CA1200519A - Process for the production and immobilization of modified proteins - Google Patents

Abstract

ABSTRACT

Disclosed is a process for chemically modifying a native protein to produce a biologically active enzyme-like modified protein. In one embodiment, a native protein is partially denatured in the presence of an inhibitor of a model enzyme whose activity is to be modeled. The partially denatured native protein-inhibitor complex is deposited on a solid support and cross-linked to produce a new enzyme-like modified protein. The modified protein shows the enzymatic catalytic behavior characteristic of the model enzyme.

Description

~20~

This invention relates to a process for chemically modify-ing a native protein to produce a bioloaic~lly active enzyme-li~e modified protein.
Proteins are biologically synthesized macromolecules having various roles in living systems. Enzymes are particular va~iet es of biologically-active proteins which catalyze ~pecific reactions. Presently, enz~me technology is used in many areas in ind~stry and research, such as, or example, med~cal research, food processing and preservation, the ~roduction o~ ~ermented beverages, the production of pharmaceuticals and the analytical determination of the concentration of various metabolites and food components by ar~alytical en~yme techniques.
En.ymes are highly specific in their biological activity and gener~lly catal~ze a particular reaction at a very ~i~h rate compared to the corresponding reac~ion occurring at room temperature without biological catalysis~ One enæyme may show catalytic activity with respect to a number of substrates upon ~hich it can act. Accordingly, a given enzyme may catalyze the ~ynthe3is or degradation of more than one substrate. Some proteins which are not considered classical enzymes, such as bovine serum albumin, show very limited catalytic activity with LeSpect to one or more substrates.
~ any enzymes are found in nature in very small quantities~
Acc~r;3ingly, their isolat;on, purification and use is limi~ed to a small scale operation in view of the expen~e and ~ime needed ~' -~, `.

:~2(~ 5~3 to isolate them in ~ useful Eorm.
Some enzymes occur in natu~e in relatively large quantities and are relativel~ easy to isolate, purify an~ use.
Unfortunately, due to the precise catalytic behavior of the enzymes, the enzymes available in large quantities can only catalyze certain select reactions.
Much effort has been directed in the recent past toward the synthesis of synthetic biological catalysts ~hich exhibit enzymatic behavior similar to the enzymatic behavior exhibited by native enzymes which are either scarce or expensive to isolate. Further, some attempts have been made to modify native enzymes to change their enzymatic specificity so that they may function to catalyze a reaction which they previously could not catalyze.

One technique known ~o achieve enz~me behavior to catalyze a specific desired reaction is the synthesis of so-called enzyme model molecules. For example, low ~olecular weight compounds may be covalently bonded to functional groups which exhibit the activity of the active site of an enzyme. Examples of such preparations are described in the publications: Breslo~, R.
Advances in Chemistry Series , R. ~ Gould, Ed., American Chemical Society, Washing~on, D. C. 21-43 (1971~ and Tang, C.
C.; Davalian, D.; Ha~ng, P. and Breslow, R., J. Amer. Chem.

Soc~ , 100, 3918 (1978) and Breslow, R., ~oherty, J.~ Guillo~
G. and Lipsey, C., J~ Amer. Chemv Soc., 100, 3227 ~1978)v Another technique involves the use of a syntheti~ polymer matrix w~ich is modified alon~ its backbone to provide functional groups which exhibit the function oE the active site ~`' ' , 5~9 o~ a given enzyme. ~xamples oE such techniques can be found in the following articles: Wul~f, G. and Schulza, I., Israel J.
Chem. , 17, 291 (1978) and Suh, J. and Klot~, I. M., Bioorganic , 6, 165 (1977).
Another technique involves the attachment of a new chemical moiety to a native enzyme near the active site of the enzyme to attempt to cause such enzyme to react with a different catalytic activity. One example of this is the conversion of papain, a proteolytic enzyme to an oxidase t~pe enzyme by the covalent attachment of a flavin near the active site of the native papain enzyme, as illustrated in the articles: Levine, El. L. and Kaiser, E. T., J. Amer. Chem. Soc. , 100, 7670 (1978), Kaiser, E. T., et al, Adv. In Chemistry Series , No. 191, Biomime~ic Chemistry, 1930; and Otsuki, T.; Nakagawa, Y. and Kaiser, E. T., J.C.~. Chem. Comm. , 11,457 (1978). Other examples of such enzymatic modification may be found in the article: Wilson, M.E.
and Whitesides, G. M., J. Amer. Chem. Soc. , 100, 306 ~1978).
Still another attempt to change enzyme specificity is the immobilization of a n~tive enzyme into ~ gel matrix. For example, trypsin enzyme has been immobilized in polyacrylamide gel. The polyacrylamide gel allows amino acid esters to diffuse through the gel matrix to react with the enzyme but will not allow larger proteins to diffuse through. Thus, the enzyme specificity is changed by eliminating access of one of the ~ubstrate molecules to the enzyme.
The immobilization of native enzymes is well established in the art. Also~ examples of enzyme specificity changes by immobilization are known in the art. Both immobiliza~ion and enzyme speci~icity changes are described in the Kirk-Othmer Encyclopedia of Chemical Technology , 3 Ed., 9, 14B (19Bn) ~t~5~

published by Wiley and Son, Inc.
Two other methods relating to enzyme immobilization are disclosed in U. S. Patents 3,802,997 and 3,930,950. In U. S.
Patent 3,802,997, a metl~od of stabilizin~ enzymes by bondin~ the enzymes to inorganic carriers, in the presence of their substrates, whereby the enzyme is immobilized, is disclosed. In U. S. Patent 3,930,950, a method of enzyme immobilization is disclosed wherein an active support member is provided ~hich is capable of reacting with an enzyme to become che~ically bonded thereto. Subsequently, the active support is contacted with an enzyme-substrate complex which has been formed by mixing toyether an enzyme and a specific substrate, while minimi2ing the transformation of substrate to product. Thus, the enzyme component of the complex becomes chemically bonded to the support member.
Also, it has been kno~n that a native lysine ~ono-oxygenase can be reacted to block the sulfhydryl groups on the enzyme.
When the specific enzyme lysine mono-oxygenase is so treated, it shows new catalytic activity toward amino acids and catalyses oxidative deamination instead of its natural oxygenative decarboxylation. However, the reporters cannot account ~or the modiied behavior. See the article by Yamauchi, T~; Yamamoto, S. and Hayaishi, O., in The Journal of Biological Chemistry , 248, 10, 3750-3752 (19733. Also, it has been reported that by reacting a native enzyme, for example trypsin, with its natural inhibitor, ana subse~uently cross-linlsing the enzyme, its activity with respect to its natural substrates can be modified. See the article by Beaven, G. H. and Gratzer, W. B.
in Int. J~ Peptide Res. , 5, 215-18 (1973).
~lso, synthetic proteins have been synthesized by the . .~ _ ~3~S~

anchoriny of an amino acid residue on a solid suppor-t and subsequently adding amino acid residues one after ano-ther.
Further, semisynthetic proteins have been synthesized by a method wherein a native protein is subjec-ted to limited hydrolysis to produce protein fragments. The fragments of the native protein are then subjected to a process whereby one or more amino acid residues are added or removed from the fragments to form modified fragmen-ts. The resultant modi-fied fragments are then reattached to form the semisynthetic protein with an altered amino acid residue composition. Ex-amples of the synthetic and semisynthetic protein technologies cited immdiately above are found in the book Semisynthetic Pro-teins by R.E. Offord, published by John Wiley and Sons Ltd., copyrighted in 1980.
While these techniques are suitable for many applications, a need exists for a simple, efficient, economical and syste-matic method for chemically modifying an inexpensive and commer-cially available native protein to produce an enzyme-like modified protein. The protein can show a catalytic enzymatic activity with respect -to a desired chemical reaction which was not previously a commercially-useful reaction catalyzed by the native enzyme and which new reaction can be predetermin-ed in a systematic fashion. The methods disclosed in the above-disclosed references simply subject an enzyme to a set of conditions and attempt to eludicate its behavior. They fail to provide a systematic method to modify protein charac-teristics.
Accordingly, one aspect of the present invention provides a process for chemically altering the substra-te specificity of a native protein to produce an immobilized enzyme-like 5 _ , ~

3~5~L~

modified protein comprising, selecting an enzyme to be modeled, partially denaturing a native pro-tein in the presence of an inhibi-tor for the model enzyme to form a partially denatured native prote.in-model enzyme inhibitor complex, contacting the partially denatured protein-model enzyme inhibitor complex with a solid support for a time sufficient and at a tempera-ture sufficient to adsorb and immobilize the partially denatured protein-model enzyme inhibitor comple~ on the solid support and cross-linking the adsorbed immobilized protein-model enzyme inhibitor complex to form an immobilized enzyme-like modified protein.
Another aspect of the invention provides a process for chemicall~ altering the substrate specificity of a native protein to produce an immobilized enzyme-like modified protein comprising, selecting an enzyme to be modeled, adsorbing a native protein onto a solid support to immobilize the native protein, partially denaturing the adsorbed native protein and cross-linking the partially denatured adsorbed protein in the presence of an inhibitor for the model enzyme to form an immobilized enzyme-like modified protein.
A further aspect of the invention provides a process for chemically altering the substrate specificity of a native protein to produce an immobilized enzyme-like modified protein comprising, selecting an enzyme to be modeled, partially denatur-ing a native protein, contacting the partially denatured nativeprotein with a solid support for a time sufficient and at a temperature sufficient to adsorb and immobilize -the partially denatured protein on the solid support and cross-linking the partially denatured adsorbed protein in the presence of an inhibitor for the model enzyme to form an immobilized enzyme-like modified pro-tein.
Thus, the presen-t inven-tion achieves an enzyme-like modi-fied protein by conver-ting a na-turally occurring so-called native - 5b -'5~9 protein to an enzyme-like modified protein ~xhibiting diff~rent characteristics than the native protein starting material.
In one embodiment of the invention, a native protein is partially denatured in the presence of an inhibitor for the predetermined mQdel enzyme, whose activity is to be ~odeled.
Next, the partially denatured native protein, in the presence of the inhibitor of the model enzyme, is deposited on a solid support and cross-linked to deEine a new enzyme-like modified protein conformation which is defined by the inhibitor of the model en~yme and is preserved in an immobilized fashion on a solid carrier or support.
Subsequently, the inhibitor of the model enzyme a~d any excess cross-linkin~ agent are removed from the newly formed, immobilized enzyme-like modified protein to yield a f~nctional, stable, easy to process and isolate analogue to the model enzyme. The modified protein thusly produced exhibits the activity characteristics of the model enzyme.

In attaining the advantages of the present invention, it has now been discovered that a protein can be modified from its native conformation to a modified conformation by practicing the process of the present invention. The new conformational state defines an enzyme-like modified protein.
As used herein, the word "enzyme" is definea as a protein which has well-known catalytic activity toward specific ~ubstra~es. The term l'prvtein" as used herein is defined as generally accepted in the ~rt, to wit, a polypep~ide formed of amino acids to yield a biological molecule~
The process of the prescnt invention comprises chemically S~3 modifying a native protein from one conformation, its natural or native state, to a second conformation, a new rnodified state.
The process produces a new, enzyme-like modified protein which is produced and immobilized virtually simultaneously to yield a stable new enzyme-like modified protein which models one or more of the enæymatic activity characteri~tics of the 6elected model enzyme I have discovered that a native protein can be converted to a modified protein and virtually simultaneously immobilized without any substantial adverse e~fects upon the conversion process or the immobilization process. No adverse steric or other structural problems militate against the preparation of a new enzyme-like modified protein when following my new process.
~n the preferred embodiment of the invention, a native protein is selected which is to be chemically modified to produce the new enzyme-like modified protein analogue of a desired model enzyme. The process of the present invention converts the soluble native pro~ein, which does not possess the desired catalytic activity, namely the enzymatic catalysis behavior of the model enzyme, into a stable, immobiliæed enzyme-like modified protein which mimics or copies the biological catalytic activity characteristics of the model enzyme.
A preferred way of carrying out the novel process of the invention for chemically modifying a native protein to produce an immobilized predetermined enzyme-like modifi~d protein comprises the ~teps of: partially denaturing the native protein in the presence of an inhibitor for the predetermined model enzyme, contacting the inhibitor bound partially dena~red native protein with a solid immobilization support for a time ~Z~)~5~

sufEicient and a temperature sufficient to prcduce an adsorption-immobilized native protein-inhibitor co~plex and su~equently cross-linking the support-adsorbed, partially denatured protein-inhibitor complex to form the new, immobilized enzyme-like modified prote.in. Any excess cross-linking agent and the inhibitor are removed from the newly created immobilized modified protein to isolate the new catalytically active enzyme-like modified product protein.
. While in the preferred embodiment, a non-enzymatic native protein is converted into an en~ymatically reactive, immobilized enzyme-like modified protein, the conversion of any native prote.in to an enzyme-like modified.protein analogue of a model cn~yme is contemplated herein.
As used herein, the phrase "partial denaturation" means a change in the conformation of a protein so as to perturb the : natural s~lape or conformation of the protein without causing the irreversable, gross denaturation of the protein. As used herein, the word "conformation" is defined as generally accepted in the art, to wi.t, that combination of secendary an~ tertiary amino acid structure which produces a characteristic protein shape.
The partial denaturation of proteins is well known in the art and discussed in detail in the following references:

The book Biochemistry by A. L. Lehninger, Worth Publishers, Inc., New YorkJ New York, 1970, pg. 58; the article by P~ L.

Privalov entitled "Stability of Proteins" in Advances in P~otein Chemistry , Vol. 33, pg. 167-192; the article by C.
Sanford entitled "Protein Denaturation, Part C" in Advances in Protein Chemistry ~ Vol. 24, pg. ~-g7; the article by F. R. N.
Gurd, et al. entitled "Motions in Proteins" in Advances in ~_ ~V~S~3 Protein Ch~mistry , Vol. 33, pg. 74-166; the article by O.
Jardetzky in BB~ , Vol. 621, pg. 227-232; the article by R.
Huber in TIBS , Deeember 1979, pg. 271, and the article by D.
S. Markovich, et al. in Molekulyarnaya Biologiya , Vol. 8, No.
6, p~. 857-863.
Also, as used herein, the phrase "denaturing agent" refers to process eonditions or reagents which cause the partial denaturation of a protein. For example, the partial denaturation ~f a protein may be achieved by soaking the protein in an aqueous solution in an elevated temperature, for example, in the range of 25~C to 6Q~C. For most protein, 25-60~C will so perturb the eonformational structure of the protein as to result in the partial denaturation of the protein. However, it is well kno-.~n in the art, some proteins from thermophilie baeterial sourees are stable to near the boiling point of water and would require higher elevated temperatures than those generally -diselosed above.
The partial denaturation o~ a protein can be aceomplished by soaking the pr~tein in an aqueous solution eontaining an inorganie salt, in inorganie or organie aeid or a water-miscible organie solvent.
Suitable inorganie salts whieh served to destabilize the protein strueture for partial denaturation inel~de: NaF, (NH4)2SO4~ (CH3)4Ncl~ (CH3)4~Br, KCH3COo, NH4Cl, KCl, NaCl, CsCl, LiCl, KBr, NaBr, KNO3, MgC12, NaNO3, CaC12, RSCN, NaSCN, BaC12, NaI and LiI.
Suitable inorganie aeids inelude: hydroehlorie, nitric, sulfuric, phos~horie and similar proton donating strong inorganic aeids.
Suitable organic acids include: acetict formic, propionic _g_ . . . . , . _ .

s~

and citric acid.
Suitable water-miscible solvents for protein denaturation include: t-butanol, acetonitrile, dioxane, acetone, methanol, ethanol and dimethylsulfoxide.
As used herein, the term "inhibitor" means any compound with sufficient structural similarity to the natural substrate oE a model enzyme to serve as a template for the catalytic site of the enzyme-like modified protein. In the preferred embodiment oE the preparation of an enzyme-like modified protein, the inhibitor is any of the known classical inhibitors ~or a given model enzyme. ~owever, as used herein l'inhibitor"
can include any molecule with sufficient structural similarity to the classical inhibitor to preserve an inhibitor like site on the modified protein. The natural substrate of the model enzyme can act as the inhibitor or templa~e for the modified protein in many cases. Inhibitors are generally not degraded by the enzyme, as are substrates, and serve to more readily preserve a catalytic site than the natural substrate. One example of the str~ctural simi~arity of an enzyme inhlhitor ar.~ the natural

2~ subs~rate of an enzyme is ~he case of glucose oxidase. Glucose is the natural substrate of glucose oxidase ~hile D-glucal is the inhibitor for glucose oxidaseO Glucose and D-glucal are very structura]ly similar.
In the preferred embodiment of the present invention, the ~orming enzyme-like modified protein is immobilized ~n a solid support which is usually inorganic in composition. Particularly pre~erred are inor~anic water insoluble supports such as refractory ceramic oxides. Suitable ceramic oxides include porous, particulate ceramic oxides which can be formed by cvmpacting and sintering refractory ceramic oxide powders such :~Z~Sl~

as alumina powder, zirconia powder, magnesia po~fler, silica powder and thoria powder. Alumina powder is particularly preferred due to its chemical durability and lo-~ cost. The preparation and use of such ceramic alumina and other ceramic oxide supports such as disclosed in United States Patent 4,001,085.
As used herein, the term "cross-linking" means the formation of covalent bonds between reactive sites on a protein.
Generally, protein cross-linking is accomplished by the us~ of multifunctional reagents such as glutaraldehyde. Other examples of suitable cross-linking reagents to effect the cross-linking of a protein areo 2-amino-4, 6--dichloro-s-triazine, diazonium salts; N-hydroxysuccinamide; p-benzoylazide and those reagents disclosed in the followin~ re~erences: Wold, F., Methods Enzymol , 11, edited by C; H. W. ~irs, C~HoW~ ~ A~ademic Press~ 1967, 617; Fasold, H. et al., Augen. Chem. Int. Ed.
Enyl. , 10, 795, 197 and Keyes, M. H., in the Kirk-Othmer Encyclopedia of Chemical Technology , 9, 3rd Ed., 1980, J. Wiley & Son~, Inc r ~ 148- - 72~
~ In an alternative embodiment of cross-linking of the partially denatured protein, ~he cross-linking of the protein a~ter it has been partially denatured and subjected to inhibitor contact may be achieved by disulphide rearrangement when the native protein being converted to an enzyme-like modified protein analogue of a model enzyme is rich in disulphide bridges. ~uch disulphide rearrangemen~ is accomplished by subjecting the native disulphide bridge rich protein, at about neutral pH, to various reagents to break the disulphide bridges to yield sulphydryl groups. A preferred reayent is beta-mercaptoethanol. The beta-mercaptoethanol ?5~9 cleaves the disulphide bridcJes, thereby loosening the conformational structure of the protein and partially denaturing the protein by the formation of sulphydryl groups. After the protein hac been subjected to contact with the inhibitor of the model enzyme, the sulphydryl groups may be reduced to the disulphide form to relink the protein into a new, stable enzyme-like modified protein. Such relinking of sulphydryl groups in disulphides may be easily accomplished by raising the sulphydryl containing protein to an elevated pH. A pH value of between 9 and 10 is usually quite acceptable. It should be noted, however, that molecular oxygen is usually a reactant in a sulphydryl reaction to form disulphide bridges so the hiyh p~ reaction should be carried out in the presence of molecular oxygen. Other oxidizing agents which are known to oxidize sulphydryl ~unctions to the corresponding disulphide are equally operative.
In the preEerred embodiment of the invention, a native or host protein showing little or no catalytic activity is con.erted chemically by the process of the present invention into an enzyme-like modified pro~ein analogue of a model enzyme. Many enzymes are susceptible to modeling by the present process to produce their enzyme-like modified protein analogues from selected native protein starting materials. Examples o~

6uch model enzymes which are subject to enzyme-like modified protein analogue production are hydrolytic enzymes, redox enzymes and transferase enzymes. By way of example: The first group, hydrolytic enzymes include proteolytic enzymes which hydrolyze peoteins, e.g., papain, ficin, pepsin, ~rypsin, chymotrypsin, bromelin, keratinase; carbohydrases which hydrolyze carbohydrates~ e~g., cellulase~ amylase, maltase, ~2~6~53~

pectinase, chitanase; esterases which hydrolyze esters, e.~., lipase, cholinesterase, lecithinase, alkaline and acid phosphateases; nucleases which hydrolyze nucleic acid, e.g., ribonuclease, deoxyribonuclease; and amidases which hydrolyze amines, e.g., arginase, asparaginase, glutinase, histidase, and urease. The second group are redox enzymes that catalyze oxidation or reduction reactions. These include glucose oxidase, xanthine oxidase, catalase, peroxidase, lipoxidase, and cytochrome reductase. In the third group are transferase 1~ enzymes that transfer groups from one molecule to another.
Examples of these are glutamicpyruvic transaminase, glutamicoxalacetic transaminase, transmethylase, phosphopyruvic transphosphorylase.
In the usual practice of the present invention, one selects a first or model enzyme. One then selects a second native host protein to be modeled after the model enzyme to produce an enzyme-like modificd protein. In many cases the native protein is itself enzymatically active since many common enzymes are available in large quantities at fairly low costs in homogeneous sample form. However, nonenzymatic proteins are equally useful when they can be purified for use with the present process. One example of such a nonenzymatic protein which may be used as a native protein for the starting material of the bovine serum albumin (BSA), BSA is available in relatively pure form at - 25 fairly low cost from numerous sommercial sources.
; By practicing the process of the present invention, one can cust~m-tailor the native protein to a different immobilized, ~table enzyme-like modified protein form which shows the catalytic a~tivity characteristics of the enzyme which has been modeled. The ability to custom-tailor a -13~

native protein into a predetermined catalytic activity provides ~reater advantages in a wide range of chemical and industrial situations. ~or example, if one wishes to use an enzyme which is in short supply, is very expensive or very difficult to isolate and/or purify, such an enzyme may serve as a model enzyme for the preparation of an enzyme-like modified protein analogue by the present process to mimic its activity.
The present invention advantageously converts a native protein in a virtually simultaneous conversion and immobilization se~uence of steps into an immobilized, stable easil~ recoverable, modified protein form which mimics the desired characteristics of the model enzyme.
Thus, a native protein which is available in large quantities or at low cost can be reformed or modified by the process oE the present invention to convert the available native protein into an enzyme-like ~odified protein form of a less available and/or more expensive enzyme.
In the preferred embodiment of the invention, a native protein is purified and dissolved in a near neutral aqueous solvent in the presence of a suitable buffer to maintain the solution near neutrality. Subsequently, the native protein is partially denatured by any of the expedients described hereinabove, for example, loweriny the pH or subjecting it to partial denaturation agents, to produce a partially denatured, ~oluble form of the native protein. Subsequently, an inhibitor for the model enzyme is admixed with the partially denatured protein. Next, a support, for example~ a particulate porous alumina material, is admixed with the partially denatured native prot~in in the p~esence of the inhibitor for the model enzy~e. Sufficient ~ime and sufficient temperature are provided ~(3~

for the partially denatured protein-inhibitor complex to form and absorb onto the surface of the porous particulate su~port.
Subsequently, to preserve the new, enzyme-like modified protein, the partially denatured native protein-inhibitor complex must be stabilized.
~he new protein is stabili2ed by cross-lin~ing o~ the protein to produce the enzyme-like modified protein. Often, cross-linking is done as disclosed above by glutaraldehyde cross-linking agent or sulphydryl rearrangement since both are relatively inexpensive to achieve. However, any of the above-described cross-linking agents can be utilized e~fectively in the conventional manner.
In an alternative embodiment of the simultaneous preparation and immobilization of an enzyme-like modified protein, a native, no~partially denatured protein is adsorbed onto the surface, interi~r and/or exterior, of a porous particulate ceramic oxide support. The immobilized, native protein is subsequently subjected to partial denaturation by any of the above-described re~gents. The partially denatured, adsorption immobilized protein is subsequently admixed with the inhibitor of the model ~nzyme to form the partially denatured protein-inhibitor complex. After the partially denatured native protein-inhibitor compler. is formed, already being immobili~ed on the support, cross-linking is conducted as disclosed above.
In yet another embodiment of the invention, it has ~een ~iscovered that the native protein may be partially denatured, adsorbed onto a porous particulate support~ and thereby mmobilized, with subsequent contacting of the inhibitor to ~he partially denatured immobilized protein. Subsequently, the partially denatured native protein-inhib;tor complex is cross-3Lf~ 5~

linked to produce a new enzyme-like modified protein.
In still another embodiment of the invention, it has been discovered that the stable, immobilized, enzyme-like modified protein of the present invention may be produced by admixing together a partially denatured native protein with the innibitor of the model enzyme to form a soluble partially denatured native protein-inhibitor complex. Subsequently the complex is adsorbed onto the support. Thereafter, the complex is cross-lin~ed to produce the new, enzym2-like modified protein according to the present invention.
It has been discovered according to the present invention tha~ it is possible to prepare an enzyme-like modified protein in still another embodiment. It has been discovered that a native protein may be reacted with succinic anhydride, at low pH, for example about pH 4, to produce new negatively charged carboxylic acid sites on the protein. The carboxylic acid sites which are produced by succinic anhydride reaction are formed by the reaction of the anhydride function on the succinic anhydride with ~ree ~mine sroups on the protein. Tl;is reaction _onverts positive amino groups on the protein to negative carboxylic acid moieties. This conversion is advantageous since generally ceramic oxide supports, the pre~erred supports of the invention~
at low pH are positively charged. By forming more negatively charged yroups on the surface of the native protein, a greater the electrostatic interaction then exists between the support and the protein, which is to be immobilized~ thereby assisting in the formation and maintenance of protein immobilization.

Also, it has been discovered that enzyme~like modified protein prodllction can be enhanced by the use of various bridging groups to span between carboxylic acid residues 5~

on the protein to help cross-link p~otein (whether the caebo~yl groups are native to the protein or are anhydride derived) to form the new, stable, enzyme-like modified protein structure.
One such bridgin~ group is diaminopropane-HCl in the presence of a carbodiimide. Suitable carbodiimides include ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Diaminopropane supplies amine groups which react with the native or newly formed, succinic anhydride derived, carboxyl groups on the native protein to produce covalent bonds. Accordingly, the succinic anhydride created carboxyl groups on the protein serve not only to electrostatically anchor the protein to the support but a portion of such groups also provide reactive sites to cross-link the protein by reacting with the diaminopropane amino groups.
Also, it has been discovered that enzyme-like modified protein production is enhanced by the use of carbodiimides.
Such carbod;imides r~act with protein amine and carboxylic acid f~lnctions to produce covalent bonds. Such peptide bond ~ormation assists in the cross-linkin~ of the newly formed, modified protein to stabilize their structure.
It has been discovered according to the present invention that it is not particularly critical to admix succinic anhydride and the diaminopropane in any particular order with the native protein. For example, the native protein may be reacted with succinic anhydride to produce carboxyl groups, immobilized, reacted with diaminopropane and subsequently reacted with a carbodiimide to produce an cross-linked enzyme-like modified protein. Alternatively, the native protein may be reacted with the succinic anhydride, subsequently with the diaminopropane, then immo~ilized and subsequen~ly reacted with ~he carbodiimide.

~17-~20~P5~

Also, the native protein may be paetially denatured, contacted with the inhibitor, subsequently immobilized and then in a one step process reacted with a succinic anhydride and carbodiimide cornbination reagent to prod~ce the cross-linked new enzyme-like rnodified protein.
The process of the present invention produces a new, enz~me-like modified protein wlich exhibits a number of advantages and uses~ By the discoveries of the present invention, an immobilized, enzyme-like modified protein can be p~oduced which is stable, easily recoverable and recyclable and e~:hibits a new enzyme-like catalytic activity w'nich was not presenL in the native protein. Such modified proteins showing ~nz~.ne-like catalyt c behavior are useful to perform catalytic anabolic and catabolic reactions instead of a naturally occurring enz~ne.
In all embodiments of the present invention the inhibitor of th~ model enzyme is removed after synthesis of the enzyme-like mo~ified protein. Typically repeated washings of the i~mobilized m~dified ~rotein is su~Eicient to remove the inhibitor. ~uffered aqueous solution can also be used to remove the inhibitor, such buffers are exemplified hereinafter.

Other embodiment~ of the present invention will be apparent to those of ordinary skill in the art from a consideration of this specification or practice of invention disclosed herein.

It is intended that ~he Examples in the specification be considered as exemplary only with the scope and spirit of the inver,~ion being indica~ed by the claims~ The following ~xa:nples ~Z~3~5~3 are exemplary of the various embodiments of the process oE
the present invention discussed hereinabove.

Ten g of KimalTM (-80~100 mesh) porous particulate alumina immobilization support from Owens-Illinois, Inc. is washed three times ~7ith distilled water.
Porous, inert, rigid, dimensionally stable refractory fluid permeable support particle can be prepared by compacting such refractory oxide powders to form a "green compact" of the desired eonfigura'ion. The green compacts are then fired for a time and at a temperature sufficient for sintering to yield porous, inert, rigid, dimensionally stable, fluid permeable refractory particulate support. The sintering should not be at ~ temperature or for a time which would cause collapsing or coalescence of the particles to form a non-porous body. A
convenient indication of the degree of sintering is a comparison of the actual density of the fired compact as compared to the theoretical density of the oxide being fired. O~ the many oxides whi~h can be u~ed for the ~r~52nt purposes, alumina i~ preferred ~or its chemical durability and ease of fabrication.
In forming the particulate support from the powdered refractory oxide, the powdered particle size is selected to yield a sintered compact having a porosity and pore si~e in the desire~ range. The techniques for compaction and sinteriny of th~ porous supports are well-~nown in the art and form no part of the present invention. 5uffice it tc say that co~pacting pressures in the range o~ 1,000 p.s~i. to 1~,000 pvs.i. and sintering temperatures in the range of 1~300 to 1,700C. are ~omrnercially expedientO Additional details on compacting and sintering of re~ractory oxides can be obtained from the boo~

5~''3 "Oxide Ceramics" by E. Ryshkewitch, published in 1960 by Ac~demic Press, New York, N. Y.
Next, 0.5 g of bovine serum albumin (BSA) from Sigma Chemical Company (Sigma) Type A-6003, lot 110F - 9305 is ~issolved in 50 ml of 1 mM (ionic strength) 2-amino-2 (hydroxymethyl)-1,3-propanediol (tris) buffer at pH 7.5. The 50 ml of solution is added to the washed alumina support with a resulting pH of 7.5. The absorbance at 280 nm is 6.62. Then, 0.5 ml of a 0.2 M solution of beta-mercaptoethanol (BME) is added to the solution above the KimalTM alumina support and the resulting solution is shaken for 0.5 hours. After 0.5 hours, the p~I is 7. Next 0.333 g of tryptamine inhibitor ~or esterase is added and the resulting pH is 7.2 The solution is then stirred for 0.5 hours and then the pH is raised to 8.7 with 0.01 M IlaOH.
The solution is then shaken for an additional 1.5 hours.
After 1.5 hours of shaking the support with the bound esterase-li~e modified protein is washed five times with 1 mM tr;s at pH
7.5 contair,ing 1~ tryptamine inbibitor an~ 30~ sucrose an~
stored in the same solution until needed.
The esterase-like modified protein peepared above is assayed as follows. Twenty~five ml of 1 mM L-tryptophan solution is pre~ared by dissolviny 0 00~ 9 in distilled water and adjusting to volume in a 25 ml volumetric flask. Then, 25 ml of 0.01 M L-tryptophan methyl ester (TME) substrate is prepared by dissolviny 0.0637 9 in distilled water and adjusting to volume in a 25 ml volumetric flask. One 1 of 1 mM tris buffer is prepared ky adjusting one 1 of distilled water to pH 3 with 1 N
HCl thc-n readjusting to pH 7.5 with tris buffer. Three 1 of 0.03 r~ acetate eluant is prepared by dissolving 12 g of sodium s~

ace~ate trihydrate and 0.2 ~1 of glacial acetic acid in 3 1 of distilled ~ater. ~he resultin~ eluant solution is at pl~ 6.
~he assay is performed on a high pressure liquid chromatograph. The column is packed with porous silica containing carboxyl side chains from Baker Chemical CompanyO
The injected sample is delivered from a 0.02 ml sample loop.
The column retention times for l mM L-tryptophan; 0.01 M TME
and 50/50 mixture of each is determined as standards.
Next a control solution for the assay is pxepared by mixing 1~ 1 ml of 0.1 M TME into 9 ml o~ 1 mM tris buffer pH 7Ø The pH
of the solution is 5.8.
The sample solution of 10 ml is prepared by adding 0.5 to 1 g (dry wei~ht) of the immobilized enzyme-like modified protein prepared above to 9 ml of 1 mM tris buffer pH 9~0 and 1 ml of 0.1 M TME. The pR of the solution is 5.8.
The pH of the sample and control solution must be the same for the chromatographic assay. Generallyl the sample shows a higher pH than the control after being stored in refrigeration for a period OL time. The highe~ pH can be lowered ~y decanting the solution ~rom the solid support material and replacin~ it with new control solution. This is repeated until the pH of the sample and controls are nearly the same. Since the new esterase en~yme-like modified protein is immobilized on the support, no adverse effect is seen by multiple solution ohanges.
Injection of control and sample are made onto the column.
The concentration of L-tryptophan is plotted on the y-axis and time of elution on the x-axis of a graph.
Activity for the support immobilized esterase enzyme-like modified protein i5 calculated according to the following ~ormula.

3~Z~5~ ~3 [(change in S~m) x 10-5~[10~6]110-Activity =
dry weight of support in g 2~6 wherein^
Activity is in Units/ml of support;
change in S is the change in slope in molarity;
~ is the sum standard deviation of the slopes for the sample -and control;
10~6 is micromoles;
10 2 is the volume of sample in 1; 2.6 is the density of the support in g/ml; and U is micromoles/minute The assay results are as follows:
Substrate TME ~U/ml support) Initial ~ctivity 0.00 Final Activity Assay 1 3.9 + 1.9 x 10 ~
Assay 2 5.3 ~ O.Sl x 10 3 Assay 3 8.8 ~ 1.19 x 10-3 Assay 4 4~9 * 1.14 x 10 3 The ~esults show that the ~odified esterase enzyme like protein prepared according to the invention exhibits activity toward esterase substrate TME where no activity is previo~sly detected in the native BSA. This illustrates the conversion oE
one ~enus of nonenzymatic protein~ an albumin, to another genus OL protein, an enzymatically active esterase enzyme-like modified protein.

~3~53~

EX~IrLE 2 Sixty mg of ribonuclease, from Sigrna, No.R 5503 Type lAS, lot 20-F 81001, is diss~lved in 100 ml of distilled water. The resulting solution is at pH 4.7 and has an absorbance at 2~0 nm at 0.313. Next, 0.3 ml of 0.2 M beta-mercaptoethanol disulphide brid~e cleavin~ re~gent is added, with the pH of the solution lowering to 4.4. Next, the p~3 of the solution i5 returned to 7.9 by the addition of 1.3 ml of 0.01 N NaOH and maintained thereat ~or 2 hours. After the two hours, 40 mg of indole inhibitor is added which requires 1.25 hours to dissolve. Also, 3 ul (microliters) of 0.01 N NaOH is added to maintain the pH at 7Ø
Subsecluently, the pH of the solution is raised to 9.45 with the addition of 4.7 ml of 0.01 M NaOH. The solution is maintained at pH 9.45 for three hours which requires the addition of 0.9 ml of the 0 01 M NaOH over the 3 hour period.
I~xt, 20 9 of KimalTM ~-80 ~ 100 mesh)is added to one-half of the above solution. ~ext, 0.~ g of 0.01 N NaOH is added to ~ring the pH of the resulting support-protein mixture to 9.4~.
Next, 0.01 ml of 1 mM succinic acid re~gent (u$ed tcJ relink sulfhydryl groups into disulphide bridges) and 0.025 g of ethyl-3-(3 dimethylaminopropyl)-carbodiimide (EDC~ (cross-linking agent) is added to the solution. The pH of the solution after 15 minutes is 7.15. After the 15 minute period, 5 ml of 5 percent (W~W) EDC is added at a rate of 0.1 ml per min. The solution is ~llowed to react overnight (about 17 hours) on ~
cold lO-5C) shaker~ After the reaction time~ the pH of the solu~ion containing the newly synthesized, cross-linked, immobilized enzyme-like modiied protein is 6~4. ~rhe pE] of the solution is then re~urned to pH 7 by the addition of 0.4 ml of 0~01 N NaOH. The immobilized enzyme-like modified protein is S~

washed 5 times with one m~l tris buffer at pll 7 containin~ 0. O~Q
indole inhibitor. The inhibitor solution is believed to stabilize the desired conformation of the newly synthesized enzyme-like modified protein during storage.
The assay for enzymatic activity of a new synthesized enzyme-like modified protein is perfo~med on a high pressure liquid chromatograph. The analysis is identical to the analysis disclosed in Example 1, above, except that the eluant is 5 mM
tris at pH 8.0 and the substrate is 0.0115 M tryptophan methyl ester ~TME).
The assay results are as follows:
Substrate TME (U/ml support) Initial Activity 0.00 ~inal Activity - after 1 day 0.51 - after 2 days Q.43 The above results demonstrate that the esterase-like modif;ed protein prepared according to the present invention exhibits enzymatic activity ~ith respect to esterase substrate Tt~E. No such activity is previously detected in the native ribonuclease. This illustrates the conversion of one genus of enzyme, a nucleaseJ to another genus of protein/ an esterase enzyme-like modified protein.

~5 Sixty mg of ribonuclease, from Sigma, Type R-5000-IIA, lot llO-F02;'Sl, is dissolved in 50 ml of distilled water. The initial pH of the solution of ribonuclease is 4.6 with a 280 nm - absorbance of 0.~93. After a 15 minute waiting period, 0.1 ml of a 0.1 M BME solution is added with slow stirring at 25 C.
~he pil of the solution is immediately raised to 7.0 with the -2~-~z~s~

addition of 0.1 M NaOH. The resultinc~ neutral solution is stirred for one hour at about 25 C. Next, 10 mg of indole inhibitor is added and the solution stirred for 1.5 hours.
During the stirring period, the pH of the s~lution is slowly raised to 8.2 with the dropwise addition of 0.1 M NaOH. After the 1.5 hour stirring period, the p~l of the solution is slo~ly raised to 9.45 over a one hour period and maintained thereafter for an additional 2 hours with the dropwise addition of 0.1 M
NaOH.
1~ Subsequently, 10 9 of KimalT~ alumina support (-80 + 100 mesh) is added to the partially denatured inhibitor bound enzyme solution. The resulting mixture is placed on a cold shaker at 0-5C. When the temperature of the solution reaches 5C, 0.1 ml o~ 8~ glutaraldehyde solution is added. The mixture is allowed to react overnight ~about 17 hours) at 0-5 C with slow shaking.
The assay for enzymatic activity of the newly synthesized esterase enzyme-like modified protein prepared according to the present invention is per~ormed on a high pre~sure liquid c~rc;~a~ograph. The procedure used in this Example is identical ~ to the procedure used in Example 1.
The assay results are as follows-Sub~trat2 TME (U/ml support) Initial Activity 0.00 Final Activity 0.5~
T~e resul~s show that the esterase enzyme-like m~dified prote.n prepared according to the present invention exhibits enzyma~ic activity with respect to esterase substrate Tt~E. No activity is previously detected in a native ribonuclease. This illustrates the conversion of one genus of enzyme, namely~ a ?5-~z~ 9 ~luclease, to a second genus of protein, n~mely, an esterase enzyme-like modified protein.
EXAMPLE ~
Sixty mg of ribonuclease, from Sigma, lot no. 20F-81001, is dissolved in 100 ml of distilied water. The absorbance is 0.333 at 280 nm, with a pH of 4.6. Next, 0.3 ml of 0.2 M B~IE is added The solution is allowed to stand for 5 minutes. After 5 minutes, the pH of the solution is 4.7. The solution is then adjusted to pH 7 by adding 2~8 m~ of 0.01 N NaOH. The solution is maintained at pH 7 for 2 hours by the addition of 0.01 N
Na~H. The a~dition is occasionally unnecessary due to the natural raise in pH from about 7 to about 7.2. However, if the pH does not ~pontaneously elevate, the ad~ition of the NaOH is necessary. Next, 20 g of washed KimalTM ~-80 ~ 100 mesh~ is added to the solution at pH 7.2. The pH of the solution slowly rose to 7.7 after the addition of the solid alumina support.
The ribonuclease enzyme in the presence of the alumina support, is bound to th~ inhibitor for the model enzyme as follows. About 0.04 g of indole inhi~itor is added to the solution at p~ 7.7. The solution is maintained at pH 7.7 for 15 minutes. Then, the solution is raised to pH 9~45 by the addition o ~0 ml o~ 0.01 ~ of NaOH.
The support bound partially denatured enzyme, in the ; presence of indole inhibitor, is cross-linked by maintaining the ~olution a~ 9.45 for 2 hours at room temperature~ The 2 hour period at elevated pR allows the sulfhydryl groups to rearrange to form disulphide bridges to intramolecularly cross-link the newly synthesized esterase-like modified protein.
After the 2 hour period, the 501ution containing the immobilized esterase~like modified protein is decanted and the -26~

?5~3 support, with the bound new enzyme-like protein thereupon is washed repeatedly with two 1 of a 1 mM solution of tris buffer at pl~ 7 containing 0.0~ indole. The thusly produced solid support bound esterase-like modified protein can be stored under refrigeration in the tris buffer indole solution until used.
The activity of the support immobilized esterase-like modified protein prepared according to the present invention is determined by high pressure liquid chromatography. The procedure use~ to determine the activity of the newly synthesized enzyme-like modified protein prepared according to the present invention used in this Example is the same procedure used in Example 1, above.
The assay results are as ollows:

Substrate TME (U/ml support) Ini~ial Activity 0.00 Final Activity(days) 111.97 + 0.937 x 10-3 35.78 + 1.64 x 10-3 87.56 + 1.90 x 10-3 107.2~ ~ 1.80 x 10-3 118.65 + 1.60 x 10-3 The results illustrate that the esterase enzyme-like modified protein prepared according to the presen~ invention exhibits enzymatic activity with respect to esterase substrate TME. No activity is previously detecte~ in the native ribonuclease. This illustrates the eonversion of one enzymatic genus, namely~ a nuclease, to a second genus of protein, namely, an esterase-lilce ~odified protein.

~z~)~s~

EX~IJPLE 5 Five hundred mg of bovine liver catalase enzyme, from Sigma type C-40, lot 109C-737D, is dissolved in 200 ml of distilled deionized water with slo~ stirring at 25 C. The p}] of the resulting solution is 6.5. Next, to partially denatuee the catalase enzyme, 10 mg of succinic anhydride is dissolved in one ml of acetone. Three hundred ul of the succinic anhydride-acetone solution is added to the catalase solution every 10 minutes. After the commplete addition of all of the succinic anhydride-acetone solution, the pH of the resulting solution is

4.0 and the solution is slightly turbid.
Next, galactosidase inhibitor, namely, D-galactal is added to the above solution as follows. Two hundred fifty mg of the D-galactal, from Koch-Light Laboratories, Ltd. lot 5~129, code lS 2836h, is added and the solution is stirred ~or 15 minutes at 25C. Next, 25 g of KimalTM solid alumina support 1~70 + 80 mesh) from Owens-Illinois, Inc. Toledo, Ohio, coated with diethylaminoethyl-dextran (DEAE-dextran) is then added and the mixture is gently shaken for 30 ~inutes at 25C.
The DEAE-dextran coated support is prepared as follows.Two hundred g of porous alumina (-40 ~ 50 mesh, KimalTM brand from Owens-Illinois, IncO) is washed repeatedly with distilled deionized water until clean of fines. Then 20 g of sodium sulfate is dissolved in 500 ml of distilled,deionized water and mixed with the 200 g of alumina support while stirring at 25~C.
Then, 10 g o~ sodium hydroxide is dissolved in 250 ml ~f distilled deionized water and 32 g of DEAE-dextran ~from Sigma type 0-4885,1Ot 87 C-0139) is slowly added. The alumina-sodium sulfate mixture and the DEAE-dextran solution are then mixed to~ether and shaken gcntly for 2 hours at 2S C. Next, 10 9 of -2~-3 ~ S ~ ~

sodium hydroxide is dissolved in 250 ml of distilled, deior~iz~7 water and 40 ~1 of epichlorohydrin is ad3ed ~hile stirrin~ at 25C. The epichlorohydrin solution is then miced with the DE~-dextran alumina support mixture and the entire mixture is shaken yently for 24 hours at 25C. After the 24 hour shaking period, the preparation was extensively washed with distilled, deionized water until clear. The newly formed DEAE-dextran coate~ alumina support can be stored under refrigeration (at 0-5~C) in distilled, deionized water until ready for use.
After the addition of inhibitor and absorption of the partially denatured native catalase enzyme onto the alumina support, the catalase is cross-linked onto the ~upport as follows.
Ten mg of diaminopropane-HCl is dissolved in 25 ml of distilled, deionized water. One ml o~ this solution is then added to the preparation containing the support and shaken for one hour. The pH of the preparation is raised from about 4.0 to about 7.0 with the dropwise addition of 0.1 M NaOH. The DEAE-dextran coated alumina support is green in color and the t~rbidity of the supernatant liquid disappears. Next, 500 mg of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is then added and allowed to react for 3 hours at 25 C, with gentle shaking. After the 3 hour reaction period, the supernatant liquid is clear~ The support with the immo~ ed and cross-linked en~yme-like modi~ied protein thereupon i5 then washed with 1 1 of dis~illed~ deionized water; then 1 1 of 0.2 ~l ~NH4)2 SO4 and finally with 1 1 of distilled, deionizQd water~ The preparation is stored under 1% D-galactal inhibitor solution at 0-5CJ pH 6, in order to stabilize the newly fQrmed enzyme-like modified protein which is cross-linked and ~Z~5~9 im~nobilized on the support, as discussed above.
The assay for the enzyme-like modified protein activity is performed as follows. A reaction solution for the assay is prepared by mixing 10 ml of 0.002 M sodium phosphate buffer at pH 7.5 with 15 ml of distilled, deionized water and 5 ml of 0.014 ~ o-nitrophenyl-~-D-galactoside substrate solution in a 50 ml beaker.
Three ml of the solution is then placed in a cuvette and the cu~ette is placed in a spectrophotometer (Beckman Instruments Company, Model ACTA III) wherein a baseline is recorded for three minutes at 405 nm to establish a stable baseline. Next, approximately 1 g of the immobilized enzyme-like modified protein prepared according to the present inventions is washed with distilled, deionized water and added to the substrate sclution. Every minute, 3 ml of substrate solution is withdrawn and the absorbance measured at 405 nm and the results recorded.
The 3 ml of substrate solution is then returned to the reaction flask. This procedure is repeated 6 times. The resulting slope due to the increase of absorbance per minutes gives an activity for the enzyme-like modified protein prepared according ~o the present invention vs. the substrate o-nitrophenyl-beta-D-~alactoside. The increase in absorbance over a 6 minute periodis measured to be 0.015.
~he control assay for DEAE-coated alumina support vs. ortho-nitrophenyl-B-D-galactoside was identical to the above procedure except that one gram of DEAE-coated KimalTM is substituted for the immobilized modified protein. No absorbance change is ~bserve~ over a 15 minute period.
The activity for the support immobilized modified 3~ galactosidase-like protein is calculated according to the S~9 following formula:

(change in A/minute) (106)(VS)(2.6) ~ctivity =
(3200) (weight of sample) wherein:
change in A/minute is change in absorbance per minute; 106 is micromoles/mole; Vs is the volume of the sample in liters;
2.6 is the density of the alumina in grams per liters; and 3200 is the extinction coefficie~t of o-nitrophenol in liters/mole~
The assay results are as follows:
Substrate Galactoside (U/ml support) Initial Activity 0.0 Final Activity 0.59 The results show that the galactosidase-like modified protein prepared according to the present invention exhibited enzymatic activity with respect to o~nitrophenyl-beta-D-galactoside substrate. No activity is oreviously detected in the native catalase. This illustrates the conversion of one genus of enzyme, a bovine catalase of the oxidoreductase family to a galactosidase enzyme-like modi~ied protein.

Two hundred fifty mg cf catalase, from Sigma type C-40, lot 109 C-7370 is dissolved in 200 ml of distilled, deionized water. ~ext, 250 mg of D~galactal inhibitor for s~lactosidasepurc~ased from Koch-Liyht Laboratories, Ltd~ lot 56129, code 2~36 h is added to the oatalase. The solution is slowly stirred for one hour at 25C with the resulting pT~ of

5~9

6~5.
The inhibitor-native catalase solution is subjected to partial denaturation conditions as follows. Fifty mg o~
succinic anhydride is dissolved in one ml of acetone. Next, 0.3 5 ml of the succinic anhydride solution is added to the enzyme-inhibi~or solution prepared above with slow stirring at 25~C.
The solution is maintained at slow stir for one hour over which time the solution turns green in color. Next, 7.5 mg of diaminopropane-HCl is added and the pF] is determined to be 4.7.
The solution is then allowed to stir one additional hour.
The partially denatured inhibitor bound enzyme produced above is immobilized as follows. The pH of the above solution is raised to 7 over a 15 minute period with the addition o~
about 3 ml of 0.1 M NaOH. Over the next one hour, the p~] is raised to 8 with the dropwise addition of approximately 2 ml of 0.1 M NaOH. ~ext, ~5 g of KimalTM DEAE-dextran alumina support ~-40 ~ 50 mesh) from Owens-Illinois, Inc., Toledo, Ohio (prepared as in Example S) is washed with distilled, deioni~ed water and the p~ cf the alunlina supernatant liquid is raised to 2~ 8, with 0.01 M NaOH. The enzyme containing solution and ~he DEAE-dextran coated alumina support are then admixed and sha~en slos~ly for about an hour. Next, 700 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is added and the result mixture is placed in a water bath at 0-5C. The solution is shaken slowly for about 1 hour with the te~perature of the solution being lowered to 3 over the one hour. Next, one additional g of the EDC is added. The resulting mixture is allowed to react overnight (about 17 hours) and is ~ubsequently washed with distilled, deionized water and assayed for enzymatic activity~

5~'3 The sample prepared according to the process of the present invention is assayed Eor enzyme-like modified protein activity according to the procedure disclosed in Example 5 above. The sole change from the Example 5 procedue, formulas and particulars disclosed in the procedure for Example 5 is the substitution ~f 10 ml of 0.003 M sodium phosphate buffer at p~l

7.5 for the 10 ml of 0.002 M sodium phosphate b~ffer at 7.5 used in Example ~. All other details of the assay for the present Example are identical to the procedures disclosed in Example 5 above.
Substrate Galactoside (U/ml Support) Initial Activity 0.00 Final Ac~ivity Assay ~1 O.S6 Assa~ ~2 0.62 The results show that the galactosidase enzyme-like modified protein prepared according to the present invention exhibits activity toward the galactoside substrate for galactosidase enzymes. No activity was detected in the native catalase enzyme. This illustrates the conversion of one ~enus of enzymatic protein, namely a catalase, of the oxidoreductase amily to a galactosidase enzyme-like modified protein~

EXAMP~E 7 To demonstrate that natlve ribonuclease enzyme shows no catalytio activity with respect to L-tryptophan methyl ester ~TME) substrate of Examples 2-4 above, the follGwin~ control is perEormed~
Sixty mg of ribonuclease enzyme, Çrom Sigma Chemical Company, Type R-5000~ lot 20F-2010, is dissolved in 100 ml of 1 m~l tris buffer, at pH 7, with slow stirring at 25C.
The ribonuclease containing solution is then dialyzed using a No. 3 Spectra/Po ~ dialysis tube against 3500 ml o~ 1 m`~
tris buffer, pH 7, for 17 hours at 0-5~. The No. 3 tubing has a molecular weight exclusion range of 35-4500 daltons. Mext, 1 m]
of the ribonuclease containing solution is assayed versus TME
substrate for possible enzymatic activity with respect to TME.
The assay is conducted as follows.
A high pressure liquid chromatography system is used for the assay. The column is a 2 mm x 25 cm stainless steel column packed with porous silica containing carboxyl side chains having an average pore size of 40 microns, purchased from Baker Chemical Company. The column eluant is 0.03 M acetate, pH 6, at a Elow rate of 7 ml/min. The absorbance of the column out~low is monitored at 280 nm.
The assay sample solution containing na~ive ribonuclease is prepared as follows. Eight ml of one mM tris buffer, pH 7 50, is admixed with one ml of 0.1 M TME, pH 3.2 and 1 ml of the r~bonuclease solution prep~red a~ove. T~e ass~y c~ntrol solution is prepared by admixing 8 ml of 1 mM tris buffer, pH
7.5; one ml of 0.1 M TM~, pH 3.2 and one ml of one mM tris buffer, pH 7Ø The pH of the control and assay solutions is 5.8.
Next, one ml of the assay sample solution is removed from the sample beaker using a 2 ml hypodermic syringe with an 18 gauge needle and injected onto the column through a 20 microliter injec~ion sample loop. The time of injection is recorded.
This procedure is then repeatcd using the assay control solution. Both sample and control solutions are chcomatographed ~z~s~

four ti~es in order to obtain a plot of molarity of T~IE versus time for sample and control solutions.
~ statistical analysis of the data obtained sho~7s virtually identical slopes of 0.0620 ~ .005 for both control and sample solutions. Accordingly, the native ribonuclease shows no measurable catalytic activity properties with respect to the T~E
substrate.

To demonstrate that native bovine serum albumin (BSA) shows no catalytic activity with respect to L-tryptophan methyl ester (TME) substrate of Example 1 above, the following control is performed.
one hundred mg of BSA from Sigma Chemical Company, No. 7511, lot 90F-8351~ is dissolved in 10~ ml of 1 m~ tris buffer at pH
7, with slow stirring at 25C.
The BSA containing solution is then dialyzed using a No. 2 Spectra/Po ~ dialysis tube against 3500 ml o 1 mM tris buffer, pH 7, for 17 hours at 0-5C. The No. 2 tubing has a molecular weight exclusion range of 12-1~,000 daltons. Next, one ml of the ribonuclease containing solution is assayed versus TME substrate for possible enæymatic activity with respect to TME. The assay is conducted as follows.
; A high pressure liquid chromatography system is used for the assay. The column is a 2 mm x 25 cm stainless steel column packed with porous silica containing car~oxyl side chains having an average pore size of 4a microns, purchased from Baker Chemical Company. The column eluant is 0.03 M acetate, pH 6, at a flow rate of 7 ml/min. The absorbance of the column outflow is monitored at 280 nm.
The assay sample solution con~aining na~ive BS~ is prep~red ~2~35~5~9 as follows. Eight ml of one m~ tris buffer, p~l 7.5 is admixed witn one ml of 0.1 M T~l~, pH 3.2 and one ml of the ribonucleasé
solution prepared above. The assay control solution is prepared by admixing 8 ml oE one m~ tris buffer, pH 7.5; one ml of 0.1 M
TME, pH 3.2 and one ml of one mM tris bu f ~er, pH 7Ø The pH of the control and assay solu~ions is 5.8.
Next, one ml of the assay sample solution is removed from the sample beaker using a 2 ml hypodermic syringe with an 18 ~auge needle and injected onto the column through a 20 microliter injectio.~ sample loop. The time of injection is recorded.
This procedure is then repeated using the assay control solution. Both sample and control solutions are chromatographed four times in order to obtain a plot of molarity of TME versus time for sample and control solutions.
A statistical analysis of the data obtained shows virtually identical slopes of 0.06 -~ .0065 for both control and sample solutions. Accordingly, the native ribonuclease shows no mea~urable catalytic acti~ity properties with respect to the T~E
substrate.

To demonstrate that native catalase shows no catalytic activity with respect to p-nitrophenyl-beta-D-yalactoside (NBG) ; substrate of Examples 5 and 6 above the following control is performed.
Two hundred fifty mg of catalase, purchased from Sigma Chemical Compa~y, Type 727S, is dissolved in 25 ml of 0.5 mM
acetate buffer, pH 4, w;th slow stirring at 25C. The solution is plac~ in a No. 2 Spectra~Po ~ dialysis tube and dialyzed against 3500 ml of 5 mM acetate buffer, at pH ~ for 17 ho~rs at ~3G-~3~5~

0.5C. The No. 2 tubing has a molecular weight exclusion range of 12-14,000 daltons. Next, 0.2 ml of this as5ay sample is withdrawn and assayed versus NBG substrate for possible enz~atic activity of ribonuclease toward NBG. The assay is conducted as follows.
The assay sample mixture ~or assay is prepar~d by admixing 2.~ ml oE 0.03 M sodium phosphate buffer containing 50 ppm antibacterial agent (Bioban brand antibacterial agent purchased from International Minerals and Chemicals Corp.) with 0.5 ml of 0.014 M NBG in a 3 ml cuvette. The cuvette is next placed in a ACTA III Spectrophotometer (from Beck~an Instruments Company) wherein a straight base line is recorded for three min. The absorbance is monitored at 405 nm. Next, 0.1 ml of catalase so~ution is added to the NBG substrate solution in the c~vette.
The cuvette is inverted four times to insure a homogeneous mixture in the cuvette and is placed back in the spectrophotometer for further measurement. The absorbance change is recorded over a 20 minute experimental period, no mea~lrable catalytic activity ~roperties with respect to NBG
substrate are found in the native catalase.

Claims (49)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for chemically altering the substrate specifity of a native protein to produce an immobilized enzyme-like modified protein comprising:
a. selecting an enzyme to be modeled;
b. partially denaturing a native protein in the presence of an inhibitor for said model enzyme to form a partially denatured native protein-model enzyme inhibitor complex;
c. contacting said partially denatured protein-model enzyme inhibitor complex with a solid support for a time sufficient and at a temperature sufficient to absorb and immobilize said partially denatured protein-model enzyme inhibitor complex on said solid support and d. cross-linking said absorbed, immobilized protein-model enzyme inhibitor complex to form an immobilized enzyme-like modified protein.

2 . A process as defined in claim 1 wherein said native protein is partially denatured by forming an aqueous solution of said native protein and maintaining said aqueous solution at a temperature and for a time sufficient to partially denature said native protein.

3. A process as defined in claim 1 wherein said native protein is partially denatured by admixing said native protein with water to form an aqueous solution and admixing the resulting solution with a denaturing agent.

4. A process as defined in claim 3 wherein said denaturing agent is an inorganic acid.

5. A process as defined in claim 3 wherein said denaturing agent is an organic acid.

6. A process as defined in claim 3 wherein said denaturing agent is an inorganic salt.

7. A process as defined in claim 3 wherein said denaturing agent is a water-miscible organic solvent.

8. A process as defined in claim 1 wherein said solid support in a porous, particulate alumina ceramic oxide.

9. A process as defined in claim 1 wherein said immobilized protein is cross-linked by admixing said immobilized protein with a cross-linking agent.

10. A process as defined in claim 9 wherein said cross-linking agent is glutaraldehyde.

11. A process as defined in claim 9 wherein said cross-linking agent is ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

12. A process as defined in claim 9 wherein said cross-linking reagent is a mixture of and organic diamine and an organic carbodiimide.

13. A process of claim 12 wherein said diamine is diaminopropane-HCl and said carbodiimide is ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

14. A process as defined in claim 1 wherein said partially denatured protein-model enzyme inhibitor complex is admixed with an organic acid anhydride prior to admixture with said solid support.

15. A process for chemically altering the substrate specificity of a native protein to produce an immobilized enzyme-like modified protein comprising:
a. selecting an enzyme to be modeled;
b. adsorbing a native protein onto a solid support to immobilize the native protein;
c. partially denaturing said adsorbed native protein and d. cross-linking said partially denatured adsorbed protein in the presence of an inhibitor for said model enzyme to form an immobilized enzyme-like modified protein.

16. A process as defined in claim 15 wherein said native protein is partially denatured by forming an aqueous solution of said native protein and maintaining said aqueous solution at a temperature and for a time sufficient to partially denature said native protein.

17. A process as defined in claim 15 wherein said native protein is partially denatured by admixing said native protein with water to form an aqueous solution and admixing the resulting solution with a denaturing agent.

18. A process as defined in claim 17 wherein said denaturing agent is an inorganic acid.

19. A process as defined in claim 17 wherein said denaturing agent is an organic acid.

20. A process as defined in claim 17 wherein said denaturing agent is an inorganic salt.

21. A process as defined in claim 17 wherein said denaturing agent is a water-miscible organic solvent.

22. A process as defined in claim 15 wherein said solid support in porous, particulate alumina ceramic oxide.

23. A process as defined in claim 15 wherein said immobilized protein is cross-linked by admixing said immobilized protein with a cross-linking agent.

24. A process as defined in claim 23 wherein said cross-linking agent is glutaraldehyde.

25. A process as defined in claim 23 wherein said cross-linking agent is ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

26. A process as defined in claim 23 wherein said cross-linking agent is a mixture of an organic diamine and an organic carbodiimide.

27. A process as defined in claim 26 wherein said organic diamine is diaminopropane-HCl and said organic carbodiimide is ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

28. A process as defined in claim 15 wherein said native protein is admixed with an organic anhydride prior to admixture with said solid support.

29. A process for chemically altering the substrate specificity of a native protein to produce an immobilized enzyme-like modified protein comprising:
a. selecting an enzyme to be modeled;
b. partially denaturing a native protein;
c. contacting said partially denatured native protein with a solid support for a time sufficient and at a temperature sufficient to adsorb and immobilize said partially denatured protein on said solid support and d. cross-linking said partially denatured adsorbed protein in the presence of an inhibitor for said model enzyme to form an immobilized enzyme-like modified protein.

30. A process as defined in claim 29 wherein said native protein is partially denatured by forming an aqueous solution of said native protein and maintaining said aqueous solution at a temperature and for a time sufficient to partially denature said native protein.

31. A process as defined in claim 29 wherein said native protein is partially denatured by admixing said native protein with water to form an aqueous solution and admixing the resulting solution with a denaturing agent.

32. A process as defined in claim 31 wherein said denaturing agent is an inorganic acid.

33. A process as defined in claim 31 wherein said denaturing agent is an organic acid.

34. A process as defined in claim 31 wherein said denaturing agent is an inorganic salt.

35. A process as defined in claim 31 wherein said denaturing agent is a water-miscible organic solvent.

36. A process as defined in claim 29 wherein said solid support in a porous, particulate alumina ceramic oxide.

37. A process as defined in claim 29 wherein said immobilized protein is cross-linked by admixing said immobilized protein with a cross-linking agent.

38. A process as defined in claim 37 wherein said cross-linking agent is glutaraldehyde.

39. A process as defined in claim 37 wherein said cross-linking agent is ethyl 3-(3-dimethylaminopropyl)-carbodiimide.

40. A process as defined in claim 37 wherein said cross-linking agent is a mixture of an organic diamine and an organic carbodiimide.

41. A process as defined in claim 40 wherein said organic diamine is diaminopropane-HCl and said organic carbodiimide is ethyl-3-(3-dimethylaminopropyl)-carbodiimide.

42. A process as defined in claim 29 wherein said native protein is admixed with an organic anhydride after partial denaturation and prior to adsorption on said solid support.

43. A process for chemically altering the substrate specificity of a native protein to produce an immobilized enzyme-like modified protein, wherein said native protein has at least three disulphide groups per molecule, comprising:
a. selecting an enzyme to be modeled;
b. admixing said disulphide group containing native protein with a disulphide bridge reduction agent in the presence of an inhibitor for said enzyme to form a partially denatured native protein-model enzyme inhibitor complex;
c. contacting said complex with a solid support for a time sufficient at a temperature sufficient to adsorb and immobilize said complex on said support and d. admixing said immobilized native protein-model enzyme inhibitor complex with a sulfhydryl oxidation agent for a time and at a temperature sufficient to form disulphide bonds in said protein to produce a modified enzyme-like protein.

44. A process as defined in claim 43 wherein said disulphide bridge reduction agent is beta-mercaptoethanol.

45. A process as defined in claim 43 wherein said disulphide bridge oxidation agent is an aqueous solution containing molecular oxygen wherein said solution is above pH 7.

46. The product of the process of claim 1.

47. The product of the process of claim 15.

48. The product of the process of claim 29.

49. The product of the process of claim 43.