鲜地黄中α-半乳糖苷酶和β-葡萄糖苷酶的提取与初步纯化

目的:从鲜地黄中提取并初步纯化α-半乳糖苷酶和β-葡萄糖苷酶。方法:以总酶活为考察指标,通过正交试验确定两种酶的最佳提取条件。以酶活力和蛋白质含量为考察指标,确定两步盐析法的硫酸铵饱和度。结果:提取温度和溶剂量对α-Gal的提取具有显著性影响,而提取溶剂对-βG lu的提取具有显著性影响,因此确定以3倍量(v/w)磷酸盐缓冲液,4℃提取4 h为两种酶的最佳提取条件。以30%和60%为两步盐析的最佳硫酸铵饱和点。结论:鲜地黄中存在α-半乳糖苷酶和β-葡萄糖苷酶,两步盐析法可以使二者得到初步纯化。

Extraction and Salting-out Purification of α-galactosidase and β-glucosidase from Fresh Roots of Rehmannia glutinosa

OBJECTIVE: To extract and preliminarily purify alpha-galactosidase and beta-glucosidase from the fresh roots of Rehmannia glutinosa. METHODS: With the enzyme activity as a criterion, the best procedure of extraction was selected though orthogonal design method, and the desired saturation of ammonium sulfate in two-step salting-out was settled by gradient sedimentation of root extract according to enzyme activity and protein content. RESULTS: Temperature and solvent volume affect the extraction of alpha-galactosidase significantly, while solvent type to beta-glucosidase. Therefore the procedure for extracting two enzymes was decided as mixing comminuted fresh root with 3 times phosphate buffer, and placing the mixture in refrigeratory at 4 degrees C for 4 hours, and then obtaining the enzyme liquid by centrifuging at 4 degrees C. 30% and 60% saturation was defined as the lower and upper point for two-step ammonium sulfate salting-out of the two enzymes. CONCLUSION: alpha-Galactosidase and beta-glucosidase exist in the fresh roots, and can be preliminarily purified through two-step salting-out.