摘要
采用硫酸铵盐析、SephadexG-100凝胶过滤、DEAE-52纤维素柱层析一系列纯化步骤,得到电泳纯的亚硫酸盐还原酶,纯化倍数为115.30,酶回收率为18.36%。用SDS-PAGE测得亚基相对分子质量为66kDa。酶反应的最适温度和pH分别为30℃和7.7。热稳定性较好,80℃时酶活仍能维持在55%左右。在pH4～10之间均较稳定,残余酶活均在60%以上。Fe3+,Zn2+,Cu2+,Mn2+,Co2+对亚硫酸盐还原酶有明显的抑制作用,Ca2+,Zn2+,Ag+,Fe2+对亚硫酸盐还原酶有轻微的抑制作用,其他离子在实验浓度范围内对酶活无明显影响。以亚硫酸钠为底物,该酶的Km为1.23mmol/L,Vmax为6.80u/mL。
The sulfite reductase was purified to electrophoretic homogeneity by ammonium sulfate salting-out, Sephadex G-100 gel filtration and DEAE-52 cellulose column chromatography. A 115.30-fold-purification was achieved and the recovery was 18.36 %. Its subunit molecular weight was 66 kDa measured by SDS-PAGE. The optimal temperature and pH for enzyme reactivity were 30℃ and 7.7 respectively. At 80 ℃, there still remained about 55 % enzyme activity （good thermal stability）. It was in stable state under the condition of pH value between 4 and 10 and the activity of residual reductase was above 60 % on average. Besides, the sulfite reductase was strongly inhibited by Fe^3＋, Zn^2＋, Cu^2＋, Mn^2＋, Co^2＋, and moderately inhibited by Ca^2＋, Zn^2＋,Ag^＋, Fe^2＋ （other ions had no evident effects on it within the range of experimental concentration）. Its Km and Vmax were 1.23 mmol/L and 6.80 u/mL respectively with sodium sulfite as substrate.
出处
《酿酒科技》
北大核心
2006年第6期28-31,共4页
Liquor-Making Science & Technology
基金
国家自然科学基金(20076030)
关键词
微生物
亚硫酸盐还原酶
分离纯化
酶学性质
microbe
sulfite reductase
purification ＆ separation
zymological properties
