Xie Jingcong, Zhao Dongxia, Zhao Linguo, Pei Jianjun, Xiao Wei, Ding Gang, Wang Zhenzhong
College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing, 210037, China.
J Ind Microbiol Biotechnol. 2015 Jun;42(6):839-50. doi: 10.1007/s10295-015-1608-7. Epub 2015 Apr 3.
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 Ca(2+). 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 Ca(2+) 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.
克隆了嗜热栖热袍菌DSM 13995的耐热β-葡萄糖苷酶基因，并在大肠杆菌中进行了过量表达。重组β-葡萄糖苷酶在LB培养基中的活性为21 U/mL。对重组β-葡萄糖苷酶进行了纯化，其分子量约为81 kDa。最佳活性在pH 5.0和90℃条件下，Ca(2+)可提高该酶的热稳定性。该β-葡萄糖苷酶对人参皂苷Rb1 C-20位碳上的外葡萄糖吡喃糖基和内葡萄糖吡喃糖基具有高选择性切割作用，可产生具有药理活性的次要人参皂苷20(S)-Rg3。在90℃和pH 5.0的反应中，10 g/L人参皂苷Rb1在90分钟内转化为6.93 g/L的Rg3，相应的摩尔转化率为97.9%，Rg3产率为4620 mg/L/h。本研究首次报道了一种利用Ca(2+)提高热稳定性的GH3家族酶，也是首次报道在高温下利用耐热β-葡萄糖苷酶将人参皂苷Rb1高底物浓度生物转化为人参皂苷20(S)-Rg3 。
