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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 (k cat ) of BsGT110 toward GAA at pH 6 (k cat = 11.2 min -1 ) was 3-fold higher than that at pH 7 (k cat = 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.

8-Hydroxydaidzein (8-OHDe), an ortho -hydroxylation derivative of soy isoflavone daidzein isolated from some fermented soybean foods, has been demonstrated to possess potent anti-inflammatory activity. However, the isoflavone aglycone is poorly soluble and unstable in alkaline solutions. To improve the aqueous solubility and stability of the functional isoflavone, 8-OHDe was glucosylated with recombinant amylosucrase of Deinococcus geothermalis (DgAS) with industrial sucrose, instead of expensive uridine diphosphate-glucose (UDP-glucose). One major product was produced from the biotransformation, and identified as 8-OHDe-7-α-glucoside, based on mass and nuclear magnetic resonance spectral analyses. The aqueous solubility and stability of the isoflavone glucoside were determined, and the results showed that the isoflavone glucoside was almost 4-fold more soluble and more than six-fold higher alkaline-stable than 8-OHDe. In addition, the anti-inflammatory activity of 8-OHDe-7-α-glucoside was also determined by the inhibition of lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells. The results showed that 8-OHDe-7-α-glucoside exhibited significant and dose-dependent inhibition on the production of nitric oxide, with an IC 50 value of 173.2 µM, which remained 20% of the anti-inflammatory activity of 8-OHDe. In conclusion, the well-soluble and alkaline-stable 8-OHDe-7-α-glucoside produced by recombinant DgAS with a cheap substrate, sucrose, as a sugar donor retains moderate anti-inflammatory activity, and could be used in industrial applications in the future.

Functional bacteria that could biotransform triterpenoids may exist in the diverse microflora of fish intestines. Ganoderic acid A (GAA) is a major triterpenoid from the medicinal fungus Ganoderma lucidum . In studying the microbial biotransformation of GAA, dozens of intestinal bacteria were isolated from the excreta of zebrafish. The bacteria's ability to catalyze GAA were determined using ultra-performance liquid chromatography analysis. One positive strain, GA A07, was selected for functional studies. GA A07 was confirmed as Bacillus sp., based on the DNA sequences of the 16S rRNA gene. The biotransformed metabolite was purified with the preparative high-performance liquid chromatography method and identified as GAA-15- O -β-glucoside, based on the mass and nuclear magnetic resonance spectral data. The present study is the first to report the glycosylation of Ganoderma triterpenoids. Moreover, 15- O -glycosylation is a new microbial biotransformation of triterpenoids, and the biotransformed metabolite, GAA-15- O -β-glucoside, is a new compound.

Biotransformation of the soy isoflavone genistein by sequential 3'-hydroxylation using recombinant Escherichia coli expressing tyrosinase from Bacillus megaterium and then methylation using another recombinant E. coli expressing O -methyltransferase from Streptomyces peucetius was conducted. The results showed that two metabolites were produced from the biotransformation, identified as 5,7,4'-trihydroxy-3'-methoxyisoflavone and 5,7,3'-trihydroxy-4'-methoxyisoflavone, respectively, based on their mass and nuclear magnetic resonance spectral data. 5,7,4'-Trihydroxy-3'-methoxyisoflavone showed potent antiproliferative activity toward mouse B16 melanoma cells with an IC 50 value of 68.8 μM. In contrast, the compound did not show any cytotoxicity toward mouse normal fibroblast cells, even at 350 μM concentration. The results of the present study offer insight on the production of both 5,7,4'-trihydroxy-3'-methoxyisoflavone and 5,7,3'-trihydroxy-4'-methoxyisoflavone by two recombinant E. coli strains and the potential anti-melanoma applications of 5,7,4'-trihydroxy-3'-methoxyisoflavone.

The present study describes the biotransformation of a commercially available crude extract of soy isoflavones, which contained significant amounts of the soy isoflavone glycosides daidzin and genistin, by recombinant Escherichia coli expressing tyrosinase from Bacillus megaterium . Two major products were isolated from the biotransformation and identified as 3'-hydroxydaidzin and 3'-hydroxygenistin, respectively, based on their mass and nuclear magnetic resonance spectral data. The two 3'-hydroxyisoflavone glycosides showed potent 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity with IC 50 values of 7.4 and 9.8 μM for 3'-hydroxydaidzin and 3'-hydroxygenistin, respectively. The free radical scavenging activities of the two 3'-hydroxyisoflavone glycosides were, respectively, 120 and 72 times higher than the activity of their precursors, daidzin and genistin, and were also stronger than the activity of ascorbic acid, which showed an IC 50 value of 15.1 μM. This is the first report of the bio-production and potential antioxidant applications of both 3'-hydroxydaidzin and 3'-hydroxygenistin.