Overexpression and characterization of a Ca2+ activated thermostable β-glucosidase with high ginsenoside Rb1 to ginsenoside 20(S)-Rg3 bioconversion productivity

Abstract

The thermostable β-glucosidase gene from Thermotoga petrophila DSM 13995 was cloned and overexpressed in Escherichia coli. The activity of the recombinant β-glucosidase was 21 U/mL in the LB medium. Recombinant β-glucosidase was purified, and its molecular weight was approximately 81 kDa. The optimal activity was at pH 5.0 and 90 °C, and the thermostability of the enzyme was improved by Ca2+. The β-glucosidase had high selectivity for cleaving the outer and inner glucopyranosyl moieties at the C-20 carbon of ginsenoside Rb1, which produced the pharmacologically active minor ginsenoside 20(S)-Rg3. In a reaction at 90 °C and pH 5.0, 10 g/L of ginsenoside Rb1 was transformed into 6.93 g/L of Rg3 within 90 min, with a corresponding molar conversion of 97.9 %, and Rg3 productivity of 4620 mg/L/h. This study is the first report of a GH3-family enzyme that used Ca2+ to improve its thermostability, and it is the first report on the high substrate concentration bioconversion of ginsenoside Rb1 to ginsenoside 20(S)-Rg3 by using thermostable β-glucosidase under high temperature.