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

Abstract

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 (Tm). Single point mutations Asn2βThr, Asn2βVal, Cys470βSer, Leu154βPhe, and Leu180βPhe in hydrophobic core regions, and Ala100αSer and Ala37βSer in segment-segment interface regions, increased the Tm by 4.7–19.7° C, while combining these confirmed single mutations increased the Tm by up to 20.5° C.