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The production of rare ginsenosides has been attracting more and more interests worldwide because of the significant pharmaceutical activities compared with major ginsenosides in wild ginseng.Here, we report the cloning and characterization of a novel ginsenoside-transformation endoglucanase in the glycoside hydrolase family 2 for the first time.The purified recombinant protein catalyzed the conversion of ginsenosides Rbl, Rb2,Re, and Rd into four rare ginsenosides GypXVII, Compound O, Compound MCl, and F2, respectively.Each of them was identified by tandem mass spectrometry.Structure investigation indicated that the recombinant enzyme specifically hydrolyzed the terminal glucose which attached to the C-3 position of ginsenosides Rbl, Rb2, Rc and Rd.Further investigation on enzyme kinetics suggested that the catalytic efficiency of the enzyme followed the order of Rc (7.9 mM-1h-1)>Rb2 (5.6 mM-1h-1)>Rd (0.45 mM-1h-1)>Rb1 (0.21 raM-1n-1).Moreover, 0.5% (w/v)ginseng root extract containing the major ginsenosides Rb1, Rb2, Re and Rd could be almost totally converted into the corresponding rare ginsenosides with a 99% molar yield after 8h treatment at 60℃ and pH8.0.Our results highlight that this novel recombinant thermostable endoglucanase could be used to produce rare ginsenosides Gy(Ⅹ)(Ⅴ)(Ⅱ)pXVII, C-O, C-Mc1, and F2 with a unique specificity and hydrolyzing mechanism.