Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. beta-glucosidase that transforms ginsenoside Rb1 into the rare gypenosides XVII and LXXV.
A new beta-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082(T)) obtained from the soil of a ginseng farm was characterized, and the gene, bgpA (1,947 bp), was cloned in Escherichia coli. The enzyme catalyzed the conversion of ginsenoside Rb1 {3-O-[beta-D-glucopyranosyl-(1-2)-beta-D-glucopyranosyl]-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-beta-D-glucopyranosyl-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[beta-v-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, beta-glucosidase had apparent K(m) values of 4.2 +/- 0.8 and 0.14 +/- 0.05 mM and V(max) values of 100.6 +/- 17.1 and 329 +/- 31 micromol x min(-1) x mg of protein(-1) against p-nitrophenyl-beta-D-glucopyranoside and Rb1, respectively. The enzyme catalyzed the hydrolysis of the two glucose moieties attached to the C-3 position of ginsenoside Rb1, and the outer glucose attached to the C-20 position at pH 7.0 and 37 degrees C. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose of C-20. These results indicated that BgpA selectively and sequentially converts ginsenoside Rb1 to the rare ginsenosides gypenoside XVII, gypenoside LXXV, and then C-K. Herein is the first report of the cloning and characterization of a novel ginsenoside-transforming beta-glucosidase of the glycoside hydrolase family 3.
An
Dong-Shan
DS
Biological Resource Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Republic of Korea.
Cui
Chang-Hao
CH
Lee
Hyung-Gwan
HG
Wang
Liang
L
Kim
Sun Chang
SC
Lee
Sung-Taik
ST
Jin
Fengxie
F
Yu
Hongshan
H
Chin
Young-Won
YW
Lee
Hyeong-Kyu
HK
Im
Wan-Taek
WT
Kim
Song-Gun
SG
D000192:Actinomycetales / Q000201:enzymology* / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D036145:Ginsenosides / Q000378:metabolism*; D029774:Gynostemma / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D015394:Molecular Structure; D005894:Panax; D010802:Phylogeny; D010936:Plant Extracts / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D012988:Soil Microbiology; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups); D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D036145:Ginsenosides; D008565:Membrane Proteins; D010936:Plant Extracts; C474918:gypenoside; C442759:ginsenoside Rb1; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase; D001617:beta-Glucosidase