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Ganoderic acid A (GAA) is a lanostane-type triterpenoid, isolated from medicinal fungus Ganoderma lucidum , and possesses multiple bioactivities. In the present study, GAA was sequentially biotransformed by 2 recently discovered Bacillus glycosyltransferases (GT), BtGT_16345 and BsGT110, and the final product was purified and identified as a new compound, GAA-15,26- O -β-diglucoside, which showed 1024-fold aqueous solubility than GAA. [ABSTRACT FROM AUTHOR]

Ganoderma lucidum is a medicinal fungus whose numerous triterpenoids are its main bioactive constituents. Although hundreds of Ganoderma triterpenoids have been identified, Ganoderma triterpenoid glycosides, also named triterpenoid saponins, have been rarely found. Ganoderic acid A (GAA), a major Ganoderma triterpenoid, was synthetically cascaded to form GAA-15-O-β-glucopyranoside (GAA-15-G) by glycosyltransferase (BtGT_16345) from Bacillus thuringiensis GA A07 and subsequently biotransformed into a series of GAA glucosides by cyclodextrin glucanotransferase (Toruzyme® 3.0 L) from Thermoanaerobacter sp. The optimal reaction conditions for the second-step biotransformation of GAA-15-G were found to be 20% of maltose; pH 5; 60 °C. A series of GAA glucosides (GAA-G2, GAA-G3, and GAA-G4) could be purified with preparative high-performance liquid chromatography (HPLC) and identified by mass and nucleic magnetic resonance (NMR) spectral analysis. The major product, GAA-15-O-[α-glucopyranosyl-(1→4)-β-glucopyranoside] (GAA-G2), showed over 4554-fold higher aqueous solubility than GAA. The present study demonstrated that multiple Ganoderma triterpenoid saponins could be produced by sequential actions of BtGT_16345 and Toruzyme®, and the synthetic strategy that we proposed might be applied to many other Ganoderma triterpenoids to produce numerous novel Ganoderma triterpenoid saponins in the future. [ABSTRACT FROM AUTHOR]

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-β-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-β-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-β-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min−1) was 3-fold higher than that at pH 7 (kcat = 3.8 min−1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids. [ABSTRACT FROM AUTHOR]

Celastrol is a quinone-methide triterpenoid isolated from the root extracts of Tripterygium wilfordii (Thunder god vine). Although celastrol possesses multiple bioactivities, the potent toxicity and rare solubility in water hinder its clinical application. Biotransformation of celastrol using either whole cells or purified enzymes to form less toxic and more soluble derivatives has been proven difficult due to its potent antibiotic and enzyme-conjugation property. The present study evaluated biotransformation of celastrol by four glycosyltransferases from Bacillus species and found one glycosyltransferase (BsGT110) from Bacillus subtilis with significant activity toward celastrol. The biotransformation metabolite was purified and identified as celastrol-29- O -β-glucoside by mass and nuclear magnetic resonance spectroscopy. Celastrol-29- O -β-glucoside showed over 53-fold higher water solubility than celastrol, while maintained 50% of the free radical scavenging activity of celastrol. When using zebrafish as the in vivo animal model, celastrol-29- O -β-glucoside exhibited 50-fold less toxicity than celastrol. To our knowledge, the present study is not only the first report describing the biotransformation of celastrol, but also the first one detailing a new compound, celastrol-29 -O -β-glucoside, that is generated in the biotransformation process. Moreover, celastrol-29 -O -β-glucoside may serve as a potential candidate in the future medicine application due to its higher water solubility and lower toxicity. [ABSTRACT FROM AUTHOR]