Computational design of thermostable mutants for cephalosporin C acylase from Pseudomonas strain SE83

Computational protein design strategies can be used to increase enzyme stability without the need for high-throughput screening. In this report, computational methods were used to redesign cephalosporin C acylase from Pseudomonas strain SE83 to enhance its stability by repacking the hydrophobic core regions and reconstructing the protein-protein interactions in the segment interface regions. A nine-fold mutant with enhanced catalytic activity in the hydrolysis of cephalosporin C to 7-aminocephalosporanic acid, but with low stability, was used as a starting point. A computational enzyme design strategy was used to identify target regions to increase the protein melting temperature (T_m). Single point mutations Asn2βThr, Asn2βVal, Cys470βSer, Leu154βPhe, and Leu180βPhe in hydrophobic core regions, and Ala100αSer and Ala37βSer in segment-segment interface regions, increased the T_m by 4.7–19.7° C, while combining these confirmed single mutations increased the T_m by up to 20.5° C.