DESIGN AND STRUCTURAL-ANALYSIS OF AN ENGINEERED THERMOSTABLE CHICKEN LYSOZYME

A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (Delta T-m approximate to +10.5 degrees C) and chemical (Delt a C-m for guanidine hydrochloride denaturation = +1.3 M) stabilities r elative to wild-type (WT) was constructed by combining several individ ual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 degre es C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mo l(-1) greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came fro m diverse sources: (1) The 155L/S91T core construct with Delta T-m = 3 .3 degrees C from WT was available from the accompanying study (Shih P , Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysoz yme structure where the Ala 31 equivalent is Leu. (3) The H15L and R11 4H substitutions were selected on the basis of sequence comparisons wi th pheasant lysozymes that are more stable than the chicken enzyme. (4 ) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.