Studies on Purification and Active Site of Escherichia coli UDP- galactose 4-epimerase

• Main Authors: Chia-Tsung Hung, 洪嘉聰
• Other Authors: Cheng-Tzung Tsai,Ph.D.
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/29304520359659642714

碩士 === 東海大學 === 食品科學系 === 82 === UDP-galactose 4-epimerase isolated from E.coli ATCC 27797 by using preparative electrophoresis instead of the last three steps of purification showed specific activity of 500 units per milli- gram,two hundred and ninety folds,and overall avarage activity recovery of 7 %.Synthetic diastereoisomers Rp-and Sp- UDPα S- glucose have been seperated by using semipreparative uBondpak C18 column and 50 mM phosphate buffer,pH 6.0 with high performance liquid chromatography.The diastereospecificity of UDP-galactosepimerase.The results showed that the epimerase showed a rigid conformation of binding site around the Pα of the substrate that .Rp-UDPαS-glucose (Km 2.7) seemed to be a much better substrate than Sp-UDPα S-glucose (Km 25).The location of Cysteine around the active site have been tested by reacting with thiol reagentB and diamide.Base on the change in activity and,fluoresence and electrophoresis result,if was tentatively concluded that there are two thiols around the active site and it is far fromother for making a disulfide bond by oxidation.