接骨草蔗糖酶同工酶的分离纯化及其部分性质研究

Isolation, Purification and Some Properties of the Isozymes of Invertase in Sambucus chinensis Lindl

从接骨草幼叶中提取的蔗糖酶粗酶液经硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、SephacrylS-200 凝胶过滤层析纯化,得到3种同工酶．对3种同工酶进行SDS-PAGE和IEF电泳均显示一条蛋白带,其亚基分子量分别为45．8 KD,44．3 KD,43．2 KD;等电点分别为pH 4．0,pH 3．8,pH 3．75．凝胶过滤测得3种同工酶全酶分子量分别为91．2 KD,89．1 KD,87．4 KD,表明3种同工酶均由2个相同亚基组成,其表观Km分别为30 mmol／L, 57．1 mmol／L,65 mmol／L;Vmax分别为98μg／min,48 μg／min,35μg／min．同工酶Ⅰ和同工酶Ⅲ在pH4．4-5．0 时有最大活性,同工酶Ⅱ则在pH3．9-4．2时有最大活性．3种同工酶均在pH3-6范围内比较稳定．同工酶Ⅰ和同工酶Ⅱ在48-52℃有最大活性,同工酶Ⅲ在50-55℃有最大活性．3种同工酶均在50℃以下有较好的稳定性．

Invertase solution extracted from the leaves of Sambucus chinensis Lindl has been isolated and purified by ammonium sulphate precipitation, DEAE-Sepharose column chromatography, and SephacrylS200 gel filtration, and three isozyme of invertase were obtained. The purified isozymes were homogeneous as shown in SDS-PAGE and IEF electrophoresis. The molecular weight of the isozymes determined by SDS-PAGE were 45.7 KD, 44. 9 KD, 44. 1 KD and gel filtration were 91.2 KD, 89.1 KD, 88. 1 KD respectively. The PI determined by IEFelectrophoresis were pH4.0, pH3.8, pH3.75 respectively. The Km of the isozymes were 30 mmol/L, 57.1 mmol/L, 65 mmol/L and Vmax were 98 μg/min, 48 μg/min, 35 μg/min respectively. The optimum pH range of isozyme Ⅰ and isozymeⅢ was pH4. 4 - pH5. 0, and isozymeⅡ was pH3. 9 - pH4. 2. The optimum temperature range of isozymeIand isozymeⅡwas 48 ℃- 52 ℃, and isozymeⅢ was 50℃- 55℃. Three isozymes were stable at the range of pH3-pH6 and under 50℃.