Zheng Fei, Zhao Huanxi, Wang Nan, Zhong Peng, Zhou Kailu, Yu Shanshan
Jilin Ginseng Academy, Changchun University of Chinese Medicine, 130117, Changchun, China.
AMB Express. 2022 Oct 28;12(1):136. doi: 10.1186/s13568-022-01473-z.
A novel endoglucanase (BcelFp) was identified from Fervidobaterium pennivorans DSM9078 which had biotransformation activity for protopanaxadiol (PPD)-type ginsenosides. Sequence analysis of BcelFp revealed that it could be classified into glycoside hydrolase family 5 (GH5). The gene encoding a 323-amino acid protein was cloned and expressed in Escherichia coli. The recombinant enzyme was purified, and its molecular weight was approximate 37 kDa. The recombinant BcelFp exhibited an optimal activity at 95 C and pH 5.5 and showed high thermostability. The endoglucanase had high selectivity for cleaving the outer glucose moiety at the C3 carbon of ginsenoside Rb1, Rb2, Rc and Rd, which produced stronger pharmacologically active gypenoside XVII (GypXVII), Compound O (CO), Compound Mc1 (CMc1) and F2, respectively. The K values for Rb1, Rb2, Rc and Rd were 3.66 ± 0.04 µM, 4.02 ± 0.12 µM, 5.95 ± 0.03 µM, 0.67 ± 0.006 µM, respectively. The k/K value of BcelFp for ginsenoside Rd was 27.91 mMs, which was much higher than that of the previously enzymes. This study was the first report of the highly efficient and selective transformation of GypXVII, CO, CMc1 and F2 from Rb1, Rb2, Rc and Rd by a GH5-family thermophilic endoglucanase.
从嗜热栖热菌DSM9078中鉴定出一种新型内切葡聚糖酶（BcelFp），其对原人参二醇（PPD）型人参皂苷具有生物转化活性。BcelFp的序列分析表明，它可归类于糖苷水解酶家族5（GH5）。编码一种323个氨基酸蛋白质的基因被克隆并在大肠杆菌中表达。重组酶被纯化，其分子量约为37 kDa。重组BcelFp在95℃和pH 5.5时表现出最佳活性，并具有高耐热性。该内切葡聚糖酶对人参皂苷Rb1、Rb2、Rc和Rd的C3碳上的外侧葡萄糖部分具有高选择性切割作用，分别产生药理活性更强的绞股蓝皂苷XVII（GypXVII）、化合物O（CO）、化合物Mc1（CMc1）和F2。Rb1、Rb2、Rc和Rd的K值分别为3.66±0.04 μM、4.02±0.12 μM、5.95±0.03 μM、0.67±0.006 μM。BcelFp对人参皂苷Rd的k/K值为27.91 mM⁻¹s⁻¹，远高于先前的酶。本研究首次报道了一种GH5家族嗜热内切葡聚糖酶高效、选择性地将Rb1、Rb2、Rc和Rd转化为GypXVII、CO、CMc1和F2。
