Luo S L, Dang L Z, Zhang K Q, Liang L M, Li G H
Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China.
Lett Appl Microbiol. 2015 Jan;60(1):72-8. doi: 10.1111/lam.12339. Epub 2014 Nov 16.
Bacillus subtilis CCTCC AB 2012913 can transform ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. Based on its genome sequence, strain B. subtilis 168 contains three UDP-glycosyltransferase genes. Here, we cloned the three UDP-glycosyltransferase genes (ydhE1, yojK1 and yjiC1) from B. subtilis CCTCC AB 2012913 and expressed in Escherichia coli BL21 (DE3) with His-tag. The crude enzyme extracts were assayed, respectively, for their activities to transform ginsenoside Rh1. Extracts containing enzymes YojK1 and YjiC1 could use ginsenoside Rh1 as a substrate to produce 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, which had an additional glucopyranosyl linked with C-3 over the substrate. Enzyme YjiC1 was purified by affinity chromatography on Ni-NTA His Binding resin. The molecular mass of purified YjiC1 was c. 47 kDa as determined by SDS-PAGE. This is the first report of an in vitro biotransformation of ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol using the recombinant UDP-glycosyltransferase.
The Chinese traditional medicinal plant Panax is reported to have multiple health benefits. Its main active ingredient is saponin, and different saponins have different activity spectrum. In the study, three UDP-glycosyltransferase genes, ydhE1, yojK1 and yjiC1, were cloned from Bacillus subtilis CCTCC AB2012913 and the three genes were expressed in Escherichia coli BL21 (DE3). The enzyme YjiC1 was purified and converted ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol in vitro. The compound is the first saponin possessing β-glucopyranosyl at both C-3 and C-6 sites. We showed that the in vitro biotransformation was effective, and the reaction condition was easy to control. Our research suggests that a diversity of saponins could be generated through efficient and directed enzymatic biotransformation.
枯草芽孢杆菌CCTCC AB 2012913可将人参皂苷Rh1转化为3 - O - β - D - 吡喃葡萄糖基 - 6 - O - β - D - 吡喃葡萄糖基 - 20(S) - 原人参三醇。基于其基因组序列，枯草芽孢杆菌168菌株包含三个尿苷二磷酸糖基转移酶基因。在此，我们从枯草芽孢杆菌CCTCC AB 2012913中克隆了这三个尿苷二磷酸糖基转移酶基因（ydhE1、yojK1和yjiC1），并在带有His标签的大肠杆菌BL21(DE3)中表达。分别测定粗酶提取物将人参皂苷Rh1转化的活性。含有YojK1和YjiC1酶的提取物可以用人参皂苷Rh1作为底物生成3 - O - β - D - 吡喃葡萄糖基 - 6 - O - β - D - 吡喃葡萄糖基 - 20(S) - 原人参三醇，该产物在底物的C - 3位上额外连接了一个吡喃葡萄糖基。通过在Ni - NTA His结合树脂上进行亲和层析纯化YjiC1酶。经SDS - PAGE测定，纯化后的YjiC1分子量约为47 kDa。这是首次报道使用重组尿苷二磷酸糖基转移酶在体外将人参皂苷Rh1生物转化为3 - O - β - D - 吡喃葡萄糖基 - 环 - 6 - O - β - D - 吡喃葡萄糖基 - 20(S) - 原人参三醇。
据报道，中国传统药用植物人参具有多种健康益处。其主要活性成分是皂苷，不同的皂苷具有不同的活性谱。在本研究中，从枯草芽孢杆菌CCTCC AB2012913中克隆了三个尿苷二磷酸糖基转移酶基因ydhE1、yojK1和yjiC1，并在大肠杆菌BL21(DE3)中表达。纯化了YjiC1酶，并在体外将人参皂苷Rh1转化为3 - O - β - D - 吡喃葡萄糖基 - 6 - O - β - D - 吡喃葡萄糖基 - 20(S) - 原人参三醇。该化合物是首个在C - 3和C - 6位点均具有β - 吡喃葡萄糖基的皂苷。我们证明了体外生物转化是有效的，且反应条件易于控制。我们的研究表明，通过高效且定向的酶促生物转化可以产生多种皂苷。
