Structure-Functionality of Egg-White Lysozyme by Protein Engineering

通过蛋白质工程研究蛋清溶菌酶的结构-功能

• 批准号: 02660143
• 负责人: KATO Akio
• 金额: $ 1.28万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660143/
• 关键词: Protein Engineering; Lysozyme; Deamidation; Emulsifying Properties; Foaming Properties; 変異型リゾチ-ム

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.

为了研究溶菌酶的结构和功能特性，通过蛋白质工程构建了在位置103(N103D)和106(N106D)脱酰胺、裂解Lys 13和leu 129(K13D)之间的盐键并删除SS的多种突变酶。制备了位置 96 和 74 (C96/74A) 之间的键。突变体溶菌酶在酿酒酵母中表达，并通过CM-Toyopearl上的阳离子交换层析分两步从培养基中纯化。每种突变体溶菌酶的裂解活性几乎与野生溶菌酶相同，尽管其活性的最适pH为1。通过脱酰胺作用稍微改变至较低的 pH 值 -20°C 时的展开吉布斯自由能变化 (DELTAG)。 N103D 和 N106D 的结构灵活性与野生型几乎相同。另一方面，通过蛋白酶消化估计，溶菌酶的结构灵活性通过脱酰胺显着增加。相比之下，脱酰胺溶菌酶的表面功能特性显着增强。这些结果表明，无论 DELTA 的结构稳定性如何，结构灵活性都是蛋白质表面功能特性的重要控制因素。突变体K13D和C96/74A的G远低于野生型溶菌酶，表明它们的不稳定结构比脱酰胺溶菌酶具有更好的表面特性，因此，蛋白质稳定性似乎比其表面灵活性更有效。蛋白质的功能特性。