-action product of pentaethylenehexamine with glutardialdehyde.
12. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said enzyme is glucoamylase (3.2.1.3).
13. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said enzyme is lactase (3.2.1.23). J
14. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said enzyme is fungal protease.
15. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said enzyme is glucose isomerase (5.3.1.18).
16. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said enzyme is glucose oxidase(1.1.3.4).
17. The immobilized enzyme conjugate as set forth in Claim 1 or 2 in which said inorganic porous support material has pore diameters ranging from 100 to 55,000 Angstroms with as much as 25-60% of the porous support material having diameters above 20,000 Angstroms and surface areas ranging from 150 to 200 m /gm.
18. A method for preparing the immobilized enzyme conjugate of Claim 1, which method comprises treating said in-organic support material with a first solution of a bi- or polyfunctional monomer, a polymer hydrolysate or a preformed polymer; removing the unadsorbed solution; contacting said inorganic support material with a relatively large excess of a second bifunctional monomer solution to form said organic-inorganic matrix; adding an enzyme solution to the resulting organic-inorganic matrix; and removing the unreacted materials.
19. The method of Claim 19 in which said first sol-ution, said second solution, and said enzyme solution are aqueous solutions.
20. The method of Claim 19 in which inexpensive organic solvents such as acetone and tetrahydrofuran are used as carrier for said bi- or polyfunctional monomer, polymer hy-drolysate or preformed polymer.
21. The method of Claim 19 in which said second bi-functional monomer has reactive groups which are separated by a chain containing from 4 to 10 carbon atoms.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US55089875A | 1975-02-18 | 1975-02-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1058538A true CA1058538A (en) | 1979-07-17 |
Family
ID=24199032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA245,940A Expired CA1058538A (en) | 1975-02-18 | 1976-02-17 | Enzyme bound to polymer which is entrapped in inorganic carrier |
Country Status (26)
| Country | Link |
|---|---|
| JP (1) | JPS571988B2 (en) |
| AR (1) | AR215240A1 (en) |
| AT (1) | AT363890B (en) |
| AU (1) | AU497243B2 (en) |
| BE (1) | BE838684A (en) |
| BR (1) | BR7600989A (en) |
| CA (1) | CA1058538A (en) |
| CH (1) | CH634876A5 (en) |
| CS (1) | CS209424B2 (en) |
| DD (1) | DD123346A5 (en) |
| DE (1) | DE2605797C3 (en) |
| DK (1) | DK145104C (en) |
| EG (1) | EG11923A (en) |
| ES (1) | ES445248A1 (en) |
| FR (1) | FR2301533A1 (en) |
| GB (1) | GB1537086A (en) |
| IE (1) | IE42482B1 (en) |
| IT (1) | IT1055978B (en) |
| LU (1) | LU74379A1 (en) |
| NL (1) | NL7601580A (en) |
| NO (1) | NO148600C (en) |
| PL (1) | PL102119B1 (en) |
| PT (1) | PT64790B (en) |
| SE (1) | SE434064B (en) |
| YU (1) | YU37476A (en) |
| ZA (1) | ZA76871B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4335017A (en) | 1975-12-15 | 1982-06-15 | United Kingdom Atomic Energy Authority | Composite materials comprising deformable xerogel within the pores of particulate rigid supports useful in chromatography |
| DE2636206C3 (en) * | 1976-08-12 | 1981-06-04 | C.H. Boehringer Sohn, 6507 Ingelheim | Carrier-fixed enzymes and their manufacture and use |
| CA1128917A (en) * | 1978-10-16 | 1982-08-03 | Ronald P. Rohrbach | Support matrices for immobilized enzymes |
| FR2531452B1 (en) * | 1982-08-05 | 1985-06-28 | Uop Inc | MAGNETIC SUPPORT MATRIX AND IMMOBILIZED ENZYME SYSTEM HAVING APPLICATION |
| US4539294A (en) * | 1982-09-30 | 1985-09-03 | Akzona Incorporated | Immobilization of proteins on polymeric supports |
| MY167240A (en) | 2009-05-20 | 2018-08-14 | Xyleco Inc | Bioprocessing |
| CN117402234A (en) * | 2023-09-27 | 2024-01-16 | 重庆芳禾生物科技有限公司 | Collagen extraction process for removing pepsin residues |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3556945A (en) * | 1968-02-05 | 1971-01-19 | Corning Glass Works | Enzyme stabilization |
| CH538003A (en) * | 1968-03-29 | 1973-01-31 | Anvar | Process for obtaining textile articles carrying enzymes |
| GB1238280A (en) * | 1968-11-06 | 1971-07-07 | ||
| US3715278A (en) * | 1970-02-11 | 1973-02-06 | Monsanto Co | Enzyme-polymer product attached to surface of siliceous materials thereof |
| US3796634A (en) * | 1970-03-19 | 1974-03-12 | Us Health Education & Welfare | Insolubilized biologically active enzymes |
| CH533139A (en) * | 1971-06-21 | 1973-01-31 | Nestle Sa | Process for preparing a product endowed with enzymatic activity, insoluble in aqueous medium |
| US3802909A (en) * | 1971-11-09 | 1974-04-09 | American Hospital Supply Corp | Bonding of organic materials to inorganic particles |
| GB1484565A (en) * | 1972-07-13 | 1977-09-01 | Koch Light Labor Ltd | Binding of biologically active macromolecules |
| GB1444539A (en) * | 1972-09-11 | 1976-08-04 | Novo Industri As | Immobilised enzymes |
| CH579109A5 (en) * | 1973-02-22 | 1976-08-31 | Givaudan & Cie Sa | |
| SE7410542L (en) * | 1974-01-29 | 1976-01-12 | Givaudan & Cie Sa | CONDENSATION PRODUCTS. |
-
1976
- 1976-02-12 PT PT64790A patent/PT64790B/en unknown
- 1976-02-13 ZA ZA871A patent/ZA76871B/en unknown
- 1976-02-13 DE DE2605797A patent/DE2605797C3/en not_active Expired
- 1976-02-16 CH CH187576A patent/CH634876A5/en not_active IP Right Cessation
- 1976-02-17 FR FR7604271A patent/FR2301533A1/en active Granted
- 1976-02-17 AU AU11173/76A patent/AU497243B2/en not_active Expired
- 1976-02-17 SE SE7601757A patent/SE434064B/en not_active IP Right Cessation
- 1976-02-17 PL PL1976187276A patent/PL102119B1/en unknown
- 1976-02-17 CS CS761011A patent/CS209424B2/en unknown
- 1976-02-17 ES ES445248A patent/ES445248A1/en not_active Expired
- 1976-02-17 DD DD191282A patent/DD123346A5/xx unknown
- 1976-02-17 CA CA245,940A patent/CA1058538A/en not_active Expired
- 1976-02-17 BR BR7600989A patent/BR7600989A/en unknown
- 1976-02-17 NO NO760511A patent/NO148600C/en unknown
- 1976-02-17 GB GB6118/76A patent/GB1537086A/en not_active Expired
- 1976-02-17 YU YU00374/76A patent/YU37476A/en unknown
- 1976-02-17 DK DK63776A patent/DK145104C/en not_active IP Right Cessation
- 1976-02-17 NL NL7601580A patent/NL7601580A/en active Search and Examination
- 1976-02-18 IT IT20283/76A patent/IT1055978B/en active
- 1976-02-18 BE BE164417A patent/BE838684A/en not_active IP Right Cessation
- 1976-02-18 JP JP1608176A patent/JPS571988B2/ja not_active Expired
- 1976-02-18 AR AR262289A patent/AR215240A1/en active
- 1976-02-18 LU LU74379A patent/LU74379A1/xx unknown
- 1976-02-18 IE IE317/76A patent/IE42482B1/en unknown
- 1976-02-18 EG EG101/76A patent/EG11923A/en active
- 1976-02-18 AT AT0116176A patent/AT363890B/en not_active IP Right Cessation
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