Title of Invention

A PROCESS FOR THE DERIVATISATION OF MACROPOROUS BEADED CROSSLINKED COPOLYMERS

Abstract

The present invention relates to a process for the derivatisation of copolymers that are useful in the preparation of chromatography supports. In particular, the invention relates to a process for the derivatisation of macroporous beaded crosslinked copolymers useful as affinity chromatography supports for purification of enzymes such as penicillin G acylase. The protein capacity of immobilized dye adsorbents exceed those of natural biospecific media by a factor of 10 to 100 and thus large scale purification is possible.

Full Text

This invention relates to a process for derivatisation of copolymers that are useful in the preparation of chromalography supports. The invention more particularly relates to a process for the derivatisation of macroporous beaded crosslinked copolymers useful as affinity chromalography supports for purification of enzymes such as penicillin G acylase. Penicillin G acylase finds applications as a biocatalyst in the preparation of 6-aminopenicillanic acid (6-APA) and 7-amino dcsacctoxycephalosporanic acid (7-ADCA). 6-APA and 7-ADCA are further processed for the production of semisynthetic penicillins and cephalosporins those are potent antimicrobial agents. These applications arc developed due to absolute specificity of penicillin G acylase in hydrolysing linear amide bond present in penicillins and cephalosporins to form corresponding beta-lactam nuclei. Penicillin G acylase is used as an immobilized enzyme preparation. The protocol for preparation of immobilised penicillin G acylase constitutes fermentation of a microorganism producing penicillin G acylase (usualy Escherichia coli), extraction of the penicillin G acylase from the cells into aqueous extract, purification of the penicillin G acylase and immobilisation of Ihe purified enzyme onto a solid support. The specific activity of the immobilised penicillin G acylase is determined by the degree of purity of the enzyme used for immobilisation. Thus, purification of penicillin G acylase is an important operation in the production of immobilised penicillin G acylase. Different protocols are developed for purification of penicillin G acylase involves use of ion exchange, hydrophobia interactions and/or affinity chromatography either individually or in various combinations as described in Applied Biochemistry and Biotechnology vol.9, p. 573, 1984. Sepharose (agarose) activated with cyanogen bromide and derivatised with some amino acids, aromatic amines, aliphatic amines, ampicillin and amoxycillin are used for purification of penicillin G acylase as described in Applied Biochemistry and Biotechnology, vol. 9, p. 421, 1984; Biotechnology Letters, vol. 9, p. 539, 1987 and Hindustan Antibiotics Bulletin, vol. 31, p. 25, 1989. These protocols for purification of penicillin G acylase suffer from one or more of the following disadvantages; use of column chromatography procedures, bed compression, attrition of the chromatography support when used in batch mode. The use of reactive dyes as a ligand for immobilization offers the advantage of Ihe ability of the dye to bind to the active site of an enzyme by virtue of its similarity to naturally occurring substrate. In our copending application No. — we have described and claimed a process for the preparation of macroporous beaded crosslinked copolymers. In our another copending application No. NF 322/2000 —, we have described and claimed a process for the derivatization of macroporous beaded crosslinked allyl glycidyl ether copolymers useful as affinity supports for purification of penicillin G acylase. It is the object of the present invention to provide a process for the purification of penicillin G acylase from enzyme extract of Escherichia coli using derivatised macroporous beaded crosslinked copolymers by using synthetic dye as the ligand. Another object of the invention is to develop a process, which is simple, rapid and does not involve many steps. Yet another object is to provide a process by which purified penicillin G acylase preparation is useful for its immobilisation onto the solid supports prepared as per the process of this invention. Accordingly, the present invention provides a process for the derivatisation of macroporous beaded crosslinked copolymers winch comprises suspending macroporous beaded crosslinked copolymers in water at a temperature in the range of 50 to 60"C, adding a solution of synthetic dye solution while maintaining the contents at the same temperature in the in presence of an alkali metal chloride, adding a base such as alkaline metal carbonate, continuing the reaction at a temperature in the range of 60 to 80°C for a period ranging from 2 to 4 hours, isolating the beads by conventional methods and washing the derivatized beads with water and drying. In an embodiment of the invention, the macroporous beaded crosslinked copolymers were prepared as described and claimed in our copending application No. — ' L' In another embodiment of the invention the synthetic dye used is selected from cibacron blue, basilcn blue, reactive green, reactive red. In yet another embodiment of the invention, the alkali metal chloride used is such as sodium chloride, potassium chloride. In still another embodiment of the invention, the alkali metal carbonate used is such as sodium carbonate, potassium carbonate. The process of the present invention is described with reference to the following examples, which are given by way of illustration only and should not be construed to limit the scope of the invention, in any manner. Examples for the preparation of macroporous beaded crosslinkcd copolymcrs Example 1 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.72 grams of allyl glycidyl ether, 8.38 grams of pentaerythritol triacrylate and 22.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 17.10 grams. Example 2 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 4.30 grams of allyl glycidyl ether, 14.50 grams of pentaerythritol trimethacrylate and 22.0 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methunol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 14.5 grams. Example 3 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 8.6 grams of allyl glycidyl ether, 7.8 grams of divinyl benzene and 23 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.8 gram of azo bis isobutyronitrile was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70oC for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 50"C. The yield of copolymer obtained was 14.25 grams. Example 4 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.0 grams of allyl glycidyl ether, 8 grams of ethylene glycol dimethacrylate and 43.5 ml of cyelohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of methyl ethyl ketone peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 60(1C for 4 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 15.25 grams. Example 5 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 8.5 grams of allyl glycidyl ether, 8.25 grams of trimethylol propane triacrylate and 43.5 ml of cyelohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.5 gram of benzoyl peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 70°C for 3 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 15.35 grams. Example 6 In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 9.0 grams of allyl glycidyl ether, 8.75 grams of trimethylol propane trimethacrylate and 43.5 ml of cyclohcxanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of beiv/oyl peroxide was added to this suspension and the resultant polymerization reaction mixture was heated with agitation at a temperature of 80°C for 2 hours. The copolymer separated out as beads during the polymerization. The copolymer beads were isolated by filtration, washed with distilled water, followed by washing by ethanol and dried in a vacuum oven at 50°C. The yield of copolymer obtained was 15.75 grams. Examples for the derivatisation of macroporous beaded crosslinked copolymers Example 7 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 4 was suspended in 75 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 200 mg of cibacron blue dye in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 30 minutes. 9.0 grams of sodium chloride were to the above solution and the temperature of was increased to 80°C. 0.9 gram of sodium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 8 15 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 4 was suspended in 150 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 400 mg of cibacron blue dye in 50 ml of water was added to the above solution and the mixture was maintained at the temperature of 55°C for a period of 60 minutes. 20.0 grams of potassium chloride were to the above solution and the temperature of was increased to 75°C. 2.0 grams of potassium carbonate were added to the reaction mixture and the temperature was maintained at 75°C for 3 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 9 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 5 was suspended in 75 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 200 mg of basilen blue dye in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 30 minutes. 9.0 grams of sodium chloride were to the above solution and the temperature of was increased to 80°C. 0.9 gram of sodium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 10 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 3 was suspended in 75 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 200 mg of cibacron blue dye in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 30 minutes. 9.0 grams of sodium chloride were to the above solution and the temperature of was increased to 80°C. 0.9 gram of sodium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 11 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 2 was suspended in 75 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 200 mg of cibacron blue in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 30 minutes. 9.0 grams of sodium chloride were to the above solution and the temperature of was increased to 80°C. 0.9 gram of sodium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 12 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 4 was suspended in 75 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 200 mg of reactive red dye in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 30 minutes. 9.0 grams of sodium chloride were to the above solution and the temperature of was increased to 80°C. 0.9 gram of sodium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 13 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 6 was suspended in 75 ml of water. The mixture was heated to a temperature of 55°C in a shaker bath. A solution of 200 mg of reactive red dye in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 55°C for a period of 45 minutes. 10.0 grams of potassium chloride were to the above solution and the temperature of was increased to 75°C. 1.0 gram of potassium carbonate was added to the reaction mixture and the temperature was maintained at 75°C for 4 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 14 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 1 was suspended in 75 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 200 mg of reactive red dye in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 30 minutes. 9.0 grams of sodium chloride were to the above solution and the temperature of was increased to 80°C. 0.9 gram of sodium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 15: 10 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 4 was suspended in 75 ml of water. The mixture was heated to a temperature of 60°C in a shaker bath. A solution of 200 mg of reactive green dye in 25 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 30 minutes. 9.0 grams of sodium chloride were to the above solution and the temperature of was increased to 80°C. 0.9 gram of sodium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Example 16 15 g of the macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in Example 5 was suspended in 150 ml of water. The mixture was heated to a temperature of-60°C in a shaker bath. A solution of 300 mg of reactive green dye in 50 ml of water was added to the above solution and the mixture was maintained at the temperature of 60°C for a period of 45 minutes. 15.0 grams of potassium chloride were to the above solution and the temperature of was increased to 80°C. 2.0 gram of potassium carbonate was added to the reaction mixture and the temperature was maintained at 80°C for 2 hours. The reaction mixture was then cooled to ambient temperature and the product separated by conventional methods and washed thoroughly with water. Advantages of the invention The invention provides a process for derivatisation of macroporous beaded crosslinked copolymers useful for purification of enzymes such as penicillin G aeylase by affinity chromatography. Preparation of the affinity chromatography supports by the process of this invention offers the following advantages: i) The process does not involve the use of toxic chemicals like cyanogen bromide. ii) Protein capacity of immobilized dye adsorbents exceed those of natural biospecific media by a factor of 10 to 100 and hence large scale purification is possible iii) Synthetic dyes are resistant to chemical and enzymatic degradation. iv) The macroporous beaded crosslinked copolymers are rigid crosslinked copolymers and hence bed compression at varying pH and ionic strength does not occur. v) Characteristic spectral properties of the dyes permit ready monitoring of ligand concentrations and identification of column material. vi) The beads are resistant for attrition thereby enables its use in a batch mode on large scale. vi) The operation procedure is simple due the ease of coupling the dye onto the matrix and the process is easy to scale-up. vii) Elution of bound protein is easy with very good recoveries. viii) The adsorbents are stable over the temperature range of operation in the purification process. ix) The process offers attractive techno-economic features. We Claim: 1. A process for the derivatisation of macroporous beaded crosslinked copolymers characterized in that the macroporous beaded crosslinked copolymers of the kind such as herein described are suspended in water at a temperature in the range of 50 to 60 degree C, adding a solution of known synthetic dye solution while maintaining the contents at the same temperature in the presence of an alkali metal chloride, adding a base such as alkali metal carbonate, continuing the reaction at a temperature in the range of 60 to 80 degree C for a period ranging from 2 to 4 hours, isolating the beads by conventional methods, washing the derivatized beads with water and drying. 2. A process as claimed in claim 1, wherein the synthetic dye used is selected from the group consisting of cibacron blue, basilen blue, reactive green and reactive red. 3. A process as claimed in claim 1, wherein the alkali metal chloride used is selected from sodium chloride, potassium chloride. 4. A process as claimed in claim 1, wherein the alkali metal carbonate used is selected from sodium carbonate, potassium carbonate. 5. A process for the derivatisation of macroporous beaded crosslinked copolymers substantially as herein described with reference to the foregoing examples.

Documents:

272-del-2001-abstract.pdf

272-del-2001-claims.pdf

272-del-2001-correspondence-others.pdf

272-del-2001-correspondence-po.pdf

272-del-2001-description (complete).pdf

272-del-2001-form-1.pdf

272-del-2001-form-18.pdf

272-del-2001-form-2.pdf

272-del-2001-form-3.pdf