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

  • Biocatalysis
  • Published:
Journal of Industrial Microbiology & Biotechnology

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.

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Acknowledgments

This work was supported by the 11th Six Talents Peak Project of Jiangsu Province (Grant No. 2014-JY-011), the Natural Science Foundation of Jiangsu Higher Education Institutions (Grant No. 12KJB220001), the Natural Science Foundation of Jiangsu Province of China (Grant No. BK20131423), the Open Fund of Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals (Grant No. JSBGFC12003), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Postdoctoral Science Foundation of Jiangsu Province (Grant No. 1302022B) as well as the Doctorate Fellowship Foundation of Nanjing Forestry University.

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Authors and Affiliations

  1. College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing, 210037, China

    Jingcong Xie, Dongxia Zhao, Linguo Zhao & Jianjun Pei

  2. Jiangsu Key Laboratory of Biomass Based Green Fuels and Chemicals, 159 Long Pan Road, Nanjing, 210037, China

    Linguo Zhao & Jianjun Pei

  3. Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South Road, Lianyungang, 222001, China

    Wei Xiao, Gang Ding & Zhenzhong Wang

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  1. Jingcong Xie
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  2. Dongxia Zhao
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  3. Linguo Zhao
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Correspondence to Linguo Zhao or Wei Xiao.

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Xie, J., Zhao, D., Zhao, L. et al. Overexpression and characterization of a Ca2+ activated thermostable β-glucosidase with high ginsenoside Rb1 to ginsenoside 20(S)-Rg3 bioconversion productivity. J Ind Microbiol Biotechnol 42, 839–850 (2015). https://doi.org/10.1007/s10295-015-1608-7

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  • DOI: https://doi.org/10.1007/s10295-015-1608-7

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