Biotransformation of ginsenosides Re and Rg1 into ginsenosides Rg2 and Rh1 by recombinant β-glucosidase

Abstract

Ginsenosides Re and Rg1 were transformed by recombinant β-glucosidase (Bgp1) to ginsenosides Rg2 and Rh1, respectively. The bgp1 gene consists of 2,496 bp encoding 831 amino acids which have homology to the glycosyl hydrolase families 3 protein domain. Using 0.1 mg enzyme ml(-1) in 20 mM sodium phosphate buffer at 37°C and pH 7.0, the glucose moiety attached to the C-20 position of ginsenosides Re and Rg1, was removed: 1 mg ginsenoside Re ml(-1) was transformed into 0.83 mg Rg2 ml(-1) (100% molar conversion) after 2.5 h and 1 mg ginsenoside Rg1 ml(-1) was transformed into 0.6 mg ginsenoside Rh1 ml(-1) (78% molar conversion) in 15 min. Using Bgp1 enzyme, almost all initial ginsenosides Re and Rg1 were converted completely to ginsenosides Rg2 and Rh1. This is the first report of the conversion of ginsenoside Re to ginsenoside Rg2 and ginsenoside Rg1 to ginsenoside Rh1 using the recombinant β-glucosidase.

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides ( S )-Rh 1 and ( S )-Rg 2

“…strain QM49 was isolated. In addition, the near-neutral optimal pH and mild optimal temperature of BglQM are similar to those of other ginsenoside-hydrolyzing family 3 ␤-glucosidases from bacteria (23,32,56).…”

“…The results, which are summarized in Table 3, showed that BglQM was maximally active against PNPGlc followed by ONPGlc but had little effect on various other PNP-and ONP-glycosides, with the exception of PNP-␣-L-arabinofuranoside and PNP-␤-D-fucopyranoside. The specific activity of BglQM against PNP-␣-L-arabinofuranoside is not seen in ginsenoside-transforming glycoside hydrolase family 3 members from Terrabacter ginsenosidimutans (23) and Microbacterium esteraromaticum (32). However, BglQM cannot hydrolyze the arabinofuranoside at the outer position of the C-20 of the ginsenoside Rc, perhaps due to structural differences between the molecules.…”

“…4A). In addition to these PPT-type ginsenosides, BglQM could also transform the PPD-type ginsenosides Rb 1 The metabolic pathways of ginsenoside hydrolysis by BglQM are similar to those of a ␤-glucosidase (Bgpl) from Microbacterium esteraromaticum (32,57), except that Bgpl directly hydrolyzes Rb 1 to (S)-Rg 3 , whereas BglQM hydrolyzed Rb 1 into (S)-Rg 3 via Rd.…”

“…However, these approaches produce nonspecific stereoisomeric mixtures of rare ginsenosides that have low yields and are very difficult to separate. As an alternative, enzymatic methods have been explored as a way to more specifically convert major ginsenosides into pharmacologically active rare ginsenosides (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)52). Furthermore, methods have been developed to efficiently separate a PPT-type ginsenoside mixture (PPTGM) and a PPD-type ginsenoside mixture (PPDGM) from ginseng extracts (34,35).…”

“…Furthermore, methods have been developed to efficiently separate a PPT-type ginsenoside mixture (PPTGM) and a PPD-type ginsenoside mixture (PPDGM) from ginseng extracts (34,35). The total contents of ginsenosides Rg 1 and Re in PPTGM can reach over 82%, and they can be transformed into Rh 1 and Rg 2 , respectively, by cleavage of the glucose moiety at the C-20 position of the aglycon, often by heating or enzymatic hydrolysis (19,32,36,37). To date, however, low product yield and purity, the need for expensive substrates, and inefficient enzyme productivity have limited the mass production of Rh 1 and Rg 2 .…”