Substrate specificity and hydrolytic pathways of DT-bgl and PF-bgl for all typical glycosylated PPT ginsenosides
The specific activities of DT-bgl and PF-bgl for all typical glycosylated PPT ginsenosides as substrates are presented in Fig. 1. DT-bgl produced APPT but PF-bgl did not. PF-bgl had activity for Rg2 and F3 but DT-bgl did not. In contrast, DT-bgl had activity for Rg1, Rh1, F1, F5 but PF-bgl did not. The specific activity of PF-bgl for the common substrates, including ginsenosides R1, R2, Re, and Rf, were 7.8-, 19.3-, 6.0-, and 65-fold higher than those of DT-bgl, respectively. DT-bgl converted R1 and Re to R2 and Rg2, respectively, whereas PF-bgl converted both of these ginsenosides to Rg1. In particular, DT-bgl showed the lowest activity for R2 among the PPT-type ginsenosides, indicating that the limiting step for APPT production is the conversion of R2 to Rh1.
DT-bgl had the following hydrolytic pathways of glycosylated PPT-type ginsenosides: R1 → R2 → Rh1 → APPT, Rg1/Rf → Rh1 → APPT, F5 → F1 → APPT, and Re → Rg2. DT-bgl could not hydrolyze the outer sugar rhamnose residue at C-6 in ginsenosides Re and Rg2 and the outer sugar arabinopyranose residue at C-20 in ginsenoside F3. Since PF-bgl could hydrolyze the rhamnose at C-6 in PPT-type ginsenosides and the arabinopyranose at C-20, PF-bgl converted these ginsenosides to Rg1, Rh1, and F1, which were transformed to APPT by DT-bgl. Thus, DT-bgl combined with PF-bgl converted all typical glycosylated PPT ginsenosides to APPT via the hydrolytic pathways of R1 → R2/Rg1 → Rh1 → APPT, Re → Rg1/Rg2 → Rh1 → APPT, Rf → Rh1 → APPT, and F5/F3 → F1 → APPT (Fig. 2).
Conversion of ginsenoside R1 and glycosylated PPT-type ginsenosides in notoginseng root extract to APPT by DT-bgl under the optimized conditions
The optimal temperature and pH for APPT production from R1 using DT-bgl were previously determined to be 80 °C and 6.0, respectively (Lee et al. 2014). APPT production was investigated at enzyme concentrations ranging from 1.0 to 8.0 mg ml−1 at 1.0 mg ml−1 R1 as a substrate for 1.5 h (Additional file 1: Figure S1a). APPT production from R1 increased with increasing DT-bgl concentration up to 4.0 mg ml−1 and reached a plateau at concentrations above 4.0 mg ml−1. APPT production was tested by varying the concentration R1 ranging from 0.5 to 2.0 mg ml−1 at 4.0 mg ml−1 enzyme for 1.5 h (Additional file 1: Figure S1b). APPT production increased with increasing R1 concentration up to 1.0 mg ml−1 and reached a plateau above this level. Thus, the optimal concentrations of DT-bgl and R1 were determined to be 4.0 and 1.0 mg ml−1, respectively. Under the optimized conditions, the time-course reactions for the biotransformation of R1 to APPT were performed with 1.0 mg ml−1 R1 and 4.0 mg ml−1 DT-bgl for 8 h (Fig. 3a). After 6 h, DT-bgl produced 0.40 mg ml−1 APPT, with a productivity of 67 mg l−1 h−1 and a molar conversion of 80.6%, and also produced 0.14 mg ml−1 R2, with a molar conversion of 16.9%. The intermediate R2 was not decreased after 6 h due to the significantly low activity of DT-bgl for R2.
Notoginseng root extract containing 3.78 mg ml−1 total PPT-type ginsenosides was obtained by extraction with 80% methanol. The content of specific PPT-type ginsenosides among total PPT-type ginsenosides in notoginseng root extract followed the order R1 (54.7%, w/w) > Rg1 (37.7%) > Re (7.6%), indicating that efficient hydrolysis of ginsenoside R1 is essential for the increased biotransformation of glycosylated PPT-type ginsenosides in notoginseng root extract to APPT. APPT production was investigated at enzyme concentrations ranging from 1.0 to 8.0 mg ml−1 at 1.0 mg ml−1 total PPT-type ginsenosides in notoginseng root extract as a substrate for 5 h (Additional file 1: Figure S2a). APPT production from PPT-type ginsenosides in ginseng root extract increased proportionally with enzyme concentration up to 5.0 mg ml−1 and then reached a plateau at higher concentrations. APPT production was tested by varying the R1 concentration in notoginseng root extract from 0.5 to 2.0 mg ml−1 at 5.0 mg ml−1 enzyme for 5 h (Additional file 1: Figure S2b). APPT production was maximal at 1.0 mg ml−1 total PPT-type ginsenosides in notoginseng root extract. Thus, the optimal concentrations of DT-bgl and R1 in notoginseng root extract were determined to be 5.0 and 1.0 mg ml−1, respectively. Under the optimized conditions, the time-course reactions of APPT production were performed with 1.0 mg ml−1 total PPT-type ginsenosides and 5.0 mg ml−1 DT-bgl for 6 h (Fig. 3b). After 4 h, APPT production reached a plateau. At this time, DT-bgl produced 0.40 mg ml−1 APPT, with a productivity of 100 mg l−1 h−1 and a molar conversion of 74.9%. The enzyme also produced 0.17 mg ml−1 R2 and 0.06 mg ml−1 Rg2 as by-products, with molar conversions of 18.8 and 6.3%, respectively.
Determination of the added concentration of PF-bgl to DT-bgl for the complete conversion of ginsenoside R1 and glycosylated PPT-type ginsenosides in notoginseng root extract to APPT
The optimal pH and temperature values of DT-bgl and PF-bgl were reported to be 6.0 and 80 °C; and 5.5 and 95 °C, respectively (Lee et al. 2014; Oh et al. 2014). However, the activity of DT-bgl was completely abolished at pH 5.5 and 95 °C. Thus, all reactions were performed at pH 6.0 and 80 °C. DT-bgl at 4.0 mg ml−1 converted 1.0 mg ml−1 R1 to 0.40 mg ml−1 APPT with 0.17 mg ml−1 R2 and 0.02 mg ml−1 Rh1 as by-products for 6 h. To completely convert R2 and Rh1 to APPT, various concentrations of PF-bgl were added to the reaction solution. The residual concentration of R2 decreased with increasing PF-bgl concentration, and neither R2 nor Rh1 was detected at PF-bgl concentrations above 5 µg ml−1 (Fig. 4a). Therefore, 5 µg ml−1 was the optimal concentration of PF-bgl to completely produce APPT from R1 along with 4.0 mg ml−1 DT-bgl.
DT-bgl at 5.0 mg ml−1 converted 1.0 mg ml−1 total PPT-type ginsenosides in notoginseng root extract to 0.40 mg ml−1 APPT with 0.17 mg ml−1 R2 and 0.05 mg ml−1 Rg2 as by-products for 4 h. To completely convert R2 and Rg2 to APPT, various concentrations of PF-bgl were added to the reaction solution. The residual concentrations of R2 and Rg2 decreased with increasing PF-bgl concentration (Fig. 4b). R2 and Rg2 were not detected at concentrations above 0.4 mg ml−1 PF-bgl. Therefore, 0.4 mg ml−1 was the optimal concentration of PF-bgl along with 5.0 mg ml−1 DT-bgl for the complete production of APPT from glycosylated PPT ginsenoside in notoginseng root extract.
Complete conversion of ginsenoside R1 and PPT-type ginsenosides in notoginseng root extract to APPT by DT-bgl combined with PF-bgl under the optimized conditions
The time-course reactions for the biotransformation of ginsenoside R1 to APPT were carried out for 6 h under the optimized conditions of pH 6.0, 80 °C, 1.0 mg ml−1 R1, 4.0 mg ml−1 DT-bgl, and 5 µg ml−1 PF-bgl. DT-bgl combined with PF-bgl converted 1.0 mg ml−1 R1 to 0.5 mg ml−1 APPT, with a productivity of 125 mg l−1 h−1 and a molar conversion of 100% after 4 h (Fig. 5a). The HPLC profiles at 0, 2, and 4 h are presented in Additional file 1: Figure S2.
The time-course reactions for the biotransformation of glycosylated PPT-type ginsenoside in notoginseng root extract to APPT were carried out for 4 h under the optimized conditions of pH 6.0, 80 °C, 1.0 mg ml−1 total PPT-type ginsenosides, 5.0 mg ml−1 DT-bgl, and 0.4 mg ml−1 PF-bgl. DT-bgl combined with PF-bgl converted 1.0 mg ml−1 total PPT-type ginsenosides in notoginseng root extract to 0.63 mg ml−1 APPT, with a productivity of 210 mg l−1 h−1 and a molar conversion of 100% after 3 h (Fig. 5b). The HPLC profiles at 0, 2, and 3 h are presented in Additional file 1: Figure S5.
Complete conversion of ginsenosides F3 and F5 to APPT by DT-bgl combined with PF-bgl
Under the optimized conditions used for the complete conversion of ginsenoside R1 to APPT, the transformations of ginsenosides F3 and F5 to APPT were performed with 1.0 mg ml−1 of each ginsenoside by 4.0 mg ml−1 DT-bgl and 5 µg ml−1 PF-bgl for 8 h. DT-bgl combined with PF-bgl converted 1.0 mg ml−1 F3 and F5 to 0.62 mg ml−1 APPT for 4 and 6 h, with productivities of 155 and 103 mg l−1 h−1 and a molar conversion of 100%, respectively (Additional file 1: Figure S3).