Jiang Mingxing, Zhu Ling, Xie Shuhan, Ren Zhen, Chen Xiu, Liu Minjiao, Yin Genshen
College of Agriculture and Life Sciences, Kunming University, 2 Pu Xin Road, Kunming, 650214, Yunnan, China.
College of Biological Resources and Food Engineering, Qujing Normal University, 222 San Jiang Road, Qujing, 655000, Yunnan, China.
Curr Microbiol. 2024 Dec 24;82(1):56. doi: 10.1007/s00284-024-04012-0.
β-Glucosidase plays a pivotal role in transforming ginsenosides into specific minor ginsenosides. In this study, total ginsenosides from Panax notoginseng leaves were used as substrates to stimulate the growth of Aspergillus niger NG1306. Transcriptome analysis identified a β-glucosidase gene, Anglu04478 (1455 bp, 484 amino acids, 54.5 kDa, pI = 5.1), as a participant in the ginsenosides biotransformation process. This gene was cloned and expressed in Escherichia coli BL21 Transetta (DE3). The AnGlu04478 protein was purified using a Ni column, and its enzymatic properties were characterized. Purified AnGlu04478 exhibited a specific activity of 32.97 U/mg when assayed against pNPG. Under optimal conditions (pH 4.5, temperature 40 °C), the kinetic parameters, K and V, for pNPG were 1.55 mmol/L and 0.014 mmol/min, respectively. Cu displayed an inhibitory effect on AnGlu04478, whereas Ca, Co, and Ni ions had minimal impact. The enzyme showed tolerance to ethanol and was largely unaffected by glucose feedback inhibition. Testing with ginsenosides as substrates revealed selective hydrolysis at the C3 position of ginsenosides Rb1, Rb2, Rb3, and Rc, with the metabolic pathway delineated as Rb1 → GypXVII, Rb2 → C-O, Rb3 → C-Mx1 → C-Mx, and Rc → C-Mc1. The conversion rates of ginsenosides Rb1, Rb2, Rb3, and Rc varied from 2.58 to 20.63%. With 0.5 U purified enzyme and 0.5 mg total ginsenosides, incubated at 40 °C for 12 h, the conversion rates were 42.6% for GypXVII, 10.4% for C-O, 6.27% for C-Mx1, 26.96% for C-Mx, and 90% for Rc. These results suggest that AnGlu04478 displays substrate promiscuity as a β-glucosidase, thus broadening the potential for ginsenoside biotransformation.
β-葡萄糖苷酶在将人参皂苷转化为特定的次要人参皂苷过程中起着关键作用。在本研究中，三七叶中的总人参皂苷被用作底物来刺激黑曲霉NG1306的生长。转录组分析确定了一个β-葡萄糖苷酶基因Anglu04478（1455 bp，484个氨基酸，54.5 kDa，pI = 5.1），它参与人参皂苷的生物转化过程。该基因被克隆并在大肠杆菌BL21 Transetta（DE3）中表达。使用镍柱纯化AnGlu04478蛋白，并对其酶学性质进行了表征。纯化后的AnGlu04478以对硝基苯-β-D-葡萄糖苷（pNPG）为底物进行测定时，比活性为32.97 U/mg。在最佳条件（pH 4.5，温度40°C）下，pNPG的动力学参数K和V分别为1.55 mmol/L和0.014 mmol/min。铜对AnGlu04478有抑制作用，而钙、钴和镍离子的影响最小。该酶对乙醇具有耐受性，并且在很大程度上不受葡萄糖反馈抑制的影响。用人参皂苷作为底物进行测试时，发现该酶对人参皂苷Rb1、Rb2、Rb3和Rc的C3位有选择性水解作用，其代谢途径为Rb1→GypXVII、Rb2→C-O、Rb3→C-Mx1→C-Mx和Rc→C-Mc1。人参皂苷Rb1、Rb2、Rb3和Rc的转化率在2.58%至20.63%之间。用0.5 U纯化酶和0.5 mg总人参皂苷在40°C下孵育12小时，GypXVII的转化率为42.6%，C-O为10.4%，C-Mx1为6.27%，C-Mx为26.96%，Rc为90%。这些结果表明，AnGlu04478作为一种β-葡萄糖苷酶表现出底物选择性，从而拓宽了人参皂苷生物转化的潜力。
