title: Overexpression and characterization of a Ca(2+) 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 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.
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing, 210037, China.
authors.forename: Jingcong
authors.initials: J
authors.lastname: Xie
authors.forename: Dongxia
authors.initials: D
authors.lastname: Zhao
authors.forename: Linguo
authors.initials: L
authors.lastname: Zhao
authors.forename: Jianjun
authors.initials: J
authors.lastname: Pei
authors.forename: Wei
authors.initials: W
authors.lastname: Xiao
authors.forename: Gang
authors.initials: G
authors.lastname: Ding
authors.forename: Zhenzhong
authors.initials: Z
authors.lastname: Wang
mesh_terms: D001711:Biotransformation; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D036145:Ginsenosides / Q000378:metabolism*; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D036145:Ginsenosides; C442759:ginsenoside Rb1; D001617:beta-Glucosidase; D002118:Calcium
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