Background

The minor ginsenosides such as F₂, C-K, Rh2, and Rg3 make up less than 1% of the total ginseng extract; however, many studies have shown that ginsenoside F₂ has anti-cancer and antioxidant effects and improves dementia and atopic dermatitis. Therefore, the enhanced production of this minor ginsenoside is a promising approach for the pharmaceutical industry. In this study, we found and cloned a novel glycoside hydrolase gene for the gram-scale production of ginsenoside F₂.

Methods and Results

Novosphingobium aromaticivorans, which was isolated from deep-terrestrial-subsurface sediments, has shown ginsenoside-converting ability. From this bacterium, a novel glycoside hydrolase, named BglNar, was found that can efficiently biotransform the protopanaxatriol (PPD-mix-type) major ginsenosides (Rb₁, Rb₂ Rc, and Rd) into the minor ginsenoside F₂. This enzyme was cloned, expressed in Escherichia coli BL21, and characterized. The BglNar comprises 439 amino acid and belongs to the glycoside hydrolase family 1. The K_m value of p-nitrophenyl-β-D-glucopyranoside was 9.06 ± 0.28 and the V_max value was 24.0 ± 0.34 µmol/min/mg of protein. For gram-scale production of minor ginsenoside F₂, crude PPD-mix-type ginsenosides (2 g/400 mL) were treated with BglNar and 1.14 g of F₂ with final purity of 82.5 ± 1.3%, was obtained after purification using a column packed with HP20 resin.

Conclusion

Our preliminary data demonstrate that the gram production of ginsenoside F₂ using a recombinant enzyme will enhance the health benefits of Panax ginseng. This is the first study describing the gram-scale production of F₂ from PPD-mix using a single novel ginsenoside-transforming β-glucosidase of the GH family 1. Significance and Impact of Study: In most of the previous studies, researchers have been used the combination of enzymes for production of F₂, while in this study we found the possibility of gram-scale production of F₂ from PPD-mix-type major ginsenosides using a single recombinant enzyme, thus simplifying the production and reducing the cost.

中文翻译：

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使用来自新鞘氨醇的新型重组糖苷水解酶从 PPD 混合型主要人参皂苷生产次要人参皂苷 F2

背景

F ₂、CK、Rh2、Rg3等次要人参皂苷占人参总提取物的比例不到1%；然而，许多研究表明，人参皂苷F ₂具有抗癌和抗氧化作用，改善痴呆和特应性皮炎。因此，提高这种次要人参皂苷的产量对于制药行业来说是一种很有前途的方法。在这项研究中，我们发现并克隆了一种用于克级规模生产人参皂苷 F ₂的新型糖苷水解酶基因。

方法和结果

从深地地下沉积物中分离出的新鞘氨醇菌已显示出人参皂苷转化能力。从这种细菌中，发现了一种名为 BglNar 的新型糖苷水解酶，它可以有效地将原人参三醇（PPD 混合型）主要人参皂苷（Rb ₁、Rb ₂ Rc 和 Rd）生物转化为次要人参皂苷 F ₂。该酶被克隆，在大肠杆菌BL21中表达，并进行了表征。所述BglNar包括439氨基酸，属于糖苷水解酶家族1 ķ_米的值p -nitrophenyl- β -D-吡喃葡糖苷为9.06±0.28和V_最大值为 24.0 ± 0.34 µmol/min/mg 蛋白质。对于少量人参皂苷 F _{2 的}克级生产，将粗 PPD 混合型人参皂苷 (2 g/400 mL) 用 BglNar 处理，并在使用纯化后获得最终纯度为 82.5 ± 1.3% 的1.14 g F ₂用 HP20 树脂填充的色谱柱。

结论

我们的初步数据表明，使用重组酶生产人参皂苷 F ₂将增强人参的健康益处。这是第一项研究，描述了使用 GH 家族 1 的单一新型人参皂苷转化 β-葡萄糖苷酶从 PPD-mix 中克级生产 F _2。研究的意义和影响：在之前的大多数研究中，研究人员已经使用酶的组合来生产 F ₂，而在本研究中，我们发现使用单一重组酶从 PPD 混合型主要人参皂苷中进行克级规模生产 F ₂的可能性，从而简化了生产并降低了成本。