Title of Invention

"AN IMPROV ED PROCESS FOR THE PRO-DUCTION OF IMMOBILIZED D-AMINO ACID OXIDASE"

Abstract

An improved process for the production of immobilized D-amino. oxidase, which comprises suspending crosslinked macropo-rous glyofrdyl methacrylate - divinyl benzene copolymers in phosphate buffer, adding D-amino acid oxidase solution, incubating the suspension at a temperature in the range of 20 to 24° c for a period in the range of 9 to 12 hours with agitation ranging from 60 to 120 rpm, separating the immobili-' zed D-amino acid oxidase by conventional filtration methods. >

Full Text

This invention relates to an improved process for the production of immobilized D-amino acid oxidase. The invention particularly relates to immobilization of D-amino acid oxidase on crosslinked macroporous glycidyl methacrylate-divinyl benzene copolymer matrix useful for the preparation of glutaryl-7-aminocephalosporanicacid. D-Amino acid oxidase finds application in the pharmaceutical industry in bioconversion of cephalosporin C to 7-amino cephalosporanic acid (7-ACA) which is a key intermediate in production of various clinically important semisynthetic cephalosporins. This bioconversion involves use of two enzymes namely D-amino acid oxidase and glutaryl acylase. D-Amino acid oxidase converts cephalosporin C to keto-adipyl-7-amino cephalosporanic acid which in presence of generated hydrogen peroxide is spontaneously converted to glutaryl-7-amino cephalosporanic acid(glutaryl-7-ACA). Glutaryl-7-ACA is then deacylated by glutaryl acylase to 7-ACA. Thus, D-amino acid oxidase is an enzyme of industrial importance. D-Amino acid oxidase from a variety of microorganisms find application in production of keto acids from respective amino acids. However, very few D-amino acid oxidase preparations accept cephalosporin C as substrate. Trigonopsis variabilis, Rhodotorula gracilis, Fusarium solani M-0718, Bacillus Licheniformis, Rhodotorula glutinis, Aspergillus sp. strain O20 produce D-amino acid oxidase in high yields which is active on cephalosporin C. Two types of biocatalysts are used in the production of glutaryl-7-ACA: i) Immobilized whole cells of the microorganism containing the D-amino acid oxidase as described in Japan Kokai Koho JP 79 134 592 and Bioprocess Engineering, vol. 12, p. 249-252, 1995; and ii) Immobilized D-amino acid oxidase on commercial matrices as described in German Patent DE 4028 119; Czeck CS 251 457; European Patents Appl. EP 492 495 and 496 993; Annals of New York Academy of Sciences, vol. 672, p. 502-509, 1992 and Enzyme and Microbial Technology, vol. 17, p. 324-329, 1995. Of these, the immobilized enzyme preparations are prefered. This is because the immobilized whole cells have shorter operational life, the product is contaminated by the metabolites secreted by the cells and the diffusion of substrate and products is hindered by the cell wall components. For immobilization of D-amino acid oxidase, the matrix should provide appropriate microenvironment for availability of oxygen, availability of hydrogen peroxide released during the reaction and required for chemical reaction, availability of the enzyme molecules for the reaction and instant diffusion of substrate (cephalosporin C) and products such as glutaryl-7-ACA, ammonia, carbon dioxide etc. In our copending Application No. 2937/DEL/97, we have described and claimed a proces for the preparation of crosslinked macroporous glycidyl methacrylate - divinyl benzene copolymers which are designed to have physical and chemical properties desired for immobilization of D-amino acid oxidase. The main object of the present invention is to provide an improved process for production of immobilized D-amino acid oxidase using crosslinked macroporous glycidyl methacrylate-divinyl benzene copolymers. Accordingly, the present invention provides an improved process for the production of immobilized D-amino acid oxidase, which comprises suspending crosslinked macroporous glycidyl methacrylate-divinyl benzene copolymers in phosphate buffer, adding D-amino acid oxidase solution, incubating the suspension at a temperature in the range of 20 to 24oC for a period in the range of 9 to 12 hours with agitation ranging from 60 to 120 rpm, separating the immobilized D-amino acid oxidase by conventional filtration methods. In an embodiment of the invention, the molarity of the phosphate buffer may be in the range of 0.05 to 0.10. In another embodiment of the invention, the pH of the phosphate buffer may be in the range of 6.8 to 7.2. In yet another embodiment of the invention, the glycidyl methacrylate-divinyl benzene copolymers are prepared as described and claimed in our copending application number 2937/DEL/97. D-Amino acid oxidase solution used is obtained from Aspergillus sp. strain O20 by fermentation. The soluble D-amino acid oxidase preparation has a specific activity of 1.2 ID. The D-amino acid oxidase activity was determined by using cephalosporin C as substrate at 37oC 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 herein below 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 gylcidyl methacrylate-divinyl benzene 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. 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 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 31.78 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 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 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 40°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 40°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 40°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 diviny| 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 40°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 30.85 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 40°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 40°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 were 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 40°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 40°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 40°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 results are summarised in Table 1 below : Table -1 (Table Removed) 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 results are summarised in Table-2 below : Table-2 (Table Removed) 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 ID of D-amino acid oxidase. The binding mixture was incubated at 24oC 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 results are summarised in Table-3 below: Table - 3 (Table Removed) 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 ID of D-amino acid oxidase. The binding mixture was incubated at 24oC 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. (Table Removed) 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 24oC 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 ILJ/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 lU/g. Example 21 1 gram of immobilized D-amino acid oxidase having activity of 24 ILJ/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 ID/g. The advantages of the invention are: 1. The crosslinked macroporous glycidyl methacrylate - divinyl benzene copolymers prepared according to Examples 1 - 14 do not need any derivatization or treatment prior to immobilization of D-amino acid oxidase. 2. Immobilization on the copolymer prepared according to the Example 10 is achieved to the extent of 96 %. 3. The D-amino acid oxidase immobilized inside the pores of copolymer prepared according to Example 10 is available for the reaction with cephalosporin C. 4. The pore size and pore size distribution of the copolymer prepared according to Example 10 provides microenvironment suitable for enzymatic reaction and spontaneous chemical reaction those take place during conversion of cephalosporin C to glutaryl-7-ACA. 5. The immobilized D-amino acid oxidase does not develop attrition when used in stirred tank reactor. 6. The immobilized D-amino acid oxidase does not retain or absorb water. In our copending Application No. 2945/DEL/97, we have described and claimed a process for the production of glutaryl-7-amino cephalosporanic acid using the immobilized D-amino acid oxidase prepared by the process described and claimed in the present Application. We claim: 1. An improved process for the production of immobilized D-amino acid oxidase, which comprises suspending crosslinked macroporous glycidyl methacrylate - divinyl benzene copolymers in phosphate buffer, adding D-amino acid oxidase solution, incubating the suspension at a temperature in the range of 20 to 24°C for a period in the range of 9 to 12 hours with agitation ranging from 60 to 120 rpm, separating the immobilized D-amino acid oxidase by conventional filtration methods. 2. An improved process as claimed in claim 1, wherein the molarity of the phosphate buffer used is in the range of 0.05 to 0.1. 3. An improved process as claimed in claims 1 and 2, wherein the pH of the phosphate buffer used is in the range of 6.8 to 7.2. 4. An improved process for the production of immobilized D-amino acid oxidase substantially as described herein with reference to Examples 18,19, 20 and 21.

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