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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

基本信息

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

项目摘要

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，利用蛋白质工程技术构建了多种突变体溶菌酶。制备在位置103（N103 D）和106（N106 D）处脱酰胺、在Iys 13和leu 129之间裂解的盐键（K13 D）和在位置96和74之间缺失的SS键（C96/74 A）的溶菌酶突变体。将野生型和突变型溶菌酶在酿酒酵母中表达，并通过在CM-Toyopolymer上的阳离子交换层析分两步从培养基中纯化。各突变体溶菌酶的裂解活性与野生型几乎相同，但最适pH值略有下降。- N103 D和N106 D在20 ° C下的吉布斯展开自由能变化（DELTAG）与野生型几乎相同。另一方面，溶菌酶的结构灵活性，估计蛋白酶消化，脱酰胺显着增加。与野生型溶菌酶相比，脱酰胺溶菌酶的表面功能特性得到了显著增强。这些结果表明，结构的灵活性是一个重要的控制因素，在表面功能的蛋白质，无论其结构的稳定性。另一方面，突变体K13 D和C96/74 A的Δ G远低于野生型溶菌酶，表明它们的结构不稳定。这些突变体具有比脱酰胺溶菌酶更好的表面性质。因此，蛋白质的稳定性似乎是比它的灵活性更有效的因素，为蛋白质的表面功能properlies。