重组嗜热β-葡萄糖苷酶转化稀有人参皂苷Rd和CK

doi:10.7503/cjcu20150536

高等学校化学学报 ›› 2016, Vol. 37 ›› Issue (2): 281.doi: 10.7503/cjcu20150536

重组嗜热β-葡萄糖苷酶转化稀有人参皂苷Rd和CK

许春春¹, 于渤浩¹, 王红蕾², 李晶¹, 刘淑莹^1,³(), 于珊珊^1,⁴()

1. 长春中医药大学吉林省人参科学研究院, 长春 130000
2. 长春工业大学化学与生命科学学院, 长春 130024
3. 中国科学院长春应用化学研究所长春质谱中心, 长春 130022
4. 上海交通大学生命科学技术学院, 上海 200240

收稿日期:2015-07-09 出版日期:2016-02-10 发布日期:2015-12-26
作者简介:联系人简介: 于珊珊, 女, 博士, 讲师, 主要从事人参皂苷生物转化研究. E-mail:yushanshan001@aliyun.com;刘淑莹, 女, 博士, 研究员, 博士生导师, 主要从事质谱学研究. E-mail:syliu@ciac.jl.cn
基金资助:
国家自然科学基金(批准号: 31400682)和长春市重大科技攻关计划项目(批准号: 13KG60)资助

Transformation of Minor Ginsenoside Rd and CK by Recombinant Thermostable β-Glucosidase^†

XU Chunchun¹, YU Bohao¹, WANG Honglei², LI Jing¹, LIU Shuying^1,^3,^*(), YU Shanshan^1,^4,^*()

1. Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130000, China
2. School of Chemistry and Bioscience, Changchun University of Technology, Changchun 130024, China
3. Changchun Center of Mass Spectrum, Changchun Institute of Applied Chemistry,Chinese Academy of Sciences, Changchun 130022, China
4. School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China

Received:2015-07-09 Online:2016-02-10 Published:2015-12-26
Contact: LIU Shuying,YU Shanshan E-mail:syliu@ciac.jl.cn;yushanshan001@aliyun.com
Supported by:
† Supported by National Natural Science Foundation of China(No.31400682) and the Key Technologies R & D Programme of Changchun, China(No.13KG60)

摘要/Abstract

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K_m值分别为0.318, 1.840和5.269 mmol/L; 酶的转换数(k_cat)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(K_m)与对接的相互作用能量值存在线性关系.

关键词: β-葡萄糖苷酶, 生物转化, 稀有人参皂苷, 动力学

Abstract:

Ginsenosides are the principle components that are responsible for the biological and pharmacological activities of ginseng. In this study, biotransformation of ginsenside Rd and CK by recombinant β-glucosidasse(Fpglu1) was studied by high performance liquid chromatography(HPLC). Structure modeling and molecular 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 K_m values for Rb₁, Rb₂ and Rc were 0.318, 1.840 and 5.269 mmol/L, and the k_cat 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 experimental K_m values and k_cat values.

Key words: β-Glucosidase, Biotransformation, Ginsenoside, Kinetics

中图分类号:

O629
O641

TrendMD:

许春春, 于渤浩, 王红蕾, 李晶, 刘淑莹, 于珊珊. 重组嗜热β-葡萄糖苷酶转化稀有人参皂苷Rd和CK. 高等学校化学学报, 2016, 37(2): 281.

XU Chunchun, YU Bohao, WANG Honglei, LI Jing, LIU Shuying, YU Shanshan. Transformation of Minor Ginsenoside Rd and CK by Recombinant Thermostable β-Glucosidase^†. Chem. J. Chinese Universities, 2016, 37(2): 281.

图/表 8

Fig.1 SDS-PAGE analysis of Fpglu1 after purification Lane M: standard protein molecular mass makers;lane 1: Fpglu1 purified by Ni-NTA chromatography.

Fig.2 HPLC analysis of biotransformation of the ginseng root extract at different time (A) Standard ginsenosides; (B)—(H) Fpglu1 reacted with 2 mg/mL ginseng root extract for 0 , 1/6, 2, 6, 66, 78 and 102 h, respectively in 20 mmol/L sodium phosphate buffer(pH=7.5) at 70 ℃.

Fig.3 Transformation pathways of the ginsenosides Rb1, Rb2 and Rc by Fpglu1 The transformation pathways were Rb1→Rd→CK, Rb2→Rd→CK and Rc→Rd/CMc→CK.

Fig.4 Production of Rd and CK from the ginseng root extract by Fpglu1 ●: Ginsenoside Rb1; ○: Ginsenoside Rb2; ▲: Ginsenoside Rc; △: Ginsenoside Rd; ■: CMc; □: CK.

Fig.5 Fitting curve of ginsenosides Rb1(A), Rb2(B) and Rc(C)

Fig.6 Ramachandran plot analysis and predicted 3D structure of Fpglu1 (A) Ramachandran plot showing different regions of the modeled enzyme; (B) the overall structure of the Fpglu1 model.

Fig.7 Docking model of Fpglu1 with ginsenoside Rb1 (A) A zooming view showing the docking model with ginsenoside Rb1. The hydrophobic amino acids around ginsenoside Rb1 and the key catalytic amino acids were indicated. The ginsenoside Rb1 and the surrounding amino acids are colored yellow and blue, respectively; (B) amino acid residues close to ginsenoside Rb1 inside the binding pocket of Fpglu1(2D view).

Table 1 Kinetic parameters of Fpglu1 for Ginsenosides

Substrate	K_m/(mmol·L^-1)	k_cat/s^-1	(k_cat/K_m)/(L·s^-1·mmol^-1)	E/(kJ·mol^-1)
Rb₁	0.318	144.191	453.016	-91.7085
Rb₂	1.840	0.572	0.311	-80.1428
Rc	5.269	0.011	0.002	-74.1413

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重组嗜热β-葡萄糖苷酶转化稀有人参皂苷Rd和CK

Transformation of Minor Ginsenoside Rd and CK by Recombinant Thermostable β-Glucosidase^†

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重组嗜热β-葡萄糖苷酶转化稀有人参皂苷Rd和CK

Transformation of Minor Ginsenoside Rd and CK by Recombinant Thermostable β-Glucosidase†

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Transformation of Minor Ginsenoside Rd and CK by Recombinant Thermostable β-Glucosidase^†