title: Identification and characterization of a ginsenoside-transforming β-glucosidase from Pseudonocardia sp. Gsoil 1536 and its application for enhanced production of minor ginsenoside Rg2(S).
abstract: The ginsenoside Rg2(S), which is one of the pharmaceutical components of ginseng, is known to have neuroprotective, anti-inflammation, and anti-diabetic effects. However, the usage of ginsenoside Rg2(S) is restricted owing to the small amounts found in white and red ginseng. To enhance the production of ginsenoside Rg2(S) as a 100 gram unit with high specificity, yield, and purity, an enzymatic bioconversion method was developed to adopt the recombinant glycoside hydrolase (BglPC28), which is a ginsenoside-transforming recombinant β-glucosidase from Pseudonocardia sp. strain Gsoil 1536. The gene, termed bglPC28, encoding β-glucosidase (BglPC28) belonging to the glycoside hydrolase family 3 was cloned. bglPC28 consists of 2,232 bp (743 amino acid residues) with a predicted molecular mass of 78,975 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The optimum conditions of the recombinant BglPC28 were pH 7.0 and 37 °C. BglPC28 can effectively transform the ginsenoside Re to Rg2(S); the Km values of PNPG and Re were 6.36 ± 1.10 and 1.42 ± 0.13 mM, respectively, and the Vmax values were 40.0 ± 2.55 and 5.62 ± 0.21 µmol min-1 mg-1 of protein, respectively. A scaled-up biotransformation reaction was performed in a 10 L jar fermenter at pH 7.0 and 30°C for 12 hours with a concentration of 20 mg/ml of ginsenoside Re from American ginseng roots. Finally, 113 g of Rg2(S) was produced from 150 g of Re with 84.0 ± 1.1% chromatographic purity. These results suggest that this enzymatic method could be usefully exploited in the preparation of ginsenoside Rg2(S) in the cosmetics, functional food, and pharmaceutical industries.
authors.affiliation: KAIST Institute for Biocentury, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea; College of Biotechnology, Dalian Polytechnic University, Ganjingzi-qu, Dalian, P. R. China.
authors.forename: Juan
authors.initials: J
authors.lastname: Du
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Chang-Hao
authors.initials: CH
authors.lastname: Cui
authors.affiliation: Intelligent Synthetic Biology Center, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Sung Chul
authors.initials: SC
authors.lastname: Park
authors.affiliation: KAIST Institute for Biocentury, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Jin-Kwang
authors.initials: JK
authors.lastname: Kim
authors.affiliation: College of Biotechnology, Dalian Polytechnic University, Ganjingzi-qu, Dalian, P. R. China.
authors.forename: Hong-Shan
authors.initials: HS
authors.lastname: Yu
authors.affiliation: College of Biotechnology, Dalian Polytechnic University, Ganjingzi-qu, Dalian, P. R. China.
authors.forename: Feng-Xie
authors.initials: FX
authors.lastname: Jin
authors.affiliation: Institute for Brain Disorders, Dalian Medical University, Dalian, P.R. China.
authors.forename: Changkai
authors.initials: C
authors.lastname: Sun
authors.affiliation: KAIST Institute for Biocentury, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea; Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea; Intelligent Synthetic Biology Center, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Sun-Chang
authors.initials: SC
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Hankyoung National University, Chungang-no Anseong-si, Republic of Korea.
authors.forename: Wan-Taek
authors.initials: WT
authors.lastname: Im
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D036145:Ginsenosides / Q000096:biosynthesis*; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D036145:Ginsenosides; D011994:Recombinant Proteins; C026474:ginsenoside Rg2; C440192:ginsenoside-beta-glucosidase; D001617:beta-Glucosidase