Title of Invention

"AN IMPROVED PROCESS FOR THE PRODUCTION OF GLUTARYL-7-AMINO CEPHALOSPORANIC ACID"

Abstract

An improved process for the production of glutary 1-7-Amino cephalosporanic acid, which comprises suspending D-Amino acid oxidase immobilized on crosslinked glycidyl methacrylate-divinyl benzene copolymers in phosphate buffer, adding to this suspension the solution of cephalosporin C, agitating the resultant reaction mixture at rpm between 150 to 250, maintaining a temperature in the range of 20 to 24°C, aerating the reaction mixture, maintaining the pH between 7.6 to 7.8 by conventional method followed by seperation of the immobilized D-Amino acid oxidase and recovering the glutaryl-7-amino cephalosporanic acid by conventional methods.

Full Text

This invention relates to an improved process for the production of glutaryl- 7-amino cephalosporanic acid. More particularly it relates to the production of glutaryl-7-amino cephalosporanic acid using D-amino acid oxidase immobilized on crosslinked glycidyl metha-crylate-divinyl benzene copolymers. D-Amino acid oxidase catalyses the conversion of D-amino acids to corresponding alpha-keto acids as described in Applied Biochemistry and Biotechnology, vol.6, p.293, 1981. It is now established that this enzyme can be used in the production of 7-amino cepha¬losporanic acid(7-ACA) via glutaryl-7-amino cephalospo¬ranic acid(glutaryl-7-ACA). 7-ACA is a key intermediate in the preparation of many cepham antibiotics such as cefazolin, cephalogly-cine and cephalothin. The sequence of chemical reac¬tions that occur during the conversion of cephalosporin C to 7-ACA and the enzymes involved are as follows: Cephalosporin C D-Amino acid oxidase Keto-adipyl-7-ACA + H202 + NH3 Spontaneous Glutaryl-7-ACA + C02 + H20 Glutaryl acylase 7-ACA + Glutaric acid Conversion of cephalosporin C to 7-ACA is per¬formed in two different reactors : the first one con¬tains D-amino acid oxidase which produces glutaryl-7-ACA and second contains glutaryl acylase which hydroly-ses glutaryl-7-ACA to generate 7-ACA. Currently both immobilized whole cells of a microorganism containing D-amino acid oxidcise and immobilized D-amino acid oxidase are used for the production of glutaryl-7-ACA as described in the following literature : German Patent DE 4 028 119; Japan Kokai Tokkyo Koho JP 79 154 592; Czeck CS 251 457; European Patent Appl. EP 492 495, EP 496 993 and EP 517 200; Annals of New York Academy of Sciences vol. 672, p. 502-509, 1992 and Bioprocess Engineering vol.12, p. 249-252, 1995. Of these, processes that use immobilized whole cells containing D-amino acid oxidase suffer from disadvantages such as shorter operational life, lower catalytic efficiency and leaching of the metabolites from the cells whereas the matrices used in the prepa¬ration of immobilized D-amino acid oxidases are not designed to provide ideal microenvironment for the chemical reactions and hence the conversion of cepha¬losporin C to glutaryl-7-ACA is less than 95% in these processes. The conversion of cephalosporin C to gluta- ryl-7-ACA is comprised of chemical reactions involving many reactants. Thus, for a commercially efficient process, it is essential that the D-amino acid oxidase is immobilized on a matrix whose structure is tailored so that the chemical reactions are driven promptly. In our copending application No. 2937/DEL/97, we have described and claimed a process for the prepara¬tion of crosslinked macroporous glycidyl methacrylate -divinyl benzene copolymers which are designed to have physical and chemical properties desired for the immo¬bilization of D-amino acid oxidase. We have also de¬scribed and claimed a process for the production of immobilized D-amino acid oxidase using the crosslinked macroporous glycidyl methacrylate - divinyl benzene copolymers in our copending Application No. 2946/DEL/97. It is the object of present invention to provide a process for production of glutaryl-7-ACA using immobi¬lized D-amino acid oxidase. Yet another object is to provide a process wherein the conversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid is more than 95%. Accordingly, the present invention provides an improved process for the production of glutaryl-7-Amino cephalosporanic acid, which comprises suspending D-Amino acid oxidase immobilized on crosslinked glycidyl methacrylate-divinyl benzene copolymers in phosphate buffer, adding to this suspension the solution of cephalosporin C, agitating the resultant reaction mixture at rpm between 150 to 250, maintaining a tem¬perature in the range of 20 to 24°C, aerating the reaction mixture, maintaining the pH between 7.6 to 7.8 by conventional method followed by separation of the immobilized D-Amino acid oxidase and recovering the glutaryl-7-amino cephalosporanic acid by conventional methods. In an embodiment of the present invention, the molarity of the buffer used may be in the range 0.05 to 0.1. In another embodiment of the invention, the pH of the solution is maintained by controlled addition of alkaline solution, preferably ammonia solution having a strength of 0.05 to 0.1 M. In yet another embodiment of the invention, the immobilized D-amino acid oxidase used is prepared as described and claimed in our copending application number 2 94 6/DEL/97. The D-amino acid oxidase activity is determined by using cephalosporin C as the substrate at 3 7°C and pH 8.0 as described in Biocatalysis and Biotransformation vol. 12, p.215-219, 1995 and in Methods in Enzymology vol. 3, p. 414-418, 1957. The process of the present invention is described with reference to the following examples which are illustrative only and should not be construed to limit the scope of the invention in any manner. Preparation of crosslinked macroporous glycidyl metha-crylate - divinyl benzene copolymers. Example1: 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. 25.3 grams of glycidyl methacrylate, 7.8 grams of divinyl benzene 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 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 filtrarion, washed with distilled water, followed by wanning by methanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 31.78 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. 20.0 grams of glycidyl methacrylate, 12.8 grams of divinyl benzene and 4 3.5 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 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 methanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 31.8 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. 18.2 grams of glycidyl methacrylate, 13.8 grams of divinyl benzene and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.9 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 4 0°C. The yield of copolymer obtained was 31.2 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. 16.5 grams of glycidyl methacrylate, 15.2 grams of divinyl benzene and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.9 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 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 4 0°C. The yield of copolymer obtained was 30.75 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. 14.6 grams of glycidyl methacrylate, 17.0 grams of divinyl benzene and 43.5 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.8 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 30.50 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. 18.1 grams of glycidyl methacrylate, 13.75 ml of divinyl benzene and 32.7 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.8 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 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 ethanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 30.92 grams. Example 7 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. 18.1 grams of glycidyl methacrylate, 13.75 grams of divinyl benzene and 54.6 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 31.06 grams. Example 8.: 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. 18.1 grams of glycidyl methacrylate, 13.75 ml of divinyl benzene and 65.4 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 3 0.85 grams. Example 9: In an inert atmosphere of nitrogen, 0.5 gram of poly vinyl pyrrolidone was dissolved in 100 ml of distilled water in a 250 ml capacity glass reactor. 18.1 grams of glycidyl methacrylate, 13.75 grams of divinyl benzene and 54.6 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 ethanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 31.15 grams. Example 10: In an inert atmosphere of nitrogen, 1.5 grams of poly vinyl pyrrolidone was dissolved in 100 ml of distilled water in a 250 ml capacity glass reactor. 18.1 grams of glycidyl methacrylate, 13.75 grams of divinyl benzene and 54.6 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.6 gram of azobis 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 ethanol and dried in a vacuum oven at 4 0°C. The yield of copolymer obtained was 30.86 grams. Example 11: In an inert atmosphere of nitrogen, 3.0 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 18.1 grams of glycidyl methacrylate, 13.75 grams of divinyl benzene and 54.6 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 1.20 grams 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 ethanol and dried in a vacuum oven at 40°C. The yield of copolymer obtained was 31.10 grams. Example 12: In an inert atmosphere of nitrogen, 2.25 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 18.1 grams of glycidyl methacrylate, 13.75 grams of divinyl benzene and 54.6 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.2 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 4 0°C. The yield of copolymer obtained was 30.75 grams. Example 13: In an inert atmosphere of nitrogen, 2.25 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 ml capacity glass reactor. 18.1 grams of glycidyl methacrylate, 13.75 grams of divinyl benzene and 54.6 ml of cyclohexanol were added to the aqueous solution of poly vinyl pyrrolidone. The suspension was stirred at 300 rpm. 0.4 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 4 0°C. The yield of copolymer obtained was 31.18 grams. Example 14: In an inert atmosphere of nitrogen, 2.25 grams of poly vinyl pyrrolidone was dissolved in 150 ml of distilled water in a 250 « ml capacity glass reactor. 18.1 grams of glycidyl methacrylate, 13.75 grams of divinyl benzene and 56.4 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 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 4 0°C. The yield of copolymer obtained was 31.07 grams. Preparation of immobilized D-amino acid oxidase using crosslinked macroporous glycidyl methacrylate - divinyl benzene copolymers. Example 15: 1.0 gram of copolymer prepared as described in Examples 1 to 5 were suspended in 25 ml of phosphate buffer having molarity of 0.05 and pH 7.0 containing 50 IU of D-amino acid oxidase. The binding mixture was incubated at 24°C for 10 hours with agitation at 80 rpm. The immobilized D-amino acid oxidase was separated by filtration. The D-amino acid oxidase activity of immobilized enzyme was determined. The activity of immobilized D-amino acid oxidase prepared using copolymer having 50% crosslink density prepared as described in Example 3 was 19.35 which was more when compared to the copolymers prepared as described in Examples 1,2,4 and 5. Example 16: 1.0 gram of copolymer prepared as described in Examples 3, 6, 7 and 8 were suspended in 25 ml of phosphate buffer having molarity of 0.05 and pH 7.0 containing 50 IU of D-amino acid oxidase. The binding mixture was incubated at 24°C for 10 hours with agitation at 80 rpm. The immobilized D-amino acid oxidase was separated by filtration. The D-amino acid oxidase activity of immobilized enzyme was determined. The activity of immobilized D- amino acid oxidase prepared using the copolymer as described in Example 7 was 21.17 which was more compared to copolymers prepared as described in Examples 3, 6 and 8. Example 17: 1.0 gram of copolymer prepared as described in Examples 7, 9, 10 and 11 were suspended in 25 ml of phosphate buffer having molarity of 0.05 and pH 7.0 containing 50 IU of D-amino acid oxidase. The binding mixture was incubated at 24°C for 10 hours with agitation at 80 rpm. The immobilized D-amino acid oxidase was separated by filtration. The D-amino acid oxidase activity of immobilized enzyme was determined. The activity of immobilized D-amino acid oxidase prepared using the copolymer as described in Example 10 was 22.97 which was more when compared to the copolymers prepared as described in Examples 7, 9 and 11. Example 18: 1.0 gram of copolymer prepared as described in Examples 10, 12 13 and 14 were suspended in 25 ml of phosphate buffer having molarity of 0.05 and pH 7.0 containing 50 IU of D-amino acid oxidase. The binding mixture was incubated at 24°C for 10 hours with agitation at 80 rpm. The immobilized D-amino acid oxidase was separated by filtration. The D-amino acid oxidase activity of immobilized enzyme was determined. The activity of immobilized D-amino acid oxidase prepared using the copolymer as described in Example 10 was 22 . 97 which was more when compared to the copolymers prepared as described in Examples 12, 13 and 14. Example 19: 1.0 g of copolymer prepared as described in Example 10 was suspended in 25 ml of phosphate buffer having molarity of 0.05 and pH 7.0 containing 25 IU of D-amino acid oxidase. The binding mixture was incubated at 24°C for 10 hours with agitation at 80 rpm. The immobilized D-amino acid oxidase was separated by filtration. The D-amino acid oxidase activity of immobilized enzyme was determined. The activity of immobilized D-amino acid oxidase was 24 IU/g. Example 20: 25 grams of the copolymer prepared as described in Example 10 was suspended in 500 ml of phosphate buffer having molarity of 0.05 and pH 7.0 containing 625 IU of D-amino acid oxidase. The binding mixture was agitated at 150 rpm at 24°C for 10 hours. The immobilized D-amino acid oxidase was separated by filtration and stored at 4°C as a suspension in phosphate buffer having molarity of 0.05 and pH 7.5 containing 0.02% w/v of sodium azide. The activity of the immobilized D-amino acid oxidase was 24 IU/g. Example 21: 1 gram of immobilized D-amino acid oxidase having activity of 24 IU/g was suspended in 10 ml of phosphate buffer having molarity of 0.05 and pH 7.5 containing 0.02% w/v of sodium azide and incubated at 24°C for 240 hours. The residual activity was 22.8 IU/g. C.Production of glutaryl-7-amino cephalosporanic acid using immobilized D-amino acid oxidase Example 22 168 IU (7.0 g) of immobilized D-amino acid oxidase prepared as described in Example 2 0 was suspended in 50 ml of phosphate buffer having molarity of 0.05 and pH 7.8 in a 250 ml capacity beaker. The immobilized D-amino acid oxidase was kept under suspension at 24°C by agitation at 150 rpm. In a separate container, 1.0 g of sodium cephalosporin C was dissolved in 25 ml of phosphate buffer haviny molarity of 0.05 and pH 7.8. The solution of cephalosporin c was added to the suspension of immobilized D-amino acid oxidase. Final volume of the reaction mixture was made to 100 ml. The reaction mixture was aerated at 4.0 litre/minute. The pH of the reaction mixture was maintained between 7.6 - 7.8 by controlled addition of 2.0 N ammonia solution. The reaction was allowed to proceed at the aforesaid temperature, agitation, aeration and pH values. More than 95% conversion was achieved in 100 minutes. The immobilized D-amino acid oxidase was collected by filtration, washed with 50 ml of phosphate buffer having molarity of 0.05 and pH 7.5. Example 23 168 IU (7.0 g) of immobilized D-amino acid oxidase prepared as described in Example 20 was suspended in 50 ml of phosphate buffer having molarity of 0.05 and pH 7.8 in a 250 ml capacity beaker. The immobilized D-amino acid oxidase was kept under suspension at 24°C by agitation at 150 rpm. In a separate container, 3.0 g of sodium cephalosporin C was dissolved in 25 ml of phosphate buffer having molarity of 0.05 and pH 7.8. The solution of cephalosporin c was added to the suspension of immobilized D-amino acid oxidase. Final volume of the reaction mixture was made to 100 ml. The reaction mixture was aerated at 4.0 litre/minute. The pH of the reaction mixture was maintained between 7.6 - 7.8 by controlled addition of 2.0 N ammonia solution. The reaction was allowed to proceed at the aforesaid temperature, agitation, aeration and pH values. More than 95% conversion of cephalosporin C to glutaryl-7-ACA was achieved in 180 minutes. The immobilized D-amino acid oxidase was collected by filtration, washed with 50 ml of phosphate buffer having molarity of 0.05 and pH 7.5. Example 24 168 IU (7.0 g) of immobilized D-amino acid oxidase prepared as described in Example 20 was suspended in 50 ml of phosphate buffer having molarity of 0.05 and pH 7.8 in a 250 ml capacity beaker. The immobilized D-amino acid oxidase was kept under suspension at 24°C by agitation at 150 rpm. In a separate container, 5.0 g of sodium cephalosporin C was dissolved in 25 ml of phosphate buffer having molarity of 0.05 and pH 7.8. The solution of cephalosporin c was added to the suspension of immobilized D-amino acid oxidase. Final volume of the reaction mixture was made to 100 ml. The reaction mixture was aerated at 4.0 litre/minute. The pH of the reaction mixture was maintained between 7.6 - 7.8 by, controlled addition of 2.0 N ammonia solution. The reaction was allowed to proceed at the aforesaid temperature, agitation, aeration and pH values. More than 95% conversion of cephalosporin c to glutaryl-7-ACA was achieved in 380 minutes. The immobilized D-amino acid oxidase was collected by filtration, washed with 50 ml of phosphate buffer having molarity of 0.05 and pH 7.5. Example 25 576 IU (24 g) of immobilized D-amino acid oxidase prepared as described in Example 20 was suspended in 150 ml of phosphate buffer having molarity of 0.05 and pH 7.8 in 1 litre capacity container. The suspension was agitated at 250 rpm at 24°C. 12 g of sodium cephalosporin C was dissolved in 150 ml of phosphate buffer having molarity of 0.05 and pH 7.8 and added to the suspension of immobilized D-amino acid oxidase. Final volume of the reaction mixture was made to 400 ml. The reaction mixture was aerated at 6.0 litre/minute. The pH of the reaction mixture was maintained between 7.6 to 7.8 by controlled addition of 2.0 N ammonia solution. The reaction was allowed to proceed at aforesaid temperature, agitation, aeration and pH values. More than 95% conversion of cephalosporin C was achieved in 150 minutes. The immobilized D-amino acid oxidase was collected by filtration, washed with 100 ml of phosphate buffer having molarity of 0.05 and pH 7.5 and used for next experiment. Example 26 60 experiments were performed for the production of glutaryl-7-ACA as described in Example 25 wherein same batch of immobilized D-amino acid oxidase was used. The quantity of sodium cephalosporin C used and final volume of the reaction mixture during the 60 experiments are summmarised in Table-1 below : Table - 1 Experiments Sodium Final Time required cephalospo- volume for >95% rin C conversion g ml minutes 1 to 17 12.0 400 150 18 to 30 9.0 300 160 31 to 40 7.5 300 180 41 to 50 6.0 300 180 51 to 60 5.0 300 180 The time required for more than 95% conversion of sodium cephalosporin c to glutaryl-7-ACA increased from 150 minutes to 180 minutes. The activity of immobilized D-amino acid oxidase decreased from 24 IU/g to 8.55 IU/g after 60 experiments. Total amount of glutaryl-7-ACA produced by 576 IU of immobilized D-amino acid oxidase in total operational time of 167 hours was 427 grams. The solution of glutaryl-7-ACA obtained from the process described in this example is useful for production of 7-ACA. Advantages of the Invention 1. The immobilized D-amino acid oxidase is used for production of glutaryl-7-ACA for 60 consecutive batches. 2. The operational life of the immobilized D-amino acid oxidase for production of glutaryl-7-ACA is 167 hours. 3. The immobilized D-amino acid oxidase does not adsorb either cephalosporin c or glutaryl-7-ACA. 4. The immobilized D-amino acid oxidase being beaded in nature does not hinder the filtration during the process. 5. Glutaryl-7-ACA is free from the metabolites of microbial cells. 6. Conversion of cephalosporin C to glutaryl-7-ACA is more than 95%. WE CLAIM: 1. An improved process for the production of glutaryl-7-Amino cephalosporanic acid, which comprises suspending D-Amino acid oxidase immobilized on cross-linked glycidyl methacrylate-divinyl benzene copolymers in phosphate buffer, adding to this suspension the solution of cephalosporin C, agitating the resultant reaction mixture at rpm between 150 to 250, maintaining a temperature in the range of 20 to 24°C, aerating the reaction mixture, maintaining the pH between 7.6 to 7.8 by conventional method followed by separation of the immobilized D-Amino acid oxidase and recovering the glutaryl-7-amino cephalosporanic acid by conventional methods. 2. An improved process as claimed in claim 1, wherein the molarity of the buffer used is in the range 0.05 to 0.1. 3. An improved process as claimed in claims 1 & 2, wherein the pH of the solution is maintained by con¬trolled addition of alkaline solution, preferably ammonia solution having a strength of 0.05 to 0.1 M. 4. An improved process for the production of gluta- ryl-7-Amino cephalosporanic acid substantially as herein described with reference to the examples 22 to 26.

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