Characterization and partial purification of fructose-1,6-bisphosphate aldolase from Deinococcus radiodurans

• Main Authors: Meng-Chi Lee, 李孟芝
• Other Authors: Jong-Kang Liu
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/40579298529824090256

碩士 === 國立中山大學 === 生物學系 === 87 === The Gram positive Deinococcus radiodurans is characterized by its strongly resistant to ionized radiation and ultraviolet. There is little documentation on the studies of its physiology and sugar metabolism and some studies even conducted to inconsistent results. We had desmonstrated that glucose and fructose could be used as the substrate for the growth of this bacterium but with a low efficiency. We also found that D. radiodurans contained two classes of aldolase, in which the class II aldolase is an Mn-dependent enzyme. When the growth culture was supplemented with Mn(II), the class II aldolase performed a higher activity in the stationery phase cells. This enzyme exhibited the highest activity at neutral pH and was more stable in lower temperature. The enzyme activity was propotional to the concentration of Mn(II). However, too much Mn(II) could also inhibit the activity of this enzyme. For the purification of class II aldolase, we had ever assessed the efficacy of the following chromatographic columns：DEAE Sepharose CL-6B, DEAE cellulose, Q-Sepharose Fast-Flow（QAE）, Superdex 200, Superose 12, Sephadex G-100, and Phenyl-Sepharose. We found out that the QAE chromatrography column and Amicon ultrafiltration derived the best purification result and the enzyme activity was triple than those of the originally S2 crude extract. The effectiveness of protein purification was also monitored and confirmed by the SDS-PAGE electrphoresis.