Evaluation of Different Glutaryl Acylase Mutants to Improve the Hydolysis of Cephalosporin C in the Absence of Hydrogen Peroxide

2-Oxoadipoyl-7-ACA is an intermediate in the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) when using a new route involving D-amino acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7-ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2-oxoadipoyl-7-ACA, different mutants of glutaryl acylase from Pseudomonas SY-77 with an improved activity towards adipoyl-7-ACA were evaluated. The best results on 2-oxoadipoyl-7-ACA hydrolysis were found with the double mutant Y178F+F375H, which showed a K(cat) increase of 6.5-fold and a K(m) decrease of 3-fold compared to the wild-type (wt) enzyme. When this enzyme was tested in the tri-enzymatic system to convert CPC into 7-ACA, this mutant permitted us to reach more than an 80% yield of 7-ACA using a 3-fold mass excess compared to DAAO; while the wt enzyme gave only a 40% yield. Therefore, the application of this new mutant to the one-pot conversion of CPC to 7-ACA gives very good result in terms of efficiency, yield and rate of the process.