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Engineering Enzymes for Enhanced Thermostability

  • Phoebe Shih (a1), Bruce A. Malcolm (a1) and Jack F. Kirsch (a1)


Chicken egg-white lysozyme (CEWL) is used as a model to attempt to engineer proteins for enhanced thermostability. Site-directed mutagenesis is employed for selective amino acid substitution to probe the contribution of an individual amino acid in a given sequence to thermostability. A linear correlation is found between the side-chain volume of a triplet of amino acid residues located at the interior core of the protein and its thermostability. Additional mutant constructs at the core position reveal that hyperpacking can disrupt other intramolecular contacts and offset the hydrophobic stabilization due to denser packing. Multiple substitutions at different loci of the protein are combined to analyze the additivity of thermostability mutations.



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