Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China.
College of Chemical Engineering, Nanjing Forestry University, Nanjing, China.
J Appl Microbiol. 2020 Mar;128(3):721-734. doi: 10.1111/jam.14513. Epub 2019 Dec 12.
The aim of this work was to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3 by three thermostable glycosidases.
The GH1 thermostable beta-glucosidase Tpebgl1 from Thermotoga petrophlia was found to have the ability to convert ginsenosides Rb1 and Rb2. Its properties concerning ginsenoside conversion were systematically investigated. It had high specific activity on pNPG (162·20 U mg ) and pNPArp (22·14 U mg ). The Km and Vmax of Tpebgl1 for pNPG were 0·28 mmol l and 470·2 U mg and for pNPArp were 17·30 mmol l and 74·28 U mg . Therefore, it could successfully convert ginsenosides Rb1 and Rb2 into ginsenoside Rd, which has been proven by experiments in this paper then. Tpebgl1 also had good tolerance to glucose and some organic solvents. These made Tpebgl1 a good catalyst candidate for industrial application. Finally, it was applied to convert ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, combined with thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase Tt-Afs and ginsenoside Rd converting β-glucosidase Tpebgl3. A quantity of 10 g l of ginsenoside extract was transformed into 3·93 g l of Rg3 at 90°C, pH 5·0 for 3 h, with a corresponding molar conversion of 98·19%.
The thermostable enzyme Tpebgl1 was found to be a ginsenoside-converting enzyme and successfully applied in the preparation of ginsenoside 20(S)-Rg3 from ginsenoside extract. The three-step cooperate transformation system of ginsenoside extract was established by using Tpebgl1, Tt-Afs (a thermostable ginsenoside Rc converting α-1,6-l-arabinofranosidase) and Tpebgl3 (a thermostable ginsenoside Rb1 converting β-glucosidase).
Converting all the major ginsenosides into protopanaxadiol-type ginsenoside extract would greatly reduce the cost of ginsenoside Rg3 preparation. Enzymes from thermophilic bacteria can meet the requirement of higher reaction temperatures in industrial reactions for substrate solubility promotion and bacterial contamination prevention.
本工作旨在通过三种耐热糖苷酶将人参皂苷提取物转化为具有药理活性的低极性人参皂苷 20(S)-Rg3。
从嗜热栖热菌中发现的 GH1 耐热β-葡萄糖苷酶 Tpebgl1 具有转化人参皂苷 Rb1 和 Rb2 的能力。系统研究了其与人参皂苷转化相关的性质。它对 pNPG（162.20 U mg）和 pNPArp（22.14 U mg）具有高比活性。Tpebgl1 对 pNPG 的 Km 和 Vmax 分别为 0.28 mmol l 和 470.2 U mg，对 pNPArp 的 Km 和 Vmax 分别为 17.30 mmol l 和 74.28 U mg。因此，它可以成功地将人参皂苷 Rb1 和 Rb2 转化为人参皂苷 Rd，本文中的实验证明了这一点。Tpebgl1 对葡萄糖和一些有机溶剂也有良好的耐受性。这使得 Tpebgl1 成为工业应用的良好催化剂候选物。最后，它被应用于将人参皂苷提取物转化为具有药理活性的低极性人参皂苷 20(S)-Rg3，与耐热的人参皂苷 Rc 转化α-1,6-l-阿拉伯呋喃糖苷酶 Tt-Afs 和人参皂苷 Rd 转化β-葡萄糖苷酶 Tpebgl3 结合使用。在 90°C、pH 5.0 下反应 3 小时，10 g l 的人参皂苷提取物转化为 3.93 g l 的 Rg3，相应的摩尔转化率为 98.19%。
发现耐热酶 Tpebgl1 是一种人参皂苷转化酶，并成功应用于从人参皂苷提取物中制备人参皂苷 20(S)-Rg3。通过使用 Tpebgl1、Tt-Afs（一种耐热的人参皂苷 Rc 转化α-1,6-l-阿拉伯呋喃糖苷酶）和 Tpebgl3（一种耐热的人参皂苷 Rb1 转化β-葡萄糖苷酶），建立了人参皂苷提取物的三步协同转化体系。
将所有主要的人参皂苷转化为原人参二醇型人参皂苷提取物将大大降低人参皂苷 Rg3 制备的成本。来自嗜热菌的酶可以满足工业反应中更高反应温度的要求，以促进底物溶解度和防止细菌污染。
