Erwinia+rhapontici+NX4中蔗糖异构酶的纯化及酶学性质研究.pdfVIP

11．生物转化与催化 Erwinia rhapontici NX-4 中蔗糖异构酶的纯化及酶学性质 任贲 李莎 冯小海 蔡恒 徐虹＊ （南京工业大学制药与生命科学学院，南京工业大学材料化学工程国家重点实验室，江苏南京 210009 ） 摘 要：对Erwinia rhapontici NX-4 中的蔗糖异构酶（SIase ）进行了纯化和酶学性质的研究。粗酶经硫酸铵 沉淀、DEAE弱阴离子层析和SP Fast Flow强阳离子层析三步获得电泳纯的蔗糖异构酶，其分子量约为 65.6kDa 。纯化倍数、酶比活和活力收率分别为317倍、423 U/mg和27.7% 。该酶的最适pH值为6.0，最适反 应温度为30℃，在20～40 ℃范围内酶活力相对稳定，60℃保存30min 以上酶失活；酶的最大反应速率为 2+ Vmax=10.14mmol/(L ⋅min), 米氏常数Km=988.24 mmol/L 。蔗糖异构酶不是一种金属酶，酶活力能够被Ca ， Mn2+促进，被Cu2+ ，Zn2+抑制。该酶只作用于单一底物蔗糖，果糖和葡萄糖对该酶具有抑制作用，后者能 完全抑制酶对底物蔗糖的转化。 关键词：蔗糖异构酶（SIase ）；阴离子交换层析；阳离子交换层析；酶学性质 中图分类号： Q815 文献标识码：A 文章编号： Purification and Properties of Sucrose Isomerase from Erwinia rhapontici NX-4 Ren Ben, Li SHa,Feng Xiaohai, Cai Heng, Xu Hong ＊ （College of Life Science and Pharmaceutical Engineering, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing 210009) Abstract: A sucrose isomerase (SIase) from Erwinia rhapontici NX-4 was purified and characterized. The enzyme was purified through three steps including ammonium sulphate fractionation, DEAE-Sepharose and SP Fast Flow anion exchange chromatography. The molecular weight of holoenzymes was 65.6 kDa which was detected by SDS−PAGE. The final purified fold, specific activity and the yield of the purified enzyme are 317, 423U/mg and 27.7 ％, respectively. The optimal pH value and temperature were 7.0 and 30 ℃ respectively. The activity of SIase was kept stable when the enzyme was conserved in the temperature range from 20 to 40 ℃, however, it declined rapidly when conserved at 60℃ for about 30min. The Km value was 988.24 mmol/L and the Vmax 10.14 mm