尿甘二磷酸半乳糖4-差向異構酵素（UDP-galactose 4-epimerase）係純化、分離 自突變種E. coli ATCC 27797。以製備型電泳取代Hydroxylapatite管柱，得純化後酵素之 比活性500unit/mg；純度為290倍，純化過程活性回收率為7%。合成之尿甘-5[1-硫二磷酸] 葡萄糖（UDPαS-glucose），係自然基質尿甘二磷酸葡萄糖（UDP-glucose）的類似物。因 為Pα含硫而具不對稱性，而產生Rp-和Sp-非對映異構物。二異構物能以半製備型μ Bondpak C?琠M磷酸緩衝溶液（50mM pH6.0），經高效率液態層析管柱分離。其純度高，可 運用作為反應基質，以鑑定UDP-galactose 4-epimerase對基質之立體專一性。實驗結果顯 示，epimerase會催化Rp-異構物，而Sp-異構物之反應速率極低，可證實其活化部位與基質 Pα結合的位置，構象很嚴緊，具有高度的立體專一性。 E. coli ATCC27797 UDP-galactose 4-epimerase purified by preparative electrophoresis method rater then hydroxylaptite column showed a specific activity of 500 units per milligram protein, the enzyme purification fold was 290 and the percentage of activity recovery 7%. Synthetic diastereoisomers Rp-and Sp-UDPαS-glucose could be separated by using HPLC semipreparative μBondpak C18 column and 50 mM phosphate buffer, pH 6.0 as elution buffer. The sterospecificity of UDP-galactose 4-epimerase to Rp-UDPαS-Glucose indicated that the epimerase showed a rigid conformation of binding site around the Pα of the substrate.
