重组嗜热β-葡萄糖苷酶转化稀有人参皂苷 Rd 和 CK

摘要

利用高效液相色谱(HPLC)法,对重组嗜热β-葡萄糖苷酶(Fpglu1)转化稀有人参皂苷(Rd 和 CK)进行研究,并表征了其催化动力学参数.利用同源模建和分子动力学模拟等生物信息学技术,探究了 Fpglu1转化人参皂苷的结构基础及其相互作用.结果表明, Fpglu1能够水解人参总皂苷生成稀有皂苷 Rd 和 CK,其催化人参皂苷 Rb1, Rb2和 Rc 的 Km 值分别为0.318,1.840和5.269 mmol/ L；酶的转换数( kcat )值分别为144.191,0.572和0.011 s-1.当转化时间分别为6和102 h 时, Rd 和 CK 的产率达到最大,分别为60%和93%.通过对该酶的结构预测及皂苷分子的对接研究发现,底物位于由疏水性氨基酸构成的底物口袋中,氨基酸残基 Glu194和 Glu367是参与催化作用的关键,且实验测得的酶促反应动力学参数(Km )与对接的相互作用能量值存在线性关系.%Ginsenosides are the principle components that are responsible for the biological and pharmacologi-cal activities of ginseng. In this study, biotransformation of ginsenside Rd and CK by recombinant β-glucosi-dasse(Fpglu1) was studied by high performance liquid chromatography(HPLC). Structure modeling and mo-lecular docking studies were performed to study the interaction between the protein and the ginsenosides. The results showed that ginsenoside Rd and CK could be transformed from major ginsenosides Rb1 , Rb2 and Rc by Fpglu1. The Km values for Rb1 , Rb2 and Rc were 0. 318, 1. 840 and 5. 269 mmol/ L, and the kcat values were 144. 191, 0. 572 and 0. 011 s-1 , respectively. The highest productivity of ginsenoside Rd and CK were 60%and 93% with biotransformation time of 6 and 102 h, respectively. Molecular docking studies show that Glu194 and Glu367 are key active site residues and the predicted inter-energy exhibits linear relations to ex-perimental Km values and kcat values.