Title of Invention	A PROCESS FOR THE PREPARATION OF MOLECULARLY IMPRINTED POYMERS USEFUL FOR SEPARATION OF ENZYMES
Abstract	This invention relates to the process for the preparation of molecularly imprinted polymers useful for separation of enzymes. Molecularly imprinted polymers prepared by the process of the present invention exhibit selective binding of imprinted enzyme which is useful in the imprinted enzyme form aqueous solution of the imprinted enzyme or a mixture containing imprinted enzyme and other enzymes. Process steps comprises; reacting the complex of enzyme and affinity monomer, a co monomer and a cross linker with a polymerization initiator and a polymerization accelerator at ambient temperature and pressure for a period ranging between 2 to 24 hrs, to obtain a cross linked polymer, crushing the said cross linked polymer to fine particles, adding a solvent and extracting imprinted enzyme from the cross linked polymer to obtain the molecularly imprinted polymer.

Title of Invention

A PROCESS FOR THE PREPARATION OF MOLECULARLY IMPRINTED POYMERS USEFUL FOR SEPARATION OF ENZYMES

Abstract

Full Text	This invention relates to the process for the preparation of molecularly imprinted polymers useful for separation of enzymes. Molecularly imprinted polymers prepared by the process of the present invention exhibit selective binding of imprinted enzyme which is useful in separating the imprinted enzyme from aqueous solution of the imprinted enzyme or a mixture containing imprinted enzyme and other enzymes. Purification and isolation of biological macroraolecules like proteins and enzymes are critical multi-step process that result in very low yields and thus a high cost. As a better alternative to the conventional processes, researchers have developed various techniques for purification of enzymes which can broadly be classified as follows. 1. Affinity chromatography (Y. Li, G.Kunyu, C.Lubai Z.Hanfa, Z.Yunkui Sepu, 14, 415 (1996), T.Makriyannis, Y.D.Clonis, BiotecLBioengg. 53, 49 (1997)). 2. Affinity precipitation (C.Senstad, B.Mattiasscn. Biiotech.Bioengg. 33, 216 (1989), M.Schneider, C.Guillot, B.Lamy, AnnNlY.Acad.Sci. 369, 257 (1981)). 3. Affinity crossflow ultrafiltration ( K.Sigmundsson, H.Filippusson, Polyimer int. 41, 355 (1996), T.B.Choe, P.Masse, A.Verdier, Biotech.Lett. 8, 163 (19S6)). 4. Affinity partitioning ( G.Takerkart, E.Segard, M.Monsigny, FEBS Dett. 42, 218 (1972), B.A.Andrews, D.M.Head, P.Dunthorne, J.A.Asenjo, Biotscih.Tech. 4, 49 (1990)). 5. Immobilized metal affinity chromatography ( G.Ehteshami. J.Porath, » R.Guzman, G.Ehteshami, J.Mol.Recognit. 9, 733 (1996), A.L.BIonnkalns, M.R.Gomez, Prep.Biochem.Biotechnol. 27, 219 (1997)). 6. Molecularly imprinted immobilized metal affinity chromarography (F.H.Arnold, P.Dhal, D.Shnek, S.Plunkett, US patent 5,310,648 (1994)).. These techniques exploit either the binding interaction between actiive site of enzyme and its inhibitor i.e. affinity monomer or between surface histidine, tyrosine, cysteine groups of enzymes with metal ions immobilized on a suipport. But these techniques also suffer from one or other disadvantages as follows. Affinity chromatography uses a column containing an inhibitor or a dye or an antibody for a given enzyme for its separation from a mixture of enzymes. The solution of enzymes is poured over the affinity column to retain the desired enzyme on column for subsequent isolation. This technique is efficient omly for small capacity columns. With the scale up of columns, the problems of sample pretreatment and plugging of packed column become severe. Affinity precipitation technique needs a complex to be formed between a macro-ligand and a protein both containing multiple binding sites. When such a •i complex is formed, it precipitates. Thus, this technique is restricted mainly for multimeric proteins. Beside such macro-ligands are often costly. Therefore, certain synthetic inhibitors or dyes are linked to stimuli responsive polymer which can be precipitated by pH or temperature stimuli after it forms complex with the enzyme. In this case along with the desired enzyme, a substantial amount of other enzymes bind to the polymer by means of nonspecific adsorption. In case of affinity crossflow ultrafiltration, a solution of enzymes is filtered through a membrane containing affinity group under pressure. This technique is suitable in the cases where difference between molecular weights of two enzymes is high. Also, with increase in the filtration time, denaturation of enzymes as well as clogging of membrane takes place due to the pressure applied. Affinity partitioning of two- phase aqueous systems is widely used technique as compared to those mentioned above, hi this technique concentrated aqueous solution of poly (ethylene glycol) (PEG) with or without linking affinity group is mixed with enzyme solution containing moderate to high salt concentration. The two phases are mixed well and allowed to separate. Enzyme gets predominantly partitioned in one phase, which subsequently can be isolated Disadvantages of this technique are nonspecific extraction of other protenacious molecules along with desired enzyme and also poor interactions between enzyme and affinity group due to high ionic strength. Immobilized metal affinity chromatography is a technique in which columns of polymeric support containing chelated metal ions are used. These metal ions form coordination complex with histidine, tyrosine, cysteine etc. present on the surface of the enzyme. Although this technique has advantages like high column capacity, ease in the enzyme elution etc. it is not very selective in descriminating between two closely related (with respect to their amino acid sequences) enzymes. Molecular imprinting of matrices containing metal chelates is a recently developed technique, which increases the selectivity (F.H.Amold, P.Dhal, D.Shnek, S.Plunkett, US patent 5,310,648 (1994)). In this technique complex of monomer containing chelated metal ion and enzyme is polymerized with crosslinker in order to imprint the polymer with enzyme. Although this technique exhibits a substantial selectivity, it is not as selective as that of biological antibodies or active site inhibitor of enzymes. Synthetic inhibitors are molecules which exhibit affinity to the active site of a given enzyme. This interaction is very specific for a given pair of enzyme and inhibitor and such inhibitors bind very strongly to a given enzyme. Molecular imprinting is a simple technique which allows synthesis of polymers capable of recognizing specific molecules. These polymers are stable and can withstand harsh conditions such as temperature and pH etc ( G.Wulff, Molecular interactions in bioseparations Ed. by T.T.Ngo, Plenum press, N.Y. (1993)). Thus these polymers are finding wide range of applications. A polymer containing monomer based on enzyme inhibitor Le. affinity monomer which is also imprinted for corresponding enzyme is expected to exhibit a high selectivity as well as high capacity for binding a specific enzyme. This synergistic affinity -imprinting effect will not be available in the above mentioned techniques. Such affinity- imprinted polymers have not been reported so far. The object of the present invention is to provide a process for the preparation of molecularly imprinted polymers comprising affinity monomers imprinted for various enzymes, by the above mentioned affinity imprinting technique, useful for having high selectivity as well as high capacity for separation of enzymes. Accordingly the present invention provides a process for the preparation of molecularly imprinted polymers useful for separation of enzymes which comprises; a) reacting the complex of enzyme and affinity monomer in the ratio of 1:1 to 1:10 (w/w) a co monomer and a cross linker such as herein described in 5 to 80 % and 10 to 90 % by wt. of the total feed respectively with a polymerization initiator and a polymerization accelerator such as herein described at ambient temperature and pressure for a period ranging between 2 to 24 hrs, to obtain a cross linked polymer, Jo) crushing the said cross linked polymer to fine particles, e) adding a solvent and extracting imprinted enzyme from the cross linked polymer to obtain the molecularly imprinted polymer. In one of the embodiments of the present invention, enzymes may be selected from a wide range of enzymes and proteins such as trypsin, chymotrypsin, lysozyme, ova albumin (no enzyme nutritional protein) etc. and the complexing affinity monomers for these enzymes may be selected from acryloyl derivatives of inhibitor s for respective enzymes such as N-acryloyl para amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine, N- oleoylacrylamide respectively, and the enzyme-affinity monomer complex may be prepared by contacting aqueous solutions of enzyme and the respective affinity monomer. In an another embodiment, affinity monomers- N-acryloyl para amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine and N-oleoylacrylamide may be prepared by the reaction of acryloyl chloride with para amino benzamidine, 4-phenyl butyl amine, N-acetyl glucosamine, and acrylamide respectively. In an another embodiment of the present invention the ratio of enzyme to affinity monomer may be in the range of 1:1 to 1:10, preferably 1:1. (w/w). In yet another embodiment, the comonomer may be selected from compounds such as acrylamide, methacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, acrylic acid, methacrylic acid and the like. In another embodiment, the amount of comonomer in the polymerization mixture may be in the range of 5 to 80%, more particularly 45 to 55% by weight of the total feed (i.e. enzyme-affinity monomer complex+comonomer-tcrosslmker, hereinafter.) In still another embodiment, crosslinker may be selected from compounds such as methylene bisacrylamide, N, N diacryloyl 1,2 diaminoethane. N, N dimethacryloyl 1,2 diaminoethane N, N diacryloyl 1,6 diaminohexane. ethylene glycol dimethacryalte and the like. In still another embodiment, the amount of crosslinker may be between 10% to 90%, more particularly 30 to 50% by weight of the total feed. In yet another embodiment, the polymerization initiator used may be selected from compounds such as ammonium per sulfate. eerie ammonium sulfate, potassium persulfate, azobisisobutyronitrile and the like. In yet another embodiment, the polymerization initiator may be 0.1% to 1% by weight of the total feed. In still another embodiment, the polymerization accelerator may be selected from compounds such as ethylene diamine, N. N, V, N"tetramethy ethylenediamine and the like. In still another embodiment, the polymerization accelerator may be 1% to 4% by weight of the total feed. In still another embodiment, the solvent used to extract the engine from the imprinted polymer may be selected from solvents which break the enzyme-inhibitor complex such as phenol, chloroform, glacialacetic acid and the like. In a feature of the present invention, the molecularly imprinted polymer is typically prepared under mild conditions as follows. Affinity monomer and its respective enzyme are dissolved in water and stirred for 15 to 30 minutes at room temperature to form the enzyme-affinity monomer complex. Then comonomer, crosslinker and polymerization initiator are added to the solution containing enzyme-affinity monomer complex and the solution is purged with nitrogen for 10 minutes. Polymerization accelerator is then added and the solution is kept at 37 ° C for 18 hours for polymerization. The crosslinked polymer so obtained is treated with acetone to remove water and crushed to small particles. Template enzyme is extracted from the particles by 3 to 4 alternative treatments of the solvent which breaks the complex between enzyme and the inhibitor. Polymer particles are then sieved and used for enzyme separations. In an another feature of the present invention, enzyme imprinted polymers are contacted with aqueous solution containing the respective enzyme or a mixture of enzymes and allowed to shake at room temperature for 1 hour. Polymer particles with bound enzyme are then filtered and isolated. Although, the present invention describes a process for the preparation of molecularly imprinted polymers useful for the separation of enzymes namely trypsin, chymotrypsin, lysozyme and ovaalbumin (nonenzyme nutritional protein), the scope of the present invention is not and should not be construed to limit only to such molecularly imprinted polymers for separation of trypsin, chymotrypsin, lysozyme and ovaalbumin only but it may extend to such combinations of affinity monomers and their respective enzymes. The ranges and limitations provided in the instant specification and claims are those which are believed to particularly point out and distinctly claim the present invention. It is however understood that other ranges and limitations which perform substantially the same function in me same or substantially the same manner to obtain the same or substantially the same results are intended to be within the scope of the instant invention as defined by the instant specification and claims. Example 1 Preparation of trypsin imprinted polymer. In an air tight round bottom teflon tube 50 mg N-acryloyl para aminobenzamidine hydrochloride is dissolved in 3 ml. water containing stoichiometric amount of sodium hydroxide so as to free gnanidine groirp from its hydrochloride form. To this solution 50 mg trypsin is added; and the resulting solution is shaken gently on a shaker bath for 15 min.; to form trypsin-N-acryloyl para aminobenzamidine complex. The comonomer acrylamide 400 mg; crosslinker methylene bis acrylamide 500 mg; and initiator ammonium per sulfate 10 mg are dissolved in 1 ml. dimethyl formamide and this solution is added to above aqueous solution containing trypsin-N-acryloyl para aminobenzamidine complex. This is mixed well and nitrogen gas is passed through this solution for 10 min. and 40µl tetramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bath at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remove water. It is then crushed to fine particles. Template trypsin is extracted from the particles by 3-4 alternative treatments of acetone and chloroform. The resulting crypsin imprhied polymer is then dried in vacuum oven at 50° C for 12 hrs. Dried polymer piracies are then sieved through standard test sieves and particles in the size range of 250-500 µ are used for enzyme separation. Example 2 Preparation of chymotrypsin imprinted pohmer. In an air tight round bottom teflon tube 50 mg N-acryloyl 4 phenyl butylamine is dissolved 3 ml. dimethyl formamide water mixture (10:80). To this solution 50 mg chymotrypsin is added; and the resulting solution if shaken gently on a shaker bath for 15 min.; to form chymotrypsin-N-acryloyl 4 phenyl butyl amine complex. The comonomer acrylamide 400 rug; crosslinker rnethylene bis acrylamide 500 mg; and initiator ammonium per sulfase 10 mg are dissolved in 1 ml.dimethyl formamide and this solution is added to above aqueous solution containing chymotrypsin-N-acryloyl 4 phenyl butylsmine complex This is mixed well and nitrogen gas is passed through this solution for 10 min and 40 µl tetramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bath at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remove water. It is then crushed to fine parades. Template chymotrypsin is extracted from the particles by 5-4 alternative treatments of acetone and chloroform. The resulting chymotrypsin imprinted polymer is then dried in vacuum oven at 50° C for 12 hrs. Dried polymer panicles are then sieved through standard test sieves and particles in the size range of 250-500µ are used for enzyme separation. Example 3 Preparation of lysozyme imprinted polymer. In an air tight round bottom teflon tube 50 mg acrloyl N-acetyl glucosamine is dissolved in 3 ml. water. To this solution 20 mg lysozyme is added; and the resulting solution is shaken gently on a shaker bath for 15 min.; to form lysozyme -acryloyl N-acetyl glucosamine complex. The comonomer acrylamide 400 mg; crosslinker methylene bis acrylamide 500 mg: and initiator ammonium per sulfate 10 mg are dissolved in 1 ml.dimetfeyl formamide and this solution is added to above aqueous solution containing h-sozyme-acryloyl N-acetyl glucosamine complex. This is mixed well and nitrogen gas is passed through this solution for 10 min. and 40 µl tetramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bath at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remove water. It is then crushed to fine particles. Template lysozyme is extracted from the particles by 3-4 treatments of glacial acetic acid. The resulting lysozyme imprinted polymer is tken dried in vacuum oven at 50° C for 12 hrs. Dried polymer particles are then sieved through standard test sieves and particles in the size ranct of 250-500 n are used for enzyme separation. Example 4 Preparation of ovaalbumin imprinted polymer. In an air tight round bottom teflon tube 5) mg N-oleoyl acrylamide is dissolved in 3 ml. Dimethyl formamide/water mixture (20:80). To this solution 50 mg ovaalbumin is added; and the resulting solution is shaken gently on a shaker bath for 15 min.; to form ovaalbumin-N-oleoyl acrylamide complex. The comonomer acrylamide 400 mg; crosslinker methykne bis acrylamide 500 mg; and initiator ammonium per sulfate 10 mg are dissolved in 1 ml.dimethyl formamide and this solution is added to above aqueous solution containing ovaalbumin-N-oleoyl acrylamide.complex. This is nixed well and nitrogen gas is passed through this solution for 10 min. and 40 µl teramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bam at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remover water. It is then crushed to fine particles. Template albumin is extracted from the particles by 3-4 treatments of phenol - chloroform The resulting ovaalbumin imprinted polymer is men dried in vacuum oven at 50° C for 12 hrs. Dried polymer paricles are then sieved through standard test sieves and particles in the size range of 250-500 µ are used for enzyme separation. In all the above-mentioned examples, formation of enzyme-affinity monomer complex was confirmed by the active site titration of respective complexes. Example-5 This example describes the process for selective binding of trypsin by trypsin imprinted polymer from trypsin and chymotrypsin mixture. In a 50 ml. capacity conical flask; 250 mg trypsin imprinted polymer is suspended in 25 ml 10 mM Ca+2 aqueous solution containing trypsin and chymotrypsin with equal initial activities. The flask is allowed to shake at 50 rpm speed at room temperature on a rotary shaker for 1 hr. The swollen polymer particles are then filtered and in the filtrate the activities of trypsin and chymotrypsin are determined by using following standard substrates. For trypsin DL-Benzoyl-Arginyl.-Para nitroanilide, and for chymotrypsin L- Benzoyl-Tyrosyl-Para nitroanilide are used. Uptake of trypsin by the polymer was determined by subtracting the activity of trypsin in the filtrate from the initial activity of trypsin in the solution. It was found that activity wise 40 percent trypsin and only 2.6 percent chymotrypsin was adsorbed by trypsin imprinted polymer. Example-6 This example describes the process for selective binding of chymotrypsin by chymotrypsin imprinted polymer from chymotrypsin and trypsin mixture. hi a 50 ml. capacity conical flask; 250 mg chymotrypsin imprinted polymer is suspended in 25 ml 10 mM Ca+2 aqueous solution containing trypsin and chymotrypsin with equal initial activities. The flask is allowed to shake at 50 rpm speed at room temperature on a rotary shaker for 1 hr. The swollen polymer particles are then filtered and in the filtrate the activities of trypsin and chymotrypsin are determined by using following standard substrates. For trypsin DL-Benzoyl-Arginyl.-Para nitroanilide, and for chymotrypsin L- Benzoyl-Tyrosyl-Para nitroanilide are used. Uptake of chymotrypsin by the polymer was determined by subtracting the activity of chymotrypsin in the filtrate from the initial activity of chymotrypsin in the solution. It was found that activity wise 52 percent chymotrypsin and only 4 percent trypsin was adsorbed by chymotrypsin imprinted polymer. Example-7 This example describes the process for binding of lysozyme by lysozyme imprinted polymer from lysozyme solution. In a 50 ml. capacity conical flask; 250 mg lysozyme imprinted polymer is suspended in 25 ml 0.06 M phosphate buffer. PH = 6,8 containing 2.5 mg lysozyme. The flask is allowed to shake at 50 rpn speed at room temperature on a rotary shaker for 1 hr. The swollen polymer particles are men filtered and in the filtrate the activity of lysozyme is determined by using micrococus lyzodeikticus, a standard substrate for lysozyme. Uptake of lysozyme by the polymer was determined by subtracting the activity of lysozyme in the filtrate from the initial activity of lysozyme in the solution. It was found that activity wise 60% percent lysozyme was adsorbed by lysozyme imprinted polymer. Example-8 This example describes the process fcr binding of ovaalbumin by ovaalbumin imprinted polymer from evaalbumin solution. In a 50 ml. capacity conical flask; 250 mg cvaalbumin imprinted polymer is suspended in 25 ml water containing 2.5 mg ovaalbumin (determined by absorbance at 280 nm). The flask is allowed to shake at 50 rpm speed at room temperature on a rotaiy shaker for 1 hr. The swollen polymer partides are then filtered and in the filtrate the ovaalbumin in terms of protein (determined from standard graph at 280 nm) is determined by using pure ovaalbumin as a standard. Uptake of ovaalbumin by the polymer was determined by subtracting the protein present in the filtrate from the initial protein added in the solution. It was found that protein wise 45% percent ovaalbumin was adsorbed by ovaalbumin imprinted polymer. The advantages of the present invention are as follows: 1) The process of the present invention, unlike the other conventional processes, exhibits a very high selectivity as well as very high capacity for separating the imprinted enzyme either from a mixture of enzymes or from their aqueous solutions. 2) The process of the present invention is applicable in general for separating various other enzymes by preparing affinityimprinted polymers as mentioned herein above using appropriate complexing affinity monomer and the respective enzyme. 3) The process of the present invention is convenient and the polymers synthesized by following the present process are resistant to denaturation by the action of organic solvent, high temperature and pressure. We Claim: 1. A process for the preparation of molecularly imprinted polymers useful for separation of enzymes which comprises; a) reacting the complex of enzyme and affinity monomer in the ratio of 1:1 to 1:10 (w/w), a co-monomer and a cross linker such as herein described in 5 to 80 % and 10 to 90 % by wt. of the total feed respectively with a polymerization initiator and a polymerization accelerator such as herein described at ambient temperature and pressure for a period ranging between 2 to 24 hrs, to obtain a cross linked polymer, b) crushing the said cross linked polymer to fine particles, c) adding a solvent and extracting imprinted enzyme from the cross linked polymer to obtain the molecularly imprinted polymer. 2. A process as claimed in claim 1 wherein enzymes used are selected from tryspin, chymotrypsing, lysozyme, ovaalbumin; the complexing affinity monomers for these enzymes used are selected from N-acryloyl para amino benzamidine, N- acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine, N- oleoylacyrylaminde. 3. A process as claimed in claims 1 and 2 wherein, the enzyme-affinity monomer complex is prepared by contacting aqueous solutions of enzyme and the respective afinity monomer. 4. A process as claimed in claims 1 to 3 wherein, affinity monomers-N-acryloyl para amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine and N-oleoylacrylamide are prepared by the reaction of acryloyl chloride with para amino benzamidine, 4-phenyl butyl amine, N-acetyl glucosamine, and acrylamide. 5. A process as claimed in claims 1 to 4 wherein, the ratio of enzyme to affinity monomer is preferably 1:1 (w/w). 6. A process as claimed in claims 1 to 5 wherein, the comonomer used is selected from acrylamide, methacrylamide, N-isopropylacrylamide, N- isopropylmethacrylamide, acrylic acid, methacrylic acid. 7. A process as claimed in claims 1 to 6 wherein, the amount of co monomer in the polymerization mixture is preferably 45 to 55% by weight of the total feed 8. A process as claimed in claims 1 to 7 wherein, cross linker used is selected from methylene bisacrylamide, N, N diacryloyl 1,2 diaminoethane, N,N dimethacryloyl 1,2 diaminoethane, N, N diacryloyl 1,6 diaminohexane, ethylene glycol dimethacrylate. 9. A process as claimed in claims 1 to 8 wherein, the amount of cross linker is preferably 30 to 50% by weight of the total feed. 10. A process as claimed in claims 1 to 9 wherein, the polymerization initiator used is selected from ammonium per sulfate, eerie ammonium sulfate, potassium per sulfate, azobisisobutyronitrile. 11. A process as claimed in claims 1 to 10 wherein, the polymerization initiator is 0.1 to 1% by weight of the total feed. 12. A process as claimed in claims 1 to 11 wherein, the polymerization accelerator used is selected from ethylene diamine, N, N, N', N"tetramethylenediamine. 13. A process as claimed in claims 1 to 12 wherein, the polymerization accelerator is 1 to 4% weight of the total feed. 14. A process as claimed in claims 1 to 13 wherein, the solvent used to extract the enzyme from the imprinted polymer is which break the enzyme-inhibitor complex is selected from chloroform, glacial acetic acid. 15. A process for the preparation of molecularly imprinted polymers useful for separation of enzymes as described hereinabove with reference to the examples.

Full Text

This invention relates to the process for the preparation of molecularly imprinted polymers useful for separation of enzymes. Molecularly imprinted polymers prepared by the process of the present invention exhibit selective binding of imprinted enzyme which is useful in separating the imprinted enzyme from aqueous solution of the imprinted enzyme or a mixture containing imprinted enzyme and other enzymes.
Purification and isolation of biological macroraolecules like proteins and
enzymes are critical multi-step process that result in very low yields and thus a
high cost. As a better alternative to the conventional processes, researchers have developed various techniques for purification of enzymes which can broadly be classified as follows.
1. Affinity chromatography (Y. Li, G.Kunyu, C.Lubai Z.Hanfa, Z.Yunkui Sepu,
14, 415 (1996), T.Makriyannis, Y.D.Clonis, BiotecLBioengg. 53, 49 (1997)).
2. Affinity precipitation (C.Senstad, B.Mattiasscn. Biiotech.Bioengg. 33, 216
(1989), M.Schneider, C.Guillot, B.Lamy, AnnNlY.Acad.Sci. 369, 257
(1981)).

3. Affinity crossflow ultrafiltration ( K.Sigmundsson, H.Filippusson, Polyimer int.
41, 355 (1996), T.B.Choe, P.Masse, A.Verdier, Biotech.Lett. 8, 163 (19S6)).
4. Affinity partitioning ( G.Takerkart, E.Segard, M.Monsigny, FEBS Dett. 42,
218 (1972), B.A.Andrews, D.M.Head, P.Dunthorne, J.A.Asenjo, Biotscih.Tech.
4, 49 (1990)).
5. Immobilized metal affinity chromatography ( G.Ehteshami. J.Porath,
»
R.Guzman, G.Ehteshami, J.Mol.Recognit. 9, 733 (1996), A.L.BIonnkalns, M.R.Gomez, Prep.Biochem.Biotechnol. 27, 219 (1997)).
6. Molecularly imprinted immobilized metal affinity chromarography
(F.H.Arnold, P.Dhal, D.Shnek, S.Plunkett, US patent 5,310,648 (1994))..
These techniques exploit either the binding interaction between actiive site
of enzyme and its inhibitor i.e. affinity monomer or between surface histidine,
tyrosine, cysteine groups of enzymes with metal ions immobilized on a suipport. But these techniques also suffer from one or other disadvantages as follows.
Affinity chromatography uses a column containing an inhibitor or a dye or an antibody for a given enzyme for its separation from a mixture of enzymes. The solution of enzymes is poured over the affinity column to retain the desired enzyme on column for subsequent isolation. This technique is efficient omly for

small capacity columns. With the scale up of columns, the problems of sample pretreatment and plugging of packed column become severe.
Affinity precipitation technique needs a complex to be formed between a
macro-ligand and a protein both containing multiple binding sites. When such a
•i
complex is formed, it precipitates. Thus, this technique is restricted mainly for multimeric proteins. Beside such macro-ligands are often costly. Therefore, certain synthetic inhibitors or dyes are linked to stimuli responsive polymer which can be precipitated by pH or temperature stimuli after it forms complex with the enzyme. In this case along with the desired enzyme, a substantial amount of other enzymes bind to the polymer by means of nonspecific adsorption.
In case of affinity crossflow ultrafiltration, a solution of enzymes is filtered through a membrane containing affinity group under pressure. This technique is suitable in the cases where difference between molecular weights of two enzymes is high. Also, with increase in the filtration time, denaturation of enzymes as well as clogging of membrane takes place due to the pressure applied.
Affinity partitioning of two- phase aqueous systems is widely used technique as compared to those mentioned above, hi this technique concentrated

aqueous solution of poly (ethylene glycol) (PEG) with or without linking affinity group is mixed with enzyme solution containing moderate to high salt concentration. The two phases are mixed well and allowed to separate. Enzyme gets predominantly partitioned in one phase, which subsequently can be isolated Disadvantages of this technique are nonspecific extraction of other protenacious molecules along with desired enzyme and also poor interactions between enzyme and affinity group due to high ionic strength.
Immobilized metal affinity chromatography is a technique in which columns of polymeric support containing chelated metal ions are used. These metal ions form coordination complex with histidine, tyrosine, cysteine etc. present on the surface of the enzyme. Although this technique has advantages like high column capacity, ease in the enzyme elution etc. it is not very selective in descriminating between two closely related (with respect to their amino acid sequences) enzymes.
Molecular imprinting of matrices containing metal chelates is a recently developed technique, which increases the selectivity (F.H.Amold, P.Dhal, D.Shnek, S.Plunkett, US patent 5,310,648 (1994)). In this technique complex of monomer containing chelated metal ion and enzyme is polymerized with crosslinker in order to imprint the polymer with enzyme. Although this technique

exhibits a substantial selectivity, it is not as selective as that of biological antibodies or active site inhibitor of enzymes.
Synthetic inhibitors are molecules which exhibit affinity to the active site of a given enzyme. This interaction is very specific for a given pair of enzyme and inhibitor and such inhibitors bind very strongly to a given enzyme.
Molecular imprinting is a simple technique which allows synthesis of polymers capable of recognizing specific molecules. These polymers are stable and can withstand harsh conditions such as temperature and pH etc ( G.Wulff, Molecular interactions in bioseparations Ed. by T.T.Ngo, Plenum press, N.Y. (1993)). Thus these polymers are finding wide range of applications. A polymer containing monomer based on enzyme inhibitor Le. affinity monomer which is also imprinted for corresponding enzyme is expected to exhibit a high selectivity as well as high capacity for binding a specific enzyme. This synergistic affinity -imprinting effect will not be available in the above mentioned techniques. Such affinity- imprinted polymers have not been reported so far.
The object of the present invention is to provide a process for the preparation of molecularly imprinted polymers comprising affinity monomers

imprinted for various enzymes, by the above mentioned affinity imprinting technique, useful for having high selectivity as well as high capacity for separation of enzymes.
Accordingly the present invention provides a process for the preparation of molecularly imprinted polymers useful for separation of enzymes which comprises;
a) reacting the complex of enzyme and affinity monomer in the ratio of 1:1 to 1:10 (w/w) a co monomer and a cross linker such as herein described in 5 to 80 % and 10 to 90 % by wt. of the total feed respectively with a polymerization initiator and a polymerization accelerator such as herein described at ambient temperature and pressure for a period ranging between 2 to 24 hrs, to obtain a cross linked polymer,
Jo) crushing the said cross linked polymer to fine particles,
e) adding a solvent and extracting imprinted enzyme from the cross linked polymer to obtain the molecularly imprinted polymer.
In one of the embodiments of the present invention, enzymes may be selected from a wide range of enzymes and proteins such as trypsin, chymotrypsin, lysozyme, ova albumin (no enzyme nutritional protein) etc. and the complexing affinity monomers for these enzymes may be selected from acryloyl derivatives of inhibitor s for respective enzymes such as N-acryloyl para amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine, N-

oleoylacrylamide respectively, and the enzyme-affinity monomer complex may be prepared by contacting aqueous solutions of enzyme and the respective affinity monomer.
In an another embodiment, affinity monomers- N-acryloyl para amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine and N-oleoylacrylamide may be prepared by the reaction of acryloyl chloride with para amino benzamidine, 4-phenyl butyl amine, N-acetyl glucosamine, and acrylamide respectively.
In an another embodiment of the present invention the ratio of enzyme to affinity monomer may be in the range of 1:1 to 1:10, preferably 1:1. (w/w).
In yet another embodiment, the comonomer may be selected from compounds such as acrylamide, methacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, acrylic acid, methacrylic acid and the like.
In another embodiment, the amount of comonomer in the polymerization mixture may be in the range of 5 to 80%, more particularly 45 to 55% by weight of the total feed (i.e. enzyme-affinity monomer complex+comonomer-tcrosslmker,

hereinafter.)
In still another embodiment, crosslinker may be selected from compounds such as methylene bisacrylamide, N, N diacryloyl 1,2 diaminoethane. N, N dimethacryloyl 1,2 diaminoethane N, N diacryloyl 1,6 diaminohexane. ethylene glycol dimethacryalte and the like.
In still another embodiment, the amount of crosslinker may be between 10% to 90%, more particularly 30 to 50% by weight of the total feed.
In yet another embodiment, the polymerization initiator used may be selected from compounds such as ammonium per sulfate. eerie ammonium sulfate, potassium persulfate, azobisisobutyronitrile and the like.
In yet another embodiment, the polymerization initiator may be 0.1% to 1% by weight of the total feed.
In still another embodiment, the polymerization accelerator may be selected from compounds such as ethylene diamine, N. N, V, N"tetramethy

ethylenediamine and the like.
In still another embodiment, the polymerization accelerator may be 1% to 4% by weight of the total feed.
In still another embodiment, the solvent used to extract the engine from the imprinted polymer may be selected from solvents which break the enzyme-inhibitor complex such as phenol, chloroform, glacialacetic acid and the like.
In a feature of the present invention, the molecularly imprinted polymer is typically prepared under mild conditions as follows. Affinity monomer and its respective enzyme are dissolved in water and stirred for 15 to 30 minutes at room temperature to form the enzyme-affinity monomer complex. Then comonomer, crosslinker and polymerization initiator are added to the solution containing enzyme-affinity monomer complex and the solution is purged with nitrogen for 10 minutes. Polymerization accelerator is then added and the solution is kept at 37 ° C for 18 hours for polymerization. The crosslinked polymer so obtained is treated with acetone to remove water and crushed to small particles. Template enzyme is extracted from the particles by 3 to 4 alternative treatments of the solvent which breaks the complex between enzyme and the inhibitor. Polymer particles are then

sieved and used for enzyme separations.
In an another feature of the present invention, enzyme imprinted polymers are contacted with aqueous solution containing the respective enzyme or a mixture of enzymes and allowed to shake at room temperature for 1 hour. Polymer particles with bound enzyme are then filtered and isolated.
Although, the present invention describes a process for the preparation of molecularly imprinted polymers useful for the separation of enzymes namely trypsin, chymotrypsin, lysozyme and ovaalbumin (nonenzyme nutritional protein), the scope of the present invention is not and should not be construed to limit only to such molecularly imprinted polymers for separation of trypsin, chymotrypsin, lysozyme and ovaalbumin only but it may extend to such combinations of affinity monomers and their respective enzymes.
The ranges and limitations provided in the instant specification and claims are those which are believed to particularly point out and distinctly claim the present invention. It is however understood that other ranges and limitations which perform substantially the same function in me same or substantially the same manner to obtain the same or substantially the same results are intended to be

within the scope of the instant invention as defined by the instant specification and claims.
Example 1
Preparation of trypsin imprinted polymer.
In an air tight round bottom teflon tube 50 mg N-acryloyl para aminobenzamidine hydrochloride is dissolved in 3 ml. water containing stoichiometric amount of sodium hydroxide so as to free gnanidine groirp from its hydrochloride form. To this solution 50 mg trypsin is added; and the resulting solution is shaken gently on a shaker bath for 15 min.; to form trypsin-N-acryloyl para aminobenzamidine complex. The comonomer acrylamide 400 mg; crosslinker methylene bis acrylamide 500 mg; and initiator ammonium per sulfate 10 mg are dissolved in 1 ml. dimethyl formamide and this solution is added to above aqueous solution containing trypsin-N-acryloyl para aminobenzamidine complex. This is mixed well and nitrogen gas is passed through this solution for 10 min. and 40µl tetramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bath at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remove water. It is then crushed to fine particles.
Template trypsin is extracted from the particles by 3-4 alternative

treatments of acetone and chloroform. The resulting crypsin imprhied polymer is then dried in vacuum oven at 50° C for 12 hrs. Dried polymer piracies are then sieved through standard test sieves and particles in the size range of 250-500 µ are used for enzyme separation.
Example 2
Preparation of chymotrypsin imprinted pohmer.
In an air tight round bottom teflon tube 50 mg N-acryloyl 4 phenyl butylamine is dissolved 3 ml. dimethyl formamide water mixture (10:80). To this
solution 50 mg chymotrypsin is added; and the resulting solution if shaken gently
on a shaker bath for 15 min.; to form chymotrypsin-N-acryloyl 4 phenyl butyl
amine complex. The comonomer acrylamide 400 rug; crosslinker rnethylene bis acrylamide 500 mg; and initiator ammonium per sulfase 10 mg are dissolved in 1 ml.dimethyl formamide and this solution is added to above aqueous solution containing chymotrypsin-N-acryloyl 4 phenyl butylsmine complex This is mixed well and nitrogen gas is passed through this solution for 10 min and 40 µl tetramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bath at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remove water. It is then crushed to fine parades.
Template chymotrypsin is extracted from the particles by 5-4 alternative treatments of acetone and chloroform. The resulting chymotrypsin imprinted

polymer is then dried in vacuum oven at 50° C for 12 hrs. Dried polymer panicles are then sieved through standard test sieves and particles in the size range of 250-500µ are used for enzyme separation.
Example 3
Preparation of lysozyme imprinted polymer.
In an air tight round bottom teflon tube 50 mg acrloyl N-acetyl glucosamine is dissolved in 3 ml. water. To this solution 20 mg lysozyme is added; and the resulting solution is shaken gently on a shaker bath for 15 min.; to form lysozyme -acryloyl N-acetyl glucosamine complex. The comonomer acrylamide 400 mg; crosslinker methylene bis acrylamide 500 mg: and initiator ammonium per sulfate 10 mg are dissolved in 1 ml.dimetfeyl formamide and this solution is added to above aqueous solution containing h-sozyme-acryloyl N-acetyl glucosamine complex. This is mixed well and nitrogen gas is passed through this solution for 10 min. and 40 µl tetramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bath at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remove water. It is then crushed to fine particles.
Template lysozyme is extracted from the particles by 3-4 treatments of glacial acetic acid. The resulting lysozyme imprinted polymer is tken dried in vacuum oven at 50° C for 12 hrs. Dried polymer particles are then sieved through

standard test sieves and particles in the size ranct of 250-500 n are used for enzyme separation.
Example 4
Preparation of ovaalbumin imprinted polymer.
In an air tight round bottom teflon tube 5) mg N-oleoyl acrylamide is dissolved in 3 ml. Dimethyl formamide/water mixture (20:80). To this solution 50 mg ovaalbumin is added; and the resulting solution is shaken gently on a shaker bath for 15 min.; to form ovaalbumin-N-oleoyl acrylamide complex. The comonomer acrylamide 400 mg; crosslinker methykne bis acrylamide 500 mg; and initiator ammonium per sulfate 10 mg are dissolved in 1 ml.dimethyl formamide and this solution is added to above aqueous solution containing ovaalbumin-N-oleoyl acrylamide.complex. This is nixed well and nitrogen gas is passed through this solution for 10 min. and 40 µl teramethylene ethylenediamine is added. The teflon tube containing this solution is kept in a water bam at 37 ° C; for 18 hrs. The gel so synthesized is treated with acetone to remover water. It is then crushed to fine particles.
Template albumin is extracted from the particles by 3-4 treatments of phenol - chloroform The resulting ovaalbumin imprinted polymer is men dried in vacuum oven at 50° C for 12 hrs. Dried polymer paricles are then sieved through standard test sieves and particles in the size range of 250-500 µ are used for

enzyme separation.
In all the above-mentioned examples, formation of enzyme-affinity monomer complex was confirmed by the active site titration of respective complexes.
Example-5
This example describes the process for selective binding of trypsin by trypsin imprinted polymer from trypsin and chymotrypsin mixture.
In a 50 ml. capacity conical flask; 250 mg trypsin imprinted polymer is suspended in 25 ml 10 mM Ca+2 aqueous solution containing trypsin and chymotrypsin with equal initial activities. The flask is allowed to shake at 50 rpm speed at room temperature on a rotary shaker for 1 hr. The swollen polymer particles are then filtered and in the filtrate the activities of trypsin and chymotrypsin are determined by using following standard substrates. For trypsin DL-Benzoyl-Arginyl.-Para nitroanilide, and for chymotrypsin L- Benzoyl-Tyrosyl-Para nitroanilide are used.
Uptake of trypsin by the polymer was determined by subtracting the activity of trypsin in the filtrate from the initial activity of trypsin in the solution. It was found that activity wise 40 percent trypsin and only 2.6 percent chymotrypsin was adsorbed by trypsin imprinted polymer.

Example-6
This example describes the process for selective binding of chymotrypsin by chymotrypsin imprinted polymer from chymotrypsin and
trypsin mixture.
hi a 50 ml. capacity conical flask; 250 mg chymotrypsin imprinted polymer is suspended in 25 ml 10 mM Ca+2 aqueous solution containing trypsin and chymotrypsin with equal initial activities. The flask is allowed to shake at 50 rpm speed at room temperature on a rotary shaker for 1 hr. The swollen polymer particles are then filtered and in the filtrate the activities of trypsin and chymotrypsin are determined by using following standard substrates. For trypsin DL-Benzoyl-Arginyl.-Para nitroanilide, and for chymotrypsin L- Benzoyl-Tyrosyl-Para nitroanilide are used.
Uptake of chymotrypsin by the polymer was determined by subtracting the activity of chymotrypsin in the filtrate from the initial activity of chymotrypsin in the solution. It was found that activity wise 52 percent chymotrypsin and only 4 percent trypsin was adsorbed by chymotrypsin imprinted polymer.

Example-7
This example describes the process for binding of lysozyme by lysozyme imprinted polymer from lysozyme solution.
In a 50 ml. capacity conical flask; 250 mg lysozyme imprinted polymer is suspended in 25 ml 0.06 M phosphate buffer. PH = 6,8 containing 2.5 mg lysozyme. The flask is allowed to shake at 50 rpn speed at room temperature on a rotary shaker for 1 hr. The swollen polymer particles are men filtered and in the filtrate the activity of lysozyme is determined by using micrococus lyzodeikticus, a standard substrate for lysozyme.
Uptake of lysozyme by the polymer was determined by subtracting the activity of lysozyme in the filtrate from the initial activity of lysozyme in the solution. It was found that activity wise 60% percent lysozyme was adsorbed by lysozyme imprinted polymer.
Example-8
This example describes the process fcr binding of ovaalbumin by ovaalbumin imprinted polymer from evaalbumin solution.
In a 50 ml. capacity conical flask; 250 mg cvaalbumin imprinted polymer is suspended in 25 ml water containing 2.5 mg ovaalbumin (determined by

absorbance at 280 nm). The flask is allowed to shake at 50 rpm speed at room temperature on a rotaiy shaker for 1 hr. The swollen polymer partides are then filtered and in the filtrate the ovaalbumin in terms of protein (determined from standard graph at 280 nm) is determined by using pure ovaalbumin as a standard.
Uptake of ovaalbumin by the polymer was determined by subtracting the protein present in the filtrate from the initial protein added in the solution. It was found that protein wise 45% percent ovaalbumin was adsorbed by ovaalbumin imprinted polymer.
The advantages of the present invention are as follows:
1) The process of the present invention, unlike the other conventional processes,
exhibits a very high selectivity as well as very high capacity for separating the
imprinted enzyme either from a mixture of enzymes or from their aqueous
solutions.
2) The process of the present invention is applicable in general for separating
various other enzymes by preparing affinityimprinted polymers as mentioned
herein above using appropriate complexing affinity monomer and the respective
enzyme.
3) The process of the present invention is convenient and the polymers synthesized
by following the present process are resistant to denaturation by the action of

organic solvent, high temperature and pressure.

We Claim:
1. A process for the preparation of molecularly imprinted polymers useful for
separation of enzymes which comprises;
a) reacting the complex of enzyme and affinity monomer in the ratio of 1:1 to 1:10
(w/w), a co-monomer and a cross linker such as herein described in 5 to 80 % and
10 to 90 % by wt. of the total feed respectively with a polymerization initiator
and a polymerization accelerator such as herein described at ambient temperature
and pressure for a period ranging between 2 to 24 hrs, to obtain a cross linked
polymer,
b) crushing the said cross linked polymer to fine particles,
c) adding a solvent and extracting imprinted enzyme from the cross linked polymer
to obtain the molecularly imprinted polymer.

2. A process as claimed in claim 1 wherein enzymes used are selected from tryspin,
chymotrypsing, lysozyme, ovaalbumin; the complexing affinity monomers for
these enzymes used are selected from N-acryloyl para amino benzamidine, N-
acryloyl 4-phenyl butyl amine, acryloyl N-acetyl glucosamine, N-
oleoylacyrylaminde.
3. A process as claimed in claims 1 and 2 wherein, the enzyme-affinity monomer
complex is prepared by contacting aqueous solutions of enzyme and the
respective afinity monomer.
4. A process as claimed in claims 1 to 3 wherein, affinity monomers-N-acryloyl para
amino benzamidine, N-acryloyl 4-phenyl butyl amine, acryloyl N-acetyl
glucosamine and N-oleoylacrylamide are prepared by the reaction of acryloyl
chloride with para amino benzamidine, 4-phenyl butyl amine, N-acetyl
glucosamine, and acrylamide.
5. A process as claimed in claims 1 to 4 wherein, the ratio of enzyme to affinity
monomer is preferably 1:1 (w/w).

6. A process as claimed in claims 1 to 5 wherein, the comonomer used is selected
from acrylamide, methacrylamide, N-isopropylacrylamide, N-
isopropylmethacrylamide, acrylic acid, methacrylic acid.
7. A process as claimed in claims 1 to 6 wherein, the amount of co monomer in the
polymerization mixture is preferably 45 to 55% by weight of the total feed
8. A process as claimed in claims 1 to 7 wherein, cross linker used is selected from
methylene bisacrylamide, N, N diacryloyl 1,2 diaminoethane, N,N dimethacryloyl
1,2 diaminoethane, N, N diacryloyl 1,6 diaminohexane, ethylene glycol
dimethacrylate.
9. A process as claimed in claims 1 to 8 wherein, the amount of cross linker is
preferably 30 to 50% by weight of the total feed.
10. A process as claimed in claims 1 to 9 wherein, the polymerization initiator used is
selected from ammonium per sulfate, eerie ammonium sulfate, potassium per
sulfate, azobisisobutyronitrile.
11. A process as claimed in claims 1 to 10 wherein, the polymerization initiator is 0.1
to 1% by weight of the total feed.
12. A process as claimed in claims 1 to 11 wherein, the polymerization accelerator
used is selected from ethylene diamine, N, N, N', N"tetramethylenediamine.
13. A process as claimed in claims 1 to 12 wherein, the polymerization accelerator is
1 to 4% weight of the total feed.
14. A process as claimed in claims 1 to 13 wherein, the solvent used to extract the
enzyme from the imprinted polymer is which break the enzyme-inhibitor complex
is selected from chloroform, glacial acetic acid.
15. A process for the preparation of molecularly imprinted polymers useful for
separation of enzymes as described hereinabove with reference to the examples.

Documents:

1199-del-1999-abstract.pdf

1199-del-1999-claims.pdf

1199-del-1999-correspondence-others.pdf

1199-del-1999-correspondence-po.pdf

1199-del-1999-description (complete).pdf

1199-del-1999-form-1.pdf

1199-del-1999-form-19.pdf

1199-del-1999-form-2.pdf

1199-del-1999-form-3.pdf

1199-del-1999-petition-138.pdf

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Patent Number

215686

Indian Patent Application Number

1199/DEL/1999

PG Journal Number

12/2008

Publication Date

21-Mar-2008

Grant Date

29-Feb-2008

Date of Filing

08-Sep-1999

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ALANKAR ARUN VAIDYA	POLYMER SCIENCE & ENGINEERING UNIT, NATIONAL CHEMICAL LABORATOTY, PUNE INDIA-411 008.
2	BHALCHANDRA SHRIPAD LELE	POLYMER SCIENCE & ENGINEERING UNIT, NATIONAL CHEMICAL LABORATORY, PUNE, INDIA-411 008.
3	MOHAN GOPALKRISHNA KULKARNI	POLYMER SCIENCE & ENGINEERING UNIT, NATIONAL CHEMICAL LABORATORY, PUNE, INDIA-411 008.
4	RAGHUNATH ANANT MASHELKAR	POLYMER SCIENCE & ENGINEERING UNIT, NATIONAL CHEMICAL LABORATORY, PUNE INDIA-411 008.

PCT International Classification Number

C08J 005/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA