Ganoderic acid is one of most common types of Ganoderma
triterpenoids and primarily contains five functional groups that are available for modification, including the C-3, C-7, C-12, and C-15 hydroxyl groups and the C-26 carboxyl group 
. In this study, the four Bacillus
GTs (Figure 1
) were assayed for their glycosylation activity toward GAG containing C-3, C-7, and C-12 hydroxyl groups and the C-26 carboxyl group (Figure 2
). After the completion of the biotransformation reaction, the reaction mixture was analyzed using high-performance liquid chromatography (HPLC), the results of which are shown in Figure 3
. The results indicate that BsUGT489 and BsGT110 biotransformed GAG to compound (1
) and compound (2
), with yields of 74% and 35%, respectively. Both BsUGT398 and BtGT_16345 showed little to no activity toward GAG, respectively.
Figure 2. The chemical structure of ganoderic acid G (GAG).
Figure 3. High-performance liquid chromatography (HPLC) results of the biotransformation products of GAG using the four Bacillus GT enzymes. The biotransformation and HPLC conditions are described in the Materials and Methods section.
3. Purification and Identification of Biotransformed Products
The biotransformation reactions of GAG with BsUGT489 and BsGT110 were selected to purify compound (1) and compound (2), respectively. The two biotransformation reactions were scaled up to 20 mL and the products were purified by preparative HPLC. From the 20 mL reaction mixture, 14.4 mg of compound (1) and 7.8 mg of compound (2) were purified. The molecular weights of the purified products were then determined by mass spectrometry. The mass spectrometer showed an [M−H]− ion peak at m/z: 693.5 in the electrospray ionization mass spectrum (ESI-MS), corresponding to the molecular formula C36H54O13. The mass data imply that both compound (1) and compound (2) contain one glucosyl moiety attached to the GAG structure. To identify the structures in advance, the structures of both products were determined using nuclear magnetic resonance (NMR) spectroscopy. The 1H and 13C NMR, including the distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple bond connectivity (HMBC), heteronuclear single quantum coherence (HSQC), nuclear Overhauser effect spectroscopy (NOESY), and correlation spectroscopy (COSY) spectra were obtained. The NMR spectra of compound (1) exhibited characteristic glucosyl signals, with the anomeric proton signal at δH 3.98 (1H, ddd, J = 8.6, 5.6, 2.1 Hz, H-5′), 4.03 (1H, t, J = 8.6 Hz, H-2′), 4.22 (1H, t, J = 8.6 Hz, H-4′), 4.24 (1H, t, J = 8.6 Hz, H-3′), 4.40 (1H, dd, J = 11.9, 5.6 Hz, H-6′a), 4.57 (1H, dd, J = 11.9, 2.1 Hz, H-6′b), and 4.91 (1H, d, J = 8.6 Hz, H-1′); and the anomeric carbon signal at δC 63.0 (C-6′), 71.8 (C-4′), 75.7 (C-2′), 78.4 (C-5′), 78.7 (C-3′), and 107.0 (C-1′). The large coupling constant (8.6 Hz) of the anomeric proton H-1′ (4.91 ppm) indicated the β-configuration. An ether linkage between the H-1′ of glucose and C-3 (4.91/88.3 ppm) of GAG was proven by the HMBC and NOESY (H-3/H-1′) spectra. The structure of compound (1) was thus confirmed to be GAG-3-o-β-glucoside. The signals of compound (2) were attributed to a glucose moiety, with δH 4.03 (1H, ddd, J = 8.8, 4.9, 2.8 Hz, H-5′), 4.20 (1H, t, J = 8.8 Hz, H-2′), 4.29 (1H, t, J = 8.8 Hz, H-3′), 4.35 (1H, t, J = 8.8 Hz, H-4′), 4.36 (1H, dd, J = 11.9, 4.9 Hz, H-6′a), 4.46 (1H, dd, J = 11.9, 2.8 Hz, H-6′b), and 6.33 (1H, d, J = 8.8 Hz, H-1′); and δC 62.1 (C-6′), 71.0 (C-4′), 74.2 (C-2′), 78.5 (C-3′), 79.5 (C-5′), and 96.3 (C-1′). The cross peak of H-1′ with C-26 (6.33/175.0 ppm) in the HMBC spectrum demonstrated the structure of compound (2) to be GAG-26-o-β-glucoside. The structures of the GAG saponins and the biotransformation process are shown in Figure 4.
Figure 4. The biotransformation process of GAG to GAG saponins by the Bacillus GTs.