Design and creation of a Ca2+ binding site in human lysozyme to enhance structural stability.

Abstract

A Ca2+ binding site like an EF-hand motif was designed and created in human lysozyme by replacing both Gln-86 and Ala-92 with aspartic acids by site-directed mutagenesis. The mutant human lysozyme (D86/92-lysozyme) was expressed and secreted by yeast. One Ca2+ was found to bind one molecule of the purified protein with the binding constant 5.0 .times. 106 M-1. The enzymatic activity of holo-D86/92-lysozyme against glycol chitin at 40.degree. C was 2-fold higher than that of the native lysozyme. Maximal activity of the holo-D86/92-lysozyme was observed at 80.degree. C, where its relative activity normalized to the value at 40.degree. C was 6-fold and 17-fold higher than those of the native and apoenzymes, respectively. The activities of the native lysozyme and apo-D86/92-lysozyme were maximum at 65.degree. C-70.degree. C. Moreover, D86/92-lysozyme was more stable against protease digestion than the native lysozyme. These results indicate that the creation of the calcium binding site like an EF-hand motif in the human lysozyme enhances its structural stability.