Evolution of an acylase active on cephalosporin C.

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.

Engineering the substrate specificity of D-amino-acid oxidase.

The high resolution crystal structure of D-amino-acid oxidase (DAAO) from the yeast Rhodotorula gracilis provided us with the tool to engineer the substrate specificity of this flavo-oxidase. DAAO catalyzes the oxidative deamination of D-amino acids, with the exception of D-aspartate and D-glutamate (which are oxidized by D-aspartate oxidase, DASPO). Following sequence homology, molecular modeling, and simulated annealing docking analyses, the active site residue Met-213 was mutated to arginine. The mutant enzyme showed properties close to those of DASPO (e.g. the oxidation of D-aspartate and the binding of l-tartrate), and it was still active on D-alanine. The presence of an additional guanidinium group in the active site of the DAAO mutant allowed the binding (and thus the oxidation) of D-aspartate, but it was also responsible for a lower catalytic activity on D-alanine. Similar results were also obtained when two additional arginines were simultaneously introduced in the active site of DAAO (M213R/Y238R mutant, yielding an architecture of the active site more similar to that obtained for the DASPO model), but the double mutant showed very low stability in solution. The decrease in maximal activity observed with these DAAO mutants could be due to alterations in the precise orbital alignment required for efficient catalysis, although even the change in the redox properties (more evident in the DAAO-benzoate complex) could play a role. The rational design approach was successful in producing an enzymatic activity with a new, broader substrate specificity, and this approach could also be used to develop DAAO variants suitable for use in biotechnological applications.