Title of Invention

A PROCESS FOR THE PURIFICATION OF PENICILLIN G ACYLASE FROM CRUDE ENZYME EXTRACT.

Abstract

The present invention provides a process for purification of penicillin G acylase from crude enzyme extract, using derivatized macroporous beaded crosslinked copolymers . The derivatized macroporous beaded crosslinked copolymers acts as solid support and adsorbent of penicillin G acylase . Said process comprises the step of suspending the derivatized macroporous beaded crosslinked copolymers as described herein , in a crude enzyme extract from Escherichia coli cells and eluting the adsorbed penicillin G acylase using phosphate buffer having molarity ranging between 0.02 to 1 M containing 0.5 M of phenylacetic acid followed by dialyzing the eluted solution to get desired purified penicillin G acylase

Full Text

This invention relates to the purification of industrially important enzyme such as Penicillin G acylase. More particularly it relates to the process for the purification of penicillin G acylase from crude enzyme extract of Escherichia coli using derivatised macroporous beaded crosslinked copolymers. The purification achieved is effected in a single operation by the process of this invention and high yields are obtained. Penicillin G acylase finds applications as a biocatalyst in the preparation of 6-aminopenicillanic acid (6-APA) and 7-amino desacetoxycephalosporanic 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 are 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 the 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, hydrophobic 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.P2571989. 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. In our copending application No.1059/DEL/2000 we have described and claimed a process for the preparation of macroporous beaded crosslinked copolymers. In our another copending application No.1059/DEL/2000 we have described and claimed a process for the derivatization of macroporous beaded crosslinked copolymers by binding of dyes as the ligands 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. Another object of the invention is to develop a process, which is simple , rapid an does not involve many steps. Yet another object is to provide a process by which purified penicillin G acylase preparation is useful for its immobilization onto solid supports. Accordingly, the present invention provides a process for purification of penicillin G acylase from crude enzyme extract, which comprises suspending the derivatized macroporous beaded crosslinked copolymers as described herein , in a crude enzyme extract from Escherichia coli cells prepared in phosphate buffer having a molarity ranging between; 0.02 to 1 M , at a pH ranging from 7 to 8, agitating the suspension for a period of 30 minutes at a temperature 25 °C at a rpm of 1100, filtering the suspension to get beads having adsorbed penicillin G acylase , washing the beads with phosphate buffer having molarity ranging between 0.02 to 1 M containing 0.5 M of phenylacetic acid to elute the adsorbed penicillin G acylase, dialyzing the eluted solution containing penicillin G acylase by conventional methods to get desired purified penicillin G acylase . Penicillin G acylase and total protein content in the enzyme extract used was 5 IU/ml and 2 mg/ml, respectively. Thus, the specific activity of penicillin G acylase in the enzyme extract was 2.5 IU/mg. The enzyme activity was determined using penicillin G as substrate as described in Hindustan Antibiotics Bulletin Vol. 20, p. 80, 1978. Protein was determined as described in-Journal of Biological Chemistry vol. 193, pp.265, 1951. The process of the present invention is described with reference to following examples, which are given by way of illustration only and should not construed to limit the scope of the invention in any manner. Examples for the preparation of macroporous beaded crosslinked copolymers 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 methanol 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 cyclohexanol 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°C 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 nil capacity glass reactor. 8.5 grams of allyl glycidyl ether, 8.25 grams of trimethylol propane triacrylate and 43.5 ml of cyclohexanol 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 cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of benzoyl 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 copolymcrs 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 80oC. 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 80oC 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 rng of cibacrpn 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 healed 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. Examples for the purification of penicillin G acylase by using dcrivatizcd macroporous beaded crosslinked copolymers Example 17 20 mis of crude enzyme extract of penicillin G acylase from Escherichia coli prepared in phosphate buffer having molarity of 0.05 and pH 7.5 containing 100 IU of pencillin G acylase was taken in 100 ml capacity container. 1.5 gram of derivatized macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in example 7 was added to the enzyme solution and the suspension was agitated at 100 rpm at 25oC for a period of 30 minutes. The beads were separated by filtration and washed with 30 mis of phosphate buffer having a molarity of 0.05 and pH 7.5. The penicillin G acylase adsorbed onto the beads was eluted by washing in 5 lots of 5 mls of phosphate buffer having molarity of 0.05, pH 7.5 containing phenylacetic acid to a concentration of 0.5 M. The fractions containing penicillin G acylase activity were pooled and subjected to dialysis against phosphate buffer having molarity of 0.05 M and pH of 7.5. The adsorption and recovery of penicillin G acylase was 100 and 88 %, respectively. The process offered 4.1-fold purification. Example18 20 mis of crude enzyme extract of penicillin G acylase from Escherichia coli prepared in phosphate buffer having molarity of 0.05 and pH 7.5 containing 100 IU of pencillin G acylase was taken in 100 ml capacity container. 1.5 gram of derivatized macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in example 8 was added to the enzyme solution and the suspension was agitated at 100 rpm at 25°C for a period of 30 minutes. The beads were separated by filtration and washed with 30 mis of phosphate buffer having a molarity of 0.05 and pH 7.5. The penicillin G acylase adsorbed onto the beads was eluted by washing in 5 lots of 5 mis of phosphate buffer having molarity of 0.05, pH 7.5 containing phenylacetic acid to a concentration of 0.5 M. Fractions containing penicillin G acylase activity were pooled and dialysed against phosphate buffer having molarity of 0.05 M and pH of 7.5. The adsorption and recovery of penicillin G acylase was 100 and 92 %, respectively. The process offered 4.9-fold purification of penicillin G acylase. Example 19 20 mis of crude enzyme extract of penicillin G acylase from Escherichia coli prepared in phosphate buffer having molarity of 0.05 and pH 7.5 containing 100 IU of pencillin G acylase was taken in 100 ml capacity container. 1.5 gram of derivatized macroporous beaded crosslinked ally! glycidyl ether copolymer prepared as described in example 9 was added to the enzyme solution and the suspension was agitated at 100 rpm at 25°C for a period of 30 minutes. The beads were separated by filtration and washed with 30 mis of phosphate buffer having a molarity of 0.05 and pH 7.5. The penicillin G acylase adsorbed onto the beads was eluted by washing in 5 lots of 5 mis of phosphate buffer having molarity of 0.05, pH 7.5 containing phenylacelic acid to a concentration of 0.5 M. The fractions containing penicillin G acylase activity were pooled and dialysed against phosphate buffer having molarity of 0.05 M and pH of 7.5. The adsorption and recovery of penicillin G acylase was 100 and 86 %, respectively. By the use of this methodology a 4.2-fold purification was achieved. Example 20 20 mis of crude enzyme extract of penicillin G acylase from Escherichia coli prepared in phosphate buffer having molarity of 0.05 and pH 7.5 containing 100 IU of pencillin G acylase was taken in 100 ml capacity container. 1.5 gram of derivatized macroporous beaded crosslinked allyl glycidyl ether copolymer prepared as described in example 10 was added to the enzyme solution and the suspension was agitated at 100 rpm at 25°C for a period of 30 minutes. The beads were separated by filtration and washed with 30 mls of phosphate buffer having a molarity of 0.05 and pH 7.5. The penicillin G acylase adsorbed onto the beads was eluted by washing in 5 lots of 5 mls of phosphate buffer having molarity of 0.05, pH 7.5 containing phenylacetic acid to a concentration of 0.5 M. The fractions containing penicillin G acylase activity were pooled and dialysed against phosphate buffer having molarity of 0.05 M and pH of 7.5. The adsorption and recovery of penicillin G acylase was 96 and 78 %, respectively. By the use of this methodology a 3.7-fold purification was achieved. Advantages of the invention The invention provides a process for the purification of penicillin G acylase from an enzyme extract of Escherichia coli cells and offers the following advantages. 1. High recovery up to 92 % of penicillin G acylase can be obtained. 2. The operations involve single step of affinity interaction chromatography. 3. The process developed is a batch operation and use of column chromatography equipments is not required. 4. . The purified penicillin G acylase obtained is useful for immobisation onto other solid matrices. We Claim: 1. A process for purification of of penicillin G acylase from crude enzyme extract, which comprises suspending the derivatized macroporous beaded crosslinked copolymers as described herein , in a crude enzyme extract from Escherichia coli cells prepared in phosphate buffer having a molarity ranging between 0.02 to 1 M , at a pH ranging from 7 to 8, agitating the suspension for a period of 30 minutes at a temperature 25 °C at a rpm of 1100, filtering the suspension to get beads having adsorbed penicillin G acylase , washing the beads with phosphate buffer having molarity ranging between 0.02 to 1 M containing 0.5 M of phenylacetic acid to elute the adsorbed penicillin G acylase, dialyzing the eluted solution containing penicillin G acylase by conventional methods to get desired purified penicillin G acylase . 2. A process for the purification of penicillin G acylase from crude enzyme extract using derivatized macroporous beaded crosslinked copolymers as herein described with reference to the Examples 17 to 20.

Documents:

271-del-2001-abstract.pdf

271-del-2001-claims.pdf

271-del-2001-correspondence-others.pdf

271-del-2001-correspondence-po.pdf

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

271-del-2001-form-1.pdf

271-del-2001-form-18.pdf

271-del-2001-form-2.pdf

271-del-2001-form-3.pdf