A highly active adipyl‐cephalosporin acylase obtained via rational randomization

There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study, the active site of the glutaryl acylase from Pseudomonas SY‐77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adipyl‐7‐aminodesacetoxycephalosporanic acid (ADCA) and cephalosporin C have now been combined, and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine‐deficient Escherichia coli strain and subjected to growth selection with adipyl‐leucine or amino‐adipyl‐leucine as sole leucine source. The mutants growing on these media were selected and purified, and their hydrolysis activities towards adipyl‐7‐ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected from a library of totally randomized residues: 178, 266, and 375. This mutant comprises two mutations, Y178F + F375H, which synergistically improve the catalytic efficiency towards adipyl‐7‐ADCA 36‐fold. The activity of this mutant towards adipyl‐7‐ADCA is 50% of the activity of the wild‐type enzyme towards the preferred substrate glutaryl‐7‐aminocephalosporanic acid, and therefore the characteristics of this mutant approach those needed for industrial application.