蓖麻β—半乳糖苷酶的研究

PROPERTIES OF β-GALACTOSIDASES FROM CASTOR BEAN

蓖麻种子黄化苗提取液中存在高活性的β—半乳糖苷酶。经硫酸铵分级分离、DEAE—纤维素(DE—52)离子交换层析分离得到具有β—半乳糖苷酶活性的2种酶,分别称为β—半乳糖苷酶Ⅰ和Ⅱ。分别经Sephadex G—150分子筛层析后用PAGE分析表明二者纯度均达到60以上。以邻硝基苯酚—β—半乳糖苷为底物,β—半乳糖苷酶Ⅰ和Ⅱ的Km分别为5.9mmol/L和2.9mmol/L。以乳糖为竞争性抑制剂,其Ki分别为150mmol/L和175mmol/L。β—乳糖苷糖Ⅰ和Ⅱ最适pH分别为4.5和3.8。酸碱稳定区域分别在pH 3.6—7.0和4.6—7.0。二者的最适温度均为50℃。用Sephadex G—200分子筛层析测得β—半乳糖苷酶Ⅰ和Ⅱ的相对分子量分别为67000和85000。不同离子强度缓冲液,不同种类缓冲液以及金属离子对酶活性的影响也进行了讨论。

The germinating seedlings of castor bean contained a high leval of β-galactosidase activity, and the two components found to be responsible for this activity, β-galactosidase Ⅰ and Ⅱ , were separated and partially purified by ammonium sulphate precipitation, DEAE-cellulose(DE -52) ion-exchange chromatography and Sephadex G -150 gel filtration. The two forms appear to be more than 60% in purity as judged by PAGE and differ in the molecular weights and ionic charges. The two forms showed optimal activity in the range pH3. 8-4. 5 and optimal temperature 50℃. The apparent relative molecular weights of β-galactosidase Ⅰ and Ⅱ as determined by Sephadex G -200 gel filtration were 67000 and 85000 respectively. The two forms followed typical Michaelis-Menten kinetics with Km of 5. 9×10-3M and 2. 9×10-3M, respectively. The Ki for laptose determined by Michaelis-Menten kinetics were 1. 5×10-1M and 1. 75×10-1M, respectively. The effects of various metal ions and buffers on enzyme activity were also tested.