Title of Invention

A PROCESS FOR THE PREPARATION OF THERMOPRECIPITATING AFFINITY POLYMERS

Abstract This invention relates to the process for the preparation of thermo precipitating affinity polymers, which are useful for separation of enzymes of protease type exemplified by trypsin. Affinity polymers prepared by the process of the present invention exhibit stronger binding with trypsin which is useful in enhancing the recovery of trypsin from dilute aqueous solutions and from a mixture of trypsin and chymotrypsin or a mixture of trypsin and other enzymes. The process of the present invention enables synthesis of thermo precipitating affinity polymers exhibiting enhanced interactions with the enzyme and thereby giving high recovery of the desired enzyme. Unlike high molecular weight affinity polymers reported in the literature, low molecular weight affinity polymers containing high concentration of inhibitors, synthesized by the process of the present invention, eliminate the crowding effect. This feature is very useful in achieving higher yields of separations. Affinity polymers exhibiting a wide range of lower critical solution temperature are synthesized using the process of the present invention. Thus affinity polymers with low LCST could be used for separations at very low temperatures. This is an added advantage for recovery of heat sensitive enzymes.
Full Text This invention relates to the process for the preparation of (hermoprccipitatmg affinity polymers. More particularly, it relates to the preparation of these polymers which are useful for separation of enzymes of protease type exemplified by trypsin. Affinity polymers prepared by the process of the present invention exhibit stronger binding with trypsin which is useful in enhancing the recoveiy of trypsin from dilute aqueous solutions and from a mixture of trypsin and chymotrypsin or a mixture of trypsin and other enzymes.
Isolation and purification of biologically active macromolecules such as enzymes, from natural sources is a tedious, multi-step process, which results in very low yields and thus higher costs. As a better alternative to the conventional processes, researchers have developed affinity separations based techniques for selective and enhanced separations of enzymes. The basic principle used in these techniques is to form a complex between the active site of an enzyme and its inhibitor. Since enzyme-inhibitor interactions are very strong and are specific for a given parr of enzyme and inhibitor, selective and high separations are possible. Most of the affinity based separations involved polymers to which inhibitors are chemically linked. The complex formed between polymeric inhibitor and the enzyme is subsequently processed to isolate the enzyme.

Various techniques such as affinity chromatography, affinity partitioning, affinity ultrafiltration, immobilized metal affinity chromatography, affinity imprinting and affinity precipitation have been developed so far. Although all these techniques use the same basic principle of forming an enzyme-inhibitor complex, they suffer from one or the 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 only for small capacity columns. With the scale up of columns, the problems of sample pretrearment and plugging of packed column become severe. [Y. Li, G.Kunyu, C.Lubai, Z.Hanfa, Z.Yunkui Sepu,14, 415 (1996), T.Makriyannis, Y.D.Clonis, Biotech.Bioengg. 53, 49 (1997)].
In case of affinity crossflow ultrafiltration, a mixture 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 thanes is high Al^o, with increase in the filtration time, tU.aio

of enzymes as well as clogging of membrane takes place due to the pressure applied. [K.Sigmundsson, H.Filippusson, Polymer int. 41, 355 (1996), T.B.Choe, P.Masse, A.Verdier, Biotech.Lett. 8, 163 (1986)].
Affinity partitioning of two- phase aqueous systems is widely used technique as compared to those mentioned above. In 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. The desired 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 en2yme and also poor interactions between enzyme and affinity group due to high ionic strength. [ G.Takerkart, E.Segard, M.Monsigny, FEBS Lett. 42, 218 (1972), BAAndrews, D.M.Head, P.Dunthome, J.A.Asenjo, Biotech.Tech. 4, 49 (1990)].
Immobilized metal affinity chromatography is a technique in which the columns of polymeric support containing chelated metal ions are used. These metal io?:;* form coordination complex with histidine, tyrosine, cysteine etc.

present on the surface of (he enzyme. Although this technique has advantages like high column capacity, ease in the enzyme elution etc. it is not very selective. [Ehteshami, J.Porath, R.Guzman, G.Ehteshami, J.Mol.Recognit. 9, 733 (1996), A.L.Blomkalns, M.R.Gomez, Prep.Biochem.Biotechnol. 27, 219 (1997)].
Molecular imprinting of matrices containing metal chelates is a recently developed technique, which increases the selectivity [F.H.Arnold, P.Dhal, D.Slmek, S.Plunkert, US patent 5,310,648 (1994)]. In this technique complex of monomer containing chelated metal ion and enzyme is polymerized wiA crosslinker in order to imprint the polymer with enzyme. Although this technique exllibits a substantial selectivity, it is not as selective as mat of biological antibodies or active site inhibitors of enzymes.
The inventors of the present invention have recently developed a novel technique viz. affinity-imprinting which combines advantages of both molecular imprinting and affinity interactions. In this, enzyme-inhibitor complex is polymerized with crosslinker to form an imprinted gel. Subsequently, the template enzyme is eluted out from the imprinted gel. The synergis-:ic affinity-imprinting effect exhibited by These gels enables exclusive

recognition of the imprinted-enzyme. But the-capacity of these gels is very low and also the recovery of the adsorbed en2yme is difficult due to the rigid and highly crosslinked nature of the gels. [A.A.Vaidya, B.S.Lele, M.G.Kulkarni, R.A.Mashelkar, Polymer (communicated)].
As compared to the herein above described techniques, from the
application point of view, affinity precipitation is an attractive technique.
[C.Senstad, B.Mam'asson, BioteckBioengg. 33, 216 (1989), M.Schneider,
C.Guillot, B.Lamy, Ann.KY.Acad.Sci. 369, 257 (1981)), B. Martiasson, R.
Kaul, "Affinity precipitation", In: Molecular interactions in bioseparations,
T.T.Ngo Ed. Plenum Press, New York, p 469-477 (1993), J.P.Chen, J.Ferment.
and Bioengg. 70, 119 (1990), LY.Galaev, B.Mattisson, Biotech. Bioeng. 41,
1101 (1993), M.Pecs, M.Eggert, K. Schnegerl, New Polymeric Mater. 4, 19
(1993)]. It involves formation of complex between an enzyme and a stimuli
sensitive polymeric inhibitor. This complex is precipitated by pH or
temperature stimulus and isolated. It is then dissociated, polymer is separated
by pH or temperature stimulus and the enzyme is isolated. Thus the recovery of
enzyme by this technique is much simpler and the scale up of the process is
also easy. Hitherto, affinity precipitation suffers from the following problems.
viz. restrictions on the accessibility cf the enzyme towards the polymer bound
inhibitor and the crowding effect.

The strength of the complex formed between inhibitor and enzyme decreases 20 to 300 folds when it is bound to the polymer. [K.B. Male, J.H.T. Luong, A.L. Nguyen, Enzy. Microb. Tech. 9, 374 (1987), Yu, I. Galeav, B. Mattisson, Biotech Bioengg. 41, 1101 (1993), J.H.T. Luong, K.B. Male, A.L. Nguyen, Biotech. Bioengg. 31, 439 (1988), M. Pecs, M. Eggert, K. Schuegerl, J. Biotech. 21, 137 (1991)]. This weakening of the complex is attributed mainly to the restrictions on the free access of enzyme to the polymer bound inhibitor. The strength of the complex is expressed in terms of inhibition constant (K,-). The lower the value of K;, the higher is the inhibition and stronger is the complex formed. Higher K; values of polymeric inhibitors result in poor recovery of enzymes. Also, increased concentration of inhibitors on the high molecular weight polymers results in high K; values (crowding effect).
Introduction of spacers between polymer backbone and the inhibitor is a well-known methodology used in affinity chromatography to enhance the interactions between inhibitor and the enzyme. But in affinity precipitation, the use of spacer containing polymers has not been reported so far, because the complex formation between polymer bound inhibitor and the enzyme takes place in homogeneous solution and it has been suggested that m homogenous solution spacers are not required. Also, the crowding effect described above was observed for high molecular weight polymers. Low molecular weight

Polymers are expected to eliminate this crowding effect and exhibit lower K values which, will subsequently increases the recovery of enzyme.
It is therefore an object of the present invention to provide, a process for the preparation of thermo precipitating affinity polymers comprising spacers between the polymer backbone, and the inhibitor, useful in enhanced recovery of trypsin by affinity precipitation.
Another object of the present invention is to provide a process for the preparation of low molecular weight thermo precipitating affinity polymers with increasing concentration of inhibitors, useful in eliminating the crowding effect i.e. provide lower K; values.
Accordingly the present invention provide a process for the preparation of thermo precipitating affinity polymers containing spacer of 1 to 5 methylene group which comprises; polymerizing a monomer selected from compounds of the formula CH2=CR-CO-NH-(CH2)n-COOH wherein, R is hydrogen or methyl group and n is an integer of 1 to 10 and a co monomer selected from compounds N-isopropyl acrylamide, N-butyl acrylmide, N-isopropyl methacrylamide, N-vinyl caprolactam in the range of 1:10 to 1:1 with a polymerization initiator 10 to 20% by wt.

of the monomers and a polymerization accelerator 1 to 5% by wt. of the monomers at ambient temperature and pressure for a period ranging between 2 to 24 hours to obtain a polymer, linking an inhibitor to pendant carboxyl groups of the spacers via condensing regent in the above said polymer wherein the molar ratio of inhibitor to carboxyl groups in the polymer is in the range of 1:1 to 10:1. and the molar ratio of condensing reagent to carboxyl groups is in the rage of 1:1 to 100:1 to obtain an affinity polymer by conventional methods.
In one of the embodiments of the present invention, the spacer monomer may be selected from compounds of the formula CH2=CR-CO-NH-(CH2)n-COOH wherein, R is hydrogen or methyl group and n is an integer of 1 to 10.
In an another embodiment, the co-monomer may be selected from compounds like N-isopropyl acrylamide, N-butyl acrylamide, N-isopropyl methacrylamide, N-vinyl caprolactam.
In an another embodiment, the molar ratio of spacer monomer to co monomer used in the polymerization mixture may be in the range of 1:10 to 1:1.
In an another embodiment, the polymerization initiator used may be selected from compound such as ammonium per sulfate, potassium per sulfate.

In yet an another embodiment, the polymerization initisrcrmy be 10° a to 20% by wt. of the monomers.
In still another embodiment, the polymerization accelerased may be selected from compounds like N,N,N',N" tetramethylene etlib.yij!:? diamine. sodium meta bisulfate, potassium meta bisulfate.
In an another embodiment, the polymerization acceleratesry be 1% to 5% by wt. of the monomers.
In an another embodiment, the inhibitor may be *.:2£ted from compounds like meta araino benzamidine, para amino benzamiL/ss and their hydrochlorides.
In an another embodiment, the molar ratio of inhibitor carboxy groups in the polymer may be in the range of 1:1 to 10:1.
In ar another embodiment, the condensing reagent used for linking

inhibitor to pendant carboxyl groups of the polymer may be 1-cyclohexyl 3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), l-ethyl-3-(3-Dimethylamino-propyl) carbodiimide (EDC).
In an another embodiment the molar ratio of condensing reagent to carboxyl groups may be in the range of 1:1 to 100:1.
In a feature of the present invention, the thermoprecipitating affinity polymer comprising spacers is typically prepared under mild conditions as follows. Spacer monomer, co-monomer and polymerization initiator are dissolved in water and the solution is purged with nitrogen for 10 to 20 minutes. Then polymerization accelerator is added and the solution is kept at 37 °C for 24 hours for polymerization. After polymerization, temperature of the solution is raised above lower critical solution temperature (LCST) of the polymer and precipitated polymer is isolated.
In an another feature of the present invention, the polymer is dissolved in water at 10 °C. One to ten fold molar excess of inhibitor and condensing reagent over the carboxyl groups in the polymer is added in this solution. The

solution is stirred for 1 to 12 hours at 10 °C. Inhibitor-linked polymer i.e. affinity polymer is then precipitated by raising the temperature above its LCST (37 to 65 °C) and precipitated affinity polymer is isolated.
In an another feature of the present invention, affinity polymers synthesized by the process of the present invention are used for trypsin recovery. A solution of affinity polymer is mixed with a solution of trypsin and chymotrypsin. This is allowed to stand at 4 to 25 °C fqr 15 minutes to 1 hour.
*
Then the temperature of the solution is raised above LGST of the affinity polymer (37 to 65°C) to precipitate the polymer-trypsin complex. The complex is separated by centrifugation. Polymer-trypsin complex is dissociated by dissolving it in an acidic buffer. Temperature of this solution is then raised above LCST of the affinity polymer. The polymer is separated by centrifugation and the clear filtrate exhibiting trypsin activity is isolated.
Although the present invention describes a process for the preparation of thermoprecipitating affinity polymers useful in the enhanced recovery of trypsin from a mixture of trypsin and chymotrypsin, the scope of the present invention is not and should not be construed to limit only to such affinity polymers for separation of trypsin, but it may extend to such combinations of

polymer bound inhibitors and their respective enzymes.
The ranges and limitations provided hi 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 the 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
This example illustrates preparation of poly (N-isopropyl acrylamide -co-N-acryloyl glycyl- para aminobenzamidine) (affinity polymer containing spacer of 1 methylene group)
1.29 g (0.01 M) N-acryloyl glycine (spacer monomer), 10.17 g (0.09 M) N-isopropyl acrylamide (co-monomer) was dissolved hi 50 ml water. To this 1.15 g (10 % w/w) ammonium per sulfate was added and the solution was purged with nitrogen for 15 min. Then, polymerization was initiated by adding 0.5 ml of N,N,N',N" tetramethyl ethylene diamine (TEMED) to it. Polymerization

was allowed to proceed at 37 °C for 18 hrs. The polymer so synthesized was precipitated by increasing the temperature of the aqueous solution above its lower critical solution temperature (LCST). It was washed with cold double distilled water twice and once with cold Tris-HCl buffer. Then the polymer was dried under vacuum at room temperature. The amount of carboxyl groups incorporated in the polymer was estimated from its acid value. Data are listed in Table 1. Para amino benzamidine was covalently linked to pendant carboxyl groups of the polymer via amide bond using 1-cyclohexyl 3-{2-morpholinoethyl) carbodiimde metho p-toluenesulfonate (CMC). Para aminobenzamidine dihydrochloride was treated with 40 fold molar excess of sodium acetate in water to free the para-amino groups. Then 4 g polymer was dissolved in 40 ml of double distilled water at 10 °C. Ten fold molar excess of CMC and para aminobenzamidine solution, over the carboxyl groups was added and the reaction mixture was stirred for 12 hrs at 10 °C. Para amrn'obenzamidine linked polymer was precipitated out by increasing the temperature of the solution above its LCST. It was washed three times wrtfa cold double distilled water and once with Tris.HCl buffer. The polymer was dried in vacuo at room temperature. Para aminobenzamidine loading in the polymers was estimated spectrophotometrically. The loading data are summarized in Table 1.

Example 2
This example illustrates the preparation of poly (N-isopropyl acrylamide -co- N-acrylcyl β alanyl-para aminobenzamidine) (affinity polymer containing spacer of 2 methylene groups)
1.43 g (0.01 M) N-aciyloyl 0 alanine (spacer monomer), 10.17 g (0.09 M) N-isopropyl acrylamide (co-monomer) was dissolved in 50 ml water. To this 1.16 g (10 % w/w) ammonium per sulfate was added and the solution was purged with nitrogen for 15 min. Then, polymerization was initiated by adding 0.5 ml of TEMED to it. Polymerization was allowed to proceed at 37 °C for 18 hrs. The polymer so synthesized was precipitated by increasing the temperature of the aqueous solution above its lower critical solution temperature (LCST). It was washed with cold double distilled water twice and once with cold Tris-HCl buffer. Then the polymer was dried under vacuum at room temperature. The amount of carboxyl groups incorporated in the polymer was estimated from its acid value. Data are listed in Table 1. Para amino benzamidine was covalently linked to pendant carboxyl groups of the polymer via amide bond using 1-cyclohexyl 3-(2-morpholinoethyl) carbodiimde metho p-toluenesulfonate (CMC). Para aminobenzamidine dihydrochloride was treated with 40 fold molar excess of sodium acetate in water to free the para-amino groups. Then 4

g polymer was dissolved in 40 ml of double distilled water at 10 °C. Ten fold molar excess of CMC and para aminobenzamidine, over the carboxyl groups was added in polymer solution and the reaction mixture was stirred for 12 hrs at 10 °C. Para amniobenzamidine linked polymer was precipitated out by increasing the temperature of the solution above its LCST. It was washed three times with cold double distilled water and once with Tris.HCl buffer. The polymer was dried in vacuo at room temperature. Para aminobenzamidine loading in the polymers was estimated spectrophotometrically. The loading data are summarized in Table 1.
Example 3
This example illustrates the preparation of poly (N-isopropyl acrylamide -co- N-acryloyl 4 arnino butyryl-para aminobenzamidine) (affinity polymer containing spacer of 3 methylene groups)
1.57 g (0.01 M) N-acryloyl 4 amino butyric acid (spacer monomer), 10.17 g (0.09 M) N-isopropyl acrylamide (co-monomer) was dissolved in 50 ml water. To this 1.17g(10% w/w) ammonium per sulfate was added and the solution was purged with nitrogen for 15 min Then, polymerization was initiated by adding 0.5 ml of TEMED to it. Polymerization was allowed to proceed at 37

°C for 18 hrs. The polymer so synthesized was precipitated by increasing the temperature of the aqueous solution above its lower critical solution temperature (LCST). It was washed with cold double distilled water twice and once with cold Tris-HCl buffer. Then the polymer was dried under vacuum at room temperature. The amount of carboxyl groups incorporated in the polymer was estimated from its acid value. Data are listed in Table I. Para amino beozamidine was covalently linked to pendant carboxyl groups of the polymer via amide bond using 1-cyclohexyl 3-(2-morpholinoethyl) carbodiimde metho p-toluenesulfonate (CMC). Para aminobenzamidine dihydrochloride was treated with 40 fold molar excess of sodium acetate in water to free the para-amino groups. Then 4 g polymer was dissolved in 40 ml of double distilled water at 10 °C. Ten fold molar excess of CMC and para aminobenzamidine over the carboxyl groups was added in polymer solution and the reaction mixture was stirred for 12 hrs at 10 °C. Para aminobenzamidine linked polymer was precipitated out by increasing the temperature of the solution above its LCST. It was washed three times with cold double distilled water and once with Tris.HCl buffer. The polymer was dried in vacuo at room temperature. Para aminobenzamidine loading in the polymers was estimated spectrophotometrically. The loading data are summarized in Table 1.

Example 4
This example illustrates the preparation of poly (N-isopropyl acrylamide -co- N-acryloyl 6 amino caproyl-para aminobenzamidine) (affinity polymer containing spacer of 5 methylene groups)
1.85 g (0.01 M) N-acryloyl 6 amino caproic acid (spacer monomer), 10.17 g (0.09 M) N-isopropyl acrylamide (co-monomer) was dissolved in 50 ml water. To this 1.20 g (10 % w/w) ammonium per sulfare was added and the solution was purged with nitrogen for 15 min. Then, polymerization was initiated by adding 0.5 ml of TEMED to it. Polymerization was allowed to proceed at 37 °C for 18 hrs. The polymer so synthesized was precipitated by increasing the temperature of the aqueous solution above its lower critical solution temperature (LCST). It was washed with cold double distilled water twice and once with cold Tris-HCl buffer. Then the polymer was dried under vacuum at room temperature. The amount of carboxyl groups incorporated in the polymer was estimated from its acid value. Data are listed in Table 1. Para amino benzamidine was covalently linked to pendant carboxyl groups of the polymer via amide bond using 1-cyclohexyl 3-(2-morpholinoethyl) carbodiimde metho p-toluenesulfonate (CMC). Para aminobenzamidine dihydrochloride was treated with 40 fold molar excess of sodium acetate in water to free the para-arnino groups. Then 4 g polymer was dissolved in 40 ml of double .distilled

water at 10 °C. Ten fold molar excess of CMC and para aminobenzamidine over the carboxyl groups was added in polymer solution and the reaction mixture was stirred for 12 hrs at 10 °C. Para amniobenzamidine linked polymer was precipitated out by increasing the temperature of the solution above its LCST. It was washed three times with cold double distilled water and once with Tris.HCl buffer. The polymer was dried in vacuo at room temperature. Para aminobenzamidine loading in the polymers was estimated spectrophotometrically. The loading data are summarized in Table 1.
Example 5
This example illustrates the preparation of poly (N-isopropyl methacrylamide -co- N-acryloyl para aminobenzamidine)
1.89 g (0.01 M) N-acryloyl para aminobenzamidine, 11.43 g (0.09 M) N-isopropyl methacrylamide (co-monomer) was dissolved in 50 ml water. To this 1.33 g (10 % w/w) ammonium per sulfate was added and the solution was purged with nitrogen for 15 min. Then, polymerization was initiated by adding 0.5 ml of TEMED to it. Polymerization was allowed to proceed at 37 °C for 18 hrs. The polymer so synthesized was precipitated by increasing the temperature of the aqueous solution above its lower critical solution temperature (LCST). It was washed with cold double distilled water twice and once with cold Tris-HCl

buffer. Then the polymer was dried under vacuum at room temperature. The amount of carboxyl groups incorporated in the polymer was estimated from its acid value. Data are listed in Table 1. Para am inn benzamidine was covalently linked to pendant carboxyl groups of the polymer via amide bond using 1-cyclohexyl 3-(2-morpholinoethyl) carbodiimde metho p-toluenesulfonate (CMC). Para aminobenzamidine dihydrochloride was treated with 40 fold molar excess of sodium acetate in water to free the para-amino groups. Then 4 g polymer was dissolved in 40 ml of double distilled water at 10 °C. Ten fold molar excess of CMC and para aminobenzamidine over the carboxyl groups was added in polymer solution and the reaction mixture was stirred for 12 hrs at 10°C. Para aminobenzamidine linked polymer was precipitated out by increasing the temperature of the solution above its LCST. It was washed three times with cold double distilled water and once with Tris.HCl buffer. The polymer was dried in vacuo at room temperature. Para aminobenzamidine loading in the polymers was estimated spectrophotometrically. The loading data are summarized in Table 1.
Example 6
This example illustrates the preparation of poly (N-butylacryiamide -co-N-acryloyl 4 ammo butyryl-para aminobenzamidine) (affinity polymer containing spacer of 3 methylene groups)

1.57 g (0.01 M) N-acryloyl 4 amino butyric acid (spacer monomer), 11.44 g (0.09 M) N-butylaciylamide (co-monomer) was dissolved in 50 ml water. To this 1.30 g (10 % w/w) ammonium per sulfate was added and the solution was purged with nitrogen for 15 min. Then, polymerization was initiated by adding 0.5 ml of TEMED to it. Polymerization was allowed to proceed at 37 °C for 18 hrs. The polymer so synthesized was precipitated by increasing the temperature of the aqueous solution above its lower critical solution temperature (LCST). It was washed with cold double distilled water twice and once with cold Tris-HCl buffer. Then the polymer was dried under vacuum at room temperature. The amount of carboxyl groups incorporated in the polymer was estimated from its acid value. Data are listed in Table 1. Para amino benzamidine was covalently linked to pendant carboxyl groups of the polymer via amide bond using 1-cyclohexyl 3-(2-morpholinoethyl) carbodiimde metho p-toluenesulfonate (CMC). Para aminobenzamidine dihydrochloride was treated with 40 fold molar excess of sodium, acetate in water to free the para-ammo groups. Then 4 g polymer was dissolved in 40 ml of double distilled water at 10 °C. Ten fold molar excess of CMC and para aminobenzainidine over the carboxyl groups was added in polymer solution and the reaction mixture was stirred for 12 hrs at 10 °C. Para aminoberizamidine linked polymer was precipitated out by increasing the temperature of the solution above its LCST. It was washed three times with cold double distilled water and once with Tris.HCl builer. The

polymer was dried in vacuo at room temperature. Para aminobenzamidine loading in the polymers was estimated spectrophotometrically. The loading data are summarized in Table 1.
Examples 7-12
These examples illustrate the preparations of polymers for eliminating crowding effect Poly (N-isopropylacrylamide-co-N-acryloyl 6 aminocaproyl- para aminobenzamidine) [poly(NTPAM-co-Ac.6ACA-PABA)]
Copolymers of N-isopropyl acrylamide (NEPAM)with increasing concentration of N-acryloyl 6 amino caproic acid (Ac.6ACA) in the feed were synthesized and para aminobenzamidine was linked to pendant carboxyl groups of these polymers as per the procedure described in the example 1. Data for the feed composition of monomers and the amount of para aminobenzamidine incorporated hi the affinity polymers are given in Table 2.

Example 13 Estimation of inhibition constant (Ki) of affinity polymers
The inhibition constant (Kj) was determined from Dixon plot. Ten percent polymer solution was diluted serially by adding double distilled water to get 9% , 8%, 7 % and 6 % solutions. The substrate N-benzoyl arginine para nitroanilide was dissolved in distilled dimethyl formamide to get following different concentrations 0.6 mM, 0.48 mM, 0.36 mM, 0.24 mM and 0.12 mM. Trypsin solution (1 jiM = 24 fig / ml) was prepared in 0.05 M Tris-HCl buffer, pH 8.1, containing 10 mM Ca*2.
One ml of polymer solution was mixed with 1 ml of trypsin solution. After vortexing the mixture, it was allowed to incubate at 25 °C for 15 min. Then this was added to 1 ml substrate solution taken in a 3 ml capacity cuvette. The rate of substrate hydrolysis (V) was monitored by following the absorbance at 410 nm on UV spectrophotometer for 1 min. Dixon plot of 1/V vs [I] at various substrate and inhibitor concentrations was plotted and Ki was graphically obtained. The intersect on x axis gives -Ki value. Data for Ki values of all polymers are listed in Table 1 and 2.

Example 14
This example illustrates the use of the product for the recovery of trypsin from a mixture of trypsin and chymotrypsin.
One ml of 10 % (w/v) polymer solution was mixed with 1 ml of trypsin-chymotrypsin synthetic mixture ( trypsin = 24 µg / ml and chymotrypsin = 58 ug / ml which corresponds to identical initial activities of the two). It was incubated at 25 ° C for 15 min. Then the temperature of the solution was raised above its LCST. The polymer-enzyme complex was separated by centrifugation at 10,000 rpm for 20 min. The supernatant was stored at 4 ° C. Trypsin was dissociated from complex by the treatment of glycine-HCl buffer pH 2.8. Again the temperature of solution was raised above LCST and the supernatant was separated after centrifugation at 10,000 rpm for 15 min. The activities of trypsin and chymotrypsin was estimated using standard substrates viz. N-benzoyl DL arginyl para nitro anilide (DL-BAPNA) for trypsin and N-benzoyl L-tryosyl para nitro anilide (BTPNA) for chymotrypsin respectively. The percent recoveries of the activities of the enzymes are summarized in Table 3.
Data in Table 1 show that with increase in the spacer chain length from 1 to 5 methylene groups K; of affinity polymers decreased from 250 * l0-6M to 25 * l0-6M i.e. the strength of trypsin-polymer complex increased 10 folds.

(Examples 1 to 4). Data in Table 2 show that in the case of low molecular weight affinity polymers with increasing concentration of para aminobenzamidine (Examples 7-12), i.e. crowding of inhibitor molecules on the polymer, it does not exhibit increase in Ki values, on the contrary decrease in the K; values were observed. Thus, crowding effect is eliminated by low molecular weight affinity polymers synthesized by the process of the present invention. Data listed in Table 3 show that with increase in the spacer chain length from 1 to 5 methylene groups trypsin recovery by affinity polymers increased from 38% to 68%.
Table 1 Polymers which demonstrate spacer effect
(Table Removed)
Table 2 Polymers synthesized to demonstrate crowding effect
(Table Removed)
Table 3 Separation of trypsin from trypsin and chymotrypsin mixture.
The main advantages of the present invention are:
1. The process of the present invention enables synthesis of thermoprecipitating affinity polymers exhibiting enhanced interactions with the enzyme and thereby giving high recovery of the desired enzyme.
2. Unlike high molecular weight affinity polymers reported in the literature, low molecular weight affinity polymers containing high concentration of inhibitors, synthesized by the process of the present invention, eliminate the crowding effect. This feature is very useful in achieving higher yields of separations.
3. Affinity polymers exhibiting a wide range of lower critical solution temperature are synthesized using the process of the present invention. Thus affinity polymers with low LCST could be used for separations ai very low temperatures. This is an added advantage for recovery of hear sensitive enzymes.



We Claim:

1. A process for the preparation of thermo precipitating affinity polymers containing
spacer of 1 to 5 methylene group which comprises; polymerizing a monomer
selected from compounds of the formula CH2=CR-CO-NH-(CH2)n-COOH
wherein, R is hydrogen or methyl group and n is an integer of 1 to 10 and a co
monomer selected from compounds N-isopropyl acrylamide, N-butyl acrylmide,
N-isopropyl methacrylamide, N-vinyl caprolactam in the range of 1:10 to 1:1
with a polymerization initiator 10 to 20% by wt. of the monomers and a
polymerization accelerator 1 to 5% by wt. of the monomers at ambient
temperature and pressure for a period ranging between 2 to 24 hours to obtain a
polymer, linking an inhibitor to pendant carboxyl groups of the spacers via
condensing regent in the above said polymer wherein the molar ratio of inhibitor
to carboxyl groups in the polymer is in the range of 1:1 to 10:1. and the molar
ratio of condensing reagent to carboxyl groups is in the rage of 1:1 to 100:1 to
obtain an affinity polymer by conventional methods.
2. A process as claimed in claim 1 wherein, the polymerization initiator used is
ammonium per sulfate, potassium per sulfate.
3. A process as claimed in claims 1 to 2 wherein, the polymerization accelerator
used is N,N,N',N" tetramethyl ethylene diamine, sodium meta bisulfate,
potassium meta bisulfate.
4. A process as claimed in claims 1 to 3 wherein, the inhibitor is meta amino
benzamidine, para amino benzamidine and their hydrochlorides.
5. A process as claimed in claims 1 to 4 wherein, the condensing reagent used for
linking inhibitor to pendant carboxyl groups of the polymer is 1- cyclohexyl -3 (2-
morpholinoethyl) carbodiimide, l-ethyl-3-(3-Dimethylamino-propyl)
carbodiimide (EDC).
6. A process for the preparation of thermo precipitating affinity polymers containing
spacer of 1 to 5 methylene group as fully described herein before with reference
to the examples.


Documents:

1200-del-1999-abstract.pdf

1200-del-1999-claims.pdf

1200-del-1999-correspondence-others.pdf

1200-del-1999-correspondence-po.pdf

1200-del-1999-description (complete).pdf

1200-del-1999-form-1.pdf

1200-del-1999-form-19.pdf

1200-del-1999-form-2.pdf

1200-del-1999-form-3.pdf

1200-del-1999-petition-138.pdf


Patent Number 216559
Indian Patent Application Number 1200/DEL/1999
PG Journal Number 13/2008
Publication Date 28-Mar-2008
Grant Date 14-Mar-2008
Date of Filing 08-Sep-1999
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001,INDIA
Inventors:
# Inventor's Name Inventor's Address
1 ALANKAR ARUN VAIDYA POLYMER SCIENCE & ENGINEERING UNIT, NATIONAL CHEMICAL LABORATORY, 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 C08F 120/58
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA