Structure-Functionality of Egg-White Lysozyme by Protein Engineering

为了研究溶菌酶的结构和功能特性，利用蛋白质工程技术构建了各种突变酶。制备了103(N103D)和106(N106D)位去胺化、IYS 13与Leu 129(K13D)盐键断裂、96-74位SS键缺失(C96/74A)的溶菌酶突变体。在酿酒酵母中表达野生型和突变型溶菌酶，并用CM-TOYOPEL阳离子交换层析两步纯化。各突变体溶菌酶的裂解活性与野生型溶菌酶基本相同，但酶活的最适pH略有降低。-N103D和N106D在20ﾟC时展开的吉布斯自由能变化与野生型基本相同。另一方面，用酶消化法测定的溶菌酶的结构柔韧性在脱酰胺后显著增加。与野生型溶菌酶相比，去酰胺化溶菌酶的表面功能性质有了显著的提高。这些结果表明，结构柔性是蛋白质表面功能性质的一个重要控制因素，与其结构稳定性无关。另一方面，突变体K13D和C96/74A的Delta G明显低于野生型溶菌酶，表明其结构不稳定。这些突变体具有比去胺化溶菌酶更好的表面性质。因此，对于蛋白质的表面功能特性来说，蛋白质的稳定性似乎比它的灵活性更有效。

To investigate the structural and functional properties of lysozyme, various mutant enzymes were constructed by protein engineering. Lysozyme mutants deamidated at positions 103 (NlO3D) and 106 (NlO6D), cleaved salt linkage between Iys 13 and leu 129 (K13D), and deleted SS bond between positions 96 and 74 (C96/74A) were prepared. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation exchange chromatography on CM-Toyopearl. The lytic activities of each mutant lysozymes were almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. -The Gibbs free energy changes of unfolding(DELTAG)at 20ﾟC for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability. On the other hand, DELTA G of mutant K13D and C96/74A was much lower than that of wild-type lysozyme, suggesting their unstable structure. These mutants had better surface properties than deamidated lysozymes. Thus, protein stability seems to be more effective factor than its flexibility for the surface functional properlies of proteins.