A beta-glucosidase from Gordonia terrae was cloned and expressed in Escherichia con. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb-1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg(3) from Rb-1 by the enzyme were pH 6.5, 30 degrees C, 20 mg/ml enzyme, and 4 mg/ml Rb-1. Under these conditions, G. terrae beta-glucosidase completely converted Rb-1 and Re to Rg(3) and Rg(2), respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg(1) to Rh-1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg(3), 1.47 mg/ml Rg(2), and 1.17 mg/ml Rh-1 from Rb-1, Re, and Rg(1), respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30 degrees C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg(2), Rg(3), and Rh-1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg(3), Rg(2), and Rh-1. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.