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 Asn2 beta Thr, Asn2 beta Val, Cys470 beta Ser, Leu154 beta Whe, and Leu180 beta phe he in hydrophobic core regions, and Ala100 alpha Ser and Ala37 beta Ser in segment-segment interface regions, increased the T-m by 4.7-19.7 degrees C, while combining these confirmed single mutations increased the T. by up to 20.5 degrees C. (C) 2018 Elsevier Ltd. All rights reserved.