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

• Published in: Journal of industrial microbiology & biotechnology Vol. 42; no. 6; pp. 839 - 850
• Main Authors: Xie, Jingcong, Zhao, Dongxia, Zhao, Linguo, Pei, Jianjun, Xiao, Wei, Ding, Gang, Wang, Zhenzhong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01-06-2015; Springer Berlin Heidelberg
• Subjects: beta-glucosidase; Biocatalysis; Biochemistry; Bioinformatics; Biomedical and Life Sciences; Biotechnology; biotransformation; calcium; carbon; Escherichia coli; gene overexpression; genes; Genetic Engineering; Inorganic Chemistry; Life Sciences; Microbiology; molecular weight; temperature; thermal stability; Thermotoga; Recombinant enzyme; Enzymatic characterization; Biotransformation; Ginsenoside 20; Rg3 preparation
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-015-1608-7

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.