Characterization of the ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides
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This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum KACC 20028T 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., Rb1 and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F2, and Rh2(S) with various pathways. BglAm can effectively transform the ginsenoside Rb1 to gypenoside XVII and Rd to F2; the K m values of Rb1 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 Rg1 into Rg2(S) and Rh1(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.
KeywordsGinsenoside Biotransformation Glycoside hydrolase Actinosynnema mirum
This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ008193), Rural Development Administration, and by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011-0031967), Republic of Korea.
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