摘要
蓖麻籽黄化苗中存在高活性β-半乳糖苷酶。经硫酸铵分级分离、DEAE-纤维素离子交換层析、Sephadex G-100、CM-Sephadex和DEAE-Sephadex层析纯化。活性收率为6.4％,纯化倍数达107倍。纯化了的酶经聚丙烯酰胺凝胶电泳显示单一蛋白带,SDS-PAGE显示两条蛋白带,其相应分子量分别为3.25×10~4和2.94×10~4。用Sephadex G-200分子筛层析法测得分子量为6.7×10~4。综合上述结果推测该酶是由两个不同的亚基构成。以邻硝基苯酚-β-半乳糖苷为底物测得该酶的表观Km为5.9×10^(-3)mol/L。最适pH和最适温度分别为4.5和50℃。酸碱稳定区域在pH4.6—7.5之间。不同浓度缓冲液以及不同种类缓冲液、不同金属离子对酶活性影响均进行了讨论。
The major β-galactosidase was purified some 107-fold from etiolated seedlings of castor bean, The procedure used ammonium sulphate precipitation followed by chromatography on DEAE-cellulose-52, Sephadex G-100 gel filtration, CM-Sephadex G-25 and DEAE-Sephadex A-50. The purified enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and Sephadex G-200 molecular sieve chromatography. The molecular weight of the native enzyme was determined by gel filtration was 6.7×10~4. The enzyme consists of two subunits with apparent subunit weight of 3.25×10~4 and 2.94×10~4 as determined by SDS-PAGE. The enzyme followed typical Michaelis-Menten Kinetics with apparent Km of 5.9mmol/L for α-nitrophenyl-β-galactopyranoside. The optimum pH was 4.5 (stable from 4.6—7.5). The temperature optimum was 50℃(stable to 50℃). Effects of various metal ions and buffers on enzyme activity were also examined.
