Penicillin G acylases (PGAs) are industrially important enzymes primarily used for the synthesis of first- and second-generation cephalosporins or penicillins. This study aims to establish a high-efficiency biosynthetic system for cefradine on the purpose of significantly enhancing its catalytic efficiency in cefradine synthesis and developing its potentials for industrial application. In this study, we identified and engineered penicillin G acylase and obtained a highly active mutant KsPGA M7(M168F/F313G) for the synthesis of cefradine. The mutant achieved a conversion rate over 95% in the scaled-up reaction. To validate its industrial applicability, we immobilized both the wild-type and mutant enzymes and applied them in continuous flow reactions, which achieved a space-time yield of 2 800 g/(L·d). This study lays a foundation for the future applications of penicillin G acylases in the industrial synthesis of cefradine.
Penicillin Amidase/biosynthesis* ; Protein Engineering/methods* ; Cephradine/metabolism* ; Escherichia coli/metabolism* ; Enzymes, Immobilized/metabolism* ; Recombinant Proteins/biosynthesis*

The effects of cephradinum and ceftazidime on the metabolism of Escherichia coli (E. coli) DH5alpha was determined by microcalorimetry. The microbial activity was recorded as power-time curves through an ampoule method with a TAM Air Isothermal Microcalorimeter at 37 degrees C. The parameters such as the growth rate constant (k), inhibitory ratio (I), the maximum power output (Pm) and the time (tm) corresponding to the maximum power output were calculated. The results show that the ceftazidime has a better inhibitory effect on E. coli DH5alpha than cephradinum.
Anti-Bacterial Agents ; administration & dosage ; pharmacology ; Calorimetry ; methods ; Ceftazidime ; administration & dosage ; pharmacology ; Cephradine ; administration & dosage ; pharmacology ; Escherichia coli ; drug effects ; growth & development ; metabolism ; Microbial Sensitivity Tests