Enzymatic process for preparing 7-aminocephalosporanic acid and derivatives

In an enzymatic process for producing 7-amino cephalosporanic acid or its derivatives from Cephalosporin C or its derivatives in accordance with the following reaction scheme:

• ##STR5## where R is --OCOCH.sub.3, --H, --OH, --OCONH.sub.2, by means of the enzyme D-amino acid oxidase (DAO) and subsequent transformation of the glutaryl derivatives (II) into 7-amino cephalosporanic acid (III) by means of the enzyme glutaryl-7-amino cephalosporanic acid acylase (Gl-7-ACA acylase), the improvement consisting of:
• 1a) preparing an enzyme DAO, free frown interfering enzymes selected from the group consisting of catalase, esterase and .beta.-lactamase, from a Rhodotorula gracilis ATCC 26217 at 26.degree.-30.degree. C. and pH 4.8-5.5, by a process comprising the cells and obtaining a cell paste, subjecting the cell paste to lysis, separating the enzyme from the cell debris and purifying the enzyme by chromatography over an ion exchange resin with diethylaminoethyl groups as ionizable functions (DEAE resin);
• 1b) immobilizing the purified enzyme on an inert solid support which is insoluble in an aqueous medium and contains functional groups suitable for forming cross-linkages with the enzymatic protein, such support being selected from the group consisting of:
• strongly basic resins of macroreticular polystyrene structure with a quaternary amine function;
• weakly basic resins of macroreticular polystyrene structure with a primary amino function;
• porous alumina impregnated with a complex of polyethylenimine and glutaraldehyde;
• phenolformaldehyde resins with secondary or tertiary amino functional groups;
• and polyacrylic resins with terminal epoxide functional groups;
• 1c) carrying out the oxidative deamination of the Cephalosporin C or its derivatives (I) by bringing the immobilized enzyme obtained in 1b) into contact with an aqueous solution of said compound at a concentration of 20-60 g/l, at pH 7-8, and at a temperature of 20.degree.-30.degree. C. in the presence of oxygen or air;
• 1d) separating the supported enzyme from the aqueous reaction mixture and adding hydrogen peroxide in an amount equal to or in excess of the stoichiometric quantity required to convert the residual ketoadipylcephalosporanic acid into glutaryl-7-aminocephalosporanic acid;
• 1e) eliminating the excess hydrogen peroxide by adding to the solution reducing agents selected from the group consisting of pyruvic acid, pyruvic acid salts, and alkaline sulphites;
• 1f) preparing the enzyme Gl-7-ACA acylase from cultures of the microorganism Escherichia coli P-3 (p JC 200) registration number NCIMB 40433 through fermenting under aerobic conditions at a temperature of 21.degree.-28.degree. C., subjecting a cell mass obtained from the culture to lysis and purifying the enzyme by chromatography over DEAE resin;
• 1g) immobilizing the purified enzyme on an inert solid support which is insoluble in an aqueous medium and contains functional groups for forming cross-linkages with the enzymatic protein, such support being selected from the group consisting of:
• resins of macroreticular strongly basic polystyrene structure;
• phenolformaldehyde resins with secondary or tertiary amino functional groups;
• resins of polyacrylic structure crosslinked with divinylbenzene and functionalized with primary amino groups;
• polyacrylic resins with epoxide functional groups;
• and porous alumina impregnated with a complex a polyethylenimine and glutaraldehyde;
• and
• 1h) deacylating the glutaryl-7-aminocephalosporanic acid or its derivatives (II) by bringing the immobilized enzyme obtained in 1 g) into contact with an aqueous solution of said compound at a concentration of 10-30 g/l, at a temperature of 20.degree.-30.degree. C. and a pH of 7-9.