Characterization and engineering of cephalosporin C acylases to produce 7-Aminocephalosporanic acid

7-Aminocephalosporanic acid (7-ACA) is the core nuclei for the synthesis of β-lactam antibiotics. Cephalosporin C acylases (CCAs) are a class of enzymes that enable the hydrolysis of cephalosporin C (CPC) to 7-ACA. In this study, we identified, characterized and engineered new CCAs to prepare 7-ACA. The best mutant A14 (L159S/A419V) was obtained after several rounds of semi-rational enzyme design using the combinatorial active-site saturation testing (CAST) and iterative evolution. A 2.8-fold increase in specific enzymatic activity (6.1 U/mg) was obtained compared to the wild-type enzyme. The engineered variant also enabled the hydrolysis of a variety of β-lactam antibiotics including Penicillin G and phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA). Overall, this study discovered new CCAs from distinct bacterial origins and enabled the synthesis of various core β-lactam antibiotics nuclei.