Effect of disulfide-bonds on the thermostability of epimerase

• Main Authors: Guan-Yi Wu, 吳冠毅
• Other Authors: Sung-Chyr ,Lin
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/08159668085217387339

碩士 === 國立中興大學 === 化學工程學系所 === 94 === Abstract GlcNAc 2-epimerase is a valuable enzyme for production of sialic acid which is useful in medicine. The activity of epimerase purified in our study is relatively low. Therefore, it is necessary to enhance the enzyme activity. Although it has been shown that epimerase does not have disulfide-bonds, the presence of 10 cysteine residues in each subunit might imply disulfide-bonds. It is thus proposed that the activity and/or stability of epimerase may be promoted by introducing inter or intra chain disulfide-bonds. To oxidize the cysteine to form disulfide-bonds, oxidized glutathione (GSSG) was used in this study. The result of nondenature SDS-PAGE analysis indicates that disulfided epimerase can be obtained with this approach. The formation of disulfided epimerase facilitates the relative activity and thermostability. For example, the relative activity and thermostability of disulfided protein are higher than native protein. In this study , the optimal conditions of pH and temperature are pH=8 and 40℃ The relative activity and temperature of thermostability of disulfided epimerase are higher than native epimerase about 60~75℃. These results demonstrate that the relative activity and thermostability of epimerase are higher when GSSG is added.