利用重组酶高效转化制备稀有人参皂苷C-Mc1和C-Mc

Preparation of rare ginsenoside C-Mc1 and C-Mc by recombinant ginsenosidase

为了高效制备稀有人参皂苷C-Mc和CMc1,调取了Terrabacter ginsenosidimutans中的人参皂苷酶基因,构建载体GST-bgpA,并转化到Escherichia coli C41(DE3)中表达,得到人参皂苷3-O-β-D-葡萄糖苷酶。该酶分子质量为72.4 kDa,转化人参皂苷Rc的最适pH为7.0,最适温度为40℃,米氏常数K_(m)值为6.25 mmol/L,V_(m)值为19.6 mmol/(h·L)。为了降低成本,利用粗酶转化人参皂苷Rc制备C-Mc1和C-Mc。粗酶制备C-Mc1的反应条件:20 g/L的人参皂苷Rc在40℃下酶反应24 h;制备C-Mc的反应条件:5 g/L的人参皂苷Rc在40℃下酶反应60 h。此条件下,粗酶转化制备并分离纯化得到C-Mc1和C-Mc的单体,并经HPLC检测,其纯度均在90%以上。C-Mc1的酶转化制备:产物得到C-Mc和C-Mc1,其摩尔得率分别为74.9%和11.1%。C-Mc的酶转化制备:C-Mc的摩尔得率为93.2%。该研究为稀有人参皂苷C-Mc和C-Mc1产业化提供了依据。

For efficient preparation of rare ginsenoside C-Mc1 and C-Mc from ginsenoside Rc,a ginsenoside-3-O-β-D-glucosidase from Terrabacter ginsenosidimutans Gsoil 3082was cloned and expressed in Escherichia coli C41(DE3).The molecular weight of the purified enzyme was 72.4 kDa with the optimum pH and temperature of 7.0 and 40℃.Its Kvalue was 6.25 mmol/L,Vwas 19.6 mmol/(h·L).To lower the cost,preparation of ginsenoside C-Mc1 and C-Mc was carried out with the crude enzyme preparation.The results showed that when 20 g/L ginsenoside Rc was enzymatically catalyzed at 40℃for 24 h,C-Mc1 and C-Mc monomers were obtained with a molar yield of 74.9%for C-Mc1,and 11.1%for C-Mc.When 5 g/L ginsenoside Rc was enzymatically catalyzed at 40℃for 60 h,ginsenoside C-Mc was obtained with a molar yield of 93.2%.The purities of resultant C-Mc1 and C-Mc were both over 90%according to HPLC analysis.The results provide references for industrial production of C-Mc1 and C-Mc.