Specific and efficient hydrolysis of all outer glucosyls in protopanaxadiol type and protopanaxatriol type ginsenosides by a beta-glucosidase from Thermoclostridium stercorarium

To develop a new method for enzymatic preparation of minor ginsenosides, T. stercorarium beta-glucosidase (Tsbgl) was characterized and its activities of deglycosylation towards natural ginsenosides were examined. The sub-strates of 1 mmol l-1 were incubated with the enzyme of 38.3 U ml -1 at 65 celcius and pH 5.0. The Km values of Tsbgl for ginsenosides Rb1, Rg1 and pNPG were 0.37 +/- 0.03, 3.26 +/- 0.19, and 1.24 +/- 0.03 mmol l- 1, and the Vmax values were 183.63 +/- 7.15, 85.03 +/- 4.90, and 117.66 +/- 1.96 mu mol mg-1 min-1, respectively. The molar con -version of ginsenosides Rb1, Rb2, Rb3, Rc, Re, Rg1, and Rf by Tsbgl within 6 h was 100%, 50.1%, 42.7%, 92.0%, 57.3%, 67.9%, and 76.8%, respectively. The yield of aglycone protopanaxadiol was 35.5 mu mol l- 1 h-1 for Rb1, while the yields of aglycone protopanaxatriol were 64.2 and 70.4 mu mol l- 1 h-1 for Rg1 and Rf. Tsbgl with good organic solvent tolerance, mild reaction conditions and broad substrate specificity, could completely remove all outer glucosyls at the C-3 and C-20 hydroxyls of protopanaxadiol-type ginsenosides, and the C-6 and C-20 hy-droxyls of protopanaxatriol-type ginsenosides through various pathways, providing a specific and efficient way to produce minor ginsenosides.