Modifications of stability and function of lysozyme

Abstract

Approaches to improving the functionality of lysozyme are presented. Lysozyme was variously modified and the stabilities of the derivatives were determined by thermal denaturation experiments. Contributions of salt bridge(s), hydrophobic interactions(s), and cross-linkage(s) were evaluated. The stabilities against proteolysis were also considered. For the latter stability, it might be important to depress the rate of unfolding, i.e., to stabilize the native conformation. As a rule, salt bridges and hydrophobic interactions stabilize the native conformation and cross-linkages destabilize the denatured conformation. However, cross-linkages are apt to introduce strains in the native conformation and only suitable lengths of cross-linkages can stabilize the protein. The stabilization was shown to be generally effective in improving the functionality of proteins. Catalytic groups in lysozyme (Glu-35 and Asp-52) were variously modified and finally converted to the respective amides. The participation of these groups in the catalytic function was confirmed. The specificity of lysozyme was modified. Asp-101, which lies on the top of the active site cleft of lysozyme, was variously modified and the effects on the hydrolysis patterns of a hexamer of N-acetylglucosamine were analyzed. Some approaches to endowing lysozyme with altered functions are also presented. In order to give higher esterase activity to lysozyme, the complementarity of enzyme and substrate was investigated by modifying substrate and the active site cleft of lysozyme. An attempt was made to convert lysozyme into a transaminase by introducing pyridoxamine to the active site cleft of lysozyme. Finally, we have started to apply genetic engineering to this kind of investigation and would like to see how far we can go with protein engineering to improve the nature of proteins.