THE HYDROLYSIS OF CEPHALOSPORIN-G BY FREE AND IMMOBILIZED PENICILLIN-G ACYLASE FROM A MUTANT OF ESCHERICHIA-COLI ATCC-11105

The hydrolysis of cephalosporin G to 7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) by free and immobilized penicillin G acylase (PGA) was studied in a mutant of Escherichia coli ATCC 11105. V(m) and K(m) values of soluble enzyme (specific activity: 25.0 U mg-1, protein concentration: 0.4 mg ml-1) were 15.92 U ml-1 min-1 and 2.94 mM cephalosporin G (Cep G) respectively at pH 8.0 and 40-degrees-C. Optimal pH and temperature values of Cep G hydrolysis by the soluble enzyme were 8.0 and 55-degrees-C. The K(m) value of immobilized enzyme obtained by covalent attachment on oxirane acrylic beads (specific activity: 120 U g-1 dry matrix at pH 8.0 and 40-degrees-C, bound protein: 12.8 mg) was 3.32 +/- 0.06 mm Cep G. The immobilized enzyme showed highest activity at pH 8.0 and at 50-degrees-C. A 95% conversion rate to 7-ADCA was observed in 40 min at 40-degrees-C and pH 8.0 with 1.0% (w/v) Cep G. Excess Cep G, PAA and 7-ADCA were uncompetitive, competitive and non-competitive inhibitors respectively for both soluble and immobilized PGA. The activation energy of Cep G hydrolysis was calculated to be 7.83 kcal mol-1 for soluble PGA and 5.25 kcal mol-1 for immobilized PGA.