Characterization of the ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides.
This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum KACC 20028(T) in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a β-glucosidase (BglAm) belonging to the glycoside hydrolase family 3 was cloned. bglAm consisted of 1,830 bp (609 amino acid residues) with a predicted molecular mass of 65,277 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The recombinant BglAm was purified with a GST·bind agarose resin and characterized. The optimum conditions of the recombinant BglAm were pH 7.0 and 37 °C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb(1) and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F(2), and Rh(2)(S) with various pathways. BglAm can effectively transform the ginsenoside Rb(1) to gypenoside XVII and Rd to F(2); the K (m) values of Rb(1) and Rd were 0.69 ± 0.06 and 0.45 ± 0.02 mM, respectively, and the V (max) values were 16.13 ± 0.29 and 51.56 ± 1.35 μmol min(-1) mg(-1) of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg(1) into Rg(2)(S) and Rh(1)(S) hydrolyzing the attached glucose moiety at the C6 and C20 positions, respectively. These various ginsenoside-hydrolyzing pathways of BglAm may assist in producing the minor ginsenosides from abundant major ginsenosides.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Chang-Hao
CH
Cui
Sun-Chang
SC
Kim
Wan-Taek
WT
Im
D000192:Actinomycetales / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D036145:Ginsenosides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D036145:Ginsenosides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase